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Which is the binding site motif of Sp1? | Sp1 binds to a GC-rich sequence element containing the decanucleotide consensus sequence 5′-(G/T)GGGCGG(G/A)(G/A)(C/T)-3′ (GC box element) in double stranded DNA (dsDNA). Gel shift competition studies and DNase I footprinting analyses revealed that Sp1 specifically interacts with the CACCC motif. | We have previously shown that mutations in the GGAA core motif of the Ets1
binding site, EBSI, or deletion of EBSI, reduced basal and Tax1 transactivation
of the PTHrP P2 promoter. Here we demonstrate that, in addition to EBSI, a
CACCC-like motif located between -53 and -58 is required for full basal activity
of this promoter in Jurkat T-cells. Site-specific mutations in the CACCC motif
decreased promoter activity approximately 5-fold. In an effort to identify
transcription factors that bind to the CACCC element, we found that purified
human Sp1, as well as Sp1 in HeLa nuclear extract, can specifically bind to a
DNA probe that corresponds to the PTHrP-specific sequence between -94 and -34.
Gel shift competition studies and DNase I footprinting analyses revealed that
Sp1 specifically interacts with the CACCC motif. In the presence of Ets1, the
mobility of the Sp1-specific gel shift complex with the PTHrP DNA decreased.
DNase I footprint analysis of this gel shift complex showed an extended
footprint over both the Sp1 and the Ets1 binding site, demonstrating that Sp1
and Ets1 form a ternary complex with the PTHrP DNA. Cotransfection of an Ets1
and Sp1 expression vector into Drosophila Schneider cells demonstrated that Sp1
can functionally cooperate with Ets1 to transactivate the PTHrP promoter. We
conclude from these data that Ets1 and Sp1 can cooperatively regulate PTHrP P2
promoter activity. We have previously shown that the proximal 2-kb sequence of the fatty acid
synthase (FAS) promoter transfected into rat adipocytes was highly sensitive to
the cellular context, displaying an overactivity in obese (fa/fa) versus lean
Zucker rat adipocytes. Using deletional analysis, we show here that FAS promoter
activity mainly depends on a region from -200 to -126. This sequence exerts a
strong negative effect on FAS promoter in adipocytes from lean rats but not in
those from obese rats, resulting in a marked overtranscriptional activity in the
latter cells. This region, fused to a heterologous promoter, the E1b TATA box,
induced differential levels of gene reporter activity in lean and obese rat
adipocytes, indicating it harbors fa-responsive element(s). Whatever the rat
genotype, adipocyte nuclear proteins were shown to footprint the same protected
sequence within the fa-responsive region, and supershift analysis demonstrated
that Sp1 or Sp1-like proteins were bound to this DNA subregion. Compelling
evidence that the Sp1 binding site contained in this sequence was implicated in
the differential promoter activity in lean versus obese rats, was provided by
the observation that mutations at this Sp1 site induced a 2.5-fold increase in
FAS promoter activity in adipocytes from lean rats, whereas they had no effect
in adipocytes from obese rats. The polycythemic strain of the spleen focus-forming virus (SFFVp) contains the
most potent murine retroviral enhancer configuration known so far for gene
expression in myeloerythroid hematopoietic cells. In the present study, we
mapped two crucial elements responsible for the high activity of the SFFVp
enhancer to an altered upstream control region (UCR) containing a GC-rich motif
(5'-GGGCGGG-3') and to a unique enhancer core (5'-TGCGGTC-3'). Acquisition of
these motifs accounts for half of the activity of the complete retroviral
enhancer in hematopoietic cells, irrespective of the developmental stage or
lineage. Furthermore, the UCR motif contains the major determit for the
enhancer activity of SFFVp in embryonic stem (ES) cells. Using electrophoretic
mobility shift assays, we show that the UCR of SFFVp, but not of Friend murine
leukemia virus, is targeted by the ubiquitous transcriptional activator, Sp1.
The core motif of SFFVp creates a specific and high-affinity target for
polyomavirus enhancer binding protein/core binding factor (PEBP/CBF) and
excludes access of CAAT/enhancer binding protein. Cotransfection experiments
with ES cells imply that PEBP/CBF cooperates with the neighboring element, LVb
(the only conserved Ets consensus in the SFFVp enhancer), and that the Sp1 motif
in the UCR stimulates transactivation through the Ets-PEBP interaction. Putative
secondary structures of the retroviral enhancers are proposed based on these
data. Adipocyte amino acid transporter (AAAT) is induced during the 3T3-L1
preadipocyte differentiation process. In the -1819-bp 5'-upstream flanking
region of the AAAT genomic gene, six DNase I protected sites were identified by
using the 3T3-L1 adipocyte nuclear extract. Results of chloramphenicol
acetyltransferase (CAT) expression from the chimeric AAAT promoter-driven CAT
reporter gene indicated that one protein binding site, from -68 to -26, was
essential for the promoter activity. However, this protein binding site does not
contain recognition sites of the transcription factors important for adipocyte
differentiation, i.e., the C/EBP or PPAR family. Further analysis revealed that
the DNA sequence, TTCAAGTCCCGCCCTCCGCT from -65 to -46, was the cis-element
essential and partially sufficient for inducible activity of the AAAT gene
promoter. SP1 is a ubiquitous transcription factor that is involved in the regulation of
various house-keeping genes. It is known that it acts by binding to a
double-stranded consensus motif. Here, we have discovered that SP1 binds also to
a non-canonical DNA structure, a G-quadruplex, with high affinity. In
particular, we have studied the SP1 binding site within the promoter region of
the c-KIT oncogene and found that this site can fold into an anti-parallel
two-tetrad G-quadruplex. SP1 pull-down experiments from cellular extracts,
together with biophysical binding assays revealed that SP1 has a comparable
binding affinity for this G-quadruplex structure and the canonical SP1 duplex
sequence. Using SP1 ChIP-on-chip data sets, we have also found that 87% of SP1
binding sites overlap with G-quadruplex forming sequences. Furthermore, while
many of these immuoprecipitated sequences (36%) even lack the minimal SP1
consensus motif, 5'-GGGCGG-3', we have shown that 77% of them are putative
G-quadruplexes. Collectively, these data suggest that SP1 is able to bind both,
canonical SP1 duplex DNA as well as G-quadruplex structures in vitro and we
hypothesize that both types of interactions may occur in cells. |
List autoimmune disorders associated with GAD65 autoantibodies. | Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. | The processes that lead to the production of islet cell autoantibodies in
insulin-dependent (type 1) diabetes mellitus (IDDM) are largely unknown. Humoral
autoimmunity may be the result of an antigen-independent polyclonal B cell
activation, or a consequence of an antigen driven B cell activation and
selection for the antigen. We have analysed the gene elements encoding the
immunoglobulin variable regions of seven human monoclonal islet cell antibodies
(MICA) 1-7 directed to the major islet autoantigen glutamate decarboxylase
(GAD65). These autoantibodies were derived from two patients with newly
diagnosed IDDM. The variable gene regions of the MICA revealed different
sequences, and no relation between V gene usage and shared epitope recognition
of the MICA was evident. An elevated usage of VH 1, VH 4 and Vlambda 2 gene
segments was observed. The underrepresentation of VH 3 family members in the
MICA discriminated them from most autoantibodies. The high relative avidities
for GAD65 of MICA 1, 3, 4 and 6 and their high, nonrandom ratio of replacement
versus silent mutations in the antigen binding regions indicated that the
humoral response to GAD65 is driven by the antigen. MICA 2, 5 and 7 showed as
well an excess of replacement mutations in the antigen binding regions, but
revealed lower relative avidities for their antigen. Since these clones
accumulated many somatic mutations in their variable gene regions, they may be
characteristic for later stages of the autoimmune disease. The results suggest
that, in humans, an antigen driven B cell activation and affinity maturation
process may contribute to the production of GAD65-autoantibodies found in
patients with IDDM. The GM2-1 islet ganglioside has been sequenced, found to be a novel ganglioside
structure with a sialic acid moiety in the terminal position and two residues of
non-acetylated galactosamine and also shown to be a target of autoantibodies in
a subset of ICA+ relatives of type 1 diabetic patients who subsequently
progressed to the overt disease. In the present study we determined whether
antibodies to GM2-1 or to other pancreatic gangliosides (a) are also expressed
at disease onset and (b) are correlated with other diabetes-associated
autoantibodies. Pancreatic gangliosides were extracted from human pancreas and
purified by thin layer chromatography (TLC). Anti-ganglioside autoantibodies
were determined using an indirect immunoperoxidase technique performed directly
on TLC plates in the following groups of patients: (a) newly diagnosed type 1
diabetic subjects before insulin therapy (n = 45); all were tested for GAD65
autoantibodies in a fluid-phase RIA using 35S-methionine-labelled recombit
human GAD65. Of these patients, 24 were also tested for insulin autoantibodies
(IAA) by a competitive fluid phase radioimmunoassay and 21 were tested for GAD67
reactivity. (b) Forty-two age- and sex-matched normal control subjects.
Autoantibodies to GM2-1, but not to other pancreatic gangliosides (GM3, GD3,
GD1a), were expressed in 31 of 45 new-onset type 1 diabetic subjects and in one
of 42 normal controls (P < 0.01), while anti-GAD65, IAA and anti-GAD67 were
found in 31 of 45, 12 of 24 and three of 21 patients respectively, but not in
the control group of subjects. Interestingly, occurrence of GM2-1 autoantibodies
was significantly correlated (P < 0.005) with positivity for GAD65
autoantibodies, but not for IAA or GAD67 autoantibodies. It is of note that both
GAD and gangliosides are mainly expressed in islets and in neuronal tissues and,
therefore, type 1 diabetes may be regarded as a neuroendocrine autoimmune
disease. BACKGROUND: Anti-glutamic acid decarboxylase antibody (GAD-ab)-associated
cerebellar ataxia is a rare neurological disorder characterized by cerebellar
symptoms concomitant with high GAD-ab levels in serum and cerebrospinal fluid
(CSF).
CASE REPORT: We report on 2 female siblings (aged 74 and 76 years) presenting
with gradual progression of rotational vertigo, gait ataxia and vertical
diplopia, continuously progressing for 6 months and 6 years, respectively.
Autoimmune laboratory examinations showed remarkably increased serum and CSF
GAD-ab levels. Their medical histories revealed late-onset type 1 diabetes
mellitus (T1DM) and other concomitant autoimmune disorders (Grave's disease,
Hashimoto's thyroiditis). Cerebral MRI and laboratory examinations were
unremarkable. The diagnosis of GAD-ab-associated cerebellar ataxia with
particular brainstem involvement was established in both women. After the
exclusion of an underlying maligcy, immunosuppressive therapy has been
initiated in both patients, which resulted in stabilization in one and in
clinical improvement in the other patient.
DISCUSSION: The unique association of autoantibody-mediated cerebellar ataxia
and late-onset T1DM in 2 siblings with similar clinical and paraclinical
phenotypes strengthens the concept that hereditary factors might play a relevant
role also in autoimmune diseases so far considered to be sporadic. Moreover, the
occurrence of continuous vertical diplopia broadens the clinical spectrum of
GAD-ab-associated neurological syndromes. Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be
found in patients with type 1 diabetes and a number of neurological disorders,
including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The
detection of disease-specific autoantibody epitopes led to the hypothesis that
distinct GAD autoantibodies may elicit specific neurological phenotypes. We
explored the in vitro/in vivo effects of well-characterized monoclonal GAD
antibodies. We found that GAD autoantibodies present in patients with stiff
person syndrome (n = 7) and cerebellar ataxia (n = 15) recognized an epitope
distinct from that recognized by GAD autoantibodies present in patients with
type 1 diabetes mellitus (n = 10) or limbic encephalitis (n = 4). We
demonstrated that the administration of a monoclonal GAD antibody representing
this epitope specificity; (1) disrupted in vitro the association of GAD with
γ-Aminobutyric acid containing synaptic vesicles; (2) depressed the inhibitory
synaptic transmission in cerebellar slices with a gradual time course and a
lasting suppressive effect; (3) significantly decreased conditioned eyelid
responses evoked in mice, with no modification of learning curves in the
classical eyeblink-conditioning task; (4) markedly impaired the facilitatory
effect exerted by the premotor cortex over the motor cortex in a paired-pulse
stimulation paradigm; and (5) induced decreased exploratory behavior and
impaired locomotor function in rats. These findings support the specific
targeting of GAD by its autoantibodies in the pathogenesis of stiff-person
syndrome and cerebellar ataxia. Therapies of these disorders based on selective
removal of such GAD antibodies could be envisioned. |
Elaborate on the TREAT-NMD initiative for DMD patients | TREAT-NMD is a worldwide network for neuromuscular diseases that provides an infrastructure to support the delivery of promising new therapies for patients in Europe. TREAT-NMD has worked on the generation of brief standards of care for DMD. Guidelines are presented for diagnostics, neurological follow up, gastrointestinal and nutritional issues, respiratory and cardiac care as well as orthopaedics, rehabilitation, psychosocial interventions and oral care. | Care for patients with Duchenne muscular dystrophy (DMD) is poorly standardised.
There are many interventions in different systems which are known to improve
outcomes in DMD but these are not uniformly applied. This leads to inequality in
access to treatment, as well as problems for planning controlled trials of
future therapeutics. A worldwide effort is underway to generate care guidelines
for DMD, which involves the Centre for Disease Control in the USA and the
TREAT-NMD network of excellence for neuromuscular diseases in Europe. In advance
of the full consensus document, TREAT-NMD has worked on the generation of brief
standards of care for DMD, which are presented here and are available via the
TREAT-NMD website (http://www.treat-nmd.eu). Guidelines are presented for
diagnostics, neurological follow up, gastrointestinal and nutritional issues,
respiratory and cardiac care as well as orthopaedics, rehabilitation,
psychosocial interventions and oral care. Clinical trials for new therapeutic strategies are now being planned for
Duchenne and Becker muscular dystrophies (DMD/BMD); however, many challenges
exist in the planning and conduction of a clinical trial for rare diseases. The
epidemiological data, total number of patients, natural history, and clinical
outcome measures are unclear. Adequate numbers of patients are needed to achieve
significant results in clinical trials. As solutions to these problems, patient
registries are an important infrastructure worldwide, especially in the case of
rare diseases such as DMD/BMD. In Europe, TREAT-NMD, a clinical research network
for neuromuscular disorders, developeda global database for dystrophinopathy
patients. We developed a national registry of Japanese DMD/BMD patients in
collaboration with TREAT-NMD. The database includes clinical and molecular
genetic data as well as all required items for the TREAT-NMD global patient
registry. As of July 2011, 750 patients were registered in the database. The
purpose of this registry is the effective recruitment of eligible patients for
clinical trials, and it may also provide timely information to individual
patients about upcoming trials. This registry data also provides more detailed
knowledge about natural history, epidemiology, and clinical care. In recent
years, drug development has become dramatically globalized, and global clinical
trials (GCTs) are being conducted in Japan. It is appropriate, particularly with
regard to orphan diseases, to include Japanese patients in GCTs to increase
evidence for evaluation, because such large-scale trials would be difficult to
conduct solely within Japan. GCTs enable the synchronization of clinical drug
development in Japan with that in Western countries, minimizing drug approval
delays. Duchenne muscular dystrophy (DMD) is an X-linked genetic disease, caused by the
absence of the dystrophin protein. Although many novel therapies are under
development for DMD, there is currently no cure and affected individuals are
often confined to a wheelchair by their teens and die in their
twenties/thirties. DMD is a rare disease (prevalence <5/10,000). Even the
largest countries do not have enough affected patients to rigorously assess
novel therapies, unravel genetic complexities, and determine patient outcomes.
TREAT-NMD is a worldwide network for neuromuscular diseases that provides an
infrastructure to support the delivery of promising new therapies for patients.
The harmonized implementation of national and ultimately global patient
registries has been central to the success of TREAT-NMD. For the DMD registries
within TREAT-NMD, individual countries have chosen to collect patient
information in the form of standardized patient registries to increase the
overall patient population on which clinical outcomes and new technologies can
be assessed. The registries comprise more than 13,500 patients from 31 different
countries. Here, we describe how the TREAT-NMD national patient registries for
DMD were established. We look at their continued growth and assess how
successful they have been at fostering collaboration between academia, patient
organizations, and industry. BACKGROUND: Rare diseases pose many research challenges specific to their
scarcity. Advances in potential therapies have made it more important than ever
to be able to adequately identify not only patients with particular genotypes
(via patient registries) but also the medical professionals who provide care for
them at particular specialist centres of expertise and who may be competent to
participate in trials. Work within the neuromuscular field provides an example
of how this may be achieved.
METHODS: This paper describes the development of the TREAT-NMD Care and Trial
Site Registry (CTSR), an initiative of an EU-funded Network of Excellence, and
its utility in providing an infrastructure for clinical trial feasibility,
recruitment, and other studies.
RESULTS: 285 CTSR-registered centres, reporting 35,495 neuromuscular patients,
are described alongside an analysis of their provision for DMD. Site
characteristics vary by country: the average number of DMD patients seen per
site in the United States (96) is more than in Germany (25), and
paediatric/adult breakdown is also markedly distinct. Over 70% of sites have
previous trial experience, with a majority including a Clinical Trials Unit.
Most sites also have MLPA diagnostic capability and access to a range of medical
specialists. However, in the three countries reporting most sites (US, the UK
and Germany), few had access to all core DMD specialists internally. Over 60% of
sites did not report any form of transition arrangement.
CONCLUSIONS: Registries of care and trial sites have significant utility for
research into rare conditions such as neuromuscular diseases, demonstrated by
the significant engagement by industry and other researchers with the CTSR. We
suggest that this approach may be applicable to other fields needing to identify
centres of expertise with the potential to carry out clinical research and
engage in clinical trials. Such registries also lend themselves to the
developing context of European Reference Networks (ERNs), which seek to build
networks of centres of expertise which fit specific criteria, and which may
themselves aid the sustainability of such registries. This is particularly the
case given the utility of registries such as the CTSR in enabling networks of
best-practice care centres. |
Is muscle regeneration possible in mdx mice with the use of induced mesenchymal stem cells? | Purified induced mesenchymal stem cells (iMSCs) display fibroblast-like morphology, form three-dimensional spheroid structures, express characteristic mesenchymal stem cell surface markers such as CD29, CD33, CD73, CD90, and CD105, and are capable of differentiating into adipogenic, osteogenic, and chondrogenic lineages. Transplantation of iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice lowers oxidative damage, and restores the expression levels of normal dystrophin, leading to skeletal muscle regeneration. | Duchenne muscular dystrophy (DMD) is a severe hereditary disease characterized
by the absence of dystrophin on the sarcolemma of muscle fiber. This absence
results in widespread muscle damage and satellite cell activation. After
depletion of the satellite cell pool, skeletal muscle is then invariably
replaced by connective tissue, leading to progressive muscle weakness. Herein,
we isolated Flk-1(+) mesenchymal stem cells (MSCs) from adult adipose tissue and
induced them to differentiate into skeletal muscle cells in culture. Within mdx
mice, an animal model of DMD, adipose tissue-derived Flk-1(+) MSCs (AD-MSCs)
homed to and differentiated into cells that repaired injured muscle tissue. This
repair correlated with reconstitution of dystrophin expression on the damaged
fibers. Flk-1(+) AD-MSCs also differentiated into muscle satellite cells. This
differentiation may have accounted for long-term reconstitution. These cells
also differentiated into endothelial cells, thereby possibly improving fiber
regeneration as a result of the induced angiogenesis. Therefore, Flk-1(+) AD-MSC
transplants may repair muscular dystrophy. Mesenchymal stem cells (MSCs) may be used as powerful tools for the repair and
regeneration of damaged tissues. However, isolating tissue specific-derived MSCs
may cause pain and increased infection rates in patients, and repetitive
isolations may be required. To overcome these difficulties, we have examined
alternative methods for MSC production. Here, we show that induced pluripotent
stem cells (iPSCs) may be differentiated into mesenchymal stem cells (iMSCs)
following exposure to SB431542. Purified iMSCs were administered to mdx mice to
study skeletal muscle regeneration in a murine model of muscular dystrophy.
Purified iMSCs displayed fibroblast-like morphology, formed three-dimensional
spheroid structures, and expressed characteristic mesenchymal stem cell surface
markers such as CD29, CD33, CD73, CD90, and CD105. Moreover, iMSCs were capable
of differentiating into adipogenic, osteogenic, and chondrogenic lineages.
Transplanting iMSC cells to tibialis anterior skeletal muscle tissue in mdx mice
lowered oxidative damage as evidenced by a reduction in nitrotyrosine levels,
and normal dystrophin expression levels were restored. This study demonstrates
the therapeutic potential of purified iMSCs in skeletal muscle regeneration in
mdx mice, and suggests that iPSCs are a viable alternate source for deriving
MSCs as needed. |
List the different subtypes of thyroid cancer. | The different histologic subtypes of thyroid cancer include papillary, follicular, anaplastic, medullary, and Hürthle cell carcinomas. | Follicular thyroid cancer is the second most common thyroid maligcy after
PTC. There are marked geographical variations in the relative proportions of FTC
and PTC, most likely related to dietary iodine content. In iodine-deficient
areas, the relative rate of FTC tends to be increased. Other risk factors for
FTC include age over 50 years and female sex. Genetic factors may also have a
role in determining disease susceptibility but remain ill-defined.
Histologically, FTC is characterized by follicle formation and the absence of
any papillary elements in the tumor. Differential diagnosis from a benign
adenoma can be difficult. The degree of vascular invasiveness seems to correlate
with tumor aggressiveness, and two histologic subtypes, oxyphilic FTC and
insular FTC, may be associated with increased morbidity and mortality. Primary
treatment for FTC is complete surgical tumor removal. Extensive bilateral
surgery beyond this goal may not confer additional benefit but can facilitate
adjuvant treatment and follow-up. Postoperative levothyroxine treatment is
almost universally used, and patients deemed at high risk of recurrence may
benefit from radioiodine remt ablation. Treatment of metastatic disease
involves operation, radioiodine, and, in selected cases, external beam radiation
and chemotherapy. Prognosis for patients with metastatic disease is guarded, but
most other patients have good outcomes comparable to that in PTC. For
nonoxyphilic FTC, high-risk features other than initial metastases include
advanced age, locally extensive disease, and the presence of marked
angioinvasion. In oxyphilic FTC, DNA aneuploidy is also important. Follow-up
should be most intense during the first 5 years after primary treatment and
needs to be tailored to the patient's risk of disease progression. For patients
at low risk of recurrence (young, small lesions, minimally invasive tumor),
serum thyroglobulin measurements may largely suffice, whereas higher risk
patients with elevated serum thyroglobulin levels and patients with significant
titers of interfering anti-thyroglobulin autoantibodies may also need to undergo
periodic diagnostic radioiodine scanning. OBJECTIVE: To determine the efficacy of gefitinib in patients with advanced
thyroid cancer.
DESIGN: In this open-label phase II trial, 27 patients with
radioiodine-refractory, locally advanced, or metastatic thyroid cancer were
treated with 250 mg of daily gefitinib. Histologic subtypes included papillary
(41%), follicular (22%), anaplastic (19%), medullary (15%), and Hürthle cell
carcinomas (4%). The primary endpoint was overall response rate. Secondary
endpoints were toxicity, progression-free survival (PFS), and overall survival
(OS).
MAIN OUTCOMES: There were no objective responses among the 25 patients
evaluated. After 3, 6, and 12 months of treatment, 48%, 24%, and 12% of patients
had stable disease (SD), respectively. Median PFS and OS were 3.7 and 17.5
months, respectively. Five patients with SD had a decrease in thyroglobulin (Tg)
to <90% of baseline that was maintained for at least 3 months.
CONCLUSIONS: Although gefitinib therapy did not result in any tumor responses,
32% of patients had reductions in tumor volume that did not meet criteria for
partial response rate. Along with falling Tg levels and prolonged SD in a subset
of patients, this may indicate biologic activity. Papillary thyroid carcinoma is the most common type of thyroid maligcy. The
diagnostic features of these tumors include characteristic nuclear cytology.
However, many variants have been reported with different morphology and
molecular profiles. Although the vast majority of papillary thyroid carcinomas
have an excellent prognosis, some variants of papillary thyroid carcinoma can
have a more aggressive course. With this increased attention to papillary
thyroid carcinoma variants has come the need to sort out which variants are
clinically important and should be recognized by practicing pathologists. The
main objectives of this review article are to (1) summarize the gross and
histopathologic features of papillary thyroid carcinoma; (2) provide an overview
of the subtypes of papillary thyroid carcinoma and their prognosis; (3) discuss
established and emerging data on the immunohistochemical findings that are
helpful in differential diagnosis; and (4) summarize molecular findings and
pathogenesis of these lesions. Although thyroid cancer is a comparatively rare maligcy, it represents the
vast majority of endocrine cancers and its incidence is increasing. Most
differentiated thyroid cancers have an excellent prognosis if diagnosed early
and treated appropriately. Aggressive histologic subtypes and variants carry a
worse prognosis. During the last 2 decades positron emission tomography (PET)
and PET/computed tomography (CT), mostly with fluorodeoxyglucose (FDG), has been
used increasingly in patients with thyroid cancers. Currently, the most valuable
role FDG-PET/CT exists in the work-up of patients with differentiated thyroid
cancer status post thyroidectomy who present with increasing thyroglobulin
levels and a negative (131)I whole-body scan. FDG-PET/CT is also useful in the
initial (post thyroidectomy) staging of high-risk patients with less
differentiated (and thus less iodine-avid and clinically more aggressive)
subtypes, such as tall cell variant and Hürthle cell carcinoma, but in
particular poorly differentiated and anaplastic carcinoma. FDG-PET/CT may help
in defining the extent of disease in some patients with medullary thyroid
carcinoma and rising postoperative calcitonin levels. However, FDOPA has emerged
as an alternate and more promising radiotracer in this setting. In aggressive
cancers that are less amenable to treatment with (131)iodine, FDG-PET/CT may
help in radiotherapy planning, and in assessing the response to radiotherapy,
embolization, or experimental systemic treatments. (124)Iodine PET/CT may serve
a role in obtaining lesional dosimetry for better and more rationale planning of
treatment with (131)iodine. Thyroid cancer is not a monolithic disease, and
different stages and histologic entities require different approaches in imaging
and individualized therapy. BACKGROUND: The incidence of thyroid cancer is increasing worldwide. The
findings of up to 30% of thyroid fine-needle aspiration biopsies (FNAB) are
inconclusive, primarily as a result of several thyroid histologic subtypes with
overlapping cytologic features. MicroRNAs (miRNAs) are small noncoding RNAs and
have been implicated in carcinogenesis. We hypothesized that there are miRNAs
that are differentially expressed between benign and maligt thyroid tumors
that are difficult to distinguish by FNAB.
METHODS: The expression of 1263 human miRNAs was profiled in 47 tumor samples
representing difficult to diagnose histologic subtypes of thyroid neoplasm (21
benign, 26 maligt). Differentially expressed miRNAs were validated by
quantitative real-time reverse transcriptase-polymerase chain reaction. The area
under the receiver operating characteristic curve (AUC) was used to determine
the diagnostic accuracy of differentially expressed miRNAs.
RESULTS: Supervised hierarchical cluster analysis demonstrated grouping of 2
histologies (papillary and follicular thyroid carcinoma). A total of 34 miRNAs
were differentially expressed in maligt compared to benign thyroid neoplasms
(P<0.05). A total of 25 of the 34 nonproprietary miRNAs were selected for
validation, and 15 of the 25 miRNAs were differentially expressed between benign
and maligt samples with P-value<0.05. Seven miRNAs had AUC values of >0.7.
miR-7 and miR-126 had the highest diagnostic accuracy with AUCs values of 0.81
and 0.77, respectively.
CONCLUSION: To our knowledge, this is the first study to evaluate the diagnostic
accuracy of miRNAs in thyroid histologies that are difficult to distinguish as
benign or maligt by FNAB. miR-126 and miR-7 had high diagnostic accuracy and
could be helpful adjuncts to thyroid FNAB. Thyroid cancer is the most common cancer of the endocrine system and is
responsible for the majority of deaths from endocrine maligcies. Although a
large proportion of thyroid cancers belong to well differentiated histologic
subtypes, which in general show a good prognosis after surgery and radioiodine
ablation, the treatment of radio-resistant papillary-type, of undifferentiated
anaplastic, and of medullary-type thyroid cancers remains unsatisfactory.
Autophagy is a vesicular process for the lysosomal degradation of protein
aggregates and of damaged or redundant organelles. Autophagy plays an important
role in cell homeostasis, and there is evidence that this process is
dysregulated in cancer cells. Recent in vitro preclinical studies have indicated
that autophagy is involved in the cytotoxic response to chemotherapeutics in
thyroid cancer cells. Indeed, several oncogenes and oncosuppressor genes
implicated in thyroid carcinogenesis also play a role in the regulation of
autophagy. In addition, some epigenetic modulators involved in thyroid
carcinogenesis also influence autophagy. In this review, we highlight the
genetic and epigenetic factors that mechanistically link thyroid carcinogenesis
and autophagy, thus substantiating the rationale for an autophagy-targeted
therapy of aggressive and radio-chemo-resistant thyroid cancers. |
What are the characteristics of Christianson syndrome? | Christianson syndrome (CS) is caused by mutations in the X-linked Na(+) /H(+) exchanger 6 (NHE6). Patients present with prominent neurological, medical, and behavioral symptoms. All CS participants were nonverbal and had intellectual disability, epilepsy, and ataxia. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%), and magnetic resonance imaging evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, are common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%), and a majority exhibited high pain threshold. | Interstitial deletions of chromosome band Xq26.3 are rare. We report on a
2-year-old boy in whom array comparative genomic hybridization analysis revealed
an interstitial 314 kb deletion in Xq26.3 affecting SLC9A6 and FHL1. Mutations
in SLC9A6 are associated with Christianson syndrome (OMIM 300243), a syndromic
form of X-linked mental retardation (XLMR) characterized by microcephaly, severe
global developmental delay, ataxia and seizures. FHL1 mutations cause
Emery-Dreifuss muscular dystrophy (OMIM 310300), X-linked myopathy with postural
muscle atrophy (XMPMA, OMIM 300696), scapuloperoneal myopathy (OMIM 300695), or
reducing body myopathy (OMIM 300717, 300718). The clinical problems of the
patient reported here comprised severe intellectual disability, absent speech,
ataxia, epilepsy, and gastroesophageal reflux, and could mostly be attributed to
SLC9A6 insufficiency. In contrast to the majority of reported Christianson
syndrome patients who were microcephalic, this patient was normocephalic, but
his head circumference had decelerated from the 50th centile at birth to the
25th centile at the age of 2 ²/¹² years. Muscle problems due to the FHL1
deletion are not to be expected before late childhood, which is the earliest age
of onset for FHL1 associated Emery-Dreifuss muscular dystrophy. This patient
broadens the spectrum of SLC9A6 mutations and contributes to the clinical
delineation of Christianson syndrome. This is also the first patient with a
deletion affecting both SLC9A6 and the complete FHL1 gene. Neuronal arborization is regulated by cell-autonomous and nonautonomous
mechanisms including endosomal signaling via BDNF/TrkB. The endosomal Na⁺/H⁺
exchanger 6 (NHE6) is mutated in a new autism-related disorder. NHE6 functions
to permit proton leak from endosomes, yet the mechanisms causing disease are
unknown. We demonstrate that loss of NHE6 results in overacidification of the
endosomal compartment and attenuated TrkB signaling. Mouse brains with disrupted
NHE6 display reduced axonal and dendritic branching, synapse number, and circuit
strength. Site-directed mutagenesis shows that the proton leak function of NHE6
is required for neuronal arborization. We find that TrkB receptor colocalizes to
NHE6-associated endosomes. TrkB protein and phosphorylation are reduced in NHE6
mutant neurons in response to BDNF signaling. Finally, exogenous BDNF rescues
defects in neuronal arborization. We propose that NHE6 mutation leads to circuit
defects that are in part due to impoverished neuronal arborization that may be
treatable by enhanced TrkB signaling. Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized
by severe intellectual disability, absent speech, microcephaly, ataxia,
seizures, and behavioral abnormalities. The clinical phenotypes of CS and
Angelman syndrome (AS) are similar. Differentiation between CS and AS is
important in terms of genetic counseling. We report on two children with CS and
confirmed mutations in SLC9A6 focusing on neuroimaging findings and review the
available literature. Cerebellar atrophy (CA) occurs in approximately 60% of the
patients with CS and develops after the age of 12 months. Hyperintense signal of
the cerebellar cortex (CbC) is less common, and may be diffuse, patchy, or
involve only the inferior part of the cerebellum and is best seen on coronal
fluid attenuation inversion recovery images. CA and CbC-hyperintensity are not
neuroimaging features of AS. In a child with the phenotype of AS, CA and/or
CbC-hyperintensity are rather specific for CS and should prioritize sequencing
of SLC9A6. Angelman syndrome (AS) is caused by a lack of expression of the maternally
inherited UBE3A gene in the brain. However, about 10% of individuals with a
clinical diagnosis of AS do not have an identifiable molecular defect. It is
likely that most of those individuals have an AS-like syndrome that is
clinically and molecularly distinct from AS. These AS-like syndromes can be
broadly classified into chromosomal microdeletion and microduplication
syndromes, and single-gene disorders. The microdeletion/microduplication
syndromes are now easily identified by chromosomal microarray analysis and
include Phelan–McDermid syndrome (chromosome 22q13.3 deletion), MBD5
haploinsufficiency syndrome (chromosome 2q23.1 deletion), and KANSL1
haploinsufficiency syndrome (chromosome 17q21.31 deletion). The single-gene
disorders include Pitt–Hopkins syndrome (TCF4), Christianson syndrome (SLC9A6),
Mowat–Wilson syndrome (ZEB2), Kleefstra syndrome (EHMT1), and Rett (MECP2)
syndrome. They also include disorders due to mutations in HERC2,
adenylosuccinase lyase (ADSL), CDKL5, FOXG1, MECP2 (duplications), MEF2C, and
ATRX. Although many of these single-gene disorders can be caused by chromosomal
microdeletions resulting in haploinsufficiency of the critical gene, the
individual disorders are often caused by intragenic mutations that cannot be
detected by chromosomal microarray analysis. We provide an overview of the
clinical features of these syndromes, comparing and contrasting them with AS, in
the hope that it will help guide clinicians in the diagnostic work-up of
individuals with AS-like syndromes. Several genetic disorders are characterized by normal head size at birth,
followed by deceleration in head growth resulting in postnatal microcephaly.
Among these are classic disorders such as Angelman syndrome and MECP2-related
disorder (formerly Rett syndrome), as well as more recently described clinical
entities associated with mutations in CASK, CDKL5, CREBBP, and EP300
(Rubinstein-Taybi syndrome), FOXG1, SLC9A6 (Christianson syndrome), and TCF4
(Pitt-Hopkins syndrome). These disorders can be identified clinically by
phenotyping across multiple neurodevelopmental and neurobehavioral realms, and
enough data are available to recognize these postnatal microcephaly disorders as
separate diagnostic entities in their own right. A second diagnostic grouping,
comprised of Warburg MICRO syndrome, Cockayne syndrome, and
Cerebral-oculo-facial skeletal syndrome, share similar features of somatic
growth failure, ophthalmologic, and dysmorphologic features. Many postnatal
microcephaly syndromes are caused by mutations in genes important in the
regulation of gene expression in the developing forebrain and hindbrain,
although important synaptic structural genes also play a role. This is an
emerging group of disorders with a fascinating combination of brain
malformations, specific epilepsies, movement disorders, and other complex
neurobehavioral abnormalities. OBJECTIVE: Recently, Christianson syndrome (CS) has been determined to be caused
by mutations in the X-linked Na(+) /H(+) exchanger 6 (NHE6). We aimed to
determine the diagnostic criteria and mutational spectrum for CS.
METHODS: Twelve independent pedigrees (14 boys, age = 4-19 years) with mutations
in NHE6 were administered standardized research assessments, and mutations were
characterized.
RESULTS: The mutational spectrum was composed of 9 single nucleotide variants, 2
indels, and 1 copy number variation deletion. All mutations were
protein-truncating or splicing mutations. We identified 2 recurrent mutations
(c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were
unique. In our study, 7 of 12 mutations (58%) were de novo, in contrast to prior
literature wherein mutations were largely inherited. We also report prominent
neurological, medical, and behavioral symptoms. All CS participants were
nonverbal and had intellectual disability, epilepsy, and ataxia. Many had prior
diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included
eye movement abnormalities (79%), postnatal microcephaly (92%), and magnetic
resoce imaging evidence of cerebellar atrophy (33%). Regression was noted in
50%, with recurrent presentations involving loss of words and/or the ability to
walk. Medical symptoms, particularly gastrointestinal symptoms, were common.
Height and body mass index measures were below normal ranges in most
participants. Behavioral symptoms included hyperkinetic behavior (100%), and a
majority exhibited high pain threshold.
INTERPRETATION: This is the largest cohort of independent CS pedigrees reported.
We propose diagnostic criteria for CS. CS represents a novel neurogenetic
disorder with general relevance to autism, intellectual disability, Angelman
syndrome, epilepsy, and regression. |
Is the SDHAF2 gene encoding a protein necessary for flavination of SDHA? | Yes, SDHAF2 or hSDH5, is the gene encoding the enzyme responsible for the flavination of SDHA. | Paragangliomas have been linked to mutations affecting the succinate
dehydrogenase complex. In a recent issue of Science, Rutter and coworkers showed
that SDH5 is required for the flavination of SDHA, which is necessary for SDH
assembly and function. Moreover, they detected SDH5 mutations in a large kindred
with familial paraganglioma. |
What is the physiological role of LKB1 involved in Peutz-Jeghers syndrome? | LKB1 plays a physiological role in controlling the Wnt-signaling. | |
What heterotachy states about molecular evolutionary processes? | Functional constraints may account for heterogeneity in the evolutionary rates among different sites of amino acid or DNA sequences. Apart from variations of substitution rates among different sites (spatial variation), heterotachy states that there are variations of substitution rates of a given site throughout time. Heterotachy is the within-site evolutionary rate variations across different lineages over time. Heterotachy (temporal rate variation) occurs with varying severity because the intensity of purifying selection and adaptive forces acting at a given position of a homologous amino acid or DNA sequence are not the same in different species. Heterotachy may mislead phylogenetic inferences. | Because of functional constraints, substitution rates vary among the positions
of a protein but are usually assumed to be constant at a given site during
evolution. The distribution of the rates across the sequence positions generally
fits a Gamma distribution. Models of sequence evolution were accordingly
designed and led to improved phylogenetic reconstruction. However, it has been
convincingly demonstrated that the evolutionary rate of a given position is not
always constant throughout time. We called such within-site rate variations
heterotachy (for "different speed" in Greek). Yet, heterotachy was found among
homologous sequences of distantly related organisms, often with different
functions. In such cases, the functional constraints are likely different, which
would explain the different distribution of variable sites. To evaluate the
importance of heterotachy, we focused on amino acid sequences of mitochondrial
cytochrome b, for which the function is likely the same in all vertebrates.
Using 2,038 sequences, we demonstrate that 95% of the variable positions are
heterotachous, i.e., underwent dramatic variations of substitution rate among
vertebrate lineages. Heterotachy even occurs at small evolutionary scale, and in
these cases it is very unlikely to be related to functional changes. Since a
large number of sequences are required to efficiently detect heterotachy, the
extent of this phenomenon could not be estimated for all proteins yet. It could
be as large as for cytochrome b, since this protein is not a peculiar case. The
observations made here open several new avenues of research, such as the
understanding of the evolution of functional constraints or the improvement of
phylogenetic reconstruction methods. Study of structure/function relationships constitutes an important field of
research, especially for modification of protein function and drug design.
However, the fact that rational design (i.e. the modification of amino acid
sequences by means of directed mutagenesis, based on knowledge of the
three-dimensional structure) appears to be much less efficient than irrational
design (i.e. random mutagenesis followed by in vitro selection) clearly
indicates that we understand little about the relationships between primary
sequence, three-dimensional structure and function. The use of evolutionary
approaches and concepts will bring insights to this difficult question. The
increasing availability of multigene family sequences that has resulted from
genome projects has inspired the creation of novel in silico evolutionary
methods to predict details of protein function in duplicated (paralogous)
proteins. The underlying principle of all such approaches is to compare the
evolutionary properties of homologous sequence positions in paralogs. It has
been proposed that the positions that show switches in substitution rate over
time--i.e., 'heterotachous sites'--are good indicators of functional divergence.
However, it appears that heterotachy is a much more general process, since most
variable sites of homologous proteins with no evidence of functional shift are
heterotachous. Similarly, it appears that switches in substitution rate are as
frequent when paralogous sequences are compared as when orthologous sequences
are compared. Heterotachy, instead of being indicative of functional shift, may
more generally reflect a less specific process related to the many intra- and
inter-molecular interactions compatible with a range of more or less equally
viable protein conformations. These interactions will lead to different
constraints on the nature of the primary sequences, consistently with theories
suggesting the non-independence of substitutions in proteins. However, a
specific type of amino acid variation might constitute a good indicator of
functional divergence: substitutions occurring at positions that are generally
slowly evolving. Such substitutions at constrained sites are indeed much more
frequent soon after gene duplication. The identification and analysis of these
sites by complementing structural information with evolutionary data may
represent a promising direction to future studies dealing with the functional
characterization of an ever increasing number of multi-gene families identified
by complete genome analysis. Evolutionary rates vary among sites and across the phylogenetic tree
(heterotachy). A recent analysis suggested that parsimony can be better than
standard likelihood at recovering the true tree given heterotachy. The authors
recommended that results from parsimony, which they consider to be
nonparametric, be reported alongside likelihood results. They also proposed a
mixture model, which was inconsistent but better than either parsimony or
standard likelihood under heterotachy. We show that their main conclusion is
limited to a special case for the type of model they study. Their mixture model
was inconsistent because it was incorrectly implemented. A useful nonparametric
model should perform well over a wide range of possible evolutionary models, but
parsimony does not have this property. Likelihood-based methods are therefore
the best way to deal with heterotachy. Heterotachy occurs when the relative evolutionary rates among sites are not the
same across lineages. Sequence alignments are likely to exhibit heterotachy with
varying severity because the intensity of purifying selection and adaptive
forces at a given amino acid or DNA sequence position is unlikely to be the same
in different species. In a recent study, the influence of heterotachy on the
performance of different phylogenetic methods was examined using computer
simulation for a four-species phylogeny. Maximum parsimony (MP) was reported to
generally outperform maximum likelihood (ML). However, our comparisons of MP and
ML methods using the methods and evaluation criteria employed in that study, but
considering the possible range of proportions of sites involved in heterotachy,
contradict their findings and indicate that, in fact, ML is significantly
superior to MP even under heterotachy. The nature of heterotachy at the center of recent controversy over the relative
performance of tree-building methods is different from the form of heterotachy
that has been inferred in empirical studies. The latter have suggested that
proportions of variable sites (p(var)) vary among orthologues and among
paralogues. However, the strength of this inference, describing what may be one
of the most important evolutionary properties of sequence data, has remained
weak. Consequently, other models of sequence evolution have been proposed to
explain some long-branch attraction (LBA) problems that could be attributed to
differences in p(var). For an empirical case with plastid and eubacterial RNA
polymerase sequences, we confirm using capture-recapture estimates and
simulations that p(var) can differ among orthologues in anciently diverged
evolutionary lineages. We find that parsimony and a least squares distance
method that implements an overly simple model of sequence evolution are
susceptible to LBA induced by this form of heterotachy. Although homogeneous
maximum likelihood inference was found to be robust to model misspecification in
our specific example, we caution against assuming that it will always be so. BACKGROUND: Probabilistic methods have progressively supplanted the Maximum
Parsimony (MP) method for inferring phylogenetic trees. One of the major reasons
for this shift was that MP is much more sensitive to the Long Branch Attraction
(LBA) artefact than is Maximum Likelihood (ML). However, recent work by
Kolaczkowski and Thornton suggested, on the basis of simulations, that MP is
less sensitive than ML to tree reconstruction artefacts generated by
heterotachy, a phenomenon that corresponds to shifts in site-specific
evolutionary rates over time. These results led these authors to recommend that
the results of ML and MP analyses should be both reported and interpreted with
the same caution. This specific conclusion revived the debate on the choice of
the most accurate phylogenetic method for analysing real data in which various
types of heterogeneities occur. However, variation of evolutionary rates across
species was not explicitly incorporated in the original study of Kolaczkowski
and Thornton, and in most of the subsequent heterotachous simulations published
to date, where all terminal branch lengths were kept equal, an assumption that
is biologically unrealistic.
RESULTS: In this report, we performed more realistic simulations to evaluate the
relative performance of MP and ML methods when two kinds of heterogeneities are
considered: (i) within-site rate variation (heterotachy), and (ii) rate
variation across lineages. Using a similar protocol as Kolaczkowski and Thornton
to generate heterotachous datasets, we found that heterotachy, which constitutes
a serious violation of existing models, decreases the accuracy of ML whatever
the level of rate variation across lineages. In contrast, the accuracy of MP can
either increase or decrease when the level of heterotachy increases, depending
on the relative branch lengths. This result demonstrates that MP is not
insensitive to heterotachy, contrary to the report of Kolaczkowski and Thornton.
Finally, in the case of LBA (i.e. when two non-sister lineages evolved faster
than the others), ML outperforms MP over a wide range of conditions, except for
unrealistic levels of heterotachy.
CONCLUSION: For realistic combinations of both heterotachy and variation of
evolutionary rates across lineages, ML is always more accurate than MP.
Therefore, ML should be preferred over MP for analysing real data, all the more
so since parametric methods also allow one to handle other types of biological
heterogeneities much better, such as among sites rate variation. The confounding
effects of heterotachy on tree reconstruction methods do exist, but can be
eschewed by the development of mixture models in a probabilistic framework, as
proposed by Kolaczkowski and Thornton themselves. The principle of heterotachy states that the substitution rate of sites in a
gene can change through time. In this article, we propose a powerful statistical
test to detect sites that evolve according to the process of heterotachy. We
apply this test to an alignment of 1289 eukaryotic rRNA molecules to 1)
determine how widespread the phenomenon of heterotachy is in ribosomal RNA, 2)
to test whether these heterotachous sites are nonrandomly distributed, that is,
linked to secondary structure features of ribosomal RNA, and 3) to determine the
impact of heterotachous sites on the bootstrap support of monophyletic
groupings. Our study revealed that with 21 monophyletic taxa, approximately
two-thirds of the sites in the considered set of sequences is heterotachous.
Although the detected heterotachous sites do not appear bound to specific
structural features of the small subunit rRNA, their presence is shown to have a
large beneficial influence on the bootstrap support of monophyletic groups.
Using extensive testing, we show that this may not be due to heterotachy itself
but merely due to the increased substitution rate at the detected heterotachous
sites. Despite the advances in understanding molecular evolution, current phylogenetic
methods barely take account of a fraction of the complexity of evolution. We are
chiefly constrained by our incomplete knowledge of molecular evolutionary
processes and the limits of computational power. These limitations lead to the
establishment of either biologically simplistic models that rarely account for a
fraction of the complexity involved or overfitting models that add little
resolution to the problem. Such oversimplified models may lead us to assign high
confidence to an incorrect tree (inconsistency). Rate-across-site (RAS) models
are commonly used evolutionary models in phylogenetic studies. These account for
heterogeneity in the evolutionary rates among sites but do not account for
changing within-site rates across lineages (heterotachy). If heterotachy is
common, using RAS models may lead to systematic errors in tree inference. In
this work we show possible misleading effects in tree inference when the
assumption of constant within-site rates across lineages is violated using
maximum likelihood. Using a simulation study, we explore the ways in which gamma
stationary models can lead to wrong topology or to deceptive bootstrap support
values when the within-site rates change across lineages. More precisely, we
show that different degrees of heterotachy mislead phylogenetic inference when
the model assumed is stationary. Finally, we propose a geometry-based approach
to visualize and to test for the possible existence of bias due to heterotachy. BACKGROUND: The evolutionary rate at a given homologous position varies across
time. When sufficiently pronounced, this phenomenon - called heterotachy - may
produce artefactual phylogenetic reconstructions under the commonly used models
of sequence evolution. These observations have motivated the development of
models that explicitly recognize heterotachy, with research directions proposed
along two main axes: 1) the covarion approach, where sites switch from variable
to invariable states; and 2) the mixture of branch lengths (MBL) approach, where
alignment patterns are assumed to arise from one of several sets of branch
lengths, under a given phylogeny.
RESULTS: Here, we report the first statistical comparisons contrasting the
performance of covarion and MBL modeling strategies. Using simulations under
heterotachous conditions, we explore the properties of three model comparison
methods: the Akaike information criterion, the Bayesian information criterion,
and cross validation. Although more time consuming, cross validation appears
more reliable than AIC and BIC as it directly measures the predictive power of a
model on 'future' data. We also analyze three large datasets (nuclear proteins
of animals, mitochondrial proteins of mammals, and plastid proteins of plants),
and find the optimal number of components of the MBL model to be two for all
datasets, indicating that this model is preferred over the standard homogeneous
model. However, the covarion model is always favored over the optimal MBL model.
CONCLUSION: We demonstrated, using three large datasets, that the covarion model
is more efficient at handling heterotachy than the MBL model. This is probably
due to the fact that the MBL model requires a serious increase in the number of
parameters, as compared to two supplementary parameters of the covarion
approach. Further improvements of the both the mixture and the covarion
approaches might be obtained by modeling heterogeneous behavior both along time
and across sites. Sequence alignments of multiple genes are routinely used to infer phylogenetic
relationships among species. The analysis of their concatenation is more likely
to give correct results under an assumption of homotachy (i.e., the evolutionary
rates within lineages in each of the concatenated genes are constant during
evolution). Here, we examine how the violation of homotachy (i.e., presence of
within-site rate variation, called heterotachy) distorts species phylogenies. A
theoretical examination has been conducted using a four taxon case and the
neighbor joining (NJ) method, concluding that NJ recovers the incorrect tree
when concatenated genes exhibit heterotachy. The application of average and
weighted-average distance approaches, where gene boundaries are kept intact,
overcomes the detrimental effect of heterotachy in multigene analysis using the
NJ method. Heterotachy is a general term to describe positions in a sequence that evolve at
different rates in different lineages. Kolaczkowski and Thornton (2004.
Performance of maximum parsimony and likelihood phylogenetics when evolution is
heterogeneous. Nature 431:980-984.) recently described an intriguing heterotachy
model that leads to topological bias for likelihood-based methods and parsimony
methods. In this article, we show that heterotachy can generally be viewed as
multivariate rates-across-sites variation, which can be described as randomly
drawing rates (or branch lengths) from a multivariate distribution for each
branch at each site. Motivated by this idea, we propose a pairwise alpha
heterotachy adjustment model, which gives us much improved topological
estimation in the settings by Kolaczkowski and Thornton (2004). Heterotachy, the variation of substitution rate at a site across time, is a
prevalent phenomenon in nucleotide and amino acid alignments, which may mislead
probabilistic-based phylogenetic inferences. The covarion model is a special
case of heterotachy, in which sites change between the "ON" state (allowing
substitutions according to any particular model of sequence evolution) and the
"OFF" state (prohibiting substitutions). In current implementations, the switch
rates between ON and OFF states are homogeneous across sites, a hypothesis that
has never been tested. In this study, we developed an infinite mixture model,
called the covarion mixture (CM) model, which allows the covarion parameters to
vary across sites, controlled by a Dirichlet process prior. Moreover, we combine
the CM model with other approaches. We use a second independent Dirichlet
process that models the heterogeneities of amino acid equilibrium frequencies
across sites, known as the CAT model, and general rate-across-site heterogeneity
is modeled by a gamma distribution. The application of the CM model to several
large alignments demonstrates that the covarion parameters are significantly
heterogeneous across sites. We describe posterior predictive discrepancy tests
and use these to demonstrate the importance of these different elements of the
models. There is widespread evidence of lineage-specific rate variation, known as
heterotachy, during protein evolution. Changes in the structural and functional
constraints acting on a protein can lead to heterotachy, and it is plausible
that such changes, known as covarion shifts, may affect many amino acids at
once. Several previous attempts to model heterotachy have used covarion models,
where the sequence undergoes covarion drift, whereby each site may switch
independently among a set of discrete classes having different substitution
rates. However, such independent switching may not capture biologically
important events where the selective forces acting on a protein affect many
sites at once. We describe a new class of models that allow the rates of
substitution and switching to vary among branches of a phylogenetic tree. Such
models are better able to handle covarion shifts. We apply these models to a set
of genes occurring in nonphotosynthetic bacteria, cyanobacteria, and the
plastids of green and red algae. We find that 4/5 genes show evidence of some
form of rate switching and that 3/5 genes show evidence that the relative
switching rate differs among taxonomic groups. We conclude that covarion shifts
may be frequent during the deep evolution of plastid genes and that our
methodology may provide a powerful new tool for investigating such shifts in
other systems. Heterotachy is a general term to describe positions that evolve at different
rates in different lineages. Heterotachy also can generally be viewed as
multivariate rates-across-sites variation, which can be described as randomly
drawing rates (or branch lengths) from a multivariate distribution for each
branch at each site (Wu J, Susko E. 2009. General heterotachy and distance
method adjustments. Mol Biol Evol. 26:2689-2697). Motivated by this result, we
propose three new distance-based tests: a heterogeneity test, a heterotachy
test, and a within-gene heterotachy test and demonstrate with simulations that
they perform well under a wide range of conditions. We also applied the first
two tests to two real data sets and found that although all these data sets
showed significant evidence of heterotachy, there were subtrees for which the
data were consistent with an equal rates or rates-across-sites
model.heterogeneity, heterotachy, within-gene heterotachy, covarion model,
distance method, hypothesis test. BACKGROUND: Model violations constitute the major limitation in inferring
accurate phylogenies. Characterizing properties of the data that are not being
correctly handled by current models is therefore of prime importance. One of the
properties of protein evolution is the variation of the relative rate of
substitutions across sites and over time, the latter is the phenomenon called
heterotachy. Its effect on phylogenetic inference has recently obtained
considerable attention, which led to the development of new models of sequence
evolution. However, thus far focus has been on the quantitative heterogeneity of
the evolutionary process, thereby overlooking more qualitative variations.
RESULTS: We studied the importance of variation of the site-specific amino-acid
substitution process over time and its possible impact on phylogenetic
inference. We used the CAT model to define an infinite mixture of substitution
processes characterized by equilibrium frequencies over the twenty amino acids,
a useful proxy for qualitatively estimating the evolutionary process. Using two
large datasets, we show that qualitative changes in site-specific substitution
properties over time occurred significantly. To test whether this unaccounted
qualitative variation can lead to an erroneous phylogenetic tree, we analyzed a
concatenation of mitochondrial proteins in which Cnidaria and Porifera were
erroneously grouped. The progressive removal of the sites with the most
heterogeneous CAT profiles across clades led to the recovery of the monophyly of
Eumetazoa (Cnidaria+Bilateria), suggesting that this heterogeneity can
negatively influence phylogenetic inference.
CONCLUSION: The time-heterogeneity of the amino-acid replacement process is
therefore an important evolutionary aspect that should be incorporated in future
models of sequence change. |
What is known about depression in caregivers of brain tumor patients? | Depression is common affecting up to 40% of caregivers of brain tumor patients. Depression is associated with poor quality of life of caregivers of brain tumor patients. Greater anxiety, patients’ emotional distress, economic hardship, lower caregivers’ age, lower income, less social support and lower patient functioning were associated with more caregivers’ depressive symptoms. Reports of caregiver depressive symptoms were lower when paired with higher reports of spirituality. It is important to monitor and treat caregiver's depression. | PURPOSE: To identify the characteristics of adult patients with newly diagnosed
primary brain tumors associated with identifiable deficits in neuropsychologic
function to target interventions to improve function and quality of life (QOL).
MATERIALS AND METHODS: Adult patients with newly diagnosed primary brain tumors
and their caregivers were enrolled and underwent a battery of standardized
neuropsychologic tests, allowing for qualitative and quantitative assessment and
sensitive to the effects of the brain tumor, QOL, or caregiver stress.
RESULTS: We enrolled 68 patients with no prior radiotherapy. Patients with left
hemisphere tumors reported significantly more memory problems and depressive
symptoms. They also exhibited poorer attention and were more distractible, with
poorer verbal fluency and poorer verbal learning. Patients with glioblastoma
multiforme demonstrated poorer psychomotor speed and visual tracking than
patients with non-glioblastoma multiforme histologic features. Patients and
caregivers perceived QOL in a similar fashion, with significant correlation
between patient and caregiver on hope testing and general QOL on the Linear
Analog Self-Assessment Scale.
CONCLUSIONS: Patients with left hemisphere tumors and glioblastoma multiforme
histologic features demonstrated testable differences in neuropsychologic
function and QOL that may be amenable to improvement with medical therapy or
tailored rehabilitation programs. Caregiver assessments can predict patient QOL,
which may be useful in patients with declining status. The purpose of this cross-sectional, descriptive study was to identify
predictors of distress for family caregivers of persons with a primary maligt
brain tumor (PMBT). The effect of the care recipient's functional, cognitive,
and neuropsychiatric status on caregiver burden and depressive symptoms was
examined through telephone interviews with 95 caregivers. Care recipients'
neuropsychiatric status consistently affected caregivers' depressive symptoms
and burden, and assisting with activities of daily living affected burden
related to caregivers' schedules and health. The care recipient's cognitive
status and need for assistance with instrumental activities of daily living did
not affect any outcome variable. Results may help identify caregivers at risk
for negative outcomes, and suggest interventions to improve caregivers'
emotional health. PURPOSE: The purpose of this study was to explore how the relationship between
care recipients' problem behaviors and caregivers' depressive symptoms varies as
a function of caregiver mastery, controlling for the effects of caregiver age,
gender, and relationship to the care recipient in caregivers of people with
primary maligt brain tumor (PMBT).
DESIGN: A cross-sectional design was used to gather data via telephone
interviews from 95 caregivers of people with primary maligt brain tumor,
recruited from 2003 to 2004 from a brain tumor treatment center, two national
support groups, and a statewide cancer registry.
METHODS: Measures for the study included the Neuropsychiatric
Inventory-Questionnaire, Caregiver Mastery, and the Center for Epidemiologic
Studies-Depression. A stepwise regression procedure was used to evaluate
potential moderating and mediating relationships.
FINDINGS: Data did not indicate that caregiver mastery was a moderating
variable. The analysis showed caregiver mastery as a partial mediator, with both
a direct effect of care recipients' problem behaviors on caregivers' depressive
symptoms and an indirect effect through caregiver mastery. Concerning the
indirect effect, care recipients' problem behaviors were related to lower levels
of caregiver mastery, which in turn were related to more depressive symptoms in
caregivers.
CONCLUSIONS: Findings showed a link between care recipients' problem behaviors
and caregivers' depressive symptoms, a relationship that has not been well
established in oncology. This association indicates one mechanism through which
problem behaviors in the care recipient might lead to caregiver depressive
symptoms. The incidence of primary maligt brain tumors has remained stable over the
past 10 years, with median survival reported as 12 months. Once the patient has
been diagnosed, providing care for him or her is primarily performed by family
members. Although previous research has documented the stress, depression,
anxiety, and burden associated with caregiving, when these conditions occur is
not known. The purpose of this study was to explore the caregiver perspective of
providing care while the patient was receiving chemotherapy as initial treatment
for the disease. Using phenomenological techniques, data were obtained from
semistructured interviews with family caregivers and self-disclosed demographic
data. Each interview occurred while the patient was receiving treatment; all
patients were within 6 months of initial diagnosis. Interview data were analyzed
using Colaizzi's method, which allowed themes universal to the participants to
be uncovered. Interview data from 10 participants provided saturation and
identified three themes: (a) the diagnosis of a brain tumor is a shock; (b)
immediate family role changes occur; and (c) there are psychosocial effects for
the caregiver, his or her family, and the person with the brain tumor. Although several studies have quantified costs of cancer care; none to date have
examined how cancer costs impact family caregivers' emotional health. This study
was designed to evaluate how perceptions of economic hardship influence burden,
depressive symptoms, and anxiety in family caregivers of persons with a primary
maligt brain tumor. Caregiver (CG)/patient dyads (n = 33) were recruited at
the time of diagnosis; data were collected at diagnosis and 4 months, and linear
regression determined the impact of economic hardship on caregivers' emotional
health. Economic hardship did not predict CG burden-schedule at diagnosis or 4
months. Economic hardship predicted burden-abandonment at diagnosis (P < 0.01),
but not 4 months. There was a trend for economic hardship to predict CG
depressive symptoms at 4 months (P = 0.09), but not at diagnosis. Economic
hardship predicted CG anxiety at 4 months (P = 0.06), but not diagnosis. Results
suggest caregivers' economic hardship is an important and dynamic aspect of the
emotional health of neuro-oncology family caregivers. Quality of life and well-being in caregivers are usually partly neglected since
all attention is focused on patients and the way they react to the illness.
Carers also usually neglect their own needs, especially when the illness of the
patient is as complex as a brain tumor. The aim of this study is to investigate
how caregivers deal with a diagnosis of brain tumor in their relatives and how
they manage their quality of life and psychosocial well-being. One hundred
primary caregivers of patients with brain tumors were interviewed and were asked
to fill in self-administered questionnaires detecting multidimensional levels of
quality of life, anxiety, depression, and psychosocial reaction to the patient's
illness. Data were related with some functional and psychosocial information
collected about the patient's disease. Caregivers try to react to the illness of
their relatives by mobilizing their physical reaction and growing their
self-esteem, but they live with a clinically significant impairment of their
quality of life, and experience a deep level of anxiety and depression. The
caregivers' burden appears mainly in their ability to provide care and in
ficial strain. The length of disease and the functional status of patients
significantly influence caregivers' psychosocial well-being. Despite the
appearance they want to show their affected relatives, caregivers suffer from
deep limitation in their quality of life. The relevance of caregivers' burden
suggests the importance of psychological support to improve reaction to the
illness. In adults, primary maligt brain tumors (PMBT) are rare, but they have a
devastating impact and the chances for survival are limited. UK clinical
guidance on supportive care for patients with brain and central nervous system
tumors was published in 2006 and relied on very limited evidence. The current
article reviews literature from 2005 through 2011 on the psychosocial and
supportive needs of patients with PMBT and their families or caregivers.
Searches were conducted in PubMed, Web of Science, Psychinfo, Cochrane, Scopus,
ASSIA, and Sciencedirect. The search initially yielded 6220 articles, of which
60 were found to be eligible (1%). Eleven qualitative and 49 quantitative
studies are reviewed here and mapped onto the structure of the existing UK
clinical guidance. Studies suggest rates of depression and anxiety up to 48% in
patients and up to 40% in caregivers, with many unmet needs and dissatisfaction
with health care provider communication and information. Cognitive deficits
increase as the disease progresses, hampering communication and decision-making.
A range of neurological and physical symptoms at the end of life need
recognition. Some successful supportive and neuropsychological interventions are
reported. Although the volume of available studies has increased since UK
guidance, many remain observational in nature, with few trials of interventions.
However, this review provides an up to date resource for clinicians involved
with patients with PMBT, describing current knowledge on patients' psychosocial
needs, the type of care which has been found to be beneficial, and highlighting
areas where more research needs to be done. BACKGROUND: Competing theories of adaptation and wear-and-tear describe
psychological distress patterns among family caregivers.
PURPOSE: This study seeks to characterize psychological distress patterns in
family caregivers and identify predictors.
METHODS: One hundred three caregivers of care recipients with primary maligt
brain tumors were interviewed within 1, 4, 8, and 12 months post-diagnosis
regarding psychological distress; care recipients were interviewed regarding
clinical/functional characteristics. Group-based trajectory modeling identified
longitudinal distress patterns, and weighted logistic/multinomial regression
models identified predictors of distress trajectories.
RESULTS: Group-based trajectory modeling identified high-decreasing (51.1 % of
caregivers) and consistently low (48.9 %) depressive symptom trajectories,
high-decreasing (75.5 %) and low-decreasing (24.5 %) anxiety trajectories, and
high (37.5 %), moderate (40.9 %), and low-decreasing (21.6 %) caregiver burden
trajectories. High depressive symptoms were associated with high trajectories
for both anxiety and burden, lower caregivers age, income, and social support,
and lower care recipient functioning.
CONCLUSIONS: Our data support the adaptation hypothesis; interventions should
target those at risk for persistent distress. PURPOSE: The psychological burden induced by brain tumor is profound both for
the sick person and for their own family. This particular tumor not only impacts
patients' quality of life, but also reduces seriously the caregivers' quality of
life. We aim to describe brain tumor patients and their caregivers' quality of
life during the illness and assess the existing relation between clinical and
psychological features of patients and their caregivers.
METHODS: The study involved 72 patients/caregivers couples. We used the
following tools: Hospital Anxiety and Depression Scale (HADS), Functional
Assessment of Cancer Therapy--Brain (FACT-Br) for patients and HADS, Caregiver
Reaction Assessment Scale (CRA), 36-Item Short-Form Health Survey (SF-36) for
caregivers.
RESULTS: Quality of life was more compromised in caregivers than in their loved
ones. The impairment of caregivers' quality of life appeared mainly in a
significant reduction in their mental health. Most caregivers experienced more
depressive and anxiety symptoms, as compared with patients. Clinical and
psychological features of patients did not correlate with psychological patterns
of their own caregivers.
CONCLUSIONS: It is important to give caregivers appropriate help, care and
support. Therefore, it is necessary to monitor and treat, if necessary,
caregivers' anxious or depressive symptomatology that impacts their quality of
life, making them more helpless, frustrated and less able to handle the
situation of disease and caregiving situation. It would be desirable to give
caregivers the possibility of a psychological support and equally important
would be a continuous teamwork aimed to promote a better caregivers' adaptation
to the patient's illness. OBJECTIVE: First, we sought to determine if parents of children with cancer or a
brain tumor had greater stress compared to parents of healthy children and to
evaluate the correlates of stress among parents of children with cancer or brain
tumors. Second, we sought to examine the relationship between perceived stress
and symptoms of stress and how that relationship may differ for parents of
children with cancer.
METHODS: In-person, interviewer-assisted surveys were administered to 73 case
dyads (children with cancer or a brain tumor and their parents) and 133
comparison dyads (children without health problems and their parents from a
community sample). Descriptive analyses and multivariable logistic regressions
were performed for case-comparison and case-only analyses to distinguish
correlates of parental stress.
RESULTS: Parents of children with cancer exhibited higher levels of
physiological symptoms of stress than parents of healthy children. Poor sleep
quality and greater social stress (negative social interactions) were
significant correlates of increased levels of stress in parents of children with
cancer (odds ratio 4.23, 95% confidence interval 1.15-15.60; and odds ratio
1.07, 95% confidence interval 1.00-1.14, respectively). A subset of parents
reported symptoms of stress but not perceived stress, and this discordance was
more pronounced among cancer caregivers.
CONCLUSIONS: Implementation of screening tools that include symptoms of stress
may help clinicians to comprehensively identify parents of children with cancer
who are in need of additional services. Targeted stress-reduction interventions
that address sleep quality and negative social interactions may mitigate the
deleterious effects of caregiving, improving the psychosocial well-being of both
parents and children with cancer. PURPOSE/OBJECTIVES: To determine whether the perceived level of spirituality in
family caregivers of patients with primary maligt brain tumors (PMBTs)
changes across the disease trajectory.
DESIGN: Ongoing descriptive, longitudinal study.
SETTING: Southwestern Pennsylvania.
SAMPLE: 50 family caregivers of patients with PMBT.
METHODS: Caregivers and care recipients were recruited at time of diagnosis.
Participants were interviewed at two subsequent time points, four and eight
months following diagnosis.
MAIN RESEARCH VARIABLES: Care recipients' symptoms, neuropsychologic status, and
physical function, as well as caregiver social support.
FINDINGS: Results showed no significant difference in spirituality scores
reported at baseline and eight months (p = 0.8), suggesting that spirituality
may be a stable trait across the disease trajectory.
CONCLUSIONS: Spirituality remains relatively stable along the course of the
disease trajectory. Reports of caregiver depressive symptoms and anxiety were
lower when paired with higher reports of spirituality.
IMPLICATIONS FOR NURSING: Clinicians can better identify caregivers at risk for
negative outcomes by identifying those who report lower levels of spirituality.
Future interventions should focus on the development and implementation of
interventions that provide protective buffers such as increased social support.
KNOWLEDGE TRANSLATION: Spirituality is a relatively stable trait. High levels of
spirituality can serve as a protective buffer from negative mental health
outcomes. Caregivers with low levels of spirituality may be at risk for greater
levels of burden, anxiety, and stress. Sleep loss places caregivers at risk for poor health. Understanding correlates
of sleep loss and relationships to health may enable improvement of health of
caregivers of individuals with primary maligt brain tumors (PMBT). In this
cross-sectional, descriptive study of 133 caregivers, relationships were
examined between sleep loss and physical, mental, emotional, and social health
at time of patient diagnosis. Sleep loss was not related to physical health.
Shorter total sleep time was associated with greater fatigue and social support.
Sleep quality was positively associated with quality of life. Further study is
needed of the role of sleep loss in the PMBT caregiving trajectory and its
long-term relationship with health outcomes. PURPOSE: Brain tumors are associated with neurological sequelae and poor
survival, contributing to distress in patients and their families. Our
institution has conducted separate support groups for brain tumor patients and
caregivers since 1999. This retrospective cohort study aimed to identify
characteristics of brain tumor group participants in relation to attendance
frequency, and compare themes of discussion in patient and caregiver groups.
METHODS: Demographic and medical characteristics were obtained from patient and
caregiver group registration sheets and medical chart review. We quantified
discussion topics recorded by group facilitators between 1999 and 2006,
extracted themes, and examined similarities and differences in the way these
themes were expressed.
RESULTS: A total of 137 patients and 238 caregivers attended the groups; about
half attended more than one session. The chart review of a randomly selected
subset of patient participants revealed that 57.5 % were married, 58.8 % had
high-grade gliomas, and 55 % attended their first group within 3 months of
diagnosis or at tumor progression. Both groups discussed physical and cognitive
consequences, emotional reactions, relationships, coping, end of life, and
practical issues. Caregivers discussed difficulties achieving self-care and
caregiver burden.
CONCLUSIONS: Brain tumor support group facilitators can expect to encounter a
range of medical and psychosocial issues in accommodating patients' and
caregivers' diverse concerns. Separate brain tumor patient and caregiver groups
may allow participants to explore those concerns without worrying about effects
on their friends or family. It remains to be seen whether the groups meet the
needs of attendees, and whether those who do not attend the groups have unmet
needs. PURPOSE OF REVIEW: Patients with brain tumours show a high symptom burden, and
symptoms are difficult to treat and prone to be overlooked. This review of
publications dealing with advanced stages of brain tumours tries to assess the
knowledge gained in the past 2 years and to develop an outlook for further
investigations.
RECENT FINDINGS: We searched for publications on advanced brain tumours in a
palliative medicine setting. Of 138 publications retrieved by search in PubMed,
22 publications met our criteria for inclusion. We predefined categories of
interest: epidemiology and treatment of symptoms; quality of life; and impact on
next of kin, caregivers, medico-social system and decision-making.
SUMMARY: Data suggest that patients with primary or metastatic brain tumours
often have a high symptom burden and unmet needs for palliative care, and
symptoms are hard to diagnose; patients suffer often and early from cognitive
impairment but are rarely appropriately prepared concerning end-of-life wishes.
This reflects on their caregivers' burden as well. For symptomatic treatment of
common symptoms such as fatigue, depression and cognitive impairment,
methylphenidate has established an important role. For assessment of these
symptoms, a shortened questionnaire Quality of Life Questionnaire-15-Palliative
shows potential. Cancer-directed therapy in advanced stages of brain tumours has
to be weighed critically. To assess adequate strategies to help patients and
caregivers with the challenges of brain tumour-specific symptoms, randomized
intervention studies are necessary. The same accounts for cancer-directed
treatment in relation to quality of life in advanced stages of brain tumours. |
What are the effects of homozygosity of EDNRB mutations in addition to Hirschsprung disease? | Three susceptibility genes have been recently identified in HSCR, namely the RET proto-oncogene, the endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene mutations were found in significant proportions of familial (50%) and sporadic (15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients | Hirschsprung disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Two susceptibility genes
have been recently identified in HSCR, namely the RET proto-oncogene and the
endothelin B receptor (EDNRB) gene. Hitherto however, homozygosity for EDNRB
mutations accounted for the HSCR-Waardenburg syndrome (WS) association. Here, we
report heterozygous EDNRB missense mutations (G57S, R319W and P383L) in isolated
HSCR. These data might suggest that EDNRB mutations could be dosage sensitive:
heterozygosity would predispose to isolated HSCR with incomplete penetrance,
while homozygosity would result in more complex neurocristopathies associating
HSCR and WS features. In addition, the present data give further support to the
role of the endothelin-signalling pathway in the development of neural
crest-derived enteric neurons. Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Three susceptibility
genes have been recently identified in HSCR, namely the RET proto-oncogene, the
endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene
mutations were found in significant proportions of familial (50%) and sporadic
(15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the
rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous
EDNRB and EDN3 missense mutations have been reported in isolated HSCR patients.
Some of these results were obtained after the identification of mouse genes
whose natural or site-directed mutations resulted in megacolon and coat color
spotting. There is also conclusive evidence for the involvement of other
independent loci in HSCR. In particular, the recent identification of
neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide
additional candidate genes for HSCR. The dissection of the genetic etiology of
HSCR disease may then provide a unique opportunity to distinguish between a
polygenic and a genetically heterogeneous disease, thereby helping to understand
other complex disorders and congenital malformations hitherto considered as
multifactorial in origin. Finally, the study of the molecular bases of HSCR is
also a step towards the understanding of developmental genetics of the enteric
nervous system giving support to the role of the tyrosine kinase and
endothelin-signaling pathways in the development of neural crest-derived enteric
neurons in human. Hirschsprung's disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Three susceptibility
genes have been recently identified in HSCR, namely the RET proto-oncogene, the
endothelin B receptor (EDNRB) gene, and the endothelin 3 (EDN3) gene. RET gene
mutations were found in significant proportions of familial (50%) and sporadic
(15-20%) HSCR, while homozygosity for EDNRB or EDN3 mutations accounted for the
rare HSCR-Waardenburg syndrome (WS) association. More recently, heterozygous
EDNRB an EDN3 missense mutations have been reported in isolated HSCR patients.
Some of these results were obtained after the identification of mouse genes
whose natural or site-directed mutations resulted in megacolon and coat color
spotting. There is also conclusive evidence for the involvement of other
independent loci in HSCR. In particular, the recent identification of
neurotrophic factors acting as RET ligands (GDNF and Neurturin) provide
additional candidate genes for HSCR. The dissection of the genetic etiology of
HSCR disease may then provide a unique opportunity to distinguish between a
polygenic and a genetically heterogeneous disease, thereby helping to understand
other complex disorders and congenital malformations hitherto considered as
multifactorial in origin. Finally, the study of the molecular bases of HSCR is
also a step towards the understanding of developmental genetics of the enteric
nervous system giving support to the role of the tyrosine kinase and
endothelin-signaling pathways in the development of neural crest-derived enteric
neurons in human. |
What is the prognostic role of alterred thyroid profile after cardiosurgery? | Altered thyroid profile after cardiosurgery is associated with high incidence of atrial fibrillation e delay in recovery (prolonged hospitalisation) in adults and higher score on The Pediatric Risk of Mortality (PRISM; P < 0.042) and a longer duration of ventilation in children.Impportantly in transplanted patients altered thyroid metabolism,low T3 syndrome, is characterized by highest mortality, highest incidence of acute rejection or reoperations and infections | The cardiovascular system is an important target for thyroid hormones. The
present study evaluates the changes affecting thyroid hormone metabolism during
and 6 days after coronary artery bypass and their relationship with the
post-operative outcome of the patients. Thirty-three patients were enrolled in
the study; their thyroid hormone profiles were determined at 13 sampling points
during surgery and for 6 days afterwards. Serum total tri-iodothyronine (T3) and
free T3 (FT3) concentrations decreased significantly after surgery (P<0.001) and
they remained significantly low until the end of the study. Free thyroxine (FT4)
and T4 declined significantly immediately after surgery (P<0.05 for FT4, P<0.001
for T4) but they returned to baseline values (24 h and 96 h post-surgery
respectively). Serum reverse T3 increased remarkably 36 h after surgery
(P<0.001) and remained significantly higher than the baseline value throughout
the study. A relevant finding was that the days of post-operative
hospitalization (10+/-3 days, means+/-S.D.) was inversely correlated with the
slope of the recovery of T3 concentration (P<0.001) or with the area under the
plasma curves of T3 (P=0.024, time range 72-144 h) and the FT3/FT4 ratio
(P=0.037, time range 72-144 h) during the post-operative period. Our data
suggest a prolonged reduction of T4 to T3 conversion in patients undergoing
cardiac surgery and indicate that the recovery period is the most critical in
the evaluation of a possibly successful approach for T3 substitutive therapy. OBJECTIVE: Despite improved perioperative management, atrial fibrillation (AF)
after coronary artery bypass grafting (CABG) remains a relevant clinical
problem, whose pathogenetic mechanisms remain incompletely explained. A reduced
incidence of postoperative AF has been described in CABG patients receiving IV
tri-iodothyronine (T3). This study was designed to define the role of thyroid
metabolism on the genesis of postoperative AF.
METHODS AND RESULTS: Free T3 (fT3), free thyroxine (fT4), and thyroid
stimulating hormone were assayed at admission in 107 consecutive patients
undergoing isolated CABG surgery. Patients with thyroid disease or taking drugs
known to interfere with thyroid function were excluded. A preoperative rhythm
other than sinus rhythm was considered an exclusion criterion. Thirty-three
patients (30.8%) had postoperative AF. An older age (P=0.03), no therapy with
beta-blockers (P=0.08), chronic obstructive pulmonary disease (P=0.08), lower
left ventricle ejection fraction (P=0.09) and lower fT3 concentration (P=0.001),
were univariate predictors of postoperative AF. On multivariate analysis, low
fT3 concentration and lack of beta-blocking therapy were independently related
with the development of postoperative AF (odds ratio, OR, 4.425; 95% confidence
interval, CI, 1.745-11.235; P=0.001 and OR 3.107; 95% CI 1.087-8.875; P=0.03,
respectively). Postoperative AF significantly prolonged postoperative hospital
stay (P=0.002).
CONCLUSIONS: Low basal fT3 concentration can reliably predict the occurrence of
postoperative AF in CABG patients. OBJECTIVE: The purpose of this study was to evaluate serum triiodothyronine
levels as a trigger of postoperative atrial fibrillation (AF) in elderly
patients undergoing cardiac surgery and to study the possible association of
serum triiodothyronine levels with preoperative and postoperative hemodynamics.
DESIGN: Prospective study.
SETTING: University hospital.
PARTICIPANTS: Forty-six consecutive nonemergency patients 65 years or older
undergoing cardiac surgery during 1999 to 2000 in Tampere University Hospital,
Tampere, Finland.
INTERVENTIONS: Free serum T3 concentration was used as a measure of serum
triiodothyronine levels. Samples were taken preoperatively, on the fourth
postoperative day, and at the 3-month follow-up. The hemodynamic state of the
patients was estimated by whole-body impedance cardiography preoperatively,
during the intensive care unit period, daily until the fourth postoperative day,
and at the 3-month follow-up.
MEASUREMENTS AND MAIN RESULTS: AF occurred in 43% of the patients. The patients
in the AF group had significantly more grafts (3.9 v 3.1, p = 0.02), and there
was a small difference in age between the AF and non-AF groups (73 years v 69
years, p = 0.06). The free T3 concentration on the fourth postoperative day was
significantly lower in the AF group (3.5 nmol/L v 4.6 nmol/L, p = 0.04). In
logistic regression analysis, the independent predictors of AF were age, number
of grafts, and serum free T3 concentration on the fourth postoperative day. In
the group with low T3 concentration, the cardiac index was lower (1.4 v 1.8, p =
0.05) and the systemic vascular resistance index was higher (4,064 v 2,969, p =
0.04) but only immediately after the operation. Although the AF mostly appeared
during the second to fourth postoperative days, there were no longer any
differences in the hemodynamic state at that time.
CONCLUSIONS: In a group of elderly patients undergoing cardiac surgery, there
was a strong association between a postoperative decrease of serum
triiodothyronine levels and atrial fibrillation. The decrease of serum
triiodothyronine levels was related to the changes of hemodynamic parameters
only in the immediate postoperative period. BACKGROUND: Our aim was to determine whether the changes in thyroid function
after open-heart surgery in neonates depend on the postoperative course.
METHODS: Twenty neonates undergoing open-heart surgery for congenital heart
disease were prospectively studied in the cardiac intensive care unit of a
university-affiliated children's hospital. The patients were divided into two
groups by level of inotropic support (high or mild).
RESULTS: The groups were similar in age, bypass time and ultrafiltration volume.
In both groups, there was a significant reduction in levels of
thyroid-stimulating hormone and FT4 at 24 h postoperatively. However, in the
high inotropic support group, FT4 was lower for a longer time. This group also
had a significantly higher score on The Pediatric Risk of Mortality (PRISM; P <
0.042) and a longer duration of ventilation (P < 0.014).
CONCLUSIONS: Neonates after open-heart surgery undergo changes in thyroid
function characteristic of euthyroid sick syndrome. The degree of
hypothyroxinemia may be related to the severity of illness and the postoperative
course. OBJECTIVE: Cardiopulmonary bypass (CPB) is associated with thyroid hormone
changes consistent with euthyroid sick syndrome. Similar changes have been
observed after general surgical operations. Thyroid hormone changes and their
association with global oxygen consumption were studied in low-risk patients
undergoing coronary artery bypass grafting (CABG) with and without CPB.
METHODS: Fifty-two patients undergoing primary CABG by the same surgeon were
randomised into either on-pump (ONCAB, n=26) or off-pump (OPCAB, n=26) groups.
Thyroid-stimulating hormone (TSH), free thyroxine (fT4) and free
triiodothyronine (fT3) levels were measured at sequential time-points using
chemiluminescence assays. Global oxygen consumption was measured at sequential
time-points using a continuous cardiac output Swan-Ganz catheter.
RESULTS: In both groups TSH and fT4 remained within normal range throughout the
study. There was a similar and progressive decline in fT3 levels with no
significant difference between the groups over time (p=0.42). Mean fT3 levels at
24h were below the normal range and significantly lower than baseline values
(ONCAB, 3.3+/-0.69 pmol/L vs 5.1+/-0.41 pmol/L, p<0.001; OPCAB, 3.3+/-0.51
pmol/L vs 5.0+/-0.46 pmol/L, p<0.001). There was a significant inverse
relationship between fT3 levels and global oxygen consumption.
CONCLUSIONS: Off-pump surgery is associated with thyroid hormone changes similar
to conventional surgical revascularisation. The data suggest that further
studies into T3 administration during OPCAB may be warranted. BACKGROUND: Some studies have proposed that subclinical hypothyroidism (SCH) has
adverse effects on the cardiovascular system, but little is known about the
effect on patients undergoing cardiovascular operations. We examined the
influence of preoperative SCH on postoperative outcome in patients undergoing
coronary artery bypass grafting (CABG).
METHODS: Among patients who underwent CABG between July 2005 and June 2007 at
Seoul National University Bundang Hospital, 224 with normal thyroid function and
36 with SCH were enrolled. Preoperative risks and postoperative outcomes were
evaluated prospectively without thyroid hormone replacement.
RESULTS: There were no significant differences in primary outcomes (major
adverse cardiovascular events) and secondary outcomes such as wound problems,
mediastinitis, leg infection, respiratory complications, delirium, or
reoperation during the same hospitalization. However, patients with SCH had a
higher incidence of postoperative atrial fibrillation than those with normal
thyroid function after adjustment for age, gender, body mass index, and other
independent variables such as emergency operation, the use of cardiopulmonary
bypass, combined valvular operation, preoperative creatinine levels, left
ventricular systolic dysfunction, and nonuse of beta-blockers (45.5% vs 29%;
odds ratio, 2.552; 95% confidence interval, 1.117 to 5.830; p = 0.026).
CONCLUSIONS: SCH appears to influence the postoperative outcome for patients by
increasing the development of postoperative atrial fibrillation. However, it is
still unproven whether preoperative thyroxine replacement therapy for patients
with SCH might prevent postoperative atrial fibrillation after CABG. BACKGROUND: Thyroid hormones are essential for the correct functioning of the
entire body. Diagnosis of thyroid disorders in patients after heart transplant
in the early post-operative period and the implementation of correct treatment
may prevent life-threatening complications.
MATERIAL/METHODS: The aim of the study was to determine whether the complicated
postoperative course (ie, hypothyroidism, hyperthyroidism or low fT3 syndrome)
in patients in the first month after heart transplantation was connected with
impaired thyroid hormone management. The analysis encompassed material from 98
patients treated with heart transplantation between February 9, 2004 and January
4, 2010. Hyperthyroidism was diagnosed in 21 patients (19M/2F, 52±7 years of
age), hypothyroidism in was diagnosed in 13 patients (10M/3F, 46±12 years old),
and low fT3 syndrome was diagnosed in 18 patients (14M/4F, 43±12 years old).
RESULTS: Patients with fT3 syndrome had the highest mortality (16.7%, NS),
highest incidence of acute rejection (38.9%, NS), highest number of reoperations
(27.8%, NS), and highest incidence of bacterial (16.7%, NS) and fungal
infections (11.1%, NS). Cytomegaloviral infections occurred most frequently in
patients with hyperthyroidism (23.8%, NS). Patients with hypothyroidism (84.6%,
NS) were hospitalized the longest (>30 days), had the highest tendency towards
pleural effusion (23.1%, NS) and pericardial effusion with tamponade (15.5%,
NS), bradycardia with pacemaker (15.4%, NS) and renal failure requiring
hemodiafiltration (15.4%, NS) (Table 1).
CONCLUSIONS: 1. The thyroid test panel should be performed in all patients in
the early post-heart transplant period. 2. The diagnosis of thyroid disorders
should be immediately followed by correct treatment aimed at restoring the
euthyroid state, with a view to facilitate recovery and rehabilitation as well
as to shorten the hospitalization time, thereby lowering treatment costs.<br /> |
List Pentalogy of Fallot. | Pentalogy of Fallot consists of a pulmonic stenosis, a ventricular septal defect, an overriding aorta, a right ventricular hypertrophy and a patent foramen ovale. | Pentalogy of Fallot is a rare cyanotic congenital heart disease characterized by
biventricular origin of the aorta above a large ventricular septal defect,
obstruction of the pulmonary outflow, right ventricular hypertrophy (tetralogy
of Fallot), and an atrial septal defect. Mortality due to syncope, arrhythmia or
congestive heart failure is about 75% by the age of 10 years and 97% by the age
of 40. We report a 69-year old female with uncorrected pentalogy of Fallot who
suffered from recurrent syncope during classic cyanotic spells. Treatment with
bisoprolol is recommended in symptomatic children and also improved symptoms in
our patient. We conclude that single patients with pentalogy of Fallot reach the
seventh decade and that cyanotic congenital heart disease is a rare cause of
syncope in the elderly. An infant, showing peripheral cyanosis, was born after lower abdominal
peripheral caesarian section of the pregt women having TORCH positive test
with the infection of Toxoplasma gondiiand Cytomegalovirus. She had three
abortions prior to this pregcy. Doppler echocardiography of the baby showed
profound intracardiac defects. After birth, echocardiography was carried out for
diagnosis of associated cardiac anomalies. Doppler echocardiography showed
pentalogy of Fallot, and the present case represents the Pentalogy of Fallot
having pulmonary atresia. The baby's heart anomalies were ASD (Atrial Septal
Defect - 6 mm RT to LT Shunt), VSD (Ventricular Septal Defect - bidirectional
shunt), PDA (Patent Ductus Arteriosus - filling both the pulmonary arteries),
and Overriding of Aorta with pulmonary atresia. In conclusion, whenever the
diagnosis pentalogy of fallot is suspected, a multidisciplinary approach is
essential.
KEYWORDS: Pentalogy of fallot; Overriding Aorta; Ventricular Septal Defect;
Atrial Septal Defect; Pulmonary Atresia; Doppler Echocardiography. In a nearly two-year-old ram, descending from a breeding trial to investigate
the effects of shortness of the lower jaw (brachygnathia inferior), a congenital
cardiac anomaly was observed. At the age often months the affected animal, a
cross breed of Cameroon Sheep and East Friesian Milk Sheep, showed
exercise-induced respiratory distress for the first time. Auscultation revealed
a loud systolic heart murmur (grade 5) on both sides of the thorax, most
prominent over the left third intercostal space at shoulder height. Postmortem
examination of the ram's heart showed a pentalogy of Fallot, consisting of a
pulmonic stenosis, a ventricular septal defect, an overriding aorta, a right
ventricular hypertrophy and a patent foramen ovale. A genetic defect has to be
considered as a possible reason. A 3-week-old female white Bengal Tiger cub (Panthera tigris tigris) presented
with acute onset tachypnoea, cyanosis and hypothermia. The cub was severely
hypoxaemic with a mixed acid-base disturbance. Echocardiography revealed severe
pulmonic stenosis, right ventricular hypertrophy, high membranous ventricular
septal defect and an overriding aorta. Additionally, an atrial septal defect was
found on necropsy, resulting in the final diagnosis of Tetralogy of Fallot with
an atrial septal defect (a subclass of Pentalogy of Fallot). This report is the
first to encompass arterial blood gas analysis, thoracic radiographs,
echocardiography and necropsy findings in a white Bengal Tiger cub diagnosed
with Tetralogy of Fallot with an atrial septal defect. Children with uncorrected cyanotic congenital heart diseases can present for non
cardiac surgeries. They pose several challenges to the Anaesthesiologist,
especially when they are posted for emergency surgery, due to the complex
haemodynamic changes secondary to the heart disease. Pentalogy of Fallot (POF)
is a rare form of congenital heart disease characterized by the association of
Tetralogy of Fallot (TOF) with an atrial septal defect (ASD). TOF is the leading
cause of intracardiac right to left shunt and is the commonest type of cyanotic
congenital heart disease to cause a brain abscess. Children with POF presenting
with brain abscess pose several challenges to the anaesthesiologist due to the
altered haemodynamics and warrant a meticulous anaesthetic plan. There are very
few case reports of Anaesthesia management of a child with Pentalogy of Fallot
(POF) presenting for non cardiac surgery. We report the anaesthetic management
of a rare case of a 5-year-old child with uncorrected POF, who presented to our
Superspeciality hospital with a brain abscess and underwent an emergency
craniotomy with drainage of the brain abscess successfully. |
Which is the major function of sororin? | Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. | Sister chromatid cohesion is essential for chromosome segregation and is
mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by
the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin
is thought to mediate cohesion. In vertebrates, Sororin is essential for
cohesion and stable cohesin-DNA interactions, but how Sororin performs these
functions is unknown. We show that DNA replication and cohesin acetylation
promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its
binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for
cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's
ability to dissociate cohesin from DNA. Sororin has only been identified in
vertebrates, but we show that many invertebrate species contain Sororin-related
proteins, and that one of these, Dalmatian, is essential for cohesion in
Drosophila. The mechanism we describe here may therefore be widely conserved
among different species. Sororin is a positive regulator of sister chromatid cohesion that interacts with
the cohesin complex. Sororin is required for the increased stability of the
cohesin complex on chromatin following DNA replication and sister chromatid
cohesion during G(2). The mechanism by which sororin ensures cohesion is
currently unknown. Because the primary sequence of sororin does not contain any
previously characterized structural or functional motifs, we have undertaken a
structure-function analysis of the sororin protein. Using a series of mutant
derivatives of sororin, we show that the ability of sororin to bind to chromatin
is separable from both its role in sister chromatid cohesion and its interaction
with the cohesin complex. We also show that derivatives of sororin with
deletions or mutations in the conserved C terminus fail to rescue the
loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also
abrogate the association of sororin with the cohesin complex. Our data suggest
that the interaction of the highly conserved motif at the C terminus of sororin
with the cohesin complex is critical to its ability to mediate sister chromatid
cohesion. Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is
essential for chromosome segregation. In mammalian cells, cohesion establishment
and Sororin recruitment to chromatin-bound cohesin depends on the
acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a
developmental disease in which mitotic chromosomes have a 'railroad' track
morphology. Here, we show that Esco2 deficiency leads to termination of mouse
development at pre- and post-implantation stages, indicating that Esco2
functions non-redundantly with Esco1. Esco2 is transiently expressed during
S-phase when it localizes to pericentric heterochromatin (PCH). In interphase,
Esco2 depletion leads to a reduction in cohesin acetylation and Sororin
recruitment to chromatin. In early mitosis, Esco2 deficiency causes changes in
the chromosomal localization of cohesin and its protector Sgo1. Our results
suggest that Esco2 is needed for cohesin acetylation in PCH and that this
modification is required for the proper distribution of cohesin on mitotic
chromosomes and for centromeric cohesion. The maintece of sister chromatid cohesion from S phase to the onset of
anaphase relies on a small but evolutionarily conserved protein called Sororin.
Sororin is a phosphoprotein and its dynamic localization and function are
regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of
Sororin with chromatin requires cohesin to be preloaded to chromatin and
modification of Smc3 during DNA replication. Sororin antagonizes the function of
Wapl in cohesin releasing from S to G 2 phase and promotes cohesin release from
sister chromatid arms in prophase via interaction with Plk1. This review focuses
on progress of the identification and regulation of Sororin during cell cycle;
role of post-translational modification on Sororin function; role of Sororin in
the maintece and resolution of sister chromatid cohesion; and finally
discusses Sororin's emerging role in cancer and the potential issues that need
be addressed in the future. Sister chromatid cohesion depends on Sororin, a protein that stabilizes
acetylated cohesin complexes on DNA by antagonizing the cohesin release factor
Wings-apart like protein (Wapl). Cohesion is essential for chromosome
biorientation but has to be dissolved to enable sister chromatid separation. To
achieve this, the majority of cohesin is removed from chromosome arms in
prophase and prometaphase in a manner that depends on Wapl and phosphorylation
of cohesin's subunit stromal antigen 2 (SA2), whereas centromeric cohesin is
cleaved in metaphase by the protease separase. Here we show that the mitotic
kinases Aurora B and Cyclin-dependent kinase 1 (Cdk1) destabilize interactions
between Sororin and the cohesin subunit precocious dissociation of sisters
protein 5 (Pds5) by phosphorylating Sororin, leading to release of acetylated
cohesin from chromosome arms and loss of cohesion. At centromeres, the cohesin
protector shugoshin (Sgo1)-protein phosphatase 2A (PP2A) antagonizes Aurora B
and Cdk1 partly by dephosphorylating Sororin and thus maintains cohesion until
metaphase. We propose that the stepwise loss of cohesion between chromosome arms
and centromeres is caused by local regulation of Wapl activity, which is
controlled by the phosphorylation state of Sororin. Cohesin mediates sister chromatid cohesion and contributes to the organization
of interphase chromatin through DNA looping. In vertebrate somatic cells,
cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional
factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic
association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A
and Pds5B, but their functional specificity remains unclear. Here, we
demonstrate that Pds5 proteins are essential for cohesion establishment by
allowing Smc3 acetylation by the cohesin acetyl transferases (CoATs) Esco1/2 and
binding of Sororin. While both proteins contribute to telomere and arm cohesion,
Pds5B is specifically required for centromeric cohesion. Furthermore, reduced
accumulation of Aurora B at the inner centromere region in cells lacking Pds5B
impairs its error correction function, promoting chromosome mis-segregation and
aneuploidy. Our work supports a model in which the composition and function of
cohesin complexes differs between different chromosomal regions. Sister chromatid cohesion, which depends on cohesin, is essential for the
faithful segregation of replicated chromosomes. Here, we report that splicing
complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently
for the proper progression of the cell through mitosis. Inactivation of splicing
factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex
inactivation. Our data indicate that these splicing factors are all required for
the accumulation of cohesion factor Sororin, by facilitating the proper splicing
of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects
defective cohesion caused by Prp19 complex inactivation. We propose that the
Prp19 complex and the splicing machinery contribute to the establishment of
cohesion by promoting Sororin accumulation during S phase, and are, therefore,
essential to the maintece of genome stability. Author information:
(1)Ubiquitin Signaling Group, Department of Disease Biology, The Novo Nordisk
Foundation Center for Protein Research University of Copenhagen, Copenhagen,
Denmark.
(2)Bioinformatics Centre, Biotech Research and Innovation Centre, University of
Copenhagen, Copenhagen, Denmark.
(3)Department of Proteomics, The Novo Nordisk Foundation Center for Protein
Research, University of Copenhagen, Copenhagen, Denmark Institute of Molecular
Biology (IMB), Mainz, Germany.
(4)Department of Cell Death and Metabolism, Danish Cancer Society Research
Center, Copenhagen, Denmark.
(5)Institute of Molecular and Translational Medicine, Palacky University,
Olomouc, Czech Republic.
(6)Department of Proteomics, The Novo Nordisk Foundation Center for Protein
Research, University of Copenhagen, Copenhagen, Denmark.
(7)Ubiquitin Signaling Group, Department of Disease Biology, The Novo Nordisk
Foundation Center for Protein Research University of Copenhagen, Copenhagen,
Denmark [email protected]. Orderly termination of sister-chromatid cohesion during mitosis is critical for
accurate chromosome segregation. During prophase, mitotic kinases phosphorylate
cohesin and its protector sororin, triggering Wapl-dependent cohesin release
from chromosome arms. The shugoshin (Sgo1)-PP2A complex protects centromeric
cohesin until its cleavage by separase at anaphase onset. Here, we report the
crystal structure of a human cohesin subcomplex comprising SA2 and Scc1.
Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes
extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for
binding to a conserved site on SA2-Scc1. At this site, mutations of SA2 residues
that disrupt Wapl binding bypass the Sgo1 requirement in cohesion protection.
Thus, in addition to recruiting PP2A to dephosphorylate cohesin and sororin,
Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism
between Sgo1 and Wapl augments centromeric cohesion protection. BACKGROUND: Cohesin mediates sister chromatid cohesion by topologically
entrapping sister DNA molecules inside its ring structure. Cohesin is loaded
onto DNA by the Scc2/NIPBL-Scc4/MAU2-loading complex in a manner that depends on
the adenosine triphosphatase (ATPase) activity of cohesin's Smc1 and Smc3
subunits. Subsequent cohesion establishment during DNA replication depends on
Smc3 acetylation by Esco1 and Esco2 and on recruitment of sororin, which "locks"
cohesin on DNA by inactivating the cohesin release factor Wapl.
RESULTS: Human cohesin ATPase mutants associate transiently with DNA in a manner
that depends on the loading complex but cannot be stabilized on chromatin by
depletion of Wapl. These mutants cannot be acetylated, fail to interact with
sororin, and do not mediate cohesion. The absence of Smc3 acetylation in the
ATPase mutants is not a consequence of their transient association with DNA but
is directly caused by their inability to hydrolyze ATP because acetylation of
wild-type cohesin also depends on ATP hydrolysis.
CONCLUSIONS: Our data indicate that cohesion establishment involves the
following steps. First, cohesin transiently associates with DNA in a manner that
depends on the loading complex. Subsequently, ATP hydrolysis by cohesin leads to
entrapment of DNA and converts Smc3 into a state that can be acetylated.
Finally, Smc3 acetylation leads to recruitment of sororin, inhibition of Wapl,
and stabilization of cohesin on DNA. Our finding that cohesin's ATPase activity
is required for both cohesin loading and Smc3 acetylation raises the possibility
that cohesion establishment is directly coupled to the reaction in which cohesin
entraps DNA. Although splicing is essential for the expression of most eukaryotic genes,
inactivation of splicing factors causes specific defects in mitosis. The
molecular cause of this defect is unknown. Here, we show that the spliceosome
subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human
cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion
affects the splicing of specific introns in a subset of pre-mRNAs, including
pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1
depletion causes cohesion defects predomitly by reducing sororin levels,
which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces
APC/C activity and contributes to cohesion defects indirectly by delaying
mitosis and causing "cohesion fatigue". Simultaneous expression of sororin and
APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These
results indicate that the spliceosome is required for mitosis because it enables
expression of genes essential for cohesion. Our transcriptome-wide
identification of retained introns in SNW1- and PRPF8-depleted cells may help to
understand the aetiology of diseases associated with splicing defects, such as
retinosa pigmentosum and cancer. Sister chromatid cohesion mediated by the cohesin complex is essential for
chromosome segregation during cell division. Using functional genomic screening,
we identify a set of 26 pre-mRNA splicing factors that are required for sister
chromatid cohesion in human cells. Loss of spliceosome subunits increases the
dissociation rate of cohesin from chromatin and abrogates cohesion after DNA
replication, ultimately causing mitotic catastrophe. Depletion of splicing
factors causes defective processing of the pre-mRNA encoding sororin, a factor
required for the stable association of cohesin with chromatin, and an associated
reduction of sororin protein level. Expression of an intronless version of
sororin and depletion of the cohesin release protein WAPL suppress the cohesion
defect in cells lacking splicing factors. We propose that spliceosome components
contribute to sister chromatid cohesion and mitotic chromosome segregation
through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion
as an early cellular consequence of compromised splicing. This may have clinical
implications because SF3B1, a splicing factor that we identify to be essential
for cohesion, is recurrently mutated in chronic lymphocytic leukaemia. |
Which gene fusion is the result of the "philadelphia translocation" or the "philadelphia chromosome" mutation? | Chronic myeloid leukemia (CML) is genetically characterized by the presence of the reciprocal translocation t(9;22)(q34;q11), resulting in a BCR/ABL gene fusion on the derivative chromosome 22 called the Philadelphia (Ph) chromosome. The Philadelphia chromosome and its corresponding fusion gene, BCR-ABL, is one of the best-known genetic abnormalities in hematological malignancies. Major BCR-ABL translocation is much more common in chronic myelogenous leukemia (CML) and minor BCR-ABL in acute lymphoblastic leukemia. | In the great majority of patients with chronic myelogenous leukemia (CML) the
reciprocal translocation between chromosomes 9 and 22, t(9;22)(q34;q11),
resulting in the Philadelphia (Ph) chromosome produces fusion DNA sequences
consisting of the 5' part of the major breakpoint cluster region-1 (M-BCR-1) and
the ABL protooncogene which encodes for the P210BCR-ABL phosphoprotein with
tyrosine kinase activity implicated in the pathogenesis of CML. Molecular
analysis was performed on 25 patients with Ph-positive CML using 2 breakpoint
cluster region (bcr) probes within the M-BCR-1 DNA sequences, and two of them
did not contain either detectable rearranged DNA homologous to the 5' side bcr
probe or ABL-related fusion mRNA. The chromosomal in situ hybridization
technique revealed that these two Ph-positive CML cases did not carry DNAs
homologous to the 5' bcr or ABL probes on the Ph chromosome. Furthermore, one of
the two Ph-positive CML cases did not show either rearranged DNA or regions
homologous to the 3' bcr probe on a 9q+ chromosome, while the other CML case
showed a rearrangement detected by the 3' bcr probe and transposition of the 3'
bcr homologous to the 9q+ chromosome. Thus, the possibility is raised that the
BCR/ABL fusion DNA has been deleted in rare CML cases, and that the deletion
possibly occurred in a stepwise manner following the formation of the Ph
chromosome at any stage of the disease. The Philadelphia (Ph) chromosome, the product of t(9:22), is the cytogenetic
hallmark of chronic myelogenous leukemia. The c-abl oncogene on chromosome 9 is
translocated to the Ph chromosome and linked to a breakpoint cluster region
(bcr), which is part of a large bcr gene. This results in the formation of a
bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA
encoding a 210 kd bcr-c-abl fusion protein. The Ph chromosome is also found in
acute lymphoblastic leukemia (Ph+ ALL). Although the c-abl is translocated and a
new 190 kd c-abl protein has been identified, no breakpoints are observed in the
bcr (Ph+bcr- ALL). Here we show that in Ph+bcr- ALL, breakpoints in chromosome
22 occur within the same bcr gene, but more 5' of the bcr. Cloning of a chimeric
bcr-c-abl cDNA demonstrates that the fusion gene is transcribed into a 7 kb
mRNA, encoding a novel fusion protein. The translocation of the c-abl oncogene from chromosome 9 to the bcr gene on
chromosome 22 in cases of Philadelphia chromosome-positive chronic myelogenous
leukemia (CML) generates an aberrant bcr-abl fusion transcript which may be
intimately related to the pathogenesis of CML. Because factors controlling
normal bcr expression might also be involved in the expression of this aberrant
bcr-abl transcript, we studied the patterns of expression of the normal bcr gene
in different cell types. We found that the normal bcr gene was expressed in many
different types of human cells. Moreover, the bcr gene was evolutionarily
conserved, and homologous bcr genomic sequences and RNA transcripts were readily
detected in chick tissue. The highest level of bcr expression in chick tissue
was in brain tissue, the lowest level was in liver tissue, and a truncated bcr
mRNA was noted in chick testes. Normal bcr transcripts, in addition to the
aberrant bcr-abl hybrid transcripts, have been found in all Philadelphia
chromosome-positive CML cells studied to date. Within a given CML sample, the
relative amounts of normal bcr RNA and aberrant bcr-abl RNA were similar. In
addition, the normal bcr and the aberrant bcr-abl hybrid transcripts
demonstrated similarly prolonged half-lives compared with that of the normal
abl-related transcripts in CML cells. These findings suggest that in CML cells,
similar cellular mechanisms control the steady-state levels of both the normal
bcr and the bcr-abl fusion RNAs. Chronic myeloid leukaemia (CML) is characterized cytogenetically by a
t(9;22)(q34;ql1) reciprocal translocation which gives origin to a hybrid BCR-ABL
gene, encoding a p2lO(BCR-ABL) fusion protein with elevated tyrosine kinase
activity and transforming abilities. The t(9;22) was suggested to be associated
with genomic imprinting of centromeric regions of chromosomes 9 and 22, but the
genes directly affected by the translocation, ABL and BCR, were shown not to be
imprinted. For most diagnostic and research purposes the BCR-ABL gene can be
efficiently identified by reverse-transcription and polymerase chain reaction
(RT/PCR) amplification of its fusion transcripts, which can be quantified by
competitive PCR and similar assays for assessment of residual disease in the
follow-up of therapy. In the great majority of CML patients the BCR-ABL
transcripts exhibit a b2a2 and/or a b3a2 junction; in rare cases, the only
detectable BCR-ABL transcripts have unusual junctions, such as b2a3, b3a3, e1a2
or e6a2. There is a recent suggestion that the BCR-ABL gene may not be always
'functional', since extremely low levels of BCR-ABL transcripts can be found in
leucocytes from normal individuals and, conversely, it appears that no BCR-ABL
transcription can be detected in a proportion of Ph-positive haematopoietic
progenitors from some CML patients. The role, if any, of the reciprocal ABL-BCR
hybrid gene in CML is unknown. Although its mRNA message is in frame, no ABL-BCR
fusion protein has yet been identified in CML patients. The blast crisis of CML
has been variably associated with abnormalities of proto-oncogenes, such as RAS
and MYC, or of tumour suppressor genes, in particular RB, p53 and p16, or with
the generation of chimeric transcription factors, as in the AML1-EVI1 gene
fusion. It is likely, therefore, that multiple and alternative molecular
defects, as opposed to a single universal mechanism, underlie the acute
transformation of the disease. Fluorescence in situ hybridization (FISH) technique has been successfully used
to detect the BCR-ABL gene fusion in chronic myeloid leukemia (CML) with the
classic form of the Philadelphia chromosome (Ph). We applied FISH to study three
CML patients showing variant Ph chromosome (either complex or simple type). The
results demonstrate that the use of a yeast artificial chromosome (YAC)-derived
probe (D107F9) and a cosmid probe (cos-abl 8), specific for BCR and ABL genes
respectively, allows also the detection of the BCR-ABL fusion in CML patients
with variant Ph. Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder that is
characterized by splenomegaly and marked elevation of the blood leukocyte count
with granulocyte in maturity. Ph chromosome was identified in CML in 1960 and
was found to clearly result from reciprocal translocation between chromosome 9
and chromosome 22 (t(q;22)) (q34;q11). CML arises from a single pluripotent
hematopoietic stem cell with the Ph chromosome and demonstration of the Ph
chromosome in blood or marrow cells establishes and unequivocal diagnosis of
CML. The Ph chromosome is recognized as the cytogenetic result of a
rearrangement of the ABL gene on chromosome 9 and the BCL gene on chromosome 22,
which leads to the creation of a BCR/ABL fusion gene on chromosome 22. Abnormal
ABL-related protein with increased tyrosine kinase activity suggested a
molecular mechanism of CML. The BCR/ABL fusion gene can be found not only in the
chromosome but in interphase nuclei by fluorescence in situ hybridization
(FISH). We employed both fluorescence activated cell sorter (FACS) and FISH to
study the lineage involvement of individual stem cells and progenitor cells in
patients with CML. Evidence of BCR/ABL fusion was found in pluripotent stem
cells (CD34+, Thy1+), myeloid cells, B progenitor cells (CD34+, CD19+) and T/NK
progenitor cells (CD34+, CD7+, CD5+) but not mature T cells (CD3+) or natural
killer cells (CD3-, CD56+). These data suggested that BCR/ABL gene fusion occurs
in pluripotent stem cells and that Ph+ T cells and natural killer cells are
eliminated during differentiation. Chronic myeloid leukemia (CML) is characterized by formation of the BCR-ABL
fusion gene, usually as a consequence of the Philadelphia (Ph) translocation
between chromosomes 9 and 22. Large deletions on the derivative chromosome 9
have recently been reported, but it was unclear whether deletions arose during
disease progression or at the time of the Ph translocation. Fluorescence in situ
hybridization (FISH) analysis was used to assess the deletion status of 253
patients with CML. The strength of deletion status as a prognostic indicator was
then compared to the Sokal and Hasford scoring systems. The frequency of
deletions was similar at diagnosis and after disease progression but was
significantly increased in patients with variant Ph translocations. In patients
with a deletion, all Ph(+) metaphases carried the deletion. The median survival
of patients with and without deletions was 38 months and 88 months, respectively
(P =.0001). By contrast the survival difference between Sokal or Hasford
high-risk and non-high-risk patients was of only borderline significance (P
=.057 and P =.034). The results indicate that deletions occur at the time of the
Ph translocation. An apparently simple reciprocal translocation may therefore
result in considerable genetic heterogeneity ab initio, a concept that is likely
to apply to other maligcies associated with translocations. Deletion status
is also a powerful and independent prognostic factor for patients with CML. The
prognostic significance of deletion status should now be studied prospectively
and, if confirmed, should be incorporated into management decisions and the
analysis of clinical trials. BACKGROUND: The t(9;22)(q34;q11) translocation leading to the Philadelphia (Ph)
chromosome resulting in BCR-ABL gene fusion is associated with a poor prognosis
in acute lymphoblastic leukemia (ALL).
PROCEDURE: We studied the relation between t(9;22), determined by karyotype,
fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR),
and in vitro drug resistance, measured by the MTT assay, in precursor B-cell ALL
at diagnosis. The findings in twenty-one Ph-positive (Ph+) childhood
common/precursorB (c/preB) cases were compared with 254 Ph-negative (Ph-) ALL
cases.
RESULTS: A large range of LC(50) values was found within the Ph+ patients.
Moreover, LC(50) values did not differ significantly between Ph+ and Ph- samples
for prednisolone, dexamethasone, L-asparaginase, vincristine, anthracyclines,
thiopurines, epipodophyllotoxins, and 4H00-ifosfamide, even after matching for
important prognostic features (age, white blood cell count (WBC), and
immunophenotype). Adult Ph+ (n = 12) ALL was more resistant to prednisolone (>
270-fold, P = 0.030), and displayed an overall tendency to resistance when
compared to matched cases of Ph- (n = 15) adult precursor B-cell ALL. Within Ph+
ALL, in vitro prednisolone resistance increased significantly with age (P =
0.006). The expression of lung resistance protein (LRP), but not P-glycoprotein
(P-gp) or multidrug resistance protein (MRP), was significantly higher in all
Ph+ patients.
CONCLUSIONS: Both childhood and adult Ph+ precursor B-cell ALL samples display a
heterogeneous in vitro resistance profile, with relatively sensitive and
resistant cases. The adult Ph+ samples, however, are generally more resistant
compared to matched Ph- controls, reaching significance for prednisolone. The
correlation of prednisolone resistance with age within the Ph+ cases might help
explain the poorer prognosis of adult Ph+ ALL. Chronic myeloid leukemia (CML) is characterized by formation of a BCR-ABL fusion
gene, usually as a consequence of the Philadelphia (Ph) translocation between
chromosomes 9 and 22. Recently the development of new fluorescence in-situ
hybridization (FISH) techniques has allowed identification of unexpected
deletions of the reciprocal translocation product, the derivative chromosome 9,
in 10% to 15% of patients with CML. These deletions are large, span the
translocation breakpoint, and occur at the same time as the Ph translocation.
Such deletions therefore give rise to previously unsuspected molecular
heterogeneity from the very beginning of this disease, and there is mounting
evidence for similar deletions associated with other translocations. Several
studies have demonstrated that CML patients who carry derivative chromosome 9
deletions exhibit a more rapid progression to blast crisis and a shorter
survival. Deletion status is independent of, and more powerful than, the Sokal
and Hasford/European prognostic scoring systems. The poor prognosis associated
with deletions is seen in patients treated with hydroxyurea or interferon, and
preliminary evidence suggests that patients with deletions may also have a worse
outcome than nondeleted patients following stem cell transplantation or
treatment with imatinib. Poor outcome cannot be attributed to loss of the
reciprocal ABL-BCR fusion gene expression alone, and is likely to reflect loss
of one or more critical genes within the deleted region. The molecular
heterogeneity associated with the Philadelphia translocation provides a new
paradigm with potential relevance to all maligcies associated with reciprocal
chromosomal translocations and/or fusion gene formation. Chronic myelogenous leukemia (CML) is genetically characterized by the
reciprocal translocation of chromosome 9 and 22, t(9;22)(q34;q11) which results
in the fusion of BCR/ABL gene observed on the derivative chromosome 22 called
Philadelphia (Ph') chromosome. About 5-8% of Philadelphia positive patients with
CML show various complex translocations involving one or more other chromosomes,
in addition to chromosome 9 and 22. In our report we discuss one case with CML,
his cytogenetic study revealed a complex translocation t(5;9;22)(p15.1; q34;
q11.2), del 5p15.1-->pter, translocation BCR(22q11.2-->qter) to der(5), positive
Ph-chromosome and positive t(BCR\ABL). Further confirmation of complex
translocation was done by FISH study using the LSI BCR/ABL dual color dual
fusion (DF) translocation probe, chromosome 5 and 22 whole paint probes. Chronic myelogenous leukemia (CML) is characterized by Philadelphia (Ph)
chromosome with a chimeric gene BCR-ABL created by reciprocal t(9:22) (q34;q11)
translocation. Variant Ph chromosome translocations involving chromosomes other
than 9 and 22 are found in 5-10% of CML cases. We here report a CML patient who
carries a four-way Ph chromosome translocation, t(9;22;15;19) (q34;q11;q15;q13).
The patient was diagnosed in 1997 and initially treated with hydroxyurea. In
2002, treatment with imatinib, a selective BCR-ABL tyrosine kinase inhibitor
(TKI), was started but Ph-positive chromosomes remained at the levels of 42-65%,
indicating imatinib failure. In 2006, the point mutations of F359I and L387M
were detected in BCR/ABL gene, which may be related to imatinib failure.
Treatment with nilotinib, a TKI with high target specificity, was then started
which resulted in durable major molecular response. Administration of nilotinib
offered an effective treatment in a CML patient with variant Ph chromosome
translocations and BCR-ABL point mutations after imatinib failure. BACKGROUND: The t(9;22) translocation leads to the formation of the chimeric
breakpoint cluster region/c-abl oncogene 1 (BCR/ABL) fusion gene on der22, the
Philadelphia chromosome. The p185(BCR/ABL) or the p210(BCR/ABL) fusion proteins
are encoded as a result of the translocation, depending on whether a "minor" or
"major" breakpoint occurs, respectively. Both p185(BCR/ABL) and p210(BCR/ABL)
exhibit constitutively activated ABL kinase activity. Through fusion to BCR the
ABL kinase in p185(BCR/ABL) and p210(BCR/ABL) "escapes" the auto-inhibition
mechanisms of c-ABL, such as allosteric inhibition. A novel class of compounds
including GNF-2 restores allosteric inhibition of the kinase activity and the
transformation potential of BCR/ABL. Here we investigated whether there are
differences between p185(BCR/ABL) and p210(BCR/ABL) regarding their sensitivity
towards allosteric inhibition by GNF-2 in models of Philadelphia
chromosome-positive acute lymphatic leukemia.
DESIGN AND METHODS: We investigated the anti-proliferative activity of GNF-2 in
different Philadelphia chromosome-positive acute lymphatic leukemia models, such
as cell lines, patient-derived long-term cultures and factor-dependent lymphatic
Ba/F3 cells expressing either p185(BCR/ABL) or p210(BCR/ABL) and their
resistance mutants.
RESULTS: The inhibitory effects of GNF-2 differed constantly between
p185(BCR/ABL) and p210(BCR/ABL) expressing cells. In all three Philadelphia
chromosome-positive acute lymphatic leukemia models, p210(BCR/ABL)-transformed
cells were more sensitive to GNF-2 than were p185BCR/ABL-positive cells. Similar
results were obtained for p185(BCR/ABL) and the p210(BCR/ABL) harboring
resistance mutations.
CONCLUSIONS: Our data provide the first evidence of a differential response of
p185(BCR/ABL)- and p210(BCR/ABL)- transformed cells to allosteric inhibition by
GNF-2, which is of importance for the treatment of patients with Philadelphia
chromosome-positive acute lymphatic leukemia. |
Is zolpidem an antibiotic? | No, zolpidem is a short-acting imidazopyridine hypnotic drug | The imidazopyridine zolpidem is a short-acting hypnotic chemically distinct from
benzodiazepines (BZs). According to its peculiar neuropharmacologic activity
(selectivity for the omega 1-BZ receptors), zolpidem is expected to be a pure
hypnotic, without the other effects of BZs. In particular, it has been stressed
that zolpidem is well tolerated in adults and in the elderly, and that
tolerance, abuse, dependence, rebound insomnia, and other withdrawal effects do
not develop in relation to zolpidem administration. However, despite these
assumptions, zolpidem abuse, dependence, and withdrawal effects have been
recently discussed and reviewed herein. In addition, the case of a 43-year-old
woman who had an epileptic attack after abrupt interruption of an abused, high
dose of zolpidem (600 mg/d), is reported and discussed. At the clinical level,
it is stressed that the subjective effects ofzolpidem are comparable to those of
other BZs, and that abuse, dependence, and withdrawal seizures cannot be avoided
simply shifting the regimen of a BZ abuser to zolpidem. At the pharmacologic
level, it is important to note that zolpidem's clinical effects cannot be
explained on the basis of the old distinction between omega I and 2 receptors
because this distinction is no longer valid; the new classification ofGABAA
receptor subtypes is reported and zolpidem activity at this level is discussed
herein. |
Can desvenlafaxine be used at a dose of 50mg/day? | Yes, desvenlafaxine can be at 50mg/day to treat patients with major depressive disorder. Studies suggest that 50 mg is the minimum effective dose of desvenlafaxine for the treatment of major depressive disorder. The recommended dose of DVS ranges from 50 to 100 mg. | OBJECTIVE: To assess the efficacy, safety, and tolerability of 50- and
100-mg/day doses of desvenlafaxine (administered as desvenlafaxine succinate), a
serotonin-norepinephrine reuptake inhibitor, for the treatment of major
depressive disorder (MDD).
RESEARCH DESIGN AND METHODS: Patients with Diagnostic and Statistical Manual of
Mental Disorders, Fourth Edition (DSM-IV) MDD and 17-item Hamilton Rating Scale
for Depression (HAM-D(17)) scores > or =20 were randomly assigned to
double-blind placebo or desvenlafaxine treatment (fixed dose of 50 mg/day or 100
mg/day) for 8 weeks. The primary efficacy measure was the HAM-D(17). Changes
from baseline in HAM-D(17) scores were analyzed using analysis of covariance.
The final on-therapy evaluation was the primary endpoint for efficacy analyses,
using last-observation-carried-forward data. MAIN OUTCOMES MEASURES AND RESULTS:
The intent-to-treat population included 447 patients. Desvenlafaxine 50 mg was
associated with a significantly greater adjusted mean change from baseline on
the HAM-D(17) (-11.5) compared with placebo (-9.5, p=0.018); the 100-mg dose
group (-11.0) did not achieve statistical significance (p=0.065). The 100-mg
dose group experienced significant improvements compared with placebo on several
secondary efficacy measures, including the 6-item Hamilton Depression Rating
Scale (p=0.038) and the Visual Analog Scale-Pain Intensity total score
(p=0.041). Both desvenlafaxine doses were generally well-tolerated. The most
common adverse events (incidence > or =10% in either desvenlafaxine group and
twice the rate of placebo) were dry mouth, constipation, insomnia, decreased
appetite, hyperhidrosis, and dizziness.
CONCLUSIONS: These results demonstrate efficacy, safety, and tolerability of
desvenlafaxine 50 mg/day for treating MDD. The significant findings on secondary
measures support the efficacy of desvenlafaxine 100 mg, as seen in other trials.
Conclusions may be limited by the exclusion of MDD patients with comorbid
conditions and the short-term desvenlafaxine treatment duration. INTRODUCTION: To assess the efficacy of desvenlafaxine (administered as
desvenlafaxine succinate) in outpatients with major depressive disorder.
METHODS: A meta-analysis of individual patient data was performed on the
complete set of registration trials (nine randomized, double-blind,
placebo-controlled 8-week studies) of desvenlafaxine. Patients received fixed
(50, 100, 200, or 400 mg/day; n=1,342) or flexible doses (100-400 mg/day; n=463)
of desvenlafaxine or placebo (n=1,108). The primary efficacy variable was the
17-item Hamilton Rating Scale for Depression (HAM-D(17)); the primary intent to
treat analyses used the last-observation-carried-forward method.
RESULTS: Significantly greater improvement with desvenlafaxine versus placebo on
the HAM-D(17) total score was observed for the full data set (difference in
adjusted means: -1.9; P<.001), each fixed-dose group (all P<.001), and the
flexible-dose group (P=.024). Overall rates of HAM-D1(17) response (> or =150%
decrease from baseline score: 53% vs 41%) and remission (HAM-D(17) < or =7: 32%
vs 23%) were significantly greater for desvenlafaxine versus placebo (all
P<.001). Discontinuation rates due to adverse events increased with dose (4% to
18%; placebo: 3%).
CONCLUSION: Desvenlafaxine demonstrated short-term efficacy for treating major
depressive disorder across the range of doses studied. No evidence of greater
efficacy was observed with doses >50 mg/day; a strong dose-response effect on
tolerability was observed. INTRODUCTION: The safety and tolerability profiles of antidepressants can often
influence the treatment choices of clinicians treating major depressive
disorder. The purpose of this investigation was to characterize the safety and
tolerability of desvenlafaxine (administered as desvenlafaxine succinate) in
treating depression.
METHODS: An integrated analysis of all short-term, randomized, double-blind,
placebo-controlled registration studies for major depressive disorder (four
flexible-dose and five fixed-dose studies) was performed. Adult outpatients with
major depressive disorder received desvenlafaxine doses ranging from 50-400
mg/day or placebo for 8 weeks. Treatment-emergent adverse events, laboratory
values, vital signs, and discontinuation symptoms were evaluated. In the subset
of fixed-dose studies, dose-related effects were analyzed.
RESULTS: In the overall population (placebo: n=1,116; desvenlafaxine: n=1,834),
adverse events resulted in discontinuations in 3% of placebo-treated patients
and 12% of desvenlafaxine-treated patients; in the subset of fixed-dose studies,
the rates were 4% with placebo and increased with desvenlafaxine dose (50
mg/day: 4%; 400 mg/day: 18%). The most common treatment-emergent adverse event
was transient nausea that was generally mild to moderate. The most common sexual
dysfunction associated with desvenlafaxine treatment was erectile dysfunction in
men (7% vs 1% with placebo) and anorgasmia in women (1% and 0%). One
desvenlafaxine-treated patient died of a completed suicide; there were four
suicide attempts (three desvenlafaxine, one placebo) and eight cases of suicidal
ideation (five desvenlafaxine, three placebo) during the on-therapy period.
Small but statistically significant changes in mean blood pressure occurred at
all desvenlafaxine doses; clinically meaningful changes were observed in 1% of
placebo-treated patients and 2% of desvenlafaxine-treated patients.
Desvenlafaxine was associated with small but statistically significant mean
changes in laboratory assessments, particularly lipid and liver enzyme
elevations, and electrocardiograms; few cases of these changes were clinically
relevant.
CONCLUSION: Desvenlafaxine in the treatment of major depressive disorder
exhibited a safety and tolerability profile generally consistent with the
serotonin-norepinephrine reuptake inhibitor class. The most common adverse event
was transient nausea. At the recommended therapeutic dose of 50 mg/day,
discontinuation due to adverse events was similar to placebo. Desvenlafaxine succinate (DVS) is one of several serotonin-norepinephrine
reuptake inhibitors (SNRIs). Others are venlafaxine hydrochloride, milnacipran,
and duloxetine. Desvenlafaxine has been approved by the US Food and Drug
Administration (FDA) for the treatment of major depressive disorder (MDD) based
on a number of randomized, placebo-controlled clinical trials. Clinical studies
have investigated the efficacy of DVS in doses ranging from 50 to 400 mg/day for
the treatment of MDD in adult outpatients. The effects of DVS 50 mg/day have
been clearly distinguished from placebo in the reduction of MDD symptoms in such
clinical trials. No additional therapeutic benefits were found at doses > 50
mg/day. The recommended dose of DVS ranges from 50 to 100 mg. Desvenlafaxine is
currently the third SNRI approved by the FDA for this indication. Preliminary
evidence also suggests the clinical usefulness of DVS in the treatment of
vasomotor symptoms of menopause, anxiety symptoms, and painful physical
symptoms. The modified pharmacokinetic and pharmacodynamic profiles of DVS
differentiate this drug from the original product, venlafaxine. Significant
points of difference, compared with venlafaxine, are once-daily dosing and the
achievement of steady-state plasma concentrations within 4 to 5 days. To
summarize, current evidence indicates that DVS has proven efficacy, acceptable
safety and tolerability profiles, convenient dosing, and minimal impact on the
cytochrome P450 enzyme system. A reduced risk for pharmacokinetic drug
interactions is a potential advantage over other selective serotonin
noradrenaline reuptake inhibitors. Desvenlafaxine succinate has demonstrated its
efficacy for treating MDD but its variable efficacy, as shown in individual
studies, limited long-term data, and its different risk-to-benefit ratio
compared with earlier antidepressants, means that further investigation of this
drug is necessary. PURPOSE: To assess the effects of venlafaxine extended-release (XR) capsules and
desvenlafaxine extended-release (XR) tablets upon indinavir pharmacokinetic
properties when co-administrated to healthy volunteers.
METHODS: This was an open-label, two-period, fixed-dose study conducted at the
clinical research unit located on a university campus. Twenty-four healthy
volunteers enrolled in the study (mean age 28.3 ± 8.0 years). Each subject
received a single dose of indinavir 800 mg on day 1. Subsequently, subjects were
then randomly assigned to either the venlafaxine XR group (N = 12) or the
desvenlafaxine XR group (N = 12). Starting on day 2, venlafaxine XR was dosed at
37.5 mg/day for 4 days and increased to 75 mg/day for 6 days. Desvenlafaxine XR
was dosed at 50 mg/day for 10 days. On day 12, indivanvir 800 mg was
co-administered to both the venlafaxine XR and the desvenlafaxine XR groups. The
pharmacokinetics of indinavir were determined both before and at the end of
antidepressant dosing. Plasma indinavir, venlafaxine, and desvenlafaxine
concentrations were assayed by high-performance liquid chromatography with
ultra-violet (UV) detection. Indinavir pharmacokinetic parameters were
calculated by noncompartmental analysis using validated computer software.
RESULTS: Venlafaxine XR and desvenlafaxine XR did not produce any significant
changes in indinavir disposition. Both antidepressants were well tolerated by
the subjects with only minor adverse side effects.
CONCLUSIONS: No pharmacokinetic drug-drug interaction was demonstrated between
venlafaxine XR and indinavir or between desvenlafaxine XR and indinvair. The
lack of interaction could be due to the venlafaxine and desvenlafaxine
extended-release formulation. OBJECTIVE: To evaluate the long-term (11-month) efficacy and safety of
desvenlafaxine (administered as desvenlafaxine succinate) at the recommended
50-mg/d dose in preventing relapse in patients with major depressive disorder
(MDD).
METHOD: Adult outpatients (age ≥ 18 years) with MDD (DSM-IV criteria) and a
17-item Hamilton Depression Rating Scale (HDRS17) total score ≥ 20 at screening
and baseline were enrolled in a multicenter, double-blind, placebo-controlled,
randomized withdrawal trial conducted between June 2009 and March 2011. Patients
who responded to 8-week open-label treatment with desvenlafaxine 50 mg/d with
continuing stable response through week 20 were randomly assigned to receive
placebo or desvenlafaxine 50 mg/d in a 6-month, double-blind, randomized
withdrawal period. The primary efficacy endpoint was time to relapse following
randomization to double-blind treatment, which was compared between groups using
the log-rank test. Relapse was defined as HDRS17 total score ≥ 16,
discontinuation for unsatisfactory response, hospitalization for depression,
suicide attempt, or suicide. Safety and tolerability data were collected
throughout the trial.
RESULTS: A total of 874 patients were enrolled; 548 patients were randomly
assigned to receive placebo (n = 276) or desvenlafaxine 50 mg/d (n = 272) in the
double-blind withdrawal period. Time to relapse was significantly shorter for
placebo versus desvenlafaxine (P < .001). At the end of the 6-month double-blind
treatment, the estimated probability of relapse was 30.2% for placebo versus
14.3% for desvenlafaxine 50 mg/d. Safety and tolerability results were generally
consistent with those in short-term studies of desvenlafaxine 50 mg/d.
CONCLUSIONS: Desvenlafaxine at the recommended dose of 50 mg/d was effective in
relapse prevention of depression during a 6-month period in patients who
demonstrated stable response after 20 weeks of open-label desvenlafaxine
treatment.
TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00887224. BACKGROUND: In an effort to establish the lowest effective dose of
desvenlafaxine (administered as desvenlafaxine succinate), we assessed the
efficacy, safety, and tolerability of 10- and 50-mg/day desvenlafaxine vs
placebo for the treatment of major depressive disorder.
METHODS: Adult outpatients with DSM-IV-defined major depressive disorder and a
17-item Hamilton Rating Scale for Depression (HAM-D(17)) total score ≥20 were
randomly assigned to receive placebo or desvenlafaxine (10 or 50 mg/day) after a
6- to 14-day single-blind placebo lead-in period in an 8-week, phase 3,
fixed-dose trial. The primary efficacy measure was change from baseline in the
HAM-D(17) score analyzed using analysis of covariance. Efficacy analyses were
conducted with the intent-to-treat population, using the last observation
carried forward.
RESULTS: The intent-to-treat population included 673 patients. Change from
baseline to final evaluation in adjusted HAM-D(17) total scores was not
significantly different comparing desvenlafaxine 10 mg/day (-9.28) and
desvenlafaxine 50 mg/day (-8.92) with placebo (-8.42). There were no differences
among treatment groups in the rates of treatment response or remission.
Discontinuations due to adverse events occurred in 1.8%, 0.9%, and 1.8% of
patients in the placebo and desvenlafaxine 10- and 50-mg/day groups,
respectively. Overall rates of treatment-emergent adverse events with both doses
were similar to placebo.
CONCLUSIONS: Both doses of desvenlafaxine failed to separate from placebo.
However, in a companion study reported separately, desvenlafaxine 50 mg, but not
25 mg, separated from placebo. Taken together, these studies suggest that 50 mg
is the minimum effective dose of desvenlafaxine for the treatment of major
depressive disorder. CLINICALTRIALS.GOV IDENTIFIER: NCT00863798
http://clinicaltrials.gov/ct2/show/NCT00863798?term=00863798&rank=1. This study assessed the efficacy of desvenlafaxine 50 mg/day compared with
placebo for treating moderate or severe major depressive disorder (MDD). Data
were pooled from six double-blind, placebo-controlled, desvenlafaxine 50 mg/day
fixed-dose studies in adults with MDD. The primary endpoint was improvement in
17-item Hamilton Rating Scale for Depression (HAM-D17) scores from baseline at
week 8. HAM-D17 changes were evaluated in patients with moderate (18<HAM-D17<25)
or severe (HAM-D17≥25) MDD at baseline using analysis of covariance with
treatment, study, and baseline in the model. The pooled analysis included 2189
patients (desvenlafaxine 50 mg, n=1150; placebo, n=1039). Of those, 694 (32%)
patients had severe depression at baseline. Desvenlafaxine improved HAM-D17
scores versus placebo in patients with either moderate [desvenlafaxine, adjusted
mean (±SE), -10.26±0.24; placebo, -8.87±0.26; P<0.001] or severe MDD
(desvenlafaxine, -11.91±0.40; placebo, -9.85±0.42; P<0.001). Both moderately and
severely depressed patients had significantly higher rates of response and
remission with desvenlafaxine treatment compared with placebo (all P's≤0.029).
Results were similar when baseline severity was defined by Montgomery-Åsberg
Depression Rating Scale or Sheehan Disability Scale scores. Desvenlafaxine 50
mg/day significantly improved depressive symptoms regardless of severity at
baseline and was effective in treating both moderate and severe MDD. |
Which syndrome is associated with OATP1B1 and OATP1B3 deficiency? | Complete and simultaneous deficiency of the organic anion transporting polypeptides OATP1B1 and OATP1B3 due to mutations in their corresponding genes, has been linked to Rotor syndrome. | Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic reuptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted
by the liver into bile. Failure of this system can lead to a buildup of
conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis
of bilirubin excretion and hyperbilirubinemia syndromes is largely understood,
but that of Rotor syndrome, an autosomal recessive disorder characterized by
conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic
uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8
Rotor-syndrome families and found that Rotor syndrome was linked to mutations
predicted to cause complete and simultaneous deficiencies of the organic anion
transporting polypeptides OATP1B1 and OATP1B3. These important
detoxification-limiting proteins mediate uptake and clearance of countless drugs
and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1
polymorphisms have previously been linked to drug hypersensitivities. Using mice
deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we
found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b
transporters mediate their hepatic re uptake. Transgenic expression of human
OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing
liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this
shuttle may allow flexible transfer of bilirubin conjugates (and probably also
drug conjugates) formed in upstream hepatocytes to downstream hepatocytes,
thereby preventing local saturation of further detoxification processes and
hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin
glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains
Rotor-type hyperbilirubinemia.Moreover, OATP1B1 and OATP1B3 null mutations may
confer substantial drug toxicity risks. |
Which type of lung cancer is afatinib used for? | Afatinib is a small molecule covalently binding and inhibiting the EGFR, HER2 and HER4 receptor tyrosine kinases. Trials showed promising efficacy in patients with EGFR-mutant NSCLC or enriched for clinical benefit from EGFR tyrosine kinase inhibitors gefitinib or erlotinib. | Genetic alterations in the kinase domain of the epidermal growth factor receptor
(EGFR) in non-small cell lung cancer (NSCLC) patients are associated with
sensitivity to treatment with small molecule tyrosine kinase inhibitors.
Although first-generation reversible, ATP-competitive inhibitors showed
encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR
mutations, almost all patients developed resistance to these inhibitors over
time. Such resistance to first-generation EGFR inhibitors was frequently linked
to an acquired T790M point mutation in the kinase domain of EGFR, or
upregulation of signaling pathways downstream of HER3. Overcoming these
mechanisms of resistance, as well as primary resistance to reversible EGFR
inhibitors driven by a subset of EGFR mutations, will be necessary for
development of an effective targeted therapy regimen. Here, we show that
BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2,
potently suppresses the kinase activity of wild-type and activated EGFR and HER2
mutants, including erlotinib-resistant isoforms. Consistent with this activity,
BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits
survival of cancer cell lines and induces tumor regression in xenograft and
transgenic lung cancer models, with superior activity over erlotinib. These
findings encourage further testing of BIBW2992 in lung cancer patients harboring
EGFR or HER2 oncogenes. Approximately 10 to 15% of patients with non-small cell lung cancer have tumors
that depend on activation of the epidermal growth factor receptor (EGFR), as
evidenced by mutations in EGFR. In these patients, there is often an initial
dramatic response to treatment with the first-generation EGFR tyrosine kinase
inhibitors (TKIs) erlotinib or gefitinib. A small number of patients with EGFR
mutations have primary resistance to erlotinib and gefitinib, and most patients
who initially respond to treatment with erlotinib or gefitinib will develop
resistance to first-generation EGFR TKIs. The problems with both primary and
acquired resistance to erlotinib and gefitinib support the need for development
of additional agents that inhibit EGFR signaling in such patients. This is an
overview of three representative second-generation EGFR TKIs. HKI-272, a
second-generation irreversible EGFR TKI that also inhibits HER2, has completed
accrual of a phase II trial in both untreated patients and patients with
acquired resistance to erlotinib or gefitinib. XL647 is a reversible inhibitor
of EGFR, HER2, and vascular epidermal growth factor receptor. Preclinical work
shows that XL647 can inhibit cell lines bearing mutated forms of EGFR that have
been associated with acquired resistance. BIBW2992 is an irreversible EGFR TKI
that also inhibits HER2 and vascular epidermal growth factor receptors. In vitro
work shows that this compound inhibits wild-type EGFR, EGFR exon 19 deletion,
EGFR L858R, and EGFR T790M, the mutation associated with acquired resistance.
The preliminary results from phase I and phase II trials for BIBW-2992 and XL647
are discussed. Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer
(NSCLC) represents a distinct disease entity whose molecular phenotype predicts
exquisite sensitivity to the reversible EGFR-tyrosine kinase inhibitors (TKIs)
gefitinib or erlotinib. However, primary or acquired resistance to these agents
remains a major clinical problem. Afatinib is a novel dual irreversible
EGFR/HER2 TKI that has been shown in preclinical studies to potentially prevent,
delay or overcome resistance to reversible EGFR-TKIs. On this basis, the
LUX-Lung clinical trial program has been recently launched for testing this
molecule in advanced NSCLC patients. Notably, early results from the randomized
LUX-Lung 1 trial indicate that afatinib significantly prolongs progression-free
survival compared with placebo in pretreated patients with clinically acquired
resistance to gefitinib or erlotinib. On the other hand, the LUX-Lung 2 trial
shows that afatinib is highly active in the EGFR-mutant subgroup of patients.
While these preliminary data open a new exciting scenario for the future
development of anti-EGFR therapies in NSCLC, ongoing afatinib trials will
definitively establish a role for this molecule in the treatment of advanced
NSCLC. PURPOSE: This Phase I study determined the maximum-tolerated dose (MTD) of
afatinib (Afatinib is an investigational compound and its safety and efficacy
have not yet been established) (BIBW 2992; trade name not yet approved by FDA),
an irreversible inhibitor of epidermal growth factor receptor (EGFR)/human
epidermal growth factor receptor (HER)1 and 2, up to a dose of 50 mg/day in
advanced non-small cell lung cancer (NSCLC), to establish the recommended dose
for Phase II.
METHODS: Patients with advanced NSCLC who had received prior platinum-doublet
chemotherapy and/or erlotinib/gefitinib therapy, or who were ineligible for, or
not amenable to, treatment with established therapies, received oral afatinib
once daily. The MTD was determined based on dose-limiting toxicities (DLTs);
other assessments included safety, pharmacokinetic profile, antitumour activity
according to response evaluation criteria in solid tumours and EGFR/HER1
mutation analysis where possible.
RESULTS: Twelve evaluable patients were treated at doses of 20-50 mg/day. One
DLT was observed at 50 mg/day in Course 1 (Grade 3 mucositis). The most frequent
drug-related adverse events were diarrhoea, dry skin, stomatitis, rash,
paronychia and anorexia; most were Grade 1 or 2. Six out of 12 patients had
tumour size reductions; durable stable disease was achieved in three patients
including one with EGFR/HER1 exon 19 and T790 M mutations. Peak plasma
concentrations of afatinib were reached 3-4 h after administration and declined
with a half-life of 30-40 h. Afatinib 50 mg/day was well tolerated with an
acceptable safety profile during Phase I.
CONCLUSION: Recommended dose for Phase II was defined as 50 mg/day for Japanese
patients; the same as for non-Japanese patients. BACKGROUND: Afatinib, an irreversible ErbB-family blocker, has shown preclinical
activity when tested in EGFR mutant models with mutations that confer resistance
to EGFR tyrosine-kinase inhibitors. We aimed to assess its efficacy in patients
with advanced lung adenocarcinoma with previous treatment failure on EGFR
tyrosine-kinase inhibitors.
METHODS: In this phase 2b/3 trial, we enrolled patients with stage IIIB or IV
adenocarcinoma and an Eastern Cooperative Oncology Group performance (ECOG)
performance score of 0-2 who had received one or two previous chemotherapy
regimens and had disease progression after at least 12 weeks of treatment with
erlotinib or gefitinib. We used a computer-generated sequence to randomly
allocate patients (2:1) to either afatinib (50 mg per day) or placebo; all
patients received best supportive care. Randomisation was done in blocks of
three and was stratified by sex and baseline ECOG performance status (0-1 vs 2).
Investigators, patients, and the trial sponsor were masked to treatment
assignment. The primary endpoint was overall survival (from date of
randomisation to death), analysed on an intention-to-treat basis. This study is
registered with ClinicalTrials.gov, number NCT00656136.
FINDINGS: Between May 26, 2008, and Sept 21, 2009, we identified 697 patients,
585 of whom were randomly allocated to treatment (390 to afatinib, 195 to
placebo). Median overall survival was 10·8 months (95% CI 10·0-12·0) in the
afatinib group and 12·0 months (10·2-14·3) in the placebo group (hazard ratio
1·08, 95% CI 0·86-1·35; p=0·74). Median progression-free survival was longer in
the afatinib group (3·3 months, 95% CI 2·79-4·40) than it was in the placebo
group (1·1 months, 0·95-1·68; hazard ratio 0·38, 95% CI 0·31-0·48; p<0·0001). No
complete responses to treatment were noted; 29 (7%) patients had a partial
response in the afatinib group, as did one patient in the placebo group.
Subsequent cancer treatment was given to 257 (68%) patients in the afatinib
group and 153 (79%) patients in the placebo group. The most common adverse
events in the afatinib group were diarrhoea (339 [87%] of 390 patients; 66 [17%]
were grade 3) and rash or acne (305 [78%] patients; 56 [14%] were grade 3).
These events occurred less often in the placebo group (18 [9%] of 195 patients
had diarrhoea; 31 [16%] had rash or acne), all being grade 1 or 2. Drug-related
serious adverse events occurred in 39 (10%) patients in the afatinib group and
one (<1%) patient in the placebo group. We recorded two possibly
treatment-related deaths in the afatinib group.
INTERPRETATION: Although we recorded no benefit in terms of overall survival
with afatinib (which might have been affected by cancer treatments given after
progression in both groups), our findings for progression-free survival and
response to treatment suggest that afatinib could be of some benefit to patients
with advanced lung adenocarcinoma who have failed at least 12 weeks of previous
EGFR tyrosine-kinase inhibitor treatment.
FUNDING: Boehringer Ingelheim Inc. Reversible ATP-competitive inhibitors targeting the epidermal growth factor
receptor (EGFR) have been established as the most effective treatment of
patients with advanced non-small cell lung cancer (NSCLC) harboring "activating"
mutations in exons 19 and 21 of the EGFR gene. However, clinical activity is
limited by acquired resistance which on average develops within 10 months of
continued treatment. The mechanisms for acquired resistance include selection of
the EGFR T790M mutation in approximately 50% of cases, and MET gene
amplification, PIK3CA gene mutation, transdifferentiation into small-cell lung
cancer and additional rare or unkown mechanisms. Afatinib is a small molecule
covalently binding and inhibiting the EGFR, HER2 and HER4 receptor tyrosine
kinases. In preclinical studies, afatinib not only inhibited the growth of
models with common activating EGFR mutations, but was also active in lung cancer
models harboring wild-type EGFR or the EGFR L858R/T790M double mutant. Clinical
efficacy of afatinib has been extensively studied in the LUX-Lung study program.
These trials showed promising efficacy in patients with EGFR-mutant NSCLC or
enriched for clinical benefit from EGFR tyrosine kinase inhibitors gefitinib or
erlotinib. Here we review the current status of clinical application of afatinib
in NSCLC. We also discuss clinical aspects of resistance to afatinib and
strategies for its circumvention. Lung cancer is the leading cause of cancer-related death in the world. Prior to
the era of targeted therapy, platinum-based doublet chemotherapy was the
first-line therapy of choice for patients with metastatic non-small-cell lung
cancer (NSCLC). The availability of agents that target epidermal growth factor
receptor (EGFR)-tyrosine kinase, as well as inhibitors against anaplastic
lymphoma kinase (ALK) gene rearrangement or ROS-1 gene rearrangement product,
has provided promising clinical benefits in specific subpopulations of NSCLC. At
present, only first-generation EGFR-tyrosine kinase inhibitors (TKIs) (erlotinib
and gefitinib) are available for clinical use. Second-generation irreversible
EGFR-TKIs, such as afatinib, are still in clinical trials. In current clinical
practice, EGFR-TKI is the first-line treatment of choice for metastatic NSCLC
patients with tumor EGFR mutation or as salvage therapy in NSCLC patients who
received systemic chemotherapy previously. Platinum-based doublet chemotherapy
continues to be the standard of care for those treatment-naïve patients with
EGFR wild -type tumor or unknown EGFR status. Even though all investigators
agree with the use of EGFR-TKI as the first-line treatment in tumor EGFR-mutated
patients, only 10-30% of NSCLC patients have mutated EGFR, and there was no
obvious survival difference when EGFR-TKIs were used in a second-line setting
versus a first-line treatment in EGFR-mutated patients. Thus, the molecular
complexity of lung cancer emphasizes the need for optimizing treatment by
seeking a more personalized approach to care, including searching for driver
oncogenes, managing the emergence of resistance and overcoming that resistance,
and optimizing the sequence of treatment. Numerous other novel targeted agents
are now in clinical development, including new agents targeting novel pathways
and those that may have the potential to overcome the limitations or resistance
associated with currently available EGFR-TKIs. In this report, we review the
clinical data of EGFR-TKIs as molecular-targeted therapies in NSCLC. |
Can ferric carboxymaltose be used to treat anemia in inflammatory bowel disease patients? | Ferric carboxymaltose can be used to treat anemia in patients with inflammatory bowel disease, and prevents recurrence of anemia in these patients, compared with placebo. Treatment with ferric carboxymaltose is efficious, safe and well tolerated in iron-deficient IBD patients. | BACKGROUND AIMS: Anemia is a common complication of inflammatory bowel diseases
(IBD) This multicenter study tested the noninferiority and safety of a new
intravenous iron preparation, ferric carboxymaltose (FeCarb), in comparison with
oral ferrous sulfate (FeSulf) in reducing iron deficiency anemia (IDA) in IBD.
METHODS: Two hundred patients were randomized in a 2:1 ratio (137 FeCarb:63
FeSulf) to receive FeCarb (maximum 1,000 mg iron per infusion) at 1-wk intervals
until the patients' calculated total iron deficit was reached or FeSulf (100 mg
b.i.d.) for 12 wk. The primary end point was change in hemoglobin (Hb) from
baseline to week 12.
RESULTS: The median Hb improved from 8.7 to 12.3 g/dL in the FeCarb group and
from 9.1 to 12.1 g/dL in the FeSulf group, demonstrating noninferiority (P=
0.6967). Response (defined as Hb increase of >2.0 g/dL) was higher for FeCarb at
week 2 (P= 0.0051) and week 4 (P= 0.0346). Median ferritin increased from 5.0 to
323.5 mug/L at week 2, followed by a continuous decrease in the FeCarb group
(43.5 mug/L at week 12). In the FeSulf group, a moderate increase from 6.5 to
28.5 mug/L at week 12 was observed. Treatment-related adverse events (AEs)
occurred in 28.5% of the FeCarb and 22.2% of the FeSulf groups, with
discontinuation of study medication due to AEs in 1.5% and 7.9%, respectively.
CONCLUSIONS: FeCarb is effective and safe in IBD-associated anemia. It is
noninferior to FeSulf in terms of Hb change over 12 wk, and provides a fast Hb
increase and a sufficient refill of iron stores. Ferric carboxymaltose (FCM, Ferinject) was effective and well tolerated in the
treatment of iron-deficiency anemia (IDA) in nine, Phase III, randomized,
controlled, multicenter trials in a diverse range of indications, including
patients with inflammatory bowel disease (IBD), post-partum anemia (PPA) or
abnormal uterine bleeding (AUB), chronic heart failure (CHF),
non-dialysis-dependent chronic kidney disease (CKD) and those undergoing
hemodialysis (HD). In most trials, patients received either FCM doses of < or =
1000 mg, administered intravenously (i.v.) over < or = 15 min. or oral ferrous
sulfate (FeSulf) 325 mg (65 mg iron), three times daily (t.i.d.), or 304 mg (100
mg iron), twice daily (b.i.d.). In one trial, patients on HD received 200 mg
i.v. of either FCM or iron sucrose (ISC), two-to-three times weekly. In a pilot
study in patients with CHF and CKD, patients received 200 mg of FCM by push
injection compared with 200 mg of ISC slow injection. FCM was usually
administered until the patient's calculated total iron replacement dose was
achieved. Treatment with FCM improved indices of anemia (hemoglobin [Hb],
ferritin and transferrin saturation [TSAT] values). In patients on HD with IDA
secondary to CKD, FCM demonstrated comparable efficacy to ISC in achieving an
increase in Hb. In patients with IBD or PPA, improvements in Hb levels were more
rapid with FCM than with FeSulf. Patients with PPA receiving FCM compared with
those receiving oral iron achieved an Hb rise > or = 2.0 g/dl earlier (7 days
compared with 14 days; p < 0.001), were more likely to achieve an Hb rise > or =
3.0 g/dl at any time beginning at day 14 (86.3% compared with 60.4%; p < 0.001),
and achieve an Hb > 12.0 g/dl at the end of the study (Day 42; 90.5% compared
with 68.6%, p < 0.01). Serum ferritin increased in the i.v. FCM treatment group,
but not in the oral iron group. Differences between groups were significant at
each study interval. TSAT increased significantly at every interval in both
groups; however, FCM-treated patients showed higher TSAT at each interval after
the first week. FCM improved patient quality of life to an equivalent extent to
oral FeSulf in patients with IBD or PPA, and to a greater extent than oral
FeSulf in women with AUB. FCM also improved quality of life as well as
functional symptoms and exercise capacity in patients with CHF. Safety data from
more than 3000 patients showed that FCM was well tolerated. No safety concerns
have been identified in breastfed infants of mothers receiving FCM. FCM is,
therefore, an effective and well-tolerated option in the treatment of IDA. BACKGROUND & AIMS: Iron deficiency anemia (IDA) is common in chronic diseases
and intravenous iron is an effective and recommended treatment. However, dose
calculations and inconvenient administration may affect compliance and efficacy.
We compared the efficacy and safety of a novel fixed-dose ferric carboxymaltose
regimen (FCM) with individually calculated iron sucrose (IS) doses in patients
with inflammatory bowel disease (IBD) and IDA.
METHODS: This randomized, controlled, open-label, multicenter study included 485
patients with IDA (ferritin <100 μg/L, hemoglobin [Hb] 7-12 g/dL [female] or
7-13 g/dL [male]) and mild-to-moderate or quiescent IBD at 88 hospitals and
clinics in 14 countries. Patients received either FCM in a maximum of 3
infusions of 1000 or 500 mg iron, or Ganzoni-calculated IS dosages in up to 11
infusions of 200 mg iron. Primary end point was Hb response (Hb increase ≥ 2
g/dL); secondary end points included anemia resolution and iron status
normalization by week 12.
RESULTS: The results of 240 FCM-treated and 235 IS-treated patients were
analyzed. More patients with FCM than IS achieved Hb response (150 [65.8%] vs
118 [53.6%]; 12.2% difference, P = .004) or Hb normalization (166 [72.8%] vs 136
[61.8%]; 11.0% difference, P = .015). Both treatments improved quality of life
scores by week 12. Study drugs were well tolerated and drug-related adverse
events were in line with drug-specific clinical experience. Deviations from
scheduled total iron dosages were more frequent in the IS group.
CONCLUSIONS: The simpler FCM-based dosing regimen showed better efficacy and
compliance, as well as a good safety profile, compared with the
Ganzoni-calculated IS dose regimen. Anemia is common in inflammatory bowel disease (IBD), with a prevalence ranging
from 8.8% to 73.7%. This wide range reflects the definitions used and the
populations studied. Although many patients are reported to be asymptomatic,
systematic studies have shown anemia to have a significant impact on quality of
life. Consequently treatment should be instituted early. The commonest cause of
anemia in IBD is iron deficiency, predomitly related to gastrointestinal
blood loss. Anemia of chronic disease often occurs concomitantly, due to
cytokine-mediated impaired erythropoiesis and dysregulated iron metabolism. Oral
iron is a simple and effective method for treating iron deficiency, but requires
long courses of treatment. It is also theoretically implicated with worsening
intestinal inflammation, via the production of toxic reactive oxygen species.
Intravenous iron avoids these concerns, especially with the development of
ferric carboxymaltose, which allow up to 1000mg to be given rapidly. In patients
failing to respond to intravenous iron, the anemia of chronic disease is most
likely to be causative. In this setting evidence suggests that additional
erythropoietin therapy can be effective. Blood transfusions should be avoided as
part of routine management and reserved for patients with substantial acute
gastro-intestinal bleeding, where there is a risk of hemodynamic compromise.
This article discusses the underlying physiology of anemia in IBD, and presents
the current evidence supporting treatment options available. BACKGROUND AND AIMS: We analyzed iron deficiency and the therapeutic response
following intravenous ferric carboxymaltose in a large single-center
inflammatory bowel disease (IBD) cohort.
METHODS: 250 IBD patients were retrospectively analyzed for iron deficiency and
iron deficiency anemia. A subgroup was analyzed regarding efficacy and side
effects of iron supplementation with ferric carboxymaltose.
RESULTS: In the cohort (n = 250), 54.4% of the patients had serum iron levels
≤60 μg/dl, 81.2% had ferritin ≤100 ng/ml, and 25.6% had hemoglobin (Hb) of ≤12
g/dl (females) or ≤13 g/dl (males). In the treatment subcohort (n = 80), 83.1%
of the patients had iron ≤60 μg/dl, 90.4% had ferritin ≤100 ng/ml, and 66.7% had
Hb ≤12/13 g/dl before ferric carboxymaltose treatment. After a median dose of
500 mg ferric carboxymaltose, 74.7% of the patients reached iron >60 μg/dl,
61.6% had ferritin >100 ng/ml, and 90.7% reached Hb >12/13 g/dl at follow-up (p
< 0.0001 for all parameters vs. pretreatment values). The most frequent adverse
event was a transient increase of liver enzymes with male gender as risk factor
(p = 0.008, OR 8.62, 95% CI 1.74-41.66).
CONCLUSIONS: Iron deficiency and anemia are frequent in IBD patients. Treatment
with ferric carboxymaltose is efficious, safe and well tolerated in
iron-deficient IBD patients. BACKGROUND & AIMS: Iron-deficiency anemia is the most common systemic
complication of inflammatory bowel diseases (IBD). Iron-deficiency anemia recurs
frequently and rapidly after iron-replacement therapy in patients with IBD. We
performed a randomized, placebo-controlled trial to determine if administration
of ferric carboxymaltose (FCM) prevents anemia in patients with IBD and low
levels of serum ferritin.
METHODS: We performed a single-blind, multicenter study of noemic patients
who had completed the FERGIcor study. Serum levels of ferritin were assessed
every second month, and patients were given FCM (total iron dose, 1181 ± 662 mg;
n = 105) or placebo (n = 99) when levels decreased to less than 100 μg/L. The
primary end point was time to recurrence of anemia within 8 months. Secondary
end points included changes of quality of life, disease activity, results from
laboratory tests, and adverse events.
RESULTS: Anemia recurred in 26.7% of subjects given FCM and in 39.4% given
placebo. The time to anemia recurrence was longer in the FCM group (hazard
ratio, 0.62; 95% confidence interval, 0.38-1.00; P = .049). Markers of body
levels of iron increased or remained at normal levels in subjects given FCM
(ferritin increased by 30.3 μg/L, transferrin saturation increased by 0.6%) but
decreased in the group given placebo (ferritin decreased by 36.1 μg/L,
transferrin saturation decreased by 4.0%). Changes in quality of life and
disease activity were comparable between groups. Adverse events were reported in
59.0% of the FCM group and 50.5% of the placebo group, and serious adverse
events were reported in 6.7% and 8.1%, respectively.
CONCLUSIONS: FCM prevents recurrence of anemia in patients with IBD, compared
with placebo. Nevertheless, the high rate of anemia recurrence warrants
optimization of the frequency and requirements for FCM treatment. BACKGROUND: Secondary thrombocytosis is a common clinical feature. In patients
with cancer, it is a risk factor for venous thromboembolic events. In
inflammatory bowel disease (IBD), thrombocytosis is so far considered a marker
of active disease and may contribute to the increased thromboembolic risk in
this population. Observed effects of iron therapy on normalization of platelet
counts led us to hypothesize that iron itself may regulate megakaryopoiesis.
Here, we want to test the effect of iron replacement on platelet count and
activity in IBD-associated thrombocytosis.
METHODS: We performed a randomized, single-blinded placebo-controlled trial
testing the effect of ferric carboxymaltose (FCM) in patients with IBD with
secondary thrombocytosis (platelets > 450 G/L). Changes in platelet counts,
hemoglobin, iron parameters, disease activity, megakaryopoietic growth factors,
erythropoietin, and platelet activity were assessed. Patients received placebo
or up to 1500 mg iron as FCM. Endpoints were evaluated at week 6.
RESULTS: A total of 26 patients were included in the study, 15 patients were
available for the per protocol analysis. A drop in platelets >25% (primary
endpoint) was observed in 4 of 8 (50%, iron group) and 1 of 7 patients (14%,
placebo group, P = 0.143). Mean platelet counts dropped on FCM but not on
placebo (536 G/L to 411 G/L versus 580 G/L to 559 G/L; P = 0.002). Disease
activity and megakaryopoietic growth factors remained unchanged and hemoglobin
and iron parameters increased on FCM. The normalization of platelet counts was
associated with a decrease in platelet aggregation and P-selectin expression.
CONCLUSION: FCM lowers platelet counts and platelet activation in patients with
IBD-associated secondary thrombocytosis. OBJECTIVE: Iron deficiency and anemia are being increasingly recognized as a
complication of inflammatory bowel disease (IBD). The aim of this study was to
observe, in a non-interventional way, how Swedish gastroenterologists adhere to
guidelines in IBD outpatients treated with intravenous ferric carboxymaltose
(FCM), and the result of treatment.
MATERIAL AND METHODS: Altogether 394 IBD patients (Crohn's disease (CD) 60%,
ulcerative colitis (UC) 40%) from 14 centers were included. Group A (n = 216)
was observed from November 2008 and group B (n = 178) from March 2010. Time of
observation ranged from 12 to 29 months.
RESULTS: S-Ferritin (µmol/l) and transferrin saturation (T-Sat; %) were recorded
at baseline in 62% and 50% in group A. Median values for Hb, ferritin and T-Sat
at baseline were 111 g/l, 10 µmol/l and10%, respectively, and 134 g/l, 121
µmol/l and 20% after iron treatment (p < 0.001 for all three parameters).
Similar results were found in group B. Approximately three-quarters of all
patients had only one iron infusion during the study period. Median time to
reinfusion was 6 (1-25) months. Only previously described infusion reactions
occurred in 27 (7%) patients.
CONCLUSIONS: Adherence to European guidelines was rather poor and needs to be
improved. The effect on iron parameters of intravenous FCM was significant, and
resulted in a ferritin level that indicates an effect on the iron stores. The
effect was mostly sustained for a year since only one-quarter of the patients
were given repeated iron infusions. No unforeseen safety concerns emerged during
the observation period. |
Which disease is treated with Eliglustat? | Eliglustat was developed for treatment of Gaucher's disease type 1. | Eliglustat tartrate is an investigational oral substrate reduction therapy for
Gaucher disease type 1 that is pharmacologically distinct from intravenous
enzyme replacement therapy. Eliglustat tartrate improved clinical manifestations
in patients who received 50 or 100 mg twice daily for 1 year during an
open-label phase 2 study (Blood. 2010;116(6):893-899). We report further
improvements after 2 years of treatment in 20 patients (11 females, 9 males;
mean age, 33 years) with baseline splenomegaly and thrombocytopenia and/or
anemia. Statistically significant (P < .001) percentage improvements from
baseline occurred in platelet count (mean ± SD, 81% ± 56%), hemoglobin level
(20% ± 15%), spleen volume (-52% ± 11%), and liver volume (-24% ± 13%). Mean
platelet count increased ∼ 50 000/mm(3). Mean hemoglobin level increased 2.1
g/dL overall and 3.1 g/dL in 10 patients with baseline anemia. Organ volume
reductions were greatest in patients with severe baseline organomegaly.
Seventeen (85%) patients met established therapeutic goals for ≥ 3 of the 4
parameters. Lumbar spine bone mineral density increased 7.8% ± 10.6% (P = .01)
and T-score 0.6 ± 0.8 (P = .012), with major gains in osteoporotic and
osteopenic patients. Magnetic resoce imaging assessment showed that bone
marrow infiltration by Gaucher cells was decreased (8/18 patients) or stable
(10/18 patients). No safety-related trends emerged during 2 years of treatment.
This multisite, open-label, single-arm phase 2 study is registered at
www.clinicaltrials.gov as NCT00358150. Three phase 1 studies of eliglustat tartrate (Genz-112638), an oral inhibitor of
glucosylceramide synthase under development for treating Gaucher disease type 1
(GD1), evaluated the safety, tolerability, and pharmacokinetics in healthy
volunteers after escalating single doses (n = 99), escalating multiple doses (n
= 36), and food (n = 24). Eliglustat tartrate was well tolerated at single doses
≤ 20 mg/kg and multiple doses ≤ 200 mg bid, with 50 mg bid producing plasma
concentrations in the predicted therapeutic range. No serious adverse events
occurred. Mild to moderate events of nausea, dizziness, and vomiting increased
in frequency with escalating single and multiple doses. Single doses ≥ 10 mg/kg
caused mild increases in electrocardiogram PR, QRS, and QT/QTc intervals.
Single-dose pharmacokinetics showed dose linearity but not proportionality.
Maximum plasma concentrations occurred at ~2 hours, followed by a monophasic
decline with a ~6-hour terminal half-life. Unchanged drug in 8-hour urine
collections was <1.5% of administered doses. Food did not significantly affect
the rate or extent of absorption. Multiple-dose pharmacokinetics was nonlinear,
showing higher than expected plasma drug concentrations. Steady state was
reached ~60 hours after bid dosing. Higher drug exposure occurred in slower
CYP2D6 metabolizers. Based on favorable results in healthy participants, a phase
2 trial of eliglustat tartrate was initiated in GD1 patients. Synthesis inhibition is the basis for the treatment of type 1 Gaucher disease by
the glucosylceramide synthase (GCS) inhibitor eliglustat tartrate. However, the
extended use of eliglustat and related compounds for the treatment of
glycosphingolipid storage diseases with CNS manifestations is limited by the
lack of brain penetration of this drug. Property modeling around the
D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol (PDMP) pharmacophore was
employed in a search for compounds of comparable activity against the GCS but
lacking P-glycoprotein (MDR1) recognition. Modifications of the carboxamide
N-acyl group were made to lower total polar surface area and rotatable bond
number. Compounds were screened for inhibition of GCS in crude enzyme and whole
cell assays and for MDR1 substrate recognition. One analog,
2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide
(CCG-203586), was identified that inhibited GCS at low omolar concentrations
with little to no apparent recognition by MDR1. Intraperitoneal administration
of this compound to mice for 3 days resulted in a significant dose dependent
decrease in brain glucosylceramide content, an effect not seen in mice dosed in
parallel with eliglustat tartrate. OBJECTIVE: Eliglustat is an investigational oral substrate reduction therapy for
Gaucher disease type 1 (GD1). Its skeletal effects were evaluated by prospective
monitoring of bone mineral density (BMD), fractures, marrow infiltration by
Gaucher cells, focal bone lesions, and infarcts during an open-label,
multi-site, single-arm phase 2 trial (NCT00358150).
MATERIALS AND METHODS: Institutional review board approval and patient informed
consent were obtained. Eliglustat (50 or 100 mg) was self-administered by mouth
twice daily; 19 patients completed 4 years of treatment. All were skeletally
mature (age range, 18-55 years). DXA and MRI assessments were conducted at
baseline and annually thereafter. X-rays were obtained annually until month 24,
and then every other year.
RESULTS: Lumbar spine BMD increased significantly (p = 0.02; n = 15) by a mean
(SD) of 9.9% (14.2%) from baseline to year 4; corresponding T-scores increased
significantly (p = 0.01) from a mean (SD) of -1.6 (1.1) to -0.9 (1.3). Mean
femur T-score remained normal through 4 years. Femur MRI showed that 10/18 (56%)
patients had decreased Gaucher cell infiltration compared to baseline; one
patient with early improvement had transient worsening at year 4. There were no
lumbar spine or femoral fractures and no reported bone crises during the study.
At baseline, 8/19 (42%) patients had focal bone lesions, which remained stable,
and 7/19 (37%) patients had bone infarctions, which improved in one patient by
year 2. At year 4, one new asymptomatic, indeterminate bone lesion was
discovered that subsequently resolved.
CONCLUSIONS: Eliglustat may be a therapeutic option for treating the skeletal
manifestations of GD1. Author information:
(1)Hematology Research Center of Ministry of Healthcare of the Russian
Federation, Novy Zykovsky proezd 4, 125167 Moscow, Russia. Electronic address:
[email protected].
(2)Hospital Ramos Mejia, Urquiza 609, Buenos Aires, Argentina. Electronic
address: [email protected].
(3)Instituto Argentino de Diagnostico y Tratamiento, Marcelo T. de Alvear
2346/2400, Buenos Aires, Argentina. Electronic address: [email protected].
(4)National Gaucher Disease Treatment Center: Yale Lysosomal Disease Center, 333
Cedar Street, 1080 LMP, New Haven, CT 06519, USA. Electronic address:
[email protected].
(5)Instituto Mexicano del Seguro Social, Hospital de Especialidades, Seris y
Zaachila S/N, Mexico City, Mexico. Electronic address: [email protected].
(6)Hematology Ambulatory Services, Rambam Medical Center, 8 Haaliya St, Haifa
31096, Israel. Electronic address: [email protected].
(7)Shaare Zedek Medical Center, Gaucher Unit Floor 5, POB 3235, One Bezek Road,
Jerusalem 91031, Israel. Electronic address: [email protected].
(8)Genzyme, a Sanofi company, 500 Kendall Street, Cambridge, MA, 02142, USA.
Electronic address: [email protected].
(9)Genzyme, a Sanofi company, 500 Kendall Street, Cambridge, MA, 02142, USA.
Electronic address: [email protected].
(10)Genzyme, a Sanofi company, 500 Kendall Street, Cambridge, MA, 02142, USA.
Electronic address: [email protected].
(11)Genzyme, a Sanofi company, 500 Kendall Street, Cambridge, MA, 02142, USA.
Electronic address: [email protected]. Clonal B-cell proliferation is a frequent manifestation of Gaucher disease - a
sphingolipidosis associated with a high risk of multiple myeloma and non-Hodgkin
lymphoma. Gaucher disease is caused by genetic deficiency of acid β-glucosidase,
the natural substrates of which (β-d-glucosylceramide and
β-d-glucosylsphingosine) accumulate, principally in macrophages. Mice with
inducible deficiency of β-glucosidase [Gba(tm1Karl/tm1Karl)Tg(MX1-cre)1Cgn/0]
serve as an authentic model of human Gaucher disease; we have recently reported
clonal B-cell proliferation accompanied by monoclonal serum paraproteins and
cognate tumours in these animals. To explore the relationship between B-cell
maligcy and the biochemical defect, we treated Gaucher mice with eliglustat
tartrate (GENZ 112638), a potent and selective inhibitor of the first committed
step in glycosphingolipid biosynthesis. Twenty-two Gaucher mice received
300 mg/kg of GENZ 112638 daily for 3-10 months from 6 weeks of age. Plasma
concentrations of β-d-glucosylceramide and the unacylated glycosphingolipid,
β-d-glucosylsphingosine, declined. After administration of GENZ 112638 to
Gaucher mice for 3-10 months, serum paraproteins were not detected and there was
a striking reduction in the maligt lymphoproliferation: neither lymphomas nor
plasmacytomas were found in animals that had received the investigational agent.
In contrast, 14 out of 60 Gaucher mice without GENZ 112638 treatment developed
these tumours; monoclonal paraproteins were detected in plasma from 18 of the 44
age-matched mice with Gaucher disease that had not received GENZ 112638.
Long-term inhibition of glycosphingolipid biosynthesis suppresses the
development of spontaneous B-cell lymphoma and myeloma in Gaucher mice. |
List algorithms suitable for predicting protein complexes | Protein-Protein interactions (PPI) play a key role in determining the outcome of most cellular processes. The correct identification and characterization of protein interactions and the networks, which they comprise, is critical for understanding the molecular mechanisms within the cell. Large-scale techniques such as pull down assays and tandem affinity purification are used in order to detect protein interactions in an organism. Today, relatively new high-throughput methods like yeast two hybrid, mass spectrometry, microarrays, and phage display are also used to reveal protein interaction networks. Some suitable algorithms for predicting protein complexes are Naive Bayes Classifier, Negatome, Support Vector Machine, PEWCC, iPTMClust, NDComplex, PROCOMOSS, PPI network, metaPIS, EPOF, EAGLE, NFC, MCODE, DPClus, IPCA, CPM, MCL, CMC, SPICi, Core-Attachment, ProRank, ClusterONE, CFinder, Spectral, RNSC, Affinity Propagation, HKC, NWE, CP-DR, Struct2net and PIPE. | This paper presents a framework for predicting protein-protein interactions
(PPI) that integrates structure-based information with other functional
annotations, e.g. GO, co-expression and co-localization, etc., Given two protein
sequences, the structure-based interaction prediction technique threads these
two sequences to all the protein complexes in the PDB and then chooses the best
potential match. Based on this match, structural information is incorporated
into logistic regression to evaluate the probability of these two proteins
interacting. This paper also describes a random forest classifier which can
effectively combine the structure-based prediction results and other functional
annotations together to predict protein interactions. Experimental results
indicate that the predictive power of the structure-based method is better than
many other information sources. Also, combining the structure-based method with
other information sources allows us to achieve a better performance than when
structure information is not used. We also tested our method on a set of
approximately 1000 yeast genes and, interestingly, the predicted interaction
network is a scale-free network. Our method predicted some potential
interactions involving yeast homologs of human disease-related proteins.
SUPPLEMENTARY INFORMATION: http://theory.csail.mit.edu/struct2net MOTIVATION: Protein complexes are important for understanding principles of
cellular organization and function. High-throughput experimental techniques have
produced a large amount of protein interactions, which makes it possible to
predict protein complexes from protein-protein interaction (PPI) networks.
However, protein interaction data produced by high-throughput experiments are
often associated with high false positive and false negative rates, which makes
it difficult to predict complexes accurately.
RESULTS: We use an iterative scoring method to assign weight to protein pairs,
and the weight of a protein pair indicates the reliability of the interaction
between the two proteins. We develop an algorithm called CMC (clustering-based
on maximal cliques) to discover complexes from the weighted PPI network. CMC
first generates all the maximal cliques from the PPI networks, and then removes
or merges highly overlapped clusters based on their interconnectivity. We
studied the performance of CMC and the impact of our iterative scoring method on
CMC. Our results show that: (i) the iterative scoring method can improve the
performance of CMC considerably; (ii) the iterative scoring method can
effectively reduce the impact of random noise on the performance of CMC; (iii)
the iterative scoring method can also improve the performance of other protein
complex prediction methods and reduce the impact of random noise on their
performance; and (iv) CMC is an effective approach to protein complex prediction
from protein interaction network.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. BACKGROUND: During the last years, high throughput experimental methods have
been developed which generate large datasets of protein - protein interactions
(PPIs). However, due to the experimental methodologies these datasets contain
errors mainly in terms of false positive data sets and reducing therefore the
quality of any derived information. Typically these datasets can be modeled as
graphs, where vertices represent proteins and edges the pairwise PPIs, making it
easy to apply automated clustering methods to detect protein complexes or other
biological significant functional groupings.
METHODS: In this paper, a clustering tool, called GIBA (named by the first
characters of its developers' nicknames), is presented. GIBA implements a two
step procedure to a given dataset of protein-protein interaction data. First, a
clustering algorithm is applied to the interaction data, which is then followed
by a filtering step to generate the final candidate list of predicted complexes.
RESULTS: The efficiency of GIBA is demonstrated through the analysis of 6
different yeast protein interaction datasets in comparison to four other
available algorithms. We compared the results of the different methods by
applying five different performance measurement metrices. Moreover, the
parameters of the methods that constitute the filter have been checked on how
they affect the final results.
CONCLUSION: GIBA is an effective and easy to use tool for the detection of
protein complexes out of experimentally measured protein - protein interaction
networks. The results show that GIBA has superior prediction accuracy than
previously published methods. BACKGROUND: Identification of protein complexes in large interaction networks is
crucial to understand principles of cellular organization and predict protein
functions, which is one of the most important issues in the post-genomic era.
Each protein might be subordinate multiple protein complexes in the real
protein-protein interaction networks. Identifying overlapping protein complexes
from protein-protein interaction networks is a considerable research topic.
RESULT: As an effective algorithm in identifying overlapping module structures,
clique percolation method (CPM) has a wide range of application in social
networks and biological networks. However, the recognition accuracy of algorithm
CPM is lowly. Furthermore, algorithm CPM is unfit to identifying protein
complexes with meso-scale when it applied in protein-protein interaction
networks. In this paper, we propose a new topological model by extending the
definition of k-clique community of algorithm CPM and introduced distance
restriction, and develop a novel algorithm called CP-DR based on the new
topological model for identifying protein complexes. In this new algorithm, the
protein complex size is restricted by distance constraint to conquer the
shortcomings of algorithm CPM. The algorithm CP-DR is applied to the protein
interaction network of Sacchromyces cerevisiae and identifies many well known
complexes.
CONCLUSION: The proposed algorithm CP-DR based on clique percolation and
distance restriction makes it possible to identify dense subgraphs in protein
interaction networks, a large number of which correspond to known protein
complexes. Compared to algorithm CPM, algorithm CP-DR has more outstanding
performance. BACKGROUND: Although homology-based methods are among the most widely used
methods for predicting the structure and function of proteins, the question as
to whether interface sequence conservation can be effectively exploited in
predicting protein-protein interfaces has been a subject of debate.
RESULTS: We studied more than 300,000 pair-wise alignments of protein sequences
from structurally characterized protein complexes, including both obligate and
transient complexes. We identified sequence similarity criteria required for
accurate homology-based inference of interface residues in a query protein
sequence.Based on these analyses, we developed HomPPI, a class of sequence
homology-based methods for predicting protein-protein interface residues. We
present two variants of HomPPI: (i) NPS-HomPPI (Non partner-specific HomPPI),
which can be used to predict interface residues of a query protein in the
absence of knowledge of the interaction partner; and (ii) PS-HomPPI
(Partner-specific HomPPI), which can be used to predict the interface residues
of a query protein with a specific target protein.Our experiments on a benchmark
dataset of obligate homodimeric complexes show that NPS-HomPPI can reliably
predict protein-protein interface residues in a given protein, with an average
correlation coefficient (CC) of 0.76, sensitivity of 0.83, and specificity of
0.78, when sequence homologs of the query protein can be reliably identified.
NPS-HomPPI also reliably predicts the interface residues of intrinsically
disordered proteins. Our experiments suggest that NPS-HomPPI is competitive with
several state-of-the-art interface prediction servers including those that
exploit the structure of the query proteins. The partner-specific classifier,
PS-HomPPI can, on a large dataset of transient complexes, predict the interface
residues of a query protein with a specific target, with a CC of 0.65,
sensitivity of 0.69, and specificity of 0.70, when homologs of both the query
and the target can be reliably identified. The HomPPI web server is available at
http://homppi.cs.iastate.edu/.
CONCLUSIONS: Sequence homology-based methods offer a class of computationally
efficient and reliable approaches for predicting the protein-protein interface
residues that participate in either obligate or transient interactions. For
query proteins involved in transient interactions, the reliability of interface
residue prediction can be improved by exploiting knowledge of putative
interaction partners. BACKGROUND: Protein complexes are important entities to organize various
biological processes in the cell, like signal transduction, gene expression, and
molecular transmission. In most cases, proteins perform their intrinsic tasks in
association with their specific interacting partners, forming protein complexes.
Therefore, an enriched catalog of protein complexes in a cell could accelerate
further research to elucidate the mechanisms underlying many biological
processes. However, known complexes are still limited. Thus, it is a challenging
problem to computationally predict protein complexes from protein-protein
interaction networks, and other genome-wide data sets.
METHODS: Macropol et al. proposed a protein complex prediction algorithm, called
RRW, which repeatedly expands a current cluster of proteins according to the
stationary vector of a random walk with restarts with the cluster whose proteins
are equally weighted. In the cluster expansion, all the proteins within the
cluster have equal influences on determination of newly added protein to the
cluster. In this paper, we extend the RRW algorithm by introducing a random walk
with restarts with a cluster of proteins, each of which is weighted by the sum
of the strengths of supporting evidence for the direct physical interactions
involving the protein. The resulting algorithm is called NWE (Node-Weighted
Expansion of clusters of proteins). Those interaction data are obtained from the
WI-PHI database.
RESULTS: We have validated the biological significance of the results using
curated complexes in the CYC2008 database, and compared our method to RRW and
MCL (Markov Clustering), a popular clustering-based method, and found that our
algorithm outperforms the other algorithms.
CONCLUSIONS: It turned out that it is an effective approach in protein complex
prediction to expand a cluster of proteins, each of which is weighted by the sum
of the strengths of supporting evidence for the direct physical interactions
involving the protein. With the availability of more and more genome-scale protein-protein interaction
(PPI) networks, research interests gradually shift to Systematic Analysis on
these large data sets. A key topic is to predict protein complexes in PPI
networks by identifying clusters that are densely connected within themselves
but sparsely connected with the rest of the network. In this paper, we present a
new topology-based algorithm, HKC, to detect protein complexes in genome-scale
PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to
predict protein complexes by identifying overlapping clusters. The experiments
on two data sets and two benchmarks show that our algorithm has relatively high
F-measure and exhibits better performance compared with some other methods. BACKGROUND: Protein-Protein interactions (PPI) play a key role in determining
the outcome of most cellular processes. The correct identification and
characterization of protein interactions and the networks, which they comprise,
is critical for understanding the molecular mechanisms within the cell.
Large-scale techniques such as pull down assays and tandem affinity purification
are used in order to detect protein interactions in an organism. Today,
relatively new high-throughput methods like yeast two hybrid, mass spectrometry,
microarrays, and phage display are also used to reveal protein interaction
networks.
RESULTS: In this paper we evaluated four different clustering algorithms using
six different interaction datasets. We parameterized the MCL, Spectral, RNSC and
Affinity Propagation algorithms and applied them to six PPI datasets produced
experimentally by Yeast 2 Hybrid (Y2H) and Tandem Affinity Purification (TAP)
methods. The predicted clusters, so called protein complexes, were then compared
and benchmarked with already known complexes stored in published databases.
CONCLUSIONS: While results may differ upon parameterization, the MCL and RNSC
algorithms seem to be more promising and more accurate at predicting PPI
complexes. Moreover, they predict more complexes than other reviewed algorithms
in absolute numbers. On the other hand the spectral clustering algorithm
achieves the highest valid prediction rate in our experiments. However, it is
nearly always outperformed by both RNSC and MCL in terms of the geometrical
accuracy while it generates the fewest valid clusters than any other reviewed
algorithm. This article demonstrates various metrics to evaluate the accuracy of
such predictions as they are presented in the text below. Supplementary material
can be found at: http://www.bioacademy.gr/bioinformatics/projects/ppireview.htm. The tertiary structures of protein complexes provide a crucial insight about the
molecular mechanisms that regulate their functions and assembly. However,
solving protein complex structures by experimental methods is often more
difficult than single protein structures. Here, we have developed a novel
computational multiple protein docking algorithm, Multi-LZerD, that builds
models of multimeric complexes by effectively reusing pairwise docking
predictions of component proteins. A genetic algorithm is applied to explore the
conformational space followed by a structure refinement procedure. Benchmark on
eleven hetero-multimeric complexes resulted in near-native conformations for all
but one of them (a root mean square deviation smaller than 2.5Å). We also show
that our method copes with unbound docking cases well, outperforming the
methodology that can be directly compared with our approach. Multi-LZerD was
able to predict near-native structures for multimeric complexes of various
topologies. Detecting protein complexes from protein-protein interaction (PPI) network is
becoming a difficult challenge in computational biology. There is ample evidence
that many disease mechanisms involve protein complexes, and being able to
predict these complexes is important to the characterization of the relevant
disease for diagnostic and treatment purposes. This article introduces a novel
method for detecting protein complexes from PPI by using a protein ranking
algorithm (ProRank). ProRank quantifies the importance of each protein based on
the interaction structure and the evolutionarily relationships between proteins
in the network. A novel way of identifying essential proteins which are known
for their critical role in mediating cellular processes and constructing protein
complexes is proposed and analyzed. We evaluate the performance of ProRank using
two PPI networks on two reference sets of protein complexes created from Munich
Information Center for Protein Sequence, containing 81 and 162 known complexes,
respectively. We compare the performance of ProRank to some of the well known
protein complex prediction methods (ClusterONE, CMC, CFinder, MCL, MCode and
Core) in terms of precision and recall. We show that ProRank predicts more
complexes correctly at a competitive level of precision and recall. The level of
the accuracy achieved using ProRank in comparison to other recent methods for
detecting protein complexes is a strong argument in favor of the proposed
method. High-throughput experimental technologies, along with computational predictions,
have promoted the emergence of large-scale interactome for numerous organisms.
Identification of protein complexes from these interactome networks is crucial
to understand principles of cellular organization and predict protein functions.
Protein complexes are generally considered as dense subgraphs. However, the real
protein complexes do not always have highly connected topologies. In this paper,
a novel protein complex identifying method, named EPOF, is proposed, using
essential proteins and the local metric of vertex fitness. In EPOF, cliques in
the subnetwork which is consisted by the essential proteins are firstly
considered as seeds, which are ordered according to their size and the number of
their neighbors. A protein complex is extended from a seed based on the
evaluation of its neighbors' fitness value. Then, the similar procedure is
applied to the cliques identified in the subnetwork which is consisted by the
proteins which is not clustered in the first step. When EPOF identifies
complexes by expanding essential protein cliques, the essential proteins have
higher priority and lower threshold. When it identifies complexes by expanding
nonessential protein cliques, the nonessential proteins have higher priority and
lower threshold. Finally, the last step, we output the identified complexes set.
The proposed algorithm EPOF is applied to the unweighted and weighted
interaction networks of S. cerevisiae and detects many well known protein
complexes. We compare the performances of EPOF to other ten previous algorithms,
including EAGLE, NFC, MCODE, DPClus, IPCA, CPM, MCL, CMC, SPICi, and
Core-Attachment. Experimental results show that EPOF outperforms other previous
competing algorithms in terms of matching with known complexes, sensitivity,
specificity, f-measure, function enrichment and accuracy. The program and
related files available on https://github.com/gangchen/epof. The identification of interfaces in protein complexes is effective for the
elucidation of protein function and helps us to understand their roles in
biological processes. With the exponentially growing amount of protein sequence
data, an exploration of new methods that predict protein interaction sites based
solely on sequence information is becoming increasingly urgent. Because a
combination of different methods could produce better results than a single
method, interaction site prediction can be improved through the utilization of
different methods. This paper describes a new method that predicts interaction
sites based on protein sequences by integrating five different algorithms
employing meta-method, Majority Vote and SVMhmm Regression techniques. The
'metaPIS' web-server was implemented for meta-prediction. An evaluation of the
meta-methods using independent datasets revealed that Majority Vote achieved the
highest average Matthews correlation coefficient (0.181) among all the methods
assessed. SVMhmm Regression achieved a lower score but provided a more stable
result. The metaPIS server allows experimental biologists to speculate regarding
protein function by identifying potential interaction sites based on protein
sequence. As a web server, metaPIS is freely accessible to the public at
http://202.116.74.5:84/metapis. Protein complexes play an important role in cellular mechanism. Identification
of protein complexes in protein-protein interaction (PPI) networks is the first
step in understanding the organization and dynamics of cell function. Several
high-throughput experimental techniques produce a large amount of protein
interactions, which can be used to predict protein complexes in a PPI network.
We have developed an algorithm PROCOMOSS (Protein Complex Detection using
Multi-objective Evolutionary Approach based on Semantic Similarity) for
partitioning the whole PPI network into clusters, which serve as predicted
protein complexes. We consider both graphical properties of a PPI network as
well as biological properties based on GO semantic similarity measure as
objective functions. Here three different semantic similarity measures are used
for grouping functionally similar proteins in the same clusters. We have applied
the PROCOMOSS algorithm on two different datasets of Saccharomyces cerevisiae to
find and predict protein complexes. A real-life application of the PROCOMOSS is
also shown here by applying it in the human PPI network consisting of
differentially expressed genes affected by gastric cancer. Gene ontology and
pathway based analyses are also performed to investigate the biological
importance of the extracted gene modules. To facilitate the realization of biological functions, proteins are often
organized into complexes. While computational techniques are used to predict
these complexes, detailed understanding of their organization remains
inadequate. Apart from complexes that reside in very dense regions of a protein
interaction network in which most algorithms are able to identify, we observe
that many other complexes, while not residing in very dense regions, reside in
regions with low neighborhood density. We develop an algorithm for identifying
protein complexes by considering these two types of complexes separately. We
test our algorithm on a few yeast protein interaction networks, and show that
our algorithm is able to identify complexes more accurately than existing
algorithms. A software program NDComplex for implementing the algorithm is
available at http://faculty.cse.tamu.edu/shsze/ndcomplex. MOTIVATION: Tandem mass spectrometry (MS/MS) is a domit approach for
large-scale high-throughput post-translational modification (PTM) profiling.
Although current state-of-the-art blind PTM spectral analysis algorithms can
predict thousands of modified peptides (PTM predictions) in an MS/MS experiment,
a significant percentage of these predictions have inaccurate modification mass
estimates and false modification site assignments. This problem can be addressed
by post-processing the PTM predictions with a PTM refinement algorithm. We
developed a novel PTM refinement algorithm, iPTMClust, which extends a recently
introduced PTM refinement algorithm PTMClust and uses a non-parametric Bayesian
model to better account for uncertainties in the quantity and identity of PTMs
in the input data. The use of this new modeling approach enables iPTMClust to
provide a confidence score per modification site that allows fine-tuning and
interpreting resulting PTM predictions.
RESULTS: The primary goal behind iPTMClust is to improve the quality of the PTM
predictions. First, to demonstrate that iPTMClust produces sensible and accurate
cluster assignments, we compare it with k-means clustering, mixtures of
Gaussians (MOG) and PTMClust on a synthetically generated PTM dataset. Second,
in two separate benchmark experiments using PTM data taken from a phosphopeptide
and a yeast proteome study, we show that iPTMClust outperforms state-of-the-art
PTM prediction and refinement algorithms, including PTMClust. Finally, we
illustrate the general applicability of our new approach on a set of human
chromatin protein complex data, where we are able to identify putative novel
modified peptides and modification sites that may be involved in the formation
and regulation of protein complexes. Our method facilitates accurate PTM
profiling, which is an important step in understanding the mechanisms behind
many biological processes and should be an integral part of any proteomic study.
AVAILABILITY: Our algorithm is implemented in Java and is freely available for
academic use from http://genes.toronto.edu.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics
online. 1. BACKGROUND: Protein complexes are basic cellular entities that carry out the
functions of their components. It can be found that in databases of protein
complexes of yeast like CYC2008, the major type of known protein complexes is
heterodimeric complexes. Although a number of methods for trying to predict sets
of proteins that form arbitrary types of protein complexes simultaneously have
been proposed, it can be found that they often fail to predict heterodimeric
complexes.
RESULTS: In this paper, we have designed several features characterizing
heterodimeric protein complexes based on genomic data sets, and proposed a
supervised-learning method for the prediction of heterodimeric protein
complexes. This method learns the parameters of the features, which are embedded
in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve
Bayes classifier with the parameter values obtained by maximum likelihood
estimation gives the score of a given pair of proteins to predict whether the
pair is a heterodimeric complex or not. A five-fold cross-validation shows good
performance on yeast. The trained classifiers also show higher predictability
than various existing algorithms on yeast data sets with approximate and exact
matching criteria.
CONCLUSIONS: Heterodimeric protein complex prediction is a rather harder problem
than heteromeric protein complex prediction because heterodimeric protein
complex is topologically simpler. However, it turns out that by designing
features specialized for heterodimeric protein complexes, predictability of them
can be improved. Thus, the design of more sophisticate features for
heterodimeric protein complexes as well as the accumulation of more accurate and
useful genome-wide data sets will lead to higher predictability of heterodimeric
protein complexes. Our tool can be downloaded from
http://imi.kyushu-u.ac.jp/~om/. BACKGROUND: Many computational approaches have been developed to detect protein
complexes from protein-protein interaction (PPI) networks. However, these PPI
networks are always built from high-throughput experiments. The presence of
unreliable interactions in PPI network makes this task very challenging.
METHODS: In this study, we proposed a Genetic-Algorithm Fuzzy Naïve Bayes
(GAFNB) filter to classify the protein complexes from candidate subgraphs. It
takes unreliability into consideration and tackles the presence of unreliable
interactions in protein complex. We first got candidate protein complexes
through existed popular methods. Each candidate protein complex is represented
by 29 graph features and 266 biological property based features. GAFNB model is
then applied to classify the candidate complexes into positive or negative.
RESULTS: Our evaluation indicates that the protein complex identification
algorithms using the GAFNB model filtering outperform original ones. For
evaluation of GAFNB model, we also compared the performance of GAFNB with Naïve
Bayes (NB). Results show that GAFNB performed better than NB. It indicates that
a fuzzy model is more suitable when unreliability is present.
CONCLUSIONS: We conclude that filtering candidate protein complexes with GAFNB
model can improve the effectiveness of protein complex identification. It is
necessary to consider the unreliability in this task. |
What are piggyBAC transposons? | The piggyBAC transposons are a nonviral gene delivery approach, that have been developed as tools for insertional mutagenesis. It can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications. | DNA synthesis is considered a defining feature in the movement of transposable
elements. In determining the mechanism of piggyBac transposition, an insect
transposon that is being increasingly used for genome manipulation in a variety
of systems including mammalian cells, we have found that DNA synthesis can be
avoided during piggyBac transposition, both at the donor site following
transposon excision and at the insertion site following transposon integration.
We demonstrate that piggyBac transposon excision occurs through the formation of
transient hairpins on the transposon ends and that piggyBac target joining
occurs by the direct attack of the 3'OH transposon ends on to the target DNA.
This is the same strategy for target joining used by the members of DDE
superfamily of transposases and retroviral integrases. Analysis of mutant
piggyBac transposases in vitro and in vivo using a piggyBac transposition system
we have established in Saccharomyces cerevisiae suggests that piggyBac
transposase is a member of the DDE superfamily of recombinases, an uticipated
result because of the lack of sequence similarity between piggyBac and DDE
family of recombinases. Effective gene therapy requires robust delivery of the desired genes into the
relevant target cells, long-term gene expression, and minimal risks of secondary
effects. The development of efficient and safe nonviral vectors would greatly
facilitate clinical gene therapy studies. However, nonviral gene transfer
approaches typically result in only limited stable gene transfer efficiencies in
most primary cells. The use of nonviral gene delivery approaches in conjunction
with the latest generation transposon technology based on Sleeping Beauty (SB)
or piggyBac transposons may potentially overcome some of these limitations. In
particular, a large-scale genetic screen in mammalian cells yielded a novel
hyperactive SB transposase, resulting in robust and stable gene marking in vivo
after hematopoietic reconstitution with CD34(+) hematopoietic stem/progenitor
cells in mouse models. Moreover, the first-in-man clinical trial has recently
been approved to use redirected T cells engineered with SB for gene therapy of
B-cell lymphoma. Finally, induced pluripotent stem cells could be generated
after genetic reprogramming with piggyBac transposons encoding reprogramming
factors. These recent developments underscore the emerging potential of
transposons in gene therapy applications and induced pluripotent stem generation
for regenerative medicine. Somatic cell gene transfer has permitted inducible gene expression in vivo
through coinfection of multiple viruses. We hypothesized that the highly
efficient plasmid-based piggyBac transposon system would enable long-term
inducible gene expression in mice in vivo. We used a multiple-transposon
delivery strategy to create a tetracycline-inducible expression system in vitro
in human cells by delivering the two genes on separate transposons for inducible
reporter gene expression along with a separate selectable transposon marker.
Evaluation of stable cell lines revealed 100% of selected clones exhibited
inducible expression via stable expression from three separate transposons
simultaneously. We next tested and found that piggyBac-mediated gene transfer to
liver or lung could achieve stable reporter gene expression in mice in vivo in
either immunocompetent or immune deficient animals. A single injection of
piggyBac transposons could achieve long-term inducible gene expression in the
livers of mice in vivo, confirming our multiple-transposon strategy used in
cultured cells. The plasmid-based piggyBac transposon system enables
constitutive or inducible gene expression in vivo for potential therapeutic and
biological applications without using viral vectors. Generation of cultured human cells stably expressing one or more recombit
gene sequences is a widely used approach in biomedical research, biotechnology,
and drug development. Conventional methods are not efficient and have severe
limitations especially when engineering cells to coexpress multiple transgenes
or multiprotein complexes. In this report, we harnessed the highly efficient,
nonviral, and plasmid-based piggyBac transposon system to enable concurrent
genomic integration of multiple independent transposons harboring distinct
protein-coding DNA sequences. Flow cytometry of cell clones derived from a
single multiplexed transfection demonstrated approximately 60% (three
transposons) or approximately 30% (four transposons) stable coexpression of all
delivered transgenes with selection for a single marker transposon. We validated
multiplexed piggyBac transposon delivery by coexpressing large transgenes
encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing
a pore-forming subunit and two accessory subunits while using two additional
genes for selection. Previously unobtainable robust sodium current was
demonstrated through 38 passages, suitable for use on an automated
high-throughput electrophysiology platform. Cotransfection of three large (up to
10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line
expressing two separate alleles of the pore-forming subunit and two accessory
subunits (total of four sodium channel subunits) with robust functional
expression. We conclude that the piggyBac transposon system can be used to
perform multiplexed stable gene transfer in cultured human cells, and this
technology may be valuable for applications requiring concurrent expression of
multiprotein complexes. Transposons are promising systems for somatic gene integration because they can
not only integrate exogenous genes efficiently, but also be delivered to a
variety of organs using a range of transfection methods. piggyBac (PB)
transposon has a high transposability in mammalian cells in vitro, and has been
used for genetic and preclinical studies. However, the transposability of PB in
mammalian somatic cells in vivo has not been demonstrated yet. Here, we
demonstrated PB-mediated sustained gene expression in adult mice. We constructed
PB-based plasmid DNA (pDNA) containing reporter [firefly and Gaussia luciferase
(Gluc)] genes. Mice were transfected by injection of these pDNAs using a
hydrodynamics-based procedure, and the conditions for high-level sustained gene
expression were examined. Consequently, gene expressions were sustained over 2
months. Our results suggest that PB is useful for organ-selective somatic
integration and sustained gene expression in mammals, and will contribute to
basic genetic studies and gene therapies. The TTAA-specific transposon piggyBac (PB), originally isolated from the cabbage
looper moth, Trichoplusia ni, has been utilized as an insertional mutagenesis
tool in various eukaryotic organisms. Here, we show that PB transposes in the
fission yeast Schizosaccharomyces pombe and leaves almost no footprints. We
developed a PB-based mutagenesis system for S. pombe by constructing a strain
with a selectable transposon excision marker and an integrated transposase gene.
PB transposition in this strain has low chromosomal distribution bias as shown
by deep sequencing-based insertion site mapping. Using this system, we obtained
loss-of-function alleles of klp5 and klp6, and a gain-of-function allele of dam1
from a screen for mutants resistant to the microtubule-destabilizing drug
thiabendazole. From another screen for cdc25-22 suppressors, we obtained
multiple alleles of wee1 as expected. The success of these two screens
demonstrated the usefulness of this PB-mediated mutagenesis tool for fission
yeast. BACKGROUND: DNA transposons have emerged as indispensible tools for manipulating
vertebrate genomes with applications ranging from insertional mutagenesis and
transgenesis to gene therapy. To fully explore the potential of two highly
active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have
conducted a side-by-side comparison of the two transposon systems in the same
setting to evaluate their advantages and disadvantages for use in gene therapy
and gene discovery.
RESULTS: We have observed that (1) the Tol2 transposase (but not piggyBac) is
highly sensitive to molecular engineering; (2) the piggyBac donor with only the
40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective
transposition; and (3) a small amount of piggyBac transposases results in robust
transposition suggesting the piggyBac transpospase is highly active. Performing
genome-wide target profiling on data sets obtained by retrieving chromosomal
targeting sequences from individual clones, we have identified several piggyBac
and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear
difference in targeting preferences in the human genome. Finally, we have
observed that (5) only sites with a particular sequence context can be targeted
by either piggyBac or Tol2.
CONCLUSIONS: The non-overlapping targeting preference of piggyBac and Tol2 makes
them complementary research tools for manipulating mammalian genomes. PiggyBac
is the most promising transposon-based vector system for achieving site-specific
targeting of therapeutic genes due to the flexibility of its transposase for
being molecularly engineered. Insights from this study will provide a basis for
engineering piggyBac transposases to achieve site-specific therapeutic gene
targeting. Transposons have been promising elements for gene integration, and the Sleeping
Beauty (SB) system has been the major one for many years, although there have
been several other transposon systems available, for example, Tol2. However,
recently another system known as PiggyBac (PB) has been introduced and developed
for fulfilling the same purposes, for example, mutagenesis, transgenesis and
gene therapy and in some cases with improved transposition efficiency and
advantages over the Sleeping Beauty transposon system, although improved
hyperactive transposase has highly increased the transposition efficacy for SB.
The PB systems have been used in many different scientific research fields;
therefore, the purpose of this review is to describe some of these versatile
uses of the PiggyBac system to give readers an overview on the usage of PiggyBac
system. Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) allows
the derivation of -personalized stem cells. Transposon transgenesis is a novel
and viable alternative to viral transduction methods for the delivery of
reprogramming factors (Oct4, Sox2, Klf4, c-Myc) to somatic cells. Since
transposons can be introduced as naked DNA using common plasmid transfection
protocols, they provide a safer alternative to viral methods. piggyBac
transposons are host-factor independent and integrate stably into the target
genome, yet benefit from the unique characteristic of seamless removal mediated
by transient expression of piggyBac transposase. Thus, piggyBac transposition
provides an effective means to generate human, transgene-free iPSCs. The
protocol describes the production of iPSCs from human embryonic fibroblasts,
delivering reprogramming factors via plasmid transfection and piggyBac
transposition. The development of technologies that allow the stable delivery of large genomic
DNA fragments in mammalian systems is important for genetic studies as well as
for applications in gene therapy. DNA transposons have emerged as flexible and
efficient molecular vehicles to mediate stable cargo transfer. However, the
ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here,
we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in
mouse embryonic stem (ES) cells, making it the only known transposon with such a
large cargo capacity. The integrity of the cargo is maintained during
transposition, the copy number can be controlled and the inserted giant
transposons express the genomic cargo. Furthermore, these 100-kb transposons can
also be excised from the genome without leaving a footprint. The development of
piggyBac as a large cargo vector will facilitate a wider range of genetic and
genomic applications. Mutagenesis by transposon-mediated imprecise excision is the most extensively
used technique for mutagenesis in Drosophila. Although P-element is the most
widely used transposon in Drosophila to generate deletion mutants, it is limited
by the insertion coldspots in the genome where P-elements are rarely found. The
piggyBac transposon was developed as an alternative mutagenic vector for
mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac
transposon can more randomly integrate into the genome. Previous studies
suggested that the piggyBac transposon always excises precisely from the
insertion site without initiating a deletion or leaving behind an additional
footprint. This unique characteristic of the piggyBac transposon facilitates
reversible gene-transfer in several studies, such as the generation of induced
pluripotent stem (iPS) cells from fibroblasts. However, it also raised a
potential limitation of its utility in generating deletion mutants in
Drosophila. In this study, we report multiple imprecise excisions of the
piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila.
Through imprecise excision of the piggyBac transposon inserted in the 5'-UTR of
the SR gene, we generated a hypomorphic mutant allele of the SR gene which
showed markedly decreased levels of SR expression. Our finding suggests that it
is possible to generate deletion mutants by piggyBac transposon-mediated
imprecise excision in Drosophila. However, it also suggests a limitation of
piggyBac transposon-mediated reversible gene transfer for the generation of
induced pluripotent stem (iPS) cells. The piggyBac transposon system is naturally active, originally derived from the
cabbage looper moth. This non-viral system is plasmid based, most commonly
utilizing two plasmids with one expressing the piggyBac transposase enzyme and a
transposon plasmid harboring the gene(s) of interest between inverted repeat
elements which are required for gene transfer activity. PiggyBac mediates gene
transfer through a "cut and paste" mechanism whereby the transposase integrates
the transposon segment into the genome of the target cell(s) of interest.
PiggyBac has demonstrated efficient gene delivery activity in a wide variety of
insect, mammalian, and human cells6 including primary human T cells. Recently, a
hyperactive piggyBac transposase was generated improving gene transfer
efficiency. Human T lymphocytes are of clinical interest for adoptive
immunotherapy of cancer. Of note, the first clinical trial involving transposon
modification of human T cells using the Sleeping beauty transposon system has
been approved. We have previously evaluated the utility of piggyBac as a
non-viral methodology for genetic modification of human T cells. We found
piggyBac to be efficient in genetic modification of human T cells with a
reporter gene and a non-immunogenic inducible suicide gene. Analysis of genomic
integration sites revealed a lack of preference for integration into or near
known proto-oncogenes. We used piggyBac to gene-modify cytotoxic T lymphocytes
to carry a chimeric antigen receptor directed against the tumor antigen HER2,
and found that gene-modified T cells mediated targeted killing of HER2-positive
tumor cells in vitro and in vivo in an orthotopic mouse model. We have also used
piggyBac to generate human T cells resistant to rapamycin, which should be
useful in cancer therapies where rapamycin is utilized. Herein, we describe a
method for using piggyBac to genetically modify primary human T cells. This
includes isolation of peripheral blood mononuclear cells (PBMCs) from human
blood followed by culture, gene modification, and activation of T cells. For the
purpose of this report, T cells were modified with a reporter gene (eGFP) for
analysis and quantification of gene expression by flow cytometry. PiggyBac can
be used to modify human T cells with a variety of genes of interest. Although we
have used piggyBac to direct T cells to tumor antigens, we have also used
piggyBac to add an inducible safety switch in order to eliminate gene modified
cells if needed. The large cargo capacity of piggyBac has also enabled gene
transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we
present a non-viral methodology for stable gene-modification of primary human T
cells for a wide variety of purposes. The DNA transposon piggyBac is widely used as a tool in mammalian experimental
systems for transgenesis, mutagenesis, and genome engineering. We have
characterized genome-wide insertion site preferences of piggyBac by sequencing a
large set of integration sites arising from transposition from two separate
genomic loci and a plasmid donor in mouse embryonic stem cells. We found that
piggyBac preferentially integrates locally to the excision site when mobilized
from a chromosomal location and identified other nonlocal regions of the genome
with elevated insertion frequencies. piggyBac insertions were associated with
expressed genes and markers of open chromatin structure and were excluded from
heterochromatin. At the nucleotide level, piggyBac prefers to insert into
TA-rich regions within a broader GC-rich context. We also found that piggyBac
can insert into sites other than its known TTAA insertion site at a low
frequency (2%). Such insertions introduce mismatches that are repaired with
signatures of host cell repair pathways. Transposons could be mobilized from
plasmids with the observed noncanonical flanking regions, indicating that
piggyBac could generate point mutations in the genome. Reported here is a piggyBac transposon-based expression system for the
generation of doxycycline-inducible, stably transfected mammalian cell cultures
for large-scale protein production. The system works with commonly used adherent
and suspension-adapted mammalian cell lines and requires only a single
transfection step. Moreover, the high uniform expression levels observed among
clones allow for the use of stable bulk cell cultures, thereby eliminating
time-consuming cloning steps. Under continuous doxycycline induction, protein
expression levels have been shown to be stable for at least 2 mo in the absence
of drug selection. The high efficiency of the system also allows for the
generation of stable bulk cell cultures in 96-well format, a capability leading
to the possibility of generating stable cell cultures for entire families of
membrane or secreted proteins. Finally, we demonstrate the utility of the system
through the large-scale production (140-750 mg scale) of an endoplasmic
reticulum-resident fucosyltransferase and two potential anticancer protein
therapeutic agents. Accurate and effective positive marker excision is indispensable for the
introduction of desired mutations into the plant genome via gene targeting (GT)
using a positive/negative counter selection system. In mammals, the moth-derived
piggyBac transposon system has been exploited successfully to eliminate a
selectable marker from a GT locus without leaving a footprint. Here, we present
evidence that the piggyBac transposon also functions in plant cells. To
demonstrate the use of the piggyBac transposon for effective marker excision in
plants, we designed a transposition assay system that allows the piggyBac
transposition to be visualized as emerald luciferase (Eluc) luminescence in rice
cells. The Eluc signal derived from piggyBac excision was observed in
hyperactive piggyBac transposase-expressing rice calli. Polymerase chain
reaction, Southern blot analyses and sequencing revealed the efficient and
precise transposition of piggyBac in these calli. Furthermore, we have
demonstrated the excision of a selection marker from a reporter locus in T0
plants without concomitant re-integration of the transposon and at a high
frequency (44.0% of excision events), even in the absence of negative selection. |
Is there any association of the chromosomal region harboring the gene ITIH3 with schizophrenia? | Yes, genome-wide significant associations in schizophrenia has been linked to the locus harboring the ITIH3/4 genes. | Collaborators: Sklar P, Ripke S, Scott LJ, Andreassen OA, Cichon S, Craddock N,
Edenberg HJ Jr, Nurnberger JI, Rietschel M, Blackwood D, Corvin A, Flickinger M,
Guan W, Mattingsdal M, McQuillen A, Kwan P, Wienker TF, Daly M, Dudbridge F,
Holmans PA, Lin D, Burmeister M, Greenwood TA, Hamshere ML, Muglia P, Smith EN,
Zandi PP, Nievergelt CM, McKinney R, Shilling PD, Schork NJ, Bloss CS, Foroud T,
Koller DL, Gershon ES, Liu C, Badner JA, Scheftner WA, Lawson WB, Nwulia EA,
Hipolito M, Coryell W, Rice J, Byerley W, McMahon FJ, Schulze TG, Berrettini W,
Lohoff FW, Potash JB, Mahon PB, McInnis MG, Zöllner S, Zhang P, Craig DW,
Szelinger S, Barrett TB, Breuer R, Meier S, Strohmaier J, Witt SH, Tozzi F,
Farmer A, McGuffin P, Strauss J, Xu W, Kennedy JL, Vincent JB, Matthews K, Day
R, Ferreira MA, O'Dushlaine C, Perlis R, Raychaudhuri S, Ruderfer D, Lee PH,
Smoller JW, Li J, Absher D, Bunny WE, Barchas JD, Schatzberg AF, Jones EG, Meng
F, Thompson RC, Watson SJ, Myers RM, Akil H, Boehnke M, Chambert K, Moran J,
Scolnick E, Djurovic S, Melle I, Morken G, Gill M, Morris D, Quinn E, Mühleisen
TW, Degenhardt FA, Mattheisen M, Schumacher J, Maier W, Steffans M, Propping P,
Nöthen MM, Anjorin A, Bass N, Gurling H, Kandaswamy R, Lawrence J, McGhee K,
McIntosh A, McLean AW, Muir WJ, Pickard BS, Breen G, St Clair D, Caesar S,
Gordon-Smith K, Jones L, Fraser C, Green EK, Frozeva D, Jones IR, Kirov G,
Moskvina V, Nikolov I, O'Donovan MC, Owen MJ, Collier DA, Elkin A, Williamson R,
Young AH, Ferrier IN, Stefansson K, Stefansson H, Porgeirsson P, Steinberg S,
Gustafsson Ó, Bergen SE, Nimgaonkar V, Hultman C, Landén M, Lichtenstein P,
Sullivan P, Schalling M, Osby U, Backlund L, Frisén L, Langstrom N, Jamain S,
Leboyer M, Etain B, Bellivier F, Petursson H, Sigurđsson E, Müller-Mysok B,
Lucae S, Schwarz M, Fullerton JM, Schofield PR, Martin N, Montgomery GW, Lathrop
M, Óskarsson H, Bauer M, Wright A, Mitchell PB, Hautzinger M, Reif A, Kelsoe JR,
Purcell SM. Collaborators: Ripke S, Sanders AR, Kendler KS, Levinson DF, Sklar P, Holmans
PA, Lin DY, Duan J, Ophoff RA, Andreassen OA, Scolnick E, Cichon S, St Clair D,
Corvin A, Gurling H, Werge T, Rujescu D, Blackwood DH, Pato CN, Malhotra AK,
Purcell S, Dudbridge F, Neale BM, Rossin L, Visscher PM, Posthuma D, Ruderfer
DM, Fanous A, Stefansson H, Steinberg S, Mowry BJ, Golimbet V, De Hert M,
Jönsson EG, Bitter I, Pietiläinen OP, Collier DA, Tosato S, Agartz I, Albus M,
Alexander M, Amdur RL, Amin F, Bass N, Bergen SE, Black DW, Børglum AD, Brown
MA, Bruggeman R, Buccola NG, Byerley WF, Cahn W, Cantor RM, Carr VJ, Catts SV,
Choudhury K, Cloninger CR, Cormican P, Craddock N, Danoy PA, Datta S, de Hann L,
Demontis D, Dikeos D, Djurovic S, Donnelly P, Donohoe G, Duong L, Dwyer S,
Fink-Jensen A, Freedman R, Freimer NB, Friedl M, Georgieva L, Giegling I, Gill
M, Glenthøj B, Godard S, Hamshere M, Hansen M, Hansen T, Hartmann AM, Henskens
FA, Hougaard DM, Hultman CM, Ingason A, Jablensky AV, Jakobsen KD, Jay M,
Jürgens G, Kahn RS, Keller MC, Kenis G, Kenny E, Kim Y, Kirov GK, Konnerth H,
Konte B, Krabbendam L, Krausucki R, Lasseter VK, Laurent C, Lawrence J, Lencz T,
Lerer FB, Liang KY, Lichtenstein P, Lieberman JA, Linszen DH, Lönnqvist J,
Loughland CM, Maclean AW, Maher BS, Maier W, Mallet J, Malloy P, Mattheisen M,
Mattinsgsdal M, McGhee KA, McGrath JJ, McIntosh A, McLean DE, McQuillin A, Melle
I, Michie PT, Milanova V, Morris DW, Mors O, Mortensen PB, Moskvina V, Muglia P,
Myin-Germeys I, Nertney DA, Nestadt G, Nielsen J, Nikolov I, Nordentroft M,
Norton N, Nöthen MM, O'Dushlaine CT, Olincy A, Olsen L, O'Neill FA, Ørntoft T,
Owen MJ, Pantelis C, Papadimitriou G, Pato MT, Peltonen L, Petursson H, Pickard
B, Pimm J, Pulver AE, Puri V, Quested D, Quinn EM, Rasmussen HB, Réthelyi JM,
Ribble R, Rietschel M, Riley BP, Ruggeri M, Schall U, Schulze TG, Schwab SG,
Scott RJ, Shi J, Sigurdsson E, Silverman JM, Spencer CC, Stefansson K, Strange
A, Strengman E, Stroup TS, Suvisaari J, Tereniuis L, Thirumalai S, Thygesen JH,
Timm S, Toncheva D, van den Oord E, van Os J, van Winkel R, Veldink J, Walsh D,
Wang AG, Wiersma D, Wildenauer DB, Williams HJ, Williams NM, Wormley B, Zammit
S, Sullivan PF, O'Donovan MC, Daly MJ, Gejman PV. The Schizophrenia Psychiatric Genome-Wide Association Study Consortium (PGC)
highlighted 81 single-nucleotide polymorphisms (SNPs) with moderate evidence for
association to schizophrenia. After follow-up in independent samples, seven loci
attained genome-wide significance (GWS), but multi-locus tests suggested some
SNPs that did not do so represented true associations. We tested 78 of the 81
SNPs in 2640 individuals with a clinical diagnosis of schizophrenia attending a
clozapine clinic (CLOZUK), 2504 cases with a research diagnosis of bipolar
disorder, and 2878 controls. In CLOZUK, we obtained significant replication to
the PGC-associated allele for no fewer than 37 (47%) of the SNPs, including many
prior GWS major histocompatibility complex (MHC) SNPs as well as 3/6 non-MHC
SNPs for which we had data that were reported as GWS by the PGC. After combining
the new schizophrenia data with those of the PGC, variants at three loci
(ITIH3/4, CACNA1C and SDCCAG8) that had not previously been GWS in schizophrenia
attained that level of support. In bipolar disorder, we also obtained
significant evidence for association for 21% of the alleles that had been
associated with schizophrenia in the PGC. Our study independently confirms
association to three loci previously reported to be GWS in schizophrenia, and
identifies the first GWS evidence in schizophrenia for a further three loci.
Given the number of independent replications and the power of our sample, we
estimate 98% (confidence interval (CI) 78-100%) of the original set of 78 SNPs
represent true associations. We also provide strong evidence for overlap in
genetic risk between schizophrenia and bipolar disorder. Author information:
(1)Department of Neuroscience, Faculty of Medicine, Norwegian University of
Science and Technology (NTNU), Trondheim, Norway; Østmarka Psychiatric
Department, St. Olavs Hospital, Trondheim University Hospital, Trondheim,
Norway. Electronic address: [email protected].
(2)Department of Medical Genetics, Oslo University Hospital, Oslo, Norway; K. G.
Jebsen Centre for Psychosis Research, Institute of Clinical Medicine, University
of Oslo, Oslo, Norway.
(3)Department of Medical Genetics, Oslo University Hospital, Oslo, Norway; K. G.
Jebsen Centre for Psychosis Research, Division of Mental Health and Addiction,
Oslo University Hospital, Oslo, Norway.
(4)K. G. Jebsen Centre for Psychosis Research, Division of Mental Health and
Addiction, Oslo University Hospital, Oslo, Norway; Department of Psychiatric
Research, Diakonhjemmet Hospital, Oslo, Norway.
(5)Institute of Clinical Medicine, University of Oslo, Oslo, Norway; Department
of Neuropsychiatry and Psychosomatic Medicine, Division of Surgery and
Neuroscience, Oslo University Hospital, Oslo, Norway.
(6)K. G. Jebsen Centre for Psychosis Research, Institute of Clinical Medicine,
University of Oslo, Oslo, Norway; K. G. Jebsen Centre for Psychosis Research,
Division of Mental Health and Addiction, Oslo University Hospital, Oslo, Norway.
(7)Department of Neuroscience, Faculty of Medicine, Norwegian University of
Science and Technology (NTNU), Trondheim, Norway; Department of Research and
Development, Psychiatry, St. Olavs Hospital, Trondheim University Hospital,
Trondheim, Norway.
(8)Department of Neuroscience, Faculty of Medicine, Norwegian University of
Science and Technology (NTNU), Trondheim, Norway; Østmarka Psychiatric
Department, St. Olavs Hospital, Trondheim University Hospital, Trondheim,
Norway. |
Is APOBEC3B protein predominantly cytoplasmic or nuclear? | Contrary to other APOBEC family members, APOBEC3B was found to predominantly concentrate to the cell nucleus. | The most common transposable genetic element in humans, long interspersed
element 1 (L1), constitutes about 20% of the genome. The activity of L1 and
related transposons such as Alu elements causes disease and contributes to
speciation. Little is known about the cellular mechanisms that control their
spread. We show that expression of human APOBEC3B or APOBEC3F decreased the rate
of L1 retrotransposition by 5-10-fold. Expression of two related proteins,
APOBEC3D or APOBEC3G, had little effect. The mechanism of L1 inhibition did not
correlate with an obvious subcellular protein distribution as APOBEC3B appeared
predomitly nuclear and APOBEC3F was mostly cytosolic. Two lines of evidence
indicated that these APOBEC3 proteins use a deamination-independent mechanism to
inhibit L1. First, a catalytically inactive APOBEC3B mutant maintained L1
inhibition activity. Second, cDNA strand-specific C --> T hypermutations were
not detected among L1 elements that had replicated in the presence of APOBEC3B
or APOBEC3F. In addition, lower levels of retrotransposed L1 DNA accumulated in
the presence of APOBEC3B and APOBEC3F. Together, these data combined to suggest
a model in which APOBEC3B or APOBEC3F provide a preintegration barrier to L1
retrotransposition. A particularly high level of APOBEC3F protein in human
testes and an inverse correlation between L1 activity and APOBEC3 gene number
suggest the relevance of this mechanism to mammals. APOBEC3G limits the replication of human immunodeficiency virus type 1, other
retroviruses, and retrotransposons. It localizes predomitly to the cytoplasm
of cells, which is consistent with a model wherein cytosolic APOBEC3G packages
into assembling virions, where it exerts its antiviral effect by deaminating
viral cDNA cytosines during reverse transcription. To define the domains of
APOBEC3G that determine cytoplasmic localization, comparisons were made with
APOBEC3B, which is predomitly nuclear. APOBEC3G/APOBEC3B chimeric proteins
mapped a primary subcellular localization determit to a region within the
first 60 residues of each protein. A panel of 25 APOBEC3G mutants, each with a
residue replaced by the corresponding amino acid of APOBEC3B, revealed that
several positions within this region were particularly important, with Y19D
showing the largest effect. The mislocalization phenotype of these mutants was
only apparent in the context of the amino-terminal half of APOBEC3G and not the
full-length protein, suggesting the existence of an additional localization
determit. Indeed, a panel of five single amino acid substitutions within the
region from amino acids 113 to 128 had little effect by themselves but, in
combination with Y19D, two substitutions-F126S and W127A-caused full-length
APOBEC3G to redistribute throughout the cell. The critical
localization-determining residues were predicted to cluster on a common
solvent-exposed surface, suggesting a model in which these two regions of
APOBEC3G combine to mediate an intermolecular interaction that controls
subcellular localization. Human APOBEC3B (A3B) has been described as a potent inhibitor of retroviral
infection and retrotransposition. However, we found that the predomitly
nuclear A3B only weakly restricted infection by HIV-1, HIV-1Δvif, and human
T-cell leukemia virus type 1 (HTLV-1), while significantly inhibiting LINE-1
retrotransposition. The chimeric construct A3G/B, in which the first 60 amino
acids of A3B were replaced with those of A3G, restricted HIV-1, HIV-1Δvif, and
HTLV-1 infection, as well as LINE-1 retrotransposition. In contrast to the
exclusively cytoplasmic A3G, which is inactive against LINE-1
retrotransposition, the A3G/B protein, while localized mainly to the cytoplasm,
was also present in the nucleus. Further mutational analysis revealed that
residues 18, 19, 22, and 24 in A3B were the major determits for nuclear
versus cytoplasmic localization and antiretroviral activity. HIV-1Δvif packages
A3G, A3B, and A3G/B into particles with close-to-equal efficiencies. Mutation
E68Q or E255Q in the active centers of A3G/B resulted in loss of the inhibitory
activity against HIV-1Δvif, while not affecting activity against LINE-1
retrotransposition. The low inhibition of HIV-1Δvif by A3B correlated with a low
rate of G-to-A hypermutation. In contrast, viruses that had been exposed to
A3G/B showed a high number of G-to-A transitions. The mutation pattern was
similar to that previously reported for A3B, with a preference for the GA
context. In summary, these observations suggest that changing 4 residues in the
amino terminus of A3B not only retargets the protein from the nucleus to the
cytoplasm but also enhances its ability to restrict HIV while retaining
inhibition of retrotransposition. Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic
polypeptide-like) protein family catalyze DNA cytosine deamination and underpin
a variety of immune defenses. For instance, several family members, including
APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction
activities. However, unlike the other proteins, A3B is the only family member
with steady-state nuclear localization. Here, we show that A3B nuclear import is
an active process requiring at least one amino acid (Val54) within an N-terminal
motif analogous to the nuclear localization determit of the antibody gene
diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic
conservation with AID is further suggested by A3B's capacity to interact with
the same subset of importin proteins. Despite these mechanistic similarities,
enforced A3B expression cannot substitute for AID-dependent antibody gene
diversification by class switch recombination. Regulatory differences between
A3B and AID are also visible during cell cycle progression. Our studies suggest
that the present-day A3B enzyme retained the nuclear import mechanism of an
ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our
studies also highlight the likelihood that, after nuclear import, specialized
mechanisms exist to guide these enzymes to their respective physiological
substrates and prevent gratuitous chromosomal DNA damage. Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes
allows them to restrict a variety of retroviruses and retrotransposons, but may
also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3
proteins have different subcellular localizations: cell-wide, cytoplasmic or
nuclear. This implies that only a subset of APOBEC3s have contact with nuclear
DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic
proteins may enter what was formerly a privileged zone. To address the
hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the
localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A,
APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become
cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G
are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes
nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural
motifs as well as size may be factors in regulating chromatin exclusion.
Deaminase activity was not dependent on cell cycle phase. We also analyzed
APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity
to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle
profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that
several APOBEC3 family members have access to the nuclear compartment and can
impede the cell cycle, most likely through DNA deamination and the ensuing DNA
damage response. Such genomic damage may contribute to carcinogenesis, as
demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers. Several mutations are required for cancer development, and genome sequencing has
revealed that many cancers, including breast cancer, have somatic mutation
spectra dominated by C-to-T transitions. Most of these mutations occur at
hydrolytically disfavoured non-methylated cytosines throughout the genome, and
are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B
is a probable source of these mutations. APOBEC3B messenger RNA is upregulated
in most primary breast tumours and breast cancer cell lines. Tumours that
express high levels of APOBEC3B have twice as many mutations as those that
express low levels and are more likely to have mutations in TP53. Endogenous
APOBEC3B protein is predomitly nuclear and the only detectable source of DNA
C-to-U editing activity in breast cancer cell-line extracts. Knockdown
experiments show that endogenous APOBEC3B correlates with increased levels of
genomic uracil, increased mutation frequencies, and C-to-T transitions.
Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell
death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data
suggest a model in which APOBEC3B-catalysed deamination provides a chronic
source of DNA damage in breast cancers that could select TP53 inactivation and
explain how some tumours evolve rapidly and manifest heterogeneity. |
Is the regulation of Vsr endonuclease independent of the growth phase of bacteria? | The regulation of Vsr endonuclease levels is growth phase dependent. | Using rabbit polyclonal antibodies, we have shown that the Dcm cytosine
methylase of Escherichia coli is maintained at a constant level during cell
growth, while Vsr endonuclease levels are growth phase dependent. Decreased
production of Vsr relative to Dcm during the log phase may contribute
substantially to the mutability of 5-methylcytosine. In Escherichia coli and related enteric bacteria, repair of base-base mismatches
is performed by two overlapping biochemical processes, methyl-directed mismatch
repair (MMR) and very short-patch (VSP) repair. While MMR repairs replication
errors, VSP repair corrects to C*G mispairs created by 5-methylcytosine
deamination to T. The efficiency of the two pathways changes during the
bacterial life cycle; MMR is more efficient during exponential growth and VSP
repair is more efficient during the stationary phase. VSP repair and MMR share
two proteins, MutS and MutL, and although the two repair pathways are not
equally dependent on these proteins, their dual use creates a competition within
the cells between the repair processes. The structural and biochemical data on
the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays
a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic
studies of the two repair pathways have helped eliminate certain models for MMR
and put restrictions on models that can be developed regarding either repair
process. We review here recent information about the biochemistry of both repair
processes and describe the balancing act performed by cells to optimize the
competing processes during different phases of the bacterial life cycle. |
Are there interactomes available for POU5F1 and SOX2? | Yes. Long-range chromosomal interactions on putative enhancers of POU5F1 and SOX2 genes in human embryonic stem cells (hESCs) have been assayed using 4C-Seq technique. Their frequent interacting regions mainly overlap with early DNA replication domains. The interactomes are associated with active histone marks and enriched with 5-hydroxymethylcytosine sites. | The genes POU5F1 and SOX2 are critical for pluripotency and reprogramming, yet
the chromosomal organization around these genes remains poorly understood. We
assayed long-range chromosomal interactions on putative enhancers of POU5F1 and
SOX2 genes in human embryonic stem cells (hESCs) using 4C-Seq technique. We
discovered that their frequent interacting regions mainly overlap with early DNA
replication domains. The interactomes are associated with active histone marks
and enriched with 5-hydroxymethylcytosine sites. In hESCs, genes within the
interactomes have elevated expression. Additionally, some genes associated with
the POU5F1 enhancer contribute to pluripotency. Binding sites for multiple DNA
binding proteins, including ATF3, CTCF, GABPA, JUND, NANOG, RAD21 and YY1, are
enriched in both interactomes. The RARG locus, frequently interacting with the
POU5F1 locus, has abundant RAD21 binding sites co-localized with other protein
binding sites. Thus the interactomes of these two pluripotency genes could be an
important part of the regulatory network in hESCs. |
Which tool is used for promoterome mining using CAGE data? | Despite their high resolution and functional significance, published CAGE data are still underused in promoter analysis due to the absence of tools that enable its efficient manipulation and integration with other genome data types. CAGEr is an R implementation of novel methods for the analysis of differential TSS usage and promoter dynamics, integrated with CAGE data processing and promoterome mining into a first comprehensive CAGE toolbox on a common analysis platform. | |
Is Rheumatoid Arthritis related to myopathy? | Vacuolar myopathy and statin-induced myopathy have been reported in rheumatoid arthritis patients, but this association may be due to the anti-malarial treatment received. An increased prevalence of neurogenic but not myogenic changes in patients with RA compared with controls has been reported. | OBJECTIVE: To test the hypothesis that patients with rheumatoid arthritis (RA)
have clinical or subclinical evidence of peripheral neuropathy or myopathy.
METHODS: We studied 40 seropositive women with RA, mean age 46.6 years (SD 6.4),
and 56 healthy controls, mean age 43.0 years (SD 9.1). Patients had a mean
disease duration of 13.0 years (SD 7.8). We performed electromyographic
examination of 4 muscles [extensor digitorum communis (EDC), biceps brachii
(BB), vastus lateralis (VL), and tibialis anterior (TA)] on the right side in
both groups. Quantitative data included percentage of polyphasic potentials,
motor unit potential amplitude, area, duration, turns, and number of polyphasic
potentials.
RESULTS: There were statistically significantly higher proportions of polyphasic
potentials in 3 muscles in patients compared with controls. Mean number of
phases in EDC was 4.6 (SD 0.4) in the patients and 4.1 (0.5) in controls (p =
0.0001). The values for the VL were 4.1 (SD 0.4) in patients compared with 3.6
(0.4) in controls (p = 0.0001), and in the TA 4.5 (SD 0.5) versus 4.0 (0.4) (p =
0.0001). We also found significantly increased duration of motor unit potentials
in the VL and TA of patients. The amplitudes of motor unit action potentials
were not significantly different in the 2 groups.
CONCLUSION: The study reveals an increased prevalence of neurogenic but not
myogenic changes in patients with RA compared with controls. INTRODUCTION: Chloroquine is a drug that is widely used in rheumatology and
occasionally prescribed in dermatology. From a neurotoxicological point of view,
chloroquine can have effects on the peripheral nerves, muscles, neuromuscular
junctions and the central nervous system. In this study we analyse the clinical,
neurophysiological and anatomopathological findings in two patients with
chloroquine induced neuromyopathy, which took the form of a
polyradiculoneuropathy.
CASE REPORTS: Case 1: a 75 year old female with rheumatoid arthritis treated
with daily doses of 250 mg of chloroquine for four years. The patient visited
because of several months history of predomitly proximal progressive
tetraparesis with areflexia. Analytical tests and lumbar puncture were normal.
Electromyogram (EMG): proximal myopathic and distal neuropathic patterns.
Muscular biopsy: vacuolar myopathy with accumulations of phagolysosomes, lipids,
lipofuscin, myelinic curvilinear bodies. Case 2: a 74 year old female with
arthropathy treated with daily doses of 250 mg of chloroquine for nine months.
The patient presented a progressive proximal paraparesis with generalised
areflexia. Analytical tests and lumbar puncture were normal. EMG: mixed sensory
motor polyneuropathy, myogenic pattern with high frequency discharges in the
iliac psoas and a neurogenic pattern in the distal muscles. Muscular biopsy:
vacuolar myopathy suggesting a myopathy due to chloroquine. After stopping
treatment with this drug the patients progressed favourably.
CONCLUSION: Chloroquine can induce a clinical pattern that suggests a
polyradiculoneuropathy. It is important to establish a history of having taken
this drug. If this is indeed the case, then an electromyographic study of the
most proximal muscles should be performed in order to detect a myogenic pattern
and the same exploration should be applied to the distal muscles to reveal a
neurogenic pattern. The final diagnosis will be established by muscular biopsy. The myotoxicity of chloroquine and hydroxychloroquine has been known for
decades. Limb-girdle weakness due to a vacuolar myopathy may occur occasionally
in a dose-dependent manner during the first 24 months on chloroquine. However,
we report on a case in which muscular weakness developed after a daily intake of
250 chloroquine phosphate (= 155 mg chloroquine base) for a period of 7 years.
Even after long-term and apparently well-tolerated chloroquine treatment, the
occurrence of severe side-effects is possible. |
what is the role of GATA-4 in regeneration of the heart after myocardial infarction? | GATA-4 is implicated in cardiogenic differentiation of cardiac c-kit+AT2+ cells that represent approximately 0.19% of total cardiac cells in infarcted heart. GATA-4 overexpression in mesenchymal stem cells increases both survival and angiogenic potential in ischemic myocardium and may therefore represent a novel and efficient therapeutic approach for postinfarct regenaration. In addition, interventions, such as hypergravity and 5-Aza treatment, induce expression of early muscle and cardiac markers like GATA-4 in BMSCs. A subpopulation of primitive cells from rat heart, expressing c-kit and myogenic transcriptional factors, GATA-4 and MEF 2C, exhibit a high in vitro proliferative potential. Progeny of these implanted cells have been shown to migrate along the infarcted scar, reconstitute regenerated cardiomyocytes with incorporation into host myocardium, and inhibit cardiac remodeling with decreased scar formation. In another study, TGF-beta has been shown to conduct the myogenic differentiation of stem cells by upregulating GATA-4 and NKx-2.5 expression and the intramyocardial implantation of TGF-beta-preprogrammed stem cells effectively assisted the myocardial regeneration. Furthermore, G-CSF treatment in postinfarcted murine hearts appears to be an effective approach for treating heart failure and also leads to induction of GATA-4 resulting in expression of various sarcomeric proteins. | BACKGROUND: Recent studies have shown that bone marrow-derived stem cells
differentiate into the phenotype of cardiomyocytes in vivo and in vitro. We
tried to regenerate infarcted myocardium by implanting ex vivo transforming
growth factor (TGF)-beta-preprogrammed CD117 (c-kit)-positive (CD117+) stem
cells intramyocardially.
METHODS AND RESULTS: CD117+ cells were isolated from the bone marrow mononuclear
cells of GFP-transgenic or normal C57/BL6 mice. The myogenic differentiation of
CD117+ cells was achieved by cultivation with TGF-beta. Using an acute
myocardial infarction model, we also tried to regenerate infarcted myocardium by
implanting untreated (newly isolated) or preprogrammed (24 hours of cultivation
with 5 ng/mL TGF-beta1) CD117+ cells intramyocardially. TGF-beta increased the
cellular expression of myosin, troponins, connexin-43, GATA-4, and NKx-2.5,
which suggested that it induced the myogenic differentiation of CD117+ cells.
Compared with the effects of PBS injection only, the microvessel density in the
infarcted myocardium was increased significantly 3 months after the implantation
of either TGF-beta-preprogrammed or untreated CD117+ cells. Moreover, many of
the TGF-beta-preprogrammed CD117+ cells were stained positively for myosin,
whereas few of the untreated CD117+ cells were. Histological analysis revealed
newly regenerated myocardium in the left ventricular anterior wall after the
implantation of TGF-beta-preprogrammed cells but not untreated cells.
Furthermore, the left ventricular percent fraction shortening was significantly
higher after the implantation of TGF-beta-preprogrammed cells than after the
implantation of untreated CD117+ cells.
CONCLUSIONS: TGF-beta conducted the myogenic differentiation of CD117+ stem
cells by upregulating GATA-4 and NKx-2.5 expression. Therefore, the
intramyocardial implantation of TGF-beta-preprogrammed CD117+ cells effectively
assisted the myocardial regeneration and induced therapeutic angiogenesis,
contributing to functional cardiac regeneration. Chronic heart failure remains a leading cause of mortality. Although granulocyte
colony-stimulating factor (G-CSF) is reported to have a beneficial affect on
postinfarction cardiac remodeling and dysfunction when administered before the
onset of or at the acute stage of myocardial infarction (MI), its effect on
established heart failure is unknown. We show here that subcutaneous
administration of G-CSF greatly improves the function of murine hearts failing
due to a large, healed MI. G-CSF changed the geometry of the infarct scar from
elongated and thin to short and thick, induced hypertrophy among surviving
cardiomyocytes, and reduced myocardial fibrosis. Expression of G-CSF receptor
was confirmed in failing hearts and was upregulated by G-CSF treatment. G-CSF
treatment also led to activation of signal transducer and activator of
transcription-3 and induction of GATA-4 and various sarcomeric proteins such as
myosin heavy chain, troponin I and desmin. Expression of metalloproteinase-2 and
-9 was also increased in G-CSF-treated hearts, while that of tumor necrosis
factor-alpha, angiotensin II type 1 receptor (AT1) and transforming growth
factor-beta1 was reduced. Although activation of Akt was noted in G-CSF-treated
hearts, vessel density was unchanged, and apoptosis was too rare to exert a
meaningful effect. No bone marrow-derived cardiomyocytes or vascular cells were
detected in the failing hearts of green fluorescent protein chimeric mice.
Finally, beneficial effects of G-CSF on cardiac function were found persisting
long after discontinuing the treatment (2 weeks). Collectively, these findings
suggest G-CSF administration could be an effective approach to treating chronic
heart failure following a large MI. BACKGROUND: The heart is a highly vascular organ and prolonged interruption of
myocardial blood flow initiates events that culminate in cardiac myocyte death.
Proposed experimental reparative strategies include harvesting potent cells
followed by direct injection into ischemic myocardium to achieve myogenesis and
angiogenesis.
METHODS: Accordingly, we set out to isolate and expand a purified population of
adult rat putative cardiomyocyte precursors, and to identify their
characteristics in vitro. By using an acute myocardial infarction model and
direct cell implantation, we further tested the hypothesis that these cells are
an ideal cell source for myocardial regeneration and can enhance cardiac repair
after implantation into the ischemic rat heart.
RESULTS: We describe here the identification of a subpopulation of primitive
cells from rat heart, processing stem cell marker, c-kit and myogenic
transcriptional factors, GATA-4 and MEF 2C, and cardiac specific proteins,
troponin-I, alpha-sarcomeric actinin and connexin-43. They exhibited a high in
vitro proliferative potential. These findings strongly suggest that these cells
are putative cardiomyocyte precursors. After transplantation, they were able to
be retained and proliferate (13.63 +/- 5.97% after 2 weeks) within the ischemic
heart. Progeny of implanted cells migrated along the infarcted scar,
reconstituted regenerated cardiomyocytes with incorporation into host
myocardium, and inhibited cardiac remodeling with decreased scar formation.
CONCLUSION: Our findings suggest that putative cardiomyocyte precursors isolated
from adult heart could potentially be an autologous cell source for myocardial
regeneration cell therapy. The expression pattern of angiotensin AT2 receptors with predomice during
fetal life and upregulation under pathological conditions during tissue
injury/repair process suggests that AT2 receptors may exert an important action
in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute
ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2
receptors after acute myocardial infarction. Double immunofluorescence staining
showed that cardiac AT2 receptors were mainly detected in clusters of small
c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in
response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell
population by modified magnetic activated cell sorting and fluorescence
activated cell sorting. These cardiac c-kit+AT2+ cells, represented
approximately 0.19% of total cardiac cells in infarcted heart, were
characterized by upregulated transcription factors implicated in cardiogenic
differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal
(Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells
isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in
vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo
AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the
infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute
myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell
population exists and increases after acute ischemic injury. AT2 receptor
activation supports performance of cardiomyocytes, thus contributing to
cardioprotection via cardiac c-kit+AT2+ cell population. BACKGROUND: Recent data suggest that GATA-4 is an antiapoptotic factor required
for adaptive responses and a key regulator of hypertrophy and
hypertrophy-associated genes in the heart. As a leading cause of chronic heart
failure, reversal of postinfarction left ventricular remodeling represents an
important target for therapeutic interventions. Here, we studied the role of
GATA-4 as a mediator of postinfarction remodeling in rats.
METHODS AND RESULTS: Myocardial infarction, caused by ligating the left anterior
descending coronary artery, significantly decreased the DNA binding activity of
GATA-4 at day 1, whereas at 2 weeks the GATA-4 DNA binding was significantly
upregulated. To determine the functional role of GATA-4, peri-infarct
intramyocardial delivery of adenoviral vector expressing GATA-4 was done before
left anterior descending coronary artery ligation. Hearts treated with GATA-4
gene transfer exhibited significantly increased ejection fraction and fractional
shortening. Accordingly, infarct size was significantly reduced. To determine
the cardioprotective mechanisms of GATA-4, myocardial angiogenesis, rate of
apoptosis, c-kit+ cardiac stemlike cells, and genes regulated by GATA-4 were
studied. The number of capillaries and stemlike cells was significantly
increased, and decreased apoptosis was observed.
CONCLUSION: These results indicate that the reversal of reduced GATA-4 activity
prevents adverse postinfarction remodeling through myocardial angiogenesis,
antiapoptosis, and stem cell recruitment. GATA-4-based gene transfer may
represent a novel, efficient therapeutic approach for heart failure. Transplanted mesenchymal stem cells (MSC) release soluble factors that
contribute to cardiac repair and vascular regeneration. We hypothesized that
overexpression of GATA-4 enhances the MSC secretome, thereby increasing cell
survival and promoting postinfarction cardiac angiogenesis. MSCs harvested from
male rat bone marrow were transduced with GATA-4 (MSC(GATA-4)) using the murine
stem cell virus retroviral expression system; control cells were either
nontransduced (MSC(bas)) or transduced with empty vector (MSC(Null)). Compared
with these control cells, MSC(GATA-4) were shown by immunofluorescence,
real-time PCR, and Western blotting to have higher expression of GATA-4. An
increased expression of angiogenic factors in MSC(GATA-4) and higher MSC
resistance against hypoxia were observed. Human umbilical vein endothelial cells
(HUVEC) treated with MSC(GATA-4) conditioned medium exhibited increased
formation of capillary-like structures and promoted migration, compared with
HUVECs treated with MSC(Null) conditioned medium. MSC(GATA-4) were injected into
the peri-infarct region in an acute myocardial infarction model in
Sprague-Dawley rats developed by ligation of the left anterior descending
coronary artery. Survival of MSC(GATA-4), determined by Sry expression, was
increased at 4 days postengraftment. MSC(GATA-4)-treated animals showed
significantly improved cardiac function as assessed by echocardiography.
Furthermore, fluorescent microsphere and histological studies revealed increased
blood flow and blood vessel density and reduced infarction size in
MSC(GATA-4)-treated animals. We conclude that GATA-4 overexpression in MSCs
increased both MSC survival and angiogenic potential in ischemic myocardium and
may therefore represent a novel and efficient therapeutic approach for
postinfarct remodeling. BACKGROUND: Placement of an elastic biodegradable patch onto a subacute
myocardial infarct (MI) provides temporary elastic support that may act to
effectively alter adverse left ventricular (LV) remodeling processes.
METHODS: Two weeks after permanent left coronary ligation in Lewis rats, the
infarcted anterior wall was covered with polyester urethane urea (MI + PEUU; n =
15) or expanded polytetrafluoroethylene (MI + ePTFE; n = 15) patches, or had no
implantation (MI + sham; n = 12). Eight weeks after surgery, cardiac function
and histology were assessed.
RESULTS: The ventricular wall in the MI + ePTFE and MI + sham groups was
composed of fibrous tissue, whereas PEUU implantation induced α-smooth muscle
actin-positive muscle bundles coexpressing sarcomeric α-actinin and
cardiac-specific troponin-T. This pattern of colocalization was also found in
developing embryonic myocardium. Cardiac transcription factors Nkx-2.5 and
GATA-4 were strongly expressed in the muscle bundles. In the MI + sham group,
end-diastolic LV cavity area (EDA) increased and the percentage of fractional
area change (%FAC) decreased. For ePTFE patched animals, both EDA and %FAC
decreased. In contrast, with MI + PEUU patching, %FAC increased and EDA was
maintained. With dobutamine-stress echocardiography, MI + PEUU patched LVs
possessed contractile reserve significantly larger than the MI + sham group.
CONCLUSIONS: MI + PEUU patch implantation onto subacute infarcted myocardium
induced muscle cellularization with characteristics of early developmental
cardiomyocytes as well as providing a functional reserve. Transplantation of human cardiomyoblast-like cells (hCLCs) from human adipose
tissue-derived multi-lineage progenitor cells improved left ventricular function
and survival of rats with myocardial infarction. Here we examined the effect of
intracoronary artery transplantation of human CLCs in a swine model of chronic
heart failure. Twenty-four pigs underwent balloon-occlusion of the first
diagonal branch followed by reperfusion, with a second balloon-occlusion of the
left ascending coronary artery 1 week later followed by reperfusion. Four weeks
after the second occlusion/reperfusion, 17 of the 18 surviving animals with
severe chronic MI (ejection fraction <35% by echocardiography) were
immunosuppressed then randomly assigned to receive either intracoronary artery
transplantation of hCLCs hADMPCs or placebo lactic Ringer's solution with
heparin. Intracoronary artery transplantation was followed by the distribution
of DiI-stained hCLCs into the scarred myocardial milieu. Echocardiography at
post-transplant days 4 and 8 weeks showed rescue and maintece of cardiac
function in the hCLCs transplanted group, but not in the control animals,
indicating myocardial functional recovery by hCLCs intracoronary
transplantation. At 8 week post-transplantation, 7 of 8 hCLCs transplanted
animals were still alive compared with only 1 of the 5 control (p=0.0147).
Histological studies at week 12 post-transplantation demonstrated engraftment of
the pre DiI-stained hCLCs into the scarred myocardium and their expression of
human specific alpha-cardiac actin. Human alpha cardiac actin-positive cells
also expressed cardiac nuclear factors; nkx2.5 and GATA-4. Our results suggest
that intracoronary artery transplantation of hCLCs is a potentially effective
therapeutic strategy for future cardiac tissue regeneration. |
What is the life expectancy of professional athletes in respect to the general population? | Elite endurance (aerobic) athletes and mixed-sports (aerobic and anaerobic) athletes show higher longevity than the general population, but results about power (anaerobic) athletes are inconsistent. | Nine hundred eighty-three top Italian track and field athletes (700 males and
283 females) were examined for survival, mortality and causes of death for an
average follow-up period of 18.6 years starting from their last year of
competition as members of the national team. Overall mortality rates were
compared to the rates expected on the basis of the life tables for Italian
people of the same age, sex and time period. Thirty-four deaths were observed
among males (vs 46.6 expected) with a O/E ratio of 0.73, while 3 deaths were
observed among women (vs 6.2 expected) with a O/E ratio of 0.48. Neither of
these differences was significant, but the O/E ratio for the group as a whole
was quite significant (p = 0.0296). Some of the athletes demonstrated
behavioural characteristics developed during their active careers that might
have contributed to their low mortality rate. Recently, Sarna et al. (Med. Sci. Sports Exerc. 25:237-244, 1993) reported
increased mean life expectancy in former world class athletes. Because lifestyle
is associated with longevity, we have examined whether health habits of former
Finnish male athletes (N = 1274; present mean age: 57.5, range: 36-94 yr)
differed from those of noncompetitive referents (N = 788; mean age: 55.7, range:
39-87 yr). The athletes had represented Finland in international competitions in
endurance (N = 177), power (N = 454), or other ("mixed") events (N = 643) from
1920-1965. Data on physical characteristics, sociodemographic factors, and
health habits were obtained from questionnaires. All dependent variables in an
analysis of covariance and in a logistic regression analysis were adjusted for
age and occupation. Both leisure aerobic and work activity of all athlete groups
was higher (P < 0.01) than that of referents. Compared with the referents, both
power and "mixed" athletes were more prone to eat fruits and vegetables and to
avoid vitamin supplements, but less prone to use butter and high-fat milk, and
to smoke (odds ratios different from 1.0, P < 0.05). Also endurance athletes
smoked less and drank less alcohol than the referents (P < 0.05). Higher leisure
aerobic activity and less frequent smoking after athletic years might explain
higher life expectancy of Finnish athletes. Reliable data are scanty on the incidence of chronic diseases and life
expectancy (LE) of highly trained athletes. We therefore studied Finnish male
world class athletes to estimate the LE of athletes. Finnish team members in the
Olympic games, World or European championships or intercountry competitions
during 1920-1965 in track and field athletics, cross-country skiing, soccer, ice
hockey, basketball, boxing, wrestling, weight lifting, and shooting were
included (N = 2613 men). The reference cohort, 1712 men, was selected from the
Finnish Defence Forces conscription register matched on age and area of
residence. All referents were classified completely healthy at the time of
induction to military service. The stratified Kaplan-Meier product limit method
and the Cox proportional hazards model were used to estimate the life
expectancies and the mortality odds ratios (OR) and their confidence limits. The
mean LE adjusted for occupational group, marital status, and the age at entry to
the cohort (and its 95% confidence limits) was in endurance sports (long
distance running and cross-country skiing) 75.6 (73.6, 77.5) yr; in team games
(soccer, ice hockey, basketball, as well as jumpers and short-distance runners
from track and field (73.9 (72.7, 75.1) yr; in power sports (boxing, wrestling,
weight lifting, and throwers from field athletics) 71.5 (70.4, 72.2) yr; and in
the reference group 69.9 (69.0, 70.9) yr. The increased mean life expectancies
were mainly explained by decreased cardiovascular mortality (endurance sports
mortality odds ratio OR = 0.49 (95% CL 0.26, 0.93), team sports OR = 0.61 (0.41,
0.92) compared with referents). For maximum life span no differences between the
groups were observed.(ABSTRACT TRUNCATED AT 250 WORDS) Physical activity is an important aspect of health behavior and life-style, when
considering the possibilities to prevent premature deaths and sustain functional
capacity. We studied former Finnish male athletes and controls to investigate
the effects of long-lasting participation in vigorous sports on health, and the
main findings are reviewed here. The athletes represented Finland between the
years 1920-1965 at least once in international competitions. The following
sports were selected: track and field athletics, cross-country skiing, soccer,
ice hockey, basketball, boxing, wrestling, weight lifting, and shooting. The
full name, place and date of birth were traced for 2613 (97.7%) men. The
referent subjects (N = 1712) were selected among those Finnish men who, at the
age of 20, were classified completely healthy at the medical examination for
induction into military service. In most analyses we grouped the sports
according to the type of training needed to achieve maximal results, i.e.,
principally aerobic training, principally anaerobic training or mixed. In 1985,
a questionnaire on physical activity, health and health habits was mailed to
surviving former athletes and referents (N = 2851, 65.9% of the original
cohort). Follow-up for morbidity and mortality was based on national medical
registries. We found that former aerobic sports athletes (endurance and mixed
sports) in particular have high total and active life expectancy and low risk
for ischemic heart disease and diabetes in later years. On the other hand, they
have slightly higher risk for lower-limb osteoarthritis. Overall, the benefits
of physically active life-style on health were clearly higher than the adverse
effects. The purpose of this study was to determine whether major league baseball players
live lontger than the general public. Ages of death of major league baseball
players who debuted between 1900 and 1950 were obtained, and differences between
ages of death and age-adjusted life expectancies were determined by analysis of
variance and t-tests, taking into account player position. Correlational
analysis also was conducted to determine if career length affected longevity.
Baseball players lived an average of four years longer than age-matched controls
from the general public. Career length did not affect longevity among players.
We concluded that professional athletes, as represented by major league baseball
players, have increased life expectancies. This increase cannot be explained by
increased fitness associated with working as a professionral athlete, but is
likely the result of an initial selection process for becoming a professional
athlete. We investigated the "healthy athlete effect" (HAE) in American professional
football. Ages of death of professional football players who debuted prior to
1940 were obtained and differences between those ages and age-adjusted life
expectancies were examined to determine if longevity increased with career
length, controlling for birth year, body mass index (BMI), position, and type.
Professional football players (N=1,512) lived an average of 6.1 (+/- 11.9 SD)
years longer than age-matched controls from the general public. Career length
significantly and incrementally increased longevity of players from an average
of 5.5 years for players playing one season to 6.7 years for players playing 4
or more seasons. Both birth year and BMI had a statistically significant
association with longevity, but position type did not. These data provide strong
support for the healthy worker effect (HWE) in professional football. Moderate exercise and intense physical training are associated with increased
life expectancy (LE). Boxing is characterized by intentional and repetitive head
blows, sometimes causing brain injury, possibly reducing LE. We examined a
sample of male athletes born between 1860 and 1930 selected from the
international "hall of fame" inductees in baseball (n = 154), ice hockey (n =
130), tennis (n = 83), football (n = 81), boxing (n = 81), track and field (n =
59), basketball (n = 58), swimming (n = 37) and wrestling (n = 32). In boxing,
we analyzed the number of disputed bouts/rounds and career records. Sports were
also analyzed according to physiological demand and occurrence and kind of
contact (intentional, unintentional). The Kaplan-Meier product limit method was
used to compare survival curves (significance: p <or= 0.05). Median LE of the
samples was 76.0 yrs and no differences were observed in different sports,
although it was lower in boxers (73.0 yrs) and higher in tennis players (79.0
yrs). Sports of different physiological demand were similar in respect to LE. No
differences in LE were found related to occurrence and kind of impact. Similar
LE was found in boxers of different weight or career records. In conclusion,
this study indicates that LE in top-level athletes is unaffected by the type of
discipline, and not related to physiological demand and intentional contact. The authors used duration analysis to examine the longevity of Major League
Baseball players. Using data on players who were born between 1945 and 1964, the
authors found that the hazard rate of death for players who only attended high
school was almost 2.0 times higher than those players who attended a 4-year
university, evidence that the educaton [sic]-health link applies to professional
athletes. Another important determit of longevity was race. In addition, a
player's body mass index was positively associated with a higher hazard of
death. Compared with the general population, the death rate of baseball players
was lower--the observed number of deaths in the dataset was only 31% of the
expected number. Findings in this article are likely attributable to education
being correlated with other variables that affect longevity, most likely
intelligence and time preference. Although a regular moderate physical activity is commonly accepted as an
important heath promoting factor, the role of professional sport training is
still under debate. The presented mortality analysis, concerning the period of
1946-2000, was performed for three groups of people completely healthy in youth,
but exhibiting different physical activity level in those days: all Polish
athletes who participated in the Olympic Games (1689 males, 424 females), drama
actors/actresses (2406/1938), and monks/ nuns (796/323). In all studied cohorts
mortality was lower than that of gender adjusted, Polish urban population. The
SMR values equaled 0.50 for male athletes, 0.78 for actors, 0.64 for monks, and
respectively 0.73, 0.84, and 0.77 for corresponding female cohorts. Moreover,
the direct comparison indicated that the mortality of male athletes was
significantly lower than that of actors and monks: the age-standardized rate
ratio of death were equal: 0.64 and 0.81 respectively. The sportsmen also
exhibited the longest mean survival. The similar, but not statistically
significant trends were observed for female cohorts. All the obtained results
proved that high level physical activity in youth positively influenced men's
mortality, what was observed for the former athletes even after 40 years since
the end of their professional carrier. Sudden cardiac death in athletes is an uncommon but extremely visible event
because of the high profile of amateur and professional athletes and the
expected excellent health of these athletes. However, paradoxically, athletic
performance may immediately increase the risk of ventricular arrhythmias and
sudden cardiac death while run reducing atherosclerosis, which thus improves
cardiovascular health and longevity. In athletes younger than 30 years, the most
common underlying causes are due to inherited heart disease. In the older
athletes, sudden death is generally due to arrhythmias in the context of
coronary artery disease. Many athletes with aborted sudden death,
arrhythmia-related syncope, or high-risk genetic disorders benefit from therapy
with implanted cardioverter/defibrillators (ICDs) . Although ICD therapy can
effectively abort sudden death, implantation of an ICD generally prohibits an
individual from all competitive athletics except low-intensity sports. The
screening of athletes has been notoriously inadequate; however, the optimal
screening strategies have yet to be determined. Recommendations for
participation in competitive athletics generally follow the recently published
36th Bethesda Conference Eligibility Recommendations for Competitive Athletes
with Cardiovascular Abnormalities. The health benefits of leisure-time physical activity are well known, however
the effects of engaging in competitive sports on health are uncertain. This
literature review examines mortality and longevity of elite athletes and
attempts to understand the association between long-term vigorous exercise
training and survival rates. Fourteen articles of epidemiological studies were
identified and classified by type of sport. Life expectancy, standardised
mortality ratio, standardised proportionate mortality ratio, mortality rate, and
mortality odds ratio for all causes of death were used to analyse mortality and
longevity of elite athletes. It appears that elite endurance (aerobic) athletes
and mixed-sports (aerobic and anaerobic) athletes survive longer than the
general population, as indicated by lower mortality and higher longevity. Lower
cardiovascular disease mortality is likely the primary reason for their better
survival rates. On the other hand, there are inconsistent results among studies
of power (anaerobic) athletes. When elite athletes engaging in various sports
are analysed together, their mortality is lower than that of the general
population. In conclusion, long-term vigorous exercise training is associated
with increased survival rates of specific groups of athletes. People with higher fitness and higher aerobic capacity have longer life
expectancies compared to inactive people. Longer and better life can result from
some genetic factors, as well as from higher physical capacities acquired during
younger years. However, there are several studies proving the definitive role of
life-long physical activity, too, which can be engaged in at any age. Even at
very old ages, regular physical activity results in a lot of benefits, in high
quality of life, in independence and longevity. Leisure-time physical activity is associated with better health and a reduced
risk of all-cause mortality. It is unclear if this association is also present
with a high level of physical activity as it is found in professional athletes.
In a population-based retrospective cohort study, we compared the survival
experience of all soccer players participating for Germany in international
matches between 1908 and 2006 to that of the general population. To summarize
survival experience, we calculated cumulative relative survival ratios (RSRs)
from a life table. We included data of 812 international players, of which 428
(=52.7%) died during follow-up. In all 13 intervals, cumulative observed
survival was smaller than cumulative expected survival, resulting in cumulative
RSRs being <1. The cumulative RSRs are statistically significantly different
from 1 in all but the last interval. This impaired survival experience of the
internationals translates into a loss of median residual lifetime of 1.9 years
[95% confidence interval: 0.6, 3.2] years at the entry time into the cohort.
This loss is mainly driven by the mortality of internationals from the earlier
half of the observation period. Reasons for this might be poorer medical care in
former times, internationals being killed in action during World War II, and a
changing distribution of causes of death during the 20th century. PURPOSE: The African-American-white mortality gap for males in the United States
is 6 years in favor of whites. Participation in professional sport may moderate
this ethnic disparity. The historical cohort of professional basketball players,
with nearly equal numbers of African-American and white players, can provide a
natural experiment that may control for the classic confounders of income,
education, socioeconomic status (SES), and physical factors related to
mortality. The objectives of this study are to assess mortality and calculate
survival for the overall study population and within ethnicity.
METHODS: Data were combined from several publicly available sources. The cohort
was analyzed to compare longevity among all players, and for players stratified
by ethnicity, with the general U.S. population.
RESULTS: The final dataset included 3366 individuals, of whom 56.0% were African
American. Results suggest white players live 18 months longer than their
African-American colleagues. African-American players gained 9 years on their
respective referent and live longer than white men in the general public. After
controlling for covariates, we found that African-American players have a 75%
increased risk of death compared with white players, a statistically significant
gap (p < .0001, 95% confidence interval 1.41-2.44).
CONCLUSIONS: The African-American-white mortality gap for males is largely
ameliorated (1.5 years vs. 6.1 years) in professional basketball but still
persists. Physical activity reduces many major mortality risk factors including arterial
hypertension, diabetes mellitus type 2, dyslipidemia, coronary heart disease,
stroke, and cancer. All-cause mortality is decreased by about 30% to 35% in
physically active as compared to inactive subjects. The purpose of this paper
was to synthesize the literature on life expectancy in relation to physical
activity. A systematic PubMed search on life expectancy in physically active and
inactive individuals was performed. In addition, articles comparing life
expectancy of athletes compared to that of nonathletes were reviewed. Results of
13 studies describing eight different cohorts suggest that regular physical
activity is associated with an increase of life expectancy by 0.4 to 6.9 years.
Eleven studies included confounding risk factors for mortality and revealed an
increase in life expectancy by 0.4 to 4.2 years with regular physical activity.
Eleven case control studies on life expectancy in former athletes revealed
consistently greater life expectancy in aerobic endurance athletes but
inconsistent results for other athletes. None of these studies considered
confounding risk factors for mortality. In conclusion, while regular physical
activity increases life expectancy, it remains unclear if high-intensity sports
activities further increase life expectancy. OBJECTIVE: To assess the mortality risk in subsequent years (adjusted for year
of birth, nationality, and sex) of former Olympic athletes from disciplines with
different levels of exercise intensity.
DESIGN: Retrospective cohort study.
SETTING: Former Olympic athletes.
PARTICIPANTS: 9889 athletes (with a known age at death) who participated in the
Olympic Games between 1896 and 1936, representing 43 types of disciplines with
different levels of cardiovascular, static, and dynamic intensity exercise; high
or low risk of bodily collision; and different levels of physical contact.
MAIN OUTCOME MEASURE: All cause mortality.
RESULTS: Hazard ratios for mortality among athletes from disciplines with
moderate cardiovascular intensity (1.01, 95% confidence interval 0.96 to 1.07)
or high cardiovascular intensity (0.98, 0.92 to 1.04) were similar to those in
athletes from disciplines with low cardiovascular intensity. The underlying
static and dynamic components in exercise intensity showed similar
non-significant results. Increased mortality was seen among athletes from
disciplines with a high risk of bodily collision (hazard ratio 1.11, 1.06 to
1.15) and with high levels of physical contact (1.16, 1.11 to 1.22). In a
multivariate analysis, the effect of high cardiovascular intensity remained
similar (hazard ratio 1.05, 0.89 to 1.25); the increased mortality associated
with high physical contact persisted (hazard ratio 1.13, 1.06 to 1.21), but that
for bodily collision became non-significant (1.03, 0.98 to 1.09) as a
consequence of its close relation with physical contact.
CONCLUSIONS: Among former Olympic athletes, engagement in disciplines with high
intensity exercise did not bring a survival benefit compared with disciplines
with low intensity exercise. Those who engaged in disciplines with high levels
of physical contact had higher mortality than other Olympians later in life. |
Is EZH2 associated with prostate cancer? | EZH2 is an epigenetic driver of prostate cancer. EZH2 dependent H3K27me3 is involved in epigenetic silencing of ID4 in prostate cancer. EZH2 plays an active role in this process by repressing the expression of TIMP2 and TIMP3 in prostate cancer cells. EZH2 knockdown markedly reduces the proteolytic activity of MMP-9, thereby decreasing the invasive activity of prostate cancer cells. Composite index of c-Myc, EZH2, and p27 can be valued as powerful prognosis parameter for intermediate-risk prostate cancer patients after the surgery, and postoperative adjuvant therapy can be adopted accordingly. | Prostate cancer is a leading cause of cancer-related death in males and is
second only to lung cancer. Although effective surgical and radiation treatments
exist for clinically localized prostate cancer, metastatic prostate cancer
remains essentially incurable. Here we show, through gene expression profiling,
that the polycomb group protein enhancer of zeste homolog 2 (EZH2) is
overexpressed in hormone-refractory, metastatic prostate cancer. Small
interfering RNA (siRNA) duplexes targeted against EZH2 reduce the amounts of
EZH2 protein present in prostate cells and also inhibit cell proliferation in
vitro. Ectopic expression of EZH2 in prostate cells induces transcriptional
repression of a specific cohort of genes. Gene silencing mediated by EZH2
requires the SET domain and is attenuated by inhibiting histone deacetylase
activity. Amounts of both EZH2 messenger RNA and EZH2 protein are increased in
metastatic prostate cancer; in addition, clinically localized prostate cancers
that express higher concentrations of EZH2 show a poorer prognosis. Thus,
dysregulated expression of EZH2 may be involved in the progression of prostate
cancer, as well as being a marker that distinguishes indolent prostate cancer
from those at risk of lethal progression. BACKGROUND: Molecular signatures in cancer tissue may be useful for diagnosis
and are associated with survival. We used results from high-density tissue
microarrays (TMAs) to define combinations of candidate biomarkers associated
with the rate of prostate cancer progression after radical prostatectomy that
could identify patients at high risk for recurrence.
METHODS: Fourteen candidate biomarkers for prostate cancer for which antibodies
are available included hepsin, pim-1 kinase, E-cadherin (ECAD; cell adhesion
molecule), alpha-methylacyl-coenzyme A racemase, and EZH2 (enhancer of zeste
homolog 2, a transcriptional repressor). TMAs containing more than 2000 tumor
samples from 259 patients who underwent radical prostatectomy for localized
prostate cancer were studied with these antibodies. Immunohistochemistry results
were evaluated in conjunction with clinical parameters associated with prostate
cancer progression, including tumor stage, Gleason score, and prostate-specific
antigen (PSA) level. Recurrence was defined as a postsurgery PSA level of more
than 0.2 ng/mL. All statistical tests were two-sided.
RESULTS: Moderate or strong expression of EZH2 coupled with at most moderate
expression of ECAD (i.e., a positive EZH2:ECAD status) was the biomarker
combination that was most strongly associated with the recurrence of prostate
cancer. EZH2:ECAD status was statistically significantly associated with
prostate cancer recurrence in a training set of 103 patients (relative risk [RR]
= 2.52, 95% confidence interval [CI] = 1.09 to 5.81; P =.021), in a validation
set of 80 patients (RR = 3.72, 95% CI = 1.27 to 10.91; P =.009), and in the
combined set of 183 patients (RR = 2.96, 95% CI = 1.56 to 5.61; P<.001).
EZH2:ECAD status was statistically significantly associated with disease
recurrence even after adjusting for clinical parameters, such as tumor stage,
Gleason score, and PSA level (hazard ratio = 3.19, 95% CI = 1.50 to 6.77; P
=.003).
CONCLUSION: EZH2:ECAD status was statistically significantly associated with
prostate cancer recurrence after radical prostatectomy and may be useful in
defining a cohort of high-risk patients. BACKGROUND: Antigens overexpressed in metastatic prostate cancer are appropriate
targets in anti-cancer immunotherapy, and one candidate is the polycomb group
protein enhancer of zeste homolog 2 (EZH2).
METHODS: Eleven EZH2-derived peptides were prepared based on the HLA-A24 binding
motif. These peptide candidates were screened first by their ability to be
recognized by immunoglobulin G (IgG), and then by their ability to induce
peptide-specific cytotoxic T lymphocytes (CTLs).
RESULTS: IgGs reactive to three EZH2 peptides (EZH2-243 to -252, EZH2-291 to
-299, and EZH2-735 to -;742) were detected in the plasma of almost half of
prostate cancer patients. Among them, the EZH2-291 to -299 and EZH2-735 to -742
peptides effectively induced HLA-A24-restricted and prostate cancer-reactive
CTLs from prostate cancer patients. The cytotoxicity was mainly dependent on
EZH2 peptide-specific and CD8+ T cells.
CONCLUSIONS: These EZH2-291 to -299 and EZH2-735 to -742 peptides could be
promising candidates for peptide-based immunotherapy for HLA-A24+ prostate
cancer patients with metastases. BACKGROUND: Several linkage studies have provided evidence for a prostate cancer
aggressiveness gene on chromosome 7q. This report details the results of the
first mutation screen and association study of EZH2 (located at 7q35) as a
potential candidate gene for the development of aggressive prostate cancer.
METHODS: In 10 families with linkage of chromosome 7q31-33 to aggressive
prostate cancer, we sequenced the promoter region and all 20 exons of EZH2. We
genotyped 11 variants in 287 prostate cancer probands and 96 controls.
Association between the disease and the variants/haplotypes was evaluated taking
into account clinical data and disease recurrence.
RESULTS: The individual variation sites did not show significant differences in
the allele frequencies between cases and controls. In contrast, one haplotype
had a higher frequency in controls, and another haplotype was significantly more
frequent in cases with low grade tumors (GI/II) and progression free survival
(NED).
CONCLUSION: We have possibly identified haplotypes which mark alleles that have
a beneficial effect on the development of prostate cancer. Moreover, our results
suggest that genetic variations of the EZH2 gene are not responsible for the
linkage of 7q to aggressive prostate cancer. PURPOSE: EZH2 is a member of the polycomb group of genes and important in cell
cycle regulation. Increased expression of EZH2 has been associated previously
with invasive growth and aggressive clinical behavior in prostate and breast
cancer, but the relationship with tumor cell proliferation has not been examined
in human tumors. The purpose of this study was to validate previous findings in
a population-based setting, also including tumors that have not been studied
previously.
PATIENTS AND METHODS: In our study of nearly 700 patients, we examined EZH2
expression and its association with tumor cell proliferation and other tumor
markers, clinical features, and prognosis in cutaneous melanoma and cancers of
the endometrium, prostate, and breast.
RESULTS: Strong EZH2 expression was associated with increased tumor cell
proliferation in all four cancer types. Associations were also found between
EZH2 and important clinicopathologic variables. EZH2 expression showed
significant prognostic impact in melanoma, prostate, and endometrial carcinoma
in univariate survival analyses, and revealed independent prognostic importance
in carcinoma of the endometrium and prostate. CONCLUSION Our findings point at
EZH2 as a novel and independent prognostic marker in endometrial cancer, and
validate previous findings on prostate and breast cancer. Further, EZH2
expression was associated with features of aggressive cutaneous melanoma. The
fact that EZH2 might identify increased tumor cell proliferation and aggressive
subgroups in several cancers may be of practical interest because the polycomb
group proteins have been suggested as candidates for targeted therapy. EZH2
expression should, therefore, be further examined as a possible predictive
factor. Overexpression of the polycomb group protein enhancer of zeste homologue 2
(EZH2) has been found in several maligcies, including prostate cancer, with
an aggressive phenotype. Amplification of the gene has previously been
demonstrated in several maligcies, but not in prostate cancer. Our goal was
to evaluate the gene copy number and expression alterations of EZH2 in prostate
cancer. The copy number of EZH2 in cell lines (LNCaP, DU145, PC-3, 22Rv1),
xenografts (n = 10), and clinical tumors (n = 191) was studied with fluorescence
in situ hybridization. All cell lines had a gain of EZH2. Eight of the ten
xenografts showed an increased copy number of the gene, including one case of
high-level amplification (>or=5 copies of the gene and EZH2/centromere ratio
>or=2). 34/125 (27%) of untreated prostate carcinomas showed increased copy
number, but only one case of low-level amplification (>or=5 copies of the gene
and EZH2/centromere ratio <2), whereas half (25/46) of the hormone-refractory
carcinomas showed increased copy number, including seven cases of low-level
amplification and three cases of high-level amplification (P < 0.0001).
Expression of EZH2 was significantly (P = 0.0009) higher in hormone-refractory
prostate cancer compared with that in benign prostatic hyperplasia or untreated
cancer, according to quantitative real-time RT-PCR assay. Also, the expression
of EZH2 protein was found to be higher in hormone-refractory tumors than in
hormone-naïve tumors by immunohistochemistry. The EZH2 gene amplification was
significantly (P < 0.05) associated with increased EZH2 protein expression. The
data show that amplification of the EZH2 gene is rare in early prostate cancer,
whereas a fraction of late-stage tumors contains the gene amplification leading
to the overexpression of the gene, thus indicating the importance of EZH2 in the
progression of prostate cancer. BACKGROUND: The transcriptional repressor EZH2 is implicated in control of cell
proliferation in embryonic, immortalized and transformed cells. EZH2 expression
in prostate cancer correlates with progression to hormone-refractory and
metastatic disease, but it is unknown whether EZH2 plays a specific role in the
acquisition of an advanced prostate cancer phenotype.
METHODS: Using siRNA knockdown, we investigated the role of EZH2 in maintece
of prostate cancer cell proliferation and invasiveness. Using LNCaP cells with
inducible EZH2 overexpression, we investigated whether EZH2 upregulation
promotes an aggressive phenotype.
RESULTS: Knockdown of endogenous EZH2 reduced proliferation of
androgen-responsive and androgen-independent prostate cancer cells. EZH2
knockdown also inhibited prostate cancer cell invasion. However, overexpression
of EZH2 in androgen-responsive cancer cells did not appreciably affect either
proliferation or invasiveness.
CONCLUSIONS: EZH2 promotes proliferation and invasion of prostate cancer cells,
which can account for the correlation between EZH2 expression levels and an
adverse prostate cancer prognosis. The polycomb group protein enhancer of zeste homolog 2 (EZH2) is linked to
aggressive prostate cancer and could be an appropriate target in specific
immunotherapy. In this study, we attempted to identify EZH2-derived peptides
that have the potential to generate cancer-reactive cytotoxic T lymphocytes
(CTLs) in human leukocyte antigen (HLA)-A2+ prostate cancer patients. Twelve
EZH2-derived peptides were prepared based on the HLA-A2 binding motif. These
peptide candidates were screened first by their ability to be recognized by
immunoglobulin G (IgG), and then by their ability to induce peptide-specific
cytotoxic T lymphocytes (CTLs). As a result, five EZH2 peptides recognized by
IgG (EZH2 120-128, EZH2 165-174, EZH2 569-577, EZH2 665-674, and EZH2 699-708)
were frequently detected in the plasma of prostate cancer patients. Among them,
the EZH2 120-128 and EZH2 165-174 peptides effectively induced HLA-A2-restricted
and cancer-reactive CTLs from prostate cancer patients. The cytotoxicity was
mainly dependent on EZH2 peptide-specific and HLA-A2-restricted CD8+ T cells.
These results indicate that these EZH2 120-128 and EZH2 165-174 peptides could
be promising candidates in peptide-based immunotherapy for HLA-A2+ prostate
cancer patients. BACKGROUND: The Polycomb Group protein EZH2 is implicated in prostate cancer
progression. EZH2 promotes prostate cancer cell proliferation and invasiveness.
We describe a link between EZH2 function and actin polymerization in prostate
cancer cells.
METHODS: Nuclear and cytoplasmic EZH2 expression in benign and maligt
prostate tissue samples was assessed. An association between EZH2 function and
actin polymerization in prostate cancer cells was investigated using
siRNA-mediated knock-down of EZH2. Effects of EZH2 knock-down on actin
polymerization dynamics were analyzed biochemically using immunoblot analysis of
cell lysate fractions, and morphologically using immunocytochemistry.
RESULTS: Cytoplasmic EZH2 is expressed at low levels in benign prostate
epithelial cells and over-expressed in prostate cancer cells. Cytoplasmic EZH2
expression levels correlate with nuclear EZH2 expression in prostate cancer
samples. Knock-down of EZH2 in PC3 prostate cancer cells increases the amount of
F-actin polymerization, cell size, and formation of actin-rich filaments.
CONCLUSIONS: Cytoplasmic EZH2 is over-expressed in prostate cancer cells. EZH2
function promotes a reduction in the pool of insoluble F-actin in invasive
prostate cancer cells. EZH2 may regulate actin polymerization dynamics and
thereby promote prostate cancer cell motility and invasiveness. PURPOSE: Enhancer of zeste homolog 2 (EZH2), a kind of transcriptional
repressor, is reportedly over-expressed in metastatic prostate cancer. In this
study, we analyzed EZH2 mRNA in circulating tumor cells (CTCs) in peripheral
blood as a biomarker in patients with metastatic prostate cancer.
PATIENTS AND METHODS: Ber-EP4 coated immunomagnetic beads were used to harvest
CTCs, and mRNA was isolated by oligo- dT conjugated immunomagnetic beads.
Reverse transcriptase- polymerase chain reaction for EZH2 mRNA was performed and
the expression density was measured. The sensitivity of this test for detection
of EZH2 mRNA was determined by serial dilutions of a human prostate cancer cell
line. Blood samples were collected from 20 patients each with metastatic or
localized prostate cancer and 10 healthy volunteers.
RESULTS: Sensitivity experiments showed that the test was highly sensitive as it
could detect 10 tumor cells per 5 mL. EZH2 mRNA expression was obtained from
peripheral blood samples of patients and control subjects. EZH2 mRNA expression
density in the metastatic prostate cancer group was significantly higher than in
the control (p=0.023) and localized prostate cancer groups (p=0.019). There was
no difference between the control and localized prostate cancer groups (p >
0.05).
CONCLUSION: EZH2 mRNA expression in circulating epithelial cells represents a
promising marker for detecting early metastasis in prostate cancer. However,
more specific and sensitive techniques for detection of CTCs are needed to avoid
mononuclear cell contamination. The polycomb proteins BMI-1, EZH2, and SIRT1 are characteristic components of
the PRC1, PRC2, and PRC4 repressor complexes, respectively, that modify
chromatin. Moreover, EZH2 may influence DNA methylation by direct interaction
with DNA methyltransferases. EZH2 expression increases during prostate cancer
progression, whereas BMI-1 and SIRT1 are not well investigated. Like EZH2
expression, DNA methylation alterations escalate in higher stage prostate
cancers, raising the question whether these epigenetic changes are related.
Expression of EZH2, BMI-1, SIRT1, and the DNA methyltransferases DNMT1 and
DNMT3B measured by qRT-PCR in 47 primary prostate cancers was compared to APC,
ASC, GSTP1, RARB2, and RASSF1A hypermethylation and LINE-1 hypomethylation.
SIRT1 and DNMT3B were overexpressed in cancerous over benign tissues, whereas
BMI-1 was rather downregulated and DNMT1 significantly diminished. Nevertheless,
cancers with higher DNMT1 and BMI-1 expression had worse clinical
characteristics, as did those with elevated EZH2. In particular, above median
DNMT1 expression predicted a worse prognosis. EZH2 and SIRT1 overexpression were
well correlated with increased MKI67. Immunohistochemistry confirmed limited
EZH2 and heterogeneous DNMT3B overexpression and explained the decrease in BMI-1
by pronounced heterogeneity among tumor cells. EZH2 overexpression, specifically
among all factors investigated, was associated with more frequent
hypermethylation, in particular of GSTP1 and RARB2, and also with LINE-1
hypomethylation. Our data reveal complex changes in the composition of polycomb
repressor complexes in prostate cancer. Heterogeneously expressed BMI-1 and
slightly increased EZH2 may characterize less maligt cancers, whereas more
aggressive cases express both at higher levels. SIRT1 appears to be generally
increased in prostate cancers. Intriguingly, our data suggest a direct influence
of increased EZH2 on altered DNA methylation patterns in prostate cancer. Enhancer of zeste homolog 2 (EZH2) is a subunit of the Polycomb-Repressive
Complex 2 (PRC2), which catalyses the trimethylation of histone H3 on Lys 27
(H3K27) and involves in genes repression. EZH2 is amplified and overexpressed in
a variety of cancers, including prostate and breast cancer. Overexpression of
EZH2 has been associated with the invasion and progression of maligt cancers,
especially with the progression of prostate cancer. Here, we review the
structure and biological function of EZH2, especially focused on its activities
in the tumorigenesis. EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in
multiple cancer types and has been implicated in prostate cancer initiation and
progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate
cancer and show that MYC coordinately regulates EZH2 through transcriptional and
post-transcriptional means. Although prior studies in prostate cancer have
revealed a number of possible mechanisms of EZH2 upregulation, these changes
cannot account for the overexpression EZH2 in many primary prostate cancers, nor
in most cases of high grade PIN. We report that upregulation of Myc in the mouse
prostate results in overexpression of EZH2 mRNA and protein which coincides with
reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate
cell types, albeit not in others. Further, in human prostate cancer cells, Myc
negatively regulates miR-26a and miR-26b via direct binding to their parental
Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in
prostate cancer cells results in decreased EZH2 levels and suppressed
proliferation. In human clinical samples, miR-26a and miR-26b are downregulated
in most primary prostate cancers. As a separate mechanism of EZH2 mRNA
upregulation, we find that Myc binds directly to and activates the transcription
of the EZH2 promoter. These results link two major pathways in prostate cancer
by providing two additional and complementary Myc-regulated mechanisms by which
EZH2 upregulation occurs and is enforced during prostatic carcinogenesis.
Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate
prostate tumor initiation and disease progression. BACKGROUND: c-Myc, EZH2 and p27 were defined to modulate the behavior of
prostate cancer with pro-tumoral or anti-tumoral effects and had ability in
predicting prostate cancer progression, but the research of their co-expression
value of prognosis is rarely. This study aimed to investigate the prognostic
value of combining tri-marker together in patients with intermediate-risk
prostate cancer after surgery.
METHODS: Expression levels of c-Myc, EZH2 and p27 in 129 patients with
intermediate-risk prostate cancer were assessed using immunohistochemistry in a
semi-quantitative manner. The expression profiles of these three markers were
analyzed and investigated for association with biochemical recurrence.
RESULTS: In all, fifty of 129 cases experienced biochemical recurrence during a
median follow-up time of 31 months (range, 6 - 60 months). Of these relapse
patients, one case without and 10 cases with any single positive marker were
observed; 39 cases were detected with any two or all three positive markers (22
cases with any two and 17 cases with all three positive markers). Survival
analysis showed that patients with over-expression of c-Myc or EZH2, and lower
expression of p27 manifested significantly higher biochemical recurrence rates.
Subsequent multivariate analysis revealed that c-Myc, EZH2 and p27 expression
statuses showed potential in predicting relapse, respectively. Notably,
combining three markers together as a "composite index" (0 or 1, vs. 2 or 3
positive markers) provided powerful prognostic value (HR 6.57, 95% CI 3.02 -
14.31, P < 0.001). There was a significant difference between the patient
subgroups with 0 or 1 and those with 2 or 3 positive markers expression
statuses, and tri-marker composite index was an independent risk factor for
predicting relapse in patients with intermediate-risk prostate cancer after
surgery.
CONCLUSION: Composite index of c-Myc, EZH2, and p27 can be valued as powerful
prognosis parameter for intermediate-risk prostate cancer patients after the
surgery, and postoperative adjuvant therapy can be adopted accordingly. The histone methyltransferase EZH2 has been in the limelight of the field of
cancer epigenetics for a decade now since it was first discovered to exhibit an
elevated expression in metastatic prostate cancer. It persists to attract much
scientific attention due to its important role in the process of cancer
development and its potential of being an effective therapeutic target. Thus
here we review the dysregulation of EZH2 in prostate cancer, its function,
upstream regulators, downstream effectors, and current status of EZH2-targeting
approaches. This review therefore provides a comprehensive overview of EZH2 in
the context of prostate cancer. Enhancer of zeste homolog 2 (EZH2) plays a crucial role in embryonic and somatic
stem cells for their proliferation and differentiation. However, the roles and
underlying mechanisms of EZH2 in prostate cancer stem cells (PCSCs) remain
unknown. This study aimed to investigate the effects of EZH2 on PCSCs. PCSCs
were isolated from the human prostate cancer cell line LNcap by fluorescence
activated cell sorting (FACS). EZH2 expression was compared between PCSCs and
non-PCSCs. The association between EZH2 function and PCSC growth was
investigated using siRNA-mediated knock-down of EZH2. Cell growth was
investigated by MTT, cell cycle and apoptosis of PCSCs were explored by flow
cytometric analysis. Finally, the upstream pathway miRNA level was determined
via a luciferase reporter assay, and the downstream pathway cycle regulators
were examined via reverse transcriptase-polymerase chain reaction. The results
showed that LNcap cell line comprised a greater proportion of CD44+/CD133+ cells
by comparison to the PC-3 cell line. EZH2 was up-regulated in PCSCs compared
with non-PCSCs. Silence of EZH2 inhibited cell growth and the cell cycle and
promoted the progression of apoptosis. Furthermore, EZH2 was a direct target of
miR-101 in PCSCs and EZH2's mRNA levels were inversely correlated with miR-101
expression and cyclin E2 (a cell-cycle regulator) was suppressed by siEZH2. In
conclusion, EZH2 is essential for PCSC growth, partly through a negative
regulation by miR-101 and positively regulating cyclin E2. Inhibitor of DNA binding/differentiation protein 4 (ID4) is domit negative
helix loop helix transcriptional regulator is epigenetically silenced due to
promoter hyper-methylation in many cancers including prostate. However, the
underlying mechanism involved in epigenetic silencing of ID4 is not known. Here,
we demonstrate that ID4 promoter methylation is initiated by EZH2 dependent
tri-methylation of histone 3 at lysine 27 (H3K27me3). ID4 expressing (LNCaP) and
non-expressing (DU145 and C81) prostate cancer cell lines were used to
investigate EZH2, H3K27me3 and DNMT1 enrichment on ID4 promoter by Chromatin
immuno-precipitation (ChIP). Enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and
C81 cell lines compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in
DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2,
H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent
manner. ChIP data on prostate cancer tissue specimens and cell lines suggested
EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data
indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer
through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased
EZH2 but decreased ID4 expression in prostate cancer strongly supports this
model. |
How does adrenergic signaling affect thyroid hormone receptors? | alpha1- adrenergic signalling increases TRalpha1 expression in nucleus and decreases TRalpha1 expression in cytosol. | Thyroid hormone receptor alpha1 (TRalpha1) is predomitly expressed in the
myocardium but its biological function under physiological or pathological
conditions remains largely unknown. The present study investigated possible
interactions between alpha1 adrenergic and thyroid hormone signaling at the
level of TRalpha1, potential underlying mechanisms and physiological
consequences, as well as the role of TRalpha1 in cell differentiation. This may
be of physiological relevance since both thyroid hormone and adrenergic
signalling are implicated in the pathophysiology of cardiac remodelling.
Neonatal cardiomyocytes obtained from newborn rats (2-3 days) were exposed to
phenylephrine (PE, an alpha1 adrenergic agonist) for 5 days, in the absence or
excess of T3 in the culture medium. PE, in the absence of T3, resulted in 5.0
fold increase in TRalpha1 expression in nucleus and 2.0 fold decrease in
TRalpha1 expression in cytosol, P<0.05. As a result, a fetal pattern of myosin
isoform expression with marked expression of beta-MHC was observed in PE treated
vs the untreated cells, P<0.05. PD98059 (an ERK signalling inhibitor) abrogated
this response. In the presence of T3 in the culture medium, TRalpha1 expression
was increased 1.6 fold in nucleus and 2.0 fold in cytosol in PE-T3 vs PE treated
cells, P<0.05, and the fetal pattern of myosin isoform expression was prevented.
Parallel studies with H9c2 myoblasts showed that reduction of T3 binding to
TRalpha1 receptor delayed cardiac myoblasts differentiation without affecting
proliferation. In conclusion, in neonatal cardiomyocytes, nuclear TRalpha1 is
overexpressed after prolonged activation of the alpha1- adrenergic signalling by
PE. This response seems to be an ERK kinase dependent process. Over-expression
of TRalpha1 may lead to fetal cardiac phenotype in the absence of thyroid
hormone availability. Furthermore, TRalpha1 seems to be critical in cardiac
myoblast differentiation. |
What are the computational tools for the prediction of beta-barrel transmembrane proteins? | The computational tools for the prediction of beta-barrel transmembrane proteins (TMBs) are based mainly on the following methodologies: Hidden Markov Models (HMMs), hydrophobicity, structural data, k-nearest neighbor algorithm, Neural Networks and Support Vector Machines. The state-of-the-art computational tools for the prediction of TMBs are: BETAWARE, BOCTOPUS, BOMP, BTMX, HMM-B2TMR, OMPdb,PRED-TMBB, PROB, ProfTMB, PV, TMBETA-NET, TMB finding pipeline, TMBETADISC-RBF, TMBETAPRED-RBF, TMBHMM, TMB-Hunt, TMB-Hunt2, TMBKNN, TMBpro, transFold. | Very few methods address the problem of predicting beta-barrel membrane proteins
directly from sequence. One reason is that only very few high-resolution
structures for transmembrane beta-barrel (TMB) proteins have been determined
thus far. Here we introduced the design, statistics and results of a novel
profile-based hidden Markov model for the prediction and discrimination of TMBs.
The method carefully attempts to avoid over-fitting the sparse experimental
data. While our model training and scoring procedures were very similar to a
recently published work, the architecture and structure-based labelling were
significantly different. In particular, we introduced a new definition of beta-
hairpin motifs, explicit state modelling of transmembrane strands, and a
log-odds whole-protein discrimination score. The resulting method reached an
overall four-state (up-, down-strand, periplasmic-, outer-loop) accuracy as high
as 86%. Furthermore, accurately discriminated TMB from non-TMB proteins (45%
coverage at 100% accuracy). This high precision enabled the application to 72
entirely sequenced Gram-negative bacteria. We found over 164 previously
uncharacterized TMB proteins at high confidence. Database searches did not
implicate any of these proteins with membranes. We challenge that the vast
majority of our 164 predictions will eventually be verified experimentally. All
proteome predictions and the PROFtmb prediction method are available at
http://www.rostlab.org/ services/PROFtmb/. This work describes the development of a program that predicts whether or not a
polypeptide sequence from a Gram-negative bacterium is an integral beta-barrel
outer membrane protein. The program, called the beta-barrel Outer Membrane
protein Predictor (BOMP), is based on two separate components to recognize
integral beta-barrel proteins. The first component is a C-terminal pattern
typical of many integral beta-barrel proteins. The second component calculates
an integral beta-barrel score of the sequence based on the extent to which the
sequence contains stretches of amino acids typical of transmembrane
beta-strands. The precision of the predictions was found to be 80% with a recall
of 88% when tested on the proteins with SwissProt annotated subcellular
localization in Escherichia coli K 12 (788 sequences) and Salmonella typhimurium
(366 sequences). When tested on the predicted proteome of E.coli, BOMP found 103
of a total of 4346 polypeptide sequences to be possible integral beta-barrel
proteins. Of these, 36 were found by BLAST to lack similarity (E-value score <
1e-10) to proteins with annotated subcellular localization in SwissProt. BOMP
predicted the content of integral beta-barrels per predicted proteome of 10
different bacteria to range from 1.8 to 3%. BOMP is available at
http://www.bioinfo.no/tools/bomp. The beta-barrel outer membrane proteins constitute one of the two known
structural classes of membrane proteins. Whereas there are several different
web-based predictors for alpha-helical membrane proteins, currently there is no
freely available prediction method for beta-barrel membrane proteins, at least
with an acceptable level of accuracy. We present here a web server (PRED-TMBB,
http://bioinformatics.biol.uoa.gr/PRED-TMBB) which is capable of predicting the
transmembrane strands and the topology of beta-barrel outer membrane proteins of
Gram-negative bacteria. The method is based on a Hidden Markov Model, trained
according to the Conditional Maximum Likelihood criterion. The model was
retrained and the training set now includes 16 non-homologous outer membrane
proteins with structures known at atomic resolution. The user may submit one
sequence at a time and has the option of choosing between three different
decoding methods. The server reports the predicted topology of a given protein,
a score indicating the probability of the protein being an outer membrane
beta-barrel protein, posterior probabilities for the transmembrane strand
prediction and a graphical representation of the assumed position of the
transmembrane strands with respect to the lipid bilayer. A new method is presented for identification of beta-barrel membrane proteins.
It is based on a hidden Markov model (HMM) with an architecture obeying these
proteins' construction principles. Once the HMM is trained, log-odds score
relative to a null model is used to discriminate beta-barrel membrane proteins
from other proteins. The method achieves only 10% false positive and false
negative rates in a six-fold cross-validation procedure. The results compare
favorably with existing methods. This method is proposed to be a valuable tool
to quickly scan proteomes of entirely sequenced organisms for beta-barrel
membrane proteins. BACKGROUND: Prediction of the transmembrane strands and topology of beta-barrel
outer membrane proteins is of interest in current bioinformatics research.
Several methods have been applied so far for this task, utilizing different
algorithmic techniques and a number of freely available predictors exist. The
methods can be grossly divided to those based on Hidden Markov Models (HMMs), on
Neural Networks (NNs) and on Support Vector Machines (SVMs). In this work, we
compare the different available methods for topology prediction of beta-barrel
outer membrane proteins. We evaluate their performance on a non-redundant
dataset of 20 beta-barrel outer membrane proteins of gram-negative bacteria,
with structures known at atomic resolution. Also, we describe, for the first
time, an effective way to combine the individual predictors, at will, to a
single consensus prediction method.
RESULTS: We assess the statistical significance of the performance of each
prediction scheme and conclude that Hidden Markov Model based methods,
HMM-B2TMR, ProfTMB and PRED-TMBB, are currently the best predictors, according
to either the per-residue accuracy, the segments overlap measure (SOV) or the
total number of proteins with correctly predicted topologies in the test set.
Furthermore, we show that the available predictors perform better when only
transmembrane beta-barrel domains are used for prediction, rather than the
precursor full-length sequences, even though the HMM-based predictors are not
influenced significantly. The consensus prediction method performs significantly
better than each individual available predictor, since it increases the accuracy
up to 4% regarding SOV and up to 15% in correctly predicted topologies.
CONCLUSIONS: The consensus prediction method described in this work, optimizes
the predicted topology with a dynamic programming algorithm and is implemented
in a web-based application freely available to non-commercial users at
http://bioinformatics.biol.uoa.gr/ConBBPRED. BACKGROUND: Beta-barrel transmembrane (bbtm) proteins are a functionally
important and diverse group of proteins expressed in the outer membranes of
bacteria (both gram negative and acid fast gram positive), mitochondria and
chloroplasts. Despite recent publications describing reasonable levels of
accuracy for discriminating between bbtm proteins and other proteins, screening
of entire genomes remains troublesome as these molecules only constitute a small
fraction of the sequences screened. Therefore, novel methods are still required
capable of detecting new families of bbtm protein in diverse genomes.
RESULTS: We present TMB-Hunt, a program that uses a k-Nearest Neighbour (k-NN)
algorithm to discriminate between bbtm and non-bbtm proteins on the basis of
their amino acid composition. By including differentially weighted amino acids,
evolutionary information and by calibrating the scoring, an accuracy of 92.5%
was achieved, with 91% sensitivity and 93.8% positive predictive value (PPV),
using a rigorous cross-validation procedure. A major advantage of this approach
is that because it does not rely on beta-strand detection, it does not require
resolved structures and thus larger, more representative, training sets could be
used. It is therefore believed that this approach will be invaluable in
complementing other, physicochemical and homology based methods. This was
demonstrated by the correct reassignment of a number of proteins which other
predictors failed to classify. We have used the algorithm to screen several
genomes and have discussed our findings.
CONCLUSION: TMB-Hunt achieves a prediction accuracy level better than other
approaches published to date. Results were significantly enhanced by use of
evolutionary information and a system for calibrating k-NN scoring. Because the
program uses a distinct approach to that of other discriminators and thus
suffers different liabilities, we believe it will make a significant
contribution to the development of a consensus approach for bbtm protein
detection. Mycobacterial porins and other beta-barrel outer-membrane proteins are
represented by the structure of Mycobacterium smegmatis porin MspA. On the basis
of existing knowledge of beta-barrel outer-membrane proteins, several state of
the art prediction methods, as well as a new in-house program (PROB) were
employed for the systematic exploration of Mycobacterium tuberculosis predicted
proteomes for potential beta-barrel structures. PROB allowed parameter
optimization while functioning with an adaptive algorithm for the detection of
outer-membrane beta-barrel proteins in highly divergent proteomes. As a result
of the predictions, 114 proteins in total were predicted to be beta-barrel
structures; of these, 40 were PE-PPE proteins, 8 Mce proteins, 24 hypothetical,
11 probable membrane proteins, 10 transporters, 4 lipoproteins, and 14
classified as other. The congruence among three of the predictors, PROB,
TMB-Hunt, and BOMP, was low with only three proteins (MT0318, MT0356, and
MT2423) predicted by the three. Overall, 79 new proteins for which no previous
experimental work has been performed are reported. At least 10 of these have
high potential of being not only surface-exposed but also served as putative
vaccine candidates as determined by in silico predictions of CD4T cell MHC-II
restricted epitopes. BACKGROUND: Hidden Markov Models (HMMs) have been extensively used in
computational molecular biology, for modelling protein and nucleic acid
sequences. In many applications, such as transmembrane protein topology
prediction, the incorporation of limited amount of information regarding the
topology, arising from biochemical experiments, has been proved a very useful
strategy that increased remarkably the performance of even the top-scoring
methods. However, no clear and formal explanation of the algorithms that retains
the probabilistic interpretation of the models has been presented so far in the
literature.
RESULTS: We present here, a simple method that allows incorporation of prior
topological information concerning the sequences at hand, while at the same time
the HMMs retain their full probabilistic interpretation in terms of conditional
probabilities. We present modifications to the standard Forward and Backward
algorithms of HMMs and we also show explicitly, how reliable predictions may
arise by these modifications, using all the algorithms currently available for
decoding HMMs. A similar procedure may be used in the training procedure, aiming
at optimizing the labels of the HMM's classes, especially in cases such as
transmembrane proteins where the labels of the membrane-spanning segments are
inherently misplaced. We present an application of this approach developing a
method to predict the transmembrane regions of alpha-helical membrane proteins,
trained on crystallographically solved data. We show that this method compares
well against already established algorithms presented in the literature, and it
is extremely useful in practical applications.
CONCLUSION: The algorithms presented here, are easily implemented in any kind of
a Hidden Markov Model, whereas the prediction method (HMM-TM) is freely
available for academic users at http://bioinformatics.biol.uoa.gr/HMM-TM,
offering the most advanced decoding options currently available. PROFtmb predicts transmembrane beta-barrel (TMB) proteins in Gram-negative
bacteria. For each query protein, PROFtmb provides both a Z-value indicating
that the protein actually contains a membrane barrel, and a four-state
per-residue labeling of upward- and downward-facing strands, periplasmic
hairpins and extracellular loops. While most users submit individual proteins
known to contain TMBs, some groups submit entire proteomes to screen for
potential TMBs. Response time is about 4 min for a 500-residue protein. PROFtmb
is a profile-based Hidden Markov Model (HMM) with an architecture mirroring the
structure of TMBs. The per-residue accuracy on the 8-fold cross-validated
testing set is 86% while whole-protein discrimination accuracy was 70 at 60%
coverage. The PROFtmb web server includes all source code, training data and
whole-proteome predictions from 78 Gram-negative bacterial genomes and is
available freely and without registration at
http://rostlab.org/services/proftmb. Transmembrane beta-barrel (TMB) proteins are embedded in the outer membrane of
Gram-negative bacteria, mitochondria and chloroplasts. The cellular location and
functional diversity of beta-barrel outer membrane proteins makes them an
important protein class. At the present time, very few non-homologous TMB
structures have been determined by X-ray diffraction because of the experimental
difficulty encountered in crystallizing transmembrane (TM) proteins. The
transFold web server uses pairwise inter-strand residue statistical potentials
derived from globular (non-outer-membrane) proteins to predict the
supersecondary structure of TMB. Unlike all previous approaches, transFold does
not use machine learning methods such as hidden Markov models or neural
networks; instead, transFold employs multi-tape S-attribute grammars to describe
all potential conformations, and then applies dynamic programming to determine
the global minimum energy supersecondary structure. The transFold web server not
only predicts secondary structure and TMB topology, but is the only method which
additionally predicts the side-chain orientation of transmembrane beta-strand
residues, inter-strand residue contacts and TM beta-strand inclination with
respect to the membrane. The program transFold currently outperforms all other
methods for accuracy of beta-barrel structure prediction. Available at
http://bioinformatics.bc.edu/clotelab/transFold. Accurate protein structure prediction remains an active objective of research in
bioinformatics. Membrane proteins comprise approximately 20% of most genomes.
They are, however, poorly tractable targets of experimental structure
determination. Their analysis using bioinformatics thus makes an important
contribution to their on-going study. Using a method based on Bayesian Networks,
which provides a flexible and powerful framework for statistical inference, we
have addressed the alignment-free discrimination of membrane from non-membrane
proteins. The method successfully identifies prokaryotic and eukaryotic
alpha-helical membrane proteins at 94.4% accuracy, beta-barrel proteins at 72.4%
accuracy, and distinguishes assorted non-membranous proteins with 85.9%
accuracy. The method here is an important potential advance in the computational
analysis of membrane protein structure. It represents a useful tool for the
characterisation of membrane proteins with a wide variety of potential
applications. Membrane proteins, which constitute approximately 20% of most genomes, form two
main classes: alpha helical and beta barrel transmembrane proteins. Using
methods based on Bayesian Networks, a powerful approach for statistical
inference, we have sought to address beta-barrel topology prediction. The
beta-barrel topology predictor reports individual strand accuracies of 88.6%.
The method outlined here represents a potentially important advance in the
computational determination of membrane protein topology. We have developed a novel approach for dissecting transmembrane beta-barrel
proteins (TMBs) in genomic sequences. The features include (i) the
identification of TMBs using the preference of residue pairs in globular,
transmembrane helical (TMH) and TMBs, (ii) elimination of globular/TMH proteins
that show sequence identity of more than 70% for the coverage of 80% residues
with known structures, (iii) elimination of globular/TMH proteins that have
sequence identity of more than 60% with known sequences in SWISS-PROT, and (iv)
exclusion of TMH proteins using SOSUI, a prediction system for TMH proteins. Our
approach picked up 7% TMBs in all the considered genomes. The comparison between
the identified TMBs in E. coli genome and available experimental data
demonstrated that the new approach could correctly identify all the 11 known
TMBs, whose crystal structures are available. Further, it revealed the presence
of 19 TMBs, homology with known structures, 60 TMBs similar to well annotated
sequences, and 54 TMBs that have high sequence similarity with Escherichia coli
beta-barrel proteins deposited in Transport Classification Database (TCDB).
Interestingly, the present approach identified TMBs from all 15 families in
TCDB. In human genome, the occurrence of TMBs varies from 0 to 3% in different
chromosomes. We suggest that our approach could lead to a step forward in the
advancement of structural and functional genomics. MOTIVATION: Transmembrane beta-barrel (TMB) proteins are embedded in the outer
membranes of mitochondria, Gram-negative bacteria and chloroplasts. These
proteins perform critical functions, including active ion-transport and passive
nutrient intake. Therefore, there is a need for accurate prediction of secondary
and tertiary structure of TMB proteins. Traditional homology modeling methods,
however, fail on most TMB proteins since very few non-homologous TMB structures
have been determined. Yet, because TMB structures conform to specific
construction rules that restrict the conformational space drastically, it should
be possible for methods that do not depend on target-template homology to be
applied successfully.
RESULTS: We develop a suite (TMBpro) of specialized predictors for predicting
secondary structure (TMBpro-SS), beta-contacts (TMBpro-CON) and tertiary
structure (TMBpro-3D) of transmembrane beta-barrel proteins. We compare our
results to the recent state-of-the-art predictors transFold and PRED-TMBB using
their respective benchmark datasets, and leave-one-out cross-validation. Using
the transFold dataset TMBpro predicts secondary structure with per-residue
accuracy (Q(2)) of 77.8%, a correlation coefficient of 0.54, and TMBpro predicts
beta-contacts with precision of 0.65 and recall of 0.67. Using the PRED-TMBB
dataset, TMBpro predicts secondary structure with Q(2) of 88.3% and a
correlation coefficient of 0.75. All of these performance results exceed
previously published results by 4% or more. Working with the PRED-TMBB dataset,
TMBpro predicts the tertiary structure of transmembrane segments with RMSD <6.0
A for 9 of 14 proteins. For 6 of 14 predictions, the RMSD is <5.0 A, with a
GDT_TS score greater than 60.0.
AVAILABILITY: http://www.igb.uci.edu/servers/psss.html. Discriminating outer membrane proteins (OMPs) from other folding types of
globular and membrane proteins is an important task both for identifying OMPs
from genomic sequences and for the successful prediction of their secondary and
tertiary structures. We have developed a method based on radial basis function
networks and position specific scoring matrix (PSSM) profiles generated by
PSI-BLAST and non-redundant protein database. Our approach with PSSM profiles
has correctly predicted the OMPs with a cross-validated accuracy of 96.4% in a
set of 1251 proteins, which contain 206 OMPs, 667 globular proteins and 378
alpha-helical inner membrane proteins. Furthermore, we applied our method on a
dataset containing 114 OMPs, 187 TMH proteins and 195 globular proteins obtained
with less than 20% sequence identity and obtained the cross-validated accuracy
of 95%. This accuracy of discriminating OMPs is higher than other methods in the
literature and our method could be used as an effective tool for dissecting OMPs
from genomic sequences. We have developed a prediction server, TMBETADISC-RBF,
which is available at http://rbf.bioinfo.tw/~sachen/OMP.html. BACKGROUND: Due to their role of receptors or transporters, membrane proteins
play a key role in many important biological functions. In our work we used
Grammatical Inference (GI) to localize transmembrane segments. Our GI process is
based specifically on the inference of Even Linear Languages.
RESULTS: We obtained values close to 80% in both specificity and sensitivity.
Six datasets have been used for the experiments, considering different encodings
for the input sequences. An encoding that includes the topology changes in the
sequence (from inside and outside the membrane to it and vice versa) allowed us
to obtain the best results. This software is publicly available at:
http://www.dsic.upv.es/users/tlcc/bio/bio.html
CONCLUSION: We compared our results with other well-known methods, that obtain a
slightly better precision. However, this work shows that it is possible to apply
Grammatical Inference techniques in an effective way to bioinformatics problems. Prediction of membrane spanning segments in beta-barrel outer membrane proteins
(OMP) and their topology is an important problem in structural and functional
genomics. In this work, we propose a method based on radial basis networks for
predicting the number of beta-strands in OMPs and identifying their membrane
spanning segments. Our method showed a leave-one-out cross validation accuracy
of 96% in a set of 28 OMPs, which have the range of 8-22 beta-strand segments.
The beta-strand segments in OMPs and the residues in membrane spanning segments
are correctly predicted with the accuracy of 96% and 87%, respectively. We have
developed a web server, TMBETAPRED-RBF for predicting the transmembrane
beta-strands from amino acid sequence and it is available at
http://rbf.bioinfo.tw/~sachen/tmrbf.html. We suggest that our method could be an
effective tool for predicting the membrane spanning regions and topology of
beta-barrel membrane proteins. Transmembrane beta barrel (TMB) proteins are found in the outer membranes of
bacteria, mitochondria and chloroplasts. TMBs are involved in a variety of
functions such as mediating flux of metabolites and active transport of
siderophores, enzymes and structural proteins, and in the translocation across
or insertion into membranes. We present here TMBHMM, a computational method
based on a hidden Markov model for predicting the structural topology of
putative TMBs from sequence. In addition to predicting transmembrane strands,
TMBHMM also predicts the exposure status (i.e., exposed to the membrane or
hidden in the protein structure) of the residues in the transmembrane region,
which is a novel feature of the TMBHMM method. Furthermore, TMBHMM can also
predict the membrane residues that are not part of beta barrel forming strands.
The training of the TMBHMM was performed on a non-redundant data set of 19 TMBs.
The self-consistency test yielded Q(2) accuracy of 0.87, Q(3) accuracy of 0.83,
Matthews correlation coefficient of 0.74 and SOV for beta strand of 0.95. In
this self-consistency test the method predicted 83% of transmembrane residues
with correct exposure status. On an unseen, non-redundant test data set of 10
proteins, the 2-state and 3-state TMBHMM prediction accuracies are around 73%
and 72%, respectively, and are comparable to other methods from the literature.
The TMBHMM web server takes an amino acid sequence or a multiple sequence
alignment as an input and predicts the exposure status and the structural
topology as output. The TMBHMM web server is available under the tmbhmm tab at:
http://service.bioinformatik.uni-saarland.de/tmx-site/. We introduce a graph-theoretic model for predicting the supersecondary structure
of transmembrane β-barrel proteins--a particular class of proteins that performs
diverse important functions but it is difficult to determine their structure
with experimental methods. This ab initio model resolves the protein folding
problem based on pseudo-energy minimization with the aid of a simple
probabilistic filter. It also allows for determining structures whose barrel
follows a given permutation on the arrangement of β-strands, and allows for
rapidly discriminating the transmembrane β-barrels from other kinds of proteins.
The model is fairly accurate, robust and can be run very efficiently on PC-like
computers, thus proving useful for genome screening. |
Which kinase is regulating stress granule biogenesis? | 5'-AMP-activated protein kinase alpha regulates stress granule biogenesis | Stress granule (SG) assembly represents a conserved eukaryotic defense strategy
against various insults. Although essential for the ability to cope with
deleterious conditions, the signaling pathways controlling SG formation are not
fully understood. The energy sensor AMP-activated protein kinase (AMPK) is
critical for the cellular stress response. Human cells produce two AMPK
catalytic α-subunits with not only partially overlapping, but also unique
functions. Here, we provide direct support for structural and functional links
between AMPK-α isoforms and SGs. As such, several stressors promote SG
association of AMPK-α2, but not AMPK-α1. Multiple lines of evidence link AMPK
activity to SG biogenesis. First, pharmacological kinase inhibition interfered
with SG formation. Second, AMPK-α knockdown combined with in-depth quantitative
SG analysis revealed isoform-specific changes of SG characteristics. Third,
overexpression of mutant α-subunits further substantiated that AMPK regulates SG
parameters. Finally, we identified the SG-nucleating protein G3BP1 as an AMPK-α2
binding partner. This interaction is stimulated by stress and notably occurs in
SGs. Collectively, our data define the master metabolic regulator AMPK as a
novel SG constituent that also controls their biogenesis. |
What is the association between cell phone use and glioblastoma? | The association between cell phone use and incident glioblastoma remains unclear. Some studies have reported that cell phone use was associated with incident glioblastoma, and with reduced survival of patients diagnosed with glioblastoma. However, other studies have repeatedly replicated to find an association between cell phone use and glioblastoma. | The use of cellular telephones has increased dramatically during the 1990's in
the world. In the 1980's the analogue NMT system was used whereas the digital
GSM system was introduced in early 1990's and is now the preferred system. Case
reports of brain tumours in users initiated this case-control study on brain
tumours and use of cellular telephones. Also other exposures were assessed. All
cases, both males and females, with histopathologically verified brain tumour
living in Uppsala-Orebro region (1994-96) and Stockholm region (1995-96) aged
20-80 at the time of diagnosis and alive at start of the study were included,
233 in total. Two controls to each case were selected from the Swedish
Population Register matched for sex, age and study region. Exposure was assessed
by questionnaires supplemented over the phone. The analyses were based on
answers from 209 (90%) cases and 425 (91%) controls. Use of cellular telephone
gave odds ratio (OR) = 0.98 with 95% confidence interval (CI) = 0. 69-1.41. For
the digital GSM system OR = 0.97, CI = 0.61-1.56 and for the analogue NMT system
OR = 0.94, CI = 0.62-1.44 were calculated. Dose-response analysis and using
different tumour induction periods gave similar results. Non-significantly
increased risk was found for tumour in the temporal or occipital lobe on the
same side as a cellular phone had been used, right side OR = 2.45, CI =
0.78-7.76, left side OR = 2.40, CI = 0.52-10.9 Increased risk was found only for
use of the NMT system. For GSM use the observation time is still too short for
definite conclusions. An increased risk for brain tumour in the anatomical area
close to the use of a cellular telephone should be especially studied in the
future. We included in a case-control study on brain tumours and mobile and cordless
telephones 1,617 patients aged 20-80 years of both sexes diagnosed during
January 1, 1997 to June 30, 2000. They were alive at the study time and had
histopathology verified brain tumour. One matched control to each case was
selected from the Swedish Population Register. The study area was the
Uppsala-Orebro, Stockholm, Linköping and Göteborg medical regions of Sweden.
Exposure was assessed by a questionnaire that was answered by 1,429 (88%) cases
and 1,470 (91%) controls. In total use of analogue cellular telephones gave an
increased risk with odds ratio (OR)=1.3, 95% confidence interval (CI)=1.04-1.6,
whereas digital and cordless phones did not overall increase the risk
significantly. Ipsilateral use of analogue phones gave OR=1.7, 95% CI=1.2-2.3,
digital phones OR=1.3, 95% CI=1.02-1.8 and cordless phones OR=1.2, 95%
CI=0.9-1.6. The risk for ipsilateral use was significantly increased for
astrocytoma for all studied phone types, analogue phones OR=1.8,95% CI=1.1-3.2,
digital phones OR=1.8, 95% CI=1.1-2.8, cordless phones OR=1.8, 95% CI=1.1-2.9.
Use of a telephone on the opposite side of the brain was not associated with a
significantly increased risk for brain tumours. Regarding anatomical area of the
tumour and exposure to microwaves, the risk was increased for tumours located in
the temporal area on the same side of the brain that was used during phone
calls, significantly so for analogue cellular telephones OR=2.3, 95% CI=1.2-4.1.
For acoustic neurinoma OR=4.4, 95% CI=2.1-9.2 was calculated among analogue
cellular telephone users. When duration of use was analysed as a continuous
variable in the total material, the risk increased per year for analogue phones
with OR=1.04, 95% CI=1.01-1.08. For astrocytoma and ipsilateral use the trend
was for analogue phones OR=1.10, 95% CI=1.02-1.19, digital phones OR=1.11, 95%
CI=1.01-1.22, and cordless phones OR=1.09, 95% CI=1.01-1.19. There was a
tendency of a shorter tumour induction period for ipsilateral exposure to
microwaves than for contralateral, which may indicate a tumour promotor effect. Mobile phone use and risk of acoustic neuroma: results of the interphone
case-control study in five north European countries [corrected]. DOI: 10.1038/sj.bjc.6603238
PMCID: PMC2360496
PMID: 16804530 [Indexed for MEDLINE] BACKGROUND: Use of cell phones has increased dramatically since 1992 when they
were first introduced in France. Certain electromagnetic fields (at extremely
low frequency) have been recognized as possibly carcinogenic by the
International Agency for Research on Cancer. Given the use of radiofrequency
technology in cell phones, the rapid increase in the number of cell phones has
generated concerns about the existence of a potential health hazard. To evaluate
the relationship between the use of cell phones and the development of tumors of
the head, a multicentric international study (INTERPHONE), coordinated by the
International Agency for Research on Cancer, was carried out in 13 countries.
This publication reports the results of the French part of the INTERPHONE study.
METHODS: INTERPHONE is a case-control study focused on tumors of the brain and
central nervous system: gliomas, meningiomas and neuromas of cranial nerves.
Eligible cases were men and women, residents of Paris or Lyon, aged 30-59, newly
diagnosed with a first primary tumor between February 2001 and August 2003. The
diagnoses were all either histologically confirmed or based upon unequivocal
radiological images. Controls were matched for gender, age (+/-5 years) and
place of residence. They were randomly drawn from electoral rolls. Detailed
information was collected for all subjects during a computer-assisted
face-to-face interview. Conditional logistic regression was used to estimate the
odds ratio (OR) for an association between the use of cell phones and risk of
each type of cancer.
RESULTS: Regular cell phone use was not associated with an increased risk of
neuroma (OR=0,92; 95% confidence interval=[0.53-1.59]), meningioma (OR=0,74; 95%
confidence interval=[0.43-1.28]) or glioma (OR=1.15; 95% confidence
interval=[0.65-2.05]). Although these results are not statistically significant,
a general tendency was observed for an increased risk of glioma among the
heaviest users: long-term users, heavy users, users with the largest numbers of
telephones.
CONCLUSION: No significant increased risk for glioma, meningioma or neuroma was
observed among cell phone users participating in Interphone. The statistical
power of the study is limited, however. Our results, suggesting the possibility
of an increased risk among the heaviest users, therefore need to be verified in
the international INTERPHONE analyses. There is considerable controversy surrounding the biological effects of
radiofrequency (RF) fields, as emitted by mobile phones. Previous work from our
laboratory has shown no effect related to the exposure of 1.9 GHz
pulse-modulated RF fields on the expression of 22,000 genes in a human
glioblastoma-derived cell-line (U87MG) at 6 h following a 4 h RF field exposure
period. As a follow-up to this study, we have now examined the effect of RF
field exposure on the possible expression of late onset genes in U87MG cells
after a 24 h RF exposure period. In addition, a human monocyte-derived cell-line
(Mono-Mac-6, MM6) was exposed to intermittent (5 min ON, 10 min OFF) RF fields
for 6 h and then gene expression was assessed immediately after exposure and at
18 h postexposure. Both cell lines were exposed to 1.9 GHz pulse-modulated RF
fields for 6 or 24 h at specific absorption rates (SARs) of 0.1-10.0 W/kg. In
support of our previous results, we found no evidence that nonthermal RF field
exposure could alter gene expression in either cultured U87MG or MM6 cells,
relative to nonirradiated control groups. However, exposure of both cell-lines
to heat-shock conditions (43 degrees C for 1 h) caused an alteration in the
expression of a number of well-characterized heat-shock proteins. DOI: 10.1289/ehp.116-a422
PMCID: PMC2569116
PMID: 18941554 [Indexed for MEDLINE] We investigated the mechanisms by which radiofrequency (RF) fields exert their
activity, and the changes in both cell proliferation and the gene expression
profile in the human cell lines, A172 (glioblastoma), H4 (neuroglioma), and
IMR-90 (fibroblasts from normal fetal lung) following exposure to 2.1425 GHz
continuous wave (CW) and Wideband Code Division Multiple Access (W-CDMA) RF
fields at three field levels. During the incubation phase, cells were exposed at
the specific absorption rates (SARs) of 80, 250, or 800 mW/kg with both CW and
W-CDMA RF fields for up to 96 h. Heat shock treatment was used as the positive
control. No significant differences in cell growth or viability were observed
between any test group exposed to W-CDMA or CW radiation and the sham-exposed
negative controls. Using the Affymetrix Human Genome Array, only a very small (<
1%) number of available genes (ca. 16,000 to 19,000) exhibited altered
expression in each experiment. The results confirm that low-level exposure to
2.1425 GHz CW and W-CDMA RF fields for up to 96 h did not act as an acute
cytotoxicant in either cell proliferation or the gene expression profile. These
results suggest that RF exposure up to the limit of whole-body average SAR
levels as specified in the ICNIRP guidelines is unlikely to elicit a general
stress response in the tested cell lines under these conditions. A practical mathematical model for glioblastomas (brain tumours), which
incorporates the two key parameters of tumour growth, namely the cancer cell
diffusion and the cell proliferation rate, has been shown to be clinically
useful and predictive. Previous studies explain why multifocal recurrence is
inevitable and show how various treatment scenarios have been incorporated in
the model. In most tumours, it is not known when the cancer started. Based on
patient in vivo parameters, obtained from two brain scans, it is shown how to
estimate the time, after initial detection, when the tumour started. This is an
input of potential importance in any future controlled clinical study of any
connection between cell phone radiation and brain tumour incidence. It is also
used to estimate more accurately survival times from detection. Finally, based
on patient parameters, the solution of the model equation of the tumour growth
helps to explain why certain patients live longer than others after similar
treatment protocols specifically surgical resection (removal) and irradiation. BACKGROUND: We analysed the survival of patients after glioma diagnosis in
relation to the use of wireless phones.
METHODS: All cases diagnosed between 1997 and 2003 with a maligt brain tumour
(n = 1,251) in our case-control studies were included and followed from the date
of diagnosis to the date of death or until May 30, 2012.
RESULTS: For glioma, the use of wireless phones (mobile and cordless phones)
gave a hazard ratio (HR) = 1.1 (95% confidence interval, CI = 0.9-1.2), with >
10-year latency HR = 1.2 (95% CI = 1.002-1.5, p trend = 0.02). For astrocytoma
grade I-II (low-grade), the results were, HR = 0.5 (95% CI = 0.3-0.9) and for
astrocytoma grade IV (glioblastoma), HR = 1.1 (95% CI = 0.95-1.4), with > 10
year latency HR = 1.3 (95% CI = 1.03-1.7). In the highest tertile (> 426 h) of
cumulative use, HR = 1.2 (95% CI = 0.95-1.5) was found for glioblastoma. The
results were similar for mobile and cordless phones.
CONCLUSIONS: Decreased survival of glioma cases with long-term and high
cumulative use of wireless phones was found. A survival disadvantage for
astrocytoma grade IV, but a survival benefit for astrocytoma grade I-II was
observed which could be due to exposure-related tumour symptoms leading to
earlier diagnosis and surgery in that patient group. The International Agency for Research on Cancer (IARC) at WHO evaluation of the
carcinogenic effect of RF-EMF on humans took place during a 24-31 May 2011
meeting at Lyon in France. The Working Group consisted of 30 scientists and
categorised the radiofrequency electromagnetic fields from mobile phones, and
from other devices that emit similar non-ionising electromagnetic fields
(RF-EMF), as Group 2B, i.e., a 'possible', human carcinogen. The decision on
mobile phones was based mainly on the Hardell group of studies from Sweden and
the IARC Interphone study. We give an overview of current epidemiological
evidence for an increased risk for brain tumours including a meta-analysis of
the Hardell group and Interphone results for mobile phone use. Results for
cordless phones are lacking in Interphone. The meta-analysis gave for glioma in
the most exposed part of the brain, the temporal lobe, odds ratio (OR)=1.71, 95%
confidence interval (CI)=1.04-2.81 in the ≥10 years (>10 years in the Hardell
group) latency group. Ipsilateral mobile phone use ≥1640h in total gave OR=2.29,
95% CI=1.56-3.37. The results for meningioma were OR=1.25, 95% CI=0.31-4.98 and
OR=1.35, 95% CI=0.81-2.23, respectively. Regarding acoustic neuroma ipsilateral
mobile phone use in the latency group ≥10 years gave OR=1.81, 95% CI=0.73-4.45.
For ipsilateral cumulative use ≥1640h OR=2.55, 95% CI=1.50-4.40 was obtained.
Also use of cordless phones increased the risk for glioma and acoustic neuroma
in the Hardell group studies. Survival of patients with glioma was analysed in
the Hardell group studies yielding in the >10 years latency period hazard ratio
(HR)=1.2, 95% CI=1.002-1.5 for use of wireless phones. This increased HR was
based on results for astrocytoma WHO grade IV (glioblastoma multiforme).
Decreased HR was found for low-grade astrocytoma, WHO grades I-II, which might
be caused by RF-EMF exposure leading to tumour-associated symptoms and earlier
detection and surgery with better prognosis. Some studies show increasing
incidence of brain tumours whereas other studies do not. It is concluded that
one should be careful using incidence data to dismiss results in analytical
epidemiology. The IARC carcinogenic classification does not seem to have had any
significant impact on governments' perceptions of their responsibilities to
protect public health from this widespread source of radiation. Previous studies have shown a consistent association between long-term use of
mobile and cordless phones and glioma and acoustic neuroma, but not for
meningioma. When used these phones emit radiofrequency electromagnetic fields
(RF-EMFs) and the brain is the main target organ for the handheld phone. The
International Agency for Research on Cancer (IARC) classified in May, 2011
RF-EMF as a group 2B, i.e. a 'possible' human carcinogen. The aim of this study
was to further explore the relationship between especially long-term (>10 years)
use of wireless phones and the development of maligt brain tumours. We
conducted a new case-control study of brain tumour cases of both genders aged
18-75 years and diagnosed during 2007-2009. One population-based control matched
on gender and age (within 5 years) was used to each case. Here, we report on
maligt cases including all available controls. Exposures on e.g. use of
mobile phones and cordless phones were assessed by a self-administered
questionnaire. Unconditional logistic regression analysis was performed,
adjusting for age, gender, year of diagnosis and socio-economic index using the
whole control sample. Of the cases with a maligt brain tumour, 87% (n=593)
participated, and 85% (n=1,368) of controls in the whole study answered the
questionnaire. The odds ratio (OR) for mobile phone use of the analogue type was
1.8, 95% confidence interval (CI)=1.04‑3.3, increasing with >25 years of latency
(time since first exposure) to an OR=3.3, 95% CI=1.6-6.9. Digital 2G mobile
phone use rendered an OR=1.6, 95% CI=0.996-2.7, increasing with latency >15-20
years to an OR=2.1, 95% CI=1.2-3.6. The results for cordless phone use were
OR=1.7, 95% CI=1.1-2.9, and, for latency of 15-20 years, the OR=2.1, 95%
CI=1.2-3.8. Few participants had used a cordless phone for >20-25 years. Digital
type of wireless phones (2G and 3G mobile phones, cordless phones) gave
increased risk with latency >1-5 years, then a lower risk in the following
latency groups, but again increasing risk with latency >15-20 years. Ipsilateral
use resulted in a higher risk than contralateral mobile and cordless phone use.
Higher ORs were calculated for tumours in the temporal and overlapping lobes.
Using the meningioma cases in the same study as reference entity gave somewhat
higher ORs indicating that the results were unlikely to be explained by recall
or observational bias. This study confirmed previous results of an association
between mobile and cordless phone use and maligt brain tumours. These
findings provide support for the hypothesis that RF-EMFs play a role both in the
initiation and promotion stages of carcinogenesis. |
Is there any cross-talk between the Wnt and the Akt pathways? | The Wnt/β-catenin and PI3K/Akt signaling pathways cross-talk mainly through the activity of GSK-3β, a common component of both pathways, but also through the activity of other signaling transducers, such as Cby or WISP-1. | Glucocorticoids, widely used as immune suppressors, cause osteoporosis by
inhibiting bone formation. In MC3T3-E1 osteoblast-like cultures, dexamethasone
(DEX) activates glycogen synthase kinase-3beta (GSK3beta) and inhibits a
differentiation-related cell cycle that occurs at a commitment stage immediately
after confluence. Here we show that DEX inhibition of the
differentiation-related cell cycle is associated with a decrease in beta-catenin
levels and inhibition of LEF/TCF-mediated transcription. These inhibitory
activities are no longer observed in the presence of lithium, a GSK3beta
inhibitor. DEX decreased the serum-responsive phosphorylation of protein kinase
B/Akt-Ser(473) within minutes, and this inhibition was also observed after 12 h.
When the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was inhibited by
wortmannin, DEX no longer inhibited beta-catenin levels. Furthermore,
DEX-mediated inhibition of LEF/TCF transcriptional activity was attenuated in
the presence of domit negative forms of either PI3K or protein kinase B/Akt.
These results suggest cross-talk between the PI3K/Akt and Wnt signaling
pathways. Consistent with a role for Wnt signaling in the osteoblast
differentiation-related cell cycle, wortmannin partially negated the DEX
inhibition of this cell cycle. DEX also induced histone deacetylase (HDAC) 1,
which is known to inhibit LEF/TCF transcriptional activity. Overexpression of
HDAC1 negated the inhibitory effect of DEX on LEF/TCF transcriptional activity.
In the presence of trichostatin A, a deacetylase inhibitor, DEX-mediated
inhibition of the differentiation-related cell cycle was partially negated. When
administered together, wortmannin and trichostatin A completely negated the
inhibitory effect of DEX on the differentiation-related cell cycle. These
results suggest that inhibition of a PI3K/Akt/GSK3beta/beta-catenin/LEF axis and
stimulation of HDAC1 cooperate to mediate the inhibitory effect of DEX on Wnt
signaling and the osteoblast differentiation-related cell cycle. BMP, PTEN and Wnt/beta-catenin pathways are the three signaling pathways that
control normal development and regeneration of the intestine, and contribute to
intestinal polyposis when aberrant inactivation or activation occurs in each of
these pathways. Using genetic targeting of BMPR1A in mice, we show that
inactivation of BMP signaling results in multiple polyps due to an increased
number of crypts and stem cells, accompanied by enhanced Wnt signaling in all
proliferating intestine cells. However the increased transcriptional activity of
Wnt effecter protein, beta-catenin, is found primarily in intestine stem cells
(ISCs). Concurrently, PTEN, an inhibitor of PI3K/Akt pathway, is also primarily
inactivated in the ISCs, leading to activation of Akt. Thus, Akt may contribute
to activation of beta-catenin in ISCs in coordination with Wnt signaling. By
conducting a proteomic analysis of the beta-catenin complex, we show that
14-3-3zeta exists in the beta-catenin complex and facilitates activation of
beta-catenin by Akt, which, intriguingly, appears to be predomitly in ISCs.
Thus, we propose that BMP signaling plays a role in inhibition of ISC
self-renewal through suppression of Wnt/beta-catenin signaling in ISC, and this
cross-talk is bridged, at least in part, through the PTEN/Akt pathway and
further enforced by 14-3-3zeta. WNT and FGF signaling pathways cross-talk during a variety of cellular
processes, such as human colorectal carcinogenesis, mouse mammary tumor virus
(MMTV)-induced carcinogenesis, E2A-Pbx-induced leukemogenesis, early
embryogenesis, body-axis formation, limb-bud formation, and neurogenesis.
Canonical WNT signals are transduced through Frizzled receptor and LRP5/6
coreceptor to downregulate GSK3beta (GSK3B) activity not depending on Ser 9
phosphorylation. FGF signals are transduced through FGF receptor to the
FRS2-GRB2-GAB1-PI3K-AKT signaling cascade to downregulate GSK3beta activity
depending on Ser 9 phosphorylation. Because GSK3beta-dependent phosphorylation
of beta-catenin and SNAIL leads to FBXW1 (betaTRCP)-mediated ubiquitination and
degradation, GSK3beta downregulation results in the stabilization and the
nuclear accumulation of beta-catenin and SNAIL. Nuclear beta-catenin is
complexed with TCF/LEF, Legless (BCL9 or BCL9L) and PYGO (PYGO1 or PYGO2) to
activate transcription of CCND1, MYC, FGF18 and FGF20 genes for the cell-fate
determination. Nuclear SNAIL represses transcription of CDH1 gene, encoding
E-cadherin, to induce the epithelial-mesenchymal transition (EMT). Mammary
carcinogenesis in MMTV-Wnt1 transgenic mice is accelerated by MMTV infection due
to MMTV integration around Fgf3-Fgf4 or Fgf8 loci, and mammary carcinogenesis in
MMTV-Fgf3 transgenic mice due to MMTV integration around Wnt1-Wnt10b locus.
Coactivation of WNT and FGF signaling pathways in tumors leads to more maligt
phenotypes. Single nucleotide polymorphism (SNP) and copy number polymorphism
(CNP) of WNT and FGF signaling molecules could be utilized as screening method
of cancer predisposition. cDNA-PCR, microarray or ELISA reflecting aberrant
activation of WNT and FGF signaling pathways could be developed as novel
cancer-related biomarkers for diagnosis, prognosis, and therapy. Cocktail
therapy using WNT and FGF inhibitors, such as small-molecule compounds and human
neutralizing antibodies, should be developed to increase the efficacy of
chemotherapy through the inhibition of recurrence by destructing cancer stem
cells. Prostate cancer is initially dependent on androgens for growth; hence, recurrent
prostate is treated with androgen ablation which may result in progression to
androgen independence characterized by a resistance to such therapy. Androgens
bind to and activate the androgen receptor (AR), a member of the nuclear steroid
receptor family of transcription factors, which regulates prostate cancer cell
proliferation and survival in androgen-independent, as well as -dependent,
tumors. Another pathway regulating proliferation and survival is the
phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Here we analyze reports in the
literature indicating that these two pathways cooperate to regulate prostate
tumor development and progression. Studies show that AR transcriptional activity
and expression are regulated by Akt. In addition, androgens regulate the Akt
pathway by both genomic and non-genomic effects. This explains why prostate
tumors subjected to androgen ablation experience an increase in Akt
phosphorylation, and suggest that the tumor compensates for the loss of one
pathway with another. Different modes of interaction between the two pathways,
including direct interaction, or regulation via downstream intermediates, such
as the wnt/GSK-3beta/beta-catenin pathway, NF-kappaB, and the FOXO family of
transcription factors, will be discussed. In addition, we will discuss the role
of Akt in the interaction of the AR with upstream regulators of Akt
phosphorylation, such as receptor tyrosine kinases of the EGF and IGF-1 receptor
families and the tumor suppressor PTEN. The mechanisms of granulosa cell tumor (GCT) development may involve the
dysregulation of signaling pathways downstream of follicle-stimulating hormone,
including the phosphoinosite-3 kinase (PI3K)/AKT pathway. To test this
hypothesis, a genetically engineered mouse model was created to derepress the
PI3K/AKT pathway in granulosa cells by conditional targeting of the PI3K
antagonist gene Pten (Pten(flox/flox);Amhr2(cre/+)). The majority of
Pten(flox/flox);Amhr2(cre/+) mice featured no ovarian anomalies, but
occasionally ( approximately 7%) developed aggressive, anaplastic GCT with
pulmonary metastases. The expression of the PI3K/AKT downstream effector FOXO1
was abrogated in Pten(flox/flox);Amhr2(cre/+) GCT, indicating a mechanism by
which GCT cells may increase proliferation and evade apoptosis. To relate these
findings to spontaneously occurring GCT, analyses of PTEN and phospho-AKT
expression were performed on human and equine tumors. Although PTEN loss was not
detected, many GCT (2/5 human, 7/17 equine) featured abnormal nuclear or
perinuclear localization of phospho-AKT, suggestive of altered PI3K/AKT
activity. As inappropriate activation of WNT/CTNNB1 signaling causes late-onset
GCT development and cross talk between the PI3K/AKT and WNT/CTNNB1 pathways has
been reported, we tested whether these pathways could synergize in GCT.
Activation of both the PI3K/AKT and WNT/CTNNB1 pathways in the granulosa cells
of a mouse model (Pten(flox/flox);Ctnnb1(flox(ex3)/+);Amhr2(cre/+)) resulted in
the development of GCT similar to those observed in Pten(flox/flox);Amhr2(cre/+)
mice, but with 100% penetrance, perinatal onset, extremely rapid growth and the
ability to spread by seeding into the abdominal cavity. These data indicate a
synergistic effect of dysregulated PI3K/AKT and WNT/CTNNB1 signaling in the
development and progression of GCT and provide the first animal models for
metastatic GCT. Chibby (Cby) is an evolutionarily conserved antagonist of beta-catenin, a
central player of the canonical Wnt signaling pathway, which acts as a
transcriptional coactivator. Cby physically interacts with the C-terminal
activation domain of beta-catenin and blocks its transcriptional activation
potential through competition with DNA-binding Tcf/Lef transcription factors.
Our recent study revealed a second mechanism for Cby-mediated beta-catenin
inhibition in which Cby cooperates with 14-3-3 adaptor proteins to facilitate
nuclear export of beta-catenin, following phosphorylation of Cby by Akt kinase.
Therefore, our findings unravel a novel molecular mechanism regulating the
dynamic nucleo-cytoplasmic trafficking of beta-catenin and provide new insights
into the cross-talk between the Wnt and Akt signaling pathways. Here, we review
recent literature concerning Cby function and discuss our current understanding
of the relationship between Wnt and Akt signaling. Mutational activation of the phosphatidylinositol 3-kinase (PI3K) pathway occurs
in a wide variety of tumors, whereas activating Wnt pathway mutants are
predomitly found in colon cancer. Because GSK3 is a key component of both
pathways, it is widely assumed that active PI3K signaling feeds positively into
the Wnt pathway by protein kinase B (PKB)-mediatefd inhibition of GSK3. In
addition, PKB has been proposed to modulate the canonical Wnt signaling through
direct stabilization and nuclear localization of beta-catenin. Here, we show
that compartmentalization by Axin of GSK3 prohibits cross-talk between the PI3K
and Wnt pathways and that Wnt-mediated transcriptional activity is not modulated
by activation of the PI3K/PKB pathway. Aberrant Wnt/β-catenin signaling contributes to the development of many cancers,
including glial tumorigenesis. While cross talk between the Wnt/β-catenin and
PI3K/AKT signaling pathways has been proposed, the impact of PI3K/AKT inhibition
on β-catenin signaling in glioma remains unknown. In the present study, we
report decreased cell proliferation and invasive ability upon the
LY294002-induced inhibition of PI3K in both U251 and LN229 human glioblastoma
cells in vitro. Pharmacologic inhibition of PI3K resulted in the downregulation
of several members of the β-catenin pathway, including Fra-1, c-Myc, and cyclin
D1. Downregulation impacted β-catenin-mediated transcription, as LY294002
decreased β-catenin/TCF transcriptional activity, determined by the reporter
assay. Similar results were observed in vivo, as intratumoral injection of
LY294002 downregulated the expression of the components of the β-catenin pathway
and delayed tumor growth in nude mice harboring subcutaneous LN229 xenografts.
These results suggest that the PI3K/AKT signaling pathway regulates glioma cell
proliferation, in part via repression of the Wnt/β-catenin pathway. Disturbed Wnt signaling has been implicated in numerous diseases, including type
2 diabetes and the metabolic syndrome. In the present study, we have
investigated cross-talk between insulin and Wnt signaling pathways using
preadipocytes with and without knockdown of the Wnt co-receptors LRP5 and LRP6
and with and without knock-out of insulin and IGF-1 receptors. We find that Wnt
stimulation leads to phosphorylation of insulin signaling key mediators,
including Akt, GSK3β, and ERK1/2, although with a lower fold stimulation and
slower time course than observed for insulin. These Wnt effects are
insulin/IGF-1 receptor-dependent and are lost in insulin/IGF-1 receptor double
knock-out cells. Conversely, in LRP5 knockdown preadipocytes, insulin-induced
phosphorylation of IRS1, Akt, GSK3β, and ERK1/2 is highly reduced. This effect
is specific to insulin, as compared with IGF-1, stimulation and appears to be
due to an inducible interaction between LRP5 and the insulin receptor as
demonstrated by co-immunoprecipitation. These data demonstrate that Wnt and
insulin signaling pathways exhibit cross-talk at multiple levels. Wnt induces
phosphorylation of Akt, ERK1/2, and GSK3β, and this is dependent on
insulin/IGF-1 receptors. Insulin signaling also involves the Wnt co-receptor
LRP5, which has a positive effect on insulin signaling. Thus, altered Wnt and
LRP5 activity can serve as modifiers of insulin action and insulin resistance in
the pathophysiology of diabetes and metabolic syndrome. Emerging evidence indicates that human mesenchymal stem cells (hMSCs) can be
recruited to tumor sites, and affect the growth of human maligcies. However,
little is known about the underlying molecular mechanisms. Here, we observed the
effects of hMSCs on the human cholangiocarcinoma cell line, HCCC-9810, using an
animal transplantation model, and conditioned media from human umbilical
cord-derived mesenchymal stem cells (hUC-MSCs). Animal studies showed that
hUC-MSCs can inhibit the growth of cholangiocarcinoma xenograft tumors. In cell
culture, conditioned media from hUC-MSCs inhibited proliferation and induced
apoptosis of tumor cells in a dose- and time-dependent manner. The proliferation
inhibition rate increased from 6.21% to 49.86%, whereas the apoptosis rate
increased from 9.3% to 48.1% when HCCC-9810 cells were cultured with 50% hUC-MSC
conditioned media for 24 h. Immunoblot analysis showed that the expression of
phosphor-PDK1 (Ser241), phosphor-Akt (Ser 437 and Thr308), phosphorylated
glycogen synthase kinase 3β (phospho-GSK-3β(Ser9)), β-catenin, cyclin-D1, and
c-myc were down-regulated. We further demonstrated that CHIR99021, a GSK-3β
inhibitor reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells and
increased the expression of β-catenin. The GSK-3β activator, sodium
nitroprusside dehydrate (SNP), augmented the anti-tumor effects of hUC-MSCs and
decreased the expression of β-catenin. IGF-1 acted as an Akt activator, and also
reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells. All these
results suggest that hUC-MSCs could inhibit the maligt phenotype of HCCC-9810
human cholangiocarcinoma cell line. The cross-talk role of Wnt/β-catenin and
PI3K/Akt signaling pathway, with GSK-3β as the key enzyme bridging these
pathways, may contribute to the inhibition of cholangiocarcinoma cells by
hUC-MSCs. Obesity results in reduced differentiation potential of adipocytes leading to
adipose tissue insulin resistance. Elevated proinflammatory cytokines from
adipose tissue in obesity, such as TNFα have been implicated in the reduced
adipocyte differentiation. Other mediators of reduced adipocyte differentiation
include TGFβ and wnt proteins. Although some overlap exists in the signaling
cascades of the wnt and TGFβ pathways it is unknown if TGFβ or wnt proteins
reciprocally induce the expression of each other to maximize their biological
effects in adipocytes. Therefore, we investigated the possible involvement of
TGFβ signaling in wnt induced gene expression and vice versa in 3T3-L1
adipocyte. Effect of TGFβ and Wnt pathways on differentiation was studied in
preadipocytes induced to differentiate in the presence of Wnt3a or TGFβ1 and
their inhibitors (FZ8-CRD and SB431542, respectively). Regulation of
intracellular signaling and gene expression was also studied in mature
adipocytes. Our results show that both TGFβ1 and Wnt3a lead to increased
accumulation of β-catenin, phosphorylation of AKT and p44/42 MAPK. However,
differences were found in the pattern of gene expression induced by the two
proteins suggesting that distinct, but complex, signaling pathways are activated
by TGFβ and wnt proteins to independently regulate adipocyte function. Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling
are necessary to stimulate proliferative processes in adult human β-cells.
Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt
receptor, increases β-catenin nuclear translocation and β-cell proliferation but
results in lower insulin content. Our current goal was to engage canonical and
non-canonical Wnt signaling at the receptor level to significantly increase
human β-cell proliferation while maintaining a β-cell phenotype in intact
islets. We adopted a system that utilized conditioned medium from L cells that
expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition
we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA
inhibition) in these cultures. Treatment of intact human islets with L-WRN
conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold
in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased
human β-cell proliferation ∼20 fold above glucose alone. Only the combination of
L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial
proliferation. Transcriptome-wide gene expression profiling demonstrated that
L-WRN medium provoked robust changes in several signaling families, including
enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment
also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of
Akt. Importantly, glucose-stimulated insulin secretion and content were not
downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt
signaling at the receptor level by this method leads to necessary crosstalk
between multiple signaling pathways including activation of Akt, mTOR,
Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase
human β-cell proliferation while maintaining the β-cell phenotype. |
Has the protein GFP been used in transgenesis for live protein imaging? | Yes, the stable transgenesis of genes encoding functional or spatially localized proteins, fused to fluorescent proteins such as green fluorescent protein (GFP) or red fluorescent protein (RFP), is an extremely important research tool in cell and developmental biology. | BACKGROUND: Green Fluorescent Protein (GFP) is used extensively as a reporter
for transgene expression in Drosophila and other organisms. However, GFP has not
generally been used as a reporter for circadian patterns of gene expression, and
it has not previously been possible to correlate patterns of reporter expression
with 3D movement and behavior of transgenic animals.
RESULTS: We present a video tracking system that allows tissue-specific GFP
expression to be quantified and correlated with 3D animal movement in real time.
eyeless/Pax6 reporter expression had a 12 hr period that correlated with fly
activity levels. hsp70 and hsp22 gene reporters were induced during fly aging in
circadian patterns (24 hr and 18 hr periods, respectively), and spiked in the
hours preceding and overlapping the death of the animal. The phase of hsp gene
reporter expression relative to fly activity levels was different for each fly,
and remained the same throughout the life span.
CONCLUSION: These experiments demonstrate that GFP can readily be used to assay
longitudinally fly movement and tissue-specific patterns of gene expression. The
hsp22-GFP and hsp70-GFP expression patterns were found to reflect accurately the
endogenous gene expression patterns, including induction during aging and
circadian periodicity. The combination of these new tracking methods with the
hsp-GFP reporters revealed additional information, including a spike in hsp22
and hsp70 reporter expression preceding death, and an intriguing fly-to-fly
variability in the phase of hsp70 and hsp22 reporter expression patterns. These
methods allow specific temporal patterns of gene expression to be correlated
with temporal patterns of animal activity, behavior and mortality. BACKGROUND: Fluorescent proteins such as the green fluorescent protein (GFP)
have widely been used in transgenic animals as reporter genes. Their use in
transgenic Xenopus tadpoles is especially of interest, because large numbers of
living animals can easily be screened. To track more than one event in the same
animal, fluorescent markers that clearly differ in their emission spectrum are
needed.
RESULTS: We established the transgenic Xenopus laevis strain tom3 that expresses
ubiquitously red fluorescence from the tdTomato gene through all larval stages
and in the adult animal. This new tool was applied to track transplanted
blastemas obtained after tail amputation. The blastema can regenerate ectopic
tails marked by red fluorescence in the host animal. Surprisingly, we also found
contribution of the host animal to form the regenerate.
CONCLUSION: We have established a useful new tool to label grafts in Xenopus
transplantation experiments. Transgenic animals have been used for years to study gene function, produce
important proteins, and generate models for the study of human diseases.
However, inheritance and expression instability of the transgene in transgenic
animals is a major limitation. Copy number and promoter methylation are known to
regulate gene expression, but no report has systematically examined their effect
on transgene expression. In the study, we generated two transgenic pigs by
somatic cell nuclear transfer (SCNT) that express green fluorescent protein
(GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and
bisulfite sequencing were performed to determine transgene copy number and
promoter methylation level. The correlation of transgene expression with copy
number and promoter methylation was analyzed in individual development,
fibroblast cells, various tissues, and offspring of the transgenic pigs. Our
results demonstrate that transgene expression is associated with copy number and
CMV promoter methylation in transgenic pigs. The zebrafish provides a useful experimental system for investigations of aural
development. To permit the controlled expression of transgenes in developing
hair cells, we isolated the genomic control regions of the parvalbumin 3a
(pvalb3a) and parvalbumin 3b (pvalb3b) genes. Deletion analysis and somatic-cell
transgenesis restricted the cis-acting control regions for hair cells to as
little as 484base pairs for pvalb3a and 650base pairs for pvalb3b. Using both
meganuclease-mediated and standard methods, we produced transgenic animals that
transmit transgenes through their germ lines. These fish express GFP in hair
cells in the inner ear and lateral line. Two stable transgenic lines express GFP
prior to hair-bundle formation, so the associated promoter constructs are
suitable for manipulating gene expression during bundle development. We
additionally identified a transgenic line that offers variable labeling of
supporting cells. A screen was instigated to identify novel protein components of the
Caenorhabditis elegans sarcomere. The subcellular localisation of full-length
GFP fusion proteins was examined, in transgenic animals, for 62 essentially
uncharacterized genes thought to be expressed within bodywall muscle cells.
Three genes, T03G6.3, C46G7.2 and K04A8.6, were identified for further study.
K04A8.6::GFP only displayed a regular sarcomeric distribution sporadically.
However, C46G7.2::GFP localised to the centre of A-bands and dense bodies and
T03G6.3::GFP localised in the I-band, of the bodywall muscle sarcomeres,
consistently. This success with such a small screen suggests that there are
further minor components of the C. elegans sarcomere yet to be discovered.
Fluorescence Recovery After Photobleaching (FRAP) was applied to live transgenic
individuals to assess the mobility of T03G6.3 and C46G7.2 and other well-known
constituents of the sarcomere in vivo. Proteins associated with the thin
filaments showed dynamic exchange whilst those associated with thick filaments
appeared more static. This is the first demonstration that there are sarcomeric
proteins in C. elegans muscle cells in dynamic exchange and that the rates of
exchange in vivo correspond in general terms with observations in other
experimental systems. BACKGROUND: A major technical limitation in preclinical cell replacement
research is the ability to discriminate between donor and host tissue after
transplantation. This problem has been lessened by the availability of
transgenic animals that express "reporter" genes, such as green fluorescent
protein (GFP).
OBJECTIVE: We determined the usefulness of one such transgenic reporter rat to
assess the survival of bone marrow-derived rat mesenchymal stem cells (MSCs)
following direct transplantation into the intact adult brain. We also sought to
determine if the expression of GFP in the brain affected the survival of the
MSCs or the host's neuroimmune response to the cells.
METHODS: Rats received intrastriatal injections of sterile transplantation
medium, 100 000 normal MSCs, or 100 000 GFP-MSCs and were killed humanely 1, 4,
7, 28, and 42 days posttransplantation for astrocyte and microglial
immunohistochemical staining.
RESULTS: GFP-MSCs were evident at each examination, although their survival
declined over time. Graft volume estimates comparing normal and GFP-MSCs
revealed that GFP expression did not adversely affect the survival of the stem
cells in the brain. Furthermore, immunostaining for astrocytes and microglia
revealed that expression of the reporter protein did not affect the
immunogenicity of the stem cells.
CONCLUSIONS: These data indicate the usefulness of GFP for investigating the
survival of MSCs following transplantation to the brain. However, the mechanisms
responsible for the poor survival of the stem cells must be elucidated if these
cells are to serve cell-based therapies for neurodegenerative disorders. The creation of transgenic animals is widely utilized in C. elegans research
including the use of GFP fusion proteins to study the regulation and expression
pattern of genes of interest or generation of tandem affinity purification (TAP)
tagged versions of specific genes to facilitate their purification. Typically
transgenes are generated by placing a promoter upstream of a GFP reporter gene
or cDNA of interest, and this often produces a representative expression
pattern. However, critical elements of gene regulation, such as control elements
in the 3' untranslated region or alternative promoters, could be missed by this
approach. Further only a single splice variant can be usually studied by this
means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones
likely includes most if not all elements involved in gene regulation in vivo
which permits the greater ability to capture the genuine expression pattern and
timing. To facilitate the generation of transgenes using fosmid DNA, we describe
an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other
sequences of interest into any location in the gene. The procedure uses the galK
gene as the selection marker for both the positive and negative selection steps
in recombineering which results in obtaining the desired modification with high
efficiency. Further, plasmids containing the galK gene flanked by homology arms
to commonly used GFP and TAP fusion genes are available which reduce the cost of
oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use
the R6K replication origin which precludes the need for extensive PCR product
purification. Finally, we also demonstrate a technique to integrate the unc-119
marker on to the fosmid backbone which allows the fosmid to be directly injected
or bombarded into worms to generate transgenic animals. This video demonstrates
the procedures involved in generating a transgene via recombineering using this
method. Lentiviral vectors containing the green fluorescent protein gene have been
successfully used to select transgenic embryos before transfer to a surrogate
mother. However, there are apparently no reports regarding early detection of
transgenic embryos using a lentiviral vector carrying an additional
transcription unit for tissue-specific expression of a valuable protein. In this
study, two HIV-based lentiviral vectors were constructed. The first one
contained the green fluorescent protein (GFP) coding sequence driven by the
early SV40 promoter (Lv-G), whereas the other contained an additional
transcription unit for the expression of E2 glycoprotein from classical swine
fever virus, driven by a 1.5 kb αS1casein promoter from water buffalo
(Lv-αS1cE2hisG). Microinjection of single-cell mouse embryos with Lv-G
lentiviral vector rendered embryos which were GFP-positive, beginning at the
four-cell stage. Of 33 mice born, 28 (81%) carried the transgene DNA and 15
(55.5%) were GFP-positive. Microinjection of Lv-αS1cE2hisG lentiviral vector
yielded 28 mice born; although 24 (85%) carried the transgene DNA, none were
GFP-positive, suggesting that the tissue-specific expression cassette interfered
with expression of the ubiquitous trancriptional unit. In Lv-αS1cE2hisG
transgenic mice, E2his was expressed in milk as a homodimer (at concentrations ≤
0.422 mg/mL). This was apparently the first report of expression of a
recombit protein in the milk of transgenic animals generated by lentiviral
transgenesis. The evolutionarily conserved immune system of the zebrafish (Danio rerio), in
combination with its genetic tractability, position it as an excellent model
system in which to elucidate the origin and function of vertebrate immune cells.
We recently reported the existence of antigen-presenting mononuclear phagocytes
in zebrafish, namely macrophages and dendritic cells (DCs), but have been
impaired in further characterizing the biology of these cells by the lack of a
specific transgenic reporter line. Using regulatory elements of a class II major
histocompatibility gene, we generated a zebrafish reporter line expressing green
fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes.
Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated
that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature
monocytes/macrophages and DCs, as revealed by morphologic and molecular
analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but
were most abundant in the intestine and spleen, where they up-regulate the
expression of inflammatory cytokines upon bacterial challenge. Finally,
mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in
the lymphoid fraction, enabling the delineation of the major hematopoietic
lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and
cd45:DsRed transgenic lines will be instrumental in elucidating the immune
response in the zebrafish. There is much interest in using farm animals as 'bioreactors' to produce large
quantities of biopharmaceuticals. However, uncontrolled constitutive expression
of foreign genes have been known to cause serious physiological disturbances in
transgenic animals. The objective of this study was to test the feasibility of
the controllable expression of an exogenous gene in the chicken. A retrovirus
vector was designed to express GFP (green fluorescent protein) and rtTA (reverse
tetracycline-controlled transactivator) under the control of the
tetracycline-inducible promoter and the PGK (phosphoglycerate kinase) promoter,
respectively. G0 founder chickens were produced by infecting the blastoderm of
freshly laid eggs with concentrated retrovirus vector. Feeding the chickens
obtained with doxycycline, a tetracycline derivative, resulted in emission of
green body color under fluorescent light, and no apparent significant
physiological dysfunctions. Successful germline transmission of the exogenous
gene was also confirmed. Expression of the GFP gene reverted to the
pre-induction levels when doxycycline was removed from the diet. The results
showed that a tetracycline-inducible expression system in transgenic animals
might be a promising solution to minimize physiological disturbances caused by
the transgene. Since large animal transgenesis has been successfully attempted for the first
time about 25 years ago, the technology has been applied in various lines of
transgenic pigs. Nevertheless one of the concerns with the technology--animal
welfare--has not been approached through systematic assessment and statements
regarding the welfare of transgenic pigs have been based on anecdotal
observations during early stages of transgenic programs. The main aim of the
present study was therefore to perform an extensive welfare assessment comparing
heterozygous transgenic animals expressing GFP with wildtype animals along
various stages of post natal development. The protocol used covered reproductory
performance and behaviour in GFP and wildtype sows and general health and
development, social behaviour, exploratory behaviour and emotionality in GFP and
wildtype littermates from birth until an age of roughly 4 months. The absence of
significant differences between GFP and wildtype animals in the parameters
observed suggests that the transgenic animals in question are unlikely to suffer
from deleterious effects of transgene expression on their welfare and thus
support existing anecdotal observations of pigs expressing GFP as healthy.
Although the results are not surprising in the light of previous experience,
they give a more solid fundament to the evaluation of GFP expression as being
relatively non-invasive in pigs. The present study may furthermore serve as
starting point for researchers aiming at a systematic characterization of
welfare relevant effects in the line of transgenic pigs they are working with. The ability to specify the expression levels of exogenous genes inserted in the
genomes of transgenic animals is critical for the success of a wide variety of
experimental manipulations. Protein production can be regulated at the level of
transcription, mRNA transport, mRNA half-life, or translation efficiency. In
this report, we show that several well-characterized sequence elements derived
from plant and insect viruses are able to function in Drosophila to increase the
apparent translational efficiency of mRNAs by as much as 20-fold. These
increases render expression levels sufficient for genetic constructs previously
requiring multiple copies to be effective in single copy, including constructs
expressing the temperature-sensitive inactivator of neuronal function
Shibire(ts1), and for the use of cytoplasmic GFP to image the fine processes of
neurons. BACKGROUND: Porcine endogenous retroviruses (PERVs) represent a risk of
xenotransplantation using porcine cells, tissues, or organs, as they are
integrated in the porcine genome and have been shown to be able to infect human
cells in vitro. To increase viral safety by RNA interference, transgenic pigs
expressing a PERV-specific small hairpin (sh)RNA targeted to a highly conserved
sequence in the pol gene (pol2) were generated in which expression of PERVs was
reduced (Xenotransplantation, 15, 2008, 38). However, it remains to be shown how
long expression of the shRNA and the RNA interference is effective in reducing
PERV expression.
METHODS: To analyze the long-term duration of RNA interference, expression of
the PERV-specific pol2 shRNA and inhibition of PERV expression was studied
repeatedly in fibroblasts and peripheral blood mononuclear cells (PBMCs) of
transgenic pigs over a period of 3 yr, when animals were sacrificed and
expression was studied in different organs. Expression of the PERV-specific
shRNA was measured using a newly developed real-time PCR, and expression of PERV
was measured using a PERV-specific real-time PCR.
RESULTS: Over a period of 3 yr, PERV-specific shRNA and green fluorescent
protein (GFP) as reporter of the vector system were consistently expressed in
transgenic animals. PERV expression was significantly reduced during the entire
period. Levels of PERV and shRNA expression were different in the various
organs. PERV expression was highest in the spleen and the lungs and lowest in
liver and heart. However, in all organs of the transgenic pigs, PERV expression
was inhibited compared with the vector control animals.
CONCLUSIONS: Transgenic pigs expressing PERV-specific shRNA maintained their
specific RNA interference long term, suggesting that PERV expression in the
xenotransplants will be suppressed over extended periods of time. AIM: To examine the feasibility of using the pOBCol3.6GFPtpz [3.6-green
fluorescent protein (GFP)] transgenic mice as an in vivo model for studying the
biological sequence of events during pulp healing and reparative dentinogenesis.
METHODOLOGY: Pulp exposures were created in the first maxillary molar of
12-16-week-old 3.6-GFP transgenic mice with CD1 and C57/Bl6 genetic background.
Direct pulp capping on exposed teeth was performed using mineral trioxide
aggregate followed by restoration with a light-cured adhesive system (AS) and
composite resin. In control teeth, the AS was placed in direct contact with the
pulp. Animals were euthanized at various time points after pulp exposure and
capping. The maxillary arch was isolated, fixed and processed for histological
and epifluorescence analysis to examine reparative dentinogenesis.
RESULTS: Analysis of teeth immediately after pulp exposure revealed absence of
odontoblasts expressing 3.6-GFP at the injury site. Evidence of reparative
dentinogenesis was apparent at 4 weeks in 3.6-GFP mice in CD1 background and at
8 weeks in 3.6-GFP mice with C57/Bl6 background. The reparative dentine with
both groups contained newly formed atubular-mineralized tissue resembling a
dentine bridge and/or osteodentine that was lined by cells expressing 3.6-GFP as
well as 3.6-GFP expressing cells embedded within the atubular matrix.
CONCLUSION: This study was conducted in a few animals and did not allow
statistical analysis. The results revealed that the 3.6-GFP transgenic animals
provide a unique model for direct analysis of cellular and molecular mechanisms
of pulp repair and tertiary dentinogenesis in vivo. The study also shows the
effects of the capping material and the genetic background of the mice in the
sequence and timing of reparative dentinogenesis. Specifically, gene-encoded biological probes serve as stable and
high-performance tools to visualize cellular fate in living animals. The rat, as
with the mouse, has offered important animal models for biology and medical
research, and has provided a wealth of physiological and pharmacological data.
The larger-body animals, in comparison to the mouse have allowed the application
of various physiological and surgical manipulations that may prove to have
biological significance. We have further extended the techniques of genetic
engineering to rats, rabbits, and pigs, and have created corresponding
GFP-transgenic animals. The GFP-positive organs of these animals provide
valuable sensors in preclinical settings for cell therapy and transplantation
studies. In this chapter, we highlight expression profiles in these animal
resources and describe examples of preclinical applications. The stable transgenesis of genes encoding functional or spatially localized
proteins, fused to fluorescent proteins such as green fluorescent protein (GFP)
or red fluorescent protein (RFP), is an extremely important research tool in
cell and developmental biology. Transgenic organisms constructed with
fluorescent labels for cell membranes, subcellular organelles, and functional
proteins have been used to investigate cell cycles, lineages, shapes, and
polarity, in live animals and in cells or tissues derived from these animals.
Genes of interest have been integrated and maintained in generations of
transgenic animals, which have become a valuable resource for the cell and
developmental biology communities. Although the use of Xenopus laevis as a
transgenic model organism has been hampered by its relatively long reproduction
time (compared to Drosophila melanogaster and Caenorhabditis elegans), its large
embryonic cells and the ease of manipulation in early embryos have made it a
historically valuable preparation that continues to have tremendous research
potential. Here, we report on the Xenopus laevis transgenic lines our lab has
generated and discuss their potential use in biological imaging. Transgenic technologies conventionally rely on the oocyte as a substrate for
genetic modification. Owing to their accessibility, however, male germ cells,
including mature sperm, have material advantages for use in transgenesis. Here
we have exploited lentiviruses to generate transgenic animals via the male
germline. When pseudotyped lentiviral vectors encoding green fluorescent protein
(GFP) were incubated with mouse spermatozoa, these sperm were highly successful
in producing transgenics. Lentivirally transduced mouse spermatozoa were used in
in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥
42% of founders were found to be transgenic for GFP. Inverse PCR strategy for
integration site analysis demonstrated integration of at least 1 or 2 copies of
GFP in the transgenics, mapping to different chromosomes. GFP expression was
detected in a wide range of murine tissues, including testis and the transgene
was stably transmitted to a third generation of transgenic animals. This
relatively simple, yet highly efficient, technique for generating transgenic
animals by transducing spermatozoa with lentiviral vectors in vitro is a
powerful tool for the study of fertilization/preimplantation development,
vertical viral gene transmission, gene function and regulation, and epigenetic
inheritance. Accumulating evidence suggests that up to three different molecular species of
GnRH peptides encoded by different paralogs of gnrh genes are expressed by
anatomically distinct groups of GnRH neurons in the brain of one vertebrate
species. They are called gnrh1, gnrh2, and gnrh3. Recent evidence from
molecular, anatomical, and physiological experiments strongly suggests that each
GnRH system functions differently. Here, we review recent advancement in the
functional studies of the three different GnRH neuron systems, mainly focusing
on the electrophysiological analysis of the GnRH-green fluorescent protein (GFP)
transgenic animals. The introduction of GFP-transgenic animals for the
electrophysiological analysis of GnRH neurons greatly advanced our knowledge on
their anatomy and electrophysiology, especially of gnrh1 neurons, which has long
defied detailed electrophysiological analysis of single neurons because of their
small size and scattered distribution. Based on the results of recent studies,
we propose that different electrophysiological properties, especially the
spontaneous patterns of electrical activities and their time-dependent changes,
and the axonal projections characterize the different functions of GnRH1-3
neurons; GnRH1 neurons act as hypophysiotropic neuroendocrine regulators, and
GnRH2 and GnRH3 neurons act as neuromodulators in wide areas of the brain. |
Is the Snord116 cluster associated with the Prader-Willi syndrome? | Yes, SNORD116 has a major role in Prader-Willi syndrome etiology. | Prader-Willi syndrome (PWS) is a neurobehavioral disorder manifested by
infantile hypotonia and feeding difficulties in infancy, followed by morbid
obesity secondary to hyperphagia. It is caused by deficiency of paternally
expressed transcript(s) within the human chromosome region 15q11.2. PWS patients
harboring balanced chromosomal translocations with breakpoints within small
nuclear ribonucleoprotein polypeptide N (SNRPN) have provided indirect evidence
for a role for the imprinted C/D box containing small nucleolar RNA (snoRNA)
genes encoded downstream of SNRPN. In addition, recently published data provide
strong evidence in support of a role for the snoRNA SNORD116 cluster (HBII-85)
in PWS etiology. In this study, we performed detailed phenotypic, cytogenetic,
and molecular analyses including chromosome analysis, array comparative genomic
hybridization (array CGH), expression studies, and single-nucleotide
polymorphism (SNP) genotyping for parent-of-origin determination of the 15q11.2
microdeletion on an 11-year-old child expressing the major components of the PWS
phenotype. This child had an ∼236.29 kb microdeletion at 15q11.2 within the
larger Prader-Willi/Angelman syndrome critical region that included the SNORD116
cluster of snoRNAs. Analysis of SNP genotypes in proband and mother provided
evidence in support of the deletion being on the paternal chromosome 15. This
child also met most of the major PWS diagnostic criteria including infantile
hypotonia, early-onset morbid obesity, and hypogonadism. Identification and
characterization of this case provide unequivocal evidence for a critical role
for the SNORD116 snoRNA molecules in PWS pathogenesis. Array CGH testing for
genomic copy-number changes in cases with complex phenotypes is proving to be
invaluable in detecting novel alterations and enabling better genotype-phenotype
correlations. 1. PURPOSE: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are complex
neurodevelopmental disorders caused by loss of expression of imprinted genes
from the 15q11-q13 region depending on the parent of origin.
Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA)
kits from MRC-Holland (Amsterdam, The Netherlands) were used to detect PWS and
AS deletion subtypes. We report our experience with two versions of the
MS-MLPA-PWS/AS kit (original A1 and newer B1) in determining methylation status
and deletion subtypes in individuals with PWS.
METHODS: MS-MLPA analysis was performed on DNA isolated from a large cohort of
PWS subjects with the MS-MLPA-PWS/AS-A1 and -B1 probe sets.
RESULTS: Both MS-MLPA kits will identify deletions in the 15q11-q13 region but
the original MS-MLPA-A1 kit has a higher density of probes at the telomeric end
of the 15q11-q13 region, which is more useful for identifying individuals with
atypical deletions. The newer B1 kit contains more probes in the imprinting
center (IC) and adjoining small noncoding RNAs useful in identifying small
microdeletions.
CONCLUSION: The A1 kit identified the typical deletions and smaller atypical
deletions, whereas the B1 kit was more informative for identifying
microdeletions including the IC and SNORD116 regions. Both kits should be made
available for accurate characterization of PWS/AS deletion subtypes as well as
evaluating for IC and SNORD116 microdeletions. Prader-Willi syndrome is characterized by severe infantile hypotonia with poor
suck and failure to thrive; hypogonadism causing genital hypoplasia and pubertal
insufficiency; characteristic facial features; early-childhood onset obesity and
hyperphagia; developmental delay/mild intellectual disability; short stature;
and a distinctive behavioral phenotype. Sleep abnormalities and scoliosis are
common. Growth hormone insufficiency is frequent, and replacement therapy
provides improvement in growth, body composition, and physical attributes.
Management is otherwise largely supportive. Consensus clinical diagnostic
criteria exist, but diagnosis should be confirmed through genetic testing.
Prader-Willi syndrome is due to absence of paternally expressed imprinted genes
at 15q11.2-q13 through paternal deletion of this region (65-75% of individuals),
maternal uniparental disomy 15 (20-30%), or an imprinting defect (1-3%).
Parent-specific DNA methylation analysis will detect >99% of individuals.
However, additional genetic studies are necessary to identify the molecular
class. There are multiple imprinted genes in this region, the loss of which
contribute to the complete phenotype of Prader-Willi syndrome. However, absence
of a small nucleolar organizing RNA gene, SNORD116, seems to reproduce many of
the clinical features. Sibling recurrence risk is typically <1%, but higher
risks may pertain in certain cases. Prenatal diagnosis is available. The imprinted Snurf-Snrpn chromosomal domain contains two large arrays of
tandemly repeated, paternally expressed box C/D small-nucleolar RNA (snoRNA)
genes: the SNORD115 (H/MBII-52) and SNORD116 (H/MBII-85) gene clusters believed
to play key roles in the fine-tuning of serotonin receptor (5-HT2C) pre-mRNA
processing and in the etiology of the Prader-Willi Syndrome (PWS), respectively.
SNORD115 and SNORD116 were recently proposed to undergo significant conversion
into shorter RNA species, the so-called psnoRNAs. Here, we provide evidence that
argues against the existence of abundant psnoRNAs in human or mouse brain.
Instead, we characterize a previously unsuspected low-abundance,
fibrillarin-associated SNORD115-derived smaller RNA species. Based on these
findings, we strongly recommend that PWS-encoded SNORD115 and SNORD116 be
considered as bona fide box C/D snoRNAs. The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of
imprinted genes expressed from the paternal chromosome only. Whereas loss of
function of the SNORD116 genes appears to be responsible for the major features
of PWS, the role of the other genes is less clear. One of these genes is
C15orf2, which has no orthologues in rodents, but appears to be under strong
positive selection in primates. C15orf2 encodes a 1156 amino acid protein with
six nuclear localisation sequences. By protein BLAST analysis and InterProScan
signature recognition search, we found sequence similarity of C15orf2 to the
nuclear pore complex (NPC) protein POM121. To determine whether C15orf2 is
located at nuclear pores, we generated a stable cell line that inducibly
expresses FLAG-tagged C15orf2 and performed immunocytochemical studies. We found
that C15orf2 is present at the nuclear periphery, where it colocalizes with NPCs
and nuclear lamins. At very high expression levels, we observed invaginations of
the nuclear envelope. Extending these observations to three-dimensional
structured illumination microscopy, which achieves an 8-fold improved volumetric
resolution over conventional imaging, we saw that C15orf2 is located at the
inner face of the nuclear envelope where it strongly associates with the NPC. In
nuclear envelope isolation and fractionation experiments, we detected C15orf2 in
the NPC and lamina fractions. These experiments for the first time demonstrate
that C15orf2 is part of the NPC or its associated molecular networks. Based on
our findings, we propose 'Nuclear pore associated protein 1' as the new name for
C15orf2. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted
autism-spectrum disorders with known genetic bases, but complex epigenetic
mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is
regulated by an imprinting control region that is maternally methylated and
silenced. The PWS imprinting control region is the promoter for a one megabase
paternal transcript encoding the ubiquitous protein-coding Snrpn gene and
multiple neuron-specific noncoding RNAs, including the PWS-related Snord116
repetitive locus of small nucleolar RNAs and host genes, and the antisense
transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS).
Neuron-specific transcriptional progression through Ube3a-ATS correlates with
paternal Ube3a silencing and chromatin decondensation. Interestingly,
topoisomerase inhibitors, including topotecan, were recently identified in an
unbiased drug screen for compounds that could reverse the silent paternal allele
of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus
is unknown. Here, we demonstrate that topotecan treatment stabilizes the
formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within
paternal Snord116, corresponding to increased chromatin decondensation and
inhibition of Ube3a-ATS expression. Neural precursor cells from paternal
Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated
neurons and show a reduced effect of topotecan compared with wild-type neurons.
These results demonstrate that the AS candidate drug topotecan acts
predomitly through stabilizing R loops and chromatin decondensation at the
paternally expressed PWS Snord116 locus. Our study holds promise for targeted
therapies to the Snord116 locus for both AS and PWS. Genetic analyses were performed in a male patient with suspected Prader-Willi
syndrome who presented with hypogonadism, excessive eating, central obesity,
small hands and feet and cognition within the low normal range. However, he had
no neonatal hypotonia or feeding problems during infancy. Chromosome analysis
showed a normal male karyotype. Further analysis with array-CGH identified a
mosaic 847 kb deletion in 15q11-q13, including SNURF-SNRPN, the snoRNA gene
clusters SNORD116 (HBII-85), SNORD115, (HBII-52), SNORD109 A and B (HBII-438A
and B), SNORD64 (HBII-13), and NPAP1 (C15ORF2). MLPA confirmed the deletion and
the results were compatible with a paternal origin. Metaphase-FISH verified the
mosaicism with the deletion present in 58% of leukocytes analyzed. Three smaller
deletions in this region have previously been reported in patients with
Prader-Willi syndrome phenotype. All three deletions included SNORD116, but only
two encompassed parts of SNURF-SNRPN, implicating SNORD116 as the major
contributor to the Prader-Willi phenotype. Our case adds further information
about genotype-phenotype correlation and supports the hypothesis that SNORD116
plays a major role in the pathogenesis of Prader-Willi syndrome. Furthermore, it
examplifies diagnostic difficulties in atypical cases and illustrates the need
for additional testing methods when Prader-Willi syndrome is suspected. |
Aleglitazar is agonist of which receptor? | Aleglitazar is a balanced peroxisome proliferator-activated receptor-α/γ agonist. | This multicenter, randomized, double-blind, placebo-controlled, ascending-dose
study investigated the pharmacokinetics, pharmacodynamic effects, safety, and
tolerability of aleglitazar, a novel peroxisome proliferator-activated receptor
alpha/gamma (PPARalpha/gamma) dual agonist. After a 3-week washout period, 71
patients with type 2 diabetes received either a single oral dose of aleglitazar
(20, 50, 100, 300, 600, or 900 microg) or placebo, followed by once-daily dosing
for 6 weeks. Few adverse events were reported, with no apparent relationship
between the rate of incidence or severity of the adverse events and the dose of
aleglitazar administered. Aleglitazar exposure increased in a dose-proportional
manner both after a single dose and at steady state, with no accumulation.
Aleglitazar produced dose-dependent improvements in levels of fasting and
postprandial glucose, insulin resistance, and lipid parameters. Dose-dependent
decreases from baseline in creatinine clearance exceeded 10% at doses >300
microg. The PPARalpha- and PPARgamma-related effects occurred over similar dose
ranges, indicating that aleglitazar is a balanced agonist of the two receptor
subtypes. BACKGROUND: Aleglitazar, a dual PPAR-α/γ agonist, combines the lipid benefits of
fibrates and the insulin-sensitizing benefits of thiazolidinediones.
OBJECTIVE: To investigate the pharmacokinetic effects of co-administration of
atorvastatin or rosuvastatin with aleglitazar.
RESEARCH DESIGN AND METHODS: In a two-cohort, open-label, randomised,
three-period crossover study, 44 healthy subjects received once-daily oral doses
of aleglitazar 300 μg, statin (atorvastatin 80 mg or rosuvastatin 40 mg) and
aleglitazar co-administered with each statin for 7 days. Plasma concentrations
of each drug were measured and pharmacokinetic parameters determined on day 7 in
each period.
MAIN OUTCOME MEASURES: Peak observed plasma concentration (C(max)) and total
exposures (AUC(0 - 24)) of aleglitazar, atorvastatin and rosuvastatin.
RESULTS: C(max) and AUC(0 - 24) to aleglitazar were similar, whether
administered alone or in combination with a statin. Total exposure to either
statin was unaffected by co-administration with aleglitazar. C(max) treatment
ratios for both statins exceeded the conventional no-effect boundary (1.25) when
administered with aleglitazar.
CONCLUSIONS: Co-administration of aleglitazar with a statin does not alter the
pharmacokinetic profile of either drug. BACKGROUND: Glycemic control and management of dyslipidemia to reduce
cardiovascular risk are major therapeutic goals in individuals with type 2
diabetes mellitus (T2DM). This study was performed to evaluate the effects of
aleglitazar, a balanced dual peroxisome proliferator-activated receptor α/γ
(PPARα/γ) agonist, on both lipid and glycemic parameters in obese,
hypertriglyceridemic, insulin-resistant rhesus monkeys.
METHODS: A 135-day efficacy study was performed in six rhesus monkeys. After a
28-day baseline assessment (vehicle only), monkeys received oral aleglitazar
0.03 mg/kg per day for 42 days, followed by a 63-day washout period. Plasma
levels of markers of glycemic and lipid regulation were measured at baseline, at
the end of the dosing period, and at the end of the washout period.
RESULTS: Compared with baseline values, aleglitazar 0.03 mg/kg per day reduced
triglyceride levels by an average of 89% (328 to 36 mg/dL; P = 0.0035 when
normalized for baseline levels) and increased high-density lipoprotein
cholesterol levels by 125% (46 to 102 mg/dL; P = 0.0007). Furthermore,
aleglitazar reduced low-density lipoprotein cholesterol levels (41%) and
increased levels of apolipoprotein A-I (17%) and A-II (17%). Aleglitazar also
improved insulin sensitivity by 60% (P = 0.001). Mean body weight was reduced by
5.9% from baseline values with aleglitazar at this dose (P = 0.043).
CONCLUSIONS: Aleglitazar, a dual PPARα/γ agonist, has beneficial effects on both
lipid and glucose parameters and may have a therapeutic role in modifying
cardiovascular risk factors and improving glycemic control in patients with
T2DM. BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) regulate
transcription of genes involved in glucose uptake, lipid metabolism, and
inflammation. Aleglitazar is a potent dual PPAR agonist with insulin-sensitizing
and glucose-lowering actions and favorable effects on lipid profiles and
biomarkers of cardiovascular risk. The AleCardio trial examines whether the
addition of aleglitazar to standard medical therapy reduces the risk of
cardiovascular morbidity and mortality in patients with type 2 diabetes mellitus
and recent acute coronary syndrome.
STUDY DESIGN: AleCardio is a phase 3, multicenter, randomized, double-blind,
placebo-controlled trial. A total of 7,228 patients were randomized to
aleglitazar 150 μg or placebo daily in addition to standard medical therapy. The
primary efficacy end point is time to the first event of cardiovascular death,
myocardial infarction, or stroke. Principal safety end points are
hospitalization due to heart failure and changes in renal function. Treatment
will continue until 7,000 patients are followed up for at least 2.5 years and
950 primary end point events are adjudicated.
CONCLUSIONS: AleCardio will establish whether the PPAR-α/γ agonist aleglitazar
improves cardiovascular outcomes in patients with diabetes and high-risk
coronary disease. : Aleglitazar acts through balanced activation of peroxisome
proliferator-activated receptors α and γ; warfarin is a commonly prescribed
anticoagulant. Given the extent of cardiovascular disease in patients with type
2 diabetes, cotreatment with aleglitazar and warfarin is likely in this
population. This open-label, randomized, 2-period, crossover study in 12 healthy
male subjects investigated the potential for drug-drug interactions between
warfarin and aleglitazar (final data drawn from 11 white subjects). The primary
objective was to investigate the effect of aleglitazar on the pharmacokinetic
properties of S-warfarin and on the pharmacodynamics of the racemic mixture; the
secondary objectives included the effect of aleglitazar on R-warfarin
pharmacokinetics and of racemic warfarin on aleglitazar pharmacokinetics.
Subjects were randomized to single-dose warfarin on day 1 or aleglitazar once
daily (12 days) plus single-dose warfarin on day 6 followed by a 14-day washout
period, then crossover. Coadministration of aleglitazar reduced S- and
R-warfarin exposure (AUC0-∞) by 18% and 13%, respectively, but did not change
its pharmacodynamic effects (prothrombin time and factor VII activity). After
warfarin dosing, aleglitazar trough concentrations remained within the same
range. These findings indicate that coadministration of aleglitazar and warfarin
is unlikely to affect the efficacy or safety of either agent. Collaborators: Vico ML, Conde DG, Botta CE, Gelersztein E, Ramos H, Prado AD,
Piombo A, Piredda A, Lorente C, Canella JP, Guzman P, Avaca H, Maffei LE, Vita
NA, Gorosito V, Cosman C, Caccavo A, Piskorz D, Jure HO, Ferdez RA, Toledo
SS, Nul D, Marino JC, Estrada JL, Sztejfman L, Amerena J, Arnolda L, Yadav R,
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D, Baril JF, Hatheway RJ, Roy A, Lavoie JP, Leung R, Tremblay G, Pouliot J, Hill
L, Labonte R, Lavi S, Bergin P, Pesant Y, Phaneuf DC, Nogareda G, Smith S, Huynh
T, Rolfe A, Conway J, Bertrand O, Chehayeb R, Pandey S, Petrella R, Lepage S,
Nawaz S, Johri A, Abramson B, Fay D, Searles G, Chouinard G, Luterman M, Zadra
R, Welsh R, Vizel S, Leduc C, Nault P, Beaudry P, Bakbak A, Shabani F,
Berlingieri J, Yang X, Zhao R, Peng J, Hu H, Luo M, Ke Y, Yuan Z, Zhang Y, Lv X,
Ma C, Ma G, Chen J, Li X, Li Z, Yan X, Gan X, Chen J, Zhao S, Chen Y, Li H, Li
L, Huimin L, Guo Y, Hu T, Fang W, Wang X, Yang K, Bao X, Fu G, Li H, Yan J, Wang
D, He B, Zhang F, Han Y, Liu J, Wei M, Ma H, Wu S, Li W, Shi H, Monhart Z, Solar
M, Belohlavek J, Ferkl R, Kral R, Hutyra M, Novak M, Kettner J, Podpera I, Malik
J, May O, Gohr T, Clemmensen P, Lomholdt J, Verges M, Agraou B, Montalescot G,
Probst V, Galinier M, Roudaut R, Tropeano AI, Ovize M, Elhadad S, Belhassane A,
Dhoudain F, Schiele F, Danchin N, Dutoiu T, Caussin C, Cottin Y, Münzel T,
Voeller H, Neumann FJ, Edel K, Kamke W, vom Dahl J, Ali T, Schächinger V, Natour
M, Kadel C, Löw A, Hamm C, Düngen HD, Eckert S, Hoffmann U, von Hodenberg E,
Werner N, Laufs U, Voehringer HF, Reinecke H, Mügge A, Fleck E, Reuter H, Hensen
J, Bergmann M, Tschoepe C, Kuehne U, Proskynitopoulos N, Michalko P, Kelm M,
Edes I, Koranyi L, Papp A, Vertes A, Horvath I, Lupkovics G, Deak L, Benczur B,
Sziliczei-Nemeth E, Hodi G, Csapo K, Dudas M, Somogyi A, Czuriga I, Nagy L,
Parikh K, Sethi BK, Sahay RK, Premchand R, Bhattacharya A, Hiremath J, Ghosh S,
Jawahirani A, Sharma K, Gadkari M, Dash A, Dani S, Kumar S, Khanna P, Deshpande
N, Kumar H, Arneja J, Setthuraman S, Chowdhury S, Kumble Y, Jali M, Yerra S,
Gandhi P, Mehrotra S, Bantwal G, Basavanagowdappa H, Kannampilly J, Pathanakayil
R, Malpani G, Prasad G, Sarna M, Kapoor D, Adhikari P, Hardas S, Byrapaneni R,
Suvarna T, Barton J, McAdam B, MacNeill BD, Maher VM, Sugrue D, Crean PA,
Grassia V, Morocutti G, Tortorella G, Cosentino F, Bramucci E, Filardi PP,
Mafrici A, Salvioni A, Ansani L, Pancaldi L, De Servi S, Cavallini C, Zanini R,
Musumeci G, Roncon L, Moon KW, Shin ES, Kim HY, Jeong JO, Cha TJ, Kim DK, Jeon
HK, Lim do S, Yoon JH, Park KS, Kang WC, Jeong MH, Lee NH, Kim SH, Park WJ, Hur
SH, Park CG, Kim HS, Park JS, Tahk SJ, Lee SY, Chae JK, Kim SW, Kim SJ, Jeon DW,
Hyon MS, Hong TJ, Ong TK, Ahmad WA, Ismail O, Ng KH, Mohamed M, Yahya M, Ali RM,
Ghapar AK, Cervantes J, Gambe MA, Castillo AG, Riojas C, Benavides M, Llamas G,
Pons JL, Rosas EL, Ramos G, De Los Rios M, Violante R, Mendoza E, Ruiz LN,
Lechuga A, Alva JC, Cedeño R, Herdez J, Cantu RQ, Ferdez LE, Velasco R,
Lopez AG, Cardona E, Chavez J, Alpizar M, Arechavaleta R, Micher D, Bayram E,
Sánchez RR, Herrera CH, Guerrero HG, Isunza JR, Sanchez H, Munoz L, Garza MG,
Malpica EM, Hof AV, Herrman JP, Jukema JW, Bronzwaer P, Ten Holt W, ten Berg J,
De Winter R, Basart D, de Graaf J, Gerdes VE, Bokern M, Troughton R, Fisher N,
Wong S, Tang EW, Nirmalaraj KB, O'Meeghan T, Nowak J, Kusnierz B, Pieniazek P,
Drozdz D, Zarebinski M, Ponikowski P, Kosmider M, Musial W, Zabowka M,
Hamankiewicz M, Derlaga B, Piepiorka M, Kuc K, Janion M, Kuzniar J, Wierzykowski
T, Serafin R, Kochman J, Pijanowski Z, Jaworska K, Szpajer M, Miekus P, Cwetsch
A, Ochala A, Konieczny M, Hoffmann A, Kowalski J, Walczewska J,
Krzeminska-Pakula M, Buszman P, Bryniarski L, Ogorek M, Dluzniewski M, Buszman
P, Sciborski R, Miarka J, Hiczkiewicz J, Kleinrok A, Wysokinski A, Janiak B,
Szczuka K, Lesiak M, Dryja T, Rynkiewicz A, Szwed H, Gorski J, Olszewski M,
Buszman P, Benedek IS, Popa AR, Militaru C, Morosanu M, Constantinescu S,
Tivadar S, Popescu A, Radoi M, Fruntelata AG, Veresiu IA, Dragulescu SI, Eryshev
S, Baranova E, Yakovlev A, Gordeev I, Gorelov A, Lopez MG, Pinto X, Coronado JB,
Muñoz CP, Acuna JG, Soriano FR, Navarro MJ, Sanz RR, Basilio EG, Cortada JB, Ros
JA, Macaya CM, Juanatey CG, Torrent AJ, Recena JB, Botas J, Ferdez PA,
Alegret J, Moll XG, Sanz E, Blázquez JC, Soldevila JG, Murga N, Bellido D, Plaza
I, Mellbin L, Boberg G, Mooe T, Tyden P, Linder R, Luostarinen R, Axelkvist M,
Pettersson T, Boman K, Jidbratt H, Lindgren M, Johanson P, Wongvipaporn C,
Kuanprasert S, Srimahachota S, Siriwattana K, Ngamjanyaporn P, Tresukosol D,
Promlikitchai P, Chotnoparatpat P, Taweesangsuksakul P, Thongsri T, Wongtheptian
W, Sansanayudh N, Hutayanon P, Laksomya T, Sritara P, Price D, Aggarwal R,
Purcell I, Davies C, Malik I, Violaris A, Game F, Harvey J, Robertson D, Kadr H,
Trouton TG, Kaprielian R, Dutka D, Hildick-Smith D, Butler R, MacRury S,
Millward BA, Purvis J, Reckless J, Ajjan R, Bruce D, Cox D, Malik I, Kooner J,
Muir S, Hutchinson S, Gupta D, Weinstein D, Bieniarz M, Traina M, Lieber I,
Mohart J, Prodafikas J, Laurion D, Oberoi M, Carlson E, Schmedtje J, Mayfield R,
Chandler G, Modi K, Budoff M, Zelman R, Dalton R, Lee K, Gordon P, Ayenew W,
Mathis C, Busch R, Schuchard T, Raisinghani A, Shepherd AM, Jaffrani N, Lee PV,
Chandrashekhar Y, Mohammed A, Weisz G, Almassi H, Krantzler J, Quadrel M, Martin
S, Brener S, Harris B, Eaton C, Nalluri C, Schwartz G, Gogia H, Colfer H,
Doherty J, Dang N, Blonder R, Jacobson S, Rogers W Jr, Levin E, Isa G, Chandna
H, Singh J, Forgosh L, Melucci M, Chilton R, El Hafi S, Way B, Lambert C, Korban
E, Auerbach E, Hickey K, McKenzie M, Tandon N, Mahal S, Heitner S, O'Halloran D,
Loh IK, Harris J, Gelormini J, Clavijo L, Klapholz M, Studeny M, Goswami R,
Norris R, Busui RP, Cox SL, Tak T, A T, Taghizadeh T, Pompili V, Camp A,
Khera A, Shah A, Frey A, Zakhary B, Humiston D, Griffin D, Herrington D, Perloff
D, Kereiakes D, Christofides E, Dippel E, Fung G, Marais H, Dotani I, Fidelholtz
J, Hermiller J, Naidu J, Gilbert J, El-Shahawy M, Mandviwala M, Rastogi P,
Breaux P, Norwood P, Staab P, Fish R, Sanchez R, Gupta V, Herzog W, Rabinowitz
A, Samal A, Kabour A, Lee D, Carlos E, Kosinski E, Portnay E, Rivera E, Madu IJ,
Welker J, Fialkow J, Londono J, Martinez L, Pirwitz M, Ariani M, Saklayen M,
Vijay N, Feldman R, Rosenson R, Steinhubl S, Srinivasan V, Nair V, Shalev Y,
Bouchard A, Adolphe A, Miller A, Ahmad A, Omar B, Masri B, Iteld B, Gifford C,
Scott C, Maislos F, Longo J, Rider J, Silverstein J, Miller M, Nanna M, Khan M,
Schneider R, Bashir R, Evans R, Fierer R, Teniola S, Welka S, Singh V, Randall
W, Rowe W, Salacata A, Bazzi A, Steinberg A, Mooss A, Tang A, Kahn B, Chandler
B, Bayron C, Schmalfuss C, Hirsch C, Thompson C, Thompson C, Singal D, Lo E,
Spivack E, Gopalakrish G, Lowe J, Talano J, Albu J, McClure J, McGettigan J,
Hemphill J, Vora K, Solano Mdel P, Shoukfeh M, Best P, Thompson P, Borromeo S
3rd, Barringer T, Hilton T, Knickelbine T, Palmer W. BACKGROUND: Insulin-resistant states, including type 2 diabetes (T2D) and
prediabetes, are associated with elevated cardiovascular (CV) risk. Aleglitazar
is a dual peroxisome proliferator-activated receptor α/γ agonist with favorable
insulin-sensitizing and glucose-lowering actions, favorable effects on blood
lipids, and an acceptable safety profile in short-time studies. Therefore, it
was hypothesized that aleglitazar would reduce CV morbidity and mortality in
patients with T2D mellitus and prediabetes (defined as glycosylated hemoglobin
≥5.7% to <6.5%) with previous CV complications.
STUDY DESIGN: ALEPREVENT was a phase III, multicenter, randomized, double-blind,
trial comparing aleglitazar 150 μg or placebo daily in patients with T2D or
prediabetes with established, stable CV disease. The intended sample size was
19,000 with a primary efficacy measure of major adverse CV events. However, the
trial was halted prematurely after 1,999 patients had been randomized because of
futility and an unfavorable benefit risk ratio in another CV outcomes trial
evaluating aleglitazar.
RESULTS: At study termination after 58 ± 38 days of treatment, data had been
collected from 1,996 patients (1,581 with T2D and 415 with pre-T2D). Despite the
brief duration of treatment, aleglitazar induced favorable changes in
glycosylated hemoglobin and blood lipids, similar for participants with T2D or
prediabetes. However, compared with placebo, aleglitazar increased the incidence
of hypoglycemia (86 vs 166; P < .0001), and muscular events (3 vs12; P = .012).
CONCLUSIONS: Even within a short duration of exposure, aleglitazar was
associated with excess adverse events, corroborating the findings of a larger
and longer trial in T2D. Coupled with the previous failure of several other
peroxisome proliferator-activated receptor α/γ activators, this class now holds
little promise for CV therapeutics. |
What are the main characteristics of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)? | Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias induced by physical or emotional stress, in the absence structural heart abnormalities. The phenotype of CPVT is characterized by polymorphic ventricular arrhythmias under stress and it potentially leads to syncope and/or sudden cardiac death (SCD). Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a lethal, rare hereditary disease with an estimated prevalence of 1:10 000 and is caused by mutations in proteins controlling Ca(2+) homeostasis. To date, about 189 variants in 5 genes (RYR2, CASQ2, CALM1, TRND, and KCNJ2) have been associated with CPVT pathogenesis. | BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a
severe inherited cardiac disorder caused by mutations predomitly in the
ryanodine receptor (RyR2) gene. We sought to identify mutations in genes
affecting cardiac calcium cycling in patients with CPVT and in less typical
familial exercise-related ventricular arrhythmias.
METHODS AND RESULTS: We recruited 33 consecutive patients with frequent
ventricular premature complexes (VPCs) without structural heart disease and
often history of syncope or sudden death in family. Sixteen of the patients
featured a phenotype typical of CPVT. In 17 patients, VPCs emerged also at rest.
Exercise stress test and echocardiography were performed to each patient and 232
family members. Familial background was evident in 42% of cases (n = 14). We
sequenced all the coding exons of the RyR2, FKBP1B, ATP2A2 and SLC8A1 genes from
the index patients. Single channel recordings of a mutant RyR2 were performed in
planar lipid bilayers. Two novel RyR2 missense mutations (R1051P and S616L) and
two RyR2 exon 3 deletions were identified, explaining 25% of the CPVT
phenotypes. A rare variant (N3308S) with open probabilities similar to the wild
type channels in vitro, was evident in a patient with resting VPCs. No
disease-causing variants were detectable in the FKBP1B, ATP2A2 or SLC8A1 genes.
CONCLUSION: We report two novel CPVT-causing RyR2 mutations and a novel RyR2
variant of uncertain clinical significance in a patient with abundant resting
VPCs. Our data also strengthen the previous assumption that exon 3 deletions of
RyR2 should screened for in CPVT and related phenotypes. BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a
primary electrical myocardial disease characterized by exercise- and
stress-related ventricular lachycardia manifested as syncope and sudden death
usually in child and teenager and was rarely described in adults. The management
includes betablockade, with the use of implantable cardioverter defibrillators
if medical treatment is insufficient.
AIM: Report a new case of CPVT.
OBSERVATION: We report a case of a 43 years old patient in whom CPVT diagnosis
was made during his exploration for palpitations occurring with the effort.
Registration Holter ECG revealed several episodes of supraventricular
tachycardia and episodes of nocturnal sino-atrial block. The patient had an ICD
and betablockade treatment.
CONCLUSION: The TVPC in adult can manifest with attenuated symptoms that can be
summarized with palpitations with the exertion. The supraventricular arrhythmias
and sinus dysfunction may be at the forefront of Electrocardiographic
manifestations. The prognosis of this form seems better than the TVPC of the
child. Treatment with betablockade appears to be effective but existing
dysfunction sinus facilitates decision to implant the ICD. Catecholaminergic polymorphic ventricular tachycardia (PCVT) is a rare,
congenital ventricular tachyarrhythmia which occurs in the setting of adrenergic
activation. It potentially leads to syncope and/or sudden cardiac death (SCD).
PCVT represents one of the most dangerous congenital ion channel diseases.
Mutations of the ryanodine receptor gene (RYR2), the calsequestrin gene (CASQ2),
and the triadin gene (TRDN) have been identified as an underlying correlate.
β-Blockers are employed as therapy and are sometimes combined with class IC
antiarrhythmic drugs, or calcium antagonists of the verapamil type. ICD
implantation is recommended in case of persisting syncope in the presence of
β-blocker therapy or survived SCD. Left thoracic sympathectomy represents a
subsidiary interventional therapy for individual cases. In addition,
modifications of the patient's lifestyle including avoidance of physical stress
and heart rates> 120/min are recommended. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
arrhythmogenic cardiac disorder characterized by life-threatening arrhythmias
induced by physical or emotional stress, in the absence structural heart
abnormalities. The arrhythmias may cause syncope or degenerate into cardiac
arrest and sudden death which usually occurs during childhood. Recent studies
have shown that CPVT is caused by mutations in the cardiac ryanodine receptor
type 2 (RyR2) or calsequestrin 2 (CASQ2) genes. Both proteins are key
contributors to the intracellular Ca(2+) handling process and play a pivotal
role in Ca(2+) release from the sarcoplasmic reticulum to the cytosol during
systole. Although the molecular pathogenesis of CPVT is not entirely clear, it
was suggested that the CPVT mutations promote excessive sarcoplasmic reticulum
Ca(2+) leak, which initiates delayed afterdepolarizations (DADs) and triggered
arrhythmias in cardiac myocytes. The recent breakthrough discovery of induced
pluripotent stem cells (iPSC) generated from somatic cells (e.g. fibroblasts,
keratinocytes) now enables researches to investigate mutated cardiomyocytes
generated from the patient's iPSC. To this end, in the present article we review
recent studies on CPVT iPSC-derived cardiomyocytes, thus demonstrating in the
mutated cells catecholamine-induced DADs and triggered arrhythmias. BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a
lethal, rare hereditary disease with an estimated prevalence of 1:10 000. The
genetic variants that cause CPVT are usually highly penetrant. To date, about
189 variants in 5 genes (RYR2, CASQ2, CALM1, TRND, and KCNJ2) have been
associated with CPVT pathogenesis.
METHODS AND RESULTS: The Exome Sequencing Project database (ESP; n=6503) was
systematically searched for previously published missense and nonsense
CPVT-associated variants reported in several comprehensive reviews and in 2
databases: The Human Gene Mutation Database and The Inherited Arrhythmias
Database. We used 4 different prediction tools to assess all missense variants
previously associated with CPVT and compared the prediction of protein damage
between CPVT-associated variants identified in the ESP and those variants not
identified in the ESP. We identified 11% of the variants previously associated
with CPVT in the ESP population. In the literature, 57% of these variants were
reported as novel disease-causing variants absent in the healthy control
subjects. These putative CPVT variants were identified in 41 out of 6131
subjects in the ESP population, corresponding to a prevalence of CPVT of up to
1:150. Using an agreement of ≥3, in silico prediction tools showed a
significantly higher frequency of damaging variants among the CPVT-associated
variants not identified in the ESP database (83%) compared with those variants
identified in the ESP (50%; P=0.021).
CONCLUSIONS: We identified a substantial overrepresentation of CPVT-associated
variants in a large exome database, suggesting that these variants are not
necessarily the monogenic cause of CPVT. Author information:
(1)Department of Cardiology, Japan Self-Defense Forces Central Hospital, Ikejiri
1-2-24, Setagaya-ku, Tokyo, 154-8532, Japan. [email protected].
(2)Department of Cardiology, KKR Mishuku Hospital, Tokyo, Japan.
[email protected].
(3)Department of Cardiology, Japan Self-Defense Forces Central Hospital, Ikejiri
1-2-24, Setagaya-ku, Tokyo, 154-8532, Japan.
(4)Department of Cardiology, KKR Mishuku Hospital, Tokyo, Japan.
(5)Department of Cardiovascular Medicine, National Cerebral and Cardiovascular
Center, Osaka, Japan.
(6)Laboratory of Molecular Genetics, National Cerebral and Cardiovascular
Center, Osaka, Japan.
(7)Department of Cardiovascular Medicine, Nippon Medical School, Tokyo, Japan. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited
heart rhythm disorder characterized by the occurrence of potentially
life-threatening polymorphic ventricular tachyarrhythmias in conditions of
physical or emotional stress. The underlying cause is a dysregulation in
intracellular Ca handling due to mutations in the sarcoplasmic reticulum Ca
release unit. Recent experimental work suggests that sinus bradycardia, which is
sometimes observed in CPVT patients, may be another primary defect caused by
CPVT mutations. Herein, we review the pathophysiology of CPVT and discuss the
role of sinus node dysfunction as a modulator of arrhythmia risk and potential
therapeutic target. INTRODUCTION AND OBJECTIVES: Catecholaminergic polymorphic ventricular
tachycardia is a maligt disease, due to mutations in proteins controlling
Ca(2+) homeostasis. While the phenotype is characterized by polymorphic
ventricular arrhythmias under stress, supraventricular arrhythmias may occur and
are not fully characterized.
METHODS: Twenty-five relatives from a Spanish family with several sudden deaths
were evaluated with electrocardiogram, exercise testing, and optional
epinephrine challenge. Selective RyR2 sequencing in an affected individual and
cascade screening in the rest of the family was offered. The RyR2(R420Q)
mutation was generated in HEK-293 cells using site-directed mutagenesis to
conduct in vitro functional studies.
RESULTS: The exercise testing unmasked catecholaminergic polymorphic ventricular
tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%;
negative predictive value = 93%), all of them carrying the heterozygous
RyR2(R420Q) mutation, which was also present in the proband and a young girl
without exercise testing, a 91% penetrance at the end of the follow-up.
Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant
post-effort U-waves were identified in patients. Upon permeabilization and in
intact cells, the RyR2(R420Q) expressing cells showed a smaller peak of Ca(2+)
release than RyR2 wild-type cells. However, at physiologic intracellular Ca(2+)
concentration, equivalent to the diastolic cytosolic concentration, the
RyR2(R420Q) released more Ca(2+) and oscillated faster than RyR2 wild-type
cells.
CONCLUSIONS: The missense RyR2(R420Q) mutation was identified in the N-terminus
of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is
associated with sinus bradycardia, atrial and junctional arrhythmias, and giant
U-waves. Collectively, functional heterologous expression studies suggest that
the RyR2(R420Q) behaves as an aberrant channel, as a loss- or gain-of-function
mutation depending on cytosolic intracellular Ca(2+) concentration. |
Is poly (ADP- ribosylation) involved in transcriptional control? | Yes, poly (ADP- ribosylation) plays a role in the maintenance of transcriptional fidelity. | Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is an abundant nuclear
protein that is involved in DNA repair, cell cycle control, programmed cell
death and transcriptional regulation. It also plays critical roles in the
pathogenesis of inflammatory disorders. Here we have performed a detailed
analysis of the interplay between the apicomplexan parasite Toxoplasma gondii
and host cell PARP and its consequences for the host-parasite relationship. Our
results have shown that T. gondii significantly decreased PARP expression in its
host cells within 10min of infection but that the amount of PARP normalized
during prolonged infection. Importantly, down-regulation of PARP expression
after infection abrogated the ADP-ribosylation of acceptor proteins in response
to oxidative stress. Overexpression of PARP in RAW264.7 cells revealed that
elevated amounts of PARP neither affected host cell invasion nor intracellular
development of T. gondii in non-stimulated or IFN-gamma/LPS-stimulated
monocytes/macrophages. Furthermore, measurements of the activities of effector
caspases 3 and 7 indicated that the blockade of host cell apoptosis by T. gondii
occurs independently of the inhibition of PARP after infection. These findings
suggest that the prominent decrease of host cell PARP and poly(ADP-ribos)ylation
after parasitic infection do not affect the intracellular development of T.
gondii in vitro. |
Is nimodipine recommended for prevention of vasospasm in aneurysmal subarachnoid hemorrhage patients? | Yes, nimodipine is recommended and FDA approved for prevention of vasospasm after aneurysmal subarachnoid hemorrhage. Multiple studies have demonstrated that nimodipine (administered orally or intravenously) is safe and effective for prevention of vasospasm, and reduce frequency of ischemic complications, lowered mortality and improved overall outcomes. Other preventive medication can be also effective for vasomotor prevention in aneurysmal subarachnoid hemorrhage patients. | Only 53%-58% of patients with a subarachnoid haemorrhage (SAB) following the
rupture of a cerebral aneurysm survive without neurological damage. Morbidity
and mortality are closely related to the delayed ischaemic neurological deficit
due to cerebral vasospasm. The following review gives an account of
pathophysiological mechanisms; the importance of treatment with calcium
antagonists, hypervolaemic haemodilution, and induced arterial hypertension is
discussed in light of the current literature. PATHOPHYSIOLOGY. In addition to
other vasoactive substances in the blood, haemoglobin, which is released from
lysed erythrocytes on the 2nd to 4th day after the haemorrhage, plays an
important role in inducing vasospasm. An inflammatory angiopathy ensues, with
complete resolution after 6-12 weeks. The cerebral blood flow (CBF) is reduced
depending on the extent of vasospasm. Irreversible infarction may follow the
decrease of CBF below a critical value. Severe vasospasm causes autoregulatory
disturbances and reduced responsiveness of cerebral vessels to CO2. CALCIUM
ANTAGONISTS. The calcium blocker nimodipine causes dilatation of small pial
vessels with increased CBF. However, systemic vasodilation with the subsequent
fall in blood pressure may limit the increase in CBF. Furthermore, it is known
that nimodipine decreases intracellular calcium concentrations resulting in some
protection against ischaemic cellular injury. Seven placebo-controlled clinical
studies have shown that nimodipine improves the outcome of patients with severe
neurological damage due to cerebral vasospasm. HYPERVOLAEMIC HAEMODILUTION.
Volume expansion and haemodilution to a hematocrit of 30%-33% is suggested to
improve cerebral perfusion during vasospasm. The central venous and pulmonary
capillary wedge pressures should be 10-12 mm Hg and 15-18 mm Hg, respectively.
But there is no evidence of improved outcome with this measure, and pulmonary
edema is a frequent side effect. However, impairment of cerebral perfusion and
increased neurological damage can be demonstrated with hypovolaemia and
haemoconcentration. INDUCED ARTERIAL HYPERTENSION. In the presence of cerebral
vasospasm and resulting autoregulatory disturbances, cerebral perfusion can be
increased by raising systemic arterial pressure. This measure, too, fails to
improve neurological outcome. CONCLUSION. Treatment of cerebral vasospasm
following a SAB aims to avoid any impairment of cerebral perfusion. Hypovolaemia
and haemoconcentration have to be corrected. Normoventilation should be
established to avoid hypocapnic vasoconstriction. Nimodipine should be
administered continuously after a SAB. In view of the autoregulatory
disturbances, systemic hypotension with its danger of decreased CBF must be
prevented. The importance of hypervolaemic haemodilution and/or induced arterial
hypertension is not clear. Despite therapeutic efforts, the number of patients
who have survived a SAB without a substantial neurological deficit has not
increased. OBJECTIVE: To determine the current recovery rates and causes for morbidity and
mortality in patients suffering aneurysmal subarachnoid hemorrhage (SAH).
METHODS: We reviewed a recent consecutive series of 95 patients with ruptured
intracranial aneurysms who presented to our hospital between 1994 and 1995. When
administered, active treatment consisted of early surgery for aneurysm clipping
and aggressive prevention and treatment of SAH-related complications.
RESULTS: Eighty-eight (93%) of the patients were admitted within 24 hours of
rupture. One-quarter of the patients in this series did not undergo aneurysm
clipping due to poor neurological condition on presentation. Of the 75 patients
initially considered for active treatment, 83% underwent surgery within 48 hours
of rupture, all received nimodipine, 16% received tissue plasminogen activator
to lyse subarachnoid or intraventricular clots, 40% underwent hypertensive
treatment, and 7% underwent transluminal balloon angioplasty for vasospasm. At
one year followup, 29% of patients had died, 7% had severe disabilities, 13% had
moderate disabilities, 51% had made a good recovery, and 64% of all surviving
patients had returned to their previous work status. Primary and contributing
causes of death and disability, affecting 47 patients at one year, were: direct
effects of the initial hemorrhage (79% of affected patients), surgical
complications (13%), vasopasm (11%), rebleeding (11%) and medical complications
(13%).
CONCLUSIONS: Almost two-thirds of patients suffering aneurysm rupture make a
satisfactory recovery with modern treatment. While vasospasm has become a less
common cause of poor outcome following SAH, surgical complications remain an
important problem. The efficacy and safety of fasudil hydrochloride, a novel protein kinase
inhibitor, were evaluated for the treatment of cerebral vasospasm and associated
cerebral ischemic symptoms in patients with ruptured cerebral aneurysm. This
randomized open trial with nimodipine as the control included 72 patients who
underwent subarachnoid hemorrhage surgery for ruptured cerebral aneurysm of Hunt
and Hess grades I to IV. For 14 days following surgery, patients were
administered either 30 mg of fasudil hydrochloride by intravenous injection over
a period of 30 minutes three times a day or 1 mg/hr of nimodipine by continuous
intravenous infusion. Fasudil hydrochloride and nimodipine both showed
inhibitory effects on cerebral vasospasm. The incidence of symptomatic vasospasm
was five of 33 patients in the fasudil group and nine of 32 patients in the
nimodipine group. Good recovery evaluated by the Glasgow Outcome Scale was
achieved by 23 of 33 patients in the fasudil group and 19 of 34 patients in the
nimodipine group. Both drugs significantly improved consciousness levels and
neurological deficits such as aphasia. However, fasudil hydrochloride improved
motor disturbance more than nimodipine. Adverse reactions occurred in 13 of 37
patients receiving fasudil hydrochloride and 15 of 35 patients receiving
nimodipine. There were no serious adverse events in the fasudil group. The
results of this clinical trial indicate that fasudil hydrochloride is a safe and
efficient agent for suppressing cerebral vasospasm after subarachnoid hemorrhage
surgery for ruptured cerebral aneurysm. Cerebral vasospasm is the classic cause of delayed neurological deterioration
after aneurysmal subarachnoid hemorrhage, leading to cerebral ischemia and
infarction, and thus to poor outcome and occasionally death. Advances in
diagnosis and treatment-principally the use of nimodipine, intensive care
management, hemodynamic manipulations and endovascular neuroradiology
procedures-have improved the prospects for these patients, but outcomes remain
disappointing. Recent clinical trials have demonstrated marked prevention of
vasospasm with the endothelin receptor antagonist clazosentan, yet patient
outcome was not improved. This Review considers possible explanations for this
result and proposes alternative causes of neurological deterioration and poor
outcome after subarachnoid hemorrhage, including delayed effects of global
cerebral ischemia, thromboembolism, microcirculatory dysfunction and cortical
spreading depression. Vasospasm is one of the leading causes of morbidity and mortality following
aneurysmal subarachnoid hemorrhage (SAH). Radiographic vasospasm usually
develops between 5 and 15 days after the initial hemorrhage, and is associated
with clinically apparent delayed ischemic neurological deficits (DID) in
one-third of patients. The pathophysiology of this reversible vasculopathy is
not fully understood but appears to involve structural changes and biochemical
alterations at the levels of the vascular endothelium and smooth muscle cells.
Blood in the subarachnoid space is believed to trigger these changes. In
addition, cerebral perfusion may be concurrently impaired by hypovolemia and
impaired cerebral autoregulatory function. The combined effects of these
processes can lead to reduction in cerebral blood flow so severe as to cause
ischemia leading to infarction. Diagnosis is made by some combination of
clinical, cerebral angiographic, and transcranial doppler ultrasonographic
factors. Nimodipine, a calcium channel antagonist, is so far the only available
therapy with proven benefit for reducing the impact of DID. Aggressive therapy
combining hemodynamic augmentation, transluminal balloon angioplasty, and
intra-arterial infusion of vasodilator drugs is, to varying degrees, usually
implemented. A panoply of drugs, with different mechanisms of action, has been
studied in SAH related vasospasm. Currently, the most promising are magnesium
sulfate, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, nitric oxide
donors and endothelin-1 antagonists. This paper reviews established and emerging
therapies for vasospasm. Cerebral vasospasm is the classic cause of delayed neurological deterioration
leading to cerebral ischemia and infarction, and thus, poor outcome and
occasionally death, after aneurysmal subarachnoid hemorrhage (SAH). Advances in
diagnosis and treatment, principally nimodipine, intensive care management,
hemodynamic manipulations, and endovascular neuroradiology procedures, have
improved the prospects for these patients, but outcomes remain disappointing. A
phase 2b clinical trial (CONSCIOUS-1) demonstrated marked prevention of
vasospasm with the endothelin antagonist, clazosentan, yet patient outcome was
not improved. The most likely explanation is that the study was underpowered to
detect the relatively small improvements in outcome that would be seen with
prevention of vasospasm, especially when assessed using relatively insensitive
measures such as the modified Rankin and Glasgow outcome scales. Other possible
explanations for this result are that adverse effects of treatment affected the
beneficial effects of the drug. It also is possible that alternative causes of
neurological deterioration and poor outcome after SAH, including delayed effects
of acute global cerebral ischemia, thromboembolism, microcirculatory
dysfunction, and cortical spreading depression, play a role. Clazosentan reduced
angiographic vasospasm in a dose-dependent manner in patients with aneurysmal
SAH following coiling or clipping of the aneurysm. Reducing the incidence of
vasospasm should have an important effect on clinical outcome. A phase 3
clinical trial (CONSCIOUS-2) will focus on quantifying this outcome in patients
undergoing aneurysm clipping receiving placebo or 5 mg/h of clazosentan. Fasudil is believed to be at least equally effective as nimodipine for the
prevention of cerebral vasospasm and subsequent ischemic injury in patients
undergoing surgery for subarachnoid hemorrhage (SAH). We report the final
results of a randomized, open trial to compare the efficacy and safety of
fasudil with nimodipine. A total of 63 patients undergoing surgery for SAH
received fasudil and 66 received nimodipine between 1998 and 2004. Symptomatic
vasospasm, low density areas on computed tomography (CT), clinical outcomes, and
adverse events were all recorded, and the results were compared between the
fasudil and nimodipine groups. Absence of symptomatic vasospasm, occurrence of
low density areas associated with vasospasm on CT, and occurrence of adverse
events were similar between the two groups. The clinical outcomes were more
favorable in the fasudil group than in the nimodipine group (p = 0.040). The
proportion of patients with good clinical outcome was 74.5% (41/55) in the
fasudil group and 61.7% (37/60) in the nimodipine group. There were no serious
adverse events reported in the fasudil group. The present results suggest that
fasudil is equally or more effective than nimodipine for the prevention of
cerebral vasospasm and subsequent ischemic injury in patients undergoing surgery
for SAH. OBJECTIVE: Cerebral vasospasm is a major source of morbidity and mortality
following aneurysmal subarachnoid hemorrhage (SAH). A variety of therapies have
been utilized to prevent or treat vasospasm. Despite the large number of
clinical trials, few randomized controlled trials (RCTs) of sufficient quality
have been published. We review the RCTs and meta-analyses in the literature
regarding the treatment and prevention of cerebral vasospasm following
aneurysmal SAH.
METHODS: A literature search of MEDLINE, the Cochrane Controlled Trials
Registry, and the National Institutes of Health/National Library of Medicine
clinical trials registry was performed in January 2010 using predefined search
terms. These trials were critically reviewed and categorized based on
therapeutic modality.
RESULTS: Forty-four RCTs and 9 meta-analyses met the search criteria.
Significant findings from these trials were analyzed. The results of this study
were as follows: nimodipine demonstrated benefit following aneurysmal SAH; other
calcium channel blockers, including nicardipine, do not provide unequivocal
benefit; triple-H therapy, fasudil, transluminal balloon angioplasty,
thrombolytics, endothelin receptor antagonists, magnesium, statins, and
miscellaneous therapies such as free radical scavengers and antifibrinolytics
require additional study. Tirilazad is ineffective.
CONCLUSIONS: There are many possible successful treatment options for preventing
vasospasm, delayed ischemic neurologic deficits, and poor neurologic outcome
following aneurysmal subarachnoid hemorrhage; however, further multicenter RCTs
need to be performed to determine if there is a significant benefit from their
use. Nimodipine is the only treatment that provided a significant benefit across
multiple studies. |
what is the role of prostaglandins in cardiac regenaration after myocardial infarction? | Prostaglandins are involved in tissue regeneration after myocardial infarction and inhibitors of prostanoid production, such as aspirin and COX-2 inhibitors, have negative impact in this process. Furthermore, pharmacological interference with prostaglandin synthesis following myocardial infarction is associated with reduced fibrillar collagen formation. | Bradykinin and prostaglandins are established mediators of exudative and
inflammatory phases of healing. Their contribution to the fibrogenic component
of healing in the heart is less certain. We therefore undertook the present
study in rats with acute myocardial infarction (MI) following left coronary
artery ligation. Treatment with a bradykinin B2 receptor antagonist (Hoe140, 0.5
microgram/kg/min s.c.) or a cyclooxygenase inhibitor (indomethacin, 2 mg/kg
p.o.), initiated 24 h after surgery, was examined for responses in MI topography
(size and area), MI and nonMI tissue fibrosis (fibrillar collagen specific
picrosirius red). Early (week 1) and late (week 4) phases of fibrogenesis postMI
were examined. Compared to control, we found: (1) MI size at weeks 1 and 4 was
comparable in untreated and treated rats: (2) infarct area, a measure of scar
thickness, was reduced (P < 0.05) at week 4 by each intervention; and (3)
densitometric collagen volume fraction did not reveal a reduction in collagen
accumulation at the MI site, but this was evident remote to the MI (P < 0.05) at
week 4 for each agent. Thus, pharmacological interference with
bradykinin-receptor binding or prostaglandin synthesis following MI is
associated with reduced fibrillar collagen formation. Though the mechanism
responsible for observed alteration in fibrogenesis is uncertain,
anti-inflammatory and anti-proliferative properties of these agents may be
responsible. INTRODUCTION: An emerging technology using human embryonic stem cells (hESCs) to
regenerate infarcted heart tissue has been underdeveloped. However, because
non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, are taken
during the infarction, it becomes critical to know whether the NSAIDs have
negative impacts on heart tissue regeneration when using hESCs.
METHODS: Mass spectrometry (LC/MS/MS) and high performance liquid chromatography
(HPLC) analyses were used to analyze the functional presence of the elaborate
prostanoids' biosynthesis and signaling systems in hESCs. The detected
endogenous arachidonic acid (AA) released in the hESC membranes reflects the
activity of phospholipase which directly controls the biosyntheses of the
prostanoids.
RESULTS: The complete inhibition of the endogenous prostaglandin E(2) (PGE(2))
biosynthesis by the cyclooxygenase-2 (COX-2) inhibitor, NS398, confirmed that
the major prostanoids synthesized in the hESCs are mediated by the COX-2 enzyme.
We also found that PGE(2) and the prostacyclin (PGI(2)) metabolite,
6-keto-PGF(1α), are present in the undifferentiated hESCs.
CONCLUSION: This indicated different cyclooxygenase (COX)-downstream synthases
and metabolizing enzymes are involved in the AA products' signaling through the
COX-1 and COX-2 pathways. The presence of many enzymes' and receptors' [(COX-1,
COX-2, microsomal prostaglandin E synthase (mPGES), cytosolic prostaglandin E
synthase (cPGES), prostaglandin I synthase (PGIS), the PGE(2) subtype receptors
(EP(1), EP(2), and EP(4)) and the prostacyclin receptor (IP)] involvement in the
prostanoid biosynthesis and activity was confirmed by western blot. The studies
implied the negative effects of NSAIDs, such as aspirin and COX-2 inhibitors,
which suppress prostanoid production during tissue regeneration for infarcted
heart when using hESCs. |
What is the general function of H3K79 methylation? | ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. | The gene expression pattern of differentiated oocytes is reprogrammed into that
of totipotent preimplantation embryos before and/or after fertilization. To
elucidate the mechanisms of genome reprogramming, we investigated histone H3
lysine 79 dimethylation (H3K79me2) and trimethylation (H3K79me3) in oocytes and
preimplantation embryos via immunocytochemistry. In somatic cells and oocytes,
H3K79me2 was observed throughout the genome, whereas H3K79me3 was localized in
the pericentromeric heterochromatin regions in which there are no active genes.
Because H3K79me2 is considered an active gene marker, H3K79 methylation seems to
have differing functions depending on the number of methyl groups added on the
same residues. Both H3K79me2 and H3K79me3 decreased soon after fertilization,
and the hypomethylated state was maintained at interphase (before the blastocyst
stage), except for a transient increase in H3K79me2 at mitosis (M phase).
H3K79me3 was not detected throughout preimplantation, even at M phase. To
investigate the involvement of H3K79me2 in genome reprogramming, somatic nuclei
were transplanted into enucleated oocytes. H3K79me2 in these nuclei was
demethylated following parthenogenetic activation. However, the nuclei that had
been transplanted into the parthenogenetic embryos 7 h after activation were not
demethylated. This suggests that the elimination of H3K79 methylation after
fertilization is involved in genomic reprogramming. The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic
pattern of gene expression through binding with several MLL fusion partners
found in acute leukemia. However, the normal function of DOT1L in mammalian gene
regulation is poorly understood. Here we report that DOT1L recruitment is
ubiquitously coupled with active transcription in diverse mammalian cell types.
DOT1L preferentially occupies the proximal transcribed region of active genes,
correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l
mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined,
indicating that DOT1L is the sole enzyme responsible for these marks.
Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions
necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays
revealed that levels of all degrees of genic H3K79 methylation correlate with
mRNA abundance and dynamically respond to changes in gene activity. Conversion
of H3K79 monomethylation into di- and trimethylation correlated with the
transition from low- to high-level gene transcription. We also observed
enrichment of H3K79 monomethylation at intergenic regions occupied by
DNA-binding transcriptional activators. Our findings highlight several
similarities between the patterning of H3K4 methylation and that of H3K79
methylation in mammalian chromatin, suggesting a widespread mechanism for
parallel or sequential recruitment of DOT1L and MLL to genes in their normal
"on" state. Whereas mono-, di- and trimethylation states of lysines on histones typically
have specific functions, no specific functions have been attributed so far to
the different methylation states of histone H3 Lysine 79 (H3K79) generated by
Dot1. Here we show that Dot1, in contrast to other known histone
methyltransferases, introduces multiple methyl groups via a nonprocessive
mechanism. The kinetic mechanism implies that the H3K79 methylation states
cannot be generated independently, suggesting functional redundancy. Indeed,
gene silencing in yeast, which is dependent on Dot1, relied on global H3K79
methylation levels and not on one specific methylation state. Furthermore, our
findings suggest that histone H2B ubiquitination affects H3K79 trimethylation by
enhancing synthesis of all H3K79 methylation states. Our results suggest that
multiple methylation of H3K79 leads to a binary code, which is expected to limit
the possibilities for regulation by putative demethylases or binding proteins. Dot1-like protein (DOT1L) is an evolutionarily conserved histone
methyltransferase that methylates lysine 79 of histone H3 (H3K79). Mammalian
DOT1L participates in the regulation of transcription, development,
erythropoiesis, differentiation, and proliferation of normal cells. However, the
role of DOT1L in cancer cell proliferation has not been fully elucidated. DOT1L
siRNA-transfected A549 or NCI-H1299 lung cancer cells displayed a
nonproliferating multinucleated phenotype. DOT1L-deficient cells also showed
abnormal mitotic spindle formation and centrosome number, suggesting that DOT1L
deficiency leads to chromosomal missegregation. This chromosomal instability in
DOT1L-deficient cells led to cell cycle arrest at the G(1) phase and induced
senescence as determined by enhanced activity of senescence-associated
β-galactosidase activity. Meanwhile, overexpression of a catalytically active
DOT1L, not an inactive mutant, restored DOT1L siRNA-induced phenotypes. Overall,
these data imply that down-regulation of DOT1L-mediated H3K79 methylation
disturbs proliferation of human cells. In addition, although H3K79 methylation
is down-regulated in aged tissues, it is up-regulated in lung cancer cell lines
and tumor tissues of lung cancer patients. Therefore, H3K79 methylation is a
critical histone modification that regulates cell proliferation and would be a
novel histone mark for aging and cancer. During meiosis, accurate chromosome segregation relies on the proper interaction
between homologous chromosomes, including synapsis and recombination. The
meiotic recombination checkpoint is a quality control mechanism that monitors
those crucial events. In response to defects in synapsis and/or recombination,
this checkpoint blocks or delays progression of meiosis, preventing the
formation of aberrant gametes. Meiotic recombination occurs in the context of
chromatin and histone modifications, which play crucial roles in the maintece
of genomic integrity. Here, we unveil the role of Dot1-dependent histone H3
methylation at lysine 79 (H3K79me) in this meiotic surveillance mechanism. We
demonstrate that the meiotic checkpoint function of Dot1 relies on H3K79me
because, like the dot1 deletion, H3-K79A or H3-K79R mutations suppress the
checkpoint-imposed meiotic delay of a synapsis-defective zip1 mutant. Moreover,
by genetically manipulating Dot1 catalytic activity, we find that the status of
H3K79me modulates the meiotic checkpoint response. We also define the
phosphorylation events involving activation of the meiotic checkpoint effector
Mek1 kinase. Dot1 is required for Mek1 autophosphorylation, but not for its
Mec1/Tel1-dependent phosphorylation. Dot1-dependent H3K79me also promotes Hop1
activation and its proper distribution along zip1 meiotic chromosomes, at least
in part, by regulating Pch2 localization. Furthermore, HOP1 overexpression
bypasses the Dot1 requirement for checkpoint activation. We propose that
chromatin remodeling resulting from unrepaired meiotic DSBs and/or faulty
interhomolog interactions allows Dot1-mediated H3K79-me to exclude Pch2 from the
chromosomes, thus driving localization of Hop1 along chromosome axes and
enabling Mek1 full activation to trigger downstream responses, such as meiotic
arrest. During spermiogenesis, haploid spermatids undergo extensive chromatin remodeling
events in which histones are successively replaced by more basic protamines to
generate highly compacted chromatin. Here we show for the first time that H3K79
methylation is a conserved feature preceding the histone-to-protamine transition
in Drosophila melanogaster and rat. During Drosophila spermatogenesis, the
Dot1-like methyltransferase Grappa (Gpp) is primarily expressed in canoe stage
nuclei. The corresponding H3K79 methylation is a histone modification that
precedes the histone-to-protamine transition and correlates with histone H4
hyperacetylation. When acetylation was inhibited in cultured Drosophila testes,
nuclei were smaller and chromatin was compact, Gpp was little synthesized, H3K79
methylation was strongly reduced, and protamines were not synthesized. The Gpp
isoform Gpp-D has a unique C-terminus, and Gpp is essential for full fertility.
In rat, H3K79 methylation also correlates with H4 hyperacetylation but not with
active RNA polymerase II, which might point towards a conserved function in
chromatin remodeling during the histone-to-protamine transition in both
Drosophila and rat. Epigenetic marks such as histone modifications play roles in various chromosome
dynamics in mitosis and meiosis. Methylation of histones H3 at positions K4 and
K79 is involved in the initiation of recombination and the recombination
checkpoint, respectively, during meiosis in the budding yeast. Set1 promotes
H3K4 methylation while Dot1 promotes H3K79 methylation. In this study, we
carried out detailed analyses of meiosis in mutants of the SET1 and DOT1 genes
as well as methylation-defective mutants of histone H3. We confirmed the role of
Set1-dependent H3K4 methylation in the formation of double-strand breaks (DSBs)
in meiosis for the initiation of meiotic recombination, and we showed the
involvement of Dot1 (H3K79 methylation) in DSB formation in the absence of
Set1-dependent H3K4 methylation. In addition, we showed that the histone H3K4
methylation-defective mutants are defective in SC elongation, although they seem
to have moderate reduction of DSBs. This suggests that high levels of DSBs
mediated by histone H3K4 methylation promote SC elongation. In both mammalian and Drosophila spermatids, the completely histone-based
chromatin structure is reorganized to a largely protamine-based structure.
During this histone-to-protamine switch, transition proteins are expressed, for
example TNP1 and TNP2 in mammals and Tpl94D in Drosophila. Recently, we
demonstrated that in Drosophila spermatids, H3K79 methylation accompanies
histone H4 hyperacetylation during chromatin reorganization. Preceding the
histone-to-protamine transition, the H3K79 methyltransferase Grappa is
expressed, and the predomit isoform bears a C-terminal extension. Here, we
show that isoforms of the Grappa-equivalent protein in humans, rats and mice,
that is DOT1L, have a C-terminal extension. In mice, the transcript of this
isoform was enriched in the post-meiotic stages of spermatogenesis. In human and
mice spermatids, di- and tri-methylated H3K79 temporally overlapped with
hyperacetylated H4 and thus accompanied chromatin reorganization. In rat
spermatids, trimethylated H3K79 directly preceded transition protein loading on
chromatin. We analysed the impact of bacterial infections on spermatid chromatin
using a uropathogenic Escherichia coli-elicited epididymo-orchitis rat model and
showed that these infections caused aberrant spermatid chromatin. Bacterial
infections led to premature emergence of trimethylated H3K79 and hyperacetylated
H4. Trimethylated H3K79 and hyperacetylated H4 simultaneously occurred with
transition protein TNP1, which was never observed in spermatids of mock-infected
rats. Upon bacterial infection, only histone-based spermatid chromatin showed
abnormalities, whereas protamine-compacted chromatin seemed to be unaffected.
Our results indicated that H3K79 methylation is a histone modification conserved
in Drosophila, mouse, rat and human spermatids and may be a prerequisite for
proper chromatin reorganization. Histone lysine methylation regulates gene expression and cancer initiation.
Bioinformatics analysis suggested that DOT1L, a histone H3-lysine79 (H3K79)
methyltransferase, plays a potentially important role in breast cancer. DOT1L
inhibition selectively inhibited proliferation, self-renewal, metastatic
potential of breast cancer cells and induced cell differentiation. In addition,
inhibitors of S-adenosylhomocysteine hydrolase (SAHH), such as neplanocin and
3-deazaneplanocin, also inhibited both H3K79 methylation and proliferation of
breast cancer cells in vitro and in vivo. The activity of SAHH inhibitors was
previously attributed to inhibition of H3K27 methyltransferase EZH2. However,
inhibition of EZH2 by a specific inhibitor did not contribute to cell death.
SAHH inhibitors had only weak activity against H3K27 methylation and their
activity is therefore mainly due to DOT1L/H3K79 methylation inhibition. Overall,
we showed that DOT1L is a potential drug target for breast cancer. Author information:
(1)Division of Hematology/Oncology, Children's Hospital Boston, Boston, MA
02115, USA; Department of Pediatrics and the Human Oncology and Pathogenesis
Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
(2)Department of Pediatrics and the Human Oncology and Pathogenesis Program,
Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
(3)Division of Hematology/Oncology, Children's Hospital Boston, Boston, MA
02115, USA.
(4)Laboratory of Biochemistry and Molecular Biology, The Rockefeller University,
New York, NY 10065, USA.
(5)Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer
Center, New York, NY 10065, USA.
(6)Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical
School, Boston, MA 02115, USA.
(7)Department of Pediatrics, University of Colorado Anshutz Medical Campus,
Aurora, CO 80045 USA.
(8)Division of Hematology/Oncology, Children's Hospital Boston, Boston, MA
02115, USA; Department of Pediatrics and the Human Oncology and Pathogenesis
Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
Electronic address: [email protected]. |
Where can we find the protein lacritin? | The protein lacritin can be found in lacrimal and salivary glands as well as in tear fluid and in the thyroid. | Multiple extracellular factors are hypothesized to promote the differentiation
of unstimulated and/or stimulated secretory pathways in exocrine secretory
cells, but the identity of differentiation factors, particularly those
organ-specific, remain largely unknown. Here, we report on the identification of
a novel secreted glycoprotein, lacritin, that enhances exocrine secretion in
overnight cultures of lacrimal acinar cells which otherwise display loss of
secretory function. Lacritin mRNA and protein are highly expressed in human
lacrimal gland, moderately in major and minor salivary glands and slightly in
thyroid. No lacritin message or protein is detected elsewhere among more than 50
human tissues examined. Lacritin displays partial similarity to the
glycosaminoglycan-binding region of brain-specific neuroglycan C (32 % identity
over 102 amino acid residues) and to the possibly mucin-like amino globular
region of fibulin-2 (30 % identity over 81 amino acid residues), and localizes
primarily to secretory granules and secretory fluid. The lacritin gene consists
of five exons, displays no alternative splicing and maps to 12q13. Recombit
lacritin augments unstimulated but not stimulated acinar cell secretion,
promotes ductal cell proliferation, and stimulates signaling through tyrosine
phosphorylation and release of calcium. It binds collagen IV, laminin-1,
entactin/nidogen-1, fibronectin and vitronectin, but not collagen I, heparin or
EGF. As an autocrine/paracrine enhancer of the lacrimal constitutive secretory
pathway, ductal cell mitogen and stimulator of corneal epithelial cells,
lacritin may play a key role in the function of the lacrimal gland-corneal axis. PURPOSE: The purpose of this study was to examine breast tumors and normal
breast tissues for the expression of the glycoprotein lacritin. The mRNA and
protein expression of lacritin has been reported to be restricted to the
lacrimal gland.
METHODS: We investigated 37 human primary invasive breast tumors, seven breast
cancer cell lines and 16 normal breast tissues by quantitative real-time PCR for
lacritin expression.
RESULTS: We detected lacritin transcripts in 51% (19/37) of the primary invasive
breast tumors, in 71% (5/7) of the breast cancer cell lines, and also in 56%
(9/16) of the normal breast tissues. No lacritin mRNA was detectable in
peripheral blood of healthy individuals.
CONCLUSIONS: Here we show by quantitative real-time PCR that lacritin is
expressed in human breast tumors, breast cancer cell lines, and normal breast.
The previously reported restricted expression pattern of lacritin is therefore
incorrect. Lacritin transcripts were not detected in peripheral blood which
makes lacritin a potential candidate as a breast cancer marker gene. INTRODUCTION: The proteins found in tears play an important role in maintaining
the ocular surface and changes in tear protein components may reflect changes in
the health of the ocular surface. Proteomics provides a comprehensive approach
for cataloguing all the proteins of the tear proteome, which will help to
elucidate disease pathogenesis, make clinical diagnoses and evaluate the
influence of medications on the structure, composition and secretion of tear
proteins. In this study, an alternative proteomic strategy was investigated to
explore the human tear proteome.
MATERIALS AND METHODS: Tear samples were obtained from patients who had
pterygium and were collected on the first day and third day after pterygium
surgery. Tears pooled from 6 patients were used in the analysis. Reverse-phase
high-pressure liquid chromatograph (RPHPLC) was used as the first step to
separate intact proteins into 21 peaks. Each fraction was then tryptic-digested
and analysed by oLC-o-ESI-MS/MS to characterise the protein components in
each fraction.
RESULTS: In total, 60 tear proteins were identified with high confidence,
including well-known abundant tear proteins, and tear-specific proteins such as
lacritin and proline-rich proteins. Among them, proline-rich protein 5 was found
for the first time in tear fluid. A large number of plasma proteins were also
observed in tear fluid.
CONCLUSIONS: The results showed that the proteomic strategy used in this study
was successfully applied to analyse tear proteome. Renewal of nongermative epithelia is poorly understood. The novel mitogen
"lacritin" is apically secreted by several nongermative epithelia. We tested 17
different cell types and discovered that lacritin is preferentially mitogenic or
prosecretory for those types that normally contact lacritin during its glandular
outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which
can form an alpha-helix with a hydrophobic face, as per VEGF's and PTHLP's
respective dimerization or receptor-binding domain. Lacritin targets downstream
NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic
signaling involves Galpha(i) or Galpha(o)-PKCalpha-PLC-Ca2+-calcineurin-NFATC1
and Galpha(i) or Galpha(o)-PKCalpha-PLC-phospholipase D (PLD)-mTOR in a
bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not
TRPC1. This pathway suggests the placement of transiently dephosphorylated and
perinuclear Golgi-translocated PKCalpha upstream of both Ca2+ mobilization and
PLD activation in a complex with PLCgamma2. Outward flow of lacritin from
secretory cells through ducts may generate a proliferative/secretory field as a
different unit of cellular renewal in nongermative epithelia where luminal
structures predominate. Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators
of cell migratory, mitogenic, secretory, and inflammatory activity that bind and
present soluble heparin-binding growth factors (e.g., fibroblast growth factor,
Wnt, Hh, transforming growth factor beta, amphiregulin, and hepatocyte growth
factor) to their respective signaling receptors. We demonstrate that the
deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4,
appears to target the epithelial selective prosecretory mitogen lacritin. An
important and novel step in this mechanism is that binding necessitates prior
partial or complete removal of HS chains by endogenous heparanase. This limits
lacritin activity to sites where heparanase appears to predominate, such as
sites of exocrine cell migration, secretion, renewal, and inflammation. Binding
is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N
terminus. Heparanase modification of the latter transforms a widely expressed HS
proteoglycan into a highly selective surface-binding protein. This novel example
of cell specification through extracellular modification of an HS proteoglycan
has broad implications in development, homeostasis, and disease. Lacritin is a mitogen of human salivary gland cells as well as a stimulator of
human corneal epithelial cells. It is expected to be an important factor in
maintaining the surrounding ocular surface. The monkey would be a relevant
animal model in which to study the role of lacritin in ophthalmic physiology and
pathology. However, to our knowledge, no cDNA cloning or functional analysis of
monkey lacritin has been performed. Thus, the purposes of this study were: (1)
to clone the monkey ortholog of lacritin; (2) to characterize lacritin in tears
from several species; and (3) to determine the tissues where lacritin is
produced and secreted. cDNA for lacritin from rhesus macaque contained 547 bp,
with 411 bp in an open reading frame (ORF) encoding a protein of 137 amino
acids. Monkey lacritin showed 89% amino acid homology with human lacritin; one
amino acid was deleted in all three monkey strains. The predicted MW of mature
lacritin was 12.2 kDa, and the isoelectric point was 4.99. Lacritin showed
anomalous migration at approximately 21.0 kDa on SDS-PAGE, as confirmed by
immunoblotting and amino acid sequencing. Similar to native lacritin in monkey
tears, a 21 kDa band was also detected in human tears. In contrast, no lacritin
was observed at a similar position on SDS-PAGE in rat, rabbit and dog tears. In
the monkey, lacritin mRNA was expressed highly in the lacrimal gland, moderately
in the conjunctiva and the meibomian gland, and weakly in corneal epithelium. In
primates, lacritin was produced in the lacrimal gland and secreted into tear
fluid. These results suggest that lacritin might be important for the
maintece of the ocular surface in higher animals, such as monkeys and humans. Glycoproteins are potentially important biomarkers of disease and therapeutic
targets. In particular, the N-linked glycoproteins are a focus of interest as
they can be found in the extracellular environment and body fluids. In this
study, we have sampled the tears, the extracellular fluid of the epithelial
cells covering the surface of the eye, of patients with climatic droplet
keratopathy (CDK) using tears of unaffected normal patients for comparison.
Prefractionation of the tear sample used a hydrazide-resin capture method, and
the previously N-glycosylated peptides were then subjected to two-dimensional
o-LC-o-ESI-MS/MS analysis to obtain peptide fragmentation patterns for
identification through protein database searches. We have identified a total of
43 unique N-glycoproteins, 19 of which have not previously been reported in tear
fluid. In addition, we have quantitatively compared N-glycoprotein profiles in
tear fluid of patients with CDK to tears of nondiseased controls using
glycopeptide capture, iTRAQ labeling and 2D o-LC-o-ESI-MS/MS analysis. In
tears of CDK patients, increased levels of four N-glycosylated proteins
including haptoglobin (at sites N207, N211 and N241), polymeric immunoglobulin
receptor (at sites N83, N90, N135, N186, N421, and N469), immunoglobulin J chain
(at site N49) and an uncharacterized protein DKFZp686M08189 (at site N470), as
well as a decrease in the N-glycosylation level of one N-glycosylated protein,
lacritin (at site N119) were observed. However, the overall levels of these five
proteins showed no appreciable changes between control and CDK samples. The
findings could be clinically significant in terms of disease etiology and
biomarkers. PURPOSE: Lacritin protein is highly expressed in the lacrimal gland, secreted
into tear fluid, and detected only in primates. The mechanism for lacritin
secretion has not been fully investigated, because a system for culturing
primate lacrimal acinar cells had not been established. The purposes of the
present study were (1) to develop a procedure to culture lacrimal acinar cells
from monkey and (2) to determine the mechanism for the secretion of lacritin in
the culture system.
METHODS: Acinar cells from monkey lacrimal gland were cultured and
characterized. Lacritin and other proteins were detected by
immunohistochemistry, immunocytochemistry, and immunoblot analysis. Secreted
proteins were also detected in the medium from stimulated acinar cells. mRNAs
were determined by microarray and qPCR. Intracellular calcium levels were
measured by calcium-4 assay.
RESULTS: Acinar cells cultured for 1 day contained adequate amounts of lacritin,
lactoferrin, and lipocalin for use in lacritin secretion studies. The
cholinergic agonist carbachol (Cch) stimulated the secretion of lacritin and
increased intracellular Ca2+. Cch-induced lacritin secretion was inhibited by
the store-operated calcium (SOC) channel inhibitor YM58483 and the PKC
inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin secretion was not
inhibited by MAPKK inhibitor U0126, although p42/p44 MAPK was phosphorylated.
Cch also enhanced gene transcription, which was inhibited by U0126, GF109203,
and calcium chelators.
CONCLUSIONS: Successful culture of monkey lacrimal acinar cells showed that,
among the prevalent tear proteins, the secretion of lacritin involved the
PKC/Ca2+ pathway, not the p42/p44 MAPK pathway. Induction of transcription by
Cch involved the independent p42/p44 MAPK and PKC pathways. Diabetic retinopathy is the leading cause of new cases of legal blindness among
adults in the developed countries. Approximately 40% of all people with diabetes
have diabetic retinopathy and 5% of these have sight-threatening form. As the
advanced stage, where there is a high risk for vision loss, can develop without
any serious symptoms, sometimes it is hard to detect it. A non invasive method
to detect biomarkers characteristic for diabetic retinopathy from the tear fluid
was developed. Tear samples from diabetic patients with no retinopathy, non
proliferative and proliferative stages of diabetic retinopathy were analyzed and
the protein content of each sample was compared to the protein content of tear
pool from healthy volunteers. The samples were labeled with iTRAQ fourplex
labels and were analyzed with oHPLC coupled ESI-MS/MS mass spectrometry. The
lipocalin 1, lactotransferrin, lacritin, lysozyme C, lipophilin A and
immunoglobulin lambda chain were identified as possible biomarker candidates
with significantly higher relative levels in the tear of patients with diabetic
retinopathy. PURPOSE: Lacritin is a human tear glycoprotein that promotes basal tear protein
secretion in cultured rat lacrimal acinar cells and proliferation of
subconfluent human corneal epithelial cells. When topically added to rabbit
eyes, lacritin promotes basal tearing. Despite these activities on several
species, lacritin's presence in nonprimate tears or other tissues has not been
explored. Here we probed for lacritin in normal horse tears.
METHODS: Sequences were collected from the Ensembl genomic alignment of human
LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal
horse tears were collected and assayed by Western blotting, ELISA, and mass
spectrometry. Newly generated rabbit antibodies, respectively, against N- and
C-terminal regions of human lacritin were employed.
RESULTS: Identity was 75% and 45%, respectively, at nucleotide and protein
levels. Structural features were conserved, including a C-terminal amphipathic
α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse
tears that was validated by mass spectrometry. In human tears, the same antibody
detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13
and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24
kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13
kDa species. Similar respective levels of horse C-terminal versus N-terminal
immunoreactivity were apparent by ELISA.
CONCLUSIONS: Lacritin is present in horse tears, largely as a C-terminal
fragment homologous to the mitogenic and bactericidal region in human lacritin,
suggesting potential benefit in corneal wound repair. PURPOSE: The accessory lacrimal glands are assumed to contribute to the
production of tear fluid, but little is known about their function. The goal of
this study was to conduct an analysis of gene expression by glands of Wolfring
that would provide a more complete picture of the function of these glands.
METHODS: Glands of Wolfring were isolated from frozen sections of human eyelids
by laser microdissection. RNA was extracted from the cells and hybridized to
gene expression arrays. The expression of several of the major genes was
confirmed by immunohistochemistry.
RESULTS: Of the 24 most highly expressed genes, 9 were of direct relevance to
lacrimal function. These included lysozyme, lactoferrin, tear lipocalin, and
lacritin. The glands of Wolfring are enriched in genes related to protein
synthesis, targeting, and secretion, and a large number of genes for proteins
with antimicrobial activity were detected. Ion channels and transporters,
carbonic anhydrase, and aquaporins were abundantly expressed. Genes for control
of lacrimal function, including cholinergic, adrenergic, vasoactive intestinal
polypeptide, purinergic, androgen, and prolactin receptors were also expressed
in gland of Wolfring.
CONCLUSIONS: The data suggest that the function of glands of Wolfring is similar
to that of main lacrimal glands and are consistent with secretion electrolytes,
fluid, and protein under nervous and hormonal control. Since these glands
secrete directly onto the ocular surface, their location may allow rapid
response to exogenous stimuli and makes them readily accessible to topical
drugs. PURPOSE: During inflammation of the ocular surface, increased proinflammatory
cytokines depress tear protein secretion, suggesting that a decline in lacrimal
cell function contributes to dry eye. Lacritin, a glycoprotein secreted from
lacrimal acinar cells, may function as an autocrine factor to stimulate tear
protein secretion. The purpose of the present experiment was to investigate
lacritin-induced protein secretion in normal and cytokine-pretreated
(inflammation model) monkey acinar cells.
METHODS: Acinar cells from monkey lacrimal glands were cultured with or without
tumor necrosis factor alpha (TNF-α) plus interferon gamma (IFN-γ). Protein
secretion was induced by lacritin or carbachol (Cch, positive control). Proteins
were detected and identified by immunoblotting and immunocytochemistry.
Intracellular Ca(2+) was measured with the fluorophore Calcium-4, and cell
damage was determined by LDH leakage into the culture medium.
RESULTS: In cultured monkey acinar cells, lacritin stimulated tear protein
secretion in normal cells without elevating intracellular Ca(2+). In contrast,
Cch elevated intracellular Ca(2+) and release of tear proteins. This contrast
suggested an alternate, calcium-independent mechanism for lacritin-induced
protein secretion. TNF-α plus IFN-γ caused LDH leakage from sensitive human
corneal epithelial cells, but even higher doses of TNF-α plus IFN-γ did not
cause LDH leakage from monkey acinar cells, suggesting a higher tolerance
against these cytokines. In cytokine-treated acinar cells, lacritin stimulated
protein secretion as much as that in normal cells. In contrast, Cch-induced
elevation of Ca(2+) and release of proteins were depressed by cytokines.
CONCLUSIONS: Lacritin might be a useful biotechnology-based treatment agent
against ocular surface diseases where endogenous lacritin is inadequate. |
Where can we find the protein dermcidin? | Dermcidin is a secretes protein found mainly in sweat but it is also found in serum. | Hypertension and diabetes mellitus are considered to be two major
atherosclerotic risk factors for coronary artery disease (CAD). A stress-induced
protein identified to be dermcidin isoform 2 of Mr. 11 kDa from blood plasma of
hypertensive persons when injected (0.1 μM) in rabbits increased the systolic
pressure by 77% and diastolic pressure by 45% over the controls within 2 h.
Ingestion of acetyl salicylic acid (150 mg/70 kg) by these subjects reduced
systolic (130 mm Hg) and diastolic pressures (80 mm Hg) with reduction of plasma
dermcidin level to normal ranges (9 nM). The protein was found to be a potent
activator of platelet cyclooxygenase and inhibited insulin synthesis. Aspirin
was found to reduce hypertension by reduction of plasma dermcidin level,
neutralized the effect of cyclooxygenase, and restored the pancreatic insulin
synthesis through NO synthesis. These results indicated that dermcidin could be
a novel atherosclerotic risk factor for its hypertensive and diabetogenic
effects. Eccrine sweat glands, which are distributed over the whole bodies of primates
and humans, have long been regarded mainly to have a function in
thermoregulation. However, the discovery of dermcidin-derived antimicrobial
peptides in eccrine sweat demonstrated that sweat actively participates in the
constitutive innate immune defense of human skin against infection. In the
meantime, a number of studies proved the importance of dermcidin in skin host
defense. Several reports also state that peptides processed from the dermcidin
precursor protein exhibit a range of other biological functions in neuronal and
cancer cells. This review summarizes the evidence gathered until now concerning
the expression of dermcidin and the functional relevance of dermcidin-derived
peptides. Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) has been demonstrated to
be an inhibitor of various kallikreins and is thought to play a role in the
regulation of skin desquamation. In order to identify and investigate the
potential of LEKTI to interact with other proteins, a method was developed using
immobilised proteins onto arrays and oUPLC/MALDI-TOF MS. Using various
domains of LEKTI, we demonstrated that these domains bound a number of
kallikreins (5, 13 and 14) to varied extents on the array surface. Inhibitory
assays confirmed that binding on the protein array surface corresponded directly
to levels of inhibition. The method was then tested using skin epidermal
extracts. All forms of rLEKTI with the exception of rLEKTI 12-15, demonstrated
the binding of several potential candidate proteins. Surprisingly, the major
binding partners of LEKTI were found to be the antimicrobial peptide dermcidin
and the serine protease cathepsin G and no kallikreins. Using confocal
microscopy and Netherton syndrome skin sections, we confirmed the
co-localisation of LEKTI with dermcidin and demonstrated altered trafficking of
dermcidin in these patients. This potential new role for LEKTI as a
multifunctional protein in the protection and transport of proteins in the
epidermis and its role in disease are discussed. Graft-versus-host disease (GVHD) is an important complication of bone marrow
transplantation and is known to induce sweat gland abnormalities. We employed
immunohistochemical staining for granulysin, dermcidin as well as IL-17 and
Foxp3 in the lesional skin of 7 patients with acute GVHD. Granulysin-bearing
cells were distributed in the epidermis, basal membrane zone of the dermis, and
superficial perivascular lesion of the dermis. Interestingly, granulysin-bearing
cells were also detected around the secretory portion of the eccrine glands,
which might be related to the downregulation of the expression of dermcidin on
the secretory portion of the eccrine glands. Differing from a mouse model, in
human acute GVHD, IL-17-producing cells and Foxp3(+) cells were not prominent.
Our present observation sheds light on the contribution of granulysin-bearing
cells to the decrease of the sweat in patients with acute GVHD. Though we did
not assess the function of these infiltrating lymphocytes directly, further
analysis of the mechanism of this phenomenon would offer fundamental insight
into the establishment of acute GVHD. Improved diagnostic screening has led to earlier detection of many tumors, but
screening may still miss many aggressive tumor types. Proteomic and genomic
profiling studies of breast cancer samples have identified tumor markers that
may help improve screening for more aggressive, rapidly growing breast cancers.
To identify potential blood-based biomarkers for the early detection of breast
cancer, we assayed serum samples via matrix-assisted laser desorption
ionization-time of flight mass spectrometry from a rat model of mammary
carcinogenesis. We found elevated levels of a fragment of the protein dermcidin
(DCD) to be associated with early progression of N-methylnitrosourea-induced
breast cancer, demonstrating significance at weeks 4 (p = 0.045) and 5 (p =
0.004), a time period during which mammary pathologies rapidly progress from
ductal hyperplasia to adenocarcinoma. The highest serum concentrations were
observed in rats bearing palpable mammary carcinomas. Increased DCD was also
detected with immunoblotting methods in 102 serum samples taken from women just
prior to breast cancer diagnosis. To validate these findings in a larger
population, we applied a 32-gene in vitro DCD response signature to a dataset of
295 breast tumors and assessed correlation with intrinsic breast cancer subtypes
and overall survival. The DCD-derived gene signature was significantly
associated with subtype (p < 0.001) and poorer overall survival [HR (95 % CI) =
1.60 (1.01-2.51), p = 0.044]. In conclusion, these results present novel
evidence that DCD levels may increase in early carcinogenesis, particularly
among more aggressive forms of breast cancer. The mechanism of membrane permeabilization by dermcidin (DCD-1L), an
antimicrobial peptide present in human sweat, was investigated at a
mercury-supported monolayer of dioleoylphosphatidylcholine (DOPC) or
dioleoylphosphatidylserine (DOPS) and at a mercury-supported tethered bilayer
lipid membrane (tBLM) consisting of a thiolipid (DPTL) with a DOPC or DOPS
monolayer self-assembled on top of it. In an unbuffered solution of pH 5.4,
DCD-1L is almost neutral and permeabilizes a DPTL/DOPS tBLM at transmembrane
potentials, ϕtrans, which are physiological. In a pH 7 buffer solution DCD-1L
bears two negative charges and has no effect on a DPTL/DOPC tBLM, whereas it
permeabilizes a DPTL/DOPS tBLM only outside the physiological ϕtrans range;
however, the presence of zinc ion induces DCD-1L to permeabilize the DPTL/DOPS
tBLM at physiological ϕtrans values. The effect of zinc ions suggests a DCD-1L
conformation with its positive N-terminus embedded in the lipid bilayer and the
negative C terminus floating on the membrane surface. This conformation can be
stabilized by a zinc ion bridge between the His(38) residue of the C terminus
and the carboxyl group of DOPS. Chronocoulometric potential jumps from ϕtrans ≅
+160 mV to sufficiently negative values yield charge transients exhibiting a
sigmoidal shape preceded by a relatively long "foot". This behavior is
indicative of ion-channel formation characterized by disruption of DCD-1L
clusters adsorbed on top of the lipid bilayer, incorporation of the resulting
monomers and their aggregation into hydrophilic pores by a mechanism of
nucleation and growth. As the key components of innate immunity, human host defense antimicrobial
peptides and proteins (AMPs) play a critical role in warding off invading
microbial pathogens. In addition, AMPs can possess other biological functions
such as apoptosis, wound healing, and immune modulation. This article provides
an overview on the identification, activity, 3D structure, and mechanism of
action of human AMPs selected from the antimicrobial peptide database. Over 100
such peptides have been identified from a variety of tissues and epithelial
surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary
systems. These peptides vary from 10 to 150 amino acids with a net charge
between -3 and +20 and a hydrophobic content below 60%. The sequence diversity
enables human AMPs to adopt various 3D structures and to attack pathogens by
different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial
surface into onets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are
able to block cell wall biosynthesis by binding to lipid II. Lysozyme is
well-characterized to cleave bacterial cell wall polysaccharides but can also
kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the
long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding
and disrupting the curved anionic bacterial membrane surfaces by forming pores
or via the carpet model. Furthermore, dermcidin may serve as ion channel by
forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα
can initially recognize bacterial peptidoglycans followed by pore formation in
the membrane. Finally, histatin 5 and GAPDH(2-32) can enter microbial cells to
exert their effects. It appears that granulysin enters cells and kills
intracellular pathogens with the aid of pore-forming perforin. This arsenal of
human defense proteins not only keeps us healthy but also inspires the
development of a new generation of personalized medicine to combat
drug-resistant superbugs, fungi, viruses, parasites, or cancer. Alternatively,
multiple factors (e.g., albumin, arginine, butyrate, calcium, cyclic AMP,
isoleucine, short-chain fatty acids, UV B light, vitamin D, and zinc) are able
to induce the expression of antimicrobial peptides, opening new avenues to the
development of anti-infectious drugs. In acne vulgaris, antimicrobial peptides (AMPs) could play a dual role; i.e.,
protective by acting against Propionibacterium acnes, pro-inflammatory by acting
as signalling molecules. The cutaneous expression of 15 different AMPs was
investigated in acne patients; furthermore, the impact of isotretinoin therapy
on AMP expression was analysed in skin biopsies from 13 patients with acne
vulgaris taken before, during and after a 6-month treatment cycle with
isotretinoin using quantitative real-time polymerase chain reaction. Cutaneous
expression of the AMPs cathelicidin, human β-defensin-2 (HBD-2), lactoferrin,
lysozyme, psoriasin (S100A7), koebnerisin (S100A15), and RNase 7 was upregulated
in untreated acne vulgaris, whereas α-defensin-1 (HNP-1) was downregulated
compared to controls. While relative expression levels of cathelicidin, HBD-2,
lactoferrin, psoriasin (S100A7), and koebnerisin (S100A15) decreased during
isotretinoin treatment, only those of cathelicidin and koebnerisin returned to
normal after 6 months of isotretinoin therapy. The increased expression of
lysozyme and RNase 7 remained unaffected by isotretinoin treatment. The levels
of granulysin, RANTES (CCL5), perforin, CXCL9, substance P, chromogranin B, and
dermcidin were not regulated in untreated acne patients and isotretinoin had no
effect on these AMPs. In conclusion, the expression of various AMPs is altered
in acne vulgaris. Isotretinoin therapy normalizes the cutaneous production of
distinct AMPs while the expression of others is still increased in healing acne.
Considering the antimicrobial and pro-inflammatory role of AMPs, these molecules
could serve as specific targets for acne therapy and maintece of clinical
remission. BACKGROUND: The survival promoting peptide Y-P30 has a variety of neuritogenic
and neuroprotective effects in vitro and in vivo. In previous work we reported
the expression of Y-P30/dermcidin in maternal peripheral blood mononuclear cells
(PBMCs) and the transport of the protein to the fetal brain. In this study we
analyzed hormonal regulation of Y-P30 in human immune cells and expression of
Y-P30 in the placenta. We further studied the stability and secretion of the
Y-P30 peptide.
RESULTS: We found indications that Y-P30 might be produced in human placenta.
The Y-P30 mRNA was rarely found in isolated human PBMCs and alpha-feto-protein,
human chorionic gonadotropin as well as estradiol combined with progesterone
could not induce Y-P30 expression. Y-P30 was found to be extraordinarily stable;
therefore, contamination with the peptide and the Y-P30/Dermcidin precursor mRNA
is a serious concern in experiments looking at the expression of
Y-P30/Dermcidin. In cultured cell lines and primary neurons we found that Y-P30
could be released, but neuronal uptake of Y-P30 was not observed.
CONCLUSIONS: Our data suggest that a source of Y-P30 apart from eccrine glands
might be the placenta. The peptide can be secreted together with the signaling
peptide and it might reach the fetal brain where it can exert its neuritogenic
functions by binding to neuronal membranes. The identification of human body fluids or tissues through mRNA-based profiling
is very useful for forensic investigations. Previous studies have shown mRNA
biomarkers are effective to identify the origin of biological samples. In this
study, we selected 16 tissue specific biomarkers to evaluate their specificities
and sensitivities for human body fluids and tissues identification, including
porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY)
for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for
semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion,
matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin
4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin
3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for
sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic
body fluids or tissues were used in the evaluation. Based on the evaluation, a
reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12
biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7,
TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further
validated with real casework samples and mock samples (with both single source
and mixture) and it was approved that XCYR1 is effective to identify common body
fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion,
menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic
casework samples. The aggregation of platelets on the plaque rupture site on the coronary artery
is reported to cause both acute coronary syndromes (ACS) and acute myocardial
infarction (AMI). While the inhibition of platelet aggregation by acetyl
salicylic acid was reported to produce beneficial effects in ACS, it failed to
do in AMI. The concentration of a stress induced protein (dermcidin isoform-2)
was much higher in AMI than that in ACS. Incubation of normal platelet rich
plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low
affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 μM
dermcidin, the platelets became resistant to the inhibitory effect of aspirin
similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin
antibody restored the sensitivity of the platelets to the aspirin effect.
Incubation of AMI PRP pretreated with 15 μM aspirin, a stimulator of the NO
synthesis, resulted in the increased production of NO in the platelets that
removed the bound dermcidin by 40% from the high affinity binding sites of AMI
platelets. When the same AMI PRP was retreated with 10 μM aspirin, the
aggregation of platelets was completely inhibited by NO synthesis. "Pustulosis palmaris et plantaris", or palmoplantar pustulosis (PPP), is a
chronic pustular dermatitis characterized by intraepidermal palmoplantar
pustules. Although early stage vesicles (preceding the pustular phase) formed in
the acrosyringium contain the antimicrobial peptides cathelicidin
(hCAP-18/LL-37) and dermcidin, the details of hCAP-18/LL-37 expression in such
vesicles remain unclear. The principal aim of the present study was to clarify
the manner of hCAP-18/LL-37 expression in PPP vesicles and to determine whether
this material contributed to subsequent inflammation of lesional skin. PPP
vesicle fluid (PPP-VF) induced the expression of mRNAs encoding IL-17C, IL-8,
IL-1α, and IL-1β in living skin equivalents, but the level of only IL-8 mRNA
decreased significantly upon stimulation of PPP vesicle with depletion of
endogenous hCAP-18/LL-37 by affinity chromatography (dep-PPP-VF).
Semi-quantitative dot-blot analysis revealed higher concentrations of
hCAP-18/LL-37 in PPP-VF compared to healthy sweat (2.87±0.93 µM vs. 0.09±0.09
µM). This concentration of hCAP-18/LL-37 in PPP-VF could upregulate expression
of IL-17C, IL-8, IL-1α, and IL-1β at both the mRNA and protein levels.
Recombit hCAP-18 was incubated with dep-PPP-VF. Proteinase 3, which converts
hCAP-18 to the active form (LL-37), was present in PPP-VF. Histopathological and
immunohistochemical examination revealed that early stage vesicles contained
many mononuclear cells but no polymorphonuclear cells, and the mononuclear cells
were CD68-positive. The epidermis surrounding the vesicle expresses monocyte
chemotactic chemokine, CCL2. In conclusion, PPP-VF contains the proteinase
required for LL-37 processing and also may directly upregulate IL-8 in lesional
keratinocytes, in turn contributing to the subsequent inflammation of PPP
lesional skin. In healthy human skin host defense molecules such as antimicrobial peptides
(AMPs) contribute to skin immune homeostasis. In patients with the congenital
disease ectodermal dysplasia (ED) skin integrity is disturbed and as a result
patients have recurrent skin infections. The disease is characterized by
developmental abnormalities of ectodermal derivatives and absent or reduced
sweating. We hypothesized that ED patients have a reduced skin immune defense
because of the reduced ability to sweat. Therefore, we performed a label-free
quantitative proteome analysis of wash solution of human skin from ED patients
or healthy individuals. A clear-cut difference between both cohorts could be
observed in cellular processes related to immunity and host defense. In line
with the extensive underrepresentation of proteins of the immune system,
dermcidin, a sweat-derived AMP, was reduced in its abundance in the skin
secretome of ED patients. In contrast, proteins involved in metabolic/catabolic
and biosynthetic processes were enriched in the skin secretome of ED patients.
In summary, our proteome profiling provides insights into the actual situation
of healthy versus diseased skin. The systematic reduction in immune system and
defense-related proteins may contribute to the high susceptibility of ED
patients to skin infections and altered skin colonization. Reducing the levels of toxic protein aggregates has become a focus of therapy
for disorders like Alzheimer's and Parkinson's diseases, as well as for the
general deterioration of cells and tissues during aging. One approach has been
an attempt to influence the production or activity of a class of reparative
chaperones called heat shock proteins (HSPs), of which HSP70 is a promising
candidate. Manipulation of HSP70 expression results in disposal of misfolded
protein aggregates that accumulate in aging and disease models. Recently, HSP70
has been shown to bind specifically to an amino-terminal sequence of a human
diffusible survival evasion peptide (DSEP), dermcidin. This sequence includes
CHEC-9, an orally available anti-inflammatory and cell survival peptide. In the
present study, we found that the CHEC-9 peptide also binds HSP70 in the cytosol
of the cerebral cortex after oral delivery in normal rats. Western analysis of
non-heat-denatured, unreduced samples suggested that peptide treatment increased
the level of active HSP70 monomers from the pool of chaperone oligomers, a
process that may be stimulated by potentiation of the chaperone's adenosine
triphosphatase (ATPase). In these samples, a small but consistent gel shift was
observed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a multifunctional
protein whose aggregation is influenced by HSP70. CHEC-9 treatment of an in
vitro model of α-synuclein aggregation also results in HSP70-dependent
dissolution of these aggregates. HSP70 oligomer-monomer equilibrium and its
potential to control protein aggregate disease warrant increased experimental
attention, especially if a peptide fragment of an endogenous human protein can
influence the process. |
What is the triple screening test performed during pregnancy measuring? | Alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG) and unconjugated estriol (uE3) | Between September 1st 1990 and Juli 31st 1993, 5071 pregt women were screened
prospectively by the "triple-test", including maternal serum alpha-fetoprotein,
human chorionic gonadotropin and unconjugated oestriol in order to detect
chromosomal anomalies and open neural tube defects. The serum samples were
collected in collaboration with the obstetricians of the region of
West-Mecklenburg and North-West-Brandenburg. Laboratory testing using
radioimmunoassays was performed between weeks 15 and 20 of gestation, all serum
specimens being investigated in only one institution. The original
alpha-software from Wald et al. was the basis for calculating the statistical
risk for Down's syndrome. Pregt women with a high risk for Down's syndrome
(cutoff > or = 1:250) were taken care of in a special outpatient clinic
including procedures like amniocentesis and fetal blood sampling. Amongst 5071
pregt women, 21 fetal anomalies were seen. Five cases of Down's syndrome,
three of trisomy 18, one trisomy 13, two cases of triploidy and four cases of
open neural tube defects, one 46 xy/45 x mosaic karyotype and one case of
gastroschisis could be diagnosed correctly. One case of trisomy 21, one case of
trisomy 18 and two open neural tube defects showed false negative results. Using
the cutoff of 1:250 for prenatal detection of Down's syndrome and performing
ultrasound routinely to determine gestational age, the sensitivity of the
"triple-test" was 83.33% having a specificity of 92.68%. The predictive value of
a positive test for prenatal diagnosis of Down's syndrome was 1.33%.(ABSTRACT
TRUNCATED AT 250 WORDS) The objective of this study was to evaluate economically a screening programme
within the Oxford Regional Health Authority for Down's syndrome, based on
maternal serum alpha fetoprotein, unconjugated oestriol and human chorionic
gonadotrophin as well as maternal age (the triple test) against maternal age
alone. The design of the study involved cost-effectiveness analysis of the
triple test relative to the maternal age screening programme, and the main
outcome measure was the cost per Down's birth avoided. It was found that the
triple test is more cost-effective over a wide range of assumptions concerning
detection rates and procedure costs. Indirect costs are important in considering
the cost-effectiveness of the screening programmes. The most efficient detection
rate is around 58 per cent for which the cost per Down's birth avoided is
approximately 29,600 pounds if only direct costs are evaluated, 20,100 pounds if
all NHS costs are considered and -49,800 pounds if all resource consequences are
analysed. It may be concluded that screening for Down's syndrome using the
triple test is cost-effective over a wide range of assumptions concerning
detection rate and procedure costs. If all resource costs are considered, the
programme is highly cost-effective in comparison with other health care
interventions. The demands made on prenatal care in terms of fetal anomaly recognition and full
and early counseling have become more and more sophisticated over the last few
years. Today, a range of non-invasive and invasive procedures are now available
for the detection of fetal anomalies, genetic disorders and e.g. infection. The
most important of the former are ultrasound and triple test (determination of
alpha feto-protein, HCG and unconjugated estriol), while amniocentesis remains
the major invasive measure. The indications, the information they provide, and
possible risks of these procedures are discussed. OBJECTIVE: To assess the usefulness of triple-marker screening for Down syndrome
in Venezuela.
METHOD: Maternal serum concentrations of alpha fetoprotein (AFP), beta human
chorionic gonadotropin (beta-hCG), and unconjugated estriol (uE3) were measured
weekly in 3895 women from the 15th to the 20th week of pregcy.
Population-specific likelihood ratios were determined and used to calculate the
risk of fetal Down syndrome for each pregcy.
RESULTS: The median multiple of the median values for AFP, beta-hCG, and uE3
concentrations were 0.69, 2.10, and 0.67 for the affected pregcies. The
likelihood ratio for a positive result was 1:19. The detection and
false-positive rates were 69.23% and 5.8%.
CONCLUSION: These findings were consistent with reported data and therefore
confirmed triple-marker serum screening as effective and suitable for prenatal
care in Venezuela. Latin American governments and Health Agencies should
recommend offering this screening method to all pregt women. OBJECTIVES: Uterine myomas may change the concentrations of the screening serum
markers and therefore alter the risk calculation of the fetal chromosomal
abnormalities. An increased risk leads to invasive diagnostics procedures which
in these cases can often be technically difficult due to the presence of myomas.
AIM: The aim of this study was to assess the influence of uterine myomas on the
first and second trimester serum markers concentrations and, possibly on the
test results.
MATERIAL AND METHODS: The study group consisted of 127 women between 11 and 20
weeks of normal singleton pregcy. In each case uterine myomas were
diagnosed--over 20 mm in the diameter and located in the uterine wall. 77
patients underwent the first trimester screening (PAPP-A & free beta-hCG) and 50
patients had the second trimester screening (triple test). The control group
consisted of 1020 women between 11 and 20 weeks of normal singleton pregcy
without uterine myomas. Delfia Xpress analyser was used for the serum markers
estimations. All pregt women delivered normal healthy babies.
RESULTS: In the first trimester group the PAPP-A serum concentrations were not
different from the controls while the mean median concentration of free beta-hCG
were significant higher--1.43 MoM. In the second trimester group the following
mean median values were observed: no significance for the AFP--1.18 MoM and
estriol--1.29 MoM and significantly higher mean median value for the free
beta-hCG--2.01 MoM.
CONCLUSIONS: 1. The presence of the uterine myomas is connected with the
increased maternal serum concentration of the beta-hCG, particularly in the
second trimester. 2. The uterine myomas may lead to the increased rate of the
false positive results of the prenatal screening test, especially the triple
test. |
What are the generic versions of Viagra? | Generic versions of sildenafil are Elonza, Caverta, Zenegra-100, Vega Asia, Suhagra-100, Vega, Revatio. | The appearance of counterfeit medicines in supply chains is a global public
health problem that may seriously affect patients. Counterfeit drugs do not meet
quality standards and do not declare their real composition and/or source for
the purposes of fraud. They may be generic or innovative, they may contain
genuine constituents in a fake packaging, or wrong ingredients, or inactive
ingredients, or an incorrect quantity of the active substance. In Croatia, no
cases of counterfeit medicines have been detected so far, but the Agency for
Medicinal Products and Medical Devices has received 34 samples of medicines and
other products for testing from Zagreb City Police. The samples included
medicines for erectile dysfunction: sildenafil, tadalafil, and vardenafil.
Twenty-three samples of tablets without marketing authorisation in Croatia were
tested with high-performance liquid chromatography (HPLC) for the declared
sildenafil and tadalafil content. Samples labelled 1 (batch T/33), 3 (batch
T/33), 5 (batch 4), 6 (batch M0016J), 10 (batch T-070235), 12 (batch T-070544),
15 (batch 314833201), 16 (batch 832718474), and 17 (batch 504830028) containing
sildenafil and samples labelled 20 (batch 070356), 21 (batch 05668), and 22
(batch T 378 5) containing tadalafil did not contain the active substance within
the acceptable 95 % to 105 % margin of deviation from the declared content.
While most samples cannot be described as fake with a reasonable amount of
certainty, there is still a suspicion of counterfeit. A correct conclusion can
be drawn only with the assistance of the manufacturers and by conducting
additional laboratory tests. OBJECTIVES: to categorise online suppliers of Viagra based on their legal
status, and to quantify the suppliers within each category.
METHODS: Google was used to search for websites offering to sell or supply
either proprietary Viagra tablets or generic versions containing sildenafil
citrate. Relevant websites were classified as falling into one of three
categories, which were further subclassified. Simple descriptive statistics were
calculated.
KEY FINDINGS: the number of relevant sites found within the first 100 Google
hits, following the removal of mirror and affiliate sites, was 44. Only 6.8% of
sites identified were legitimate online pharmacies. Some 34.1% of sites offered
to sell Viagra to patients in the UK without any form of medical consultation.
Whether or not the online consultation offered by 59.1% of sites had to be
completed in order to make a purchase could not be confirmed. The location of
only three pharmacies could be ascertained; the remainder made various claims as
to their location, which could not be verified.
CONCLUSIONS: we have been unable to verify that the questionnaires used for
online consultations are scrutinised by any healthcare practitioners to
determine the appropriateness of the treatment sought. This represents a serious
safety concern for UK residents who procure drugs for erectile dysfunction on
the internet. BACKGROUND: For patients, the prescription container label may be the only
source of instructions on how to take their medicines. In the United States, the
legal requirements for a prescription label are set by federal law and state
statutes. The container should be comparable to that which manufacturers use to
package drug products and should preserve a product's identity, strength,
quality, and purity and prevent contamination. Safety features such as a
child-resistant closure should be provided. Pharmaceutical products purchased
from international online pharmacies are not approved by the Food and Drug
Administration (FDA) and may not meet US guidelines for labeling and packaging.
OBJECTIVE: The study objective was to determine whether commonly purchased
pharmaceutical products obtained from international online pharmacies are
comparable to products dispensed in the United States with regard to labeling
and packaging.
METHODS: During March 2006 through January 2007, 41 pharmaceutical oral dosage
form samples were obtained from international Internet pharmacy websites for
evaluation: 18 generic simvastatin samples, 18 generic amlodipine samples, and 5
generic sildenafil samples. Contents for each package were observed and recorded
and comparison of the prescription labeling and packaging of these products was
made with prescription labeling and packaging requirements in the United States.
RESULTS: Of the 41 drug products obtained from online pharmacies from 12
different countries, only 1 product (from Canada) would meet both labeling and
packaging guidelines for products dispensed in the United States. Of those not
meeting the requirements, 7 were dispensed in paper envelopes with label affixed
that was either handwritten or typed and contained missing information such as
name and address of dispenser, name of prescriber, name of patient, and
directions for use. Another 3 products did not have a label affixed to the drug
product, but information was printed on a paper document enclosed in the
shipping package, while 28 products did not have labels affixed to the drug
product. In all, 39 of the 41 drug products' packaging would not meet the US
guidelines. Aside from the Canadian product, only 1 product from Mexico was
dispensed in a container that would meet guidelines established in the United
States. In total, 35 products were not dispensed in plastic vials but were
dispensed in unit dose packages, paper envelopes with loose dosage forms,
blister packs of drugs held together with rubber bands, or a combination of
these packaging forms.
CONCLUSIONS: Results suggest that labeling and packaging standards for
international generic drug products are not equivalent to labeling and packaging
standards in the United States. This suggests dissimilar and substandard
distribution processes compared with those in the United States, which in turn
presents a challenge to patient comprehension and health literacy and may affect
patient adherence to drug treatment regimens. These findings have strong
implications for drug product quality, patient outcomes, therapeutic
effectiveness, and safety. |
Which protein interacts with the Ragulator-RAG GTPases to control mTOR activity? | Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery that controls the activation of mTOR. | Cell growth and proliferation are tightly linked to nutrient availability. The
mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of
growth factors, energy levels, glucose and amino acids to modulate metabolic
status and cellular responses. mTORC1 is activated at the surface of lysosomes
by the RAG GTPases and the Ragulator complex through a not fully understood
mechanism monitoring amino acid availability in the lysosomal lumen and
involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human
member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal
membrane-resident protein competent in amino acid transport. Extensive
functional proteomic analysis established SLC38A9 as an integral part of the
Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells
resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired
amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional
component of the amino acid sensing machinery that controls the activation of
mTOR. Author information:
(1)Whitehead Institute for Biomedical Research and Massachusetts Institute of
Technology, Department of Biology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA. Koch Institute for Integrative Cancer
Research, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Broad Institute of
Harvard and Massachusetts Institute of Technology, 7 Cambridge Center,
Cambridge, MA 02142, USA.
(2)Harvard Medical School, 260 Longwood Avenue, Boston, MA 02115, USA.
(3)Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical
School, 220 Longwood Avenue, Boston, MA 02115, USA.
(4)Whitehead Institute for Biomedical Research and Massachusetts Institute of
Technology, Department of Biology, 9 Cambridge Center, Cambridge, MA 02142, USA.
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute
of Technology, Cambridge, MA 02139, USA. Koch Institute for Integrative Cancer
Research, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Broad Institute of
Harvard and Massachusetts Institute of Technology, 7 Cambridge Center,
Cambridge, MA 02142, USA. [email protected]. The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to
many cues, such as nutrient status and energy level. Amino acids induce mTORC1
activation on lysosomes via the small Rag GTPases and the Ragulator complex,
thereby controlling protein translation and cell growth. Here, we identify the
human 11-pass transmembrane protein SLC38A9 as a novel component of the
Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components
on lysosomes and associates with Rag GTPases in an amino acid-sensitive and
nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1
activity in the presence of amino acids and in response to amino acid
replenishment following starvation. Conversely, SLC38A9 overexpression causes
RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1
and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly,
during amino acid starvation mTOR is retained at the lysosome upon SLC38A9
depletion but fails to be activated. Together, the findings of our study reveal
SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to
mTORC1 activity. |
Against which organisms has reverse vaccinology been used? | Reverse Vaccinology (RV) was first applied to serogroup B Neisseria meningitidis. This work induced further research of other pathogens in the same way: Porphyromonas gingivalis, Streptococcus pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, group B streptococci, Helicobacter pylori and Mycobacterium tuberculosis. Concerning animal-affecting organisms, RV has been applied for vaccine design against Theileria parva, Brachyspira hyodysenteriae, Echinococcus granulosus, Ehrlichia ruminantium, Leishmania spp, Rhipicephalus microplus and Brucella melitensis. | During the last century, several approaches have been used for the development
of vaccines, going from the immunization with live-attenuated bacteria up to the
formulation of the safer subunit vaccines. This conventional approach to vaccine
development requires cultivation of the pathogen and its dissection using
biochemical, immunological and microbiological methods. Although successful in
several cases, this method is time-consuming and failed to provide a solution
for many human pathogens. Now genomic approaches allow for the design of
vaccines starting from the prediction of all antigens in silico, independently
of their abundance and without the need to grow the microorganism in vitro. A
new strategy, termed "Reverse Vaccinology", which has been successfully applied
in the last few years, has revolutionized the approach to vaccine research. The
Neisseria meningitidis serogroup B project, the first example of Reverse
Vaccinology, as well as the application of this strategy to develop novel
vaccines against other human pathogens are discussed. BACKGROUND: Since a milestone work on Neisseria meningitidis B, Reverse
Vaccinology has strongly enhanced the identification of vaccine candidates by
replacing several experimental tasks using in silico prediction steps. These
steps have allowed scientists to face the selection of antigens from the
predicted proteome of pathogens, for which cell culture is difficult or
impossible, saving time and money. However, this good example of
bioinformatics-driven immunology can be further developed by improving in silico
steps and implementing biologist-friendly tools.
RESULTS: We introduce NERVE (New Enhanced Reverse Vaccinology Environment), an
user-friendly software environment for the in silico identification of the best
vaccine candidates from whole proteomes of bacterial pathogens. The software
integrates multiple robust and well-known algorithms for protein analysis and
comparison. Vaccine candidates are ranked and presented in a html table showing
relevant information and links to corresponding primary data. Information
concerning all proteins of the analyzed proteome is not deleted along selection
steps but rather flows into an SQL database for further mining and analyses.
CONCLUSION: After learning from recent years' works in this field and analysing
a large dataset, NERVE has been implemented and tuned as the first available
tool able to rank a restricted pool (approximately 8-9% of the whole proteome)
of vaccine candidates and to show high recall (approximately 75-80%) of known
protective antigens. These vaccine candidates are required to be "safe" (taking
into account autoimmunity risk) and "easy" for further experimental,
high-throughput screening (avoiding possibly not soluble antigens). NERVE is
expected to help save time and money in vaccine design and is available as an
additional file with this manuscript; updated versions will be available at
http://www.bio.unipd.it/molbinfo. Swine dysentery (SD) is a mucohaemorrhagic colitis of pigs resulting from
infection of the large intestine with the anaerobic intestinal spirochaete
Brachyspira hyodysenteriae. Whole-cell bacterin vaccines are available to help
control SD, but their performance has been inconsistent. This study aimed to use
a reverse vaccinology approach to identify B. hyodysenteriae proteins for use as
recombit vaccine components. Nineteen open reading frames (ORFs) predicted to
encode potential vaccine candidate molecules were identified from in silico
analysis of partial genomic sequence data. The distribution of these ORFs among
strains of B. hyodysenteriae was investigated by PCR, and widely distributed
ORFs were cloned. The products were screened with a panel of immune pig sera,
and from these a subset of conserved, immunogenic proteins was selected. Mice
immunized intramuscularly with these recombit proteins developed specific
systemic antibody responses to them, and their sera agglutinated B.
hyodysenteriae cells in vitro. In a pilot experiment, eight pigs were vaccinated
twice intramuscularly with a combination of four of the proteins. The pigs
developed antibodies to the proteins, and following experimental challenge only
one developed SD compared to five of nine non-vaccinated control pigs. Although
these differences in incidence were not significant, they indicated a trend
towards protection using the recombit proteins as immunogens. This study
demonstrates that the reverse vaccinology approach has considerable potential
for use in developing novel recombit vaccines for SD. Applying reverse vaccinology strategy, we employed a sequence encoding an
enolase from Taenia asiatica to search its homolog in the expression sequence
tag (EST) database of Echinococcus granulosus and found two EST sequences
(Access number: CN653186 and CN649593) of a clone Eg_PSGRS_13B09 from E.
granulosus protoscolex full-length cDNA library, which are responding for the 5'
and 3' partial cds of E. granulosus enolase, respectively. Primers are designed
according to the 5' end and 3'end of the putative encoding sequence to amplify
the genomic DNA of E. granulosus strain isolated from sheep in Qinghai province
of China by polymerase chain reaction (PCR). A sole product of 1,449 bp in
length was obtained, which contains two little introns of 78 bp and 69 bp,
respectively. The introns were excised by unsymmetrical PCR with combined flank
sequences of introns as primers. The structural, functional, and immunological
characteristics of putative amino acid sequence were predicted by bioinformatics
analysis. The complete coding sequence was predicted to encode 433 residues and
contain a transmembrane region aa(104-124), with the N terminus outside and C
terminus inside. The inside part is quite the functional domain. SWISS-MODEL
modulated its 3D structure as a barrel which constitutes of alternatively
arranged alpha helix-beta sheet, with the key sites such as substrate binding
region, active sites, Mg(2+)-binding sites closely located at the center. The
protein contains a potential nuclear localization sequence aa(190-199) and
several linear B cell epitopes and CTL T cell epitopes, of which the outside
epitope aa(49-57) and inside epitope aa(228-236) are facultative T cell and B
cell epitope, and the linear B cell epitope aa(206-213) contains the active
center site Glu(210), suggesting the putative protein is a potential membrane
with strong immunogenicity. The complete cds was expressed in Escherichia coli,
and the recombit protein can be recognized by the serum from patient infected
with E. granulosus. Reverse vaccinology process identified E. granulosus
tegumental membrane protein enolase as vaccine candidate. Leishmaniasis is a group of diseases with a spectrum of clinical manifestations
ranging from cutaneous ulcers to visceral leishmaniasis, which results from the
bite of an infected sandfly to human. Attempts to develop an effective vaccine
have been shown to be feasible but no vaccine is in active clinical use. This
study adopts a Reverse Vaccinology approach to identify common vaccine
candidates from both highly pathogenic species Leishmania major and Leishmania
infantum. Total proteome of both species were compared to identify common
proteins, which are further taken for sub-cellular localization and
transmembrane helices prediction. Plasma membrane proteins having only one
transmembrane helix were first identified and analyzed which are non-homologous
in human and mouse in order to avoid molecular mimicry with other proteins.
Selected proteins were analyzed for their binding efficiency to both major
histocompatibility complex (MHC) class I and class II alleles. As a result, 19
potential epitopes are screened in this study using different approaches, which
can be further verified through in vivo experiments in MHC compatible animal
models. This study demonstrates that Reverse Vaccinology approach has potential
in discovering various immunogenic antigens from in silico analysis of
pathogen's genome or proteome instead of culturing the whole organism by
conventional methods. Herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) are the most common
infectious agents of humans. No safe and effective HSV vaccines have been
licensed. Reverse vaccinology is an emerging and revolutionary vaccine
development strategy that starts with the prediction of vaccine targets by
informatics analysis of genome sequences. Vaxign
(http://www.violinet.org/vaxign) is the first web-based vaccine design program
based on reverse vaccinology. In this study, we used Vaxign to analyze 52
herpesvirus genomes, including 3 HSV-1 genomes, one HSV-2 genome, 8 other human
herpesvirus genomes, and 40 non-human herpesvirus genomes. The HSV-1 strain 17
genome that contains 77 proteins was used as the seed genome. These 77 proteins
are conserved in two other HSV-1 strains (strain F and strain H129). Two
envelope glycoproteins gJ and gG do not have orthologs in HSV-2 or 8 other human
herpesviruses. Seven HSV-1 proteins (including gJ and gG) do not have orthologs
in all 40 non-human herpesviruses. Nineteen proteins are conserved in all human
herpesviruses, including capsid scaffold protein UL26.5 (NP_044628.1). As the
only HSV-1 protein predicted to be an adhesin, UL26.5 is a promising vaccine
target. The MHC Class I and II epitopes were predicted by the Vaxign Vaxitop
prediction program and IEDB prediction programs recently installed and
incorporated in Vaxign. Our comparative analysis found that the two programs
identified largely the same top epitopes but also some positive results
predicted from one program might not be positive from another program. Overall,
our Vaxign computational prediction provides many promising candidates for
rational HSV vaccine development. The method is generic and can also be used to
predict other viral vaccine targets. Reverse vaccinology (RV), the first application of genomic technologies in
vaccine research, represented a major revolution in the process of discovering
novel vaccines. By determining their entire antigenic repertoire, researchers
could identify protective targets and design efficacious vaccines for pathogens
where conventional approaches had failed. Bexsero, the first vaccine developed
using RV, has recently received positive opinion from the European Medicines
Agency. The use of RV initiated a cascade of changes that affected the entire
vaccine development process, shifting the focus from the identification of a
list of vaccine candidates to the definition of a set of high throughput screens
to reduce the need for costly and labor intensive tests in animal models. It is
now clear that a deep understanding of the epidemiology of vaccine candidates,
and their regulation and role in host-pathogen interactions, must become an
integral component of the screening workflow. Far from being outdated by
technological advancements, RV still represents a paradigm of how
high-throughput technologies and scientific insight can be integrated into
biotechnology research. Brucella is the etiologic agent of brucellosis, one of the most common and
widely distributed zoonotic diseases. Its highly infectious nature, the
insidious, systemic, chronic, debilitating aspects of the disease and the lack
of an approved vaccine for human use in the United States are features that make
Brucella a viable threat to public health. One of the main impediments to
vaccine development is identification of suitable antigens. In order to identify
antigens that could potentially be used in a vaccine formulation, we describe a
multi-step antigen selection approach. We initially used an algorithm (Vaxign)
to predict ORF encoding outer membrane proteins with antigenic determits.
Differential gene expression during acute infection and published evidence for a
role in virulence were used as criteria for down-selection of the candidate
antigens that resulted from in silico prediction. This approach resulted in the
identification of nine Brucella melitensis outer membrane proteins, 5 of which
were recombitly expressed and used for validation. Omp22 and Hia had the
highest in silico scores for adhesin probability and also conferred invasive
capacity to E. coli overexpressing recombit proteins. With the exception of
FlgK in the goat, all proteins reacted to pooled sera from exposed goats, mice,
and humans. BtuB, Hia and FlgK stimulated a mixed Th1-Th2 response in
splenocytes from immunized mice while BtuB and Hia elicited NO release from
splenocytes of S19 immunized mice. The results support the applicability of the
current approach to the identification of antigens with immunogenic and invasive
properties. Studies to assess immunogenicity and protective efficacy of
individual proteins in the mouse are currently underway. |
Galassi classification is used for which disorder? | Galassi classification system is used to classify arachnoid cysts. | The best operative intervention for children with arachnoid cysts remains the
subject of controversy. Recent reports stress that craniotomy for cyst
fenestration is associated with a low incidence of morbidity and mortality and
may leave the child shunt-independent. Among a total of 66 intracranial
arachnoid cysts operated on in the authors' department from 1985 to 1997, 44
cases (67%) were located in the middle cranial fossa. A higher incidence in the
first decade of life (53 cases) and a marked male predomice (45 cases) were
recognized. Headache, cranial deformities, symptoms of raised intracranial
pressure, and seizures constituted the most frequent features of the clinical
presentation. To determine which treatment provides the greatest benefit with
the lowest incidence of complications, the records of the 44 patients with
arachnoid cysts in the middle cranial fossa were reviewed. The mean age of these
patients was 4.6 years (range 0-16 years). Different types of initial surgical
procedures were performed. In 33 patients with middle cranial fossa arachnoid
cysts (MCFAC) the initial surgery took the form of craniotomy with excision of
the cyst walls and fenestration into the basal cisterns. Shunting procedures
were performed in 9 patients: cysto-peritoneal shunts (CPS) were placed in 4
patients and ventriculo-peritoneal shunts (VPS), in 3 patients, and cyst
excision was performed in addition to CPS in 2 patients. Excision of the cyst
membrane alone without fenestration was performed in 2 patients. The initial
treatment was successful in terms of reduced symptoms and decreased cyst size,
with no additional treatment needed for the cyst, in 79% (26/33) of patients who
had undergone excision of the cyst walls and fenestration into the basal
cisterns, compared with 66% (6/9) of patients who had undergone shunting
procedures. Cyst membrane excision was not successful in any of the patients who
underwent this procedure alone. No significant difference in morbidity was noted
between these different treatment options. On follow-up CT scan and MRI, cysts
of types I and II (Galassi classification) exhibited a steady tendency to
reduction or obliteration. These results confirm that radical excision of the
outer and inner membranes of the cyst wall with fenestration into the basal
cistern is a safe and effective shunt-independent procedure for MCFAC,
especially for those of types I and II. The authors present CT and MRI of a patient with an extremely large arachnoidal
cyst (Galassi classification type III). The cyst extended from the base of the
skull, posterior to the brain stem, on the base of the temporal lobe over the
complete convexity of the left hemisphere. The cyst consisted of multiple
compartments with intracystic septa and was accompanied by a chronic subdural
hemorrhage in the compartments. After contrast agent application, the typical
characteristics of chronic subdural membranes were found. Besides bony
deformities, a thinning of the inner table was found. The patient underwent
craniotomy for evacuation of the hemorrhage and fenestration of the septa while
he was free of symptoms. This is a remarkable case proving that chronic local
intracranial pressure does not inevitably lead to neurologic symptoms or
intellectual disabilities. BACKGROUND: The decision making process for patients with Sylvian fissure
arachnoid cysts still represents a challenge for the neurosurgeon. A high
percentage of patients are indeed asymptomatic, despite neuroimaging signs of
apparently increased intracranial pressure (ICP). The present study was
conducted to evaluate the usefulness of prolonged ICP recording in the
preoperative work-up.
METHODS: Eleven children (10 M/1 F) harboring temporal arachnoid cysts were
investigated (mean age: 5.9 years). According to Galassi classification they
were subdivided in three groups. Group I was made up of three patients with Type
I cysts; Group II comprised six children with Type II cysts; Group III consisted
of two children with Type III cysts. An extensible silicone microprocessor
(Codman), developed for continuous ICP recording, was implanted
intraparenchymally, adjacent to the major extension of the cyst. The collected
information was cable unloaded to a PC and stored. All the patients underwent a
minimum of 48 hours to a maximum of 72 hours of ICP continuous check. In resting
conditions, 10 mm Hg was arbitrarily chosen as the upper normal limit of ICP,
and the patients were classified according to the percentile distribution of
their ICP daily and nightly pressure values.
RESULTS: ICP recordings were in the normal range in all the three children with
Type I cysts (49-86%< 10 mm Hg during the entire recording), despite the fact
that two of them were apparently symptomatic. Three of the six children with a
Type II temporal cyst had elevated ICP values (69-99%> 10 mm Hg), even though
they had an incidental (1 case) or prenatal (2 cases) diagnosis. The remaining
three patients had normal ICP values for more than 70% of the recording time
period. The two patients with type III temporal arachnoid cysts had almost
constantly abnormal ICP values (95-99%> 10 mm Hg). Five patients were operated;
in four of them the surgical indication was based on ICP recordings (two of the
three children with a Type II cyst and increased ICP and the two patients with a
Type III cyst). The last child, harboring a Type I cyst, was operated upon
parents' request, as a preventive measure. At a mean follow-up of 9.3 months all
the patients operated on are in excellent clinical condition; one of them (Type
II cyst) initially undergoing a craniotomy and cystic membrane excision required
a subduro-peritoneal shunt implant 3 months after surgery for a symptomatic
subdural hygroma on the side of the cyst. Postoperative computed tomography
showed signs of brain expansion in the two patients operated on for a Type III
cyst, and in the patient operated on for a Type I cyst.
CONCLUSIONS: With the limit of the relatively small series presented here,
prolonged ICP recording appeared to be an important preoperative tool to rule
out the necessity of operating on children with Type I cysts. Furthermore, the
study confirmed that an increase of the ICP is almost constantly present in
children with Type III cysts. In cases of Type II lesions, the study results
were less discriminating, though the ICP monitoring contributed in identifying
those patients in whom surgery was indicated despite the absence of symptoms. |
Describe Mozart effect. | The Mozart effect implies the enhancement of reasoning skills solving spatial problems in normal subjects after listening to Mozart's piano sonata K 448. | The Mozart effect is an increase in spatial reasoning scores detected
immediately after listening to the first movement of a Mozart piano sonata.
Rauscher and Shaw (1998) suggested that failure to produce a Mozart effect could
arise from carryover effects of a spatial reasoning pretest which may interfere
with the effect of listening to Mozart. They cited an unpublished study in which
a verbal distractor was inserted between the pretest and listening condition,
and the manipulation produced the recovery of a Mozart effect. This experiment
attempted to confirm the unpublished study. 206 college students were exposed to
one of three sequences, pretest-Verbal distractor material-Mozart,
pretest-Mozart-Verbal distractor material, and pretest-Verbal distractor
material. An immediate posttest indicated no significant difference on solution
of paper folding and cutting items among the three groups. The results do not
support Rauscher and Shaw (1998). Our negative results are consistent with prior
failures in other laboratories to produce a Mozart effect. The "Mozart effect" refers to claims that people perform better on tests of
spatial abilities after listening to music composed by Mozart. We examined
whether the Mozart effect is a consequence of between-condition differences in
arousal and mood. Participants completed a test of spatial abilities after
listening to music or sitting in silence. The music was a Mozart sonata (a
pleasant and energetic piece) for some participants and an Albinoni adagio (a
slow, sad piece) for others. We also measured enjoyment, arousal, and mood.
Performance on tbe spatial task was better following the music than the silence
condition but only for participants who heard Mozart. The two music selections
also induced differential responding on the enjoyment, arousal and mood
measures. Moreover, when such differences were held constant by statistical
means, the Mozart effect disappeared. These findings provide compelling evidence
that the Mozart effect is an artifact of arousal and mood. The "Mozart effect" is the tendency to score higher on spatiotemporal IQ
subscales following exposure to complex music such as Mozart's Sonata K.448.
This phenomenon was investigated in 20 musicians and 20 nonmusicians. The trion
model predicts increased synchrony between musical and spatiotemporal centres in
the right cerebral hemisphere. Since increased left-hemispheric involvement in
music processing occurs as a result of music training, predictions deriving from
the possibility of increased synchrony with left-hemispheric areas in musicians
were tested. These included improved performance on language as well as
spatiotemporal tasks. Spatiotemporal, synonym generation, and rhyming word
generation tasks were employed as was the Mozart Sonata K.448. A Mozart effect
was demonstrated on the spatiotemporal task, and the facilitatory effect of
exposure to Mozart was greater for the nonmusician group. This finding adds to
the robustness of the Mozart effect since novel tasks were used. No Mozart
effect was found for either group on the verbal tasks, although the musicians
scored higher on rhyming word generation. This new finding adds to the number of
nonmusical tasks apparently showing long-term benefits from music training.
However, no systematic link was found between performance on any task and number
of years spent in music training. The failure to induce a Mozart effect in the
musician group on verbal tasks, as well as that group's limited facilitation on
spatiotemporal tasks, may be associated with either a ceiling effect due to the
long-term effects of music training or from methodological factors. Both
possibilities are discussed. Mozart's Sonata for two pianos in D major, K.448 (Mozart K.448), has been shown
to improve mental function, leading to what is known as the Mozart effect. Our
previous work revealed that epileptiform discharges in children with epilepsy
decreased during and immediately after listening to Mozart K.448. In this study,
we evaluated the long-term effects of Mozart K.448 on children with refractory
epilepsy. Eleven children with refractory epilepsy were enrolled. All of the
patients were diagnosed as having had refractory epilepsy for more than 1 year
(range =1 year to 6 years 4 months, mean =3 years 11 months) and had been
receiving at least two antiepileptic drugs (AED). During the study period, they
listened to Mozart K.448 once a day before bedtime for 6 months. Seizure
frequencies were recorded 6 months before they started listening to this music
and monthly during the study period. All of the patients remained on the same
AEDs during the 6-month study period. Frequencies of seizures were compared
before and after listening to Mozart K.448. Eight of eleven patients were
seizure free (N=2) or had very good responses (N=6) after 6 months of listening
to Mozart K.448. The remaining three (27.3%) showed minimal or no effect
(effectiveness <50%; unmodified or worsened seizure frequency). The average
seizure reduction was 53.6 ± 62.0%. There were no significant differences in
seizure reduction with IQ, etiology, or gender. We conclude that Mozart K.448
should be further studied as a potential add-on therapy in the treatment of
children with refractory epilepsy. Mozart's Sonata for Two Pianos in D major, K.448 (Mozart K.448), has been shown
to improve mental function, leading to what is known as the Mozart Effect. Our
previous work revealed that epileptiform discharges in children with epilepsy
decrease during and right after listening to Mozart K.448. However, the duration
of the effect was not studied. In the study described here, we evaluated the
long-term effect of Mozart K.448 on epileptiform discharges in children with
epilepsy. Eighteen children with epilepsy whose seizures were clinically well
controlled with antiepileptic drugs were included. For each child, EEGs had
revealed persistent epileptiform discharges for at least 6 months. These
patients listened to Mozart K.448 for 8 minutes once a day before bedtime for 6
months. Epileptiform discharges were recorded and compared before and after 1,
2, and 6 months of listening to Mozart K.448. All of the children remained on
the same antiepileptic drug over the 6 months. Relationships between number of
epileptiform discharges and foci of discharges, intelligence, epilepsy etiology,
age, and gender were analyzed. Epileptiform discharges significantly decreased
by 53.2±47.4, 64.4±47.1, and 71.6±45.8%, respectively, after listening to Mozart
K.448 for 1, 2, and 6 months. All patients except those with occipital
discharges showed a significant decrease in epileptiform discharges. Patients
with normal intelligence and idiopathic epilepsy had greater decreases than
those with mental retardation and symptomatic epilepsy. Age and gender did not
affect the results. We conclude that long-term listening to Mozart K.448 may be
effective in decreasing epileptiform discharges in children with epilepsy in a
chronologically progressive manner. The Mozart Effect is a phenomenon whereby certain pieces of music induce
temporary enhancement in "spatial temporal reasoning." To determine whether the
Mozart Effect can improve surgical performance, 55 male volunteers (mean age =
20.6 years, range = 16-27), novice to surgery, were timed as they completed an
activity course on a laparoscopic simulator. Subjects were then randomized for
exposure to 1 of 2 musical pieces by Mozart (n = 21) and Dream Theater (n = 19),
after which they repeated the course. Following a 15-minute exposure to a
nonmusical piece, subjects were exposed to one of the pieces and performed the
activity course a third time. An additional group (n = 15) that was not
corandomized performed the tasks without any exposure to music. The percent
improvements in completion time between 3 successive trials were calculated for
each subject and group means compared. In 2 of the tasks, subjects exposed to
the Dream Theater piece achieved approximately 30% more improvement (26.7 ±
8.3%) than those exposed to the Mozart piece (20.2 ± 7.8%, P = .021) or to no
music (20.4 ± 9.1%, P = .049). Distinct patterns of covariance between baseline
performance and subsequent improvement were observed for the different musical
conditions and tasks. The data confirm the existence of a Mozart Effect and
demonstrate for the first time its practical applicability. Prior exposure to
certain pieces may enhance performance in practical skills requiring spatial
temporal reasoning. OBJECTIVE: Listening to Mozart K.448 has been demonstrated to improve spatial
task scores, leading to what is known as the Mozart effect. Our previous work
revealed the positive effects of Mozart K.448 in reducing epileptiform
discharges in children with epilepsy. However, the mechanism remains unclear.
parasympathetic activation has been shown to help seizure control in many
studies. In this study, we investigated the effect of Mozart music on
epileptiform discharges and autonomic activity.
METHODS: Sixty-four epileptic children with epileptiform discharges were
included. They all received electroencephalogram and electrocardiogram
examinations simultaneously before, during, and after listening to Mozart K.448
or K.545. The total number of epileptiform discharges during each session
(before, during, and after music) were divided by the duration (in minutes) of
the session and then compared. Heart rate variability including time and
frequency domain analysis was used to represent the autonomic function.
RESULTS: The results showed that epileptiform discharges were significantly
reduced during and right after listening to Mozart music (33.3 ± 31.1%
reduction, p<0.001, during Mozart K.448 and 38.6 ± 43.3% reduction, p<0.001,
during Mozart K.545) (28.1 ± 43.2% reduction, p<0.001, after Mozart K.448 and
46.0 ± 40.5% reduction, p<0.001, after Mozart K.545). No significant difference
was noticed between the two pieces of music. The reduction was greatest in
patients with generalized seizures and discharges. Significant increases in
high-frequency (HF), the square root of the mean squared differences of
successive RR intervals (RMSSD), the standard deviation of differences between
adjacent RR intervals (SDSD), and a decrease in mean beats per minute (bpm) were
found during listening to Mozart music. Most of the patients with reduced
epileptiform discharges also showed a decreased LF/HF ratio, low-frequency
normalized units (LF nu), mean bpm, and an increased high-frequency normalized
units (HF nu).
CONCLUSIONS: Listening to Mozart music decreased epileptiform discharges in
children with epilepsy. The majority of these patients showed an increase in
parasympathetic tone during music exposure.
SIGNIFICANCE: Our results suggested that Mozart music stimuli induced
parasympathetic activation which may be involved in the effect of music in
reducing epileptiform discharges and the recurrence rate of seizures. Converging evidence suggests an association between spatial and music domains. A
cerebellar role in music-related information processing as well as in
spatial-temporal tasks has been documented. Here, we investigated the cerebellar
role in the association between spatial and musical domains, by testing
performances in embodied (EMR) or abstract (AMR) mental rotation tasks of
subjects listening Mozart Sonata K.448, which is reported to improve
spatial-temporal reasoning, in the presence or in the absence of continuous
theta burst stimulation (cTBS) of the left cerebellar hemisphere. In the absence
of cerebellar cTBS, music listening did not influence either MR task, thus not
revealing a "Mozart Effect". Cerebellar cTBS applied before musical listening
made subjects faster (P = 0.005) and less accurate (P = 0.005) in performing the
EMR but not the AMR task. Thus, cerebellar inhibition by TBS unmasked the effect
of musical listening on motor imagery. These data support a coupling between
music listening and sensory-motor integration in cerebellar networks for
embodied representations. According to the first publication in 1993 by Rauscher et al. [Nature
1993;365:611], the Mozart effect implies the enhancement of reasoning skills
solving spatial problems in normal subjects after listening to Mozart's piano
sonata K 448. A further evaluation of this effect has raised the question
whether there is a link between music-generated emotions and a higher level of
cognitive abilities by mere listening. Positron emission tomography and
functional magnetic resoce imaging have revealed that listening to
pleasurable music activates cortical and subcortical cerebral areas where
emotions are processed. These neurobiological effects of music suggest that
auditory stimulation evokes emotions linked to heightened arousal and result in
temporarily enhanced performance in many cognitive domains. Music therapy
applies this arousal in a clinical setting as it may offer benefits to patients
by diverting their attention from unpleasant experiences and future
interventions. It has been applied in the context of various important clinical
conditions such as cardiovascular disorders, cancer pain, epilepsy, depression
and dementia. Furthermore, music may modulate the immune response, among other
things, evidenced by increasing the activity of natural killer cells,
lymphocytes and interferon-γ, which is an interesting feature as many diseases
are related to a misbalanced immune system. Many of these clinical studies,
however, suffer from methodological inadequacies. Nevertheless, at present,
there is moderate but not altogether convincing evidence that listening to known
and liked music helps to decrease the burden of a disease and enhances the
immune system by modifying stress. OBJECTIVE: Listening to Mozart K.448 has been demonstrated to improve spatial
task scores, leading to what is known as the Mozart Effect. However, most of
these reports only describe the phenomena but lack the scientific evidence
needed to properly investigate the mechanism of Mozart Effect. In this study, we
used electroencephalography (EEG) and heart rate variability (HRV) to evaluate
the effects of Mozart K.448 on healthy volunteers to explore Mozart Effect.
DESIGN: An EEG-based post-intervention analysis.
SETTING: Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
PARTICIPANTS: Twenty-nine college students were enrolled. They received EEG and
electrocardiogram examinations simultaneously before, during and after listening
to the first movement of Mozart K.448.
MAIN OUTCOME MEASURE: EEG alpha, theta and beta power and HRV were compared in
each stage.
RESULTS: The results showed a significant decrease in alpha, theta and beta
power when they listened to Mozart K.448. In addition, the average root mean
square successive difference, the proportion derived by dividing NN50 by the
total number of NN intervals, standard deviations of NN intervals and standard
deviations of differences between adjacent NN intervals showed a significant
decrease, while the high frequency revealed a significant decrease with a
significantly elevated low-frequency/high-frequency ratio.
CONCLUSION: Listening to Mozart K.448 significantly decreased EEG alpha, theta
and beta power and HRV. This study indicates that there is brain cortical
function and sympathetic tone activation in healthy adults when listening to
Mozart K.448, which may play an important role in the mechanism of Mozart
Effect. |
What is the purpose of the Tokuhashi scoring system? | Tokuhashi scoring system was developed to predict life expectancy of patients with spinal metastases. The revised Tokuhashi score has been widely used to evaluate indications for surgery and predict survival in patients with metastatic spinal disease. | BACKGROUND & OBJECTIVE: Clinically, whether and how to make a surgical
interventional decision for the patients with spinal metastases is still
controversial. Life expectancy is a significant determit in the selection of
surgical procedure for spinal metastases. This study was to evaluate Tomita and
Tokuhashi scoring systems in selecting surgical procedure and predicting
prognosis of extradural spinal metastases.
METHODS: A total of 169 patients with spinal metastases, treated in the Spine
Unit of Aarhus University Hospital, Denmark, from Jan. 2001 to Apr. 2004, were
enrolled. The life expectancy was scored according to both Tomita system and
Tokuhashi system before operation, the spinal metastases were classified
according to Tomita system, and the patients underwent surgery accordingly.
Follow-up was done 6, 12, and 24 months after operation. The precise of Tomita
system and Tokuhashi system in estimating "death within 3 months", "death within
6 months", and "death within 12 months" was compared using Receiver Operating
Characteristic curves (ROC curves). The mean survival time of the patients was
calculated by Kaplan-Meier method.
RESULTS: ROC curves of "death within 3 months", "death within 6 months", and
"death within 12 months" showed no significant difference between Tomita score
and Tokuhashi score in each group (P = 0.16, P = 0.47, and P = 0.38,
respectively). Kaplan-Meier survival curves showed that Tomita system
overestimated the prognosis in scores from 4 to 7, and Tokuhashi system
underestimated the prognosis in scores from 0 to 8.
CONCLUSIONS: Both Tomita and Tokuhashi scoring systems could be used to predict
prognosis of spinal metastases after operation. Tokuhashi scoring system can
predict early death more accurately, which can be used to avoid major operation
for these patients. OBJECTIVE: To evaluate the predictive value of the Tokuhashi revised scoring
system for the life expectancy of patients with spinal metastases.
METHODS: A retrospective review of 447 patients with spinal metastases was
performed, which comprised 291 men and 156 women with a mean age of 56. 1 years.
All of the patients were scored with the Tokuhashi revised scoring system based
on the available clinical, pathological and radiographic data. The relation
between the survival time and the Tokuhashi revised score was analyzed.
RESULTS: The patients had a median survival time of 7. 9 months and a mean
Tokuhashi revised score of 8. 23 points. The median survival time of 155
patients with high grade primary tumor of lung, liver, gastrointestinal tract,
esophagus, bladder and pancreas was 4. 7 months. The median survival time of 146
patients with low grade primary tumor of thyroid, breast and prostate was 12
months. The median survival time of 146 patients with medium grade primary tumor
of kidney, lymphoma, ovary and uterus, and unknown primary tumor was 7. 1
months. The median survival time of 211 patients with the Tokuhashi revised
score of 0 to 8 points was 4 months. The median survival time of 147 patients
with the Tokuhashi revised score of 9 to 11 points was 10 months. The median
survival time of 89 patients with the Tokuhashi revised score of 12 to 15 points
was 29 months. The differences between the groups were significant (P<0. 01 or
0. 0001). The Tokuhashi revised score was positively correlated with survival
time (r=0. 833, P<0. 001).
CONCLUSION: The Tokuhashi revised score could support decision making with
reliable estimation of life expectancy of patients with spinal metastases.
Surgery could be a better choice to extend life span for those patients with
solitary spinal metastasis of slow-growth primary tumor and those with the
Tokuhashi revised score of 12 to 15 points. BACKGROUND: The spinal metastasis occurs in up to 40% of cancer patient. We
compared the Tokuhashi and Tomita scoring systems, two commonly used scoring
systems for prognosis in spinal metastases. We also assessed the different
variables separately with respect to their value in predicting postsurgical life
expectancy. Finally, we suggest criteria for selecting patients for surgery
based on the postoperative survival pattern.
MATERIALS AND METHODS: We retrospectively analyzed 102 patients who had been
operated for metastatic disease of the spine. Predictive scoring was done
according to the scoring systems proposed by Tokuhashi and Tomita. Overall
survival was assessed using Kaplan-Meier survival analysis. Using the log rank
test and Cox regression model we assessed the value of the individual components
of each scoring system for predicting survival in these patients.
RESULT: The factors that were most significantly associated with survival were
the general condition score (Karnofsky Performance Scale) (P=.000, log rank
test), metastasis to internal organs (P=.0002 log rank test), and number of
extraspinal bone metastases (P=.0058). Type of primary tumor was not found to be
significantly associated with survival according to the revised Tokuhashi
scoring system (P=.9131, log rank test). Stepwise logistic regression revealed
that the Tomita score correlated more closely with survival than the Tokuhashi
score.
CONCLUSION: The patient's performance status, extent of visceral metastasis, and
extent of bone metastases are significant predictors of survival in patients
with metastatic disease. Both revised Tokuhashi and Tomita scores were
significantly correlated with survival. A revised Tokuhashi score of 7 or more
and a Tomita score of 6 or less indicated >50% chance of surviving 6 months
postoperatively. We recommend that the Tomita score be used for prognostication
in patients who are contemplating surgery, as it is simpler to score and has a
higher strength of correlation with survival than the Tokuhashi score. STUDY DESIGN: We conducted a prospective cohort study of 448 patients with
spinal metastases from a variety of cancer groups.
OBJECTIVE: To determine the specific predictive value of the Tokuhashi scoring
system (T12) and its revised version (T15) in spinal metastases of various
primary tumors.
SUMMARY OF BACKGROUND DATA: The life expectancy of patients with spinal
metastases is one of the most important factors in selecting the treatment
modality. Tokuhashi et al formulated a prognostic scoring system with a total
sum of 12 points for preoperative prediction of life expectancy in 1990 and
revised it in 2005 to a total sum of 15 points. There is a lack of knowledge
about the specific predictive value of those scoring systems in patients with
spinal metastases from a variety of cancer groups.
METHODS: We included 448 patients with vertebral metastases who underwent
surgical treatment during November 1992 to November 2009 in Aarhus University
Hospital NBG. Data were retrieved from Aarhus Metastases Database. Scores based
on T12 and T15 were calculated prospectively for each patient. We divided all
the patients into different groups dictated by the site of their primary tumor.
Predictive value and accuracy rate of the 2 scoring systems were compared in
each cancer group.
RESULTS: Both the T12 and T15 scoring systems showed statistically significant
predictive value when the 448 patients were analyzed in total (T12, P < 0.0001;
T15, P < 0.0001). The accuracy rate was significantly higher in T15 (P < 0.0001)
than in T12. The further analyses by primary cancer groups showed that the
predictive value of T12 and T15 was primarily determined by the prostate (P =
0.0003) and breast group (P = 0.0385). Only T12 displayed predictive value in
the colon group (P = 0.0011). Neither of the scoring systems showed significant
predictive value in the lung (P > 0.05), renal (P > 0.05), or miscellaneous
primary tumor groups (P > 0.05). The accuracy rate of prognosis in T15 was
significantly improved in the prostate (P = 0.0032) and breast group (P <
0.0001).
CONCLUSION: Both T12 and T15 showed significant predictive value in patients
with spinal metastases. T15 has a statistically higher accuracy rate than T12.
Among the various cancer groups, the 2 scoring systems are especially reliable
in prostate and breast metastases groups. T15 is recommended as superior to T12
because of its higher accuracy rate. BACKGROUND: One of the most important selection criteria for spinal metastases
surgery is life expectancy and the most important system for this prediction has
been proposed by Tokuhashi. The aim of this study was to evaluate predictive
value of the Tokuhashi score for life expectancy in Iranian patients with spinal
metastases one year after diagnosis.
METHODS: From February 2007 to March 2009, of 180 patients suffering spinal
metastatic tumors, 71 patients were excluded. This left a study population of
109 patients with known maligt metastases to spine (56 females and 53 males;
mean age, 57 ± 12 years). Tokuhashi revised evaluation system for the prognosis
of metastatic spinal tumors was used for all patients. The survival period
predicted by this system for the prognosis and the actual survival period after
1 year follow up were evaluated.
RESULTS: The predicted survival according to Tokuhashi prognostic scoring system
was less than 6 months in 38(34.9%) patients, 6-12 months in 39(35.8%) patients
and 1 year or more in 32(29.4%) patients. 39 (35.8%) patients died at first
six-month of the follow up, 28(25.7%) patients at the second six-month period
and 42(38.5%) patients were alive at the end of the year. There was no
significant difference between predicted and actual survival time (p = 0.116).
CONCLUSIONS: Present study showed that the Tokuhashi revised scoring system may
be practicable and highly predictive preoperative scoring system for patients
with spinal metastases in Iran. Assessing the prognosis before treatment for metastatic spine tumor is extremely
important in therapy selection. Therefore, we review some prognostic scoring
systems and their outcomes. Articles with combinations of two keywords among
"metastatic spine tumor" and "prognosis", "score", "scoring system",
"predicting", or "life expectancy" were searched for in PubMed. As a result, 236
articles were extracted. Those referring to representative scoring systems about
predicting the survival of patients with metastatic spine tumors were used. The
significance and limits of these scoring systems, and the future perspectives
were described. Tokuhashi score, Tomita score, Baur score, Linden score, Rades
score, and Katagiri score were introduced. They are all scoring systems prepared
by combining factors that affect prognosis. The primary site of cancer and
visceral metastasis were common factors in all of these scoring systems. Other
factors selected to influence the prognosis varied. They were useful to roughly
predict the survival period, such as, "more than one year or not" or "more than
six months or not". In particular, they were utilized for decision-making about
operative indications and avoidance of excessive medical treatment. Because the
function depended on the survival period in the patients with metastatic spine
tumor, it was also utilized in assessing functional prognosis. However, no
scoring system had more than 90% consistency between the predicted and actual
survival periods. Future perspectives should adopt more oncological viewpoints
with adjustment of the process of treatment for metastatic spine tumor. BACKGROUND: We sought to identify preoperative factors significantly correlated
with survival. We also aimed to evaluate the validity of the prognostic scores
in the Tomita and Tokuhashi systems and discuss several aspects to improve the
predictive accuracy of these systems. Moreover, we suggest modified criteria for
selecting treatment strategies.
METHODS: In total, the outcomes of 112 patients with spinal metastasis who
underwent surgery between January 2006 and June 2011 were retrospectively
reviewed. The validity of the prognostic scores was assessed on the basis of
their correlation with survival. For various primary maligcies, new scoring
criteria were applied in each system according to the survival results obtained
in this study. Each revised scoring system was adjusted with a similar principle
of scoring as described previously. Patient survival according to each
preoperative factor was analyzed by the Kaplan-Meier method. The predictive
value of each scoring system was evaluated by the log-rank test and Cox
regression analysis.
RESULTS: The interval from the diagnosis of the primary maligcy to that of
spinal metastasis (p = 0.023) and the interval from the diagnosis of spinal
metastasis to surgery (p = 0.039) were significantly correlated with survival.
Regarding Tokuhashi scores, the correlation coefficient was 0.790 before
adjustment (p = 0.001) and 0.853 after adjustment (p < 0.001). For Tomita
scores, the correlation coefficient was -0.994 (p < 0.001) both before and after
adjustment.
CONCLUSIONS: Tomita scores more accurately predicted survival than Tokuhashi
scores. It is helpful to evaluate both scoring systems with adjustment for
primary maligcy depending on the clinical setting. Patients with Tomita
scores less than or equal to 8 and Tokuhashi scores greater than or equal to 6
are recommended to undergo surgical management. |
Which compounds exist that are thyroid hormone analogs? | Compoounds such as 3,5-diiodo-L-thyronine, T2, GC-24, CO23, DITPA, 3,5-diiodothyropropionic acid, GC-1, Tetrac, 3,3',5,5'-tetraiodo-thyroacetic acid, KB- 2115, KB - 141, thyronamines, T4-agarose, CGS 23425, D-T3, 3,3',5-triiodo-D-thyronine, 3,5-T2, 3,5-diiodo-L-thyronine, DIT, 3,5-diiodo-L-tyrosine, MIT, 3-monoiodo-L-tyrosine, triac, 3, 3',5-triiodo-thyroacetic acid, 3,5-Diiodo-4-hydroxyphenylpropionic acid, DIHPA, 3,5-Dimethyl-3'-isopropyl-L-thyronine, DIMIT, 3,5-diiodo-3'-isopropylthyroacetic acid and IpTA2 are compounds that are thyroid hormone analogs. | It has been reported that oligodendrocytes do not contain nuclear T3 receptors,
which is in apparent contradiction with the well-known effects of thyroid
hormones on myelination. In this study we have reexamined the presence of
receptors in this cell population, using pure rat oligodendrocyte cultures. T3
binding was also studied with the use of pure rat astrocytes as well as in mixed
neuronal-glial cultures. The latter are mainly neuronal during the first days in
culture and essentially glial thereafter. Binding studies carried out in intact
cells demonstrated the presence of high affinity-low capacity binding sites for
thyroid hormones in pure cultures of oligodendrocytes. The maximal binding
capacity was 50-60 fmol/100 micrograms DNA and the dissociation constant (Kd)
0.13 nM. Pure rat astrocyte cultures also contained high affinity sites for
thyroid hormones, although receptor concentrations was 2-3 times lower than in
oligodendrocytes or neurons. This was confirmed in pure cultures of chick
astrocytes and in neuronal-glial cultures during the astroglial period. The
relative affinity of the receptor for thyroid hormone analogs was
triiodothyroacetic acid = T3 greater than T4 greater than tetraiodothyroacetic
acid in oligodendrocyte and astrocyte nuclei, and the sedimentation coefficient
of the receptor was approximately 3.8S in both cell types. These results
demonstrate that nuclear T3 receptors similar to those found in neurons and
astrocytes are also present in oligodendrocytes. This suggests that the effects
of thyroid hormones on myelination could result from a direct action of the
hormone in the oligodendrocytes. 3,5-Dimethyl-3'-isopropyl-L-thyronine (DIMIT) and
3,5-diiodo-3'-isopropylthyroacetic acid (IpTA2), two thyroid hormone analogs,
have been tested in genetically obese Zucker rats and their lean littermates, in
comparison with thyroxine (T4) and triiodothyronine (T3) for their thyromimetic
activities on body weight gain and lipid levels in serum and liver. The
compounds were administered for 9 weeks by orogastric tube to 6- to 8-week-old
animals. While body weight gain remained practically unchanged in the lean rats,
it decreased significantly in the obese individuals, especially with IpTA2. The
serum lipid concentrations were also decreased in the obese rats in comparison
with their lean littermates, especially with DIMIT. The connection observed
between the structure of DIMIT and IpTA2 on one hand and their effects on the
other is in good agreement with previous studies. Our results confirm that the
iodine substituents are not necessary for thyromimetic activity and demonstrate
that the isopropyl substituent in 3' plays an important role in the serum
lipid-lowering effect of the thyroid hormone analogs tested. Nuclear T3 receptor (NTR) have been characterized in separated cultures of
neurons and astrocytes. Scatchard analysis indicated the presence of a single
class of high-affinity sites in both cell lines. The apparent equilibrium
association constant ranged from 1.80 +/- 0.41 X 10(10) M-1 to 3.27 +/- 0.74 +/-
10(10) M-1 in neurons and from 1.01 +/- 0.09 to 1.80 +/- 0.73 X 10(10) M-1 in
astrocytes depending on the time in culture. In neurons, the maximal binding
capacity (MBC) increased from 0.049 +/- 0.008 ng T3/mg DNA to 0.328 +/- 0.052 ng
T3/mg DNA between 3 and 12 days of culture. In astrocytes, the changes in MBC
were less pronounced ranging from a minimum of 0.095 +/- 0.024 ng T3/mg DNA at
the 7th day of culture to a maximum of 0.198 +/- 0.048 ng T3/mg DNA at the 21st
day. The relative binding affinity of the receptor for thyroid hormone analogs
was in the order TRIAC greater than L-T3 greater than D-T3 greater than L-T4 in
both cell lines. These results show that nuclear T3 receptors similar to those
found in vivo are present in primary cultures of both astrocytes and neurons. The effects on ventricular myosin isoenzyme expression of naturally occurring
and synthetic thyroid analogs (3,5,3'-L-triiodothyronine,
3,5,3'-D-triiodothyronine, 3,3',5'-L- triiodothyronine ,
3,5,3'-L-triiodothyroacetic acid and 3,5-L-diiodothyronine), catecholamines and
high carbohydrate diets have been studied in thyroidectomized and
hypophysectomized rats. Also, the effects on myosin isoenzyme expression of
adrenalectomy and hydrocortisone replacement have been studied in euthyroid
animals. Myocardial CO2 production and hepatic alpha-glycerolphosphate
dehydrogenase activity were measured to monitor the effects of these
interventions on tissue respiration. The results indicate that there was no
significant separation between the actions of thyroid analogs on metabolic
parameters and myosin isoenzyme patterns. However, high carbohydrate feeding in
hypophysectomized rats increased the isoenzyme V1 from 12% to about 36% of total
myosin; partial replacement with 3,5,3'-L-triiodothyronine and fructose feeding
had synergistic actions. In thyroidectomized rats, feeding a high carbohydrate
diet increased the V1 form from undetectable levels to about 28% of total
myosin; partial 3,5,3'-L-triiodothyronine replacement had an additive effect.
Beta adrenergic stimulation with isoproterenol and blockade with propranolol did
not affect myosin isoenzyme expression. Adrenalectomy in euthyroid rats caused a
33% decrease in the V1 form and a corresponding increase in the V3 isoenzyme,
which could be reversed by treatment with hydrocortisone. Thus, thyroid analogs
do not selectively stimulate myosin isoenzyme expression as compared with their
effects on energy production. Furthermore, the results suggest that the
mechanism for regulation of cardiac myosin isoenzymes may involve a primary
signal related to dietary carbohydrate, which is modulated by thyroid hormone,
and possibly glucocorticoids. To define the minimal structural requirements for cardiac activity of thyroid
hormone analogs, a series of substituted phenols were screened for their ability
to bind bacterially expressed thyroid hormone receptors. Compounds with binding
activity then were tested for their ability to induce expression of alpha-myosin
heavy chain mRNA in primary cultures of fetal rat cardiomyocytes, a sensitive
marker for potential inotropic activity. 3,5-Diiodo-4-hydroxyphenylpropionic
acid (DIHPA) was found to bind specifically to bacterially expressed alpha-1 and
beta-1 thyroid hormone receptors (Kaff approximately 1 to 2 x 10(5) M-1) and to
induce alpha-myosin heavy chain (EC50 approximately 5 x 10(-7)). To assess the
effects of DIHPA on cardiac performance in vivo, hemodynamic measurements were
made in three groups of hypothyroid rats treated for 5 days with s.c. doses of
DIHPA (15 mg/100 g), L-thyroxine (T4, 1.5 micrograms/100 g) or saline. Compared
to controls, DIHPA and T4 produced increases in heart rate, left ventricular
+dP/dtmax, -dP/dtmax, and isovolumic relaxation. In isometric papillary muscles
preparations, DIHPA and T4 shortened time-to-peak tension and time-from-peak
tension to 50% decline as compared with saline-treated controls. Muscles from
both drug-treatment groups showed similar responses to graded doses of
isoproterenol (10(-8) to 10(-3) M) and to variations in Ca++ concentration of
the muscle bath (0.3125 to 3.75 x 10(-3) M). Thus, DIHPA is a novel thyromimetic
compound with effects on myocardial function similar to those observed with T4. Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and
[125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day
old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was
measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse
serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37
degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac
(240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but
expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001)
greater than T3 uptake, whereas the relative nuclear binding of the two tracers
was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10
microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%;
P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05<P<0.1). In
contrast, 10 microM 3,5-T2, Triac, or tetraiodothyroacetic acid (Tetrac) did not
affect T3 uptake after 15 min or after 24 h. Diiodothyropropionic acid (DITPA)
(10 microM) reduced 15-min T3 uptake by about 24% (P<0.05), but it had a greater
effect after 4 h (56%; P<0.001). Exposure to 10 nM DITPA during culture reduced
cellular T3 uptake, as did 10 nM T3, suggesting down-regulation of the plasma
membrane T3 transporters. We conclude that i) Triac is taken up by
cardiomyocytes; ii) 3,3'-T2 and, to a lesser extent, DITPA and T4 interfere with
plasma membrane transport of T3, whereas 3,5-T2, Triac, or Tetrac do not; iii)
the transport mechanism for Triac is probably different from that for T3. The heart is an important target of thyroid hormone actions. Only a limited
number of cardiac target genes have been identified, and little is known about
their regulation by T(3) (3,3',5-triiodothyronine) and thyroid hormone analogs.
We used an oligonucleotide microarray to identify novel cardiac genes regulated
by T(3) and two thyroid hormone analogs, 3,5-diidodothyropropionic acid (DITPA)
and CGS 23425 [N-[3,5-dimethyl-4-(4'-hydroxy-3'-isopropylphenoxy)-phenyl]-oxamic
acid]. DITPA binds with lower affinity than T(3) to thyroid hormone receptor
alpha1 and beta1 isoforms, whereas CGS 23425 binds selectively to beta1.
Fluorescent-labeled cDNA was prepared from cultured heart cells maintained in
medium stripped of thyroid hormone ("hypothyroid" control) or treated with T(3),
DITPA, and CGS 23425 at concentrations 5 times their respective K(d) values for
48 h. The arrays were scanned and analyzed using an analysis of variance
program. Sixty-four genes were identified that were >1.5 times up- or
down-regulated by one of the treatments with P < 0.05. The genes regulated by
T(3) and DITPA were nearly identical. Thirteen genes were differentially
regulated by CGS 23425. Genes encoding contractile proteins, Ca(2+)-ATPase of
sarcoplasmic reticulum and several proteins of mitochondrial oxidative
phosphorylation, were up-regulated by T(3) and DITPA but not by CGS 23425. These
results indicate that some, but not all, of the actions of thyroid hormone
analogs can be explained by differences in gene activation. We have recently described the proangiogenesis effects of thyroid hormone in the
chick chorioallantoic membrane (CAM) model. Generation of new blood vessels from
existing vessels was promoted 2- to 3-fold by either T(4) or T(3) at
10(-8)-10(-7) M total hormone concentrations. In the present studies, omolar
concentrations of 3,5-diiodothyropropionic acid (DITPA), a thyroid hormone
analog with inotropic but not chronotropic properties, exhibited potent
proangiogenic activity that was comparable to that obtained with T(3) and T(4)
in both the CAM model and in an in vitro three-dimensional human microvascular
endothelial sprouting assay. The proangiogenesis effect of DITPA was inhibited
by tetraiodothyroacetic acid, a thyroid hormone analog that competes with T(4)
and T(3) for a novel cell surface hormone receptor site on integrin alphavbeta3.
The thyroid hormone analogs DITPA, T(4), and T(4)-agarose, as well as basic
fibroblast growth factor (b-FGF) and vascular endothelial cell growth factor,
demonstrated comparable proangiogenic effects in the CAM model and in the
three-dimensional human microvascular endothelial sprouting model. The
proangiogenesis effect of either DITPA or b-FGF was blocked by PD 98059, an
inhibitor of the ERK1/2 signal transduction cascade. Additionally, a specific
integrin alphavbeta3 small molecule antagonist, XT199, effectively inhibited the
proangiogenesis effect of DITPA and b-FGF. Thus, the proangiogenesis actions of
thyroid hormone and its analog DITPA are initiated at the plasma membrane,
apparently at integrin alphavbeta3, and are MAPK dependent. Thyroid hormones [predomitly 3, 5, 3 -I- iodothyronine (T3)] regulate
cholesterol and lipoprotein metabolism but cardiac effects restrict their use as
hypolipidemic drugs. New molecules have been developped which target
specifically the thyroid hormone receptor ss, predomit isoform in liver. The
first thyroid hormone agonist, called GC1, has selective actions compared to T3.
In animals, GC1 reduced serum cholesterol and serum triglycerides, probably by
stimulation important steps in reverse cholesterol transport. Other selective
thyromimetic, KB- 2115 and KB - 141 have similar effects. Another class of
thyroid hormone analogs, the thyronamines have emerged recently but the basic
biology of this new class of endogenous thyroid hormone remains to better
understood. Therefore, these molecules may be a potentially treatment for
obesity and reduction cholesterol, triglycerides and lipoprotein (a). To date
the studies in human are preliminary. Tolerance and efficacy of these drugs are
still under investigation. 3-Iodothyronamine (T(1)AM) is a naturally occurring thyroid hormone metabolite
with distinct biological effects that are opposite those of thyroid hormone. The
known molecular targets of T(1)AM include both plasma membrane and intracellular
proteins, suggesting that intracellular transport of T(1)AM may be an important
component of its action, although no uptake mechanism has yet been described.
Using various human cell lines, we show that, indeed, cellular uptake of T(1)AM
occurs in multiple cell types and that this process involves specific,
saturable, and inhibitable transport mechanisms. These mechanisms are sodium and
chloride independent, pH dependent, thyronamine specific, and do not involve the
likely candidate transporters of other monoamines, organic cations, or thyroid
hormones. A large-scale RNA interference screen targeting the entire solute
carrier superfamily of transporter genes reveals that the transport of T(1)AM
into cells involves multiple transporters, and we identify eight transporters
that may contribute to the uptake of T(1)AM in HeLa cells. This type of
transporter small interfering RNA screening approach can be used in general to
identify the constellation of transporters that participate in the intracellular
disposition of compounds. Thyroid hormone analogs with selective actions through specific thyroid hormone
receptor (TR) subtypes are of great interest. They might offer the possibility
of mimicking physiological actions of thyroid hormone with receptor subtype or
tissue specificity with therapeutic aims. They are also pharmacological tools to
dissect biochemical pathways mediated by specific receptor subtypes, in a
complementary way to mouse genetic modifications. In this work, we studied the
in vivo activity in developing rats of two thyroid hormone agonists, the
TRβ-selective GC-24 and the TRα-selective CO23. Our principal goal was to check
whether these compounds were active in the rat brain. Analog activity was
assessed by measuring the expression of thyroid hormone target genes in liver,
heart, and brain, after administration to hypothyroid rats. GC-24 was very
selective for TRβ and lacked activity on the brain. On the other hand, CO23 was
active in liver, heart, and brain on genes regulated by either TRα or TRβ. This
compound, previously shown to be TRα-selective in tadpoles, displayed no
selectivity in the rat in vivo. The worldwide prevalence of obesity-associated pathologies, including type 2
diabetes, requires thorough investigation of mechanisms and interventions.
Recent studies have highlighted thyroid hormone analogs and derivatives as
potential agents able to counteract such pathologies. In this study, in rats
receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk daily
administration of a naturally occurring iodothyronine, 3,5-diiodo-L-thyronine
(T2), on the gastrocnemius muscle metabolic/structural phenotype and insulin
signaling. The HFD-induced increases in muscle levels of fatty acid translocase
(3-fold; P<0.05) and TGs (2-fold, P<0.05) were prevented by T2 (each; P<0.05 vs.
HFD). T2 increased insulin-stimulated Akt phosphorylation levels (∼2.5-fold;
P<0.05 vs. HFD). T2 induced these effects while sparing muscle mass and without
cardiac hypertrophy. T2 increased the muscle contents of fast/glycolytic fibers
(2-fold; P<0.05 vs. HFD) and sarcolemmal glucose transporter 4 (3-fold; P<0.05
vs. HFD). Adipocyte differentiation-related protein was predomitly present
within the slow/oxidative fibers in HFD-T2. In T2-treated rats (vs. HFD),
glycolytic enzymes and associated components were up-regulated (proteomic
analysis, significance limit: 2-fold; P<0.05), as was phosphofructokinase
activity (by 1.3-fold; P<0.05), supporting the metabolic shift toward a more
glycolytic phenotype. These results highlight T2 as a potential therapeutic
approach to the treatment of diet-induced metabolic dysfunctions. |
Is lenvatinib effective for thyroid cancer? | Yes, lenvatinib is effective for thyroid cancer. | During the past two decades, several key somatic mutations associated with
development and progression of differentiated thyroid cancer (DTC) have been
revealed. Historically, the treatment for advanced DTC is challenging after
patients become refractory to radioactive iodine. The response to doxorubicin,
the only chemotherapy agent approved by the US Food and Drug Administration, is
disappointing either as monotherapy or combination therapy. Because of the lack
of effective systemic treatment coupled with increased understanding of
molecular and cellular pathogenesis, multiple kinase inhibitors (MKIs) as an
alternative therapy for the treatment of advanced DTC has generated much
interest, enthusiasm, and, most excitingly, promising results. After the
encouraging results of these agents in earlier trials, the Food and Drug
Administration approved sorafenib for the treatment of locally recurrent,
progressive, or metastatic DTC refractory to radioactive iodine treatment based
on the results of a multicenter DECISION trial. Sorafenib therefore became the
first MKI approved for this indication in more than 20 years. However, even more
impressive responses and progression-free survival benefits were seen in the
phase III SELECT trial with lenvatinib, giving even higher hopes for the future
management of what was considered just a decade ago an orphan disease. Given the
role of MKIs, a new era in the treatment of advanced DTC has begun. We review
the key therapeutic targets, oncogenic pathways, and promising clinical results
of these agents in refractory disease, as well as their roles after failure of
first line kinase inhibitors. Author information:
(1)Northwestern Medicine Developmental Therapeutics Institute, Northwestern
University, United States; Division of Endocrinology, Metabolism, and Molecular
Medicine, Feinberg School of Medicine, Northwestern University, United States;
Robert H. Lurie Comprehensive Cancer Center of Northwestern University, United
States. Electronic address: [email protected].
(2)Northwestern Medicine Developmental Therapeutics Institute, Northwestern
University, United States; Division of Hematology and Oncology, Feinberg School
of Medicine, Northwestern University, United States; Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, United States.
Electronic address: [email protected].
(3)Division of Hematology and Oncology, Feinberg School of Medicine,
Northwestern University, United States; Robert H. Lurie Comprehensive Cancer
Center of Northwestern University, United States. Electronic address:
[email protected].
(4)Division of Endocrinology, Metabolism, and Molecular Medicine, Feinberg
School of Medicine, Northwestern University, United States; Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, United States.
Electronic address: [email protected].
(5)Northwestern Medicine Developmental Therapeutics Institute, Northwestern
University, United States; Division of Hematology and Oncology, Feinberg School
of Medicine, Northwestern University, United States; Robert H. Lurie
Comprehensive Cancer Center of Northwestern University, United States.
Electronic address: [email protected]. |
What genes are related to breast cancer? | Breast cancer is a disease in which certain cells in the breast become abnormal and multiply without control or order to form a tumor. The most common form of breast cancer begins in cells lining the ducts that carry milk to the nipple (ductal cancer). Other forms of breast cancer begin in the glands that produce milk (lobular cancer) or in other parts of the breast.
Early breast cancer usually does not cause pain and may exhibit no noticeable symptoms. As the cancer progresses, signs and symptoms can include a lump or thickening in or near the breast; a change in the size or shape of the breast; nipple discharge, tenderness, or retraction (turning inward); and skin irritation, dimpling, or scaliness. These changes can occur as part of many different conditions, however. Having one or more of these symptoms does not mean that a person definitely has breast cancer.
In some cases, cancerous tumors can invade surrounding tissue and spread to other parts of the body. If breast cancer spreads, cancerous cells most often appear in the bones, liver, lungs, or brain. Tumors that begin at one site and then spread to other areas of the body are called metastatic cancers.
A small percentage of all breast cancers cluster in families. Hereditary cancers are those associated with inherited gene mutations. Hereditary breast cancers tend to occur earlier in life than noninherited (sporadic) cases and are more likely to involve both breasts.
Variations of the BRCA1, BRCA2, BRCT, and P53 genes increase the risk of developing breast cancer.
The and NFR2, HER2 and TOP2A genes are associated with breast cancer. | INTRODUCTION: Estrogen receptor (ER)-positive breast cancers are considered
prognostically more favorable than ER-negative tumors, whereas human epidermal
growth factor receptor (HER)2/neu-positive breast cancers are associated with
worse prognosis. The objective of the present study was to determine whether
ER-positive and ER-negative status relates to epigenetic changes in breast
cancer-related genes. To evaluate epigenetic differences in tumor-related genes
relating to ER and HER2/neu status of primary tumors, we examined the promoter
methylation status of the promoter region CpG islands of eight major breast
tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARbeta2, and
CDH1).
METHODS: Paired ER-positive (n = 65) and ER-negative (n = 65) primary breast
tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted
from paraffin-embedded tumor tissue after microdissection, and
methylation-specific PCR and capillary-array electrophoresis analysis were
performed.
RESULTS: In early stages of tumor progression (T1 and N0), RASSF1A and CCND2
were significantly (P < 0.05) more methylated in ER-positive than in ER-negative
tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis
positive group than in the negative group. Double negative (ER-negative,
HER2/neu-negative) breast cancers had significantly lesser frequencies of
RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035,
respectively). Both ER and HER2/neu status correlated independently with these
epigenetic alterations.
CONCLUSION: We demonstrated significant differences in tumor-related gene
methylation patterns relevant to ER and HER2/neu status of breast tumors. This
may be of significance in the assessment of targeted therapy resistance related
to ER and HER2/neu status in breast cancer patients. Author information:
(1)Division for Animal Research Resources, Institute of Health Biosciences, The
University of Tokushima Graduate School, Tokushima 770-8503, Japan.
(2)Laboratory of Veterinary Biochemistry, School of Veterinary Medicine,
Kitasato University, Aomori 034-8628, Japan.
(3)Department of Biological Resources, Integrated Centre for Science, Ehime
University, Shitsukawa, Toon City, Ehime 791-0295, Japan.
(4)Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan.
(5)Department of Veterinary Physiology, Faculty of Agriculture, Iwate
University, Morioka 020-8550, Japan; United Graduate School of Veterinary
Science, Gifu University, Yanagido, 1-1, Gifu 501-1193, Japan.
(6)Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan.
Electronic address: [email protected]. BACKGROUND: Invasion and metastasis are two important hallmarks of maligt
tumors caused by complex genetic and epigenetic alterations. The present study
investigated the contribution of aberrant methylation profiles of cancer related
genes, APC, BIN1, BMP6, BRCA1, CST6, ESR-b, GSTP1, P14 (ARF), P16 (CDKN2A), P21
(CDKN1A), PTEN, and TIMP3, in the matched axillary lymph node metastasis in
comparison to the primary tumor tissue and the adjacent normal tissue from the
same breast cancer patients to identify the potential of candidate genes
methylation as metastatic markers.
METHODS: The quantitative methylation analysis was performed using the
SEQUENOM's EpiTYPER™ assay which relies on matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
RESULTS: The quantitative DNA methylation analysis of the candidate genes showed
higher methylation proportion in the primary tumor tissue than that of the
matched normal tissue and the differences were significant for the APC, BIN1,
BMP6, BRCA1, CST6, ESR-b, P16, PTEN and TIMP3 promoter regions (P<0.05). Among
those candidate methylated genes, APC, BMP6, BRCA1 and P16 displayed higher
methylation proportion in the matched lymph node metastasis than that found in
the normal tissue (P<0.05). The pathway analysis revealed that BMP6, BRCA1 and
P16 have a role in prevention of neoplasm metastasis.
CONCLUSIONS: The results of the present study showed methylation heterogeneity
between primary tumors and metastatic lesion. The contribution of aberrant
methylation alterations of BMP6, BRCA1 and P16 genes in lymph node metastasis
might provide a further clue to establish useful biomarkers for screening
metastasis. The coordinated replication and transcription of pericentromeric repeats enable
RNA interference (RNAi)-mediated transmission of pericentromeric heterochromatin
in fission yeast, which is essential for the proper function of centromeres.
Rad3/ATR kinase phosphorylates histone H2A on serine-128/-129 to create γH2A in
pericentromeric heterochromatin during S phase, which recruits Brc1 through its
breast cancer gene 1 protein (BRCA1) C-terminal (BRCT) domains. Brc1 prevents
the collapse of stalled replication forks; however, it is unknown whether this
activity influences centromere function. Here, we show that Brc1 localizes in
pericentromeric heterochromatin during S phase, where it enhances
Clr4/Suv39-mediated H3 lysine-9 dimethylation (H3K9me2) and gene silencing. Loss
of Brc1 increases sensitivity to the microtubule-destabilizing drug
thiabendazole (TBZ) and increases chromosome missegregation in the presence of
TBZ. Brc1 retains significant function even when it cannot bind γH2A. However,
elimination of the serine-121 site on histone H2A, a target of Bub1 spindle
assembly checkpoint kinase, sensitizes γH2A-deficient and brc1Δ cells to
replication stress and microtubule destabilization. Collective results suggest
that Brc1-mediated stabilization of stalled replication forks is necessary for
fully efficient transmission of pericentromeric heterochromatin, which is
required for accurate chromosome segregation during mitosis. Although homologous recombination (HR) is an important pathway for DNA repair,
it can also be a cause for deleterious genomic rearrangements leading to
carcinogenesis. Therefore, cells have evolved elaborate mechanisms to regulate
HR, positively as well as negatively. Among many molecular components that
regulate HR are tumour suppressors p53, a negative regulator and breast cancer
early-onset (BRCA)2, a positive regulator. Both the players not only interact
with each other but also directly interact with human RAD51 (hRAD51), the key
recombinase in HR. Here, for the first time we studied HR regulation by the
combined action of p53 and BRCA2, in vitro. While BRC4 peptide inhibits ATP
hydrolysis by hRAD51, BRCA2(BRC1-8) stimulates DNA-independent and
double-stranded DNA-dependent ATPase several fold and only marginally
single-stranded DNA-dependent ATPase. Pull down assays demonstrated the
occurrence of complex comprising of all three proteins and DNA, where p53 tends
to compete out hRAD51 and BRCA2(BRC1-8), leading to not only the decline in ATP
hydrolysis but also the strand exchange function of hRAD51 that was stimulated
by BRCA2(BRC1-8). Our findings suggest a rigorous p53-mediated regulation on
hRAD51 functions in HR even in the presence of BRCA2. Cyclin E, HER-2 and p53, are considered as major prognostic markers in breast
cancer. As they are related in patho-clinical level, we aimed to check if they
have any direct interaction on expression of each other. To study the effect of
cyclin E on HER-2 expression, cell lines stably overexpressing cyclin E or its
low molecular weight (LMW) isoforms were generated. To understand the results of
p53 silencing either alone or in combination with cyclin E overexpression, we
created three different p53 stably knocked down cell lines. Protein expression
was analyzed by western blot, HER-2 expression in the established cell lines
were determined using SYBR green real time PCR and data analyzed by REST
software. Results indicate that HER-2 expression is only downregulated following
p53 silencing and none of cyclin E isoforms can alter its expression. The
presence of cyclin E isoforms in p53 silenced clones also does not altered HER-2
expression. Given the fact that p53 degradation is increased by HER-2
overexpression, these data can draw a regulatory loop in which a non-mutated
functional p53 and HER-2 can bidirectionally regulate the expression of these
two genes. This study improves our understandings of these pathways and these
proteins can be introduced either as a marker or as a target in cancer
treatment. Tripartite-motif containing 22 (TRIM22) is a direct p53 target gene and inhibits
the clonogenic growth of leukemic cells. Its expression in Wilms tumors is
negatively associated with disease relapse. This study addresses if TRIM22
expression is de-regulated in breast carcinoma. Western blotting analysis of a
panel of 10 breast cancer cell lines and 3 non-maligt mammary epithelial cell
lines with a well-characterized TRIM22 monoclonal antibody showed that TRIM22
protein is greatly under-expressed in breast cancer cells as compared to
non-maligt cell lines. Similarly, TRIM22 protein is significantly
down-regulated in breast tumors as compared to matched normal breast tissues.
Study of cell lines with methylation inhibitor and bisulfite sequencing
indicates that TRIM22 promoter hypermethylation may not be the cause for TRIM22
under-expression in breast cancer. Instead, we found that TRIM22 protein level
correlates strongly (R=0.79) with p53 protein level in normal breast tissue, but
this correlation is markedly impaired (R=0.48) in breast cancer tissue,
suggesting that there is some defects in p53 regulation of TRIM22 gene in breast
cancer. This notion is supported by cell line studies, which showed that TRIM22
was no longer inducible by p53-activating genotoxic drugs in breast cancer cell
lines and in a p53 null cell line H1299 transfected with wild type p53. In
conclusion, this study shows that TRIM22 is greatly under-expressed in breast
cancer. p53 dysfunction may be one of the mechanisms for TRIM22 down-regulation. The aim of our study was to investigate HER-2 and TOP2A gene status and their
correlation with Bcl-2, p53, Ki67, ssDNA, and clinicopathological parameters in
four molecular subtypes of breast cancer. Seventy-four paraffin-embedded samples
are immunohistochemically studied for the expression of estrogen receptor (ER),
progesterone receptor (PR), HER-2, p53, Bcl-2, ssDNA, and Ki67, while HER-2 and
TOP2A gene status by fluorescence in situ hybridization was investigated in 60
samples. Luminal A and B subtypes were characterized with small tumor size,
intermediate histological grade, negative lymph node, and metastatic status,
while triple negative and HER-2 positive subtypes were associated with larger
tumor size, poorly differentiated tumors, and positive lymph node status. p53,
Ki67, and ssDNA expression was higher in triple negative and HER-2 positive than
in luminal subtypes, while ER, PR, and Bcl-2 dominated in luminal subtypes.
HER-2 gene status was higher in luminal B and HER-2 positive than in luminal A
and triple negative subtypes, while TOP2A gene status was similar. HER-2 gene
status positively correlated with TOP2A gene status, HER-2 receptor, and
histological grade, while negative correlation characterized relationship
between HER-2 gene status and ER, PR, and Bcl-2. The shortened overall survival
period characterized patients from triple negative breast cancer subtype
(18.7 months). HER-2 and TOP2A gene amplification showed a tendency to be
associated with larger tumor size, positive lymph node status, high level of
apoptotic and proliferative indexes, and low level of p53 and Bcl-2 expression,
which all together indicate group of patients with similar outcome during the
progression of the disease. BACKGROUND: Genetic BRCA2 insufficiency is associated with breast cancer
development; however, in sporadic breast cancer cases, high BRCA2 expression is
paradoxically correlated with poor prognosis. Because DSS1, a mammalian
component of the transcription/RNA export complex, is known to stabilize BRCA2,
we investigated how the expression of DSS1 is associated with clinical
parameters in breast cancers.
METHODS: DSS1 mRNA and p53 protein were examined by RT-PCR and
immunohistochemical staining of breast cancer specimens to classify DSS1(high)
and DSS1(low) or p53(high) and p53(low) groups. Patient survival was compared
using Kaplan-Meier method. DSS1(high) or DSS1(low) breast cancer cells were
prepared by retroviral cDNA transfection or DSS1 siRNA on proliferation, cell
cycle progression, and survival by flow cytometric analyses with or without
anti-cancer drugs.
RESULTS: In comparison to patients with low levels of DSS1, high-DSS1 patients
showed a poorer prognosis, with respect to relapse-free survival period. The
effect of DSS1 was examined in breast cancer cells in vitro. DSS1
high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs,
as observed in cell cycle and apoptosis analyses. DSS1 knockdown, however,
increased the susceptibility to the DNA-damaging drugs camptothecin and
etoposide and caused early apoptosis in p53 wild type MCF7 and p53-insufficient
MDA-MB-231 cells. DSS1 knockdown suppresses the proliferation of drug-resistant
MDA-MB-231 breast cancer cells, particularly effectively in combination with
DNA-damaging agents.
CONCLUSION: Breast cancers with high DSS1 expression have worse prognosis and
shorter relapse-free survival times. DSS1 is necessary to rescue cells from DNA
damage, but high DSS1 expression increases drug resistance. We suggest that DSS1
expression could be a useful marker for drug resistance in breast cancers, and
DSS1 knockdown can induce tumor apoptosis when used in combination with
DNA-damaging drugs. Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF) is a
second-generation 2'-deoxyadenosine analogue that is structurally related to
cladribine (2-chloro-2'-deoxyadenosine, 2CdA) and fludarabine
(9-beta-d-arabinosyl-2-fluoroadenine, F-ara-A). It demonstrates potent
antitumour activity at much lower doses than parent compounds with high
therapeutic efficacy in paediatric blood cancers. Our previous studies in breast
cancer cells indicate that 2CdA and F-ara-A are involved in epigenetic
regulation of gene transcription. We therefore investigated whether ClF
influences methylation and expression of selected tumour suppressor genes, such
as adenomatous polyposis coli (APC), phosphatase and tensin homologue (PTEN),
and retinoic acid receptor beta 2 (RARbeta2), as well as expression of p53, p21
and DNA methyltransferase 1 (DNMT1) in MCF-7 and MDA-MB-231 breast cancer cell
lines with different invasive potential. Promoter methylation and gene
expression were estimated using methylation-sensitive restriction analysis
(MSRA) and real-time PCR, respectively. ClF demonstrated potent growth
inhibitory activity in MCF-7 and MDA-MB-231 cells after 96h treatment with IC50
determined as equal to 640nM and 50nM, respectively. In both breast cancer cell
lines, ClF led to hypomethylation and up-regulation of APC, PTEN and RARbeta2 as
well as increase in p21 expression. Only in non-invasive MCF-7 cells, these
changes were associated with down-regulation of DNMT1. Our results provide first
evidence of ClF implications in epigenetic regulation of transcriptional
activity of selected tumour suppressor genes in breast cancer. It seems to be a
new important element of ClF anticancer activity and may indicate its potential
efficacy in epigenetic therapy of solid tumours, especially at early stages of
carcinogenesis. BACKGROUND: Triple-negative breast cancers (TNBC) do not represent a single
disease subgroup and are often aggressive breast cancers with poor prognoses.
Unlike estrogen/progesterone receptor and HER2 (human epidermal growth factor
receptor 2) breast cancers, which are responsive to targeted treatments, there
is no effective targeted therapy for TNBC, although approximately 50% of
patients respond to conventional chemotherapies, including taxanes,
anthracyclines, cyclophosphamide, and platinum salts.
CONTENT: Genomic studies have helped clarify some of the possible disease
groupings that make up TNBC. We discuss the findings, including copy
number-transcriptome analysis, whole genome sequencing, and exome sequencing, in
terms of the biological properties and phenotypes that make up the constellation
of TNBC. The relationships between subgroups defined by transcriptome and genome
analysis are discussed.
SUMMARY: TNBC is not a uniform molecular or disease entity but a constellation
of variably well-defined biological properties whose relationship to each other
is not understood. There is good support for the existence of a basal expression
subtype, p53 mutated, high-genomic instability subtype of TNBC. This should be
considered a distinct TNBC subtype. Other subtypes with variable degrees of
supporting evidence exist within the nonbasal/p53wt (wild-type p53) TNBC,
including a group of TNBC with PI3K (phosphoinositide 3-kinase) pathway
activation that have better overall prognosis than the basal TNBC. Consistent
molecular phenotyping of TNBC by whole genome sequencing, transcriptomics, and
functional studies with patient-derived tumor xenograft models will be essential
components in clinical and biological studies as means of resolving this
heterogeneity. Nuclear factor erythroid 2-related factor 2 (NRF2 (NFE2L2)) is an important
transcriptional activator involved in the cellular defense mechanisms against
electrophilic and oxidative stress. Recent studies have demonstrated that the
expression of NRF2 protein is upregulated in several human maligcies and is
associated with worse prognosis in these patients. However, the pathological and
clinical significance of NRF2 has remained largely unknown in breast cancer
patients. Therefore, in this study, we immunolocalized NRF2 in 106 breast
carcinoma cases. NRF2 immunoreactivity was mainly detected in the nucleus of the
breast carcinoma cells and it was positive in 44% of the cases. NRF2 status was
significantly associated with histological grade, Ki-67 labeling index, p62
immunoreactivity, and
NAD(P)H: quinone oxidoreductase 1 (NQO1) immunoreactivity, and the results of
multivariate analyses revealed that NRF2 status was an independent adverse
prognostic factor for both recurrence and disease-free survival of the patients.
Subsequent in vitro studies demonstrated that the expression of NRF2
significantly increased the proliferation activity of MCF7 and SK-BR-3 breast
carcinoma cells. These results indicate that nuclear NRF2 protein plays
important roles in the proliferation and/or progression of breast carcinoma, and
nuclear NRF2 immunoreactivity is therefore considered a potent prognostic factor
in breast cancer patients. Chemokines are small molecules that when secreted by tissues under pathological
conditions such as inflammation are believed to be involved in carcinogenesis.
Recent reports have found that the genetic variation in chemokine encoding genes
are associated with risk of breast cancer.
METHODS: Using data from a population-based case-control study of 845 invasive
breast cases and 807 controls, we genotyped 34 single nucleotide polymorphisms
(SNPs) in 8 chemokine candidate genes (CCL3, CCL4, CCL5, CCL20, CCR5, CCR6,
CXCL12 and CXCR4). Associations with breast cancer were computed for individual
SNPs, groups of SNPs within genes, and in a gene-set analysis. We also performed
a meta-analysis of CXCL12 rs1801157 and a haplotype analysis for two SNPs: CXCR4
rs2228014 and CXCL12 rs1801157.
RESULTS: We found no significant associations between the risk of breast cancer
and any individual SNPs, single genes, or combined gene sets. Some individual
variants were marginally associated with some histologic subtypes, but these
associations were not significant after adjustment for multiple comparisons. In
the meta-analysis of five studies of European ancestry, CXCL12 rs1801157 was
marginally associated with breast cancer risk (OR=1.14, 95% CI: 1.00, 1.30).
CONCLUSIONS: Our findings suggest that genetic variants in the 8 candidate genes
coding for chemotactic cytokines have little influence in the risk of breast
cancer in postmenopausal women. Additional examination of the relationship
between CXCL12 rs1801157 and breast cancer risk is warranted. African Americans (AAs) have higher mortality rate from breast cancer than that
of Caucasian Americans (CAs) even when socioeconomic factors are accounted for.
To better understand the driving biological factors of this health disparity, we
performed a comprehensive differential gene expression analysis, including
subtype- and stage-specific analysis, using the breast cancer data in the Cancer
Genome Atlas (TCGA). In total, 674 unique genes and other transcripts were found
differentially expressed between these two populations. The numbers of
differentially expressed genes between AA and CA patients increased in each
stage of tumor progression: there were 26 in stage I, 161 in stage II, and 223
in stage III. Resistin, a gene that is linked to obesity, insulin resistance,
and breast cancer, was expressed more than four times higher in AA tumors. An
uncharacterized, long, non-coding RNA, LOC90784, was down-regulated in AA
tumors, and its expression was inversely related to cancer stage and was the
lowest in triple negative AA breast tumors. Network analysis showed increased
expression of a majority of components in p53 and BRCA1 subnetworks in AA breast
tumor samples, and members of the aurora B and polo-like kinase signaling
pathways were also highly expressed. Higher gene expression diversity was
observed in more advanced stage breast tumors suggesting increased genomic
instability during tumor progression. Amplified resistin expression may indicate
insulin-resistant type II diabetes and obesity are associated with AA breast
cancer. Expression of LOC90784 may have a protective effect on breast cancer
patients, and its loss, particularly in triple negative breast cancer, could be
having detrimental effects. This work helps elucidate molecular mechanisms of
breast cancer health disparity and identifies putative biomarkers and
therapeutic targets such as resistin, and the aurora B and polo-like kinase
signaling pathways for treating AA breast cancer patients. BACKGROUND: Breast tumors have been described by molecular subtypes
characterized by pervasively different gene expression profiles. The subtypes
are associated with different clinical parameters and origin of precursor cells.
However, the biological pathways and chromosomal aberrations that differ between
the subgroups are less well characterized. The molecular subtypes are associated
with different risk of metastatic recurrence of the disease. Nevertheless, the
performance of these overall patterns to predict outcome is far from optimal,
suggesting that biological mechanisms that extend beyond the subgroups impact
metastasis.
RESULTS: We have scrutinized publicly available gene expression datasets and
identified molecular subtypes in 1,394 breast tumors with outcome data. By
analysis of chromosomal regions and pathways using "Gene set enrichment
analysis" followed by a meta-analysis, we identified comprehensive mechanistic
differences between the subgroups. Furthermore, the same approach was used to
investigate mechanisms related to metastasis within the subgroups. A striking
finding is that the molecular subtypes account for the majority of biological
mechanisms associated with metastasis. However, some mechanisms, aside from the
subtypes, were identified in a training set of 1,239 tumors and confirmed by
survival analysis in two independent validation datasets from the same type of
platform and consisting of very comparable node-negative patients that did not
receive adjuvant medical therapy. The results show that high expression of 5q14
genes and low levels of TNFR2 pathway genes were associated with poor survival
in basal-like cancers. Furthermore, low expression of 5q33 genes and
interleukin-12 pathway genes were associated with poor outcome exclusively in
ERBB2-like tumors.
CONCLUSION: The identified regions, genes, and pathways may be potential drug
targets in future individualized treatment strategies. |
To the ligand of which receptors does Denosumab (Prolia) bind? | Denosumab is a monoclonal antibody against the RANKL | Osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand
(RANKL) are domit regulators of bone resorption. Many hormones, cytokines and
growth factors mediate bone resorption by altering the ratio of RANKL to OPG.
RANKL and OPG expression is also altered in numerous bone diseases, and these
changes can reflect disease etiology or compensatory responses to disease. RANKL
stimulates osteoclast formation, function and survival, and each of these
effects is inhibited by OPG. OPG suppresses bone resorption and increases the
density, area and strength of both cancellous and cortical bone. Denosumab (AMG
162), a fully human monoclonal antibody to RANKL, shares the pharmacologic
attributes of OPG but has a significantly longer half-life that allows less
frequent administration. Osteoporosis and several other bone disorders occur when there is an imbalance
between the resorption and formation components of bone remodeling activity.
Therapies available for some of these conditions modulate the activity of
osteoclasts and/or osteoblasts. The recent discoveries of receptor activator of
NF-kappaB ligand (RANKL), an endogenous activator of osteoclastogenenesis and
osteoclast activity and its inhibitor, osteoprotegerin (OPG) as pivotal
regulatory factors in the pathogenesis of bone diseases like osteoporosis
provide unique targets for therapeutic agents. In laboratory animals and now in
humans, administering forms of OPG markedly inhibits osteoclast activity and
improves bone strength, documenting that the strategy of inhibiting RANKL
activity has therapeutic promise. A highly specific, fully human antibody
against RANKL has been produced (denosumab) that in early studies in humans
reduces bone turnover and improves bone density. Attributes of denosumab in
these clinical studies include a very rapid onset of action, sustained effects
for several months after a single injection, and good tolerability. These
results provide the basis for studies evaluating the effectiveness of denosumab
in several clinical conditions characterized by increased osteoclastic activity. Patients with multiple myeloma (MM) experience pathologic fractures, bone pain,
hypercalcemia, neurologic symptoms, and renal insufficiency with substantial
morbidity and mortality. Bisphosphonates have been used successfully for the
management of MM-related bone disease. Increased incidence of osteonecrosis of
the jaw has been observed in patients with cancer receiving bisphosphonate
therapy. Recent advances in the pathobiology of MM-related bone disease and
other cancer-related bone metastases have led to the identification of novel
therapeutic targets, such as receptor activator of nuclear factor-kappaB (RANK);
its ligand (RANKL); and a decoy receptor, osteoprotegerin, for the development
of potential targeted agents. Initials studies have demonstrated that targeting
RANK/ RANKL signaling with the fully human monoclonal antibody denosumab
prevented skeletal complications in patients with MM and other cancers with bone
metastases. Ongoing studies evaluating the clinical utility of denosumab in
cancer- related bone destruction have been discussed. In addition, several
potential targets, such as macrophage inflammatory protein-1alpha, chemokine
receptors 1 and 5, interleukin-3, and Wnt signaling, are b riefly described. The skeletal and immune systems have a complex relationship. Both systems are
intimately coupled, with osteoclastogenesis and hematopoiesis occurring in the
bone marrow. Bone and immune cells also share common hematopoietic precursors.
Furthermore, the skeletal and immune systems share various cytokines, receptors,
and transcription factors that regulate signal transduction pathways involved in
osteoclastogenesis and immune system activation, including the receptor
activator of nuclear factor-κΒ ligand/receptor activator of nuclear
factor-κΒ/osteoprotegerin (RANKL-RANK-OPG) pathway. Cancer cells can disrupt
both the skeletal and immune systems. Interaction between cancer and bone cells
results in a vicious cycle of bone destruction and cancer growth. Bone
remodeling generates a growth-factor-rich environment that attracts cancer cells
and promotes their proliferation. In turn, cancer cells stimulate osteoclast
formation and activity, resulting in additional bone resorption that further
stimulates cancer cell growth. Currently available bone-targeted therapies may
also modulate the immune system. Bisphosphonates such as zoledronic acid exert
stimulating effects on the immune system, resulting in possible anticancer
activity against maligt cells. Denosumab, an anti-RANKL monoclonal antibody
with proven antiosteoclast activity, may suppress immune responses. This may
result in the reported association with an increased risk of neoplasms, as well
as serious skin and other infections as reported in some studies, mainly in the
postmenopausal setting. When assessing bone-targeted therapies, it is important
to consider the shared signaling pathways between bone and the immune system, as
well as the clinical risk:benefit ratio. Bone, as well as the lung and liver, is among the sites of predilection for
cancer metastasis. The bone stores large amounts of growth factors such as
insulin-like growth factors and transforming growth factor-b, and provides
fertile soil for metastatic cancer cells by continuously releasing these
bone-stored growth factors, which are a consequence of osteoclastic bone
resorption. Metastatic cancer cells in turn produce osteoclast-stimulating
cytokines such as parathyroid hormone-related protein( PTH-rP), prostaglandin
E2.(PGE2), and various interleukins(ILs). These cancer-produced
osteoclast-stimulating cytokines bind to their cognitive receptors and promote
the expression of ligands for the receptor activators of nuclear factor kB
(RANKL)in osteoblasts. RANKL then binds to its receptor RANK, expressed in
pre-osteoclasts, stimulates mature osteoclast formation, and subsequently,
osteoclastic bone resorption. This vicious cycle between metastatic cancer cells
and osteoclasts is critical to the development and progression of bone
metastases. In addition, it is likely that metastatic cancer cells are
influenced by bone environments(or niche)and acquire additional capacities such
as an epithelial-mesenchymal transition(EMT), allowing them to be resistant to
chemotherapy or apoptosis, to survive in a dormant state, or to aggressively
spread to distant organs including lung and liver. Thus, the bone can serve as
transit port. Disrupting this cycle by inhibiting osteoclastic bone resorption,
antagonizing bone-derived growth factors, and neutralizing RANKL or PTH-rP,
should be a promising therapeutic intervention for bone metastases.
Bisphosphonates(BP)are specific inhibitors of osteoclasts, and have been shown
to significantly reduce skeletal-related events(SRE)associated with bone
metastasis. Denosumab is a neutralizing monoclonal antibody to RANKL and has
recently been found to inhibit SRE more effectively than BP. Further
understanding of the crosstalk communication between metastatic cancer cells and
bone at the molecular level should lead us to design novel, more effective and
specific treatments for cancer patients with bone metastases. Prostate cancer is the most common cancer in men in Europe and the United
States, and the third leading cause of death from cancer in Europe. Survival of
prostate cancer cells is dependent on the activation of androgen receptors (AR),
that are overexpressed in this tumor. Furthermore, ~90% of prostate cancer
patients that respond to first-line androgen deprivation therapy (ADT) undergo
rapid progression. This condition is defined as castration-resistant prostate
cancer (CRPC). Docetaxel-based regimens significantly improve overall survival
(OS) in patients with CRPC and represent the only treatment strategy approved by
the Food and Drug Administration (FDA). Recently, abiraterone (second hormonal
therapy) and cabazitaxel (new taxane) have been shown to improve survival in
patients with CRPC who progressed following docetaxel-based chemotherapy.
Vaccine therapy has also been demonstrated to improve OS in patients with
asymptomatic or minimally symptomatic metastatic CRPC. Additional therapeutic
targets have been analyzed in prostate cancer, including apoptosis, angiogenic
receptors, vitamin D and Src pathways. Several phase II studies are ongoing. The
high frequency of prostate cancer-related metastatic bone disease has led to
consider this pathway as a therapeutic target. To this end, several
bone-targeted agents have been investigated, most notably zoledronic acid, which
is highly effective at stabilizing the bone and preventing skeletal
complications. More recently, a nuclear factor-β ligand (RANKL) inhibitor,
denosumab, has been developed for the treatment of bone metastases. Osteoporosis is characterized by reduced bone mass and disruption of bone
architecture, resulting in increased fracture risk. Several therapeutic agents
are now available to treat postmenopausal osteoporosis and prevent fractures.
Combined calcium and vitamin D supplementation reduce the relative risk of
non-vertebral fractures by about 18%. Hormone replacement therapy (HRT) should
not be prescribed for osteoporosis in women who do not experience menopausal
symptoms. The marked benefits of raloxifene on the reduction in invasive breast
cancer and vertebral fracture risk are partially counterbalanced by a lack of
effect on non-vertebral fracture risk, and an increased risk of venous
thromboembolism and stroke. All four bisphosphonates available in Belgium,
except ibandronate, have been shown to reduce the risk of vertebral,
non-vertebral and hip fractures in prospective, placebo-controlled trials.
Globally, the incidence of vertebral fractures is reduced by 41%-70%, and the
incidence of non-vertebral fractures by 25%-39%. The anti-fracture efficacy of
weekly or monthly doses of oral bisphosphonates has not been directly shown but
is assumed from bridging studies based on BMD changes. To date, the various
bisphosphonates have not been studied in head-to-head comparative trials with
fracture endpoints. There are potential concerns that long-term suppression of
bone turnover associated with bisphosphonate treatment may eventually lead to
adverse effects, especially atypical femoral fractures and osteonecrosis of the
jaw, but these cases are extremely rare. Teri-paratide (recombit human 1-34
PTH) administered by daily subcutaneous injections decreases by 65% the relative
risk of new vertebral fractures in patients with severe osteoporosis. Pivotal
trials with strontium ranelate have shown a 41% reduction in new vertebral
fractures and a 16% reduction in non-vertebral fractures over 3 years. Denosumab
is a fully human monoclonal antibody to RANK Ligand that is administered as a
60-mg subcutaneous injection every 6 months. In the pivotal phase III trial,
there was a 68% reduction in the incidence of new vertebral fractures, whereas
the incidence of non-vertebral fractures was reduced by 20%. Several new
approaches are being explored, including antibodies to sclerostin, cathepsin K
inhibitors, src kinase inhibitors, and drugs that act on calcium sensing
receptors. Osteoporosis is characterised by a progressive loss of bone mass and
microarchitecture which leads to increased fracture risk. Some of the drugs
available to date have shown reductions in vertebral and non-vertebral fracture
risk. However, in the ageing population of industrialised countries, still more
fractures happen today than are avoided, which highlights the large medical need
for new treatment options, models, and strategies. Recent insights into bone
biology, have led to a better understanding of bone cell functions and crosstalk
between osteoblasts, osteoclasts, and osteocytes at the molecular level. In the
future, the armamentarium against osteoporotic fractures will likely be enriched
by (1.) new bone anabolic substances such as antibodies directed against the
endogenous inhibitors of bone formation sclerostin and dickkopf-1, PTH and PTHrp
analogues, and possibly calcilytics; (2.) new inhibitors of bone resorption such
as cathepsin K inhibitors which may suppress osteoclast function without
impairing osteoclast viability and thus maintain bone formation by preserving
the osteoclast-osteoblast crosstalk, and denosumab, an already widely available
antibody against RANKL which inhibits osteoclast formation, function, and
survival; and (3.) new therapeutic strategies based on an extended understanding
of the pathophysiology of osteoporosis which may include sequential therapies
with two or more bone active substances aimed at optimising the management of
bone capital acquired during adolescence and maintained during adulthood in
terms of both quantity and quality. Finally, one of the future challenges will
be to identify those patients and patient populations expected to benefit the
most from a given drug therapy or regimen. The WHO fracture risk assessment tool
FRAX® and improved access to bone mineral density measurements by DXA will play
a key role in this regard. Bone and lung metastases are responsible for the majority of deaths in patients
with breast cancer. Following treatment of the primary cancer, emotional and
psychosocial factors within this population precipitate time to recurrence and
death, however the underlying mechanism(s) remain unclear. Using a mouse model
of bone metastasis, we provide experimental evidence that activation of the
sympathetic nervous system, which is one of many pathophysiological consequences
of severe stress and depression, promotes MDA-231 breast cancer cell
colonization of bone via a neurohormonal effect on the host bone marrow stroma.
We demonstrate that induction of RANKL expression in bone marrow osteoblasts,
following β2AR stimulation, increases the migration of metastatic MDA-231 cells
in vitro, independently of SDF1-CXCR4 signaling. We also show that the
stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic
activation on breast cancer bone metastasis in vivo can be blocked with the
β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells.
These findings indicate that RANKL promotes breast cancer cell metastasis to
bone via its pro-migratory effect on breast cancer cells, independently of its
effect on bone turnover. The emerging clinical implication, supported by recent
epidemiological studies, is that βAR-blockers and drugs interfering with RANKL
signaling, such as Denosumab, could increase patient survival if used as
adjuvant therapy to inhibit both the early colonization of bone by metastatic
breast cancer cells and the initiation of the "vicious cycle" of bone
destruction induced by these cells. Bone destruction is a prominent feature of multiple myeloma, but conflicting
data exist on the expression and pathophysiologic involvement of the bone
remodeling ligand RANKL in this disease and the potential therapeutic benefits
of its targeted inhibition. Here, we show that RANKL is expressed by primary
multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release
of soluble RANKL was observed exclusively with multiple myeloma cells and was
strongly influenced by posttranscriptional/posttranslational regulation.
Signaling via RANKL into multiple myeloma and CLL cells induced release of
cytokines involved in disease pathophysiology. Both the effects of RANKL on
osteoclastogenesis and cytokine production by maligt cells could be blocked
by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine
neutralization of RANKL with induction of antibody-dependent cellular
cytotoxicity of natural killer (NK) cells against RANKL-expressing maligt
cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc
fusion proteins with modified Fc moieties. The latter displayed similar capacity
compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis
in vitro, but also potently stimulated NK cell reactivity against primary
RANKL-expressing maligt B cells, which was dependent on their engineered
affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as
attractive tools to neutralize the detrimental function of RANKL while at the
same time potently stimulating NK cell antitumor immunity. The TNF family member receptor activator for NF-κB ligand (RANKL) and its
receptors RANK and osteoprotegerin are key regulators of bone remodeling but
also influence cellular functions of tumor and immune effector cells. In this
work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated
immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL
were found to be expressed on leukemia cells in 53 of 78 (68%) investigated
patients. Signaling via RANKL into the leukemia cells stimulated their metabolic
activity and induced the release of cytokines involved in AML pathophysiology.
In addition, the immunomodulatory factors released by AML cells upon RANKL
signaling impaired the anti-leukemia reactivity of NK cells and induced RANK
expression, and NK cells of AML patients displayed significantly upregulated
RANK expression compared with healthy controls. Treatment of AML cells with the
clinically available RANKL Ab Denosumab resulted in enhanced NK cell
anti-leukemia reactivity. This was due to both blockade of the release of
NK-inhibitory factors by AML cells and prevention of RANK signaling into NK
cells. The latter was found to directly impair NK anti-leukemia reactivity with
a more pronounced effect on IFN-γ production compared with cytotoxicity.
Together, our data unravel a previously unknown function of the RANK-RANKL
molecule system in AML pathophysiology as well as NK cell function and suggest
that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell
reactivity in leukemia patients. Estrogens and progesterones are major drivers of breast development but also
promote carcinogenesis in this organ. Yet, their respective roles and the
mechanisms underlying their action in the human breast are unclear. Receptor
activator of nuclear factor κB ligand (RANKL) has been identified as a pivotal
paracrine mediator of progesterone function in mouse mammary gland development
and mammary carcinogenesis. Whether the factor has the same role in humans is of
clinical interest because an inhibitor for RANKL, denosumab, is already used for
the treatment of bone disease and might benefit breast cancer patients. We show
that progesterone receptor (PR) signaling failed to induce RANKL in PR(+) breast
cancer cell lines and in dissociated, cultured breast epithelial cells. In
clinical specimens from healthy donors and intact breast tissue microstructures,
hormone response was maintained and RANKL expression was under progesterone
control, which increased RNA stability. RANKL was sufficient to trigger cell
proliferation and was required for progesterone-induced proliferation. The
findings were validated in vivo where RANKL protein expression in the breast
epithelium correlated with serum progesterone levels and the protein was
expressed in a subset of luminal cells that express PR. Thus, important hormonal
control mechanisms are conserved across species, making RANKL a potential target
in breast cancer treatment and prevention. |
List fluorescent reporter proteins. | Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells:
green fluorescent protein
Timer
red fluorescent protein
yellow fluorescent protein
beta-phycoerythrin
coral fluorescent reporter protein
enhanced green fluorescent reporter protein
mCherry | Regulation of gene expression involves sequence elements in nucleic acids. In
promoters, multiple sequence elements cooperate as functional modules, which in
combination determine overall promoter activity. We previously developed
computational tools based on this hierarchical structure for in silico promoter
analysis. Here we address the functional organization of post-transcriptional
control elements, using the HIV-1 genome as a model. Numerous mutagenesis
studies demonstrate that expression of HIV structural proteins is restricted by
inhibitory sequences (INS) in HIV mRNAs in the absence of the HIV-1 Rev protein.
However, previous attempts to detect conserved sequence patterns of HIV-1 INS
have failed. We defined four distinct sequence patterns for inhibitory motifs
(weight matrices), which identified 22 out of the 25 known INS as well as
several new candidate INS regions contained in numerous HIV-1 strains. The
conservation of INS motifs within the HIV genome was not due to overall sequence
conservation. The functionality of two candidate INS regions was analyzed with a
new assay that measures the effect of non-coding mRNA sequences on production of
red fluorescent reporter protein. Both new INS regions showed inhibitory
activity in sense but not in antisense orientation. Inhibitory activity
increased by combining both INS regions in the same mRNA. Inhibitory activity of
known and new INS regions was overcome by co-expression of the HIV-1 Rev
protein. Type 1 diabetes results from the selective destruction of insulin-producing beta
cells in the islets of Langerhans, and autoimmune T cells are thought to be the
mediators of this destruction. T cells are also responsible for allorejection
once the islets are transplanted into a patient to reduce the negative
consequences of a lack of insulin. To better understand these processes, we have
developed a transgenic mouse expressing proinsulin II tagged with a live-cell
fluorescent reporter protein, Timer. Timer protein is unique because it changes
color from green to red in the first 24 h after synthesis. With this marker,
insulin synthesis can be carefully monitored through fluorescent changes over
time. To complement this new biotechnological research tool, we designed a body
window to allow for in vivo imaging over time of the islets transplanted under
the kidney capsule. The window device, which is sutured to replace the
underlying skin and body wall over the site of islet transplantation, may be
used to simultaneously observe beta cells and T cells that have been labeled
with a fluorochrome distinguishable from Timer. The imaging of both
insulin-producing cells and T cells may be carried out repeatedly for a week or
more with no need for repeated surgery, while preserving the life of the studied
animal. Ozone, the main component of photochemical smog and air pollution, can damage
the skin by oxidizing stratum corneum enzymes, lipids and structural proteins.
We have developed a rapid screening assay to determine free radical scavenging
capacity of various active ingredients that are frequently used in personal care
products. Several known antioxidants including vitamin C, vitamin E analog
Trolox, walnut seed extract, lipoic acid and ergothioneine inner salt were
assayed for their ability to neutralize ozone-induced oxidation of
beta-phycoerythrin, a fluorescent reporter protein derived from algae. The free
radical scavenging capacities of these antioxidants were quantified and
compared. The results demonstrate that this assay is a valuable primary
screening tool for identifying antioxidant activity of natural or synthetic
substrates that can be used in personal care products to protect the uppermost
layer of our skin from oxidizing damage induced by O3. Borrelia spirochaetes are unique among diderm bacteria in their abundance of
surface-displayed lipoproteins, some of which play important roles in the
pathogenesis of Lyme disease and relapsing fever. To identify the
lipoprotein-sorting signals in Borrelia burgdorferi, we generated chimeras
between the outer surface lipoprotein OspA, the periplasmic oligopeptide-binding
lipoprotein OppAIV and mRFP1, a monomeric red fluorescent reporter protein.
Localization of OspA and OppAIV point mutants showed that Borrelia lipoproteins
do not follow the '+2' sorting rule which targets lipoproteins to the
cytoplasmic or outer membrane of Gram-negative bacteria via the Lol pathway.
Fusions of mRFP1 to short N-terminal lipopeptides of OspA, and surprisingly
OppAIV, were targeted to the spirochaetal surface. Mutagenesis of the OspA
N-terminus defined less than five N-terminal amino acids as the minimal
secretion-facilitating signal. With the exception of negative charges, which can
act as partial subsurface retention signals in certain peptide contexts,
lipoprotein secretion occurs independent of N-terminal sequence. Together, these
data indicate that Borrelia lipoproteins are targeted to the bacterial surface
by default, but can be retained in the periplasm by sequence-specific signals. There is an increasing demand for high throughput (HTP) methods for gene
analysis on a genome-wide scale. However, the current repertoire of HTP
detection methodologies allows only a limited range of cellular phenotypes to be
studied. We have constructed two HTP-optimized expression vectors generated from
the red fluorescent reporter protein (RFP) gene. These vectors produce
RFP-tagged target proteins in a multiple expression system using gateway cloning
technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing
us localize the expressed proteins in mammalian cells. The effectiveness of the
vectors was evaluated using an HTP-screening system. Sixty representative human
C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor
cells, and we studied in detail two C2 domains that promoted the neuronal
differentiation of HiB5 cells. Our results show that the two vectors developed
in this study are useful for functional gene analysis using an HTP-screening
system on a genome-wide scale. An in-depth characterization of the Aspergillus niger glucoamylase (glaA)
promoter performance was carried out on defined medium employing multi-well
high-throughput screening as well as controlled batch and fed-batch bioreactor
culture techniques with GFP as a fluorescent reporter protein. A variety of
metabolizable carbon substrates and non-metabolizable analogs were screened with
regard to their effect on the glaA expression system. The results clearly
demonstrate that only starch and its hydrolytic products, including glucose, act
as inducers. However, induction of the glaA expression system through the
monosaccharide glucose is significantly lower compared to starch and the higher
molecular weight starch degradation products. All other 26 carbon substrates
tested do not induce, or even, as in the case of the easily metabolizable
monosaccharide xylose, repress glaA-promoter controlled gene expression in the
presence of the inducing disaccharide maltose with an increase of repression
strength by increasing xylose concentrations. The complex effect of glucose on
glaA-promoter controlled expression was also analyzed using non-metabolizable
glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified
as novel and potent inducers of the glaA expression system. The results show
that the induction strength depends on the inducer concentration with a maximum
at defined concentrations and lower induction or even repression at
concentrations above. Moreover, controlled fed-batch cultivations using a high
maltose feed rate with concomitant extracellular accumulation of glucose
resulted in lower levels of the reporter protein compared to cultures with a
low-maltose feed rate without extracellular glucose accumulation, thus
supporting the conclusion that increasing the glucose concentration beyond a
critical point reduces the induction strength or may even cause repression. This
way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown
can be fine-tuned for optimal fungal growth and the metabolic burden for
glucoamylase synthesis can be limited adequately in response to nutrient
availability. Potential therapeutic applications of embryonic stem cell (ESC)-derived
hepatocytes are limited by their relatively low output in differentiating ESC
cultures, as well as by the danger of contamination with tumorigenic
undifferentiated ESCs. To address these problems, we developed transgenic murine
ESC clones possessing bicistronic expression vector that contains the
alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live"
fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under
established culture conditions these clones allowed for both monitoring of
differentiation and for puromycin selection of hepatocyte-committed cells in a
suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]).
When plated on fibronectin, the selected eGFP-positive cells formed colonies, in
which intensely proliferating hepatocyte precursor-like cells gave rise to
morphologically differentiated cells expressing alpha-1-antitrypsin,
alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in
some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte
marker genes were expressed in the culture, along with the gene and protein
expression of stem/progenitor markers, suggesting the features of both
hepatocyte precursors and more advanced differentiated cells. When cultured in
suspension, the EB-derived puromycin-selected cells formed spheroids capable of
outgrowing on an adhesive substrate, resembling the behavior of fetal mouse
hepatic progenitor cells. The established system based on the highly efficient
selection/purification procedure could be suitable for scalable generation of
ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a
potential in vitro source of cells for transplantation therapy of liver
diseases, tissue engineering, and drug and toxicology screening. We engineered a method for detecting intramolecular and intermolecular phox
protein interactions in cells by fluorescence microscopy using fusion proteins
of complementary fragments of a coral fluorescent reporter protein (monomeric
Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green
system by showing that the PX and PB1 domains of p40phox interact in intact
cells, which we suggested maintains this protein in an inactive closed
conformation. Using this system, we also explored intramolecular interactions
within p47phox and showed that the PX domain interacts with the autoinhibited
tandem Src homology 3 domains maintained in contact with the autoinhibitory
region, along with residues 341-360. Furthermore, we demonstrated sequential
interactions of p67phox with phagosomes involving adaptor proteins, p47phox and
p40phox, during FcgammaR-mediated phagocytosis. Although p67phox is not targeted
to phagosomes by itself, p47phox functions as an adaptor for the ternary complex
(p47phox-p67phox-p40phox) in early stages of phagocytosis before phagosome
closure, while p40phox functions in later stages after phagosomal closure.
Interestingly, a mutated "open" form of p40phox linked p47phox to closed
phagosomes and prolonged p47phox and p67phox retention on phagosomes. These
results indicate that binding of the ternary complex to phagosomes can be
temporally regulated by switching between adaptor proteins that have PX domains
with distinct lipid-binding specificities. Neuronal gap junctions, allowing fast intercellular electrotonic signal
transfer, have been implicated in mechanisms governing learning and memory
processes. We have examined conditional neuron-directed (Cx45fl/fl:Nestin-Cre)
connexin45 deficient mice in terms of behavioral and electrophysiological
correlates of learning and memory. Behavioral habituation to a novel environment
and motor learning were not changed in these mice. Novel object recognition
after delays of up to 60min was impaired in neuronal Cx45 deficient mice.
However, object-place recognition was not significantly different from controls.
Analysis of enhanced green fluorescent reporter protein expression controlled by
the endogenous mouse Cx45 promoter in the brain of neuronal Cx45 deficient mice
suggested that Cx45 is expressed in the perirhinal cortex and the CA3 subregion
of the hippocampus. The neuronal Cx45 deficient mice were also examined for
aberrations in the generation and synchronization of network oscillations in the
hippocampus. General excitability, synaptic short time plasticity, and
spontaneous high-frequency oscillations (sharp-wave ripples) in the hippocampus
were not different from controls. However, bath stimulation of hippocampal
slices with kainate induced significantly lower gamma-oscillation amplitudes in
the CA3, but not in the CA1 subfield of the neuronal Cx45 deficient mice.
Additionally, they exhibited a significantly larger full width half maximum of
the frequency distribution in the CA1 subfield as compared to the controls. In
conclusion, the neuron-directed deletion of Cx45 impaired one-trial novel object
recognition and altered kainate-induced gamma-oscillations possibly via the
disruption of inter-neuronal gap junctional communication in the hippocampus or
perirhinal cortex. A new experimental approach was designed to test different predictions of
current models of the nuclear architecture with respect to the topography of
transcription. We constructed a plasmid, termed pIndi, which carries a reporter
gene coding for a red cytoplasmic fluorescent reporter protein. Transcription of
the reporter gene is regulated by the inducible promoter of the human
immunodeficiency virus (HIV) and is strongly dependent on the HIV-1 Tat protein.
Expressing the red fluorescent reporter protein allowed us to distinguish
between cells with active and silent reporter genes. Importantly, transient
transfection resulted in the clustering of plasmids, forming one or several
extra-chromosomal pIndi bodies. Repetitive lac operator sequences in pIndi
allowed us to visualize these bodies in living cells by the binding of LacI
proteins tagged with a fluorescent protein. Using this model, we analyzed the
three-dimensional nuclear topography of pIndi bodies with active or silent
reporter genes. Our results are compatible with predictions of the chromosome
territory-interchromatin compartment (CT-IC) model. We demonstrate that pIndi
bodies localize in the IC, both in the silent and active state. Activation of
transgene transcription resulted in the recruitment of RNA polymerase II and
NFkappaB and a closer positioning to splicing speckles. Many debilitating diseases, including neurodegenerative diseases, involve
apoptosis. Several methods have been developed for visualizing apoptotic cells
in vitro or in fixed tissues, but few tools are available for visualizing
apoptotic cells in live animals. Here we describe a genetically encoded
fluorescent reporter protein that labels apoptotic cells in live zebrafish
embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed
on the outer leaflet of the plasma membrane. The calcium-dependent protein
Annexin V (A5) binds PS with high affinity, and biochemically purified,
fluorescently labeled A5 probes have been widely used to detect apoptosis in
vitro. Here we show that secreted A5 fused to yellow fluorescent protein
specifically labels apoptotic cells in living zebrafish. We use this fluorescent
probe to characterize patterns of apoptosis in living zebrafish larvae and to
visualize neuronal cell death at single-cell resolution in vivo. The study of plant parasitic nematodes such as Meloidogyne spp. and their
interactions with phytopathogenic bacteria remains underexplored. One of the
challenges towards establishing such interactions is the dependence on symptom
development as a measure of interaction. In this study, mCherry was employed as
a reporter protein to investigate the interaction between the soft rot
Enterobacteriaceae (SRE) Pectobacterium carotovorum subsp. brasiliensis (Pcb)
and root-knot nematode (M. incognita). Pectobacterium carotovorum subsp.
brasiliensis was transformed with pMP7604 generating Pcb_mCherry strain. This
strain was shown to attach to the surface coat of M.incognita J2 at the optimum
temperature of 28°C. This suggests that RKN juveniles may play a role in
disseminating Pcb in soils that are heavily infested with Pcb. The presence of
RKN juveniles was shown to play a role in introducing Pcb_mCherry into potato
tubers potentially acting as a source of latent tuber infections. Fluorescent reporter proteins based on flavin-binding photosensors were recently
developed as a new class of genetically encoded probes characterized by small
size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent
proteins (FbFPs) address two major limitations associated with existing
fluorescent reporters derived from the green fluorescent protein (GFP)-namely,
the overall large size and oxygen-dependent maturation of fluorescence of GFP.
However, FbFPs are at a nascent stage of development and have been utilized in
only a handful of biological studies. Importantly, a full understanding of the
performance and properties of FbFPs as a practical set of biological probes is
lacking. In this work, we extensively characterize three FbFPs isolated from
Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro
studies to assess probe brightness, oligomeric state, maturation time, fraction
of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal
stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo
studies by constructing a series of FbFP-based transcriptional constructs to
probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages
as broad-spectrum biological reporters including robust pH tolerance (4-11),
thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.).
In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a
stable and nonperturbative reporter of promoter activity in Escherichia coli.
Our results demonstrate that FbFP-based reporters have the potential to address
key limitations associated with the use of GFP, such as pH-sensitive
fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for
half maximal fluorescence recovery). From this view, FbFPs represent a useful
new addition to the fluorescent reporter protein palette, and our results
constitute an important framework to enable researchers to implement and further
engineer improved FbFP-based reporters with enhanced brightness and tighter
flavin binding, which will maximize their potential benefits. Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein,
dsRed, in which fluorescence shifts over time from green to red as the protein
matures. This molecular clock gives temporal and spatial information on protein
turnover. To visualize mitochondrial turnover, we targeted Timer to the
mitochondrial matrix with a mitochondrial-targeting sequence (coined
"MitoTimer") and cloned it into a tetracycline-inducible promoter construct to
regulate its expression. Here we report characterization of this novel
fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were
transfected with pTRE-tight-MitoTimer and production was induced with
doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence
microscopy and verified by subcellular fractionation and western blot analysis.
Dox addition for as little as 1 h was sufficient to induce MitoTimer expression
within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The
color-specific conformation of MitoTimer was stable after fixation with 4%
paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent
transition from green to red over 48 h and was amenable to analysis by
fluorescence microscopy and flow cytometry of whole cells or isolated
mitochondria. A second Dox administration 48 h after the initial induction
resulted in a second round of expression of green MitoTimer. The extent of new
protein incorporation during a second pulse was increased by administration of a
mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and
biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal
new insights into mitochondrial dynamics within cells. Coupled with organelle
flow cytometry, it offers new opportunities to investigate mitochondrial
subpopulations by biochemical or proteomic methods. |
By which mechanism MutT proteins act against DNA lesions in bacteria? | MutT proteins belong to a class of Nudix hydrolases. The common substrate structure for the proteins of the functionally diverse Nudix superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving groups. The activities of Nudix hydrolases usually result in the release of an inorganic phosphate ion or of a product bearing a terminal phosphate moiety. MutT proteins hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. MutT proteins cleave 8-oxo-dGTP (8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate) at the α-β position; they also cleave 8-oxo-dGTP at the β-γ phosphate bond at a rate of 3% of that recorded for hydrolysis at the α-β position. 8-oxo-dGTP induces A to C transversions when misincorporated in DNA opposite to template A. By hydrolyzing 8-oxo-dGTP before their incorporation into DNA, MutT proteins play a critical role in allowing bacteria to avoid A-to-C mutations, which are a hallmark of MutT deficiency. Thus, MutT proteins prevent oxidative DNA lesions, as part of the GO system. Oxidized nucleotides can occur when bacteria are exposed to reactive oxygen species. Also, reactive oxygen species are produced as side products of oxygen utilization, leading to the oxidation of nucleic acids and their precursor nucleotides. Distinct from the Escherichia coli MutT, which hydrolyzes 8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only 8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Moreover, the hydrolysis of 8-oxo-dGTP and 8-oxo-GTP in mycobacteria seems to be catalysed in a two-stage mechanism, since MutT converts these oxidized nucleoside triphosphates to their corresponding nucleoside diphosphates, and not to monophosphates. | 8-Oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate) is a potent mutagenic
substrate for DNA synthesis. The accumulation of 8-oxo-dGTP in the nucleotide
pool induces G:C-->T:A transversion as well as A:T-->C:G transversion, and
Escherichia coli cells possess mechanisms for preventing such mutations. The
mutT gene product specifically hydrolyzes 8-oxo-dGTP to the monophosphate form
while the mutM and the mutY gene products function to correct mispairs caused by
incorporation of 8-oxoguanine into DNA. From analyses of forward mutations
induced in cells lacking 8-oxo-dGTPase (MutT protein) and/or repair enzymes that
suppress mutations caused by 8-oxoguanine in DNA (MutM and MutY proteins),
cooperative functions of these proteins in control of the spontaneous
mutagenesis became evident. In mutator strains lacking MutT and/or MutM
proteins, 8-oxoguanine of DNA increased to a concentration expected from the
increased rate of mutation. The toxicity of Ni(II), Co(II) and Cu(II) in animals, and that of Cd(II) in
cultured cells, has been associated with generation of the promutagenic lesion
8-oxo-7,8-dihydroguanine (8-oxoguanine) in DNA, among other effects. One
possible source of this base may be
8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate (8-oxo-dGTP), a product of
oxidative damage to the nucleotide pool, from which it is incorporated into DNA.
To promote such incorporation, the metals would have to inhibit specific
cellular 8-oxo-dGTPases that eliminate 8-oxo-dGTP from the nucleotide pool. The
present study was designed to test such inhibition in vitro on 8-oxo-dGTPases
from two different species, the human MTH1 protein and Escherichia coli MutT
protein. In the presence of Mg(II), the natural activator of 8-oxo-dGTPases, all
four metals were found to inhibit both enzymes. For MTH1, the IC50 values (+/-
SE; n = 3-4) were 17 +/- 2 microM for Cu(II), 30 +/- 8 microM for Cd(II), 376
+/- 71 microM for Co(II) and 801 +/- 97 microM for Ni(II). For MutT, they were
60 +/- 6 microM for Cd(II), 102 +/- 8 microM for Cu(II), 1461 +/- 96 microM for
Ni(II) and 8788 +/- 1003 microM for Co(II). Thus, Cu(II) and Cd(II) emerged as
much stronger inhibitors than Ni(II) and Co(II), and MTH1 appeared to be
generally more sensitive to metal inhibition than MutT. Interestingly, in the
absence of Mg(II), the activity of the enzymes could be restored by Co(II) to
73% of that with Mg(II) alone for MutT, and 34% for MTH1, the other metals being
much less or non-effective. The difference in sensitivity to metal inhibition
between the two enzymes may reflect the differences in the amino acid ligands,
especially the cysteine ligand, outside their evolutionarily conserved
Mg(II)-binding active sites, which might indicate predomitly non-competitive
or uncompetitive mechanism of the inhibition. The overall results suggest that
inhibition of 8-oxo-dGTPases may be involved in the mechanisms of induction of
the 8-oxoguanine lesion in DNA by the metal ions studied, especially the
non-redox-active Cd(II) cation. Escherichia coli WP2 bacteria with an ochre amino acid auxotrophy show no
evidence of growth during the first few days after plating at densities above
10(8) on plates lacking the required amino acid. They lose viability for some
days, and then a subpopulation recovers and there is cell turnover. At very low
plating densities (around 10(2) per plate), almost every cell will eventually
form a small but visible colony. At intermediate plating densities (10(6) to
10(7) per plate), there is an immediate increase in the number of viable
bacteria. The results are consistent with a model that assumes that growth is
dependent on trace amounts of tryptophan or a tryptophan-complementing substance
and that death is due to extracellular toxic species in the medium, including
active oxygen species. Mutations in mutT bacteria under these conditions result
from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect
DNA synthesis associated with the increase in the number of viable cells at the
initial density used (10(7) per plate). We show that the increase in cell number
and much of this DNA synthesis can be eliminated by the presence of 10(8)
scavenger bacteria and by removal of early-arising mutant colonies that release
the required amino acid. The synthesis that remains is equivalent to less than a
quarter of a genome per day and is marginally reduced, if at all, in a polA
derivative. We cannot exclude the possibility that this residual DNA synthesis
is peculiar to mutT bacteria due to transcriptional leakiness, although there is
no evidence that this is a major problem in this strain. If such DNA synthesis
also occurs in wild-type bacteria, it may well be important for adaptive
mutation since use of a more refined agar in selective plates both eliminated
the initial increase in cell number seen at low density (10(7) per plate) and
reduced the rate of appearance of mutants at plating densities above 10(8) per
plate. MutT protein of Escherichia coli prevents the occurrence of A:T --> C:G
transversion by hydrolyzing an oxidized form of dGTP, 8-oxo-7,
8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), which is produced by
active oxygen species. In a search for mutT-related genes, we found that the
ribA gene, encoding GTP cyclohydrolase II, is able to reduce the increased level
of mutation frequency of the mutT strain to almost the normal level, provided
that the gene product is overproduced. Purified preparations of Escherichia coli
GTP cyclohydrolase II protein as well as the histidine hexamer-tagged
recombit GTP cyclohydrolase II protein efficiently hydrolyze 8-oxo-dGTP and
8-oxo-GTP, producing 8-oxo-dGMP and 8-oxo-GMP, respectively. dGTP was not
hydrolyzed by these preparations. GTP cyclohydrolase II catalyzes conversion of
GTP to 2, 5-diamino-6-hydroxy-4-(5-phosphoribosylamino)-pyrimidine, which
constitutes the first step for riboflavin synthesis. The Km values for the three
types of guanine nucleotides, GTP, 8-oxo-GTP, and 8-oxo-dGTP, were almost the
same. In the mutT- background, ribA- cells showed higher spontaneous mutation
frequencies as compared with that of ribA+ cells. Thus, GTP cyclohydrolase II,
the ribA gene product, has a potential to protect genetic material from the
untoward effects of endogenous oxygen radicals. Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2'-dGTP (8-oxo-dGTP)
to the monophosphate, thus avoiding the incorporation of
8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA. Bacterial and mammalian
homologs of MutT protein share the phosphohydrolase module (MutT:
Gly37-->Gly59). By saturation mutagenesis of conserved residues in the MutT
module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were
revealed to be essential to suppress spontaneous A:T-->C:G transversion mutation
in a mutT(-) mutator strain. For the other six residues (Lys39, Glu44, Thr45,
Arg52, Glu56 and Gly59), many positive mutants which can suppress the
spontaneous mutation were obtained; however, all of the positive mutants for
Glu44 and Arg52 either partially or inefficiently suppressed the mutation,
indicating that these two residues are also important for MutT function. Several
positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the
elevated spontaneous mutation rate, as seen with the wild-type, hence, these
four residues are non-essential for MutT function. As Lys38 and Glu55 in human
MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could
not be replaced by any other residue without loss of function, different
structural features between the two modules of MTH1 and MutT proteins are
evident. In Escherichia coli the mutT gene is one of several that acts to minimize
mutagenesis by reactive oxygen species. The bacterial MutT protein and its
mammalian homolog have been shown to catalyze in vitro the hydrolysis of the
oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding
monophosphate. Thus, the protein is thought to "sanitize" the nucleotide pool by
ridding the cell of a nucleotide whose incorporation into DNA would be intensely
mutagenic. However, because others have shown mutT mutations to be mutagenic
under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a
poor DNA polymerase substrate, there is reason to question this model. We have
devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which
involves reversed-phase high-performance liquid chromatography and
electrochemical detection, we have been unable to detect 8-oxo-dGTP in extracts
of three different mutT mutants of E. coli, even after growth of the bacteria in
the presence of hydrogen peroxide. Our estimated upper limit for 8-oxo-dGTP
content of these bacteria is about 200 molecules/cell, corresponding to a
concentration of about 0.34 microm. When 8-oxo-dGTP was added at 0.34 microm to
an in vitro DNA replication system primed with a DNA template that permits
scoring of replication errors and with the four normal dNTPs at their estimated
intracellular concentrations, there was no detectable effect upon the frequency
of replication errors. These findings lead us to question the conclusion that
8-oxo-dGTP is the most significant physiological substrate for the MutT protein. We have investigated in detail the interactions between the Escherichia coli
mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect
the cell against the effects of the oxidative stress product, 8-oxoguanine
(8-oxoG), a base analog with ambiguous base-pairing properties, pairing with
either A or C during DNA synthesis. mutT mutator strains display a specific
increase in A.T-->C.G transversions, while mutM and mutY mutator strains show
specific G.C-->T.A increases. To study in more detail the in vivo processing of
the various mutational intermediates leading to A.T-->C.G and G.C-->T.A
transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains
containing all possible combinations of these mutator alleles. We report three
major findings. First, we do not find evidence that the mutT allele
significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or
mutMmutY backgrounds. We interpret this result to indicate that incorporation of
8-oxodGTP opposite template C may not be frequent relative to incorporation
opposite template A. Second, we show that mutT-induced A.T-->C.G transversions
are significantly reduced in strains carrying mutY and mutMmutY deficiencies
suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP.
Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions
in the mutT(+) background, suggesting that, even in the presence of functional
MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP
misincorporation. MutT-related proteins, including the Escherichia coli MutT and human MutT
homologue 1 (MTH1) proteins, degrade 8-oxo- 7,8-dihydrodeoxyguanosine
triphosphate (8-oxo-dGTP) to a monophosphate, thereby preventing mutations
caused by the misincorporation of 8-oxoguanine into DNA. Here, we report that
human cells have another mechanism for cleaning up the nucleotide pool to ensure
accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses
8-oxo-dGDP to monophosphate with a K(m) of 0.77 microM, a value considerably
lower than that for ADP sugars, which were originally identified as being
substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is
able to substitute for MutT when it is defective. When NUDT5 is expressed in E.
coli mutT(-) cells, the increased frequency of spontaneous mutations is
decreased to normal levels. Considering the enzymatic parameters of MTH1 and
NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role
than MTH1 in preventing the occurrence of mutations that are caused by the
misincorporation of 8-oxoguanine in human cells. A strong chronic induction of the SOS response system occurs in E. coli BW535, a
strain defective in nth, nfo and xth genes, and hence severely deficient in the
repair of abasic sites in DNA. This was shown here by visualization of
filamentous growth of the BW535 strain and by measuring the level of
beta-galactosidase expressed in BW535/pSK1002 in comparison to the
AB1157/pSK1002 strain. The plasmid pSK1002 bears an umuC::lacZ fusion in which
lacZ is under the control of the umuC promoter and regulated under the SOS
regulon. Increases in the expression of beta-galactosidase occur in BW535
without any exogenous SOS inducer. Chronic induction of the SOS response was
observed previously in E. coli strains bearing mutations in certain genes that
have mutator activity and BW535 is a moderate mutator strain. However, not all
mutators show this property, since chronic induction of SOS was not observed in
mutT or mutY mutators. MutT and MutY proteins, when active, protect bacteria
from mutations induced by 8-oxoG lesions in DNA. This suggests that accumulation
of abasic sites, but not 8-oxoG residues in DNA, induce the SOS response. MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate
(8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool,
thereby preventing DNA replication errors. During a search of GenBank EST
database, we found a new member of MutT-related protein, MTH2, which possesses
the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed
in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein
hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA
for mMTH2 reduced significantly the elevated level of spontaneous mutation
frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the
genetic material from the untoward effects of endogenous oxygen radicals. MTH2
could act as an MTH1 redundancy factor. A search for candidates for a functional homologue of Escherichia coli MutT in
yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the
Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas
vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused
on the open reading frame YLR151c, because it had a region with approximately
76% similarity to the N-terminal half of MutT including the Nudix box. We thus
evaluated the ability of YLR151c as a functional homologue of E.coli MutT in
S.cerevisiae. Expression of YLR151c was able to suppress the transversion from
A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in
the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain
caused approximately 14-fold increase in the frequency of spontaneous mutation
compared to the wild type. Additionally, biochemical analysis indicated that
GST-YLR151c fusion protein possessed pyrophosphatase activity for both
7,8-dihydro-8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP) and
1,2-dihydro-2-hydroxy-2'-deoxyadenosine triphosphate (2-OH-dATP). The specific
activity of GST-YLR151c for 8-oxo-dGTP was 5.6 x 10(-3) microM(-1) s(-1), which
was similar to that of RibA, a backup enzyme for MutT in E.coli, but was
150-fold lower than that of hMTH1. From these results, we conclude that YLR151c
has an ability to prevent spontaneous mutagenesis via sanitization of oxidized
nucleotides, and that it may be the functional homologue of E.coli MutT in
S.cerevisiae. Nucleotide pool sanitizing enzymes Dut (dUTPase), RdgB (dITPase), and MutT
(8-oxo-dGTPase) of Escherichia coli hydrolyze noncanonical DNA precursors to
prevent incorporation of base analogs into DNA. Previous studies reported
dramatic AT-->CG mutagenesis in mutT mutants, suggesting a considerable density
of 8-oxo-G in DNA that should cause frequent excision and chromosomal
fragmentation, irreparable in the absence of RecBCD-catalyzed repair and similar
to the lethality of dut recBC and rdgB recBC double mutants. In contrast, we
found mutT recBC double mutants viable with no signs of chromosomal
fragmentation. Overproduction of the MutM and MutY DNA glycosylases, both acting
on DNA containing 8-oxo-G, still yields no lethality in mutT recBC double
mutants. Plasmid DNA, extracted from mutT mutM double mutant cells and treated
with MutM in vitro, shows no increased relaxation, indicating no additional
8-oxo-G modifications. Our DeltamutT allele elevates the AT-->CG transversion
rate 27,000-fold, consistent with published reports. However, the rate of
AT-->CG transversions in our mutT(+) progenitor strain is some two orders of
magnitude lower than in previous studies, which lowers the absolute rate of
mutagenesis in DeltamutT derivatives, translating into less than four 8-oxo-G
modifications per genome equivalent, which is too low to cause the expected
effects. Introduction of various additional mutations in the DeltamutT strain or
treatment with oxidative agents failed to increase the mutagenesis even twofold.
We conclude that, in contrast to the previous studies, there is not enough
8-oxo-G in the DNA of mutT mutants to cause elevated excision repair that would
trigger chromosomal fragmentation. Exposure to solar radiation can cause mortality in natural communities of
pico-phytoplankton, both at the surface and to a depth of at least 30 m. DNA
damage is a significant cause of death, mainly due to cyclobutane pyrimidine
dimer formation, which can be lethal if not repaired. While developing a UV
mutagenesis protocol for the marine cyanobacterium Prochlorococcus, we isolated
a UV-hyper-resistant variant of high light-adapted strain MED4. The
hyper-resistant strain was constitutively upregulated for expression of the
mutT-phrB operon, encoding nudix hydrolase and photolyase, both of which are
involved in repair of DNA damage that can be caused by UV light. Photolyase
(PhrB) breaks pyrimidine dimers typically caused by UV exposure, using energy
from visible light in the process known as photoreactivation. Nudix hydrolase
(MutT) hydrolyses 8-oxo-dGTP, an aberrant form of GTP that results from
oxidizing conditions, including UV radiation, thus impeding mispairing and
mutagenesis by preventing incorporation of the aberrant form into DNA. These
processes are error-free, in contrast to error-prone SOS dark repair systems
that are widespread in bacteria. The UV-hyper-resistant strain contained only a
single mutation: a 1 bp deletion in the intergenic region directly upstream of
the mutT-phrB operon. Two subsequent enrichments for MED4 UV-hyper-resistant
strains from MED4 wild-type cultures gave rise to strains containing this same 1
bp deletion, affirming its connection to the hyper-resistant phenotype. These
results have implications for Prochlorococcus DNA repair mechanisms, genome
stability and possibly lysogeny. Hypermutable or mutator microorganisms are those that have an increased
spontaneous mutation rate as a result of defects in DNA repair or error
avoidance systems. Over the last two decades, several studies have provided
strong evidence for a relevant role of mutators in the evolution of natural
bacterial populations, particularly in the field of infectious diseases. Among
them, chronic respiratory infection with Pseudomonas aeruginosa in cystic
fibrosis (CF) patients was the first natural environment to reveal the high
prevalence and important role of mutators. A remarkable positive selection of
mutators during the course of the chronic infection has been reported, mainly as
a result of the emergence of DNA mismatch repair system (mutS, mutL or
mutU)-deficient mutants, although strains defective in the GO system (mutM, mutY
and mutT) have also been observed. High frequencies of mutators have also been
noted among other pathogens in the CF setting, particularly Staphylococcus
aureus and Haemophilus influenzae. Enhanced antimicrobial resistance development
is the most thoroughly studied consequence of mutators in CF and other chronic
infections, although recent studies show that mutators may additionally have
important effects on the evolution of virulence, genetic adaptation to the
airways of CF patients, persistence of colonization, transmissibility, and
perhaps lung function decline. Further prospective clinical studies are
nevertheless still needed for an in-depth evaluation of the impact of mutators
on disease progression and outcome. MutT enzymes prevent DNA damage by hydrolysis of 8-oxodGTP, an oxidized
substrate for DNA synthesis and antimutagenic, anticarcinogenic, and
antineurodegenerative functions of MutT enzymes are well established. MutT has
been found in almost all kingdoms of life, including many bacterial species,
yeasts, plants and mammals. However, a Caenorhabditis elegans MutT homologue was
not previously identified. Here, we demonstrate that NDX-4 exhibits both
hallmarks of a MutT-type enzyme with an ability to hydrolyze 8-oxodGTP and
suppress the Escherichia coli mutT mutator phenotype. Moreover, we show that
NDX-4 contributes to genomic stability in vivo in C. elegans. Phenotypic
analyses of an ndx-4 mutant reveal that loss of NDX-4 leads to upregulation of
key stress responsive genes that likely compensate for the in vivo role of NDX-4
in protection against deleterious consequences of oxidative stress. This
discovery will enable us to use this extremely robust genetic model for further
research into the contribution of oxidative DNA damage to phenotypes associated
with oxidative stress. MutT protein of Escherichia coli hydrolyzes oxidized guanine nucleotides,
8-oxo-dGTP and 8-oxoGTP, to the corresponding monophosphates, thereby preventing
misincorporation of 8-oxoguanine into DNA and RNA, respectively. Although the
biological significance of the MutT has been established, how MutT protein
actually works in vivo remains to be elucidated. The current study shows the
molecular behavior of the MutT protein in vivo and in vitro with special
reference to control of spontaneous mutagenesis. A single E. coli cell carries
about 70-75 molecules of the MutT protein and that this number does not change
even when the cells were cultured in anaerobic and hyper-oxidative conditions.
Conditional gene silencing analyses revealed that about a half number of MutT
molecules are needed for keeping the spontaneous mutation frequency at the
normal level. The MutT functions are not needed under anaerobic condition, yet
the level of the MutT protein in cell is kept constant, probably for preparing
for sudden changes of oxygen pressure. There is a possibility that MutT
functions in close association with other proteins, and evidence is presented
that MutT protein can interact with some proteins in vivo. The oxidized nucleotide precursors 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) and 1,
2-dihydro-2-oxo-dATP (2-oxo-dATP) are readily incorporated into nascent DNA
strands during replication, which would cause base substitution mutations. E.
coli MutT and human homologue hMTH1 hydrolyze 8-oxo-dGTP, thereby preventing
mutations. In this study, we searched for hMTH1 homologues in the ascidian Ciona
intestinalis using the NCBI-BLAST database. Among several candidates, we focused
on one open reading frame, designated as CiMutT, because of its high degree of
identity (41.7%) and similarity (58.3%) to the overall amino acid sequence of
hMTH1, including the Nudix box. CiMutT significantly suppressed the mutator
activity of E. coli mutT mutant. Purified CiMutT had a pyrophosphohydrolase
activity that hydrolyzed 8-oxo-dGTP to 8-oxo-dGMP and inorganic pyrophosphate.
It had a pH optimum of 9.5 and Mg(++) requirement with optimal activity at 5 mM.
The activity of CiMutT for 8-oxo-dGTP was comparable to that of hMTH1, while it
was 100-fold lower for 2-oxo-dATP than that of hMTH1. These facts indicate that
CiMutT is a functional homologue of E. coli MutT. In addition, the enzyme
hydrolyzed all four of the unoxidized nucleoside triphosphates, with a
preference for dATP. The specific activity for 8-oxo-dGTP was greater than that
for unoxidized dATP and dGTP. These results suggest that CiMutT has the
potential to prevent mutations by 8-oxo-dGTP in C. intestinalis. Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is
ensured by the DNA oxidative repair system (GO). Single inactivation of mutT,
mutY and mutM involved in GO led to elevated mutation rates (MRs) that
correlated to increased development of resistance to antibiotics. In this study,
we constructed a double mutant in mutY and mutM (PAOMY-Mgm) and characterized
the phenotype and the gene expression profile using microarray and RT-PCR.
PAOMY-Mgm presented 28-fold increases in MR compared with wild-type reference
strain PAO1. In comparison, the PAOMYgm (mutY) single mutant showed only a
fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a
nonsignificant increase in MR compared with PAO1 and the single mutants.
Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux
pump were found as the mechanism of resistance to ciprofloxacin in the double
mutant. A better fitness of the mutator compared with PAO1 was found in growth
competition experiments in the presence of ciprofloxacin at concentrations just
below minimal inhibitory concentration. Up-regulation of the antimutator gene
pfpI, that has been shown to provide protection to oxidative stress, was found
in PAOMY-Mgm compared with PAO1. In conclusion, we showed that MutY and MutM are
cooperating in the GO of P. aeruginosa, and that oxidative DNA lesions might
represent an oxidative stress for the bacteria. Most of the proteins carrying the 23-residue MutT-related sequence are capable
of hydrolyzing compounds with a general structure of nucleoside diphosphate
linked to another moiety X and are called the Nudix hydrolases. Among the 22
human Nudix proteins (identified by the sequence signature), some remain
uncharacterized as enzymes without a defined substrate. Here, we reveal that the
NUDT18 protein, whose substrate was unknown, can degrade
8-oxo-7,8-dihydroguanine (8-oxo-Gua)-containing nucleoside diphosphates to the
monophosphates. Because this enzyme is closely related to MTH1 (NUDT1) and MTH2
(NUDT15), we propose that it should be named MTH3. Although these three human
proteins resemble each other in their sequences, their substrate specificities
differ considerably. MTH1 cleaves 8-oxo-dGTP but not 8-oxo-dGDP, whereas MTH2
can degrade both 8-oxo-dGTP and 8-oxo-dGDP, although the intrinsic enzyme
activity of MTH2 is considerably lower than that of MTH1. On the other hand,
MTH3 is specifically active against 8-oxo-dGDP and hardly cleaves 8-oxo-dGTP.
Other types of oxidized nucleoside diphosphates, 2-hydroxy-dADP and
8-hydroxy-dADP, were also hydrolyzed by MTH3. Another notable feature of the
MTH3 enzyme is its action toward the ribonucleotide counterpart. MTH3 can
degrade 8-oxo-GDP as efficiently as 8-oxo-dGDP, which is in contrast to the
finding that MTH1 and MTH2 show a limited activity against the ribonucleotide
counterpart, 8-oxo-GTP. These three enzymes may function together to help
maintain the high fidelity of DNA replication and transcription under oxidative
stress. Antibacterial action of silver oparticles (AgNP) on Gram-negative bacteria
(planctonic cells and biofilms) is reported in this study. AgNP of 8.3 nm in
diameter stabilized by hydrolyzed casein peptides strongly inhibited biofilms
formation of Escherichia coli AB1157, Pseudomonas aeruginosa PAO1 and Serratia
proteamaculans 94 in concentrations of 4-5 μg/ml, 10 μg/ml and 10-20 μg/ml,
respectively. The viability of E. coli AB1157 cells in biofilms was considerably
reduced by AgNP concentrations above 100 to -150 μg/ml. E. coli strains with
mutations in genes responsible for the repair of DNA containing oxidative
lesions (mutY, mutS, mutM, mutT, nth) were less resistant to AgNP than wild type
strains. This suggests that these genes may be involved in the repair of DNA
damage caused by AgNP. E. coli mutants deficient in excision repair,
SOS-response and in the synthesis of global regulators RpoS, CRP protein and Lon
protease present similar resistance to AgNP as wild type cells. LuxI/LuxR Quorum
Sensing systems did not participate in the control of sensitivity to AgNP of
Pseudomonas and Serratia. E. coli mutant strains deficient in OmpF or OmpC
porins were 4-8 times more resistant to AgNP as compared to the wild type
strain. This suggests that porins have an important function related AgNP
antibacterial effects. Mycobacterium tuberculosis, the causative agent of tuberculosis, is at increased
risk of accumulating damaged guanine nucleotides such as 8-oxo-dGTP and
8-oxo-GTP because of its residency in the oxidative environment of the host
macrophages. By hydrolyzing the oxidized guanine nucleotides before their
incorporation into nucleic acids, MutT proteins play a critical role in allowing
organisms to avoid their deleterious effects. Mycobacteria possess several MutT
proteins. Here, we purified recombit M. tuberculosis MutT2 (MtuMutT2) and M.
smegmatis MutT2 (MsmMutT2) proteins from M. tuberculosis (a slow grower) and M.
smegmatis (fast growing model mycobacteria), respectively, for their biochemical
characterization. Distinct from the Escherichia coli MutT, which hydrolyzes
8-oxo-dGTP and 8-oxo-GTP, the mycobacterial proteins hydrolyze not only
8-oxo-dGTP and 8-oxo-GTP but also dCTP and 5-methyl-dCTP. Determination of
kinetic parameters (Km and Vmax) revealed that while MtuMutT2 hydrolyzes dCTP
nearly four times better than it does 8-oxo-dGTP, MsmMutT2 hydrolyzes them
nearly equally. Also, MsmMutT2 is about 14 times more efficient than MtuMutT2 in
its catalytic activity of hydrolyzing 8-oxo-dGTP. Consistent with these
observations, MsmMutT2 but not MtuMutT2 rescues E. coli for MutT deficiency by
decreasing both the mutation frequency and A-to-C mutations (a hallmark of MutT
deficiency). We discuss these findings in the context of the physiological
significance of MutT proteins. Reactive oxygen species are produced as side products of oxygen utilization and
can lead to the oxidation of nucleic acids and their precursor nucleotides.
Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most
critical during the transfer of genetic information because it can pair with
both cytosine and adenine. During the de novo synthesis of guanine nucleotides,
GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme
hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of
GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the
formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be
produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus
formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and
adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not
discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and
by the oxidation of GTP can be used for RNA synthesis. This misincorporation is
prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well
as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to
CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated
into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess
mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA. Approximately one third of the world population is infected with Mycobacterium
tuberculosis, the causative agent of tuberculosis. A better understanding of the
pathogen biology is crucial to develop new tools/strategies to tackle its spread
and treatment. In the host macrophages, the pathogen is exposed to reactive
oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP,
respectively. Incorporation of the damaged nucleotides in nucleic acids is
detrimental to organisms. MutT proteins, belonging to a class of Nudix
hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the
corresponding nucleoside monophosphates and sanitize the nucleotide pool.
Mycobacteria possess several MutT proteins. However, a functional homolog of
Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1
and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1
converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then
converts them to the corresponding nucleoside monophosphates. This observation
suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP
detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with
a Km of ∼50 μM and Vmax of ∼0.9 pmol/min per ng of protein, and Rv1700 converts
8-oxo-dGDP to 8-oxo-dGMP with a Km of ∼9.5 μM and Vmax of ∼0.04 pmol/min per ng
of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for
its MutT deficiency by decreasing A to C mutations (a hallmark of MutT
deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a
crucial role in survival of bacteria against oxidative stress. The common substrate structure for the functionally diverse Nudix protein
superfamily is nucleotide-diphosphate-X, where X is a large variety of leaving
groups. The substrate specificity is known for less than 1% of the 29,400 known
members. Most activities result in the release of an inorganic phosphate ion or
of a product bearing a terminal phosphate moiety. Reactions have typically been
monitored by a modification of the discontinuous Fiske-SubbaRow assay, which is
relatively insensitive and slow. We report here the development of a continuous
fluorescence assay that enables the rapid and accurate determination of
substrate specificities in a 96-well format. We used this novel assay to confirm
the reported substrate characterizations of MutT and NudD of Escherichia coli
and to characterize DR_1025 of Deinococcus radiodurans and MM_0920 of
Methanosarcina mazei. Novel findings enabled by the new assay include the
following. First, in addition to the well-characterized hydrolysis of 8-oxo-dGTP
at the α-β position, MutT cleaves at the β-γ phosphate bond at a rate of 3% of
that recorded for hydrolysis at the α-β position. Second, MutT also catalyzes
the hydrolysis of 5-methyl-dCTP. Third, 8-oxo-dGTP was observed to be the best
substrate for DR_1025 of the 41 compounds screened. |
Was tamoxifen tested for treatment of glioma patients? | Yes, tamoxifen was tested for glioma treatment. However, clinical efficacy of tamoxifen in glioma patients remains unclear and should be tested in further studies. | Previous work has demonstrated the importance of protein kinase C in regulating
glioma cell proliferation in vitro. Tamoxifen, a protein kinase C inhibitor when
administered in high dosages, is currently being used as an adjuvant in the
treatment of patients with maligt gliomas. The patient in the present study
harbored a left frontal anaplastic astrocytoma adjacent to Broca's area and the
paracentral region, which limited gross resection. After a subtotal resection of
the tumor and after radiation, the patient was administered high-dose tamoxifen
therapy for gross residual gadolinium-enhancing regions that were revealed by
magnetic resoce imaging and by high glucose uptake as demonstrated by
positron emission tomography. After treatment, a decrease in gadolinium
enhancement on magnetic resoce images and a decrease in glucose uptake
revealed by positron emission tomography were noted. A laboratory examination of
the tissue obtained from the original surgical resection revealed resistance to
radiation therapy but sensitivity to tamoxifen as measured by
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. The
subsequent in vitro testing of the tumor that was removed after the recurrence
of tumor (22 months after the initiation of tamoxifen) revealed loss of
sensitivity to tamoxifen. However, the recurrent tumor remained sensitive to
growth inhibition by the potent protein kinase C inhibitor, hypericin, despite
loss of sensitivity to tamoxifen in vitro, suggesting the potential clinical
application of this agent. This close in vitro correlation with the clinical
course of the patient in the present study suggests a potential role for such in
vitro radiation and chemosensitivity testing in designing a rational
individualized clinical course of treatment. Traditional study design for treatment of maligt gliomas does not allow tumor
progression to be greater than 25-50 percent without terminating treatment. This
design may prevent recognition of patients who benefit from the treatment either
by slowed growth or delayed response. A delayed response or slowed growth may be
characteristic of biologic agents being evaluated in the treatment of maligt
glioma. Because of the low toxicity of certain biologic drugs, continued
treatment through tumor growth can be ethically considered in study design. The
effect of biologic agents on a neoplasm may include cellular differentiation,
retardation of growth, cytostasis, cytocidal effects, or apoptosis. Such effects
may clinically translate into a complete response, partial response, stable
disease or retardation of growth with or without an eventual reduction of tumor.
We present a patient with a recurrent maligt glioma who was continued on high
dose tamoxifen despite radiologic documented doubling of the tumor size and who
eventually showed a delayed response to this agent nine months after initiation
of treatment. Strong consideration should be given to the prolonged treatment of
non-toxic biologic agents in a controlled clinical trial, where agents have
shown some benefit in phase one studies. Chemotherapeutic regimens in present use for recurrent glioma have substantial
toxicity. Activity against recurrent gliomas has been reported for both
tamoxifen and interferon alpha, agents that have more acceptable toxicity
profiles and that can be administered in an outpatient setting. We tested the
efficacy and toxicity of the combination of high-dose tamoxifen and interferon
alpha in adults with recurrent glioma in a phase II trial. Eligible patients had
radiographically measurable recurrent gliomas of any grade after initial
radiation therapy. Interferon-alpha [6 x 10(6) U subcutaneously three times per
week] and tamoxifen (240 mg/m2/day orally) were administered continuously.
Treatment response was assessed at 6 week intervals using clinical and
radiographic criteria. Eighteen patients (11 males and 7 females) were enrolled.
Median age was 41 years (range 23-61 years). All patients had gliomas that
progressed after radiation therapy and nitrosourea chemotherapy. The histologic
diagnosis of the original tumor was glioblastoma multiforme in 8 patients,
anaplastic astrocytoma in 5 patients, astrocytoma in 4 patients and mixed
maligt glioma in 1 patient. Reversible moderate to severe neurological
toxicity manifested by dizziness and unsteady gait was seen at tamoxifen doses
of 240 mg/m2/day. Although the initial tamoxifen dose was reduced to 120
mg/m2/day, moderate neurotoxicity was noted at this dose as well and the trial
was closed early. The combination of oral tamoxifen (120 to 240 mg/m2/day) and
subcutaneous interferon-alpha [6 x 10(6) U three times per week] was associated
with significant neurotoxicity in this group of recurrent glioma patients,
resulting in early study closure. Of 16 evaluable patients, 12 had progressive
disease after one cycle of treatment, 3 had stable disease, and there was one
minor response. Gradual dose escalation may be required if similar patients are
to be treated with high dose tamoxifen in conjunction with interferon. Cerebral gliomas have a poor survival time even after multimodal treatment,
because of the unavoidable recurrence of tumor. Several trials with a
combination of old and new chemotherapics have been performed, but survival time
remains generally less than 12 months. Tamoxifen (TAM) has recently been shown
to inhibit the growth rate of established and low-passage human glioma cell
lines. Furthermore, this drug has enabled stabilization of the clinical and
radiographic picture in selected patients with recurrent glioma. Here we review
published data to discuss a potential role of TAM in the multimodal
postoperative treatment of cerebral gliomas. Multifocal tumor recurrence of glioblastomas occurs in up to 14% of patients. In
a parallel phase-II-study investigating post-operative treatment with tamoxifen
(TAM), carboplatin and radiation therapy for glioblastomas, 16 of 49 patients
(33%) showed multifocal recurrence, which developed after a mean of 46 weeks,
raising the question of an association with therapy. We studied the
interrelation of proliferation and migration in the presence of different
protein-kinase-C(PKC) inhibitors (TAM, staurosporine, hypericin) in 2 glioma
cell lines. In addition, 3 cell lines were selected for TAM resistance by
repeated cycles of treatment with sub-lethal concentrations of TAM. The
proliferative capacity and the invasive potential of selected sub-populations
were assessed using growth-curve experiments, monolayer migration, and
cell-adhesion assays. Treatment with all PKC inhibitors tested resulted in a
dose-dependent decrease of proliferation, while motility was altered only at
significantly higher doses. Resistance to TAM occurred in all 3 selected cell
lines. The TAM-resistant sub-populations showed significantly increased
proliferation, migration and adhesion as compared with the parental
(non-selected) cell line. The higher incidence of multifocal disease after TAM
treatment was paralleled by increased migratory potential of TAM-treated cells
in vitro. BACKGROUND: Doxorubicin exhibits high efficacy in maligt glioma cell
cultures. Nonetheless, as a standard formulation, doxorubicin has not been used
clinically, due to poor penetration of the blood-brain barrier. Furthermore,
doxorubicin is known to induce tumor resistance genes. To address both of these
issues, the authors investigated the use of pegylated liposomal doxorubicin
(Caelyx; Essex Pharma, Munich, Germany) alone (Trial 1) and in combination with
tamoxifen (Trial 2) in two sequentially performed nonrandomized prospective
Phase II trials involving patients with recurrent high-grade glioma.
METHODS: Twenty patients were included in each trial. Progression-free survival
at 6 months (PFS-6) and toxicity were the primary endpoints. Expression of the
tumor resistance proteins multidrug resistance protein 1 (MDR-1) and multiple
resistance protein (MRP) was evaluated by immunohistochemical methods and by
sestamibi-single-photon emission computed tomography (SPECT).
RESULTS: The overall response rate (including cases of disease stabilization)
was 40% in both Trial 1 and Trial 2. PFS-6 was 15%, and the median time to
disease progression was 17 weeks. It is noteworthy that 40% of patients with
Grade III tumors had long-term responses, which lasted for up to 3 years. There
was no significant difference between Trial 1 and Trial 2 in terms of efficacy.
Both regimens were well tolerated, with the main side effect being palmoplantar
erythrodysesthesia. The authors found no correlation between clinical response
and expression of tumor resistance genes or between clinical response and SPECT
data.
CONCLUSIONS: Pegylated liposomal doxorubicin administered alone or in
combination with tamoxifen is safe and moderately effective in patients with
recurrent high-grade glioma. None of the putative predictors for response that
were evaluated proved to be significant in this setting. PURPOSE: To determine the response rate, time to disease progression, survival,
and toxicity of intravenous carboplatin and chronic oral high-dose tamoxifen in
patients with recurrent maligt gliomas.
PATIENTS AND METHODS: Patients with histological confirmation of recurrent
maligt gliomas were eligible for this multicenter phase II trial. Treatment
consisted of 400 mg/m2 carboplatin intravenously every 4 weeks and oral high
dose chronic tamoxifen (80 mg bid in women and 100 mg bid in men).
RESULTS: Twenty seven patients met the eligibility criteria and were evaluable
for response. The histological subtypes were: 16 (59%) glioblastoma multiforme
(GBM), maligt astrocytoma (5 patients), maligt mixed glioma (5 patients),
and glioblastoma/gliosarcoma (1 patient). Twenty-two patients (82%) had an ECOG
performance status of 0 or 1. No complete responses were observed, 4 patients
(15%) achieved a partial response, and 14 patients (52%) had stable disease.
Median time to progression was 3.65 months (95%CI 2.56, 4.83). Median overall
survival was 14.09 months (95%CI 7.06, 19.91). One patient with a recurrent GBM
had a sustained partial response and is progression free 81 months since
starting treatment. Another patient with mixed maligt oligoastrocytoma also
had a prolonged partial response (lasting 63 months) and is alive 84 months
after treatment for recurrence. The most frequently reported grade 3 or 4
toxicities were fatigue (19%), nausea (11%) and anorexia (11%).
CONCLUSIONS: Carboplatin and high dose tamoxifen has similar response rates to
other regimens for recurrent maligt gliomas and are probably equivalent to
those found using tamoxifen as monotherapy. Long-lasting periods of disease free
survival in some patients (particularly those with maligt mixed oligo
astrocytomas) were found. Approaches to improve the prognosis of patients with high-grade glioma, which is
still grim despite combined therapy and major advances in the development of new
drugs, are highly important. Chemotherapeutic agents have not consistently
produced favorable results in the relapse setting so far, with tumor responses
reported in a minority of cases. Among them, doxorubicin has not shown
significant efficacy, despite being one of the most effective substances in
vitro and in animal models. Nevertheless, encapsulation of doxorubicin using
polyethylene-glycol significantly improves penetration across the blood-brain
barrier. Moreover, recent clinical trials in other entities have demonstrated
that similar clinical responses can be achieved using approximately half of the
dose of polyethylene-glycol-liposomes compared with conventional liposomes.
Considering these facts, polyethylene-glycol-liposomal doxorubicin with and
without tamoxifen was evaluated within two sequential Phase II trials performed
at our institution. Polyethylene-glycol-liposomal doxorubicin (Caelyx) was
efficient in a reasonable number of patients with equivalent results in
comparison with other successful Phase II studies. This article will discuss the
literature and our own results on polyethylene-glycol-liposomal doxorubicin,
with a special focus on toxicity and efficacy data raised in clinical trials on
patients with high-grade glioma. The study aimed to examine the tolerability of the combination of radiotherapy
and tamoxifen and the effect on median and event free survival as well as
collecting data on the use of steroids in this population. 31 patients with
diffuse intrinsic pontine glioma, diagnosed on clinical and radiological
criteria, were treated with high-dose oral tamoxifen (120 mg/m(2)/day) given
concomitantly with standard dose radiotherapy (54 Gy in 1.8 Gy fractions over 6
weeks). Results Tamoxifen was well tolerated with no grade 3 or 4 CTC toxicity
reported. At 1 year, the progression free and event free survival were 3.2% (95%
CI: 0.2-14.1%), and at 6 months 19.4% (CI: 7.9% to 34.6%). The overall survival
at 1 year was 16.1% (CI: 5.9-30.9%) with median survival 6.32 months. In this
study, in which tamoxifen was used in conjunction with radiotherapy, progression
free survival was shown to be less good when compared with historical data HR =
3.1 (CI: 1.7-5.7). There was no significant reduction in overall survival. The
addition of high-dose tamoxifen, although well tolerated, confers no clinical
benefit to patients treated with diffuse intrinsic pontine glioma treated with
standard radiotherapy. |
Which transcription factors are known as the four (4) "Yamanaka factors" that have been used to create induced pluripotent stem cells (iPSCs)? | Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM). In particular, the mechanisms how the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which additional components are involved are still not yet understood. | Human induced pluripotent stem cells (iPSCs) have become an intriguing approach
for neurological disease modeling, because neural lineage-specific cell types
that retain the donors' complex genetics can be established in vitro. The
statistical power of these iPSC-based models, however, is dependent on accurate
diagnoses of the somatic cell donors; unfortunately, many neurodegenerative
diseases are commonly misdiagnosed in live human subjects. Postmortem
histopathological examination of a donor's brain, combined with premortem
clinical criteria, is often the most robust approach to correctly classify an
individual as a disease-specific case or unaffected control. In this study, we
describe iPSCs generated from a skin biopsy collected postmortem during the
rapid autopsy of a 75-year-old male, whole body donor, defined as an unaffected
neurological control by both clinical and histopathological criteria. These
iPSCs were established in a feeder-free system by lentiviral transduction of the
Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Selected iPSC clones expressed
both nuclear and surface antigens recognized as pluripotency markers of human
embryonic stem cells (hESCs) and were able to differentiate in vitro into
neurons and glia. Statistical analysis also demonstrated that fibroblast
proliferation was significantly affected by biopsy site, but not donor age
(within an elderly cohort). These results provide evidence that autopsy
donor-derived fibroblasts can be successfully reprogrammed into iPSCs, and may
provide an advantageous approach for generating iPSC-based neurological disease
models. Cellular reprogramming consists of the conversion of differentiated cells into
pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are
amenable to in vitro manipulation and, in theory, direct production of any
differentiated cell type. Furthermore, iPSC can be obtained from sick
individuals and subsequently used for disease modeling, drug discovery and
regenerative treatments. iPSC production was first achieved by transducing, with
the use of retroviral vectors, four specific transcription factors: Oct4, Klf4,
Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka,
(Cell 126(4):663-676, 2006). Many alternative protocols have since been
proposed: repeated transfections of expression plasmids containing the four
pluripotency-associated genes Okita et al. (Science 322(5903):949-953, 2008),
lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543-549,
2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy.
Series B, Physical and Biological Sciences 85(8):348-362, 2009), removal of the
reprogramming vectors by 'piggyBac' transposition Woltjen et al. (Nature
458(7239):766-770, 2009); Kaji et al. (Nature 458(7239):771-775, 2009),
Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964-977, 2009),
episomal vectors Yu et al. (Science 324(5928):797-801, 2009), cell-penetrating
reprogramming proteins Zhou et al. (Stem Cells 4(5):381-384, 2009), mammalian
artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011)
synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012),
miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376-388, 2009); however, although
some of these methods are commercially available, in general they still need to
attain the reproducibility and reprogramming efficiency required for routine
applications Mochiduki and Okita (Biotechnol Journal 7(6):789-797, 2012). Herein
we explain, in four detailed protocols, the isolation of mouse and human somatic
cells and their reprogramming into iPSC. All-encompassing instructions, not
previously published in a single document, are provided for mouse and human iPSC
colony isolation and derivation. Although mouse and human iPSC share
similarities in the cellular reprogramming process and culture, both cell types
need to be handled differently. Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2
and cMyc, human somatic cells can be converted to a pluripotent state,
generating so-called induced pluripotent stem cells (iPSCs)(1-4).
Patient-specific iPSCs lack the ethical concerns that surround embryonic stem
cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have
attracted considerable attention for disease modeling studies, the screening of
pharmacological compounds, and regenerative therapies(5). We have shown the
generation of transgene-free human iPSCs from patients with different lung
diseases using a single excisable polycistronic lentiviral Stem Cell Cassette
(STEMCCA) encoding the Yamanaka factors(6). These iPSC lines were generated from
skin fibroblasts, the most common cell type used for reprogramming. Normally,
obtaining fibroblasts requires a skin punch biopsy followed by expansion of the
cells in culture for a few passages. Importantly, a number of groups have
reported the reprogramming of human peripheral blood cells into iPSCs(7-9). In
one study, a Tet inducible version of the STEMCCA vector was employed(9), which
required the blood cells to be simultaneously infected with a constitutively
active lentivirus encoding the reverse tetracycline transactivator. In contrast
to fibroblasts, peripheral blood cells can be collected via minimally invasive
procedures, greatly reducing the discomfort and distress of the patient. A
simple and effective protocol for reprogramming blood cells using a constitutive
single excisable vector may accelerate the application of iPSC technology by
making it accessible to a broader research community. Furthermore, reprogramming
of peripheral blood cells allows for the generation of iPSCs from individuals in
which skin biopsies should be avoided (i.e. aberrant scarring) or due to
pre-existing disease conditions preventing access to punch biopsies. Here we
demonstrate a protocol for the generation of human iPSCs from peripheral blood
mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector
constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are
expanded for 9 days as described(10,11) in medium containing ascorbic acid, SCF,
IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells
are then plated onto MEFs and ESC-like colonies can be visualized two weeks
after infection. Finally, selected clones are expanded and tested for the
expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This
protocol is simple, robust and highly consistent, providing a reliable
methodology for the generation of human iPSCs from readily accessible 4 ml of
blood. Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by
the application of Yamanaka factors (OSKM), but the mechanisms underlying this
reprogramming remain poorly understood. Here, we report that Sox2 directly
regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation
and that miR-29b expression is required for OSKM- and OSK-mediated
reprogramming. Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo
targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated
with miR-29b expression during reprogramming. Moreover, the effect of miR-29b on
reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression. Further
experiments indicate that miR-29b-DNMT signaling is significantly involved in
the regulation of DNA methylation-related reprogramming events, such as
mesenchymal-to-epithelial transition (MET) and Dlk1-Dio3 region transcription.
Thus, our studies not only reveal that miR-29b is a novel mediator of
reprogramming factor Sox2 but also provide evidence for a multistep mechanism in
which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA
methylation-related events during reprogramming. Delivery of the transcription factors Oct4, Klf4, Sox2 and c-Myc via integrating
viral vectors has been widely employed to generate induced pluripotent stem cell
(iPSC) lines from both normal and disease-specific somatic tissues, providing an
invaluable resource for medical research and drug development. Residual
reprogramming transgene expression from integrated viruses nevertheless alters
the biological properties of iPSCs and has been associated with a reduced
developmental competence both in vivo and in vitro. We performed transcriptional
profiling of mouse iPSC lines before and after excision of a polycistronic
lentiviral reprogramming vector to systematically define the overall impact of
persistent transgene expression on the molecular features of iPSCs. We
demonstrate that residual expression of the Yamanaka factors prevents iPSCs from
acquiring the transcriptional program exhibited by embryonic stem cells (ESCs)
and that the expression profiles of iPSCs generated with and without c-Myc are
indistinguishable. After vector excision, we find 36% of iPSC clones show normal
methylation of the Gtl2 region, an imprinted locus that marks ESC-equivalent
iPSC lines. Furthermore, we show that the reprogramming factor Klf4 binds to the
promoter region of Gtl2. Regardless of Gtl2 methylation status, we find similar
endodermal and hepatocyte differentiation potential comparing syngeneic Gtl2(ON)
vs Gtl2(OFF) iPSC clones. Our findings provide new insights into the
reprogramming process and emphasize the importance of generating iPSCs free of
any residual transgene expression. CytoTune™-iPS Reprogramming System uses vectors based on replication in
competent Sendai virus (SeV) to safely and effectively deliver and express key
genetic factors necessary for reprogramming somatic cells into iPSCs. In
contrast to many available protocols, which rely on viral vectors that integrate
into the genome of the host cell, the CytoTune™ Reprogramming System uses
vectors that are non-integrating and remain in the cytoplasm (i.e., they are
zero-footprint). In addition, the host cell can be cleared of the vectors and
reprogramming factor genes by exploiting the cytoplasmic nature of SeV and the
functional temperature sensitivity mutations introduced into the key viral
proteins. The CytoTune™-iPS Reprogramming Kit contains four SeV-based
reprogramming vectors, each capable of expressing one of the four Yamanaka
factors (i.e., Oct4, Sox2, Klf4, and c-Myc) and are optimized for generating
iPSCs from human somatic cells. The reprogramming vectors in this kit have been
engineered to increase biological and environmental safety. The recently established reprogramming of somatic cells into induced pluripotent
stem cells (iPSCs) by Takahashi and Yamanaka represents a valuable tool for
future therapeutic applications. To date, the mechanisms underlying this process
are still largely unknown. In particular, the mechanisms how the Yamanaka
factors (Oct4, Sox2, Klf4, and c-Myc) directly drive reprogramming and which
additional components are involved are still not yet understood. In this study,
we aimed at analyzing the role of ADP-ribosyltransferase diphtheria toxin-like
one (Artd1; formerly called poly(ADP-ribose) polymerase 1 [Parp1]) during
reprogramming. We found that poly(ADP-ribosylation) (PARylation) of the
reprogramming factor Sox2 by Artd1 plays an important role during the first days
upon transduction with the reprogramming factors. A process that happens before
Artd1 in conjunction with 10-11 translocation-2 (Tet2) mediates the histone
modifications necessary for the establishment of an activated chromatin state at
pluripotency loci (e.g., Nanog and Essrb) [Nature 2012;488:652-655]. Wild-type
(WT) fibroblasts treated with an Artd1 inhibitor as well as fibroblasts
deficient for Artd1 (Artd1-/-) show strongly decreased reprogramming capacity.
Our data indicate that Artd1-mediated PARylation of Sox2 favors its binding to
the fibroblast growth factor 4 (Fgf4) enhancer, thereby activating Fgf4
expression. The importance of Fgf4 during the first 4 days upon initiation of
reprogramming was also highlighted by the observation that exogenous addition of
Fgf4 was sufficient to restore the reprogramming capacity of Artd1-/- fibroblast
to WT levels. In conclusion, our data clearly show that the interaction between
Artd1 and Sox2 is crucial for the first steps of the reprogramming process and
that early expression of Fgf4 (day 2 to day 4) is an essential component for the
successful generation of iPSCs. Induced pluripotent stem cells (iPSCs) are created by the reprogramming of
somatic cells via overexpression of certain transcription factors, such as the
originally described Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc (OSKM). Here
we discuss recent advancements in iPSC reprogramming and introduce mathematical
approaches to help map the landscape between cell states during reprogramming.
Our modelization indicates that OSKM expression diminishes and/or changes
potential barriers between cell states and that epigenetic remodeling facilitate
these transitions. From a practical perspective, the modeling approaches
outlined here allow us to predict the time necessary to create a given number of
iPSC colonies or the number of reprogrammed cells generated in a given time.
Additional investigations will help to further refine modeling strategies,
rendering them applicable toward the study of the development and stability of
cancer cells or even other reprogramming processes such as lineage conversion.
Ultimately, a quantitative understanding of cell state transitions might
facilitate the establishment of regenerative medicine strategies and enhance the
translation of reprogramming technologies into the clinic. |
What is the role of anhedonia in coronary disease patients? | Anhedonia is associated with poor prognosis in patients with coronary disease. Namely, in patients with coronary disease, anhedonia was associated with increased mortality, greater risk for major cardiac event, impaired physical health status, more cardiac symptoms, more feelings of disability. These associations were independent from clinical and demographic factors. | OBJECTIVE: Type D personality has been associated with impaired health status in
chronic heart failure (CHF), but other psychological factors may also be
important.
AIM: To determine whether non-Type D patients with low positive affect and Type
D patients report lower health status, compared with non-Type D patients with
high positive affect at 12-month follow-up in chronic heart failure.
METHODS: Consecutive CHF outpatients (n = 276) filled out the Short Form-12
(health status) and Health Complaints Scale (disease-specific complaints) at
inclusion and 12-month follow-up, and the DS14 (Type D personality) and positive
affect (Global Mood Scale) at inclusion. Three groups were composed: non-Type D
patients without anhedonia, non-Type D patients with anhedonia, and Type D
patients.
RESULTS: After controlling for demographic and clinical confounders, and scores
at inclusion, anhedonic non-Type D patients reported lower mental health status
(beta = -.19, P < .004), and more feelings of disability (beta = .10, P = .04),
marginally lower physical health status (beta = -.11, P = .07), and equal levels
of cardiac symptoms (beta = .04, P = .43), when compared with non-Type D's
without anhedonia. Type D patients reported lower levels of impaired mental
health status, more cardiac symptoms and feelings of disability (-.31 < beta <
.17, all Ps < .05). A trend was shown for physical health status (beta = -.11, P
= .09).
CONCLUSION: Non-Type D patients low on positive affect and Type D patients
report lower levels of health status in CHF, compared with non-Type D patients
with high positive affect. Future studies need to determine whether lack of
positive affect is associated with impaired clinical outcome. BACKGROUND: Depression after acute coronary syndromes (ACS) is an important risk
factor for further coronary events; but the influence of anhedonia, the
decreased capacity to experience pleasure, has received little attention. The
aim of the study was to investigate the effects of anhedonia on prognosis at
3-year follow-up in patients hospitalized for ACS.
METHOD: Consecutively admitted ACS patients (n = 291) completed the Chapman
Physical Anhedonia Scale (PAS) and the Hospital Anxiety and Depression Scale
depression subscale (HAD-D) at baseline (1-4 days after their admission). Two
definitions of anhedonia were taken into account: dimensional definition using
PAS score as well as categorical definition using several cutoff scores
(hedonics: PAS less than 23 or 29; anhedonics: PAS equal to or greater than 23
or 29). Patients were followed during 3 years for adverse clinical events
divided into severe cardiac events (mortality or myocardial infarction [MI]) and
clinical events (mortality, MI, recurrence of ACS, hospital readmission, and
onset or deterioration of heart failure).
RESULTS: At follow-up, there were 176 clinical events (36 deaths, 8 MIs, 58 ACS,
55 hospital readmissions, 19 heart failures). Dimensional anhedonia and
depression were associated with poor prognosis, but anhedonia was the only
predictor of severe cardiac events and clinical events after adjusting for
demographic and clinical variables. Contrary to depression, categorical
anhedonia (PAS >23) was an independent and significant predictor of severe
cardiac events after adjusting for clinical variables. The incidence of death/MI
in hedonics vs anhedonics was 11.1% vs 22.1% (hazard ratio = 2.18; 95%
confidence interval, 1.11-4.26).
CONCLUSIONS: Dimensional and categorical anhedonias predicted independently
severe cardiac events and clinical events after ACS. OBJECTIVE: To determine which particular depressive symptom scales, derived from
three scales, predicted poorer prognosis in persons with acute coronary syndrome
(ACS).
METHODS: Hospitalized ACS patients (n=408) completed questionnaires (depression,
vital exhaustion). Mokken scaling derived unidimensional scales. Major cardiac
events (cardiac mortality, ACS, unplanned revascularization) were assessed at
median 67 weeks post event.
RESULTS: Only depressive symptoms of fatigue-sadness predicted prognosis in
univariate (hazard ratio [HR]=1.8, 95% CI 1.1-3.0, P=.025) and multivariate
analysis (HR=1.8, 95% CI 1.1-2.9, P=.025). Symptoms of anhedonia (HR=1.6, 95% CI
0.9-2.8, P=.102) and depressive cognitions (HR=1.3, 95% CI 0.7-2.2, P=.402) did
not.
CONCLUSION: Symptoms of fatigue-sadness, but not other symptoms, were associated
with increased risk of major cardiac events. Depression should be considered as
a multidimensional, rather than a unidimensional, entity when designing
interventions. AIMS: Negative mood states (e.g., anxiety and depression) have been associated
with increased cardiovascular morbidity and mortality in coronary artery disease
(CAD), but little is known about the impact of positive emotions on these health
outcomes. We examined whether anhedonia (i.e., reduced positive affect) was
associated with 7-year mortality in patients treated with percutaneous coronary
intervention (PCI).
METHODS: Consecutive PCI patients (n = 1206; 71.5% men; mean age 62.0 ± 11.1
years) from the Rapamycin-Eluting Stent Evaluated At Rotterdam Cardiology
Hospital (RESEARCH) registry completed the Hospital Anxiety and Depression Scale
(HADS) to assess anhedonia at baseline. Anhedonia was defined as a score ≤ 7
(i.e., one SD below the mean) on the positive affect scale of the HADS. The
endpoint was defined as all-cause mortality.
RESULTS: The prevalence of anhedonia was 23.7% (286/1206). After a median follow
up of 7.0 ± 1.6 years, 186 deaths (15.4%) from any cause were recorded. The
incidence of mortality in anhedonic patients was 22.7% (65/286) vs. 13.2%
(121/920) in non-anhedonic patients (HR = 1.66, 95% CI [1.19-2.32], p = 0.003).
Cumulative hazard functions were significantly different for anhedonic vs.
non-anhedonic patients (log-rank χ(2) = 16.61, p < 0.001). In multivariable
analysis, anhedonia remained independently associated with all-cause mortality
(HR = 1.51, 95% CI [1.03-2.22], p = 0.036), after adjusting for
socio-demographics, clinical characteristics, and negative and relaxed affect.
CONCLUSION: Anhedonia was independently associated with a 1.5-fold increased
risk for all-cause mortality in patients who survived the first 6 months
post-PCI. Enhancing positive emotions, in addition to reducing negative
emotions, may constitute an important target for future psychological
intervention trials in CAD patients. |
What is the clinical value of naltrexone in Parkinson's disease patients? | Naltrexone does not improve clinical features, including motor function, in Parkinson's disease patients. Naltrexone was shown to be effective for treatment of pathological gambling in Parkinson's disease patients. | Animal experiments suggest that opiate peptides might play a role in
extrapyramidal function. This hypothesis was tested by administering the opiate
antagonist, naltrexone, in doses sufficient to antagonize exogenous opiates, to
patients with parkinsonism and Huntington's disease. No improvement in the
clinical features of either disorder was noted. Pathological gambling (PG) is a potential complication related to the treatment
of Parkinson disease (PD) with dopamine agonists (DA). The cause of this
disorder is unknown, but altered dopamine neurotransmission may be involved.
OBJECTIVE: We evaluated the efficacy and tolerability of the opioid antagonist
naltrexone in the treatment of PG in PD.
METHODS: Our cases included 3 patients with PD who developed PG after DA
treatment.
RESULTS: Pathological gambling did not improve after reduction or
discontinuation of DA. These patients responded poorly to serotonin reuptake
inhibitors, whereas treatment with opioid antagonist naltrexone resulted in the
remission of PG. Naltrexone treatment was well tolerated. In one patient, higher
dose of naltrexone resulted in hepatic abnormalities, which resolved after
dosage reduction.
CONCLUSIONS: The opioid antagonist naltrexone could be an effective option for
the treatment of PG in PD. Impulse control disorders are a psychiatric condition characterized by the
failure to resist an impulsive act or behavior that may be harmful to self or
others. In movement disorders, impulse control disorders are associated with
dopaminergic treatment, notably dopamine agonists (DAs). Impulse control
disorders have been studied extensively in Parkinson's disease, but are also
recognized in restless leg syndrome and atypical Parkinsonian syndromes.
Epidemiological studies suggest younger age, male sex, greater novelty seeking,
impulsivity, depression and premorbid impulse control disorders as the most
consistent risk factors. Such patients may warrant special monitoring after
starting treatment with a DA. Various individual screening tools are available
for people without Parkinson's disease. The Questionnaire for
Impulsive-Compulsive Disorders in Parkinson's Disease has been developed
specifically for Parkinson's disease. The best treatment for impulse control
disorders is prevention. However, after the development of impulse control
disorders, the mainstay intervention is to reduce or discontinue the offending
anti-Parkinsonian medication. In refractory cases, other pharmacological
interventions are available, including neuroleptics, antiepileptics, amantadine,
antiandrogens, lithium and opioid antagonists. Unfortunately, their use is only
supported by case reports, small case series or open-label clinical studies.
Prospective, controlled studies are warranted. Ongoing investigations include
naltrexone and nicotine. OBJECTIVE: Impulse control disorders (ICDs) in Parkinson disease (PD) are common
and can be difficult to manage. The objective of this study was to determine the
efficacy and tolerability of naltrexone, an opioid antagonist, for the treatment
of ICDs in PD.
METHODS: Patients with PD (n = 50) and an ICD were enrolled in an 8-week,
randomized (1:1), double-blind, placebo-controlled study of naltrexone 50-100
mg/d (flexible dosing). The primary outcome measure was response based on the
Clinical Global Impression-Change score, and the secondary outcome measure was
change in symptom severity using the Questionnaire for Impulsive-Compulsive
Disorders in Parkinson's Disease-Rating Scale (QUIP-RS) ICD score.
RESULTS: Forty-five patients (90%) completed the study. The Clinical Global
Impression-Change response rate difference favoring naltrexone in completers was
19.8% (95% confidence interval [CI] -8.7% to 44.2%). While this difference was
not significant (odds ratio=1.6, 95% CI 0.5-5.2, Wald χ2 [df]=0.5 [1], p=0.5),
naltrexone treatment led to a significantly greater decrease in QUIP-RS ICD
score over time compared with placebo (regression coefficient for interaction
term in linear mixed-effects model=-7.37, F[df]=4.3 [1, 49], p=0.04). The
estimated changes in QUIP-RS ICD scores from baseline to week 8 were 14.9 points
(95% CI 9.9-19.9) for naltrexone and 7.5 points (95% CI 2.5-12.6) for placebo.
CONCLUSIONS: Naltrexone treatment was not efficacious for the treatment of ICDs
in PD using a global assessment of response, but findings using a PD-specific
ICD rating scale support further evaluation of opioid antagonists for the
treatment of ICD symptoms in PD.
CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that in
patients with PD and an ICD, naltrexone does not significantly increase the
probability of achieving response. However, the study lacked the precision to
exclude an important difference in response rates. |
What is the efficacy of oxaliplatin monotherapy in the management of colorectal cancer? | Oxaliplatin is a promising treatment option for patients with metastatic colorectal cancer. It appears to be particularly advantageous (in terms of response rate and duration of progression-free survival) when used in combination with fluorouracil/calcium folinate as both a first- and second-line option, although preliminary studies have failed to show any survival advantage over fluorouracil/calcium folinate alone. Promising results have been found in studies of the drug as monotherapy, and oxaliplatin may also prove useful in the neoadjuvant setting in patients with unresectable liver metastases; however, data are limited at present | PURPOSE: To evaluate the objective tumor response rate and safety profile of
oxaliplatin when administered to patients with previously untreated metastatic
colorectal adenocarcinoma.
PATIENTS AND METHODS: A total of 39 patients were entered onto this phase II
trial. One patient was excluded for having had a second cancer, so the study was
based on 38 patients. Patients were treated with oxaliplatin 130 mg/m2 as a
2-hour infusion on day 1, every 21 days. Patients were assessed for response
every three courses. All clinical and radiologic data were reviewed by an
external panel of experts, with their assessment being considered definitive.
RESULTS: Nine partial responses (PRs) were observed (response rate, 24.3%; 95%
confidence interval, 11.8% to 41.2%). The median duration of response was 216+
days. Fifteen patients (40.5%) had stable disease and 13 (35.2%) had progressive
disease. The median progression-free survival time for all patients was 126+
days (range, 21 to 447+). The main toxicity was peripheral sensory neuropathy.
Grade 3 neurotoxicity (National Cancer Institute common toxicity criteria
[NCI-CTC]) was reported in 13%. Hematologic and gastrointestinal toxicities were
mild. The incidence of grade 3 neutropenia was 5.2%, while that of grade 3 or 4
thrombopenia was 7.9%. Vomiting (grade 3 or 4) occurred in 7.9% of patients and
grade 3 diarrhea in 2.6%.
CONCLUSION: This phase II study provides clear evidence of the safety and
efficacy of oxaliplatin monotherapy at this dose and schedule in patients with
previously untreated metastatic colorectal carcinoma. Oxaliplatin is a cytotoxic agent which, like other platinum compounds, acts
primarily by causing inter- and intra-strand cross-links in DNA, thereby
inhibiting DNA synthesis. Oxaliplatin has a bulky carrier ligand which is
thought to enhance cytotoxicity and abolish cross-resistance between oxaliplatin
and other platinum compounds. Phase II and III clinical trials have found
oxaliplatin combined with fluorouracil/calcium folinate (leucovorin/folinic
acid) to be an effective first- and second-line treatment for patients with
metastatic colorectal cancer. First-line triple therapy with oxaliplatin and
fluorouracil/calcium folinate achieved significantly higher response rates than
fluorouracil/calcium folinate alone in 2 phase III studies (objective response
rates 59 vs 23% and 50.7 vs 22.3%). In addition, median progression-free
survival was longer with triple therapy in both studies (8.9 vs 5.2 and 8.75 vs
6.0 months). However, there was no significant difference in median duration of
survival between treatment groups, although this may be a consequence of the
subsequent use of oxaliplatin and/or surgery in patients who relapsed during
therapy with fluorouracil/calcium folinate alone. About 30 to 45% of patients
(whose disease progressed during or after fluorouracil-based therapy) responded
to second-line combination therapy with oxaliplatin and fluorouracil/calcium
folinate. Median progression-free survival ranged from 7 to 10 months and the
median duration of survival from 10 to 17 months. Objective responses were
achieved in 20 and 24% of patients in 2 small trials of first-line oxaliplatin
monotherapy and in about 10% of patients given the drug as a second-line option.
Peripheral sensory neuropathy is the dose-limiting toxicity associated with
oxaliplatin. Severe neurotoxicity has been estimated to occur in 10% of patients
after 6 treatment cycles and in 50% after 9 cycles of an oxaliplatin dosage of
130 mg/m2 once every 3 weeks. It is cumulative, but reversible on
discontinuation of therapy. Nausea, vomiting and diarrhoea are common, but are
usually mild to moderate. Myelosuppression is also observed, but is usually
mild.
CONCLUSION: oxaliplatin is a promising treatment option for patients with
metastatic colorectal cancer. It appears to be particularly advantageous (in
terms of response rate and duration of progression-free survival) when used in
combination with fluorouracil/calcium folinate as both a first- and second-line
option, although preliminary studies have failed to show any survival advantage
over fluorouracil/calcium folinate alone. Promising results have been found in
studies of the drug as monotherapy, and oxaliplatin may also prove useful in the
neoadjuvant setting in patients with unresectable liver metastases; however,
data are limited at present. BACKGROUND: Capecitabine and oxaliplatin are both effective and well-tolerated
monotherapies for the treatment of advanced colorectal cancer (CRC). Oxaliplatin
has also been shown to be very effective when combined with 5-FU/LV in the
first-line setting.
AIM OF THE STUDY: Assess the efficacy and safety of capecitabine plus
oxaliplatin (XELOX) in patients with previously untreated advanced CRC.
METHODS: Fifty-three patients with measurable disease received capecitabine
1,000 mg/m2 twice daily on d 1-14 and oxaliplatin 130 mg/m2 on d 1, every 3 wk.
Of these, 52 were evaluable for safety and 49 for antitumor response.
RESULTS: There was a low rate of grade 1/2 adverse events; grade 3/4 events
included leukopenia (10%), neutropenia (6%), thrombocytopenia (2%),
nausea/vomiting (4%), and diarrhea (4%). The overall response rate was 39% (95%
CI, 25-54%) and median time to disease progression was 7.8 mo.
CONCLUSIONS: XELOX is an active and well-tolerated first-line treatment for
advanced CRC. Randomized phase III studies are ongoing to compare XELOX with
FOLFOX in view of the comparable efficacy and safety but superior convenience of
XELOX therapy. BACKGROUND: Oxaliplatin, a platinum compound, has been commonly used around the
world for treating advanced colorectal cancer. The generally recommended dose
and schedule of oxaliplatin monotherapy is 130 mg/m(2) every 3 weeks. This trial
was conducted to evaluate the safety and pharmacokinetics of oxaliplatin
monotherapy in Japanese patients with solid tumors.
METHODS: Oxaliplatin was administered as a 2-h intravenous infusion every 3
weeks at a dose of 90 and 130 mg/m(2). Blood was collected to determine the
total platinum and the ultrafiltrate platinum concentrations in plasma in all
cycles.
RESULTS: Nine patients were enrolled; three were given oxaliplatin monotherapy
at 90 mg/m(2) and six received 130 mg/m(2). All tumors were colorectal cancer.
The major adverse reactions included myelosuppressive, neurological and
gastrointestinal toxicities, although most were grades 1 and 2 at both dose
levels. Peripheral sensory neuropathy of without movement disturbance (grade 1
or 2) was observed in all patients at both dose levels. The 130 mg/m(2) dose
level was not found to be the maximum tolerated dose, but was judged to be the
recommended dose. No objective responses were seen and five cases of no change
were observed. A bi-exponential open model best described the disappearance of
platinum in the plasma, and a tri-exponential open model best described the
disappearance of ultrafilterable platinum in the plasma at both dose levels. No
racial difference was suggested in the pharmacokinetics of oxaliplatin.
CONCLUSIONS: The oxaliplatin monotherapy dose schedule of 130 mg/m(2) every 3
weeks, recommended worldwide, is acceptable for Japanese patients. PURPOSE: To establish whether cetuximab, a chimeric IgG1 antibody targeting
epidermal growth factor receptor, has the potential to restore responsiveness to
oxaliplatin in preclinical cancer models, as has been shown with irinotecan in
irinotecan refractory metastatic colorectal cancer patients.
EXPERIMENTAL DESIGN: The effects of cetuximab and oxaliplatin, alone or in
combination, were tested in vitro and in vivo using human colorectal cancer cell
lines selected for oxaliplatin resistance, as well as parental control cell
lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu
athymic mice, as well as activation of mitogen-activated protein kinase
(extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair
genes, density of apurinic/apyrimidinic DNA damage, and accumulation of
platinum-DNA adducts in vitro.
RESULTS: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft
models was greater than that of monotherapies and independent of the
responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced
expression of excision repair cross-complementation group 1 and XPF, which are
key components of the nucleotide excision repair pathway involved in the
excision of platinum-DNA adducts. In addition, cetuximab reduced expression of
XRCC1, a component of the base excision repair pathway responsible for the
repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair
protein levels were downstream to effects on mitogen-activated protein kinase
and AKT pathway activation. In line with effects on DNA repair protein
expression, cetuximab increased the accumulation of platinum and
apurinic/apyrimidinic sites on DNA during oxaliplatin treatment.
CONCLUSIONS: Cetuximab has the potential to salvage the benefits of oxaliplatin
in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair
capacity. |
Are there any animal models for Niemann-Pick C1 disease? | Yes, murine models of Niemann-Pick type C disease (NPC) exist. They are either homozygous or heterozygous NPC1-deficient [NPC1 (-/-)]/ [NPC1 (+/-)] mouse models. | Niemann-Pick type C1 (NPC1) disease is a fatal neurodegenerative disease
characterized by neuronal lipid storage and progressive Purkinje cell loss in
the cerebellum. We investigated whether therapeutic approaches to bypass the
cholesterol trafficking defect in NPC1 disease might delay disease progression
in the npc1(-/-) mouse model. We show that the neurosteroid allopregolone
(ALLO) and T0901317, a synthetic oxysterol ligand, act in concert to delay onset
of neurological symptoms and prolong the lifespan of npc1(-/-) mice. ALLO and
T0901317 therapy preserved Purkinje cells, suppressed cerebellar expression of
microglial-associated genes and inflammatory mediators, and reduced infiltration
of activated microglia in the cerebellar tissue. To establish whether the
mechanism of neuroprotection in npc1(-/-) mice involves GABA(A) receptor
activation, we compared treatment of natural ALLO and ent-ALLO, a stereoisomer
that has identical physical properties of natural ALLO but is not a GABA(A)
receptor agonist. ent-ALLO provided identical functional and survival benefits
as natural ALLO in npc1(-/-) mice, strongly supporting a GABA(A)
receptor-independent mechanism for ALLO action. On the other hand, the efficacy
of ALLO, ent-ALLO, and T0901317 therapy correlated with the ability of these
compounds to activate prege X receptor-dependent pathways in vivo. These
findings suggest that treatment with prege X receptor ligands may be useful
clinically in delaying the progressive neurodegeneration in human NPC disease. Niemann-Pick type C1 (NPC1) disease is an autosomal-recessive
cholesterol-storage disorder characterized by liver dysfunction,
hepatosplenomegaly, and progressive neurodegeneration. The NPC1 gene is
expressed in every tissue of the body, with liver expressing the highest amounts
of NPC1 mRNA and protein. A number of studies have now indicated that the NPC1
protein regulates the transport of cholesterol from late endosomes/lysosomes to
other cellular compartments involved in maintaining intracellular cholesterol
homeostasis. The present study characterizes liver disease and lipid metabolism
in NPC1 mice at 35 days of age before the development of weight loss and
neurological symptoms. At this age, homozygous affected (NPC1(-/-)) mice were
characterized with mild hepatomegaly, an elevation of liver enzymes, and an
accumulation of liver cholesterol approximately four times that measured in
normal (NPC1(+/+)) mice. In contrast, heterozygous (NPC1(+/-)) mice were without
hepatomegaly and an elevation of liver enzymes, but the livers had a significant
accumulation of triacylglycerol. With respect to apolipoprotein and lipoprotein
metabolism, the results indicated only minor alterations in NPC1(-/-) mouse
serum. Finally, compared to NPC1(+/+) mouse livers, the amount and processing of
SREBP-1 and -2 proteins were significantly increased in NPC1(-/-) mouse livers,
suggesting a relative deficiency of cholesterol at the metabolically active pool
of cholesterol located at the endoplasmic reticulum. The results from this study
further support the hypothesis that an accumulation of lipoprotein-derived
cholesterol within late endosomes/lysosomes, in addition to altered
intracellular cholesterol homeostasis, has a key role in the biochemical and
cellular pathophysiology associated with NPC1 liver disease. The role of cholesterol in Alzheimer's disease (AD) has been linked to the
generation of toxic amyloid beta peptides (Abeta). Using genetic mouse models of
cholesterol loading, we examined whether mitochondrial cholesterol regulates
Abeta neurotoxicity and AD pathology. Isolated mitochondria from brain or
cortical neurons of transgenic mice overexpressing SREBP-2 (sterol regulatory
element binding protein 2) or NPC1 (Niemann-Pick type C1) knock-out mice
exhibited mitochondrial cholesterol accumulation, mitochondrial glutathione
(mGSH) depletion and increased susceptibility to Abeta1-42-induced oxidative
stress and release of apoptogenic proteins. Similar findings were observed in
pharmacologically GSH-restricted rat brain mitochondria, while selective mGSH
depletion sensitized human neuronal and glial cell lines to Abeta1-42-mediated
cell death. Intracerebroventricular human Abeta delivery colocalized with
mitochondria resulting in oxidative stress, neuroinflammation and neuronal
damage that were enhanced in Tg-SREBP-2 mice and prevented upon mGSH recovery by
GSH ethyl ester coinfusion, with a similar protection observed by
intraperitoneal administration of GSH ethyl ester. Finally, APP/PS1 (amyloid
precursor protein/presenilin 1) mice, a transgenic AD mouse model, exhibited
mitochondrial cholesterol loading and mGSH depletion. Thus, mitochondrial
cholesterol accumulation emerges as a novel pathogenic factor in AD by
modulating Abeta toxicity via mGSH regulation; strategies boosting the
particular pool of mGSH may be of relevance to slow down disease progression. Niemann-Pick type C1 (NPC) disease is an autosomal recessive neurodegenerative
disorder. One feature of the mouse model of NPC1 is it's infertility. We have
made transgenic mice which express the Npc1 protein exclusively in fibrillary
astrocytes, using the glial fibrillary acidic protein (GFAP) promoter. This
selective expression of Npc1 corrects sterility in GFAP-Npc1(-/-), Npc1(-/-)
mice. Counts of acidophils in the pituitary of GFAP-Npc1E, Npc1(-/-) mice, as
compared Npc1(-/-) mice, and measurements of dopamine D2 receptor (DRD2) mRNA in
the pituitary, suggest mechanisms for fertility enhancement. We conclude that
the correction of sterility in GFAP-Npc1E, Npc1(-/-) mice is a result of
restoring hypothalamic control of the pituitary. Niemann-Pick type C1 (NPC1) disease arises from a mutation inactivating NPC1
protein that normally moves unesterified cholesterol from the late
endosomal/lysosomal complex of cells to the cytosolic compartment for
processing. As a result, cholesterol accumulates in every tissue of the body
causing liver, lung, and CNS disease. Treatment of the murine model of this
disease, the npc1 mouse, s.c. with β-cyclodextrin (4000 mg/kg) one time each
week normalized cellular cholesterol metabolism in the liver and most other
organs. At the same time, the hepatic dysfunction seen in the untreated npc1
mouse was prevented. The severity of cerebellar neurodegeneration also was
ameliorated, although not entirely prevented, and the median lifespan of the
animals was doubled. However, in contrast to these other organs, lung showed
progressive macrophage infiltration with development of lipoid pneumonitis.
These studies demonstrated that weekly cyclodextrin administration overcomes the
lysosomal transport defect associated with the NPC1 mutation, nearly normalizes
hepatic and whole animal cholesterol pools, and prevents the development of
liver disease. Furthermore, this treatment slows cerebellar neurodegeneration
but has little or no effect on the development of progressive pulmonary disease. Niemann-Pick type C (NPC) disease is an autosomal recessive neurodegenerative
disorder characterized by intracellular accumulation of cholesterol and
glycosphingolipids in many tissues including the brain. The disease is caused by
mutations of either NPC1 or NPC2 gene and is accompanied by a severe loss of
neurons in the cerebellum, but not in the hippocampus. NPC pathology exhibits
some similarities with Alzheimer's disease, including increased levels of
amyloid beta (Abeta)-related peptides in vulnerable brain regions, but very
little is known about the expression of amyloid precursor protein (APP) or APP
secretases in NPC disease. In this article, we evaluated age-related alterations
in the level/distribution of APP and its processing enzymes, beta- and
gamma-secretases, in the hippocampus and cerebellum of Npc1(-/-) mice, a
well-established model of NPC pathology. Our results show that levels and
expression of APP and beta-secretase are elevated in the cerebellum prior to
changes in the hippocampus, whereas gamma-secretase components are enhanced in
both brain regions at the same time in Npc1(-/-) mice. Interestingly, a subset
of reactive astrocytes in Npc1(-/-) mouse brains expresses high levels of APP as
well as beta- and gamma-secretase components. Additionally, the activity of
beta-secretase is enhanced in both the hippocampus and cerebellum of Npc1(-/-)
mice at all ages, while the level of C-terminal APP fragments is increased in
the cerebellum of 10-week-old Npc1(-/-) mice. These results, taken together,
suggest that increased level and processing of APP may be associated with the
development of pathology and/or degenerative events observed in Npc1(-/-) mouse
brains. Alterations in the metabolism of amyloid precursor protein (APP) are believed to
play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate
that APP is proteolytically processed in endosomal-autophagic-lysosomal
compartments. In this study, we used both in vivo and in vitro paradigms to
determine whether alterations in macroautophagy affect APP metabolism. Three
mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1,
GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the
autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired
lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in
brains of the three mouse models; however, discrepancies between LC3-II and
APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and
secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were
proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis
and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of
NPC1 mice. Endogenous murine Aβ40 from RIPA-soluble extracts was increased in
brains of all three mice. The in vivo relationship between AV and APP-CTF
accumulation was also seen in cultured neurons treated with agents that impair
primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and
vinblastine) lysosomal flux. However, Aβ secretion was unaffected by agents that
induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs
predomitly co-localized with degradative LAMP-1-positive lysosomes. These
data suggest that neuronal macroautophagy does not directly regulate APP
metabolism but highlights the important anti-amyloidogenic role of lysosomal
proteolysis in post-secretase APP-CTF catabolism. Niemann Pick disease type C1 (NPC) is an autosomal recessive disease
characterized by progressive neurological deterioration leading to premature
death. In this study, we hypothesized that human umbilical cord blood-derived
mesenchymal stem cells (hUCB-MSCs) have the multifunctional abilities to
ameliorate NPC symptoms in the brain. To test this hypothesis, hUCB-MSCs were
transplanted into the hippocampus of NPC mice in the early asymptomatic stage.
This transplantation resulted in the recovery of motor function in the Rota Rod
test and impaired cholesterol homeostasis leading to increased levels of
cholesterol efflux-related genes such as LXRα, ABCA1, and ABCG5 while decreased
levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase were observed in NPC
mice. In the cerebrum, hUCB-MSCs enhanced neuronal cell survival and
proliferation, where they directly differentiated into electrically active
MAP2-positive neurons as demonstrated by whole-cell patch clamping. In addition,
we observed that hUCB-MSCs reduced Purkinje neuronal loss by suppression of
inflammatory and apoptotic signaling in the cerebellum as shown by
immunohistochemistry. We further investigated how hUCB-MSCs enhance cellular
survival and inhibit apoptosis in NPC mice. Neuronal cell survival was
associated with increased PI3K/AKT and JAK2/STAT3 signaling; moreover, hUCB-MSCs
modulated the levels of GABA/glutamate transporters such as GAT1, EAAT2, EAAT3,
and GAD6 in NPC mice as assessed by Western blot analysis. Taken together, our
findings suggest that hUCB-MSCs might play multifunctional roles in neuronal
cell survival and ameliorating motor deficits of NPC mice. We have identified a point mutation in Npc1 that creates a novel mouse model
(Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change
(A to G at cDNA bp 3163) that results in an aspartate to glycine change at
position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the
NPC1 protein and is highly similar to commonly occurring human mutations.
Genetic and molecular biological analyses, including sequencing the Npc1(spm)
allele and identifying a truncating mutation, confirm that the mutation in
Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC
disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight,
gait and other motor activities, as well as acoustic startle responses all
reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in
mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels
appear relatively normal, Npc1(nmf164) brain and liver display dramatic
reductions in Npc1 protein, as well as abnormal cholesterol metabolism and
altered glycolipid expression. Furthermore, histological analyses of liver,
spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol
accumulation, glial activation and Purkinje cell loss at a slower rate than in
the Npc1(nih) mouse model. Magnetic resoce imaging studies also reveal
significantly less demyelination/dysmyelination than in the null alleles. Thus,
although prior mouse models may correspond to the severe infantile onset forms
of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the
late-onset, more slowly progressing forms of NPC disease that comprise the large
majority of human cases. Niemann-Pick disease type C (NPC) is an inherited lysosomal storage disease
characterised by accumulation of cholesterol and glycosphingolipids. NPC
patients suffer a progressive neurodegenerative phenotype presenting with motor
dysfunction, mental retardation and cognitive decline. To examine the onset and
progression of neuropathological insults in NPC we have systematically examined
the CNS of a mouse model of NPC1 (Npc1(-/-) mice) at different stages of the
disease course. This revealed a specific spatial and temporal pattern of
neuropathology in Npc1(-/-) mice, highlighting that sensory thalamic pathways
are particularly vulnerable to loss of NPC1 resulting in neurodegeneration in
Npc1(-/-) mice. Examination of markers of astrocytosis and microglial activation
revealed a particularly pronounced reactive gliosis in the thalamus early in the
disease, which subsequently also occurred in interconnected cortical laminae at
later ages. Our examination of the precise staging of events demonstrate that
the relationship between glia and neurons varies between brain regions in
Npc1(-/-) mice, suggesting that the cues causing glial reactivity may differ
between brain regions. In addition, aggregations of pre-synaptic markers are
apparent in white matter tracts and the thalamus and are likely to be formed
within axonal spheroids. Our data provide a new perspective, revealing a number
of events that occur prior to and alongside neuron loss and highlighting that
these occur in a pathway dependent manner. Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage
disorder caused by mutations in the NPC1 gene which encodes a transmembrane
protein of the acidic compartment. Albeit the NPC1(-/-) mouse is available
serving as an appropriate animal model of the human disease, the precise
function of this protein remains obscure. Here, we investigated the synaptic
consequences of this disease and explored long-term potentiation (LTP) in slices
taken from the hippocampal CA1 region, the dorsomedial striatum as well as the
somatosensory neocortex in NPC1(-/-) mice using extracellular field potential
recordings. We did not observe significant changes in synaptic excitability as
well as LTP in the hippocampal CA1 region and the dorsomedial striatum of
NPC1(-/-) mice when compared to wildtype littermates. However, neocortical
excitability was significantly enhanced while LTP was abolished. These results
suggest that at least in the somatosensory neocortex NPC1 protein is
instrumental in synaptic function. BACKGROUND: Niemann-Pick type C1 disease (NPC1) is a rare progressive
neurodegenerative disorder caused by mutations in the NPC1 gene. In this
lysosomal storage disorder the intracellular transport and sequestration of
several lipids like cholesterol is severely impaired, resulting in an
accumulation of lipids in late endosomes and lysosomes. The neurological
manifestation of the disease is caused by dysfunction and cell death in the
central nervous system. Several animal models were used to analyze the impaired
pathways. However, the underlying pathogenic mechanisms are still not completely
understood and the genetic variability in humans cannot be reflected in these
models. Therefore, a human model using patient-specific induced pluripotent stem
cells provides a promising approach.
METHODS: We reprogrammed human fibroblasts from a NPC1 patient and a healthy
control by retroviral transduction with Oct4, Klf4, Sox2 and c-Myc. The obtained
human induced pluripotent stem cells (hiPSCs) were characterized by
immunocytochemical analyses. Neural progenitor cells were generated and patch
clamp recordings were performed for a functional analysis of derived neuronal
cells. Filipin stainings and the Amplex Red assay were used to demonstrate and
quantify cholesterol accumulation.
RESULTS: The hiPSCs expressed different stem cell markers, e.g. Nanog, Tra-1-81
and SSEA4. Using the embryoid body assay, the cells were differentiated in cells
of all three germ layers and induced teratoma in immunodeficient mice,
demonstrating their pluripotency. In addition, neural progenitor cells were
derived and differentiated into functional neuronal cells. Patch clamp
recordings revealed voltage dependent channels, spontaneous action potentials
and postsynaptic currents. The accumulation of cholesterol in different tissues
is the main hallmark of NPC1. In this study we found an accumulation of
cholesterol in fibroblasts of a NPC1 patient, derived hiPSCs, and neural
progenitor cells, but not in cells derived from fibroblasts of a healthy
individual. These findings were quantified by the Amplex Red assay,
demonstrating a significantly elevated cholesterol level in cells derived from
fibroblasts of a NPC1 patient.
CONCLUSIONS: We generated a neuronal model based on induced pluripotent stem
cells derived from patient fibroblasts, providing a human in vitro model to
study the pathogenic mechanisms of NPC1 disease. |
Which type of genes are modulated by SATB1? | Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. At an Associative t-test threshold of P | Chromatin modulation at various cis-acting elements is critical for V(D)J
recombination during T and B cell development. MARbeta, a matrix-associated
region (MAR) located upstream of the T cell receptor beta (TCRbeta) enhancer
(Ebeta), serves a crucial role in silencing Ebeta-mediated TCR activation. By
DNaseI hypersensitivity assays, we show here that overexpression of the MAR
binding proteins SMAR1 and Cux/CDP modulate the chromatin structure at MARbeta.
We further demonstrate that the silencer function of MARbeta is mediated
independently by SMAR1 and Cux/CDP as judged by their ability to repress
Ebeta-dependent reporter gene expression. Moreover, the repressor activity of
SMAR1 is strongly enhanced in the presence of Cux/CDP. These two proteins
physically interact with each other and colocalize within the perinuclear region
through a SMAR1 domain required for repression. The repression domain of SMAR1
is separate from the MARbeta binding domain and contains a nuclear localization
signal and an arginine-serine (RS)-rich domain, characteristic of pre-mRNA
splicing regulators. Our data suggest that at the double positive stage of T
cell development, cis-acting MARbeta elements recruit the strong negative
regulators Cux and SMAR1 to control Ebeta-mediated recombination and
transcription. Gene expression changes in CD4 + Vbeta8+ T cells energized by in vivo exposure
to staphylococcal enterotoxin B (SEB) bacterial superantigen compared to CD4 +
Vbeta8+ non-energic T cells were assessed using DNA microarrays containing 5184
murine complementary DNAs. Anergy in splenic T cells of SEB-immunized BALB/c
mice was verified by dramatically reduced proliferative capacity and an 8 x
overexpression of GRAIL mRNA in CD4 + Vbeta8+ T cells taken from mice 7 days
after injection. At an Associative t-test threshold of P<0.0005, 96 genes were
overexpressed or detected only in anergic T cells, while 256 genes were
suppressed or not detected in anergic T cells. Six of eight differential
expressions tested using real-time quantitative PCR were validated. Message for
B-Raf was detected only in non-anergic cells, while expression of the TCR
signaling modulator Slap (Src-like adapter protein) and the TCR zeta-chain
specific phosphatase Ptpn3 was enhanced. Modulation of multiple genes suggests
downregulation of Wnt/beta-catenin signaling and enhanced Notch signaling in the
anergic cells. Consistent with previous reports in a non-superantigen in vivo
anergy model, mRNA for CD18 and the transcription factor Satb1 (special
AT-rich-binding protein 1) was increased in SEB-energized T cells. This is the
first report of global transcriptional changes in CD4+ T cells made anergic by
superantigen exposure. Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune
homeostasis. Lack of effector T cell (T(eff) cell) function and gain of
suppressive activity by T(reg) cells are dependent on the transcriptional
program induced by Foxp3. Here we report that repression of SATB1, a genome
organizer that regulates chromatin structure and gene expression, was crucial
for the phenotype and function of T(reg) cells. Foxp3, acting as a
transcriptional repressor, directly suppressed the SATB1 locus and indirectly
suppressed it through the induction of microRNAs that bound the SATB1 3'
untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells
caused loss of suppressive function, establishment of transcriptional T(eff)
cell programs and induction of T(eff) cell cytokines. Our data support the
proposal that inhibition of SATB1-mediated modulation of global chromatin
remodeling is pivotal for maintaining T(reg) cell functionality. |
Does HER2 under-expression lead to favorable response to trastuzumab? | No, trastuzumab is effective only in cancers where Her2 is over-expessed. | HercepTestTM (DAKO A/S, Glostrup, Denmark) is an immunohistochemical assay that
detects HER2/neu gene products, and evaluates the overexpression status of the
HER2/neu protein in determining eligibility for the Trastuzumab (HerceptinR,
Genentech, San Francisco, CA, USA) therapy. However, practically, interobserver
variability of the HER2/neu interpretation of the immunostained results has
caused marked disagreement with regard to the intensity of tumor staining. In
this study, we quantitated HER2/neu expression by image analysis, and applied
this analyzing system to help to minimize interobserver variability of the
interpretation of the HercepTestTM. All the immunostained results were scored
semiquantitatively on a range of 0 to 3+ in accordance with the criteria
described as per the manufacturer's instructions, and quantitatively evaluated
using an image analyzing system with image processing software. Among the 92
cases, 15 were scored as 3+, six were 2+, and 32 were 1+ under intraobservers
agreement. When the cases were quantitated, a high correlation was shown between
the signal area extracted by image analysis and the corresponding score of
staining intensity with the HercepTestTM. By converting the quantitatively
extracted data into a scoring system based upon the criteria, the outcome
demonstrated a strong concordance with the scoring data obtained from
immunostaining. The results indicated that a quantitative scoring system
performed by simple image analysis may provide to improve interobserver
agreement of the interpretation of the HercepTest TM in clinical practice. BACKGROUND: The humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin)
is useful in the treatment of ErbB2-overexpressing breast cancers, but its
efficiency is limited because development of resistance is common. In order to
study the possibility of improving the efficacy of therapies directed against
HER2/erbB2, we investigated the effects of co-targeting this receptor and the
insulin-like growth factor 1 receptor (IGF-1R), a widely-expressed protein
tyrosine kinase with important roles in suppression of apoptosis and stimulation
of proliferation.
MATERIAL/METHODS: The experimental strategy involved combining trastuzumab
treatment and reduction of IGF-1R signaling through incremental heat-induced
expression of the domit-negative IGF-1 receptor 486/STOP under the control of
the heat-sensitive Drosophila HSP70 promoter, in HER2/erbB2-overexpressing
MCF7her18 breast cancer cells.
RESULTS: Isobologram analysis of combinatorial treatment data revealed a strong
synergistic interaction between trastuzumab treatment and the induction of the
domit-negative IGF-1R expression, resulting in potentiation of growth
inhibition in transfected cancer cells.
CONCLUSIONS: These observations support the concept that simultaneously
co-targeting tyrosine kinase receptors may be therapeutically useful, and
provide a specific rationale for combining IGF-1R and HER2/erbB2 targeting
strategies in anti-neoplastic approaches. BACKGROUND: Breast cancers that overexpress the human epidermal growth factor
receptor 2 (HER2) are eligible for effective biologically targeted therapies,
such as trastuzumab. However, accurately determining HER2 overexpression,
especially in immunohistochemically equivocal cases, remains a challenge. Manual
analysis of HER2 expression is dependent on the assessment of membrane staining
as well as comparisons with positive controls. In spite of the strides that have
been made to standardize the assessment process, intra- and inter-observer
discrepancies in scoring is not uncommon. In this manuscript we describe a
pathologist assisted, computer-based continuous scoring approach for increasing
the precision and reproducibility of assessing imaged breast tissue specimens.
METHODS: Computer-assisted analysis on HER2 IHC is compared with manual scoring
and fluorescence in situ hybridization results on a test set of 99 digitally
imaged breast cancer cases enriched with equivocally scored (2+) cases. Image
features are generated based on the staining profile of the positive control
tissue and pixels delineated by a newly developed Membrane Isolation Algorithm.
Evaluation of results was performed using Receiver Operator Characteristic (ROC)
analysis.
RESULTS: A computer-aided diagnostic approach has been developed using a
membrane isolation algorithm and quantitative use of positive immunostaining
controls. By incorporating internal positive controls into feature analysis a
greater Area Under the Curve (AUC) in ROC analysis was achieved than feature
analysis without positive controls. Evaluation of HER2 immunostaining that
utilized membrane pixels, controls, and percent area stained showed
significantly greater AUC than manual scoring, and significantly less false
positive rate when used to evaluate immunohistochemically equivocal cases.
CONCLUSION: It has been shown that by incorporating both a membrane isolation
algorithm and analysis of known positive controls a computer-assisted diagnostic
algorithm was developed that can reproducibly score HER2 status in IHC stained
clinical breast cancer specimens. For equivocal scoring cases, this approach
performed better than standard manual evaluation as assessed by ROC analysis in
our test samples. Finally, there exists potential for utilizing image-analysis
techniques for improving HER2 scoring at the immunohistochemically equivocal
range. Human epidermal growth factor receptor 2 (HER2/neu) is an important target for
the treatment of the breast cancers in which it is overexpressed. However, no
approved anti-HER2/neu therapy is available for the majority of breast cancer
patients, who express HER2/neu at low levels (with scores of 1+ or
2+/fluorescence in situ hybridization-negative). The trifunctional antibody
ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence
of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and
accessory cells form to cause tumor cell lysis. In a phase I trial with
metastatic breast cancer patients, ertumaxomab could be applied safely and
resulted in radiographically confirmed clinical responses. In this study, we
compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that
express HER2/neu at low and high levels. Under optimal conditions for
trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only
ertumaxomab was able to mediate the elimination of tumor cell lines that express
HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a
Th1-based cytokine release, a unique mode of action of trifunctional antibodies.
Competitive binding studies with trastuzumab and 520C9 mapped the binding site
of ertumaxomab to the extracellular regions II and III of the HER2/neu
ectodomain. This site is distinct from the binding site of trastuzumab, so that
HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence
of high amounts of trastuzumab. The ability of ertumaxomab to induce
cytotoxicity against various tumor cell lines, including those with low HER2/neu
antigen density, may provide a novel therapeutic option for breast cancer
patients who are not eligible for trastuzumab treatment. Autophagy has been emerging as a novel cytoprotective mechanism to increase
tumor cell survival under conditions of metabolic stress and hypoxia as well as
to escape chemotherapy-induced cell death. To elucidate whether autophagy might
also protect cancer cells from the growth inhibitory effects of targeted
therapies, we evaluated the autophagic status of preclinical breast cancer
models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody
trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive
SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which
optimally grow in the presence of Tzb doses as high as 200 microg/ml Tzb.
Fluorescence microscopic analyses revealed that the number of punctate LC3
structures -a hallmark of autophagy- was drastically higher in Tzb-refractory
cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses
confirmed that the lipidation product of the autophagic conversion of LC3 was
accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form
in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein
expression, a phenomenon characterizing the occurrence of increased autophagic
flux. Moreover, increased autophagy was actively used to survive Tzb therapy as
TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal
formation/function. Knockdown of LC3 expression via siRNA similarly resulted in
reduced TzbR cell proliferation and supra-additively interacted with Tzb to
re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells
accumulated LC3 punctate structures and decreased p62 expression after treatment
with high-dose Tzb, likely promoting their own resistance. This is the first
report showing that HER2-overexpressing breast cancer cells chronically exposed
to Tzb exhibit a bona fide up-regulation of the autophagic activity that
efficiently works to protect breast cancer cells from the growth-inhibitory
effects of Tzb. Therapeutic targeting autophagosome formation/function might
represent a novel molecular avenue to reduce the emergence of Tzb resistance in
HER2-dependent breast carcinomas. BACKGROUND: With the advent of targeted therapies, biomarkers provide a
promising means of individualizing therapy through an integrated approach to
prediction using the genetic makeup of the disease and the genotype of the
patient. Biomarker validation has therefore become a central topic of discussion
in the field of medicine, primarily due to the changing landscape of therapies
for treatment of a disease and these therapies purported mechanism(s) of action.
PURPOSE: In this report, we discuss the merits and limitations of some of the
clinical trial designs for predictive biomarker validation using examples from
ongoing or completed clinical trials.
METHODS: The designs are broadly classified as retrospective (i.e., using data
from previously well-conducted randomized controlled trials (RCT)) versus
prospective (enrichment or targeted, unselected or all-comers, hybrid, and
adaptive analysis). We discuss some of these designs in the context of real
trials.
RESULTS: Well-designed retrospective analysis of prospective RCT can bring
forward effective treatments to marker defined subgroup of patients in a timely
manner. An example is the KRAS gene status in colorectal cancer - the benefit
from cetuximab and panitumumab was demonstrated to be restricted to patients
with wild type status based on prospectively specified analyses using data from
previously conducted RCTs. Prospective enrichment designs are appropriate when
compelling preliminary evidence suggests that not all patients will benefit from
the study treatment under consideration; however, this may sometimes leave
questions uswered. An example is the established benefit of trastuzumab as
adjuvant therapy for breast cancer; a clear definition of HER2-positivity and
the assay reproducibility have, however, remained uswered. An all-comers
design is optimal where preliminary evidence regarding treatment benefit and
assay reproducibility is uncertain (e.g., EGFR expression and tyrosine kinase
inhibitors in lung cancer), or to identify the most effective therapy from a
panel of regimens (e.g., chemotherapy options in breast cancer).
LIMITATIONS: The designs discussed here rest on the assumption that the
technical feasibility, assay performance metrics, and the logistics of specimen
collection are well established and that initial results demonstrate promise
with regard to the predictive ability of the marker(s).
CONCLUSIONS: The choice of a clinical trial design is driven by a combination of
scientific, clinical, statistical, and ethical considerations. There is no one
size fits all solution to predictive biomarker validation. The 12th St Gallen International Breast Cancer Conference (2011) Expert Panel
adopted a new approach to the classification of patients for therapeutic
purposes based on the recognition of intrinsic biological subtypes within the
breast cancer spectrum. For practical purposes, these subtypes may be
approximated using clinicopathological rather than gene expression array
criteria. In general, systemic therapy recommendations follow the subtype
classification. Thus, 'Luminal A' disease generally requires only endocrine
therapy, which also forms part of the treatment of the 'Luminal B' subtype.
Chemotherapy is considered indicated for most patients with 'Luminal B', 'Human
Epidermal growth factor Receptor 2 (HER2) positive', and 'Triple negative
(ductal)' disease, with the addition of trastuzumab in 'HER2 positive' disease.
Progress was also noted in defining better tolerated local therapies in selected
cases without loss of efficacy, such as accelerated radiation therapy and the
omission of axillary dissection under defined circumstances. Broad treatment
recommendations are presented, recognizing that detailed treatment decisions
need to consider disease extent, host factors, patient preferences, and social
and economic constraints. Trastuzumab (Herceptin®) is a humanized monoclonal antibody designed to bind and
inhibit the function of the human epidermal growth factor receptor 2
(HER2)/erbB2 receptor. Trastuzumab has demonstrated clinical activity in several
types of HER2-overexpressing epithelial tumors, such as breast and metastatic
gastric or gastroesophageal junction cancer. Relapse and therapeutic resistance,
however, still occur in a subset of patients treated with regimens including
trastuzumab, despite significant improvements in response rates, survival and
quality of life. To investigate the potential mechanisms of acquired therapeutic
resistance to trastuzumab, we developed a preclinical model of human ovarian
cancer cells, SKOV-3 Herceptin-resistant (HR), and examined the corresponding
changes in gene expression profiles. SKOV-3 HR cells were developed by in vivo
serial passaging of parental trastuzumab-sensitive SKOV-3 cells. Following four
rounds of serial transplantation of 'break-through' xenograft tumors under
trastuzumab treatment, significant and reproducible differences in the effects
of trastuzumab treatment between SKOV-3 HR and SKOV-3 cells in vivo and in vitro
were revealed. SKOV-3 HR cells retained HER2 protein expression but were
unaffected by the antiproliferative effects of trastuzumab. The trastuzumab
binding affinity for SKOV-3 HR cells was diminished, despite these cells having
more binding sites for trastuzumab. Microarray expression profiling (MEP) was
performed to determine the genes involved in the resistance mechanism.
Functional analysis revealed the differential expression of genes potentially
involved in angiogenesis, metastasis, differentiation and proliferation, such as
mucin1 (MUC1). Immunohistochemical staining of SKOV-3 HR cells demonstrated a
marked overexpression of MUC1. Based on these data, we hypothesize that the
overexpression of MUC1 may hinder trastuzumab binding to HER2 receptors,
abrogating the antitumor effects of trastuzumab and thus could contribute to
resistance to therapy. Moreover, the resultant MEP preclinical gene signature in
this preclinical model system may provide the basis for further investigation of
potential clinical mechanisms of resistance to trastuzumab. In patients with metastatic gastric cancer, median overall survival remains
under 1 year and standard chemotherapy regimens are not able to substantially
improve the prognosis of the patients. Amplification and over-expression of HER2
is reported in approximately 20% of gastric tumours, challenging the use of
targeted therapies. There are several targeted therapies in different stages of
clinical development with trastuzumab being the first overcoming the regulatory
hurdle and getting European Medicines Agency approval. In patients with advanced
gastric or gastro-oesophageal junction cancer, addition of trastuzumab to
chemotherapy significantly improved overall survival compared with chemotherapy
alone. Addition of trastuzumab to chemotherapy did not increase the incidence of
adverse events. Other agents targeting the HER2 pathway (lapatinib) or other
domains of epidermal growth factor receptor family (cetuximab) are currently
being investigated for the treatment of an advanced gastric cancer. A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has
been suggested for breast cancer but has not been evaluated for esophageal
carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth
and metastases in an orthotopic model of esophageal carcinoma. The chemokine
receptor CXCR4 has been implicated in metastatic dissemination of various tumors
and correlates with poor survival in esophageal carcinoma. The aim of this study
was to investigate a correlation between the expression levels of HER2 and CXCR4
and to evaluate the involvement of CXCR4-expression in HER2-positive esophageal
carcinoma. The effects of HER2-inhibition with trastuzumab and of
CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and
receptor expression were evaluated in vitro and in an orthotopic model of
metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of
HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A
significant correlation of HER2- and CXCR4-expression in primary tumor and
metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led
to a significant reduction of primary tumor growth, metastases and
micrometastases. HER2-expression was significantly elevated under AMD3100
treatment in the primary tumor and particularly in the metastases. The positive
correlation between HER2- and CXCR4-expression was validated in esophageal
cancer patients. The correlation of CXCR4- and HER2-expression and the elevation
of HER2-expression and reduction of metastases through CXCR4-inhibition suggest
a possible functional linkage and a role in tumor dissemination in HER2-positive
esophageal carcinoma. |
Which are the most abundant human lincRNA? | MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. MALAT/NEAT2 highly abundant, its expression is strongly regulated in many tumor entities including lung adenocarcinoma and hepatocellular carcinoma as well as physiological processes, and it is associated with many RNA binding proteins and highly conserved throughout evolution. H19 large intergenic non-coding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extra-embryonic cell lineages, yet its physiological function is unknown. Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. | In a previous work, we have isolated the human H19 gene and shown accumulation
of transcripts in various human tumors including breast carcinomas (Douc-Rasy et
al (1993) Int J Oncol 2, 753-758). Questions arose, after Northern blot results,
about the precise H19 mRNA location, specially in normal breast tissues and
benign or malign primary breast tumors. Then we performed molecular in situ
hybridization to get insight into tissue expression of the H19 gene. Examined
resections included one normal tissue, one fibroadenoma and 13 cancers. Results
obtained with the H19 probe can be summarized as follows: 1) in normal breast
tissues signals were focally observed in epithelial cells, but more
predomitly in the palleal tissue which is sensitive to hormones; 2) in the
fibroadenoma, fibroblastic cells were extensively labeled at the
stroma-epithelium boundary, but epithelial cells were negative; and 3) in
primary cancers, eight specimens exhibited signals on stromal cells, one
specimen on epithelial cells and four on both epithelial and stromal cells. Data
provide the following evidence: 1) usually labeled cells are clustered, either
within normal or pathological tissues; 2) the labeling pattern highly differs
from one tumor to another; and 3) H19 probe displays very different signals from
one cell to another in given compartment of a given tissue section. In
conclusion, it seems that a high H19 expression matches the tumor invasion. Our
results suggest that the expression of this gene is concerned by the
relationships between epithelial and stromal cells, and can reflect peculiar
physiological states of the cells. Furthermore, we discuss results showing an
abundant expression of H19 gene in some adenocarcinomas of bad prognosis, in the
context of the otherwise established tumor-suppressor role of this gene, or the
strictly controlled gene dosage, which could be overridden in these particular
cases. The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is
expressed in fetal bladder, down-regulated postnatally and is re-expressed in
human bladder carcinoma. This study was designed to investigate the dynamics of
the expression of H19 in the mouse bladder carcinoma induced by
N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of
neoplastic transformation. BBN was administered to mice in the drinking water
for 26-28 weeks. The bladders were removed at 5-10 week intervals for
histopathological examination and for in situ hybridization for H19 RNA, using a
35S-labeled probe. Following BBN administration expression of H19 first appeared
after 5 weeks in the lamina propria adjacent to the basement membrane,
concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in
epithelial cells. Invasive carcinomas, of the transitional and squamous
sub-types, were seen after 20 weeks and more of BBN administration. At this
stage H19 expression was observed in scattered tumor cells, in the connective
tissue stroma of the tumor and in the lamina propria underlying the remaining
hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal
bladder but was absent in normal adult bladder. We conclude that, similar to
humans, the H19 gene product is an oncofetal RNA molecule in the experimental
mouse model of bladder carcinoma. In this model H19 is expressed in the
connective tissue of the lamina propria prior to its expression in epithelial
cells, concurrent with preneoplastic changes in the transitional epithelium of
the bladder. H19, which is one of the most abundantly expressed imprinted genes during
mammalian embryonic and foetal development, has been cloned from a rumit. The
sheep (Ovis aries) gene contains five exons interspersed by four exceptionally
small introns; only short stretches of the nucleotide sequence, particularly in
exon 1, show good homology with the human gene. The size of the exons and
introns and the sequences around the splice junctions however, are well
conserved between the species. The gene encodes a approximately 2.6 kb
transcript which contains several potential short open reading frames, none of
which is conserved between the ovine and human or murine transcripts, supporting
a previous hypothesis that the gene product is the untranslated RNA itself. H19
mRNA is highly abundant in most ovine embryonic and foetal tissues of mesodermal
and endodermal origins but was not detected in tissues of ectodermal origin such
as the trophectoderm and the foetal brain. Expression of H19 in the
extraembryonic membranes was detected only after the ovine conceptus began
attachment to the endometrium and the embryo itself had undergone early
organogenesis. This may be regarded as the first step in implantation; thus, in
comparison with the mouse, the initiation of H19 expression appears to be
determined by the timing of implantation rather than by the stage of development
of the embryo itself. In most tissues, H19 expression is temporally linked to
IGF2, a major foetal growth factor. The exceptions were the elongated
blastocyst, the trophectoderm and brain, where low levels of IGF2 were observed
in the absence of detectable H19. The abundance of H19 mRNA was in general,
directly correlated with IGF2 mRNA abundance in mesodermal and endodermal
tissues, suggesting that the two ovine genes share common regulatory elements
that co-ordinately regulate their expression. Though both are generally regarded
as embryonic and foetal genes, their expression was still maintained at a fairly
high level in the adult sheep liver, lung, skeletal muscle, adrenal gland and
kidney, suggesting that these organs are significant sources of IGF II in the
adult. BACKGROUND: Recent work has identified that many long mRNA-like noncoding RNAs
(lncRNAs) are expressed in the developing nervous system. Despite their
abundance, the function of these ncRNAs has remained largely unexplored. We have
investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal
cell differentiation.
RESULTS: We find that the RNCR2 is selectively expressed in a subset of both
mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated
knockdown of RNCR2 results in an increase of both amacrine cells and Müller
glia, indicating a role for this lncRNA in regulating retinal cell fate
specification. We further report that RNCR2 RNA, which is normally
nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP
sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of
shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2
induces a domit-negative phenotype. Finally, we use the IRES-GFP fusion
approach to identify specific domains of RNCR2 that are required for repressing
both amacrine and Müller glial differentiation.
CONCLUSION: These data demonstrate that the lncRNA RNCR2 plays a critical role
in regulating mammalian retinal cell fate specification. Furthermore, we present
a novel approach for generating domit-negative constructs of lncRNAs, which
may be generally useful in the functional analysis of this class of molecules. The H19 large intergenic non-coding RNA (lincRNA) is one of the most highly
abundant and conserved transcripts in mammalian development, being expressed in
both embryonic and extra-embryonic cell lineages, yet its physiological function
is unknown. Here we show that miR-675, a microRNA (miRNA) embedded in H19's
first exon, is expressed exclusively in the placenta from the gestational time
point when placental growth normally ceases, and placentas that lack H19
continue to grow. Overexpression of miR-675 in a range of embryonic and
extra-embryonic cell lines results in their reduced proliferation; targets of
the miRNA are upregulated in the H19 null placenta, including the
growth-promoting insulin-like growth factor 1 receptor (Igf1r) gene. Moreover,
the excision of miR-675 from H19 is dynamically regulated by the stress-response
RNA-binding protein HuR. These results suggest that H19's main physiological
role is in limiting growth of the placenta before birth, by regulated processing
of miR-675. The controlled release of miR-675 from H19 may also allow rapid
inhibition of cell proliferation in response to cellular stress or oncogenic
signals. Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known
as nuclear speckles, which contain a distinct set of pre-mRNA processing
factors. Previous studies in cell culture have demonstrated that Malat1
interacts with pre-mRNA splicing factors, including the serine- and
arginine-rich (SR) family of proteins, and regulates a variety of biological
processes, including cancer cell migration, synapse formation, cell cycle
progression, and responses to serum stimulation. To address the physiological
function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice
using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and
fertile, showing no apparent phenotypes. Nuclear speckle markers were also
correctly localized in cells that lacked Malat1. However, the cellular levels of
another long, noncoding RNA--Neat1--which is an architectural component of
nuclear bodies known as paraspeckles, were down-regulated in a particular set of
tissues and cells lacking Malat1. We propose that Malat1 is not essential in
living mice maintained under normal laboratory conditions and that its function
becomes apparent only in specific cell types and under particular conditions. Abundant expression of the long noncoding (lnc) PAN (polyadenylated nuclear) RNA
by the human oncogenic gammaherpesvirus Kaposi's sarcoma-associated herpesvirus
(KSHV) depends on a cis-element called the expression and nuclear retention
element (ENE). The ENE upregulates PAN RNA by inhibiting its rapid nuclear decay
through triple-helix formation with the poly(A) tail. Using structure-based
bioinformatics, we identified six ENE-like elements in evolutionarily diverse
viral genomes. Five are in double-stranded DNA viruses, including mammalian
herpesviruses, insect polydnaviruses, and a protist mimivirus. One is in an
insect picorna-like positive-strand RNA virus, suggesting that the ENE can
counteract cytoplasmic as well as nuclear RNA decay pathways. Functionality of
four of the ENEs was demonstrated by increased accumulation of an intronless
polyadenylated reporter transcript in human cells. Identification of these ENEs
enabled the discovery of PAN RNA homologs in two additional gammaherpesviruses,
RRV and EHV2. Our findings demonstrate that searching for structural elements
can lead to rapid identification of lncRNAs. Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs)
lacking protein-coding capacity. However, most lncRNAs are expressed at a very
low level, and in most cases there is no genetic evidence to support their in
vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1)
is among the most abundant and highly conserved lncRNAs, and it exhibits an
uncommon 3'-end processing mechanism. In addition, its specific nuclear
localization, developmental regulation, and dysregulation in cancer are
suggestive of it having a critical biological function. We have characterized a
Malat1 loss-of-function genetic model that indicates that Malat1 is not
essential for mouse pre- and postnatal development. Furthermore, depletion of
Malat1 does not affect global gene expression, splicing factor level and
phosphorylation status, or alternative pre-mRNA splicing. However, among a small
number of genes that were dysregulated in adult Malat1 knockout mice, many were
Malat1 neighboring genes, thus indicating a potential cis-regulatory role of
Malat1 gene transcription. The metastasis-associated lung adenocarcinoma transcript 1, MALAT1, is a long
non-coding RNA (lncRNA) that has been discovered as a marker for lung cancer
metastasis. It is highly abundant, its expression is strongly regulated in many
tumor entities including lung adenocarcinoma and hepatocellular carcinoma as
well as physiological processes, and it is associated with many RNA binding
proteins and highly conserved throughout evolution. The nuclear transcript
MALAT-1 has been functionally associated with gene regulation and alternative
splicing and its regulation has been shown to impact proliferation, apoptosis,
migration and invasion. Here, we have developed a human and a mouse knockout
system to study the loss-of-function phenotypes of this important ncRNA. In
human tumor cells, MALAT1 expression was abrogated using Zinc Finger Nucleases.
Unexpectedly, the quantitative loss of MALAT1 did neither affect proliferation
nor cell cycle progression nor nuclear architecture in human lung or liver
cancer cells. Moreover, genetic loss of Malat1 in a knockout mouse model did not
give rise to any obvious phenotype or histological abnormalities in Malat1-null
compared with wild-type animals. Thus, loss of the abundant nuclear long ncRNA
MALAT1 is compatible with cell viability and normal development. The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is
misregulated in many human cancers and produces an abundant long
nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II,
the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but
instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature
MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many
protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the
MEN β long noncoding RNAs are protected from 3'-5' exonucleases by highly
conserved triple helical structures. Surprisingly, when these structures are
placed downstream from an ORF, the transcript is efficiently translated in vivo
despite the lack of a poly(A) tail. The triple helix therefore also functions as
a translational enhancer, and mutations in this region separate this translation
activity from simple effects on RNA stability or transport. We further found
that a transcript ending in a triple helix is efficiently repressed by microRNAs
in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated
silencing. These results provide new insights into how transcripts that lack
poly(A) tails are stabilized and regulated and suggest that RNA triple-helical
structures likely have key regulatory functions in vivo. Stability of the long noncoding-polyadenylated nuclear (PAN) RNA from Kaposi's
sarcoma-associated herpesvirus is conferred by an expression and nuclear
retention element (ENE). The ENE protects PAN RNA from a rapid
deadenylation-dependent decay pathway via formation of a triple helix between
the U-rich internal loop of the ENE and the 3'-poly(A) tail. Because viruses
borrow molecular mechanisms from their hosts, we searched highly abundant human
long-noncoding RNAs and identified putative ENE-like structures in
metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple
endocrine neoplasia-β (MENβ) RNAs. Unlike the PAN ENE, the U-rich internal loops
of both predicted cellular ENEs are interrupted by G and C nucleotides and
reside upstream of genomically encoded A-rich tracts. We confirmed the ability
of MALAT1 and MENβ sequences containing the predicted ENE and A-rich tract to
increase the levels of an intronless β-globin reporter RNA. UV thermal
denaturation profiles at different pH values support formation of a
triple-helical structure composed of multiple U•A-U base triples and a single
C•G-C base triple. Additional analyses of the MALAT1 ENE revealed that robust
stabilization activity requires an intact triple helix, strong stems at the
duplex-triplex junctions, a G-C base pair flanking the triplex to mediate
potential A-minor interactions, and the 3'-terminal A of the A-rich tract to
form a blunt-ended triplex lacking unpaired nucleotides at the duplex-triplex
junction. These examples of triple-helical, ENE-like structures in cellular
noncoding RNAs, are unique. The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma
transcript 1), also known as MALAT-1 or NEAT2 (nuclear-enriched abundant
transcript 2), is a highly conserved nuclear noncoding RNA (ncRNA) and a
predictive marker for metastasis development in lung cancer. To uncover its
functional importance, we developed a MALAT1 knockout model in human lung tumor
cells by genomically integrating RNA destabilizing elements using zinc finger
nucleases. The achieved 1,000-fold MALAT1 silencing provides a unique
loss-of-function model. Proposed mechanisms of action include regulation of
splicing or gene expression. In lung cancer, MALAT1 does not alter alternative
splicing but actively regulates gene expression including a set of
metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired
in migration and form fewer tumor nodules in a mouse xenograft. Antisense
oligonucleotides (ASO) blocking MALAT1 prevent metastasis formation after tumor
implantation. Thus, targeting MALAT1 with ASOs provides a potential therapeutic
approach to prevent lung cancer metastasis with this ncRNA serving as both
predictive marker and therapeutic target. Finally, regulating gene expression,
but not alternative splicing, is the critical function of MALAT1 in lung cancer
metastasis. In summary, 10 years after the discovery of the lncRNA MALAT1 as a
biomarker for lung cancer metastasis, our loss-of-function model unravels the
active function of MALAT1 as a regulator of gene expression governing hallmarks
of lung cancer metastasis. We describe the identification and characterization of circular intronic long
noncoding RNAs in human cells, which accumulate owing to a failure in
debranching. The formation of such circular intronic RNAs (ciRNAs) can be
recapitulated using expression vectors, and their processing depends on a
consensus motif containing a 7 nt GU-rich element near the 5' splice site and an
11 nt C-rich element close to the branchpoint site. In addition, we show that
ciRNAs are abundant in the nucleus and have little enrichment for microRNA
target sites. Importantly, knockdown of ciRNAs led to the reduced expression of
their parent genes. One abundant such RNA, ci-ankrd52, largely accumulates to
its sites of transcription, associates with elongation Pol II machinery, and
acts as a positive regulator of Pol II transcription. This study thus suggests a
cis-regulatory role of noncoding intronic transcripts on their parent coding
genes. |
Which is the molecular function of the protein CCDC40? | The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation and mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms. | Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal
recessive disorder characterized by recurrent infections of the respiratory
tract associated with the abnormal function of motile cilia. Approximately half
of individuals with PCD also have alterations in the left-right organization of
their internal organ positioning, including situs inversus and situs ambiguous
(Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain
containing a protein, CCDC40, essential for correct left-right patterning in
mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in
tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with
reduced ranges of motility. We further show that CCDC40 mutations in humans
result in a variant of PCD characterized by misplacement of the central pair of
microtubules and defective assembly of inner dynein arms and dynein regulatory
complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is
required for axonemal recruitment of CCDC39, disruption of which underlies a
similar variant of PCD. BACKGROUND: Patients with congenital heart disease (CHD) and heterotaxy show
high postsurgical morbidity/mortality, with some developing respiratory
complications. Although this finding is often attributed to the CHD, airway
clearance and left-right patterning both require motile cilia function. Thus,
airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia
(PCD) may contribute to increased respiratory complications in heterotaxy
patients.
METHODS AND RESULTS: We assessed 43 CHD patients with heterotaxy for airway CD.
Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal
nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen
patients exhibited CD characterized by abnormal ciliary motion and nNO levels
below or near the PCD cutoff values. Patients with CD aged >6 years show
increased respiratory symptoms similar to those seen in PCD. Sequencing of all
14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD
disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077
novel variants per patient, respectively. One heterotaxy patient with CD had the
PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2
mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among
PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations.
CONCLUSIONS: Our studies show that CHD patients with heterotaxy have substantial
risk for CD and increased respiratory disease. Heterotaxy patients with CD were
enriched for mutations in PCD genes. Future studies are needed to assess the
potential benefit of prescreening and prophylactically treating heterotaxy
patients for CD. BACKGROUND: CCDC39 and CCDC40 genes have recently been implicated in primary
ciliary dyskinesia (PCD) with inner dynein arm (IDA) defects and axonemal
disorganisation; their contribution to the disease is, however, unknown. Aiming
to delineate the CCDC39/CCDC40 mutation spectrum and associated phenotypes, this
study screened a large cohort of patients with IDA defects, in whom clinical and
ciliary phenotypes were accurately described.
METHODS: All CCDC39 and CCDC40 exons and intronic boundaries were sequenced in
43 patients from 40 unrelated families. The study recorded and compared clinical
features (sex, origin, consanguinity, laterality defects, ages at first symptoms
and at phenotype evaluation, neonatal respiratory distress, airway infections,
nasal polyposis, otitis media, bronchiectasis, infertility), ciliary beat
frequency, and quantitative ultrastructural analyses of cilia and sperm
flagella.
RESULTS: Biallelic CCDC39 or CCDC40 mutations were identified in 30/34 (88.2%)
unrelated families with IDA defects associated with axonemal disorganisation (22
and eight families, respectively). Fourteen of the 28 identified mutations are
novel. No mutation was found in the six families with isolated IDA defects.
Patients with identified mutations shared a similar phenotype, in terms of both
clinical features and ciliary structure and function. The sperm flagellar
ultrastructure, analysed in 4/7 infertile males, showed evidence of
abnormalities similar to the ciliary ones.
CONCLUSIONS: CCDC39 and CCDC40 mutations represent the major cause of PCD with
IDA defects and axonemal disorganisation. Patients carrying CCDC39 or CCDC40
mutations are phenotypically indistinguishable. CCDC39 and CCDC40 analyses in
selected patients ensure mutations are found with high probability, even if
clinical or ciliary phenotypes cannot prioritise one analysis over the other. Human cilia were once thought merely to be important in respiratory mucociliary
clearance, with primary ciliary dyskinesia (PCD) the sole manifestation of
ciliary dysfunction. There are now known to be three types of cilia: primary,
nodal and motile. Cilia are complex, likely involving more than 1000 gene
products; in this review, recent advances in PCD genetics, and the potential
relationships with genes causing other ciliopathies, are discussed. PCD is the
most important respiratory disease, characterized by upper and lower airway
infection and inflammation and disorders of laterality. Ciliary gene mutations
are now known to cause single organ disease, as well as complex syndromes. The
focus of the review is primarily PCD, in the context of the expanding ciliopathy
spectrum. The authors consider the clinical situations in which ciliary disease
should be considered, and the implications for specialist respiratory practice. Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused
by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2
microtubule cilia structure and inner dynein arm (IDA) loss, historically termed
"radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke
defect" families, as these are the two genes identified so far to cause this
defect. We discovered biallelic mutations in a remarkable 69% (37/54) of
families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39
and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift
predicting early protein truncation, which suggests this defect is caused by
"null" alleles conferring complete protein loss. Most families (73%; 27/37) had
homozygous mutations, including families from outbred populations. A major
putative hotspot mutation was identified, CCDC40 c.248delC, as well as several
other possible hotspot mutations. Together, these findings highlight the key
role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and
these genes represent major candidates for genetic testing in families affected
by this ciliary phenotype. We show that radial spoke structures are largely
intact in these patients and propose this ciliary ultrastructural abnormality be
referred to as "IDA and microtubular disorganisation defect," rather than
"radial spoke defect." Pompe disease is an uncommon autosomal recessive glycogen storage disorder
caused by deficiency of acid α-glucosidase. Classic infantile form triggers
severe cardiomyopathy, hypotonia, and respiratory failure, leading to death
within the first two years of life. The majority of patients with Pompe disease
have been reported to have point mutations in the GAA gene. We report the first
complex deletion-insertion encompassing the complete structure of GAA gene and a
large fragment of the gene CCDC40 in a patient with very severe form of Pompe
disease. Sequencing analysis of breakpoints allowed us to determine the
potential implication of an Alu repeat in the pathogenic mechanism. We suggest
that molecular strategy of Pompe disease should include systematic analysis of
large rearrangements. Primary ciliary dyskinesia (PCD) is a rare genetic disorder leading to recurrent
respiratory tract infections. High-speed video-microscopy analysis (HVMA) of
ciliary beating, currently the first-line diagnostic tool for PCD in most
centres, is challenging because recent studies have expanded the spectrum of
HVMA findings in PCD from grossly abnormal to very subtle. The objective of this
study was to describe the diversity of HVMA findings in genetically confirmed
PCD individuals. HVMA was performed as part of the routine work-up of
individuals with suspected PCD. Subsequent molecular analysis identified
biallelic mutations in the PCD-related genes of 66 individuals. 1072 videos of
these subjects were assessed for correlation with the genotype. Biallelic
mutations (19 novel) were found in 17 genes: DNAI1, DNAI2, DNAH5, DNAH11,
CCDC103, ARMC4, KTU/DNAAF2, LRRC50/DNAAF1, LRRC6, DYX1C1, ZMYND10, CCDC39,
CCDC40, CCDC164, HYDIN, RSPH4A and RSPH1. Ciliary beat pattern variations
correlated well with the genetic findings, allowing the classification of
typical HVMA findings for different genetic groups. In contrast, analysis of
ciliary beat frequency did not result in additional diagnostic impact. In
conclusion, this study provides detailed knowledge about the diversity of HVMA
findings in PCD and may therefore be seen as a guide to the improvement of PCD
diagnostics. RATIONALE: The relationship between clinical phenotype of childhood primary
ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly
defined.
OBJECTIVES: To delineate clinical features of childhood PCD and their
associations with ultrastructural defects and genotype.
METHODS: A total of 118 participants younger than 19 years old with PCD were
evaluated prospectively at six centers in North America using standardized
procedures for diagnostic testing, spirometry, chest computed tomography,
respiratory cultures, and clinical phenotyping.
MEASUREMENTS AND MAIN RESULTS: Clinical features included neonatal respiratory
distress (82%), chronic cough (99%), and chronic nasal congestion (97%). There
were no differences in clinical features or respiratory pathogens in subjects
with outer dynein arm (ODA) defects (ODA alone; n = 54) and ODA plus inner
dynein arm (IDA) defects (ODA + IDA; n = 18) versus subjects with IDA and
central apparatus defects with microtubular disorganization (IDA/CA/MTD;
n = 40). Median FEV1 was worse in the IDA/CA/MTD group (72% predicted) versus
the combined ODA groups (92% predicted; P = 0.003). Median body mass index was
lower in the IDA/CA/MTD group (46th percentile) versus the ODA groups (70th
percentile; P = 0.003). For all 118 subjects, median number of lobes with
bronchiectasis was three and alveolar consolidation was two. However, the 5- to
11-year-old IDA/CA/MTD group had more lobes of bronchiectasis (median, 5;
P = 0.0008) and consolidation (median, 3; P = 0.0001) compared with the ODA
groups (median, 3 and 2, respectively). Similar findings were observed when
limited to participants with biallelic mutations.
CONCLUSIONS: Lung disease was heterogeneous across all ultrastructural and
genotype groups, but worse in those with IDA/CA/MTD ultrastructural defects,
most of whom had biallelic mutations in CCDC39 or CCDC40. |
Can administration of the thyrotropin releasing hormone reduce fatigue in cancer patients? | yes, in cancer patients, thyrotropin releasing hormone (TRH) administration was associated with significant improvement in cancer related fatigue levels as measured by the Visual Analog Scale-Energy, fatigue and vigor subscales of the POMS, and the fatigue subscale of FACIT-F. TRH administration was safe and tolerable in the treatment of cancer-related with a positive impact on quality of life, suggesting the need for further studies investigating TRH for treatment of fatigue in cancer patients. | BACKGROUND: Fatigue is a common and often disabling symptom for cancer patients.
To date, no pharmacological interventions have shown reliable efficacy in
treatment of cancer-related fatigue (CF). Thyrotropin-releasing hormone (TRH), a
key regulator of homeostasis, exerts arousing and analeptic actions in instances
of behavioral depression. In the present pilot, randomized, placebo-controlled,
crossover study, we investigated the efficacy and safety of TRH as a treatment
for CF.
METHODS: Patients with cancer experiencing significant fatigue without medically
reversible causes were enrolled in this study. The primary outcome measure was
the visual analog scale for energy (VAS-E) assessed at 3, 7, and 24 h post-study
medication administration. Secondary outcome measures included the profile of
mood states (POMS) questionnaire, a 6-min walking test, the hospital anxiety and
depression scale, the Leeds sleep questionnaire, and assessment of quality of
life using the Functional Assessment of Chronic Illness Therapy-Fatigue
(FACIT-F).
RESULTS: Eight patients completed the study. TRH administration was associated
with significant improvement in fatigue level as measured by the VAS-E, the
fatigue and vigor subscales of the POMS, and the fatigue subscale of FACIT-F (p
< 0.05). It was also associated with a positive impact on quality of life. TRH
administration was associated with transient increases in blood pressure and
heart rate.
CONCLUSIONS: TRH administration was efficacious, safe, and tolerable in the
treatment of CF with a positive impact on quality of life. These results provide
a crucial impetus for pursuing TRH therapeutics to treat CF. Aging and aging related illnesses such as cancer have been associated with
inflammatory changes. Cancer-related behavioral comorbidities such as fatigue,
sleep disturbances, depression have also been associated with inflammation,
hypothalamic-pituitary-adrenal (HPA) axis dysregulation and other neuroendocrine
changes. From a clinical perspective, cancer-related fatigue demonstrates
striking similarities with the cytokine-induced sickness phenomenon in animal
models. Thyrotopin-releasing hormone (TRH) plays a homeostatic role in its
interaction with several biological systems, including a critical role in its
interactions with the immune system. Considerable evidence supports a pivotal
role for TRH in the inflammatory processes with specific relevance to the
"cytokine-induced sickness behavior" paradigm. Additionally, TRH exerts arousing
and analeptic effects in instances of behavioral depression. In a small
proof-of-concept study conducted by our group, we investigated TRH
administration as a treatment fatigue in cancer survivors in comparison with
saline administration using a double-blind, crossover design. We also evaluated
impact of TRH/saline administration on the inflammatory markers in these
patients. TRH administration was associated with significant improvement (p <
0.05) in fatigue levels as measured by the Visual Analog Scale-Energy (VAS-E),
was associated with significant (p < 0.05) improvement in sleep disturbances and
improved quality of life. Notably, TRH administration was associated with
decrease in C-reactive protein (CRP) levels, a marker of inflammation. This
decrease in CRP level with TRH administration was associated with improvement in
energy levels as measured by the VAS-E. The present review supports potential
utility of TRH-based therapeutics in medical and psychiatric disorders with
underlying inflammatory processes. |
What is the role of chromomethylases in plants? | Chromomethylases (CMTs), which constitute a plant-specific DNA (cytosine-5)-methyltransferase family, are involved primarily in the maintenance of symmetrical CpNpG (N = A, T, C, or G) methylation and they also play a role in de novo methylation. CMTs are characterized by the presence of a chromatin-associated domain (chromodomain) inserted within the catalytic protein motifs I and IV. CMTs have likely evolved because of the high levels of CpNpG methylation present in plant genomes relative to animal genomes. The targeting of CMT methylation is accomplished by short interfering RNA (siRNA) pathways and histone methylation (H3K9, H3K27). It has been shown that transposons are in vivo targets of CMT-dependent methylation, suggesting that CMTs play a role in the plant genome surveillance. In Arabidopsis, CMTs play a key role in egg cell reprogramming and normal embryogenesis during the first few divisions of the zygote by mediating transposon and euchromatin epigenetic gene silencing. In tomatoes, CMTs are preferentially expressed in the pericarp during fruit development which suggests involvement of CMTs in the locus-specific control of methylation in the pericarp during fruit growth. | Chromodomains are thought to mediate protein-protein interactions between
chromatin components. We have detected a chromodomain embedded within the
catalytic region of a predicted Arabidopsis DNA methyltransferase that is
diverged from other eukaryotic enzymes. The 791 residue "chromomethylase" (CMT1)
is encoded by a floral transcript that is spliced from 20 exons and is present
at only approximately 1/10(-7) of total mRNA. Genomic sequencing reveals an
ancient haplotype split at CMT1 between Col-0 + Metz and the other ecotypes
examined. In the Col-0 + Metz haplotype, alternative mRNA processing at intron
13 truncates the coding region. In Ler, RLD, and No-0, similar truncation is
caused by insertion of an intact retrotransposon, Evelknievel, which is present
as a single copy in Ler and RLD and is currently methylated and inactive.
Evelknievel is found at this site on a single branch that connects the Ler, RLD,
and No-0 ecotypes but is absent from the genomes of all other ecotypes examined.
A stop codon within exon 6 of the Metz ecotype confirms that CMT1 is
nonessential. Nevertheless, comparison to CMT1 of Cardaminopsis arenosa, an
outcrossing relative, indicates conservation for DNA methyltransferase function.
We discuss how allelic diversity of CMT1 may reflect loosened selective
constraints in a self-fertilizing species such as Arabidopsis thaliana. Plants maintain cytosine methylation at CG and non-CG residues to control gene
expression and genome stability. In a screen for Arabidopsis mutants that alter
methylation and silencing of a densely methylated endogenous reporter gene, we
recovered 11 loss-of-function alleles in the CMT3 chromomethylase gene. The cmt3
mutants displayed enhanced expression and reduced methylation of the reporter,
particularly at non-CG cytosines. CNG methylation was also reduced at repetitive
centromeric sequences. Thus, CMT3 is a key determit for non-CG methylation.
The lack of CMT homologs in animal genomes could account for the observation
that in contrast to plants, animals maintain primarily CG methylation. A cytosine DNA methyltransferase containing a chromodomain, Zea
methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is
similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with
C-terminal motifs characteristic of eukaryotic and prokaryotic DNA
methyltransferases. We used a reverse genetics approach to determine the
function of the Zmet2 gene. Plants homozygous for a Mutator transposable element
insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel
blot analysis of these plants with methylation-sensitive restriction enzymes and
bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only
at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric
sites in heterozygous or homozygous mutant plants. Our research shows that
chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences. Proper DNA methylation patterning requires the complementary processes of de
novo methylation (the initial methylation of unmethylated DNA sequences) and
maintece methylation (the faithful replication of preexisting methylation).
Arabidopsis has two types of methyltransferases with demonstrated maintece
activity: MET1, which maintains CpG methylation and is homologous to mammalian
DNMT1, and CHROMOMETHYLASE 3 (CMT3), which maintains CpNpG (N = A, T, C, or G)
methylation and is unique to the plant kingdom. Here we describe
loss-of-function mutations in the Arabidopsis DOMAINS REARRANGED METHYLASE (DRM)
genes and provide evidence that they encode de novo methyltransferases. drm1
drm2 double mutants retained preexisting CpG methylation at the endogenous FWA
locus but blocked de novo CpG methylation that is normally associated with FWA
transgene silencing. Furthermore, drm1 drm2 double mutants blocked de novo CpNpG
and asymmetric methylation and gene silencing of the endogenous SUPERMAN (SUP)
gene, which is normally triggered by an inverted SUP repeat. However, drm1 drm2
double mutants did not show reactivation of previously established SUPERMAN
epigenetic silenced alleles. Thus, drm mutants prevent the establishment but not
the maintece of gene silencing at FWA and SUP, suggesting that the DRMs
encode the major de novo methylation enzymes affecting these genes. BACKGROUND: Going from a gene sequence to its function in the context of a whole
organism requires a strategy for targeting mutations, referred to as reverse
genetics. Reverse genetics is highly desirable in the modern genomics era;
however, the most powerful methods are generally restricted to a few model
organisms. Previously, we introduced a reverse-genetic strategy with the
potential for general applicability to organisms that lack well-developed
genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method
uses chemical mutagenesis followed by screening for single-base changes to
discover induced mutations that alter protein function. TILLING was shown to be
an effective reverse genetic strategy by the establishment of a high-throughput
TILLING facility and the delivery of thousands of point mutations in hundreds of
Arabidopsis genes to members of the plant biology community.
RESULTS: We demonstrate that high-throughput TILLING is applicable to maize, an
important crop plant with a large genome but with limited reverse-genetic
resources currently available. We screened pools of DNA samples for mutations in
1-kb segments from 11 different genes, obtaining 17 independent induced
mutations from a population of 750 pollen-mutagenized maize plants. One of the
genes targeted was the DMT102 chromomethylase gene, for which we obtained an
allelic series of three missense mutations that are predicted to be strongly
deleterious.
CONCLUSIONS: Our findings indicate that TILLING is a broadly applicable and
efficient reverse-genetic strategy. We are establishing a public TILLING service
for maize modeled on the existing Arabidopsis TILLING Project. Tomato fruit cells are characterized by a strong increase in nuclear ploidy
during fruit development. Average ploidy levels increased to similar levels
(above 50C) in two distinct fruit tissues, pericarp and locular tissue. However,
ploidy profiles differed significantly between these two tissues suggesting a
tissue-specific control of endoreduplication in tomato fruit. To determine
possible relationships between endoreduplication and epigenetic mechanisms, the
methylation status of genomic DNA from pericarp and locular tissue of tomato
fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG
and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like
retrotransposon sequences during fruit growth. A sharp decrease of the global
DNA methylation level together with a reduction of methylation at the rDNA loci
was also observed in pericarp during fruit ripening. Inversely, no major
variation of DNA methylation either global or locus-specific, was observed in
locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely
to be triggered by the induction of endoreduplication in fruit tissues, but may
reflect tissue-specific ploidy profiles. Expression analysis of eight putative
tomato DNA methyltransferases encoding genes showed that one chromomethylase
(CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed
in the pericarp during fruit growth and could be involved in the locus-specific
increase of methylation observed at this developmental phase in the pericarp. During embryogenesis there is a major switch from dependence upon
maternally-deposited products to reliance on products of the zygotic genome. In
animals, this so-called maternal-to-zygotic transition occurs following a period
of transcriptional quiescence. Recently, we have shown that the early embryo in
Arabidopsis is also quiescent, a state inherited from the female gamete and
linked to specific patterns of H3K9 dimethylation and TERMINAL FLOWER2 (TFL2)
localization. We also demonstrated that CHROMOMETHYLASE 3 (CMT3) is required for
H3K9 dimethylation in the egg cell and for normal embryogenesis during the first
few divisions of the zygote. Subsequent analysis of CMT3 mutants points to a key
role in egg cell reprogramming by controlling silencing in both transposon and
euchromatic regions. A speculative model of the CMT3-induced egg cell silencing
is presented here, based on these results and current data from the literature
suggesting the potential involvement of small RNAs targeted to the egg cell, a
process conceptually similar to the division of labor described in the male
gametophyte for which we show that H3K9 modifications and TFL2 localization are
reminiscent of the female gametophyte. In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting
through a methylation-dependent epigenetic mechanism. Here, the effects of Cd
treatment on the DNA methylation patten are examined together with its effect on
chromatin reconfiguration in Posidonia oceanica. DNA methylation level and
pattern were analysed in actively growing organs, under short- (6 h) and long-
(2 d or 4 d) term and low (10 μM) and high (50 μM) doses of Cd, through a
Methylation-Sensitive Amplification Polymorphism technique and an
immunocytological approach, respectively. The expression of one member of the
CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by
qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission
electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an
up-regulation of CMT, indicating that de novo methylation did indeed occur.
Moreover, a high dose of Cd led to a progressive heterochromatinization of
interphase nuclei and apoptotic figures were also observed after long-term
treatment. The data demonstrate that Cd perturbs the DNA methylation status
through the involvement of a specific methyltransferase. Such changes are linked
to nuclear chromatin reconfiguration likely to establish a new balance of
expressed/repressed chromatin. Overall, the data show an epigenetic basis to the
mechanism underlying Cd toxicity in plants. DNA methylation and histone modification exert epigenetic control over gene
expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9
dimethylation (H3K9me2), but the mechanism underlying this relationship is
poorly understood. Here, we report multiple lines of evidence that CMT3
interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly
perfectly correlated with H3K9me2, and CMT3 stably associated with
H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2,
in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo
adjacent homology (BAH) and chromo domains. The structures reveal an aromatic
cage within both BAH and chromo domains as interaction interfaces that capture
H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to
nucleosomes and show a complete loss of CMT3 activity in vivo. Our study
establishes dual recognition of H3K9me2 marks by BAH and chromo domains and
reveals a distinct mechanism of interplay between DNA methylation and histone
modification. |
Is curcumin a phytochemical? | Yes, curcumin is a phytochemical derived from rhizome of turmeric Curcuma longa. | PURPOSE: Curcumin, a natural phytochemical, exhibits potent anticancer
activities. Here, we sought to determine the molecular mechanisms underlying the
cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC)
cells.
METHODS: MTT assay and annexin-V/PI staining were used to analyze the effects of
curcumin on the proliferation and apoptosis of A549 cells. The expression of
microRNA-21 in curcumin-treated A549 cells was measured by quantitative
real-time polymerase chain reaction assay. The protein level of phosphatase and
tensin homolog (PTEN), a putative target of microRNA-21, was determined by
Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN
small interfering RNA was performed to modulate the expression of microRNA-21
and PTEN under the treatment of curcumin.
RESULTS: Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis
in A549 cells. Curcumin treatment produced a dose-dependent and significant
(P < 0.05) suppression of microRNA-21 expression, compared to untreated A549
cells. Moreover, the protein level of PTEN, a putative target of microRNA-21,
was significantly elevated in curcumin-treated A549 cells, as determined by
Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN
small interfering RNA significantly (P < 0.05) reversed the growth suppression
and apoptosis induction by curcumin, compared to corresponding controls.
CONCLUSIONS: Our data suggest a novel molecular mechanism in which inhibition of
microRNA-21 and upregulation of PTEN mediate the anticancer activities of
curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic
benefits against this maligcy. BACKGROUND: Traumatic brain injury (TBI) initiates a neuroinflammatory cascade
that contributes to substantial neuronal damage and behavioral impairment, and
Toll-like receptor 4 (TLR4) is an important mediator of thiscascade. In the
current study, we tested the hypothesis that curcumin, a phytochemical compound
with potent anti-inflammatory properties that is extracted from the rhizome
Curcuma longa, alleviates acute inflammatory injury mediated by TLR4 following
TBI.
METHODS: Neurological function, brain water content and cytokine levels were
tested in TLR4⁻/⁻ mice subjected to weight-drop contusion injury. Wild-type (WT)
mice were injected intraperitoneally with different concentrations of curcumin
or vehicle 15 minutes after TBI. At 24 hours post-injury, the activation of
microglia/macrophages and TLR4 was detected by immunohistochemistry; neuronal
apoptosis was measured by FJB and TUNEL staining; cytokines were assayed by
ELISA; and TLR4, MyD88 and NF-κB levels were measured by Western blotting. In
vitro, a co-culture system comprised of microglia and neurons was treated with
curcumin following lipopolysaccharide (LPS) stimulation. TLR4 expression and
morphological activation in microglia and morphological damage to neurons were
detected by immunohistochemistry 24 hours post-stimulation.
RESULTS: The protein expression of TLR4 in pericontusional tissue reached a
maximum at 24 hours post-TBI. Compared with WT mice, TLR4⁻/⁻ mice showed
attenuated functional impairment, brain edema and cytokine release post-TBI. In
addition to improvement in the above aspects, 100 mg/kg curcumin treatment
post-TBI significantly reduced the number of TLR4-positive microglia/macrophages
as well as inflammatory mediator release and neuronal apoptosis in WT mice.
Furthermore, Western blot analysis indicated that the levels of TLR4 and its
known downstream effectors (MyD88, and NF-κB) were also decreased after curcumin
treatment. Similar outcomes were observed in the microglia and neuron co-culture
following treatment with curcumin after LPS stimulation. LPS increased TLR4
immunoreactivity and morphological activation in microglia and increased
neuronal apoptosis, whereas curcumin normalized this upregulation. The increased
protein levels of TLR4, MyD88 and NF-κB in microglia were attenuated by curcumin
treatment.
CONCLUSIONS: Our results suggest that post-injury, curcumin administration may
improve patient outcome by reducing acute activation of microglia/macrophages
and neuronal apoptosis through a mechanism involving the TLR4/MyD88/NF-κB
signaling pathway in microglia/macrophages in TBI. Colon cancer is the third most leading causes of death due to cancer worldwide
and the chemo drug 5-fluorouracil's (5-FU) applicability is limited due to its
non-specificity, low bioavailability and overdose. The efficacy of 5-FU in colon
cancer chemo treatment could be improved by oencapsulation and combinatorial
approach. In the present study curcumin (CUR), a known anticancer phytochemical,
was used in combination with 5-FU and the work focuses on the development of a
combinatorial omedicine based on 5-FU and CUR in N,O-carboxymethyl chitosan
oparticles (N,O-CMC NPs). The developed 5-FU-N,O-CMC NPs and CUR-N,O-CMC NPs
were found to be blood compatible. The in vitro drug release profile in pH 4.5
and 7.4 showed a sustained release profile over a period of 4 days. The combined
exposure of the oformulations in colon cancer cells (HT 29) proved the
enhanced anticancer effects. In addition, the in vivo pharmacokinetic data in
mouse model revealed the improved plasma concentrations of 5-FU and CUR which
prolonged up to 72 h unlike the bare drugs. In conclusion, the 5-FU and CUR
released from the N,O-CMC NPs produced enhanced anticancer effects in vitro and
improved plasma concentrations under in vivo conditions. Colorectal cancer remains the most prevalent maligcy in humans. The impact of
epigenetic alterations on the development of this complex disease is now being
recognized. The dynamic and reversible nature of epigenetic modifications makes
them a promising target in colorectal cancer chemoprevention and treatment.
Curcumin (CUR), the major component in Curcuma longa, has been shown as a potent
chemopreventive phytochemical that modulates various signaling pathways. Deleted
in lung and esophageal cancer 1 (DLEC1) is a tumor suppressor gene with reduced
transcriptional activity and promoter hypermethylation in various cancers,
including colorectal cancer. In the present study, we aimed to investigate the
inhibitory role of DLEC1 in anchorage-independent growth of the human colorectal
adenocarcinoma HT29 cells and epigenetic regulation by CUR. Specifically, we
found that CUR treatment inhibited colony formation of HT29 cells, whereas
stable knockdown of DLEC1 using lentiviral short hairpin RNA vector increased
cell proliferation and colony formation. Knockdown of DLEC1 in HT29 cells
attenuated the ability of CUR to inhibit anchorage-independent growth.
Methylation-specific polymerase chain reaction (MSP), bisulfite genomic
sequencing, and methylated DNA immunoprecipitation revealed that CUR decreased
CpG methylation of the DLEC1 promoter in HT29 cells after 5 days of treatment,
corresponding to increased mRNA expression of DLEC1. Furthermore, CUR decreased
the protein expression of DNA methyltransferases and subtypes of histone
deacetylases (HDAC4, 5, 6, and 8). Taken together, our results suggest that the
inhibitory effect of CUR on anchorage-independent growth of HT29 cells could, at
least in part, involve the epigenetic demethylation and up-regulation of DLEC1. With the idea that platinum compounds that bind with DNA differently than
cisplatin may be better-able to overcome platinum resistance in ovarian tumor,
the monofunctional platinum complex tris(imidazo(1,2-α)pyridine)
chloroplatinum(II) chloride (coded as LH6) has been synthesized and investigated
for its activity, alone and in combination with the phytochemicals curcumin and
quercetin, against human ovarian A2780, A2780(cisR) and A2780(ZD0473R) cancer
cell lines. LH6 is found to be more active than cisplatin against the resistant
cell lines and its bolus combinations with curcumin and quercetin are found to
produce more pronounced cell kill. Whereas platinum accumulation from cisplatin
is found to increase almost linearly with time, that from LH6 reaches a maximum
at 4 h and is somewhat lowered at 24 h. It is possible that the presence of
bulky hydrophobic imidazo (1,2-α-pyridine) ligand in LH6 facilitates its rapid
uptake through the cytoplasmic membrane. Lower platinum accumulation at 24 h
than at 4 h for LH6 can be seen to imply that efflux processes may be more
domit as the period of incubation is increased. When platinum-DNA binding
levels at 24 h are compared, cisplatin is found to be associated with the higher
level in the parent A2780 cell line and LH6 in the resistant A2780(cisR) cell
line, in line with greater activity of cisplatin in the parent cell line and
that of LH6 in the resistant cell line. If the observed in vitro activity of LH6
is confirmed in vivo, it can be seen to have the potential for development as
novel platinum based anticancer drug. Curcuma longa (Zingiberaceae) is known for its uses in medicine, cosmetics, food
flavouring and textile industries. The secondary metabolites of turmeric like
essential oil, oleoresin and curcumin are important for its multipurpose uses.
These traits of turmeric vary from place to place due to the influence of
environment, soil and agro-climatic conditions. Here, we analyzed turmeric from
different agroclimatic regions for influence of various factors on its growth
and yield of important phytochemicals. A high curcumin yielding cultivar i.e.,
Roma was collected from high altitude research station, Koraput (HARS) and
planted in nine agroclimatic regions of Odisha. Analysis of soil texture, pH,
organic carbon, micro and macro nutrients were done from all the studied zones
up to 2nd generation. Plants grown in their released station i.e., Eastern Ghat
High Land showed 5% of curcumin and were taken as control. Plants grown in
different agroclimatic zones showed a range of 1.4-5% of curcumin and 0.3-0.7%
of rhizome essential oil and 0.3-1% of leaf essential oil content. Gas
chromatography and mass spectra analysis showed tumerone and alpha phellandrene
as the major compounds in all the zones with 10-20% variation. The present study
will be immensely helpful for standardization and management of environmental
and ecological factors for high phytochemical yield in turmeric plant. |
What is Cerebral Cavernous Malformation? | Cerebral cavernous malformation (CCM) is a disease of the central nervous system causing hemorrhage-prone multiple lumen vascular malformations and very severe neurological consequences | BACKGROUND: Cerebral cavernous malformation is a vascular disease of the brain
causing headaches, seizures, and cerebral hemorrhage. Familial and sporadic
cases are recognized, and a gene causing familial disease has been mapped to
chromosome 7. Hispanic Americans have a higher prevalence of cavernous
malformation than do other ethnic groups, raising the possibility that affected
persons in this population have inherited the same mutation from a common
ancestor.
METHODS: We compared the segregation of genetic markers and clinical cases of
cavernous malformation in Hispanic-American kindreds with familial disease; we
also compared the alleles for markers linked to cavernous malformation in
patients with familial and sporadic cases.
RESULTS: All kindreds with familial disease showed linkage of cavernous
malformation to a short segment of chromosome 7 (odds supporting linkage,
4X10(10).1). Forty-seven affected members of 14 kindreds shared identical
alleles for up to 15 markers linked to the cavernous-malformation gene,
demonstrating that they had inherited the same mutation from a common ancestor.
Ten patients with sporadic cases also shared these same alleles, indicating that
they too had inherited the same mutation. Thirty-three asymptomatic carriers of
the disease gene were identified, demonstrating the variability and age
dependence of the development of symptoms and explaining the appearance of
apparently sporadic cases.
CONCLUSIONS: Virtually all cases of familial and sporadic cavernous malformation
among Hispanic Americans of Mexican descent are due to the inheritance of the
same mutation from a common ancestor. To classify the cerebral cavernous malformations and to investigate the natural
history of cavernous malformations according to the classification, 41 patients
with 61 cavernous malformations (40 cavernous malformations from 22 patients
treated with gamma knife surgery) were regularly followed up using magnetic
resoce (MR) imaging for a mean period of 25.5 months in treated cavernous
malformations and 20.7 months in untreated cavernous malformations,
respectively. Cavernous malformations were classified into four types: type I,
extralesional gross hemorrhage beyond cavernous malformation; type II, mixture
of subacute and chronic hemorrhage; type III, area of hemosiderin with small
central core; and type IV, area of hemosiderin deposition without central core.
Follow-up MR images were analyzed to evaluate changes in size, signal intensity,
rebleeding, and perilesional adverse reaction of irradiation. A total of 61
cavernous malformations including 17 in type I, 23 in type II, 10 in type III,
and 11 in type IV showed usual degradation of blood product in 22 cavernous
malformations, no change in shape and signal intensity in 31 cavernous
malformations, and eight cavernous malformations with rebleedings in the serial
MR images. In these eight cavernous malformations with rebleedings, six occurred
in type II and two in type III, but none in type I or IV. Rebleedings were more
frequent in type II than in other types (p = 0.044). Adverse reaction of
irradiation was observed in five of 22 patients treated with gamma knife
surgery. Although most cerebral cavernous malformations showed evolution of
hemorrhage or no change in size or shape on follow-up MR images, cerebral
cavernous malformations represented as mixture of subacute and chronic
hemorrhage with hemosiderin rim (type II) have a higher frequency to rebleed
than other types of cerebral cavernous malformations. Cerebral cavernous
malformations represented as hemosiderin deposition without central core (type
IV) have a lower tendency to rebleed than other types and do not need any
treatment. Most of the adverse reaction of irradiation after gamma knife surgery
around cavernous malformations are transient findings and are considered to be
perilesional edema. Mutations in Krev1 interaction trapped gene 1 (KRIT1) cause cerebral cavernous
malformation, an autosomal domit disease featuring malformation of cerebral
capillaries resulting in cerebral hemorrhage, strokes, and seizures. The
biological functions of KRIT1 are unknown. We have investigated KRIT1 expression
in endothelial cells by using specific anti-KRIT1 antibodies. By both microscopy
and coimmunoprecipitation, we show that KRIT1 colocalizes with microtubules. In
interphase cells, KRIT1 is found along the length of microtubules. During
metaphase, KRIT1 is located on spindle pole bodies and the mitotic spindle.
During late phases of mitosis, KRIT1 localizes in a pattern indicative of
association with microtubule plus ends. In anaphase, the plus ends of the
interpolar microtubules show strong KRIT1 staining and, in late telophase, KRIT1
stains the midbody remt most strongly; this is the site of cytokinesis where
plus ends of microtubules from dividing cells overlap. These results establish
that KRIT1 is a microtubule-associated protein; its location at plus ends in
mitosis suggests a possible role in microtubule targeting. These findings,
coupled with evidence of interaction of KRIT1 with Krev1 and integrin
cytoplasmic domain-associated protein-1 alpha (ICAP1 alpha), suggest that KRIT1
may help determine endothelial cell shape and function in response to cell-cell
and cell-matrix interactions by guiding cytoskeletal structure. We propose that
the loss of this targeting function leads to abnormal endothelial tube
formation, thereby explaining the mechanism of formation of cerebral cavernous
malformation (CCM) lesions. PURPOSE: Cerebral cavernous malformations can occur sporadically or are caused
by mutations in one of three identified genes. Cerebral cavernous malformations
often remain clinically silent until a mutation carrier suffers a stroke or
seizure. Presymptomatic genetic testing has been valuable to follow and manage
cerebral cavernous malformation mutation carriers. During routine diagnostic
testing, we identified a two base pair change in seven unrelated people of
Ashkenazi Jewish heritage. Because of the location of the variant beyond the
invariant splice donor sequence, the change was reported as a variant of unknown
significance. In this study, we determined whether this change was a
disease-causing mutation and whether it represents a founder mutation in the
Ashkenazi Jewish population.
METHODS: Transcripts arising from the normal and mutant alleles were examined by
reverse transcription-polymerase chain reaction from affected and unaffected
Ashkenazi Jewish cerebral cavernous malformation family members. A synthetic
splicing system using a chimeric exon was used to visualize the effects of the
change on splice donor site utilization.
RESULTS: The two base pair change in CCM2, c.30 + 5_6delinsTT, segregated with
affected status in the study families. Reverse transcription-polymerase chain
reaction revealed loss of the transcript allele that was in phase with the
mutation. The two base pair change, when tested in an in vitro synthetic
splicing system, altered splice donor site utilization. Resequencing of the
genomic region proximal and distal to the CCM2 gene mutation revealed a common
single-nucleotide polymorphism haplotype in affected individuals.
CONCLUSIONS: The two base pair change in CCM2, c.30 + 5_6delinsTT, disrupted
proper splice donor utilization leading to a degraded transcript. Single
nucleotide polymorphism haplotype analysis demonstrated that this mutation was
due to a founder in the Ashkenazi Jewish population. These data have the
potential to simplify genetic testing for cerebral cavernous malformation in the
Ashkenazi Jewish population. Cerebral cavernous malformation (CCM) is a common vascular disease in central
nervous system that frequently predisposes to stroke, seizure, and cerebral
hemorrhage. CCM lesions are characterized by dilated and leaky intracranial
capillaries composed of a thin layer of vascular endothelial cells with abnormal
subendothelial extracellular matrix. Despite the understanding that genetic
mutation of three CCM genes (CCM1, CCM2, and CCM3) results in hereditary CCM,
the molecular mechanism underlying vascular defects in CCM lesions remains
poorly understood. Recent studies have shown that integrin cytoplasmic
domain-associated protein-1 (ICAP-1, also known as integrin β1 binding protein1,
ITGB1BP), a cytoplasmic protein interacting with both β1 integrin subunit and
CCM1 protein (also known as Krit1), is implicated in vascular development.
Analysis of data on the biochemistry and cellular biology of ICAP-1 highlights
that bidirectional interaction of ICAP-1 with CCM1 and integrin might regulate
diverse pathological processes of CCM disorder. Specifically, emerging evidence
supports the hypothesized involvement of ICAP-1 in CCM pathogenesis through its
significant effect in attenuating excessive vascular growth, its indispensable
function in activating CCM1 protein, and its essential role in regulating
integrin functions. BACKGROUND: Cerebral cavernous malformations (CCM) are enlarged vascular lesions
affecting 0.1-0.5% of the population worldwide and causing hemorrhagic strokes,
seizures, and neurological deficits. Familial CCM type 1 (CCM1) is an autosomal
domit disease caused by mutations in the Krev Interaction Trapped 1
(KRIT1/CCM1) gene, and is characterized by multiple brain lesions whose number
and size increase with age. The number of lesions varies widely for unknown
reasons, even among carriers of similar ages with the same mutation. The purpose
of this study was to investigate whether cardiovascular (CV) risk factors
influence potential markers of familial CCM1 disease severity, such as lesion
count and history of intracerebral hemorrhage.
METHODS: We analyzed baseline data from 185 Hispanic subjects, enrolled in the
Brain Vascular Malformation Consortium study between June 2010 and March 2013.
All subjects were carriers of the founder Q455X 'Common Hispanic Mutation' (CHM)
in the KRIT1 gene, and had a clinical diagnosis of CCM or had an affected first-
or second-degree relative with CCM. We performed a cross-sectional study,
collecting detailed clinical information of CCM1-CHM subjects and cerebral
susceptibility-weighted magnetic resoce imaging to assess lesion count.
Linear or logistic regression analysis of log-lesion count or history of
intracerebral hemorrhage and CV risk factors (age, gender, obesity, diabetes,
hypertension, hyperlipidemia and smoking status) and related quantitative traits
(body mass index, glycosylated hemoglobin levels, blood pressure, lipids levels
and pack-years of cigarette smoking) was performed accommodating familial
clustering.
RESULTS: CCM1-CHM subjects were mainly female (63.8%) and symptomatic at
presentation (63.2%). Lesion count was highly variable (mean ± SD: 57.7 ± 110.6;
range: 0-713); 90% of CCM1-CHM subjects had multiple lesions at enrollment. Age
(p < 0.001) was positively correlated with lesion count and male gender (p =
0.035) was associated with a greater number of lesions. Obesity (p = 0.001) and
higher body mass index (p = 0.002) were associated with fewer lesions. No
association with hypertension was detected, however, systolic blood pressure (p
= 0.002) was associated with fewer lesions. No significant association with
lesion count was observed for diabetes, hyperlipidemia, smoking status or for
related quantitative traits. History of intracerebral hemorrhage was not
significantly associated with any CV risk factors, however, we found borderline
associations of hemorrhage with obesity (p = 0.062), systolic blood pressure (p
= 0.083) and pack-years of cigarette smoking (p = 0.055). After correction for
multiple testing, age and obesity remained significantly associated with lesion
count in CCM1-CHM subjects.
CONCLUSIONS: These results suggest that several CV risk factors explain some of
the variability in lesion count in Hispanic CCM1-CHM subjects. Although age,
gender, obesity, body mass index and systolic blood pressure may influence
familial CCM1 disease severity, further longitudinal studies in larger sample
sizes are essential to confirm these findings. Cerebral cavernous malformation (CCM) is a disease of the central nervous system
causing hemorrhage-prone multiple lumen vascular malformations and very severe
neurological consequences. At present, the only recommended treatment of CCM is
surgical. Because surgery is often not applicable, pharmacological treatment
would be highly desirable. We describe here a murine model of the disease that
develops after endothelial-cell-selective ablation of the CCM3 gene. We report
an early, cell-autonomous, Wnt-receptor-independent stimulation of β-catenin
transcription activity in CCM3-deficient endothelial cells both in vitro and in
vivo and a triggering of a β-catenin-driven transcription program that leads to
endothelial-to-mesenchymal transition. TGF-β/BMP signaling is then required for
the progression of the disease. We also found that the anti-inflammatory drugs
sulindac sulfide and sulindac sulfone, which attenuate β-catenin transcription
activity, reduce vascular malformations in endothelial CCM3-deficient mice. This
study opens previously unidentified perspectives for an effective
pharmacological therapy of intracranial vascular cavernomas. Cerebral cavernous malformations (CMs) are vascular malformations of the central
nervous system, which can be detected in the absence of any clinical symptoms.
Nodules and cysts with mixed signal intensity and a peripheral hemosiderin rim
are considered brain magnetic resoce imaging (MRI) findings typical of CMs. A
48-year-old man was admitted to our hospital because of abnormal MRI findings
without significant neurological symptoms. A cyst with an internal fluid-fluid
level was found in the left basal ganglia on the initial brain MRI. We decided
to observe the natural course of the asymptomatic lesion with serial MRI
follow-up. On MRI at the 5-month follow-up, the cystic mass was enlarged and
showed findings consistent with those of cystic CM. Surgical resection was
performed and the pathological diagnosis was CM. Our experience suggests that
the initial presentation of a CM can be a pure cyst and neurosurgeons should
consider the likelihood of CMs in cases of cystic cerebral lesions with
intracystic hemorrhage. |
Is Fanconi anemia presented as a genetically and clinically heterogeneous disease entity? | Fanconi anemia (FA), an autosomal recessive disorder characterized by a progressive pancytopenia associated with congenital anomalies and high predisposition to malignancies, is a genetically and clinically heterogeneous disease. At least eight complementation groups (FA-A to FA-H) have been identified | Fanconi anemia (FA) is an autosomal recessive disorder characterized by bone
marrow failure, cancer susceptibility, and a variety of developmental defects.
The disease is clinically heterogeneous; eight different complementation groups
(FA A-H) and, thus, genetic loci have been discovered. Two genes, FAA and FAC,
have been cloned. Disease-associated mutations have been detected and rapid
mutation screening makes possible the assignment of patients without resorting
to time-consuming cell fusion and complementation analysis. Amplification of
specific cDNAs from RNA followed by direct or indirect sequence analysis is a
standard method for mutation detection. During the course of such examinations
of the FAC gene, we have noted that frequently only one of the expressed alleles
is successfully amplified. This can lead to false assignment of patients to a
complementation group. As we report here, such cases can be rapidly clarified by
retroviral gene transfer and complementation analysis. Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight
genes on the basis of complementation groups (FAA to FAH). The analysis of the
FAA gene in patients suggested the existence of deletions, none of which have
thus far been characterized at the genomic level. A detailed restriction map of
the FAA gene with the fine localization of its 43 exons is reported in this
paper. We also describe the first two genomic deletions, one of 5.0 kb and
another of at least 120 kb. The former was likely the result of a recombination
between related Alu sequences. Since these interspersed repeats could generate
deletions and insertions by mispairing, rearrangements of this gene are a
possibility in those FA families in which FAA mutations have not been
identified. Fanconi anemia (FA), an autosomal recessive disorder characterized by a
progressive pancytopenia associated with congenital anomalies and high
predisposition to maligcies, is a genetically and clinically heterogeneous
disease. At least eight complementation groups (FA-A to FA-H) have been
identified. Previously, we studied mutations of the FANCA gene, responsible for
FA-A, and found pathogenic mutations in 12 of 15 unclassified Japanese FA
patients. Here, we further studied an additional 5 FA patients for sequence
alterations of the FANCA gene and found pathogenic mutations in 2 of them. We
further analyzed mutations of the FANCC and FANCG genes, responsible for FA-C
and FA-G, respectively, in the remaining 6 FA patients. Although there was no
alterations in the FANCC gene in these 6 patients, two novel mutations of the
FANCG gene, causing aberrant RNA splicing, were detected in 2 FA patients. One
was a base substitution from G to C of the invariant GT dinucleotides at the
splice donor site of intron 3, resulting in the skipping of exon 3, as well as
the skipping of exons 3 and 4. The other was a base substitution from C to T in
exon 8, creating a nonsense codon (Q356X). This mutation resulted in the
exclusion of a sequence of 18 nucleotides containing the mutation from the mRNA,
without affecting the splicing potential of either the authentic or the cryptic
splice donor site. Collectively, 14 of the 20 unclassified Japanese FA patients
belong to the FA-A group, 2 belong to the FA-G group, and none belongs to the
FA-C group. Fanconi anaemia (FA) is a genetically heterogeneous disease with at least eight
complementation groups (A-H). In the present study, we investigated the
molecular basis of the disease in 13 unrelated Israeli Jewish (non-Ashkenazi)
patients with FA. All 43 exons of the Fanconi anaemia A (FANCA) gene were
amplified from genomic DNA and screened for mutations by single-strand
conformation polymorphism and DNA sequencing. We identified four ethnic-specific
mutations: (1) 2172-2173insG (exon 24), the first 'Moroccan mutation': (2)
4275delT (exon 43), the second 'Moroccan mutation'; (3) 890-893del (exon 10),
the 'Tunisian mutation'; and (4) 2574C > G (S858R), the 'Indian mutation'. The
tetranucleotide CCTG motif, previously identified as a mutation hotspot in FANCA
and other human genes, was found in the vicinity of 2172-2173insG and
890-893del. According to our study, the four mutations account for the majority
(88%) of the FANCA alleles in the Israeli Jewish (non-Ashkenazi) FA population.
A screening of 300 Moroccan Jews identified three carriers of the first
'Moroccan mutation', but we did not find any carrier of the second 'Moroccan
mutation' among 140 Moroccan Jews, nor any carrier of the 'Tunisian mutation'
among 50 Tunisian Jews. Two 'Indian mutation' carriers were identified among 53
Indian Jews. All carriers within each ethnic group had the same haplotype,
suggesting a common founder for each mutation. Fanconi anemia is a rare autosomal recessive disorder characterized clinically
by congenital abnormalities, progressive bone marrow failure, and a
predisposition to maligcy. FA cells are sensitive to DNA cross-linking
agents. Complementation analysis of FA cells using somatic cell fusion has
facilitated the identification of eight complementation groups, suggesting that
FA is a genetically heterogeneous disorder. Six genes (FANCA, FANCC, FANCD2,
FANCE, FANGF, FANCG) have been cloned so far. The majority of affected patients
belong to FA group A. Of the 32 unrelated Israeli patients with FA that we
studied, 6 carried the FANCC mutations and 15 the FANCA mutations. Among the
Jewish patients, ethnic-related mutations were common. Recent cumulative
evidence suggests that the FA proteins are repair proteins. FANCC, FANCA and
FANCG bind and interact in a protein complex found in the cytoplasm and nucleus
of normal cells. FANCD2 exists in two isoforms; the long active form, FANCD2-L,
is absent from FA cells of all complementation groups. FANCD2 colocalizes with
BRCA1 in nuclear foci, probably as part of a large genomic surveillance complex.
Studies using FANCA and FANCC knockout mice suggest that bone marrow precursors
express interferon-gamma hypersensitivity and show progressive apoptosis. The
definition of the molecular basis of FA in many affected families now enables
prenatal diagnosis. Fanconi anemia (FA) is a genetically heterogeneous chromosomal instability
syndrome associated with multiple congenital abnormalities, aplastic anemia, and
cancer. We report that a deletion mutation in the FANCG gene
(c.637_643delTACCGCC) was present in 82% of FA patients in the black populations
of Southern Africa. These patients originated from South Africa, Swaziland,
Mozambique, and Malawi. The mutation was found on the same haplotype and was
present in 1% of controls from the black South African population. These data
indicate that the birth incidence of FA in this population is higher than 1 in
40 000, which is much higher than previously supposed, and suggest that the
FANCG deletion is an ancient founder mutation in Bantu-speaking populations of
sub-Saharan Africa. Diagnostic screening is now possible by means of a simple
DNA test. Fanconi anemia (FA) is a genetically heterogeneous, autosomal recessive disorder
characterized by pediatric bone marrow failure and congenital anomalies. The
effect of FA gene deficiency on hematopoietic development in utero remains
poorly described as mouse models of FA do not develop hematopoietic failure and
such studies cannot be performed on patients. We have created a human-specific
in vitro system to study early hematopoietic development in FA using a
lentiviral RNA interference (RNAi) strategy in human embryonic stem cells
(hESCs). We show that knockdown of FANCA and FANCD2 in hESCs leads to a
reduction in hematopoietic fates and progenitor numbers that can be rescued by
FA gene complementation. Our data indicate that hematopoiesis is impaired in FA
from the earliest stages of development, suggesting that deficiencies in
embryonic hematopoiesis may underlie the progression to bone marrow failure in
FA. This work illustrates how hESCs can provide unique insights into human
development and further our understanding of genetic disease. Among patients with bone marrow failure (BMF) syndrome, some are happened to
have underlying Fanconi anemia (FA), a genetically heterogeneous disease, which
is characterized by progressive pancytopenia and cancer susceptibility. Due to
heterogeneous nature of the disease, a single genetic test, as in vitro response
to DNA cross-linking agents, usually is not enough to make correct diagnosis.
The aim of this study was to evaluate whether measuring repair kinetics of
radiation-induced DNA double-strand breaks (DSBs) can distinguish Fanconi anemia
from other BMF patients. An early step in repair of DSBs is phosphorylation of
the histone H2AX, generating gamma-H2AX histone, which extends over mega
base-pair regions of DNA from the break site and is visualised as foci
(gamma-H2AX foci) with specific antibodies. The primary fibroblasts, established
from FA patients, were exposed to gamma-rays, a dose of 2 Gy ((60)Co), incubated
for up to 24 hours under repair-permissive conditions, and assayed for the level
of gamma-H2AX foci and apoptosis at different recovery times after the
treatment. Cell lines originating from FA patients displayed a significant delay
in the repair of radiation-induced DNA DSBs relative to non-FA bone marrow
failure (non-FA BMF) and control cell lines. The delay is especially evident at
recovery time of 24 hours, and is seen as about 8-fold increase of residual
gamma-H2AX foci compared to self-state before irradiation. The delay in repair
kinetics of FA cells represents the unique feature of FA cellular phenotype,
which should be exploited to distinguish FA cellular phenotype. Bednar tumor is a rare pigmented variation of dermatofibrosarcoma protuberans,
present in 1 to 5% of all patients with dermatofibrosarcoma protuberans. No
significant clinicopathologic differences exist between Bednar tumor and
conventional dermatofibrosarcoma protuberans apart from the presence of
scattered nonneoplastic pigmented dendritic cells in the former. Although most
dermatofibrosarcoma protuberans occur in adults, they may be rarely present at
birth. Fanconi anemia is a genetically heterogeneous chromosomal instability
syndrome, characterized by multiple congenital anomalies, progressive bone
marrow failure, and a predisposition to maligcy. We describe here a patient
with Fanconi anemia who had a congenital Bednar tumor. To our knowledge, this is
the first such patient described with both dermatofibrosarcoma protuberans and
Fanconi anemia. Fanconi anaemia (FA) is a rare, autosomal recessive, genetically complex, DNA
repair deficiency syndrome in man. Patients with FA exhibit a heterogeneous
spectrum of clinical features. The most significant and consistent phenotypic
characteristics are stem cell loss, causing progressive bone marrow failure and
sterility, diverse developmental abnormalities and a profound predisposition to
neoplasia. To date, 15 genes have been identified, biallelic disruption of any
one of which results in this clinically defined syndrome. It is now apparent
that all 15 gene products act in a common process to maintain genome stability.
At the molecular level, a fundamental defect in DNA repair underlies this
complex phenotype. Cells derived from FA patients spontaneously accumulate
broken chromosomes and exhibit a marked sensitivity to DNA-damaging
chemotherapeutic agents. Despite complementation analysis defining many
components of the FA DNA repair pathway, no direct link to DNA metabolism was
established until recently. First, it is now evident that the FA pathway is
required to make incisions at the site of damaged DNA. Second, a specific
component of the FA pathway has been identified that regulates nucleases
previously implicated in DNA interstrand crosslink repair. Taken together, these
data provide genetic and biochemical evidence that the FA pathway is a bona fide
DNA repair pathway that directly mediates DNA repair transactions, thereby
elucidating the specific molecular defect in human Fanconi anaemia. INTRODUCTION: Fanconi anemia (FA) is a genetically and phenotypically
heterogeneous inherited disease. Many groups have established FA registries. In
Tunisia, in collaboration with the Tunisian Fanconi Anemia Study Group (TFASG),
we set up the Tunisian Fanconi Anemia Registry (TFAR).
PATIENTS AND METHODS: We contacted all hematology and pediatrics departments to
include their FA patients diagnosed between January 1983 and December 2008. The
registry is available on the TFASG web site (www.fanconi-tunisie.net).
RESULTS: Sorting the files brought out 142 patients belonging to 118 families.
The mean age at diagnosis was 11 years. There was consanguinity in 86%,
malformative syndrome in 91%, and pancytopenia at diagnosis in 69%. Of 28
patients, 95% belonged to the FANCA group. Androgen treatment was given in 109
cases and genoidentical bone marrow transplantation (BMT) in 27 patients. The
diagnosis of a myelodysplastic syndrome was retained in 4%, acute leukemia in
6%, and a solid tumor in 2%. The median overall survival time in all patients is
17 years 5 months; it is significantly better in patients having received
allografts (p=0.01).
CONCLUSION: FA seems frequent in Tunisia, which is in part explained by the high
consanguinity and endogamy in this country. Hematologic impairment is still the
most frequent revealing circumstance of the disease. It is often severe or
moderate and requires androgen treatment or bone marrow transplantation. BMT
should be proposed to all patients with an HLA-compatible donor. Fanconi anemia (FA) is a heterogeneous disease characterized by spontaneous
chromosomal breaks and abnormal DNA repair. Major clinical problems in FA
include congenital abnormalities, endocrinopathies, early onset bone marrow
failure and increased risk of myelodysplastic syndrome, acute leukemia and solid
tumors. To date, 15 different genes have been shown to cause FA, all of which
have some role in DNA double-strand break repair. Very few strict
genotype-phenotype associations have been identified and clinical manifestations
vary widely from patient to patient, most likely due to modifier genes,
environment and chance effects. Hematopoietic stem cell transplantation is the
only proven cure for the hematopoietic manifestations of FA and aggressive
lifelong surveillance for solid tumors is essential. |
Which protein pathway is regulating SGK1-mediated phosphorylation of FOXO3a to control cell proliferation? | mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FOXO3a at Ser314. | The mechanistic target of rapamycin (mTOR) functions as a component of two large
complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth
and homeostasis. However, the molecular mechanisms by which mTOR controls cell
proliferation remain elusive. Here we show that the FoxO3a transcription factor
is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in
controlling cell proliferation. To dissect mTOR signaling, mTORC1 was
specifically inactivated by depleting p18, an essential anchor of mTORC1 on
lysosomes. mTORC1 inactivation caused a marked retardation of cell
proliferation, which was associated with upregulation of cyclin-dependent kinase
inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a
was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314,
which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation
induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1),
the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated
at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1
overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1
knockdown induced CDKI expression in wild-type cells, resulting in the
suppression of cell proliferation. These results suggest that mTORC1, in
coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene
expression and SGK1-mediated phosphorylation of FoxO3a at Ser314. |
Which drugs have been found effective for the treatment of chordoma? | Established chordoma cell lines, and patient-derived primary cell cultures, as well as chordoma tumors in vivo were found to be sensitive to treatment with bortezomib, vincristine, doxorubicin, etoposide, cisplatin, fludarabine and SD-1029 Stat3 inhibitor. Moreover, percutaneous intratumoral injection with pingyangmycin lipiodol emulsion was shown to be effective against chordoma. It should be stressed that combination treatment with the use of the above drugs was always able to increase the therapeutic potency. | A major obstacle in the effective treatment of chordoma is that there are no
identifiable biomarkers capable of predicting prognosis. Recent research has
indicated that signal transducers and activators of transcription (Stat3) may be
an important prognostic marker in some cancers, but its role in chordoma tumors
has not been elucidated. In this study, the expression of Stat3 was evaluated in
chordoma tissue microarray that contains 70 chordoma samples. Cells in the
tissue microarray showed nuclear staining for phosphorylated Stat3 in all
instances. The level of phosphorylated Stat3 expression correlated with the
survival and severity of the disease. Three chordoma cell lines were exposed to
SD-1029, a novel inhibitor of Stat3 activation. MTT assay showed that the growth
of all chordoma cell lines was inhibited by SD-1029. The expression of Stat3
signaling cascade was inhibited in all chordoma cell lines after treatment with
SD-1029. The cytotoxicity of the combination of SD-1029 and chemotherapeutic
drugs is significantly better than either agent alone. Phosphorylation of Stat3
in chordoma cells in vitro and cellular proliferation in three-dimensional
culture were inhibited by SD-1029. In conclusion, the Stat3 pathway is
constitutively activated in chordomas and the level of expression may serve as a
predictor for prognosis. Blockade of the Stat3 pathway represents a potential
strategy for future treatment. This study describes fluoroscopy-guided percutaneous intratumoral injection
therapy (PIIT) with a pingyangmycin lipiodol emulsion in the management of
recurrent sacrococcygeal chordomas after surgical excision. Seven patients
underwent a total of 22 treatment sessions (3-4 sessions per patient); treatment
responses were evaluated clinically, and lesion size was determined using
computed tomography (CT). Over 10-26 months of follow-up, tumor sizes and visual
analogue scale (VAS) scores of all patients were decreased. No patients had
complications during the follow-up period. Preliminary results showed that PIIT
with pingyangmycin lipiodol emulsion under fluoroscopic guidance is effective
and safe and may be considered as a treatment option. OBJECT: Chordoma is a rare type of maligt bone tumor and is known to arise
from the remts of the notochord. Resistance to chemotherapy makes the
treatment of chordoma difficult; therefore, new approaches need to be developed
to cure this disease. Differentiation therapy, using various differentiating
agents, is attracting oncologists as a common therapeutic method to treat other
tumors. Based on forcing cells to mature into other lineages, differentiation
therapy might be an available method to treat chordomas in addition to
conventional therapies.
METHODS: In this study a chordoma cell line, U-CH1, was exposed to several
chemotherapeutic agents including vincristine, doxorubicin, cisplatin,
etoposide, fludarabine, methotrexate, nilotinib, and imatinib mesylate under
appropriate conditions. The first group of U-CH1 cells was exposed to drugs only
and the second group of cells was exposed to the simultaneous treatment of 1 μM
all-trans retinoic acid (ATRA) and chemotherapeutic agents in differentiation
therapy. The efficacy of the differentiation method was assessed by measuring
the viability of U-CH1 cells.
RESULTS: Vincristine, doxorubicin, etoposide, cisplatin, and fludarabine, each
at a concentration of 10 μM, decreased the number of chordoma cells when given
alone down to 11%, 0%, 30%, 67%, and 3%, respectively. Etoposide and cisplatin,
each at a concentration of 10 μM, reduced the percentage of viable chordoma
cells in a more effective way when given with 1 μM ATRA simultaneously, reducing
the number of viable cells to 14% and 9%, respectively. On the other hand,
imatinib and nilotinib, each at a concentration of 3 μM, as well as 10 μM
methotrexate, showed no decrease in the number of cancer cells.
CONCLUSIONS: The results suggest that chordoma cells may be treated using the
differentiation method in a more effective way than when they are treated with
chemotherapeutic agents alone. This new approach may be an alternative method to
conventional therapies in the treatment of chordoma. Chordoma is a rare, slow growing maligt tumor arising from remts of the
fetal notochord. Surgery is the first choice for chordoma treatment, followed by
radiotherapy, although postoperative complications remain significant.
Recurrence of the disease occurs frequently due to the anatomy of the tumor
location and violation of the tumor margins at the initial surgery. Currently,
there are no effective drugs available for patients with chordoma. Due to the
rarity of the disease, there is limited opportunity to test agents in clinical
trials and no concerted effort to develop agents for chordoma in the
pharmaceutical industry. To rapidly and efficiently identify small molecules
that inhibit chordoma cell growth, we screened the NCGC Pharmaceutical
Collection (NPC) containing approximately 2800 clinically approved and
investigational drugs at 15 different concentrations in chordoma cell lines,
U-CH1 and U-CH2. We identified a group of drugs including bortezomib, 17-AAG,
digitoxin, staurosporine, digoxin, rubitecan, and trimetrexate that inhibited
chordoma cell growth, with potencies from 10 to 370 nM in U-CH1 cells, but less
potently in U-CH2 cells. Most of these drugs also induced caspase 3/7 activity
with a similar rank order as the cytotoxic effect on U-CH1 cells. Cantharidin,
digoxin, digitoxin, staurosporine, and bortezomib showed similar inhibitory
effect on cell lines and 3 primary chordoma cell cultures. The combination
treatment of bortezomib with topoisomerase I and II inhibitors increased the
therapeutic potency in U-CH2 and patient-derived primary cultures. Our results
provide information useful for repurposing currently approved drugs for chordoma
and potential approach of combination therapy. |
Which components of the stress granules are known to be related to motor neuron degeneration in Amyotrophic Lateral Sclerosis? | Of note, both ALS and FTD are characterized by pathological inclusions, where some well-known SG markers localize with the ALS related proteins TDP-43 and FUS. | Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder consisting
of progressive loss of motor neurons. TDP-43 has been identified as a component
of ubiquitin-immunoreactive inclusions of motor neurons in ALS. We focused on
the diffuse cytoplasmic TDP-43 immunoreactivity in ALS neurons, and
quantitatively assessed it in comparison with skein/round TDP-43 and ubiquitin
immunostaining in motor neurons of 30 sporadic ALS cases. The percentage of
spinal motor neurons with cytoplasmic TDP-43 immunoreactivity was higher than
that of ubiquitin-immunoreactive ones. The percentage of TDP-43-positive motor
neurons was independent of neuron counts in anterior horns, while the percentage
of ubiquitinated neurons was inversely correlated. Aiming to define the
cytosolic localization of TDP-43, the immunoblot analysis of spinal cord and
frontal cortex showed that full-length TDP-43, the 45 kDa form and ubiquitinated
TDP-43 are found in the soluble inclusion-free fraction. The present data
suggest that delocalization, accumulation and ubiquitination of TDP-43 in the
cytoplasm of motor neurons are early dysfunctions in the cascade of the events
leading to motor neuron degeneration in ALS, preceding the formation of
insoluble inclusion bodies. Being cytoplasmic accumulation an ongoing event
during the course of the illness, a therapeutic approach to this incurable
disease can be envisaged. Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease
characterized by progressive motor neuron degeneration in association with
neurofilament (NF) aggregate formation. This process is accompanied by an
alteration in the stoichiometry of NF subunit protein expression such that the
steady state levels of the low molecular weight NF (NFL) mRNA levels are
selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and
mutant SOD1 can function as NFL mRNA 3'UTR binding proteins that directly affect
the stability of NFL transcripts. In this study, we demonstrate that the
interaction of TDP-43 with the NFL mRNA 3' UTR involves ribonucleotide (UG)
motifs present on stem loops of the 3'UTR as well as the RRM1 and RRM2 motifs of
TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA
stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1
co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA
transport granules (Staufen immunoreactive) in both control and ALS spinal motor
neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing
bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons
than in controls and demonstrated strong co-localization with TDP-43. Using
RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered
to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA
processing is fundamentally altered in ALS spinal motor neurons to favour
compartmentalization within both stress granules and P-bodies, and that TDP-43
plays a fundamental role in this process. OBJECTIVE: The fused in sarcoma/translated in liposarcoma (FUS/TLS) protein was
recently identified as a cause of familial amyotrophic lateral sclerosis (ALS),
as well as a major component of the inclusion bodies found in subtypes of
frontotemporal lobar degeneration (FTLD). These diseases now are collectively
known as the novel clinical spectrum, FUS proteinopathy. ALS-linked mutations of
FUS are clustered in the C-terminal region; however, the molecular properties of
mutant FUS remain unclear. To gain insight into the pathogenesis of FUS
proteinopathy, we examined the biochemical and cellular characteristics of
mutant FUS in expressing cells.
METHODS AND RESULTS: Expression of ALS-linked FUS mutations resulted in their
assembly into cytoplasmic stress granules (SGs), cellular structures that
package mRNA and RNA-binding proteins during cell stress. A deletion mutant
series revealed that the C-terminal region in FUS is critical for nuclear
retention via Ran guanosine triphosphatase-dependent transport machinery. A
parallel study of subcellular distribution revealed that ALS-linked mutants
additively disturb the function of the C-terminus for nuclear traffic, resulting
in cytoplasmic accumulation and the formation of SGs.
INTERPRETATION: This study demonstrates that mutant FUS, which is missing the
nuclear traffic activity of the C-terminus, is dislocated to cytoplasm and
assembled into SGs, indicating that disruption of translational regulation and
metabolism of mRNA via inappropriate/excessive SGs may be crucial for FUS
proteinopathies. Our findings provide new biological and pathological insights
into the FUS protein that should help our understanding of the pathogenesis of
ALS/FTLD. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron
disease with no effective long-term treatment options. Recently, TDP-43 has been
identified as a key protein in the pathogenesis of some cases of ALS. Although
the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has
been shown to accumulate in RNA stress granules (SGs) in cell models and in
spinal cord tissue from ALS patients. The SG association may be an early
pathological change to TDP-43 metabolism and as such a potential target for
therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of
mitochondrial activity can be inhibited by modulation of cellular kinase
activity. We have also found that treatment of cells and animal models of
neurodegeneration, including an ALS model, with bioavailable
bis(thiosemicarbazonato)copper(II) complexes (Cu(II)(btsc)s) can modulate kinase
activity and induce neuroprotective effects. In this study we examined the
effect of diacetylbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(atsm)) and
glyoxalbis(-methylthiosemicarbazonato)copper(II) (Cu(II)(gtsm)) on
TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the
Cu(II)(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs
induced by paraquat. The Cu(II)(btsc)s protected neurons from paraquat-mediated
cell death. These effects were associated with inhibition of ERK
phosphorylation. Co-treatment of cultures with either Cu(II)(atsm) or an ERK
inhibitor, PD98059 both prevented ERK activation and blocked formation of
TDP-43-and HuR-positive SGs. Cu(II)(atsm) treatment or ERK inhibition also
prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a
link between prolonged ERK activation and abnormal ubiquitin metabolism in
paraquat stress and inhibition by Cu. Moreover, Cu(II)(atsm) reduced
accumulation of C-terminal (219-414) TDP-43 in transfected SH-SY5Y cells. These
results demonstrate that Cu(II)(btsc) complexes could potentially be developed
as a neuroprotective agent to modulate neuronal kinase function and inhibit
TDP-43 aggregation. Further studies in TDP-43 animal models are warranted. Mutations in the gene encoding Fused in Sarcoma (FUS) cause amyotrophic lateral
sclerosis (ALS), a fatal neurodegenerative disorder. FUS is a predomitly
nuclear DNA- and RNA-binding protein that is involved in RNA processing. Large
FUS-immunoreactive inclusions fill the perikaryon of surviving motor neurons of
ALS patients carrying mutations at post-mortem. This sequestration of FUS is
predicted to disrupt RNA processing and initiate neurodegeneration. Here, we
demonstrate that C-terminal ALS mutations disrupt the nuclear localizing signal
(NLS) of FUS resulting in cytoplasmic accumulation in transfected cells and
patient fibroblasts. FUS mislocalization is rescued by the addition of the
wild-type FUS NLS to mutant proteins. We also show that oxidative stress
recruits mutant FUS to cytoplasmic stress granules where it is able to bind and
sequester wild-type FUS. While FUS interacts with itself directly by
protein-protein interaction, the recruitment of FUS to stress granules and
interaction with PABP are RNA dependent. These findings support a two-hit
hypothesis, whereby cytoplasmic mislocalization of FUS protein, followed by
cellular stress, contributes to the formation of cytoplasmic aggregates that may
sequester FUS, disrupt RNA processing and initiate motor neuron degeneration. FET family proteins consist of fused in sarcoma/translocated in liposarcoma
(FUS/TLS), Ewing's sarcoma (EWS), and TATA-binding protein-associated factor 15
(TAF15). Mutations in the copper/zinc superoxide dismutase (SOD1), TAR
DNA-binding protein 43 (TDP-43), and FET family proteins are associated with the
development of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative
disease. There is currently no cure for this disease and few effective
treatments are available. Epidemiological studies indicate that the consumption
of tea is associated with a reduced risk of developing neurodegenerative
diseases. The results of this study revealed that components of a pu-erh tea
extract (PTE) interacted with FET family proteins but not with TDP-43 or SOD1.
PTE induced the degradation of FET family proteins but had no effects on TDP-43
or SOD1. The most frequently occurring ALS-linked FUS/TLS mutant protein, R521C
FUS/TLS, was also degraded in the presence of PTE. Furthermore, ammonium
chloride, a lysosome inhibitor, but not lactacystin, a proteasome inhibitor,
reduced the degradation of FUS/TLS protein by PTE. PTE significantly reduced the
incorporation of R521C FUS/TLS into stress granules under stress conditions.
These findings suggest that PTE may have beneficial health effects, including
preventing the onset of FET family protein-associated neurodegenerative diseases
and delaying the progression of ALS by inhibiting the cytoplasmic aggregation of
FET family proteins. Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, have been
associated with familial amyotrophic lateral sclerosis (fALS), which is a fatal
neurodegenerative disease that causes progressive muscular weakness and has
overlapping clinical and pathologic characteristics with frontotemporal lobar
degeneration. However, the role of autophagy in regulation of FUS-positive
stress granules (SGs) and aggregates remains unclear. We found that the
ALS-linked FUS(R521C) mutation causes accumulation of FUS-positive SGs under
oxidative stress, leading to a disruption in the release of FUS from SGs in
cultured neurons. Autophagy controls the quality of proteins or organelles;
therefore, we checked whether autophagy regulates FUS(R521C)-positive SGs.
Interestingly, FUS(R521C)-positive SGs were colocalized to RFP-LC3-positive
autophagosomes. Furthermore, FUS-positive SGs accumulated in atg5(-/-) mouse
embryonic fibroblasts (MEFs) and in autophagy-deficient neurons. However,
FUS(R521C) expression did not significantly impair autophagic degradation.
Moreover, autophagy activation with rapamycin reduced the accumulation of
FUS-positive SGs in an autophagy-dependent manner. Rapamycin further reduced
neurite fragmentation and cell death in neurons expressing mutant FUS under
oxidative stress. Overall, we provide a novel pathogenic mechanism of ALS
associated with a FUS mutation under oxidative stress, as well as therapeutic
insight regarding FUS pathology associated with excessive SGs. TDP-43 is an RNA-binding protein linked to amyotrophic lateral sclerosis (ALS)
that is known to regulate the splicing, transport, and storage of specific mRNAs
into stress granules. Although TDP-43 has been shown to interact with
translation factors, its role in protein synthesis remains unclear, and no in
vivo translation targets have been reported to date. Here we provide evidence
that TDP-43 associates with futsch mRNA in a complex and regulates its
expression at the neuromuscular junction (NMJ) in Drosophila. In the context of
TDP-43-induced proteinopathy, there is a significant reduction of futsch mRNA at
the NMJ compared with motor neuron cell bodies where we find higher levels of
transcript compared with controls. TDP-43 also leads to a significant reduction
in Futsch protein expression at the NMJ. Polysome fractionations coupled with
quantitative PCR experiments indicate that TDP-43 leads to a futsch mRNA shift
from actively translating polysomes to nontranslating ribonuclear protein
particles, suggesting that in addition to its effect on localization, TDP-43
also regulates the translation of futsch mRNA. We also show that futsch
overexpression is neuroprotective by extending life span, reducing TDP-43
aggregation, and suppressing ALS-like locomotor dysfunction as well as NMJ
abnormalities linked to microtubule and synaptic stabilization. Furthermore, the
localization of MAP1B, the mammalian homolog of Futsch, is altered in ALS spinal
cords in a manner similar to our observations in Drosophila motor neurons.
Together, our results suggest a microtubule-dependent mechanism in motor neuron
disease caused by TDP-43-dependent alterations in futsch mRNA localization and
translation in vivo. Stress granules (SGs) are RNA-containing cytoplasmic foci formed in response to
stress exposure. Since their discovery in 1999, over 120 proteins have been
described to be localized to these structures (in 154 publications). Most of
these components are RNA binding proteins (RBPs) or are involved in RNA
metabolism and translation. SGs have been linked to several pathologies
including inflammatory diseases, cancer, viral infection, and neurodegenerative
diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia
(FTD). In ALS and FTD, the majority of cases have no known etiology and exposure
to external stress is frequently proposed as a contributor to either disease
initiation or the rate of disease progression. Of note, both ALS and FTD are
characterized by pathological inclusions, where some well-known SG markers
localize with the ALS related proteins TDP-43 and FUS. We propose that TDP-43
and FUS serve as an interface between genetic susceptibility and environmental
stress exposure in disease pathogenesis. Here, we will discuss the role of
TDP-43 and FUS in SG dynamics and how disease-linked mutations affect this
process. |
Is there evidence for de novo genesis of enhancers in vertebrates? | Yes. | Evolutionary innovation relies partially on changes in gene regulation. While a
growing body of evidence demonstrates that such innovation is generated by
functional changes or translocation of regulatory elements via mobile genetic
elements, the de novo generation of enhancers from non-regulatory/non-mobile
sequences has, to our knowledge, not previously been demonstrated. Here we show
evidence for the de novo genesis of enhancers in vertebrates. For this, we took
advantage of the massive gene loss following the last whole genome duplication
in teleosts to systematically identify regions that have lost their coding
capacity but retain sequence conservation with mammals. We found that these
regions show enhancer activity while the orthologous coding regions have no
regulatory activity. These results demonstrate that these enhancers have been de
novo generated in fish. By revealing that minor changes in non-regulatory
sequences are sufficient to generate new enhancers, our study highlights an
important playground for creating new regulatory variability and evolutionary
innovation. |
Which could be some of the possible causes of hypersomnia? | Sleep-wake disturbances (SWD) with hypersomnia are common after traumatic brain injury (TBI), with decreased CSF levels of hypocretin-1, a wake-promoting neurotransmitter, in cases of sleep apnea, as well as in up to half the patients with dementia, particularly in vascular dementia, Korsakow syndrome, Parkinson's disease, and depression, where the alteration of sleep architecture may be pronounced, whereas in Alzheimer's disease prominent hypersomnolence or insomnia is typically only found in later stages of the diseases. | A clinically relevant sleep-wake disturbance is found in up to half the patients
with dementia, and the sundowning agitation is a common cause of
institutionalisation of demented geriatric patients. The circadian rhythm of
demented patients is levelled off with increased daytime sleep and disrupted
night sleep. Particularly in vascular dementia, Korsakow syndrome, Parkinson's
disease, and depression the alteration of sleep architecture may be pronounced,
whereas in Alzheimer's disease prominent hypersomnolence or insomnia is
typically only found in later stages of the diseases. Greatly increased daytime
sleepiness or striking insomnia at the very beginning of suspected dementia
should thus prompt the search for other, possibly treatable causes of dementia.
Neuropathological and neurophysiological studies support the hypothesis of a
deteriorated hypothalamic suprachiasmatic nucleus (harbouring the biological
clock) as a cause for the deranged circadian sleep-wake system in dementia.
Management of sundowning behaviour includes restriction of daytime sleep,
exposure to bright lights, and social interaction schedules during the day. The
benzodiazepines and analogues usually not being sufficiently effectual, low
doses of mild neuroleptics are often needed. Whether recent reports on efficacy
of melatonin in elderly insomniacs also apply to demented patients is yet
uncertain. The careful search and treatment of possible extracerebral
physiologic factors causing reversible hypersomnia or insomnia is an important
requisite. Polysomnographic studies are needed to recognise treatable sleep
disturbance which could deteriorate or mimic dementia and sundowning.
Particularly, a sleep-apnea-hypopnea syndrome must be searched for at the
beginning of a suspected dementia, when successful treatment is still possible.
Sleep studies should also identify periodic leg movements of sleep with restless
legs and/or increased daytime sleepiness, and hyperkinetic parasomnias such as
REM sleep behaviour disorder which may complicate or imitate sundowning. Hypersomnia (excessive sleepiness) refers to an increased sleep propensity with
a subjective craving for sleep, involuntary naps and "sleep attacks" during the
day and/or prolonged nighttime sleep with sleep drunkeness. Excessive sleepiness
should be separated from fatigue and lack of energy associated with a variety of
medical and psychiatric diseases. Hypersomnia is reported by 2-5% of the adult
population and can lead to poor work, accidents and neuropsychiatric
disturbances. Sleep apnea syndrome (SAS), narcolepsy, chronic sleep deprivation
(insufficiency), and restless legs/periodic limb movements in sleep syndrome
(RLS/PLMS) represent the most common causes of hypersomnia. The diagnosis of
these conditions can often be suspected on clinical grounds. However, an
overnight polysomnography and a multiple sleep latency test are often
additionally required for definite diagnosis. Treatment options include nasal
CPAP for SAS, stimulants for narcolepsy, sleep prolongation for sleep
insufficiency, and dopaminergic agents for RLS/PLMS. When sleepiness is excessive, undesirable, inappropriate or unexplained, it
often indicates a clinical disorder that is generically termed hypersomnia. One
of the leading causes of hypersomnia is sleep apnea. We present the case of a
44-year-old woman with a history of bipolar spectrum disorder and epilepsy who
initially showed evidence of hypersomnia. The hypersomnia settled with changes
to her medication, but the patient was subsequently found to have severe
obstructive sleep apnea. The relation between the patient's medication and sleep
apnea is discussed, and the possible respiratory-suppressant effects of chronic
barbiturate treatment are considered. The role of other evoking factors within
the context of this case and the mechanisms by which drug interactions and
psychotropic treatment may worsen, obscure or perpetuate sleep apnea are also
examined. The discovery that hypocretins are involved in narcolepsy, a disorder associated
with excessive daytime sleepiness, cataplexy, and unusually rapid transitions to
rapid eye movement sleep, opens a new field of investigation in the area of
disorders of sleep and activation. Hypocretin-1 (hcrt-1) and hypocretin-2
(hcrt-2) (also called orexin-A and orexin-B) are newly discovered neuropeptides
processed from a common precursor. Hypocretin containing cells are located
exclusively in the lateral hypothalamus, with widespread projections within the
central nervous system. The role of the hypocretin system in other disorders
causing excessive daytime sleepiness is more uncertain. This study reports the
findings of a prospective study measuring cerebrospinal fluid concentrations of
hypocretin-1 and hypocretin-2 in HLA DQB1*0602 positive narcolepsy with
cataplexy, monosymptomatic narcolepsy, and primary hypersomnia. The results
confirmed the previous observations, that hcrt-1 is deficient in narcolepsy and
for the first time report very low levels of hcrt-1 in primary hypersomnia. It
is also reported for the first time that there is a generalised defect in hcrt-2
transmission in all three of these clinical entities compared with controls. OBJECTIVE: To determine the role of CSF hypocretin-1 in narcolepsy with and
without cataplexy, Kleine-Levin syndrome (KLS), idiopathic and other
hypersomnias, and several neurological conditions.
PATIENTS: 26 narcoleptic patients with cataplexy, 9 narcoleptic patients without
cataplexy, 2 patients with abnormal REM-sleep-associated hypersomnia, 7 patients
with idiopathic hypersomnia, 2 patients with post-traumatic hypersomnia, 4
patients with KLS, and 88 patients with other neurological disorders.
RESULTS: 23 patients with narcolepsy-cataplexy had low CSF hypocretin-1 levels,
while one patient had a normal hypocretin level (HLA-DQB1*0602 negative) and the
other two had intermediate levels (familial forms). One narcoleptic patient
without cataplexy had a low hypocretin level. One patient affected with
post-traumatic hypersomnia had intermediate hypocretin levels. The KLS patients
had normal hypocretin levels while asymptomatic, but one KLS patient (also
affected with Prader-Willi syndrome) showed a twofold decrease in hypocretin
levels during a symptomatic episode. Among the patients without hypersomnia, two
patients with normal pressure hydrocephalus and one with unclear central vertigo
had intermediate levels.
CONCLUSION: Low CSF hypocretin-1 is highly specific (99.1%) and sensitive
(88.5%) for narcolepsy with cataplexy. Hypocretin ligand deficiency appears not
to be the major cause for other hypersomnias, with a possible continuum in the
pathophysiology of narcolepsy without cataplexy and idiopathic hypersomnia.
However, partial hypocretin lesions without low CSF hypocretin-1 consequences
cannot be definitely excluded in those disorders. The existence of normal
hypocretin levels in narcoleptic patients and intermediate levels in other rare
aetiologies needs further investigation, especially for KLS, to establish the
functional significance of hypocretin neurotransmission alterations. L'hypersomnie est une plainte de somnolence diurne excessive qui affecte entre 4
et 6 % de la population générale et influe sur la vie quotidienne du patient. Le
diagnostic étiologique de l'hypersomnie bénéficie d'une panoplie de
questionnaires et de tests qui explorent la qualité de l'éveil et du sommeil du
patient (entretien dirigé, questionnaires, agenda de sommeil, polysomnographie,
test de latence multiple du sommeil, test de maintien de l'éveil) et de tests
psychomoteurs capables d'établir le retentissement de la somnolence excessive
sur les performances mentales et l'équilibre psychique du patient (par exemple,
test de vigilance psychomotrice, test de résistance au sommeil d'Oxford, test
d'Osier, etc.). Les différentes catégories d'hypersomnie sont détaillées selon
la classification conventionnelle actuelle : narcolepsie, hypersomnie
idiopathique, hypersomnie récurrente, syndrome d'insuffisance de sommeil,
somnolence liée aux médicaments et aux toxiques, hypersomnie associée à des
troubles psychiatriques, hypersomnie associée à des troubles neurologiques,
hypersomnie posttraumatique, hypersomnie liée à un syndrome infectieux (en
insistant sur les différences entre les infections virales et bactériennes, par
rapport aux infections parasitaires telles que la maladie du sommeil),
hypersomnie associée à des maladies métaboliques ou endocriniennes, hypersomnie
associée aux mouvements périodiques des membres et au syndrome des jambes sans
repos, et hypersomnie liée aux syndromes d'apnées et d'hypopnées au cours du
sommeil. Sleep-wake disturbances (SWD) are common after traumatic brain injury (TBI). In
acute TBI, we recently found decreased CSF levels of hypocretin-1, a
wake-promoting neurotransmitter. In the present study, we aimed to delineate the
frequency and clinical characteristics of post-traumatic SWD, to assess CSF
hypocretin-1 levels 6 months after TBI, and to identify risk factors for
posttraumatic SWD. A total of 96 consecutive patients were enrolled within the
first 4 days after TBI. Six months later, out of 76 TBI patients, who did not
die and who did not move to foreign countries, we included 65 patients (86%, 53
males, mean age 39 years) in our study. Patients were examined using interviews,
questionnaires, clinical examinations, computed tomography of the brain,
laboratory tests (including CSF hypocretin-1 levels, and HLA typing),
conventional polysomnography, maintece of wakefulness and multiple sleep
latency tests (MSLT) and actigraphy. Potential causes of post-traumatic SWD were
assessed according to international criteria. New-onset sleep-wake disturbances
following TBI were found in 47 patients (72%): subjective excessive daytime
sleepiness (EDS; defined by the Epworth Sleepiness Scale > or = 10) was found in
18 (28%), objective EDS (as defined by mean sleep latency < 5 min on MSLT) in 16
(25%), fatigue (daytime tiredness without signs of subjective or objective EDS)
in 11 (17%), post-traumatic hypersomnia 'sensu strictu' (increased sleep need of
> or = 2 h per 24 h compared to pre-TBI) in 14 (22%) patients and insomnia in 3
patients (5%). In 28 patients (43% of the study population), we could not
identify a specific cause of the post-traumatic SWD other than TBI. Low CSF
hypocretin-1 levels were found in 4 of 21 patients 6 months after TBI, as
compared to 25 of 27 patients in the first days after TBI. Hypocretin levels 6
months after TBI were significantly lower in patients with post-traumatic EDS.
There were no associations between post-traumatic SWD and severity or
localization of TBI, general clinical outcome, gender, pathological neurological
findings and HLA typing. However, post-traumatic SWD correlated with impaired
quality of life. These results suggest that sleep-wake disturbances,
particularly EDS, fatigue and hypersomnia are common after TBI, and
significantly impair quality of life. In almost one out of two patients,
post-traumatic SWD appear to be directly related to the TBI. An involvement of
the hypocretin system in the pathophysiology of post-traumatic SWD appears
possible. Other risk factors predisposing towards the development of
post-traumatic SWD were not identified. In the absence of sleep deprivation (either because of behavioral or medical
causes) or pharmacologically induced sleepiness, hypersomnia is a manifestation
of one of the central disorders of hypersomnolence, such as narcolepsy types 1
and 2, idiopathic hypersomnia, and recurrent hypersomnias such as Kleine-Levin
syndrome. Narcolepsy and most primary hypersomnias are chronic conditions, thus,
before committing an individual to chronic, possibly, life-long treatments, an
accurate diagnosis is important. The key to effective management of hypersomnia,
thus, lies in a thorough history, detailed physical examination, and appropriate
diagnostic tests. Secondary causes of hypersomnia are expected to resolve once
these disorders are treated. The treatment of central hypersomnias, on the other
hand, is guided by a level of diagnostic certainty as to the etiology of the
hypersomnia. Narcolepsy, for example, has well defined pathophysiologic and
diagnostic criteria, including low levels of hypocretin in cerebrospinal fluid
(CSF) and specific findings on a polysomnography/multiple sleep latency test
(PSG/MSLT). For these patients, life-long therapy is the norm and involves
initiating treatment usually with modafinil, armodafinil, or sodium oxybate,
with methylphenidate, amphetamine-like stimulants, atomoxetine, or
antidepressants used as second-line therapy. Pharmacologic therapy is usually
done in concert with behavioral modifications such as scheduled napping for the
best response. On the other hand, the etiology and pathophysiology of
non-hypocretin-related hypersomnias (eg, idiopathic hypersomnia, Kleine-Levine
syndrome) are unknown. For these reasons, treatment of these disorders is more
challenging and less well defined. A trial of modafinil or armodafinil may be
considered as first line therapy along with behavioral modifications.
Methylphenidate, amphetamine-based stimulants, and even clarithromycin have also
been used. There is no effective cure for hypersomnia, and the current therapy
is purely symptomatic. Thus, initial patient education, addressing treatment
expectations, as well as continued regular follow-up to monitor treatment
response are vital to effective management of hypersomnia. The focus of this
article is limited to a discussion of treatment of central disorders of
hypersomnolence. |
What is the function of caspases? | Caspases are intracellular proteases that propagate programmed cell death, proliferation, and inflammation. | The effect of expressing human huntingtin fragments containing polyglutamine
(polyQ) tracts of varying lengths was assessed in Caenorhabditis elegans ASH
sensory neurons in young and old animals. Expression of a huntingtin fragment
containing a polyQ tract of 150 residues (Htn-Q150) led to progressive ASH
neurodegeneration but did not cause cell death. Progressive cell death and
enhanced neurodegeneration were observed in ASH neurons that coexpressed
Htn-Q150 and a subthreshold dose of a toxic OSM-10::green fluorescent protein
(OSM-10::GFP) fusion protein. Htn-Q150 huntingtin protein fragments formed
protein aggregates in ASH neurons, and the number of ASH neurons containing
aggregates increased as animals aged. ASH neuronal cell death required ced-3
caspase function, indicating that the observed cell death is apoptotic. Of
interest, ced-3 played a critical role in Htn-Q150-mediated neurodegeneration
but not in OSM10::GFP-mediated ASH neurodegeneration. ced-3 function was
important but not essential for the formation of protein aggregates. Finally,
behavioral assays indicated that ASH neurons, coexpressing Htn-Q150 and
OSM10::GFP, were functionally impaired at 3 days before the detection of
neurodegeneration, cell death, and protein aggregates. Apoptosis research demonstrates that, even though the multitude of regulatory
circuits controlling programmed cell death might diverge, core elements of the
'apoptotic engine' are widely conserved. Therefore, studies in less complex
model systems, such as the nematode and the fly, should continue to have a
profound impact on our understanding of the process. This review explores genes
and molecules that control apoptosis in Drosophila. The death inducers Reaper,
Grim and Hid relay signals, possibly through IAPs (inhibitor of apoptosis
proteins) and Dark (an Apaf-1/Ced-4 homologue), to trigger caspase function.
This animal model promises continued insights into the determits of cell
death in 'naturally occurring' and pathological contexts. Caspases are a family of evolutionarily conserved cysteine proteases that
constitute the effector arm of the apoptotic machinery. Studies in
Caenorhabditis elegans, Drosophila melanogaster, and mouse point to
evolutionarily conserved caspase function in developmentally programmed cell
death in metazoans. Whereas in the nematode all developmental cell death is
mediated by a single caspase, in Drosophila and the mouse some caspases appear
to regulate cell death in a spatio-temporally restricted manner. This article
reviews what we currently know about the roles of various caspases in the
execution of developmentally programmed cell death and what may be expected from
future research in this field. Caspases (cysteine-containing aspartate-specific proteases) are at the core of
the cell's suicide machinery. These enzymes, once activated, dismantle the cell
by selectively cleaving key proteins after aspartate residues. The events
culminating in caspase activation are the subject of intense study because of
their role in cancer, and neurodegenerative and autoimmune disorders. Here we
present a mechanistic mathematical model, formulated on the basis of newly
emerging information, describing key elements of receptor-mediated and
stress-induced caspase activation. We have used mass-conservation principles in
conjunction with kinetic rate laws to formulate ordinary differential equations
that describe the temporal evolution of caspase activation. Qualitative
strategies for the prevention of caspase activation are simulated and compared
with experimental data. We show that model predictions are consistent with
available information. Thus, the model could aid in better understanding caspase
activation and identifying therapeutic approaches promoting or retarding
apoptotic cell death. Programmed cell death plays an important role in maintaining homeostasis during
animal development, and has been conserved in animals as different as nematodes
and humans. Recent studies of Drosophila have provided valuable information
toward our understanding of genetic regulation of death. Different signals
trigger the novel death regulators rpr, hid, and grim, that utilize the
evolutionarily conserved iap and ark genes to modulate caspase function.
Subsequent removal of dying cells also appears to be accomplished by conserved
mechanisms. The similarity between Drosophila and human in cell death signaling
pathways illustrate the promise of fruit flies as a model system to elucidate
the mechanisms underlying regulation of programmed cell death. Retinoids have been shown to be clinically useful in the biological therapy of
certain myeloid and T-cell maligcies, whereas CD20 has proven to be an
effective target in B-cell lymphoma immunotherapy. Both retinoic acid
derivatives and anti-CD20 monoclonal antibodies have also been shown to induce
apoptosis of maligt cells in vitro. Retinoid-induced apoptosis is thought to
be mediated by nuclear retinoid receptor binding and transcriptional activation,
whereas CD20 ligation appears to initiate transmembrane Ca(2+) influx with
resultant programmed cell death. In this report, we evaluate the in vitro
effects of N-(4-hydroxyphenyl) retinamide (4-HPR) with and without anti-CD20
antibodies in B-cell lymphoma lines. We demonstrate that 4-HPR inhibits the
growth of maligt B-cells beyond that of all-trans-retinoic acid and
13-cis-retinoic acid. We also show that this 4-HPR-mediated growth inhibition is
attributable to apoptosis, is consistent across a variety of maligt B-cell
lines (Ramos, Ramos AW, SU-DHL4, and Raji), peaks at 96 to 144 h, and is
attainable with concentrations as low as 2 microM. As with CD20-mediated
apoptosis, we show that the final common pathway includes caspase activation
that can be blocked by 2-val-Ala-Asp-fluoromethyl ketone (z-VAD), a specific
inhibitor of caspase function. Coincubation of a 2 microM concentration of 4-HPR
and the anti-CD20 antibodies rituximab and tositumomab exhibited a
supra-additive increase in levels of apoptosis induction of 24% (P = 0.009) and
42% (P = 0.0019) relative to expected additive levels of these same agents.
These in vitro findings suggest that the potential in vivo synergy of these
well-tolerated drugs may augment the previously demonstrated clinical activity
of anti-CD20 monoclonal antibodies in the treatment of B-cell maligcies. Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial
intermembrane space that is a central effector of many apoptotic pathways. In
resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active
site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis,
caspases are denitrosylated, allowing the catalytic site to function. In the
current studies, we sought to identify the subpopulation of caspases that is
regulated by S-nitrosylation. We report that the majority of mitochondrial, but
not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In
addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These
studies suggest that S-nitrosylation plays an important role in regulating
mitochondrial caspase function and that the S-nitrosylation state of a given
protein depends on its subcellular localization. Previous studies have argued that enhanced activity of the epidermal growth
factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway
can promote tumor cell survival in response to cytotoxic insults. In this study,
we examined the impact of MAPK signaling on the survival of primary hepatocytes
exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of
hepatocytes with DCA caused MAPK activation, which was dependent upon ligand
independent activation of EGFR, and downstream signaling through Ras and PI(3)
kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor
exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition
of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h.
Similar data were also obtained when either domit negative EGFR-CD533 or
domit negative Ras N17 were used to block MAPK activation. DCA-induced
apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase
9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis
in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that
were null for FAS receptor expression. These data argues that DCA is causing
ligand independent activation of the FAS receptor to stimulate an apoptotic
response, which is counteracted by enhanced ligand-independent EGFR/MAPK
signaling. In agreement with FAS-mediated cell killing, inhibition of caspase
function with the use of domit negative Fas-associated protein with death
domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or
domit negative procaspase 8 blocked the potentiation of bile acid-induced
apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage
of BID and release of cytochrome c from mitochondria, which were all blocked by
IETD. Despite activation of caspase 8, expression of domit negative
procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced
apoptosis. Treatment of hepatocytes with DCA transiently increased expression of
the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by
inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s)
abolished the potentiation of bile acid-induced apoptosis. Collectively, our
data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates
DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the
expression of c-FLIP isoforms. Nuclear factor kappaB (NFkappaB) regulates the expression of various genes
essential for cell survival. Here we demonstrate that suppression of NFkappaB
nuclear import with SN50 peptide carrying the nuclear localization sequence
(NLS) of the NFkappaB p50 subunit induces apoptosis in human peripheral blood T
lymphocytes (T-PBL), which can be blocked with the pan-caspase inhibitor
Z-VAD.fmk. However, even when caspase function is blocked, the addition of SN50
induces irreversible cell loss due to the reduction in the mitochondrial
transmembrane potential (DeltaPsim) followed by disruption of the cell membrane,
hallmarks of necrosis. These observations demonstrate that although inhibition
of NFkappaB nuclear translocation by SN50 peptide can induce caspase-dependent
apoptosis in T-PBL, cell death may still proceed in the absence of functional
caspase activity. The availability of downstream caspases appears to determine
the mode of cell death in NFkappaB defective cells. Neuroblastoma is the most common extracranial solid tumor of childhood. N-type
neuroblastoma cells (represented by SH-SY5Y and IMR32 cell lines) are
characterized by a neuronal phenotype. N-type cell lines are generally N-myc
amplified, express the anti-apoptotic protein Bcl-2, and do not express
caspase-8. The present study was designed to determine the mechanism by which
N-type cells die in response to specific cytotoxic agents (such as cisplatin and
doxorubicin) commonly used to treat this disease. We found that N-type cells
were equally sensitive to cisplatin and doxorubicin. Yet death induced by
cisplatin was inhibited by the nonselective caspase inhibitor
z-Val-Ala-Asp-fluoromethylketone or the specific caspase-9 inhibitor
N-acetyl-Leu-Glu-His-Asp-aldehyde, whereas in contrast, caspase inhibition did
not prevent doxorubicin-induced death. Neither the reactive oxygen species nor
the mitochondrial permeability transition appears to play an important role in
this process. Doxorubicin induced NF-kappa B transcriptional activation in
association with I-kappa B alpha degradation prior to loss of cell viability.
Surprisingly, the antioxidant and NF-kappa B inhibitor pyrrolidine
dithiocarbamate blocked doxorubicin-induced NF-kappa B transcriptional
activation and provided profound protection against doxorubicin killing.
Moreover, SH-SY5Y cells expressing a super-repressor form of I-kappa B were
completely resistant to doxorubicin killing. Together these findings show that
NF-kappa B activation mediates doxorubicin-induced cell death without evidence
of caspase function and suggest that cisplatin and doxorubicin engage different
death pathways to kill neuroblastoma cells. The activation of the extracellular signal-regulated kinases (ERKs) by tumour
necrosis factor-alpha (TNF) receptors (TNFRs) is an integral part of the
cytokine's pleiotropic cellular responses. Here we report differences in the
caspase sensitivity and TNFR subtype activation of members of the ERK family.
Inhibition in HeLa cells of caspase function by pharmacological inhibitors or
the expression of CrmA (cytokine response modifier A), a viral modifier protein,
blocks TNF-induced apoptosis or caspase-dependent protein kinase Cdelta and
poly(ADP-ribose) polymerase protein degradation. TNFR1- or TNFR2-stimulated
c-Jun N-terminal kinase (JNK) activity was attenuated in cells in which caspase
activity was inhibited either by pharmacological blockers or CrmA expression.
Both TNFR1- and TNFR2-stimulated JNK activity was caspase-sensitive; however,
only TNFR1 was capable of stimulating p42/44 mitogen-activated protein kinase
(MAPK) and p38 MAPK activities. TNFR1-stimulated p42/44 MAPK and p38 MAPK
activities were insensitive to pharmacological caspase inhibition or CrmA. These
findings were supported when measuring TNF-induced cytosolic phospholipase A(2)
activation, which is a downstream target for MAPK and p38 MAPK. Profiling
caspase enzymes activated by TNF in HeLa cells showed sequential caspase-8, -3,
-7, -6 and -9 activation, with their inhibition characteristics suggesting a
role for caspase-3 and/or caspase-6 in modulating JNK activity. Taken together
these results show delineated ERK-activation pathways employed by TNFR subtypes. PURPOSE: Rod, cone, cone-rod, and macular dystrophies eventually bring about the
death of cone photoreceptor cells. The present study explores means of
inhibiting apoptosis in addition to inducing a specific apoptotic pathway within
a photoreceptor-derived cell line.
METHODS: Retinal cell culture of murine 661W photoreceptor-derived cells was
used to assess the effect of both a synthetic peptide inhibitor of caspases
(benzyloxycarbonyl-Val-Ala-DL-Asp-[Ome] fluoromethylketone [Z-VAD.FMK]) and a
natural inhibitor, baculoviral p35. In addition, the effect of transfection of
Fas-associated death domain (FADD), a cellular protein implicated in
receptor-induced apoptosis, was assessed. Assays were performed by transient
transfection of cell cultures, and results were recorded by cell counting,
Western blot, and spectrophotometry.
RESULTS: Western blot analysis and chromogenic caspase substrate cleavage
analysis confirmed the activation of caspases within 661W cells. At a
concentration of 80 micro M, Z-VAD.FMK, 72.36% +/- 0.93% of 661W cells survived
cytotoxic insult compared with 6.99% +/- 1.35% of control cells. Transient
transfection of 1200 ng baculoviral p35 conferred a protection of 75.30% +/-
4.23%, compared with 19.61% +/-1.84% of control cells, and it was additionally
observed that as little as 50 ng transfection of FADD was capable of inducing
the death of 53.21% +/- 1.33% of cells in 661W cultures.
CONCLUSIONS: Apoptotic cell death in 661W cells is caspase dependent and may be
inhibited with both a synthetic and natural inhibitor of caspase function.
Furthermore, 661W cells are highly sensitive to the FADD protein, which may
suggest a number of novel therapeutic approaches to halt photoreceptor cell
apoptosis. Disruption of the actin cytoskeleton elicits profound changes in cell survival
and function. The actin cytoskeleton is regulated in a hierarchical manner by
Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation
of stress fibers and focal adhesions. Disruption of the actin cytoskeleton
causes apoptosis in airway epithelial cells. To examine further the relation of
cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase
would elicit apoptosis in airway epithelial cells. Inhibition with either
Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and
apoptosis in airway epithelial cells that was blocked by inhibiting caspase
function or by inhibiting protein synthesis. Cells overexpressing constitutively
active Rho kinase, but not native Rho kinase, were resistant to Rho kinase
inhibitor-induced stress fiber disruption and apoptosis. Inhibition of Rho
kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells.
We demonstrate that Rho kinase inhibition induces airway epithelial cell
apoptosis associated with changes in actin filament integrity. Our data suggest
that Rho kinase may be a regulator of early initiation of apoptosis. Autophagic programmed cell death occurs during the development of diverse animal
groups, but the mechanisms that control this genetically regulated form of cell
killing are poorly understood. Genetic studies of bulk protein degradation in
yeast have provided important advances in our understanding of autophagy, and
recent investigations of Drosophila autophagic cell death suggest that some of
these mechanisms may be conserved. In Drosophila, several steroid-regulated
genes that encode transcription regulators are required for autophagic cell
death. These transcription regulators appear to activate a large number of genes
that play a more direct role in cell killing, including genes that function in
apoptosis such as caspases. While caspase function is required for autophagic
cell death during Drosophila development, genes encoding proteins that are
similar to the yeast autophagy regulators are also induced in dying salivary
glands. Furthermore, numerous noncaspase proteases, cytoplasmic organizing
factors, signaling molecules, and unknown factors are expressed in interesting
patterns during autophagic cell death. This article reviews the current
knowledge of the regulation of autophagic programmed cell death during
development of Drosophila. The cysteinyl aspartate-specific proteases (caspases) have been identified as
key players in the cellular process termed programmed cell death or apoptosis.
During apoptosis, activated apoptotic caspases cleave selected target proteins
to execute cell death. Additionally to their established function in cell death,
a variety of recent publications have provided increasing evidence that
apoptotic caspases also participate in several non-apoptotic cellular processes.
Activated caspases exhibit functions during T-cell proliferation and cell cycle
regulation, but are also involved in the differentiation of a diverse array of
cell types. In some cell types, their differentiation can be morphologically
viewed as a kind of incomplete apoptosis. Analysis of well-known apoptotic
targets of caspases implicates that the cleavage of a limited number of selected
substrates plays a major role during non-apoptotic functions of caspases.
Selective substrate cleavage might be regulated by activation of anti-apoptotic
factors, via a compartmentalized activation of caspases, or through limited
activity of caspases during apoptosis-independent functions. The increasing
evidence for caspase function in non-apoptotic cellular events suggests that
caspases play a much more diverse role than previously assumed. The accelerated loss of lung epithelium through activation of extrinsic
apoptosis is believed to play a causative role in lung pathogenesis. Previous
investigations have shown that zinc is required to sustain lung epithelial cell
viability under stress conditions and that depletion of intracellular zinc
predisposes cells to apoptosis. In this investigation, we determined whether
intracellular zinc deficiency enhanced the susceptibility of primary,
differentiated cultures of human lung epithelium to death receptor-mediated
apoptosis, leading to barrier dysfunction. Cultures obtained from multiple
donors were exposed to stimuli that provoke death receptor-mediated apoptosis
and depleted of intracellular zinc with a zinc-specific chelating agent.
Transepithelial resistance, paracellular transport, caspase-8 and caspase-3
activity, and apoptosis were measured. Activation of extrinsic apoptosis or zinc
chelation alone resulted in a nominal increase in caspase function and apoptosis
without major evidence of barrier disruption. Activation of extrinsic apoptosis
in addition to zinc depletion resulted in an abrupt decrease in transepithelial
resistance, a substantial increase in apoptosis, and an increased paracellular
leak. Cultures were rescued by supplementation with zinc sulfate. Further
analysis revealed that exogenous zinc facilitates cell survival through
activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. We
conclude that intracellular zinc is a vital factor in lung epithelium that
protects cells from death receptor-mediated apoptosis and barrier dysfunction. Caspases are key effector components of apoptosis, the process of programmed
cell death. However, a large body of recent work has shown that caspase activity
is necessary for several nonapoptotic cellular functions, such as proliferation,
differentiation, survival, and the regulation of cytokine expression. In this
review, we concentrate on the nonapoptotic functions of caspases and, in
particular, on nonapoptotic roles of caspases in the immune system. Because of
the involvement of caspases in promoting cell death, as well as cell
proliferation and other nonapoptotic functions, studies involving the inhibition
of caspases in vivo must be interpreted with caution. The first proapoptotic caspase, CED-3, was cloned from Caenorhabditis elegans in
1993 and shown to be essential for the developmental death of all somatic cells.
Following the discovery of CED-3, caspases have been cloned from several
vertebrate and invertebrate species. As reviewed in other articles in this issue
of Cell Death and Differentiation, many caspases function in nonapoptotic
pathways. However, as is clear from the worm studies, the evolutionarily
conserved role of caspases is to execute programmed cell death. In this article,
I will specifically focus on caspases that function primarily in cell death
execution. In particular, the physiological function of caspases in apoptosis is
discussed using examples from the worm, fly and mammals. Advances in molecular biology provide an opportunity to develop detailed models
of biological processes that can be used to obtain an integrated understanding
of the system. However, development of useful models from the available
knowledge of the system and experimental observations still remains a daunting
task. In this work, a model identification strategy for complex biological
networks is proposed. The approach includes a state regulator problem (SRP) that
provides estimates of all the component concentrations and the reaction rates of
the network using the available measurements. The full set of the estimates is
utilised for model parameter identification for the network of known topology.
An a priori model complexity test that indicates the feasibility of performance
of the proposed algorithm is developed. Fisher information matrix (FIM) theory
is used to address model identifiability issues. Two signalling pathway case
studies, the caspase function in apoptosis and the MAP kinase cascade system,
are considered. The MAP kinase cascade, with measurements restricted to protein
complex concentrations, fails the a priori test and the SRP estimates are poor
as expected. The apoptosis network structure used in this work has moderate
complexity and is suitable for application of the proposed tools. Using a
measurement set of seven protein concentrations, accurate estimates for all
unknowns are obtained. Furthermore, the effects of measurement sampling
frequency and quality of information in the measurement set on the performance
of the identified model are described. Caspases are cysteine proteases that are essential for cytokine maturation and
apoptosis. To facilitate the dissection of caspase function in vitro and in
vivo, we have synthesized irreversible caspase inhibitors with biotin attached
via linker arms of various lengths (12a-d) and a 2,4-dinitrophenyl labeled
inhibitor (13). Affinity labeling of apoptotic extracts followed by blotting
reveals that these affinity probes detect active caspases. Using the strong
affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from
apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment
with 12c, which contains biotin attached to the N(epsilon)-lysine of the
inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric
avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to
rescue cells from apoptotic execution. These novel caspase inhibitors should
provide powerful probes for the study of the active caspase proteome during
apoptosis both in vitro and in vivo. Programmed cell death or apoptosis is required for the patterning and
development of multicellular organisms. However, apoptosis is a difficult
process to measure because the dead cells are rapidly degraded by their
neighbors within a few hours. The post-caspase activation events that determine
whether a cell will undergo apoptosis remain elusive. Here we report that
apoptosis-specific nuclear events that occur before DNA fragmentation can be
distinguished by monitoring the histone H1 status. In both mammals and
Drosophila, dying cells failed to be immunolabeled with an anti-H1 monoclonal
antibody, AE-4. Real-time imaging of caspase activation and H1 dynamics in
mammalian neural cells revealed that H1 changed its location in the nucleus
after caspase activation. In addition, the timing of this re-localization was
largely dependent on the apoptotic stimulus used. From the staining patterns of
AE-4 and anti-active caspase-3 antibodies, cells undergoing the transition from
caspase activation to the apoptotic H1 change could be identified as H1-positive
caspase-activated cells, providing a novel criterion for early apoptosis and
making it possible to characterize caspase-activated cells in tissues. On the
basis of these staining patterns, we found that many olfactory sensory neurons
in the developing mouse olfactory epithelium showed sustained caspase activity
without the H1 change, suggesting a unique caspase function in these neurons. We have defined some of the mechanisms by which the kinase inhibitor lapatinib
kills HCT116 cells. Lapatinib inhibited radiation-induced activation of ERBB1/2,
extracellular signal-regulated kinases 1/2, and AKT, and radiosensitized HCT116
cells. Prolonged incubation of HCT116 cells with lapatinib caused cell killing
followed by outgrowth of lapatinib-adapted cells. Adapted cells were resistant
to serum starvation-induced cell killing and were cross-resistant to multiple
therapeutic drugs. Lapatinib was competent to inhibit basal and epidermal growth
factor (EGF)-stimulated ERBB1 phosphorylation in adapted cells. Coexpression of
domit-negative ERBB1 and domit-negative ERBB2 inhibited basal and
EGF-stimulated ERBB1 and ERBB2 phosphorylation in parental and adapted cells.
However, in neither parental nor adapted cells did expression of
domit-negative ERBB1 and domit-negative ERBB2 recapitulate the cell
death-promoting effects of lapatinib. Adapted cells had increased expression of
MCL-1, decreased expression of BAX, and decreased activation of BAX and BAK.
Overexpression of BCL-XL protected parental cells from lapatinib toxicity.
Knockdown of MCL-1 expression enhanced lapatinib toxicity in adapted cells that
was reverted by knockdown of BAK expression. Inhibition of caspase function
modestly reduced lapatinib toxicity in parental cells, whereas knockdown of
apoptosis-inducing factor expression suppressed lapatinib toxicity. Thus, in
HCT116 cells, lapatinib adaptation can be mediated by altered expression of pro-
and antiapoptotic proteins that maintain mitochondrial function. Apoptosis is dependent upon caspase activation leading to substrate cleavage
and, ultimately, cell death. Although required for the apoptotic phenotype, it
has become apparent that cells frequently die even when caspase function is
blocked. This process, termed caspase-independent cell death (CICD), occurs in
response to most intrinsic apoptotic cues, provided that mitochondrial outer
membrane permeabilization has occurred. Death receptor ligation can also trigger
a form of CICD termed necroptosis. In this review, we will examine the molecular
mechanisms governing CICD, highlight recent findings demonstrating recovery from
conditions of CICD and discuss potential pathophysiological functions of these
processes. Caspases are intracellular proteases that propagate programmed cell death,
proliferation, and inflammation. Activation of caspases occurs by a conserved
mechanism subject to strict cellular regulation. Once activated by a specific
stimulus, caspases execute limited proteolysis of downstream substrates to
trigger a cascade of events that culminates in the desired biological response.
Much has been learned of the mechanisms that govern the activation and
regulation of caspases, and this minireview provides an update of these areas.
We also delineate substantial gaps in knowledge of caspase function, which can
be approached by techniques and experimental paradigms that are currently
undergoing development. Apoptosis and autophagic cell death occur during Drosophila development, and
recent advances in their mechanisms have been made. As in other organisms,
apoptosis is executed by caspases. In living cells, caspases are kept in check
through a combination of IAP-binding and proteolytic inhibition. Once a cell
commits to apoptosis, phagocytes recognize them through the immuno-receptor-like
proteins Draper and Simu, and initiate corpse engulfment. Drosophila research
has significantly contributed to the idea that autophagy is required for certain
forms of cell death, and that caspase function in autophagic cell death depends
on cell context. Surprisingly, the cell corpse engulfment receptor Draper also
functions in autophagic cell death. These advances facilitate our understanding
of the cell death mechanisms in development and disease. Caspases, initially identified as a family of proteases regulating cell death,
have been found to have nonapoptotic functions as well. Some family members are
critical for mediating programmed cell death in development. After development,
caspases are downregulated in the nervous system, but continue to perform
important nonapoptotic functions relevant for neurogenesis and synaptic
plasticity. In neurodegenerative diseases, where aberrant neuronal death is an
outstanding feature, there is an increase in caspase activity. The specific
caspase death pathways leading to dysfunction and death have still not been
fully clarified, despite the plethora of scientific literature addressing these
issues. In this chapter, we will present the current knowledge of caspase
activation and activity pathways, the current tools for examining caspases, and
functions of caspases in the nervous system in health and in disease.
Alzheimer's Disease, the most common neurodegenerative disorder, and cerebral
ischemia, the most common cause of neurologic death, are used to illustrate our
current understanding of death signaling in neurodegenerative diseases. A better
understanding of how caspases function in health and disease would provide
appropriate specific targets for the development of therapeutic interventions
for these diseases. Life and death are exquisitely regulated at the cellular
level from development through maturity. During development, neuronal death is
the major factor shaping the nervous system. This death is mainly
caspase-mediated apoptosis. Once the waves of developmental death have passed
(death occurs at different times in different parts of the nervous system),
there is downregulation of the death machinery, as the postmitotic neurons
should live for the life of the organism. Aberrant neuronal death is a major
part of neurodegenerative disorders, but there is still no clear understanding
of the processes leading to the phenotypes of the various diseases. Even the
type of death that occurs continues to be debated, whether it is apoptotic,
necrotic, or autophagic, or some combination of these death mechanisms. Here, we
will discuss the role that the caspases play in neuronal function, dysfunction,
and death. First, we will discuss the regulation of caspase activation and
activity. We will examine the current understanding of caspase function in
developmental neuronal death and then illustrate the role of caspases in
neuronal death in disease employing two diseases of neuronal loss, Alzheimer's
Disease (AD), which is the most common chronic neurodegenerative disorder, and
cerebral ischemia/stroke, the third most common cause of death in Western
society, which is an acute neuronal disorder with chronic sequelae. Members of the caspase family of cysteine proteases coordinate the morphological
and biochemical events that typify apoptosis. However, neutralization of caspase
activity in mammals fails to block death in response to most proapoptotic
stimuli. This is because many cell death triggers provoke mitochondrial
dysfunction upstream of caspase activation as a consequence of BAX/BAK channel
opening. Although genetic or pharmacological inactivation of caspases fails to
block cell death in most instances, it does convert the phenotype from apoptosis
to necrosis. This has important implications for how the immune system responds
to such cells, as necrotic cells provoke inflammation whereas apoptotic cells
typically do not. Here, we propose an alternative perspective on
apoptosis-associated caspase function by suggesting that these proteases are
activated, not to kill, but to extinguish the proinflammatory properties of
dying cells. This perspective unifies the mammalian caspase family as either
positive or negative regulators of inflammation. Apoptosis is critical for organismal homeostasis and a wide variety of diseases.
Caspases are the ultimate executors of the apoptotic programmed cell death
pathway. As caspases play such a central role in apoptosis, there is significant
demand for technologies to monitor caspase function. We recently developed a
caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its "dark"
state, where chromophore maturation of the GFP is inhibited by the presence of a
C-terminal peptide. Here we show that chromophore maturation is prevented
because CA-GFP does not fold into the robust β-barrel of GFP until the peptide
has been cleaved by active caspase. Both CA-GFP and GFP₁₋₁₀ , a split form of
GFP lacking the 11th strand, have similar secondary structure, different from
mature GFP. A similar susceptibility to proteolytic digestion indicates that
this shared structure is not the robust, fully formed GFP β-barrel. We have
developed a model that suggests that as CA-GFP is translated in vivo it follows
the same folding path as wild-type GFP; however, the presence of the appended
peptide does not allow CA-GFP to form the barrel of the fully matured GFP.
CA-GFP is therefore held in a "pro-folding" intermediate state until the peptide
is released, allowing it to continue folding into the mature barrel geometry.
This new understanding of the structural basis of the dark state of the CA-GFP
reporter will enable manipulation of this mechanism in the development of
reporter systems for any number of cellular processes involving proteases and
potentially other enzymes. |
Is TNNI3K a cardiac-specific protein? | Yes, TNNI3K is highly expressed in heart but is undetectable in other tissues. | Cardiac-restricted genes play important roles in cardiovascular system. In an
effort to identify such novel genes we identified a novel cardiac-specific
kinase gene TNNI3K localized on 1p31.1 based on bioinformatics analyses.
Sequence analysis suggested that TNNI3K is a distant family member of
integrin-linked kinase. Northern blot and 76-tissue array analyses showed that
TNNI3K is highly expressed in heart, but is undetectable in other tissues.
Immunohistochemical analysis predomitly localized TNNI3K in the nucleus of
cardiac myocytes. In vitro kinase assay showed that TNNI3K is a functional
kinase. The yeast two-hybrid system showed that TNNI3K could directly interact
with cardiac troponin I, results that were further confirmed by
coimmunoprecipitation in vivo. Our data suggest that TNNI3K is a
cardiac-specific kinase and play important roles in cardiac system. The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its
interaction with cardiac troponin I, is both a unique expression and
heart-enriched gene. To understand the mechanisms of CARK gene expression and
regulation, we first cloned the full-length mRNA sequence and mapped the
transcription start site of the mouse CARK gene and characterized its promoter
regions. Two transcriptional isoforms of the CARK gene were identified in mouse
heart tissue. Truncation analysis of the CARK promoter identified a minimal 151
bp region that has strong basal transcription activity. Mutational analysis
revealed five conserved cis-acting elements in this 151-bp long minimal
promoter. Mutational and loss-of-functional analysis and co-transfection studies
indicated that MEF2 binding region is the most critical cis-acting element in
the CARK promoter, and CARK transcription level can be down-regulated by MEF2C
antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by
electrophoretic mobility shift assay and competition and supershift
electrophoretic mobility shift assays. TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1
and that belongs to a tyrosine kinase-like branch in the kinase tree of the
human genome. In the present study we investigated the role of TNNI3K in the
cardiac myogenesis process and in the repair of ischemic injury. Pluripotent
P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to
induce differentiation into beating cardiomyocytes. TNNI3K promoted the
differentiation process, judging from the increasing beating mass and increased
number of alpha-actinin-positive cells. TNNI3K improved cardiac function by
enhancing beating frequency and increasing the contractile force and epinephrine
response of spontaneous action potentials without an increase of the single-cell
size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V(+)
cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial
administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial
infarction improved cardiac performance and attenuated ventricular remodeling
compared with injection of wild-type P19CL6 cells. In conclusion, our study
clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac
performance, and protects the myocardium from ischemic injury by suppressing
p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a
useful therapeutic approach for ischemic cardiac disease. Recently, regenerative medicine using the transplantation of embryonic stem
cells and bone marrow stem cells has been a great success but still has many
unconfirmed problems including its clinical evaluation. The aim of this article
is to review current literature and some patents regarding molecular therapeutic
agents including using MAP kinase TNNI3K for the treatment and diagnosis of
acute myocardial ischemia or infarction. TNNI3K is a novel cardiac troponin
I-interacting kinase gene and its overexpression may promote cardiac myogenesis,
improve cardiac performance, and attenuate ischemia-induced ventricular
remodeling. The modulation of embryonal stem cells with high TNNI3K activity
using a TNNI3K active peptide could be a useful therapeutic approach for
ischemic cardiac diseases. For overexpressing TNNI3K or enhancing TNNI3K
activity in cardiac precursor cells, the engraftment of bone marrow cells or
embryonic stem cells can effectively promote cardiac myogenesis, beating
frequency, and contractile functions, and decrease "silent" (no contraction)
cardiac cells after cell transplantion, indicating that the overexpression of
TNNI3K can increase the success rate of transplanting embryonic stem cells or
bone marrow cells into ischemic hearts for the treatment of ischemic cardiac
diseases. Although previous investigations showing that TNNI3K may be involved
in the development of cardiac hypertrophy, it is still unclear whether TNNI3K
has a role in cardiac hypertrophy or what mechanism is involved in the effects
of TNNI3K. To confirm this, further investigations need to be undertaken. 1. Cardiac troponin I-interacting kinase (TNNI3K) is a novel cardiac-specific
kinase gene. Quantitative real-time reverse transcription polymerase chain
reaction analysis showed a significant increase in TNNI3K mRNA expression in
hypertrophic cardiomyocytes induced by endothelin-1 (ET-1). The aim of the
present study was to investigate the effects of TNNI3K on neonate rat
cardiomyocyte hypertrophy induced by ET-1. 2. Adenoviruses were amplified in
293A cells. To determine a reasonable adenovirus infection dose cardiomyocytes
were infected with an adenovirus carrying human TNNI3K (Ad-TNNI3K) at varying
multiplicity of infection (MOI) and the expression of TNNI3K was analysed by
western blot. 3. Cardiomyocytes were infected with either a control adenovirus
carrying green fluorescent protein (Ad-GFP) or Ad-TNNI3K. Compared with Ad-GFP,
the Ad-TNNI3K induced an increase in sarcomere organization, cell surface area,
(3) H-leucine incorporation and β-MHC re-expression. This type of hypertrophic
phenomenon is similar to that observed in Ad-GFP-infected hypertrophic
cardiomyocytes induced by ET-1. To determine the functional role of TNNI3K in
ET-1-induced hypertrophic cardiomyocytes, the cells were infected with Ad-GFP or
Ad-TNNI3K. Ad-TNNI3K induced an increase in sarcomere organization, cell surface
area and (3) H-leucine incorporation compared with Ad-GFP. 4. These results
suggest that TNNI3K overexpression induces cardiomyocytes hypertrophy and
accelerates hypertrophy in hypertrophic cardiomyocytes. Therefore, TNNI3K might
be an interesting target for the clinical treatment of hypertrophy. Cardiac troponin I-interacting kinase (TNNI3K) is a cardiac-specific kinase
whose biological function remains largely unknown. We have recently shown that
TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of
cardiomyopathy, indicating an important role in modulating disease progression.
To further investigate TNNI3K kinase activity in vivo, we have generated
transgenic mice expressing both wild-type and kinase-dead versions of the human
TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity
induces mouse cardiac remodeling, and its kinase activity promotes accelerated
disease progression in a left-ventricular pressure overload model of mouse
cardiomyopathy. Using an in vitro kinase assay and proteomics analysis, we show
that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity.
TNNI3K expression induces a series of cellular and molecular changes, including
a reduction of sarcomere length and changes in titin isoform composition, which
are indicative of cardiac remodeling. Using antisera to TNNI3K, we show that
TNNI3K protein is located at the sarcomere Z disc. These combined data suggest
that TNNI3K mediates cell signaling to modulate cardiac response to stress. The phosphorylation of cardiac troponin I (cTnI) plays an important role in the
contractile dysfunction associated with heart failure. Human cardiac troponin
I-interacting kinase (TNNI3K) is a novel cardiac-specific functional kinase that
can bind to cTnI in a yeast two-hybrid screen. The purpose of this study was to
investigate whether TNNI3K can phosphorylate cTnI at specific sites and to
examine whether the phosphorylation of cTnI caused by TNNI3K can regulate
cardiac myofilament contractile function. Co-immunoprecipitation was performed
to confirm that TNNI3K could interact with cTnI. Kinase assays further indicated
that TNNI3K did not phosphorylate cTnI at Ser23/24 and Ser44, but directly
phosphorylated Ser43 and Thr143 in vitro. The results obtained for adult rat
cardiomyocytes also indicated that enhanced phosphorylation of cTnI at Ser43 and
Thr143 correlated with rTNNI3K (rat TNNI3K) overexpression, and phosphorylation
was reduced when rTNNI3K was knocked down. To determine the contractile function
modulated by TNNI3K-mediated phosphorylation of cTnI, cardiomyocyte contraction
was studied in adult rat ventricular myocytes. The contraction of cardiomyocytes
increased with rTNNI3K overexpression and decreased with rTNNI3K knockdown. We
conclude that TNNI3K may be a novel mediator of cTnI phosphorylation and
contribute to the regulation of cardiac myofilament contraction function. PURPOSE: Protein kinase plays an essential role in controlling cardiac growth
and hypertrophic remodeling. The cardiac troponin I-interacting kinase (TNNI3K),
a novel cardiac specific kinase, is associated with cardiomyocyte hypertrophy.
However, the precise function of TNNI3K in regulating cardiac remodeling has
remained controversial.
METHODS AND RESULTS: In a rat model of cardiac hypertrophy generated by
transverse aortic constriction, myocardial TNNI3K expression was significantly
increased by 1.62 folds (P<0.05) after constriction for 15 days. To investigate
the role of TNNI3K in cardiac hypertrophy, we generated transgenic mouse lines
with overexpression of human TNNI3K specifically in the heart. At the age of 3
months, the high-copy-number TNNI3K transgenic mice demonstrated a phenotype of
concentric hypertrophy with increased heart weight normalized to body weight
(1.31 fold, P<0.01). Echocardiography and non-invasive hemodynamic assessments
showed enhanced cardiac function. No necrosis or myocyte disarray was observed
in the heart of TNNI3K transgenic mice. This concentric hypertrophy maintained
up to 12 months of age without cardiac dysfunction. The phospho amino acid
analysis revealed that TNNI3K is a protein-tyrosine kinase. The yeast two-hybrid
screen and co-immunoprecipitation assay identified cTnI as a target for TNNI3K.
Moreover, TNNI3K overexpression induced cTnI phosphorylation at Ser22/Ser23 in
vivo and in vitro, suggesting that TNNI3K is a novel upstream regulator for cTnI
phosphorylation.
CONCLUSION: TNNI3K promotes a concentric hypertrophy with enhancement of cardiac
function via regulating the phosphorylation of cTnI. TNNI3K could be a potential
therapeutic target for preventing from heart failure. Percutaneous coronary intervention is first-line therapy for acute coronary
syndromes (ACS) but can promote cardiomyocyte death and cardiac dysfunction via
reperfusion injury, a phenomenon driven in large part by oxidative stress.
Therapies to limit this progression have proven elusive, with no major classes
of new agents since the development of anti-platelets/anti-thrombotics. We
report that cardiac troponin I-interacting kinase (TNNI3K), a
cardiomyocyte-specific kinase, promotes ischemia/reperfusion injury, oxidative
stress, and myocyte death. TNNI3K-mediated injury occurs through increased
mitochondrial superoxide production and impaired mitochondrial function and is
largely dependent on p38 mitogen-activated protein kinase (MAPK) activation. We
developed a series of small-molecule TNNI3K inhibitors that reduce
mitochondrial-derived superoxide generation, p38 activation, and infarct size
when delivered at reperfusion to mimic clinical intervention. TNNI3K inhibition
also preserves cardiac function and limits chronic adverse remodeling. Our
findings demonstrate that TNNI3K modulates reperfusion injury in the ischemic
heart and is a tractable therapeutic target for ACS. Pharmacologic TNNI3K
inhibition would be cardiac-selective, preventing potential adverse effects of
systemic kinase inhibition. |
How does Rif1 regulate DNA replication? | Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Overall, Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture. This function of Rif1 depends on its interaction with PP1 phosphatases and the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. | We report on the function of the human ortholog of Saccharomyces cerevisiae Rif1
(Rap1-interacting factor 1). Yeast Rif1 associates with telomeres and regulates
their length. In contrast, human Rif1 did not accumulate at functional
telomeres, but localized to dysfunctional telomeres and to telomeric DNA
clusters in ALT cells, a pattern of telomere association typical of
DNA-damage-response factors. After induction of double-strand breaks (DSBs),
Rif1 formed foci that colocalized with other DNA-damage-response factors. This
response was strictly dependent on ATM (ataxia telangiectasia mutated) and
53BP1, but not affected by diminished function of ATR (ATM- and Rad3-related
kinase), BRCA1, Chk2, Nbs1, and Mre11. Rif1 inhibition resulted in
radiosensitivity and a defect in the intra-S-phase checkpoint. The S-phase
checkpoint phenotype was independent of Nbs1 status, arguing that Rif1 and Nbs1
act in different pathways to inhibit DNA replication after DNA damage. These
data reveal that human Rif1 contributes to the ATM-mediated protection against
DNA damage and point to a remarkable difference in the primary function of this
protein in yeast and mammals. Rif1, originally recognized for its role at telomeres in budding yeast, has been
implicated in a wide variety of cellular processes in mammals, including
pluripotency of stem cells, response to double-strand breaks, and breast cancer
development. As the molecular function of Rif1 is not known, we examined the
consequences of Rif1 deficiency in mouse cells. Rif1 deficiency leads to failure
in embryonic development, and conditional deletion of Rif1 from mouse embryo
fibroblasts affects S-phase progression, rendering cells hypersensitive to
replication poisons. Rif1 deficiency does not alter the activation of the DNA
replication checkpoint but rather affects the execution of repair. RNA
interference to human Rif1 decreases the efficiency of homology-directed repair
(HDR), and Rif1 deficiency results in aberrant aggregates of the HDR factor
Rad51. Consistent with a role in S-phase progression, Rif1 accumulates at
stalled replication forks, preferentially around pericentromeric
heterochromatin. Collectively, these findings reveal a function for Rif1 in the
repair of stalled forks by facilitating HDR. The eukaryotic genome is replicated according to a specific spatio-temporal
programme. However, little is known about both its molecular control and
biological significance. Here, we identify mouse Rif1 as a key player in the
regulation of DNA replication timing. We show that Rif1 deficiency in primary
cells results in an unprecedented global alteration of the temporal order of
replication. This effect takes place already in the first S-phase after Rif1
deletion and is neither accompanied by alterations in the transcriptional
landscape nor by major changes in the biochemical identity of constitutive
heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S
transition and chromatin re-organization after DNA replication. Together, these
data offer a novel insight into the global regulation and biological
significance of the replication-timing programme in mammalian cells. DNA replication is spatially and temporally regulated during S-phase. DNA
replication timing is established in early-G1-phase at a point referred to as
timing decision point. However, how the genome-wide replication timing domains
are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1),
originally identified as a telomere-binding factor in yeast, is a critical
determit of the replication timing programme in human cells. Depletion of
Rif1 results in specific loss of mid-S replication foci profiles, stimulation of
initiation events in early-S-phase and changes in long-range replication timing
domain structures. Analyses of replication timing show replication of sequences
normally replicating early is delayed, whereas that normally replicating late is
advanced, suggesting that replication timing regulation is abrogated in the
absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at
late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1
colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes
the mid-S replication domains that are restrained from being activated at
early-S-phase. Our results indicate that Rif1 plays crucial roles in determining
the replication timing domain structures in human cells through regulating
higher-order chromatin architecture. Origins of DNA replication on eukaryotic genomes have been observed to fire
during S phase in a coordinated manner. Studies in yeast indicate that origin
firing is affected by several factors, including checkpoint regulators and
chromatin modifiers. However, it is unclear what the mechanisms orchestrating
this coordinated process are. Recent studies have identified factors that
regulate the timing of origin activation, including Rif1 which plays crucial
roles in the regulation of the replication timing program in yeast as well as in
higher eukaryotes. In mammalian cells, Rif1 appears to regulate the structures
of replication timing domains through its ability to organize chromatin loop
structures. Regulation of chromatin architecture by Rif1 may be linked to other
chromosome transactions including recombination, repair, or transcription. This
review summarizes recent progress in the effort to elucidate the regulatory
mechanisms of replication timing of eukaryotic replicons. DNA double strand breaks (DSBs) are highly toxic to the cells and accumulation
of DSBs results in several detrimental effects in various cellular processes
which can lead to neurological, immunological and developmental disorders.
Failure of the repair of DSBs spurs mutagenesis and is a driver of
tumorigenesis, thus underscoring the importance of the accurate repair of DSBs.
Two major canonical DSB repair pathways are the non-homologous end joining
(NHEJ) and homologous recombination (HR) pathways. 53BP1 and BRCA1 are the key
mediator proteins which coordinate with other components of the DNA repair
machinery in the NHEJ and HR pathways respectively, and their exclusive
recruitment to DNA breaks/ends potentially decides the choice of repair by
either NHEJ or HR. Recently, Rap1 interacting factor 1 has been identified as an
important component of the DNA repair pathway which acts downstream of the
ATM/53BP1 to inhibit the 5'-3' end resection of broken DNA ends, in-turn
facilitating NHEJ repair and inhibiting homology directed repair. Rif1 is
conserved from yeast to humans but its function has evolved from telomere length
regulation in yeast to the maintece of genome integrity in mammalian cells.
Recently its role in the maintece of genomic integrity has been expanded to
include the regulation of chromatin structure, replication timing and intra-S
phase checkpoint. We present a summary of these important findings highlighting
the various aspects of Rif1 functions and discuss the key implications for
genomic integrity. Initiation of eukaryotic DNA replication requires phosphorylation of the MCM
complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its
activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor
1) controls DNA replication genome-wide and describe how Rif1 opposes DDK
function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of
the MCM complex. Deleting RIF1 partially compensates for the limited DDK
activity in a cdc7-1 mutant strain by allowing increased, premature
phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal
domain are critical for its repressive effect on replication. We confirm that
Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation.
Remarkably, our results suggest that replication repression by Rif1 is itself
also DDK-regulated through phosphorylation near the PP1-interacting motifs.
Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting
subunit that counteracts DDK-mediated phosphorylation during replication.
Fission yeast and mammalian Rif1 proteins have also been implicated in
regulating DNA replication. Since PP1 interaction sites are evolutionarily
conserved within the Rif1 sequence, it is likely that replication control by
Rif1 through PP1 is a conserved mechanism. The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase
activities. DDK, in particular, is involved in setting the temporal program of
origin activation, a conserved feature of eukaryotes. Rif1, originally
identified as a telomeric protein, was recently implicated in specifying
replication timing in yeast and mammals. We show that this function of Rif1
depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking
motifs in Rif1 lead to early replication of telomeres in budding yeast and
misregulation of origin firing in fission yeast. Several lines of evidence
indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase.
Our data suggest that the PP1/Rif1 interaction is downregulated by the
phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the
mechanism of action of Rif1 in the control of DNA replication and demonstrate a
role of PP1 phosphatases in the regulation of origin firing. The Rif1 protein, originally identified as a telomere-binding factor in yeast,
has recently been implicated in DNA replication control from yeast to metazoans.
Here, we show that budding yeast Rif1 protein inhibits activation of
prereplication complexes (pre-RCs). This inhibitory function requires two
N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase
(Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an
RVxF/SILK-dependent manner and that two proteins implicated in pre-RC
activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK)
phosphorylation in rif1 mutants. Rif1 also interacts with Dbf4 in yeast
two-hybrid assays, further implicating this protein in direct modulation of
pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK
motif-dependent recruitment of Glc7 to telomeres and earlier replication of
these regions in cells where the motifs are mutated. Our data thus link Rif1 to
negative regulation of replication origin firing through recruitment of the Glc7
phosphatase. |
Is factor XI deficient in Hemophilia C? | Factor XI deficiency is associated with a bleeding tendency called Hemophilia C. | A 39-year-old male developed severely prolonged bleeding after periodontal
surgery because of a previously undetected clotting Factor XI deficiency
(Hemophilia C). Diagnosis and treatment of this bleeding disorder are discussed. The authors report a rare case of an acute cerebral aneurysm rupture in a
patient with a known factor XI deficiency. Aneurysmal subarachnoid hemorrhage
(SAH) accounts for a high mortality and morbidity rate. When SAH is associated
with an inherited coagulation disorder such as hemophilia C, an unexpected and
possible increase in hemorrhagic stroke and increase in bleeding during surgery
and in the postoperative period could lead to an extremely bad outcome. Clinical
management consists of rapid correction of the coagulation disorder before
undergoing any invasive intracranial procedure. Such an optimal therapeutic
strategy must be under the care of a multidisciplinary medical and surgical
team. Human factor XI concentrate (Hemoleven, Laboratoire Français du
Fractionnement et des Biotechnologies [LFB], Les Ulis, France) was used
successfully in this case report. New treatment using recombit factor VIIa is
discussed. Congenital factor XI deficiency (also known as the Rosenthal syndrome or
hemophilia C) manifests as minor bleeding, usually after trauma or surgery. We
report a case in which bilateral knee hemarthrosis was the first manifestation.
The patient presented at 32 years of age with a 2-year history of mechanical
pain and intermittent swelling in both knees. Knee aspiration recovered
blood-tinged fluid. The laboratory workup showed severe factor XI deficiency.
Replacement therapy with fresh frozen plasma was effective. Tests in the family
showed factor XI deficiency in the patient's sister. BACKGROUND AND OBJECTIVES: Factor XI deficiency is a rare hematologic disorder.
Hemophilia C (factor XI deficiency) affects both genders and it is usually
asymptomatic, manifesting only as postoperative hemorrhage. It is an autosomal
recessive, homozygous or heterozygous, disorder, and its severity depends on the
levels of factor XI. The objective of this report was to present the anesthetic
strategy in a patient with hemophilia C.
CASE REPORT: This is a 32 years old female, gravida 1/para 0, on the 39th week
of pregcy, scheduled for elective cesarean section. Physical and laboratorial
exams did not show any abnormalities. According to the recommendations of the
hematologist, on the day of the procedure, the patient was given promethazine,
25 mg, hydrocortisone, 500 mg, due to prior transfusion reaction, and plasma, 10
mL x kg(-1) for a total of 700 mL. Two hours later, the patient underwent
subarachnoid block under routine monitoring. Ringer's lactate, 2000 mL, was
administered for hydration. The anesthetic-surgical procedure proceeded without
intercurrences. Postoperatively, the patient was doing well when, on the 3rd PO
day, fresh frozen plasma (FFP), 10 mL x kg(-1), was administered to prevent late
postoperative bleeding.
CONCLUSIONS: The objective of this report was to present the anesthetic protocol
for patients with hemophilia C and to alert for the need of investigation in
patients with a history of postoperative bleeding, when a coagulation study
should e be done before any invasive procedure and, in the case of prolonged
aPTT, one should investigate the presence of factor XI deficiency. |
What is known about food intolerance and gluten ? | Celiac disease, with a prevalence around 1% of the general population, is the most common genetically-induced food intolerance in the world. Triggered by the ingestion of gluten in genetically predisposed individuals.
Emerging research into a syndrome known as nonceliac gluten sensitivity suggests a heterogeneous condition with some features of celiac disease but often categorized as FGIDs, including IBS. | Coeliac disease is a lifelong intolerance to the gluten found in wheat, barley
and rye, and some patients are also sensitive to oats. The disease is
genetically determined, with 10% of the first-degree relatives affected and 75%
of monozygotic twins being concordant. Of the patients with coeliac disease 95%
are human leucocyte antigen (HLA)-DQ2 or HLA-DQ8 positive. Characteristically,
the jejunal mucosa becomes damaged by a T-cell-mediated autoimmune response that
is thought to be initiated by a 33-mer peptide fragment in A2 gliadin, and
patients with this disorder have raised levels of anti-endomysium and tissue
transglutaminase antibodies in their blood. Coeliac disease is the major
diagnosable food intolerance and, with the advent of a simple blood test for
case finding, prevalence rates are thought to be approximately 1:100.
Classically, the condition presented with malabsorption and failure to thrive in
infancy, but this picture has now been overtaken by the much more common
presentation in adults, usually with non-specific symptoms such as tiredness and
anaemia, disturbance in bowel habit or following low-impact bone fractures.
Small intestinal biopsy is necessary for diagnosis and shows a
characteristically flat appearance with crypt hypoplasia and infiltration of the
epithelium with lymphocytes. Diet is the key to management and a gluten-free
diet effectively cures the condition. However, this commitment is lifelong and
many aisles in the supermarket are effectively closed to individuals with
coeliac disease. Compliance can be monitored by measuring antibodies in blood,
which revert to negative after 6-9 months. Patients with minor symptoms, who are
found incidentally to have coeliac disease, often ask whether it is necessary to
adhere to the diet. Current advice is that dietary adherence is necessary to
avoid the long-term complications, which are, principally, osteoporosis and
small bowel lymphoma. However, risk of these complications diminishes very
considerably in patients who are on a gluten-free diet. Coeliac disease is a multifactorial disease characterized by a dysregulated
immune response to ingested wheat gluten and related cereal proteins. With an
incidence of about 1% of the general population, it is considered the most
common food intolerance disorder. The mainstay of coeliac disease treatment is
strict lifelong adherence to a gluten-free diet. Elimination of gluten and
related proteins from the diet leads to clinical and histological improvement.
However, some patients do not respond to dietary therapy and others have poor
dietary compliance. This has prompted the search for a therapy alternative to a
gluten-free diet. Tissue transglutaminase is a crucial factor in coeliac disease
because it promotes the gluten-specific T-cell response and is also the target
of the autoimmune response. Tissue transglutaminase induces changes in gluten,
which in turn, cause the generation of a series of gluten peptides that bind to
HLA-DQ2 or DQ8 molecules with high affinity. The resulting HLA-DQ2 (DQ8)-gluten
peptide interaction triggers the proinflammatory T cell response. Tissue
transglutaminase is also involved in other non-T-cell-mediated biological
activities of gliadin peptides. For these reasons, tissue transglutaminase is a
potential target for therapeutic intervention. In this paper we review the
state-of-the-art of tissue transglutaminase inhibition, and examine known and
new-generation inhibitors and their activity in in vitro and in vivo models. We
also examine their potential as therapeutic tools for coeliac disease. Celiac disease, with a prevalence around 1% of the general population, is the
most common genetically-induced food intolerance in the world. Triggered by the
ingestion of gluten in genetically predisposed individuals, this enteropathy may
appear at any age, and is characterized by a wide variety of clinical signs and
symptoms. Among them, gastrointestinal presentations include chronic diarrhea,
abdominal pain, weight loss or failure to thrive in children; but
extra-intestinal manifestations are also common, and actually appear to be on
the rise. They include a large variety of ailments, such as dermatitis
Herpetiformis, anemia, short stature, osteoporosis, arthritis, neurologic
problems, unexplained elevation of transaminases, and even female infertility.
For the clinician interested in oral diseases, celiac disease can lead to
delayed tooth eruption, dental enamel hypoplasia, recurrent oral aphthae.
Diagnosing celiac disease requires therefore a high degree of suspicion followed
by a very sensitive screening test: serum levels of the autoantibody anti-tissue
transglutaminase. A positive subject will then be confirmed by an intestinal
biopsy, and will then be put on a strict gluten-free diet, that in most cases
will bring a marked improvement of symptoms. Newer forms of treatment which in
the future will probably be available to the non-responsive patients are
currently being actively pursued. The introduction of cereals in human nutrition 10 000 years ago caused the
occurrence of gluten induced diseases. This protein complex is involved in
pathogenesis of wheat allergy, celiac disease, and gluten sensitivity. Wheat
allergy and celiac disease are mediated by the system of adaptive immunity.
Gluten sensitivity is a recently defined entity induced by innate immune
mechanisms. These subjects present various intestinal and particularly
extraintestinal symptoms. The differences between celiac disease and gluten
intolerance include permeability of the intestinal mucosal barrier, histology of
duodenal biopsy, and mucosal gene expression. The symptoms of gluten sensitivity
may also have another genetic background of food intolerance independent of the
HLADQ2, - DQ8 system and tissue transglutaminase (eg. in some psychiatric
disorders). At present, there is no specific bio-marker of gluten sensitivity.
The diagnosis is possible only by exclusion of other causes of symptoms and
improvement on a glutenfree diet applied in a doubleblind placebo controlled
manner with optional sequence of both stages to exclude the placebo effect due
to nutritional intervention. |
Are EDNRB mutations involved in the development of Hirschsprung disease? | Although mutations in eight different genes (EDNRB, EDN3, ECE1, SOX10, RET, GDNF, NTN, SIP1) have been identified in affected individuals, it is now clear that RET and EDNRB are the primary genes implicated in the etiology of HSCR. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. Molecular genetic analyses have revealed that interactions between mutations in the genes encoding the RET receptor tyrosine kinase and the endothelin receptor type B (EDNRB) are central to the genesis of HSCR. | Hirschsprung disease (HSCR, aganglionic megacolon) is a frequent congenital
malformation regarded as a multigenic neurocristopathy. Two susceptibility genes
have been recently identified in HSCR, namely the RET proto-oncogene and the
endothelin B receptor (EDNRB) gene. Hitherto however, homozygosity for EDNRB
mutations accounted for the HSCR-Waardenburg syndrome (WS) association. Here, we
report heterozygous EDNRB missense mutations (G57S, R319W and P383L) in isolated
HSCR. These data might suggest that EDNRB mutations could be dosage sensitive:
heterozygosity would predispose to isolated HSCR with incomplete penetrance,
while homozygosity would result in more complex neurocristopathies associating
HSCR and WS features. In addition, the present data give further support to the
role of the endothelin-signalling pathway in the development of neural
crest-derived enteric neurons. To date, three genes have been identified as susceptibility genes for
Hirschsprung's disease (HSCR), the RET proto-oncogene, the endothelin-B receptor
gene (EDNRB) and the endothelin-3 gene (EDN3). However, the question of whether
these genes play a role in sporadically occurring HSCR has not been fully
clarified. In this study, the authors performed mutation analysis of these three
genes in 41 sporadic HSCR patients without any family history by using
single-strand conformational polymorphism or denaturing gradient gel
electrophoresis methods. Exon 2, 3, 5, 6, 12, 13, 15, and 17 of the RET gene, 7
exons of the EDNRB gene, and the region of the EDN3 gene including sequences
corresponding to proteolytic cleavage sites and mature endothelin-3 were
analysed. By direct sequencing, three causative RET mutations were confirmed; a
Phe to Ser substitution at codon 174, a Cys to Tyr substitution at codon 197,
and a point mutation at the splice acceptor site of intron 12, in patients with
aganglionosis confined to the rectosigmoid colon, the transverse colon, and the
total colon, respectively. In the EDNRB locus, two mutations were observed; a
nonsense mutation of Trp to stop at codon 275, and a T insertion at nucleotide
878, in patients with aganglionosis confined to the rectosigmoid colon, and the
descending colon, respectively. No mutation was detected in the EDN3 gene.
Mutation rates were 7.3% in the RET and 5% in the EDNRB gene. Our data indicate
that RET and EDNRB mutations have a role in the aetiology of some sporadically
occurring HSCR. However, the low mutation rate of susceptibility genes in
sporadically occurring HSCR suggests that other genes or environmental factors
are involved in the development of the disease. We report on mutation analysis of five genes involved in the receptor tyrosine
kinase (RET) or the endothelin-signalling pathways in 28 sporadic Japanese
patients with Hirschsprung disease. Analysis of DNA obtained from peripheral
blood cells revealed six mutations in the RET proto-oncogene, four of which were
disease-causing mutations in exon 9 (D584G), the splice donor site of intron 10
(+2T to A), exon 11 (A654T), and exon 12 (T706A). A heterozygous A to G
transition was found in 47 bases upstream from the 5' end of exon 2 in two HD
patients but was also seen in one control subject (2/28; 1/24). A silent 2307T
to G transversion was observed in exon 13. Two disease-causing mutations were
detected in the endothelin receptor (EDNRB) gene, in the non-coding region of
exon 1 (-26 G to A) and in exon 4 (A301T); the latter mutation was a novel one.
One silent mutation was observed in exon 4 (codon 277). One heterozygous T to C
mutation was found in the glial cell line-derived neurotrophic factor gene in 25
bases upstream of the coding region in exon 1. No nucleotide changes were
detected in either the endothelin-3 or neurturin genes. Disease-causing mutation
rates in the RET proto-oncogene and the EDNRB gene were estimated at 14.3%
(4/28) and 10.7% (3/28), respectively. In addition to mutations in the RET and
EDNRB genes, embryonic environmental factors and/or other genetic factors appear
to be involved in the development of Hirschsprung disease. Further systematic
studies of genetic variation in a large series of patients and controls are
necessary for elucidating the pathogenesis of this disorder.
CONCLUSION: This study provides further gene alterations as disease-causing
mutations in Japanese cases of sporadic Hirschsprung disease. However, the low
mutation rate of the susceptibility genes may indicate that Hirschsprung disease
arises from a combination of genetic and environmental factors. BACKGROUND: Enteric aganglionosis in Hirschsprung disease has been linked to
genes coding for endothelin-3 (EDN3) and the endothelin B receptor (EDNRB), but
there is no such linkage in most patients with sporadic Hirschsprung disease.
However, the similarity between the distal colonic aganglionosis in Hirschsprung
disease and that due to EDN3 or EDNRB mutations led to the hypothesis that
levels of expression of these genes might be affected in the absence of
mutation, thus causing the Hirschsprung disease phenotype. The aim of this study
was to determine EDN3 and EDNRB messenger RNA (mRNA) levels in tissue samples
from patients with sporadic Hirschsprung disease.
METHODS: RNA and DNA were isolated from the ganglionic and aganglionic colonic
segments of ten children with sporadic Hirschsprung disease, and from the colon
of ten age-matched controls. The DNA was analysed for mutations in the genes
coding for endothelin-3 (ET-3) and endothelin B receptor (ET-B) proteins.
Relative levels of EDN3 and EDNRB mRNA were determined by semi-quantitative
transcriptase-polymerase chain reaction.
RESULTS: Three children had sequence variants in EDN3 and EDNRB. In the
remaining seven patients, EDN3 mRNA levels were reduced in both the ganglionic
and aganglionic colon compared with levels in controls; there was no difference
in expression of EDNRB between groups.
CONCLUSION: In the absence of mutation, EDN3 is downregulated in short-segment
Hirschsprung disease, suggesting that this may be a common step leading to
aganglionosis. Several missense mutations of the endothelin-B receptor (EDNRB) associated with
Hirschsprung disease have recently been identified. Five mutated EDNRB (A183G,
W276C, R319W, M374I and P383L) cDNAs were transiently expressed in several cell
lines to examine the effects of these mutations. Ligand-receptor binding
experiments demonstrated that all mutants examined here accept endothelins with
a high affinity. Especially, the affinity of endothelins to P383L was increased.
However, the number of binding sites of A183G, W276C and P383L was markedly
decreased. The subcellular localization of these mutant receptors was the same
as that of wild-type EDNRB, whereas the amount of protein of each mutant
receptor was decreased. All mutant receptors were impaired in intracellular
Ca(2+) mobilization. These findings indicate that these missense mutations
result in loss of function of EDNRB, and may provide the molecular pathological
basis of Hirschsprung disease in some individuals. BACKGROUND: Hirschsprung disease (HSCR) is a frequent congenital disorder with
an incidence of 1 in 5000 live births, characterised by the absence of
parasympathetic intramural ganglion cells in the hindgut resulting in intestinal
obstruction in neonates and severe constipation in infants and adults.
Intestinal neuronal dysplasia (IND) shares clinical features with HSCR but the
submucosal parasympathetic plexus is affected. IND has been proposed as one of
the most frequent causes of chronic constipation and is often associated with
HSCR.
METHODS: We examined 29 patients diagnosed with sporadic HSCR, 20 patients with
IND, and 12 patients with mixed HSCR/IND for mutations in the coding regions of
the RET, GDNF, EDNRB, and EDN3 genes. The entire coding regions were analysed by
single strand conformational polymorphism and DNA sequencing.
RESULTS: Only three RET mutations were detected in patients with HSCR. In
patients with IND or a mixed HSCR/IND phenotype, no mutations in these genes
were observed. While HSCR and HSCR/IND showed over representation of a specific
RET polymorphism in exon 2, IND exhibited a significantly lower frequency
comparable with that of controls.
CONCLUSIONS: The mutation frequency found in our sporadic HSCR patients (10%)
and the allelic distribution of RET polymorphisms are comparable with earlier
published data. A significantly different allelic distribution in an established
HSCR associated polymorphism argues against common genetic pathways for HSCR and
IND. The study of vertebrate pigmentary anomalies has greatly improved our
understanding of melanocyte biology. One such disorder, Waardenburg syndrome
(WS), is a mendelian trait characterized by hypopigmentation and sensorineural
deafness. It is commonly subdivided into four types (WS1-4), defined by the
presence or absence of additional symptoms. WS type 4 (WS4), or Shah-Waardenburg
syndrome, is also known as Hirschsprung disease Type II (HSCR II) and is
characterized by an absence of epidermal melanocytes and enteric ganglia.
Mutations in the genes encoding the endothelin type-B receptor (EDNRB) and its
physiological ligand endothelin 3 (EDN3) are now known to account for the
majority of HSCR II patients. Null mutations in the mouse genes Ednrb and Edn3
have identified a key role for this pathway in the normal development of
melanocytes and other neural crest-derived lineages. The pleiotropic effects of
genes in this pathway, on melanocyte and enteric neuron development, have been
clarified by the embryologic identification of their common neural crest (NC)
ancestry. EDNRB and EDN3 are transiently expressed in crest-derived melanoblast
and neuroblast precursors, and in the surrounding mesenchymal cells,
respectively. The influence of EDNRB-mediated signaling on the emigration,
migration, proliferation, and differentiation of melanocyte and enteric neuron
precursors, in vivo and in vitro has recently been the subject of great
scrutiny. A major emergent theme is that EDN3-induced signaling prevents the
premature differentiation of melanocyte and enteric nervous system precursors
and is essential between 10 and 12.5 days post-coitum. We review the present
understanding of pigment cell development in the context of EDNRB/EDN3--a
receptor-mediated pathway with pleiotropic effects. BACKGROUND: Hirschsprung's disease (HSCR) is one the most common congenital
intestinal disease. It leads to aganglionic megacolon in the early childhood.
Several susceptibility genes have been identified : RET protooncogene and its
ligand, glial cell derived neutrophic factor (GDNF), Sox 10, Endothelin-3 (EDN3)
and its receptor B (EDNRB). EDNRB mutations are found in 5% of familial or
sporadic HSCR. Only few EDNRB mutations found in HSCR have been explored and
some of them seem to be non fonctional variants.
MATERIALS AND METHODS: The properties of three mutant human endothelin B
receptor (hETB) (G57S, R319W and P383L) in isolated HSCR were analyzed. Stable
recombit cells expressing the three mutants and the wild-type (WT) were
established. The hETB receptors were characterized for 125I ET-1 binding, ET-1
induced signaling: calcium transient, AP-1 transcriptional factor activation and
cAMP accumulation.
RESULTS: Immunofluorescence experiments showed normal cellular distributions of
the mutant G57S, R319W and WT hETB receptors. In contrast, the P383L hETB mutant
receptor was concentrated near the nucleus and essentially no ET-1 binding was
detected. The two other mutants (G57S and R319W) bound ET-1 normally, induced
calcium transients and activated the AP-1 pathway in the same way as wild type,
but did not inhibit adenylate cyclase. The G57S hETB mutant even stimulated cAMP
accumulation which was blocked by pertussis toxin.
CONCLUSION: The absence of the P383L mutant receptor from the membrane clearly
indicates that this mutation could be involved in HSCR. The G57S and R319W
mutant receptors, despite their normal coupling to Gaq, have a defect in the
Galphai signaling pathway and the G57S mutation couples to Galphas. These
observations allow us to hypothesize that cAMP signaling might be involved in
the differenciation of neural cells in the bowel. Hirschsprung disease is a developmental disorder resulting from the arrest of
the craniocaudal migration of enteric neurons from the neural crest along
gastrointestinal segments of variable length; see Behrman [Nelson textbook of
pediatrics, 1992:954-956]. It is a heterogeneous disorder in which familial
cases map to at least three loci whose function is necessary for normal neural
crest-derived cell development. Homozygous mutations in the endothelin-B
receptor gene (EDNRB) on 13q22 have been identified in humans and mice with
Hirschsprung disease type 2 (HSCR2). The auditory pigmentary disorder,
Waardenburg-Shah syndrome, comprises Waardenburg syndrome and Hirschsprung
disease and has also been mapped to the EDNRB locus. Hirschsprung disease,
malrotation, isochromia, a profound sensorineural hearing loss, and several
other anomalies were found in an infant with an interstitial deletion of 13q,
suggesting the existence of a contiguous gene syndrome involving developmental
genes necessary for the normal growth of the neural crest derivatives of the
eye, inner ear, and colon. We report on an additional patient with a deletion in
13q and Hirschsprung disease. Congenital anomalies associated with deletions of
the distal long arm of chromosome 13 are sufficiently consistent to suggest a
clinical syndrome. Genetic studies of Hirschsprung disease, a common congenital malformation, have
identified eight genes with mutations that can be associated with this
condition. Mutations at individual loci are, however, neither necessary nor
sufficient to cause clinical disease. We conducted a genome-wide association
study in 43 Mennonite family trios using 2,083 microsatellites and
single-nucleotide polymorphisms and a new multipoint linkage disequilibrium
method that searches for association arising from common ancestry. We identified
susceptibility loci at 10q11, 13q22 and 16q23; the gene at 13q22 is EDNRB,
encoding a G protein-coupled receptor (GPCR) and the gene at 10q11 is RET,
encoding a receptor tyrosine kinase (RTK). Statistically significant joint
transmission of RET and EDNRB alleles in affected individuals and
non-complementation of aganglionosis in mouse intercrosses between Ret null and
the Ednrb hypomorphic piebald allele are suggestive of epistasis between EDNRB
and RET. Thus, genetic interaction between mutations in RET and EDNRB is an
underlying mechanism for this complex disorder. BACKGROUND: Hirschsprung's disease (HSCR) is a congenital disorder characterised
by an absence of ganglion cells in the nerve plexuses of the lower digestive
tract. Manifestation of the disease has been linked to mutations in genes that
encode the crucial signals for the development of the enteric nervous system-the
RET and EDNRB signalling pathways. The Phox2b gene is involved in neurogenesis
and regulates Ret expression in mice, in which disruption of the Phox2b results
in a HSCR-like phenotype.
AIMS: To investigate the contribution of PHOX2B to the HSCR phenotype.
METHODS: Using polymerase chain reaction amplification and direct sequencing, we
screened PHOX2B coding regions and intron/exon boundaries for mutations and
polymorphisms in 91 patients with HSCR and 71 ethnically matched controls.
Seventy five HSCR patients with no RET mutations were independently considered.
Haplotype and genotype frequencies were compared using the standard case control
statistic.
RESULTS: Sequence analysis revealed three new polymorphisms: two novel single
nucleotide polymorphisms (A-->G(1364); A-->C(2607)) and a 15 base pair deletion
(DEL(2609)). Statistically significant differences were found for A-->G(1364).
Genotypes comprising allele G were underrepresented in patients (19% v 36%;
chi(2)=9.30; p=0.0095 and 22% v 36%; chi(2)=7.38; p=0.024 for patients with no
RET mutations). Pairwise linkage disequilibrium (LD) analysis revealed no LD
between physically close polymorphisms indicating a hot spot for recombination
in exon 3.
CONCLUSION: The PHOX2B A-->G(1364) polymorphism is associated with HSCR. Whether
it directly contributes to disease susceptibility or represents a marker for a
locus in LD with PHOX2B needs further investigation. Our findings are in
accordance with the involvement of PHOX2B in the signalling pathways governing
the development of enteric neurones. BACKGROUND: Hirschsprung disease (HSCR) is a congenital disorder characterized
by an absence of ganglion cells in the nerve plexuses of the lower digestive
tract. HSCR has a complex pattern of inheritance and is sometimes associated
with mutations in genes of the receptor tyrosine kinase (RET) and endothelin
receptor B (EDNRB) signaling pathways, which are crucial for development of the
enteric nervous system.
METHODS: Using PCR amplification and direct sequencing, we screened for
mutations and polymorphisms in the coding regions and intron/exon boundaries of
the RET, GDNF, EDNRB, and EDN3 genes of 84 HSCR patients and 96 ethnically
matched controls.
RESULTS: We identified 10 novel and 2 previously described mutations in RET, and
4 and 2 novel mutations in EDNRB and in EDN3, respectively. Potential
disease-causing mutations were detected in 24% of the patients. The overall
mutation rate was 41% in females and 19% in males (P = 0.06). RET mutations
occurred in 19% of the patients. R114H in RET was the most prevalent mutation,
representing 7% of the patients or 37% of the patients with RET mutations. To
date, such a high frequency of a single mutation has never been reported in
unrelated HSCR patients. Mutations in EDNRB, EDN3, and GDNF were found in four,
two, and none of the patients, respectively. Two patients with mutations in
genes of the EDNRB pathway also harbored a mutation in RET. Three novel and
three reported polymorphisms were found in EDNRB, EDN3, and GDNF.
CONCLUSION: This study identifies additional HSCR disease-causing mutations,
some peculiar to the Chinese population, and represents the first comprehensive
genetic analysis of sporadic HSCR disease in Chinese. Cumulative evidence suggests that Hirschsprung disease (HSCR) is the consequence
of multiple gene interactions that modulate the ability of enteric neural crest
(NC) cells to populate the developing gut. One of the essential genes for this
process is the NC transcription factor Sox10. Sox10Dom mice on a mixed genetic
background show variation in penetrance and expressivity of enteric
aganglionosis that are analogous to the variable aganglionosis seen in human
HSCR families. The phenotype of Sox10Dom mice in congenic lines indicates this
variation arises from modifiers in the genetic background. To determine whether
known HSCR susceptibility loci are acting as modifiers of Sox10, we tested for
association between genes in the endothelin signaling pathway (EdnrB, Edn3,
Ece1) and severity of aganglionosis in an extended pedigree of B6C3FeLe.Sox10Dom
mice. Single locus association analysis in this pedigree identifies interaction
between EdnrB and Sox10. Additional analysis of F2 intercross progeny confirms a
highly significant effect of EdnrB alleles on the Sox10Dom/+ phenotype. The
presence of C57BL/6J alleles at EdnrB is associated with increased penetrance
and more severe aganglionosis in Sox10Dom mutants. Crosses between EdnrB and
Sox10 mutants corroborate this gene interaction with double mutant progeny
exhibiting significantly more severe aganglionosis. The background strain of the
EdnrB mutant further influences the phenotype of Sox10/EdnrB double mutant
progeny implying the action of additional modifiers on this phenotype. Our data
demonstrates that Sox10-EdnrB interactions can influence development of the
enteric nervous system in mouse models and suggests that this interaction could
contribute to the epistatic network producing variation between patients with
aganglionosis. The endothelin signaling pathway plays a crucial role in melanocyte
differentiation and migration. In this study, we investigated whether germline
mutations of endothelin receptor B (EDNRB), a gene involved in Hirschsprung
disease (HSCR), could also predispose for maligt melanoma (MM). The coding
region of EDNRB was sequenced in 137 MM patients and in 130 ethnically matched
Caucasian control subjects. Six nonsynonymous EDNRB variants were found in 15
patients (11%), but only two were found in four control subjects (3%, odds ratio
[OR] = 3.87, 95% confidence interval [CI] = 1.25 to 12; P = .012). Overall, 14
out of 15 MM patients carried EDNRB mutations reported in HSCR, some of which
had previously been shown to lead to loss of function. In multivariable logistic
regression analysis including skin type, eye and hair color, number of nevi, and
dorsal lentigines (freckles), the association between EDNRB mutations and MM
risk remained statistically significant (OR = 19.9, 95% CI = 1.34 to 296.2; P =
.03). Our data strongly suggest that EDNRB is involved in predisposition for two
different multigenic disorders, HSCR and melanoma. Endothelin receptor B (Ednrb) plays a critical role in the development of
melanocytes and neurons and glia of the enteric nervous system. These distinct
neural crest-derived cell types express Ednrb and share the property of
intercalating into tissues, such as the intestine whose muscle precursor cells
also express Ednrb. Such widespread Ednrb expression has been a significant
obstacle in establishing precise roles for Ednrb in development. We describe
here the production of an Ednrb allele floxed at exon 3 and its use in excising
the receptor from mouse neural crest cells by use of Cre-recombinase driven by
the Wnt1 promoter. Mice born with neural crest-specific excision of Ednrb
possess aganglionic colon, lack trunk pigmentation, and die within 5 weeks due
to megacolon. Ednrb receptor expression in these animals is absent only in the
neural crest but present in surrounding smooth muscle cells. The absence of
Ednrb from crest cells also results in a compensatory upregulation of Ednrb
expression in other cells within the gut. We conclude that Ednrb loss only in
neural crest cells is sufficient to produce the Hirschsprungs disease phenotype
observed with genomic Ednrb mutations. PURPOSE: Hirschsprung disease is characterized by the absence of intramural
ganglion cells in the myenteric and submucosal plexuses within distal intestine,
because of a fail in the enteric nervous system formations process.
Endothelin-3-endothelin receptor B signaling pathway is known to play an
essential role in this process. The aim of this study was to evaluate the
implication of the EDN3 and EDNRB genes in a series of patients with
Hirschsprung disease from Spain and determinate their mutational spectrum.
METHODS: We performed the mutational screening of both genes in 196 patients
with Hirschsprung disease using denaturing high-performance liquid
chromatography technology. A case-control study using TaqMan Technology was also
carried out to evaluate some common polymorphisms and haplotypes as
susceptibility factors for Hirschsprung disease.
RESULTS: Besides several novel mutations in both genes, we found a truncating
mutation in an alternative isoform of EDNRB. Interestingly, we obtained an
overrepresentation of a specific EDN3 haplotype in cases versus controls.
CONCLUSIONS: Our results suggest that the isoform EDNRB Delta 3 might be playing
an essential role in the formation of enteric nervous system. In addition, based
on the haplotype distribution, EDN3 might be considered as a common
susceptibility gene for sporadic Hirschsprung disease in a low-penetrance
fashion. Enteric nervous system (ENS) development is relevant to Hirschsprung's disease
(HSCR; congenital aganglionosis of the terminal bowel), which is still
imperfectly treated. Mutations in genes encoding the RET receptor tyrosine
kinase and endothelin receptor type B (EDNRB) are involved in HSCR pathogenesis;
however, also important in ENS development are molecules that mediate events
that are more restricted than those of RET and EDNRB, act later in development
and which might not be HSCR-associated. Examples are molecules that function in
the guidance of enteric neural crest-derived cells (ENCDCs) and vagal axons, and
in regulating the terminal differentiation of enteric neurons from ENCDCs. It is
probable that highly prevalent disorders of gastrointestinal sensation and
motility result from subtle defects in ENS development. Hirschsprung disease (HSCR) is thought to result as a consequence of multiple
gene interactions that modulate the ability of enteric neural crest cells to
populate the developing gut. However, it remains unknown whether the single
complete deletion of important HSCR-associated genes is sufficient to result in
HSCR disease. In this study, we found that the null mutation of the Ednrb gene,
thought indispensable for enteric neuron development, is insufficient to result
in HSCR disease when bred onto a different genetic background in rats carrying
Ednrb(sl) mutations. Moreover, we found that this mutation results in serious
congenital sensorineural deafness, and these strains may be used as ideal models
of Waardenburg Syndrome Type 4 (WS4). Furthermore, we evaluated how the same
changed genetic background modifies three features of WS4 syndrome,
aganglionosis, hearing loss, and pigment disorder in these congenic strains. We
found that the same genetic background markedly changed the aganglionosis, but
resulted in only slight changes to hearing loss and pigment disorder. This
provided the important evidence, in support of previous studies, that different
lineages of neural crest-derived cells migrating along with various pathways are
regulated by different signal molecules. This study will help us to better
understand complicated diseases such as HSCR and WS4 syndrome. |
What is known about the effectiveness of electronic food diaries ? | Electronic dietary records were better than food diaries in terms of fat percentage reduction in our trials, indicating that teledietetics increases healthy-eating awareness. | BACKGROUND: Increasing healthcare costs related to lifestyle-related chronic
diseases require new solutions. Research on self-management tools is expanding
and many new tools are emerging. Recording food intake is a key functionality in
many of these tools. Nutrition monitoring is a relevant method to gain an
overview of factors influencing health. However, keeping a food diary often
constitutes a challenge for a patient, and developing a user-friendly and useful
electronic food diary is not straightforward.
PURPOSE: To gain insight into the existing approaches to recording food intake,
and to analyze current functionalities and input methods.
METHODS: We searched digital libraries, vendor markets and social networks
focusing on nutrition. Selection criteria were publications written in English,
and patient-oriented tools that offered recording of food intake or nutrition.
The system properties that we searched for were types of data, types of
terminal, target population, and types of reports and sharing functionalities.
We summarized the properties based on their frequency in the reviewed sample.
RESULTS: 31 publications met the selection criteria. The majority of the
identified food recording systems (67%) facilitated entry of food type and the
consumed quantity of food; 16% of the systems were able to record more than one
type of data. The three most frequent target populations were people with
obesity, diabetes and overweight. Mobile phones were used as terminals in 35% of
the cases, personal computers (PCs) in 29%, and personal digital assistants in
23%. Only 10% supported both PCs and mobile phones. Data sharing was provided by
71% and reports by 51% of the systems. We searched for apps in Google Play and
the Apple Store and tested 45 mobile applications that stored food intake data,
of which 62% supported recording of types of food, 24% recording of carbohydrate
intake and 15% recording of calorie intake. The majority of the mobile
applications offered some kind of reports and data sharing, mainly via All of
the tested social-network-enabled applications supported access from a personal
computer and a mobile phone, search in a food database, reports, graphical
presentation, listing of favorite foods, overview of own meals, and entering of
consumed food type and quantity.
CONCLUSION: The analyzed apps reflected a variety of approaches to recording
food intake and nutrition using different terminals--mostly mobile phones (35%),
followed by PCs (29%) and PDAs (23%) for older studies, designed mainly for
users with obesity (45%), diabetes mellitus (42%) and overweight (32%), or
people who want to stay healthy (10%). The majority of the reviewed applications
(67%) offered only input of food type and quantity. All approaches (n=31),
except for two, relied on manual input of data, either by typing or by selecting
a food type from a database. The exceptions (n=2) used a barcode scanning
function. Users of mobile phone applications were not limited to data recording,
but could view their data on the screen and send it via email. The tested web
applications offered similar functionalities for recording food intake. The
systems studied provided some degree of personalization: users can access some
systems via PCs or mobile phones and they can choose among various types of data
input content for recording food intake. Many functions, such as search in a
food database, reports, graphical presentation, listing of favorite foods, and
overview of the user's own meals, are optimized to simplify the recording
process and save time. Data sharing and reports are common features of the
reviewed systems. However, none use the user's recorded food history to make
suggestions on new nutritional intake, during the food recording process. This
may be an area for future research. BACKGROUND: Weight reduction without behavioral modification is not sustainable.
However, with a technology application such as teledietetics, the recording
process could be a cognitive cue for individuals to change their eating
behavior. This study tested obese participants to determine whether
teledietetics shows better results in weight reduction.
STUDY DESIGN AND METHODS: We conducted a double-blinded randomized controlled
trial. The participants in the food diary (FD) and electronic diary (ED) groups
recorded their dietary intakes in logbooks and on an electronic diary system,
respectively. The participants in the control group (CG) did nothing. Subjects
were adults 20-60 years of age with a body mass index (BMI) of ≥25 kg/m(2). The
ED and FD groups were the intervention groups and were compared with the CG
group. The participants' body weights, BMIs, fat percentages, waist-to-hip
ratios (WHRs), and mean arterial pressures (MAPs) were measured before the
study, at Week 6, and at Week 12. Demographic data were collected using
self-administered questionnaires. A chi-squared test and descriptive statistics
were used to describe the demographic and biomeasurement data. Repeated-measures
analysis of variance was used to evaluate the effectiveness of the three groups
over time.
RESULTS: Significant decreases in body weight (F1.705,86.950=20.508, p<0.001)
and BMI (F1.657, 84.486=21.256, p<0.001) and insignificant decreases in fat
percentage (F2,94=0.547, p=0.581), WHR (F1.785,91.052=2.888, p=0.067), and MAP
(F2,94=7.542, p=0.0001) were observed among the three measurement times.
CONCLUSIONS: Electronic dietary records were better than food diaries in terms
of fat percentage reduction in our trials, indicating that teledietetics
increases healthy-eating awareness. |
Can the apoptosis regulator BAX trigger the release of cytochrome c? | Yes, altered Bax conformation trigger its redistribution from the cytosol to mitochondria. Subsequently, cytochrome c is released from mitochondria to cytosol. | Encephalitis induced by reovirus serotype 3 (T3) strains results from the
apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated
in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic
apoptosis signaling during encephalitis is largely unknown. Bax plays a key role
in intrinsic apoptotic signaling in neurons by allowing the release of
mitochondrial cytochrome c. We found Bax activation and cytochrome c release in
neurons following infection of neonatal mice with T3 reoviruses. Bax(-/-) mice
infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue
injury and decreased apoptosis, despite viral replication that is similar to
that in wild-type (WT) Bax(+/+) mice. In contrast, in the heart, T3A-infected
Bax(-/-) mice have viral growth, caspase activation, and injury comparable to
those in WT mice, indicating that the role of Bax in pathogenesis is organ
specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax(-/-) mice had delayed
disease and enhanced survival compared to WT mice. T3D-infected Bax(-/-) mice
had significantly lower viral titers and levels of activated caspase 3 in the
brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac
release occurred in some reovirus-infected neurons in the absence of Bax;
however, this was clearly reduced compared to levels seen in Bax(+/+) wild-type
mice, indicating that Bax is necessary for efficient activation of proapoptotic
mitochondrial signaling in infected neurons. Our studies suggest that Bax is
important for reovirus growth and pathogenesis in neurons and that the intrinsic
pathway of apoptosis, mediated by Bax, is important for full expression of
disease, CNS tissue injury, apoptosis, and viral growth in the CNS of
reovirus-infected mice. Apoptosis is frequently induced to inhibit virus replication during infection of
Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt
pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle.
The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway
remains to be elucidated. In this study, we demonstrated that EV71 infection
altered Bax conformation and triggered its redistribution from the cytosol to
mitochondria in RD cells. Subsequently, cytochrome c was released from
mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was
activated during EV71 infection. The JNK specific inhibitor significantly
inhibited Bax activation and cytochrome c release, suggesting that EV71-induced
apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt
phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt
pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71
infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating
kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1
significantly decreased JNK phosphorylation, which implied that ASK1
phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively,
these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated
apoptosis by phosphorylating and inactivating ASK1 during EV71 infection. 6-Shogaol, a potent bioactive compound in ginger (Zingiber officinale Roscoe),
has been reported for anti-inflammatory and anti-cancer activity. In this study,
we investigated the effect of 6-shogaol to enhance tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-mediated apoptosis. The combined treatment
with 6-shogaol and TRAIL markedly induces apoptosis in various cancer cells
(renal carcinoma Caki cells, breast carcinoma MDA-MB-231 cells and glioma U118MG
cells), but not in normal mesangial cells and normal mouse kidney cells.
6-Shogaol reduced the mitochondrial membrane potential (MMP) and released
cytochrome c from mitochondria to cytosol via Bax activation. Furthermore, we
found that 6-shogaol induced down-regulation of c-FLIP(L) expression at the
post-translational levels and the overexpression of c-FLIP(L) markedly inhibited
6-shogaol plus TRAIL-induced apoptosis. Moreover, 6-shogaol increased reactive
oxygen species (ROS) production in Caki cells. Pretreatment with ROS scavengers
attenuated 6-shogaol plus TRAIL-induced apoptosis through inhibition of MMP
reduction and down-regulation of c-FLIP(L) expression. In addition, 6-gingerol,
another phenolic alkanone isolated from ginger, did not enhance TRAIL-induced
apoptosis and down-regulate c-FLIP(L) expression. Taken together, our results
demonstrated that 6-shogaol enhances TRAIL-mediated apoptosis in renal carcinoma
Caki cells via ROS-mediated cytochrome c release and down-regulation of
c-FLIP(L) expression. |
Which biological process in known as Endoplasmic Reticulum-Associated Protein Degradation (ERAD)? | Endoplasmic reticulum-associated protein degradation (ERAD) is the quality control system in the endoplasmic reticulum of eukaryotic cells which ensures that newly synthesized proteins that fail to fold into the correct conformation or unassembled orphan subunits of oligomeric proteins are rapidly eliminated by proteolytic degradation. This entails the export of proteins from the endoplasmic reticulum to the cytosol followed by their destruction by the cytosolic ubiquitin/proteasome pathway. While this mechanism effectively prevents the cellular accumulation of non-functional or unwanted endogenous proteins, it renders the cell vulnerable to certain viruses and toxins that are able to subvert this degradative mechanism for their own advantage. | Until recently, the degradation of aberrant and unassembled proteins retained in
the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized
proteases. We now show that the ER-associated degradation (ERAD) of two mutant
proteins that accumulate in the ER lumen is inhibited in a proteasome-defective
yeast strain and when cytosol from this mutant is used in an in vitro assay. In
addition, ERAD is limited in vitro in the presence of the proteasome inhibitors,
3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD
substrate is exported from ER-derived microsomes, and the accumulation of
exported substrate is 2-fold greater when proteasome mutant cytosol is used in
place of wild-type cytosol. We conclude that lumenal ERAD substrates are
exported from the yeast ER to the cytoplasm for degradation by the proteasome
complex. The quality control system in the endoplasmic reticulum of eukaryotic cells
ensures that newly synthesized proteins that fail to fold into the correct
conformation or unassembled orphan subunits of oligomeric proteins are rapidly
eliminated by proteolytic degradation. This entails the export of proteins from
the endoplasmic reticulum to the cytosol followed by their destruction by the
cytosolic ubiquitin/proteasome pathway. While this mechanism effectively
prevents the cellular accumulation of non-functional or unwanted endogenous
proteins, it renders the cell vulnerable to certain viruses and toxins that are
able to subvert this degradative mechanism for their own advantage. Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and
misfolded proteins may be retained and targeted for ER-associated protein
degradation (ERAD). To elucidate the mechanism by which an integral membrane
protein in the ER is degraded, we studied the fate of the cystic fibrosis
transmembrane conductance regulator (CFTR) in the yeast Saccharomyces
cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in
strains defective for proteasome activity or deleted for the
ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a
bona fide ERAD substrate in yeast. We also found that heat shock protein 70
(Hsp70), although not required for the degradation of soluble lumenal ERAD
substrates, is required to facilitate CFTR turnover. Conversely, calnexin and
binding protein (BiP), which are required for the proteolysis of ER lumenal
proteins in both yeast and mammals, are dispensable for the degradation of CFTR,
suggesting unique mechanisms for the disposal of at least some soluble and
integral membrane ERAD substrates in yeast. Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant
proteins in the secretory pathway. Protein substrates of ERAD are dislocated via
the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they
are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is
also responsible for import of nascent proteins, this bidirectional passage
should be coordinated, probably by molecular chaperones. Here we implicate the
cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of
mammalian p97 and its yeast homologue Cdc48p in complexes with two respective
ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in
yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived
ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The
involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD
substrates in the endoplasmic reticulum when Cdc48p fails to function, as
monitored by activation of the unfolded protein response. We propose that the
role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and
multiubiquitin binding capacity, is to act at the cytosolic face of the
endoplasmic reticulum and to chaperone dislocation of ERAD substrates and
present them to the proteasome. Proteins that fail to fold properly as well as constitutive or regulated
short-lived proteins of the endoplasmic reticulum are subjected to proteolysis
by cytosolic 26S proteasomes. This process is known as endoplasmic
reticulum-associated protein degradation. In order to become accessible to the
proteasome of this system substrates must first be retrogradely transported from
the endoplasmic reticulum into the cytosol, in a process termed dislocation.
This export step seems to be accompanied by polyubiquitination of such
molecules. Surprisingly, protein dislocation from the endoplasmic reticulum
seems to require at least some components that mediate import into this
compartment. However, protein import and export display differences in the
mechanism that provides the driving force and ensures directionality. Of special
interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for
the degradation of various endoplasmic reticulum-associated protein degradation
substrates and seems to function in a step after polyubiquitination but before
proteasomal digestion. In this review, we will summarize our knowledge on
protein export during endoplasmic reticulum-associated protein degradation and
discuss the possible function of certain components involved in this process. Proteins that fail to fold properly as well as constitutive or regulated
short-lived proteins of the endoplasmatic reticulum (ER) are subjected to
proteolysis by cytosolic 26 S proteasomes. This process, termed ER-associated
protein degradation (ERAD), has also been implicated in the generation of some
important human disorders, for example, cystic fibrosis. To become accessible to
the proteasome, ERAD substrates must first be retrogradely transported from the
ER into the cytosol, in a process termed dislocation. Surprisingly, protein
dislocation from the ER seems to require at least some components that also
mediate import into this compartment. Moreover, polyubiquitination of ERAD
substrates at the ER membrane as well as the cytoplasmic Cdc48p/Npl4p/Ufd1p
complex were shown to contribute to this export reaction. In this article we
will summarize our current knowledge on ERAD and discuss the possible function
of certain components involved in this process. Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a
pattern for assembly are often disposed of by a process named ER-associated
degradation (ERAD), which involves transport of the substrate protein across the
ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis.
Different ERAD substrates have been shown to be ubiquitinated during or soon
after dislocation, and an active ubiquitination machinery has been found to be
required for the dislocation of certain defective proteins. We have previously
shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A
chain of the heterodimeric toxin ricin is degraded by a pathway that closely
resembles ERAD but is characterized by an unusual uncoupling between the
dislocation and the degradation steps. Since lysine (Lys) residues are a major
target for ubiquitination, we have investigated the effects of changing the Lys
content on the retrotranslocation and degradation of ricin A chain in tobacco
protoplasts. Here we show that modulating the number of Lys residues does not
affect recognition events within the ER lumen nor the transport of the protein
from this compartment to the cytosol. Rather, the introduced modifications have
a clear impact on the degradation of the dislocated protein. While the
substitution of the two Lys residues present in ricin A chain with arginine
slowed down degradation, the introduction of four extra lysyl residues had an
opposite effect and converted the ricin A chain to a standard ERAD substrate
that is disposed via a process in which dislocation and degradation steps are
tightly coupled. The endoplasmic reticulum (ER) quality control processes recognize and remove
aberrant proteins from the secretory pathway. Several variants of the plasma
protein fibrinogen are recognized as aberrant and degraded by ER-associated
protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of
patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant
fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a
substitution of Arg375 to Trp in the gamma-chain. To understand the cellular
mechanisms behind clearance of the aberrant Aguadilla gamma-chain, we expressed
the mutant gammaD domain in yeast and found that it was cleared from the ER via
ERAD. In addition, we discovered that when ERAD was saturated, aggregated
Aguadilla gammaD accumulated within the ER while a soluble form of the
polypeptide transited the secretory pathway to the trans-Golgi network where it
was targeted to the vacuole for degradation. Examination of Aguadilla gammaD in
an autophagy-deficient yeast strain showed stabilization of the aggregated ER
form, indicating that these aggregates are normally cleared from the ER via the
autophagic pathway. These findings have clinical relevance in the understanding
of and treatment for ER storage diseases. Endoplasmic reticulum-associated protein degradation (ERAD) is a protein quality
control mechanism that minimizes the detrimental effects of protein misfolding
in the secretory pathway. Molecular chaperones and ER lumenal lectins are
essential components of this process because they maintain the solubility of
unfolded proteins and can target ERAD substrates to the cytoplasmic proteasome.
Other factors are likely required to aid in the selection of ERAD substrates,
and distinct proteinaceous machineries are required for substrate
retrotranslocation/dislocation from the ER and proteasome targeting. When the
capacity of the ERAD machinery is exceeded or compromised, multiple degradative
routes can be enlisted to prevent the detrimental consequences of ERAD substrate
accumulation, which include cell death and disease. Quality control mechanisms in the endoplasmic reticulum prevent deployment of
aberrant or unwanted proteins to distal destinations and target them to
degradation by a process known as endoplasmic reticulum-associated degradation,
or ERAD. Attempts to characterize ERAD by identifying a specific component have
revealed that the most general characteristic of ERAD is that the protein
substrates are initially translocated to the ER and eventually eliminated in the
cytosol by the ubiquitin-proteasome pathway. Hence, dislocation from the ER back
to the cytosol is a hallmark in ERAD and p97/Cdc48p, a cytosolic AAA-ATPase that
is essential for ERAD, appears to provide the driving force for this process.
Moreover, unlike many ERAD components that participate in degradation of either
lumenal or membrane substrates, p97/Cdc48p has a more general role in that it is
required for ERAD of both types of substrates. Although p97/Cdc48p is not
dedicated exclusively to ERAD, its ability to physically associate with ERAD
substrates, with VIMP and with the E3 gp78 suggest that the p97/Cdc48Ufdl/Npl4
complex acts as a coordinator that maintains coupling between the different
steps in ERAD. Terminally misfolded or unassembled proteins are degraded by the cytoplasmic
ubiquitin-proteasome pathway in a process known as ERAD (endoplasmic
reticulum-associated protein degradation). Overexpression of ER
alpha1,2-mannosidase I and EDEMs target misfolded glycoproteins for ERAD, most
likely due to trimming of N-glycans. Here we demonstrate that overexpression of
Golgi alpha1,2-mannosidase IA, IB, and IC also accelerates ERAD of terminally
misfolded human alpha1-antitrypsin variant null (Hong Kong) (NHK), and mannose
trimming from the N-glycans on NHK in 293 cells. Although transfected NHK is
primarily localized in the ER, some NHK also co-localizes with Golgi markers,
suggesting that mannose trimming by Golgi alpha1,2-mannosidases can also
contribute to NHK degradation. Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process
known as ER-associated degradation (ERAD), which starts with misfolded protein
recognition, followed by ubiquitination, retrotranslocation to the cytosol,
deglycosylation, and targeting to the proteasome for degradation. Actions of
multisubunit protein machineries in the ER membrane integrate these steps. We
hypothesized that regulation of the multisubunit machinery assembly is a
mechanism by which ERAD activity is regulated. To test this hypothesis, we
investigated the potential regulatory role of the small p97/VCP-interacting
protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin
ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that
SVIP is anchored to microsomal membrane via myristoylation and co-fractionated
with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also
physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks
unassembled CD3delta from association with gp78 and p97/VCP, which is
accompanied by decreases in CD3delta ubiquitination and degradation. Silencing
SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex,
which correlates with increased degradation of CD3delta and misfolded Z variant
of alpha-1-antitrypsin, established substrates of gp78. These results suggest
that SVIP is an endogenous inhibitor of ERAD that acts through regulating the
assembly of the gp78-p97/VCP-Derlin1 complex. Recognition and elimination of misfolded proteins are essential cellular
processes. More than thirty percent of the cellular proteins are proteins of the
secretory pathway. They fold in the lumen or membrane of the endoplasmic
reticulum from where they are sorted to their site of action. The folding
process, as well as any refolding after cell stress, depends on chaperone
activity. In case proteins are unable to acquire their native conformation,
chaperones with different substrate specificity and activity guide them to
elimination. For most misfolded proteins of the endoplasmic reticulum this
requires retro-translocation to the cytosol and polyubiquitylation of the
misfolded protein by an endoplasmic reticulum associated machinery. Thereafter
ubiquitylated proteins are guided to the proteasome for degradation. This review
summarizes our up to date knowledge of chaperone classes and chaperone function
in endoplasmic reticulum associated degradation of protein waste. Endoplasmic reticulum-associated degradation (ERAD) is an essential quality
control process whereby misfolded proteins are exported from the endoplasmic
reticulum and degraded by the proteasome in the cytosol. The ATPase p97 acts as
an essential component of this process by providing the force needed for
retrotranslocation and by serving as a processing station for the substrate once
in the cytosol. Proteins containing the ubiquitin regulatory X (UBX)
ubiquitin-like domain function as adaptors for p97 through their direct binding
with the amino terminus of the ATPase. We demonstrate that the UBX protein SAKS1
is able to act as an adaptor for p97 that negatively modulates ERAD. This
requires the ability of SAKS1 to bind both polyubiquitin and p97. Moreover, the
association between SAKS1 and p97 is positively regulated by polyubiquitin
binding of the UBX protein. SAKS1 also negatively impacts the p97-dependent
processing required for degradation of a cytosolic, non-ERAD, substrate. We find
SAKS1 is able to protect polyubiquitin from the activity of deubiquitinases,
such as ataxin-3, that are necessary for efficient ERAD. Thus, SAKS1 inhibits
protein degradation mediated by p97 complexes in the cytosol with a component of
the mechanism being the ability to shield polyubiquitin chains from
ubiquitin-processing factors. The AAA-type ATPase Cdc48 (named p97/VCP in mammals) is a molecular machine in
all eukaryotic cells that transforms ATP hydrolysis into mechanic power to
unfold and pull proteins against physical forces, which make up a protein's
structure and hold it in place. From the many cellular processes, Cdc48 is
involved in, its function in endoplasmic reticulum associated protein
degradation (ERAD) is understood best. This quality control process for proteins
of the secretory pathway scans protein folding and discovers misfolded proteins
in the endoplasmic reticulum (ER), the organelle, destined for folding of these
proteins and their further delivery to their site of action. Misfolded lumenal
and membrane proteins of the ER are detected by chaperones and lectins and
retro-translocated out of the ER for degradation. Here the Cdc48 machinery,
recruited to the ER membrane, takes over. After polyubiquitylation of the
protein substrate, Cdc48 together with its dimeric co-factor complex Ufd1-Npl4
pulls the misfolded protein out and away from the ER membrane and delivers it to
down-stream components for degradation by a cytosolic proteinase machine, the
proteasome. The known details of the Cdc48-Ufd1-Npl4 motor complex triggered
process are subject of this review article. In the secretory pathway, quality control for the correct folding of proteins is
largely occurring in the endoplasmic reticulum (ER), at the earliest possible
stage and in an environment where early folding intermediates mix with
terminally misfolded species. An elaborate cellular mechanism aims at dividing
the former from the latter and promotes the selective transport of misfolded
species back into the cytosol, a step called retrotranslocation. During
retrotranslocation proteins will become ubiquitinated on the cytosolic side of
the ER membrane by dedicated machineries and will be targeted to the proteasome
for degradation. The entire process, from protein recognition to final
degradation, has been named ER-associated protein degradation, or simply ERAD.
Ubiquitin has well known functions in aiding late steps of substrate
retrotranslocation and in targeting substrates to the proteasome. Recent results
show that several cytosolic machineries allow ubiquitinated substrates to
undergo extensive remodeling, or processing, on their poly-ubiquitin chains
(PUCs). Although still ill-defined, PUC processing might have a unique function
for ERAD in that it might provide a mechanism to generate optimal PUCs for
recognition by proteasomal ubiquitin receptors. Ubiquitination might also have a
previously uticipated role in quality control of ER membrane proteins. This
review recapitulates the current knowledge and recent findings about
ERAD-specific roles of ubiquitin. Misfolded proteins in the endoplasmic reticulum (ER) are dislocated out of the
ER to the cytosol, polyubiquitinated, and degraded by the ubiquitin-proteasome
system in a process collectively termed ER-associated degradation (ERAD). Recent
studies have established that a mammalian ER-localized transmembrane J-protein,
DNAJB12, cooperates with Hsc70, a cytosolic Hsp70 family member, to promote the
ERAD of misfolded membrane proteins. Interestingly, mammalian genomes have
another J-protein called DNAJB14 that shows a high sequence similarity to
DNAJB12. Yet, very little was known about this protein. Here, we report the
characterization of DNAJB14. Immunofluorescence study and protease protection
assay showed that, like DNAJB12, DNAJB14 is an ER-localized, single
membrane-spanning J-protein with its J-domain facing the cytosol. We used
co-immunoprecipitation assay to find that DNAJB14 can also specifically bind
Hsc70 via its J-domain to recruit this chaperone to ER membrane. Remarkably, the
overexpression of DNAJB14 accelerated the degradation of misfolded membrane
proteins including a mutant of cystic fibrosis transmembrane conductance
regulator (CFTRΔF508), but not that of a misfolded luminal protein. Furthermore,
the DNAJB14-dependent degradation of CFTRΔF508 was compromised by MG132, a
proteasome inhibitor, indicating that DNAJB14 can enhance the degradation of a
misfolded membrane protein using the ubiquitin-proteasome system. Thus, the
mammalian ER possesses two analogous J-proteins (DNAJB14 and DNAJB12) that both
can promote the ERAD of misfolded transmembrane proteins. Compared with DNAJB12
mRNA that was widely expressed in mouse tissues, DNAJB14 mRNA was expressed more
weakly, being most abundant in testis, implying its specific role in this
tissue. Early secretory and endoplasmic reticulum (ER)-localized proteins that are
terminally misfolded or misassembled are degraded by a ubiquitin- and
proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan
pathogens, including the causative agents of malaria, toxoplasmosis,
trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to
higher eukaryotic cells, and, because of this, we observe that the malaria
parasite Plasmodium falciparum is highly sensitive to the inhibition of
components of this protein quality control system. Inhibitors that specifically
target a putative protease component of ERAD, signal peptide peptidase (SPP),
have high selectivity and potency for P. falciparum. By using a variety of
methodologies, we validate that SPP inhibitors target P. falciparum SPP in
parasites, disrupt the protein's ability to facilitate degradation of unstable
proteins, and inhibit its proteolytic activity. These compounds also show low
omolar activity against liver-stage malaria parasites and are also equipotent
against a panel of pathogenic protozoan parasites. Collectively, these data
suggest ER quality control as a vulnerability of protozoan parasites, and that
SPP inhibition may represent a suitable transmission blocking antimalarial
strategy and potential pan-protozoan drug target. Endoplasmic reticulum-associated protein degradation (ERAD) removes
improperly-folded proteins from the ER membrane into the cytosol where they
undergo proteasomal degradation. Valosin-containing protein (VCP)/p97 mediates
in the extraction of ERAD substrates from the ER. BRSK2 (also known as SAD-A), a
serine/threonine kinase of the AMP-activated protein kinase family affected
VCP/p97 activity in ERAD. In addition, BRSK2 interacted with VCP/p97 via three
of the four functional domains of VCP/p97. Immunofluorescence demonstrated that
BRSK2 and VCP/p97 were co-localized and also that knockdown of endogenous BRSK2
induced increased levels of CD3δ, a substrate in ERAD for VCP/p97. Thus, BRSK2
might affect the activity of VCP/p97 in ERAD. A correct three-dimensional structure is crucial for the physiological functions
of a protein, yet the folding of proteins to acquire native conformation is a
fundamentally error-prone process. Eukaryotic organisms have evolved a highly
conserved endoplasmic reticulum-mediated protein quality control (ERQC)
mechanism to monitor folding processes of secretory and membrane proteins,
allowing export of only correctly folded proteins to their physiological
destinations, retaining incompletely/mis-folded ones in the ER for additional
folding attempts, marking and removing terminally misfolded ones via a unique
multiple-step degradation process known as ER-associated degradation (ERAD).
Most of our current knowledge on ERQC and ERAD came from genetic and biochemical
investigations in yeast and mammalian cells. Recent studies in the reference
plant Arabidopsis thaliana uncovered homologous components and similar
mechanisms in plants for monitoring protein folding and for retaining,
repairing, and removing misfolded proteins. These studies also revealed critical
roles of the plant ERQC/ERAD systems in regulating important
biochemical/physiological processes, such as abiotic stress tolerance and plant
defense. In this review, we discuss our current understanding about the
molecular components and biochemical mechanisms of the plant ERQC/ERAD system in
comparison to yeast and mammalian systems. |
List BRAF inhibitors that have been tested in clinical trials for treatment of melanoma patients | Vemurafenib and dabrafenib are BRAF inhibitors that have been tested in clinical trials for treatment of melanoma patients. | The discovery of activating BRAF V600E mutations in 50% of all melanoma patients
and the development of small molecule BRAF inhibitors looks set to revolutionize
the therapy of disseminated melanoma. However, in the recent clinical trial of
the BRAF inhibitor, vemurafenib (PLX4032), a significant percentage of BRAF
V600E mutant melanoma patients did not meet the RECIST criteria for a response.
Recent work from our lab identified loss of the tumor suppressor phosphatase and
tensin homolog (PTEN) as being a possible mediator of intrinsic BRAF inhibitor
resistance. In this commentary, we describe the development of a novel mass
spectrometry based proteomic screen of Bcl-2 family proteins that was used to
delineate the PTEN-dependent differences in apoptosis signaling observed when
BRAF was inhibited. We further discuss how use of these sensitive quantitative
proteomic methods gives unique insights into the signaling of cancer cells that
are not captured through routine biochemical techniques and how this may lead to
the development of combination therapy strategies for overcoming intrinsic BRAF
inhibitor resistance. BACKGROUND: Phase 1 and 2 clinical trials of the BRAF kinase inhibitor
vemurafenib (PLX4032) have shown response rates of more than 50% in patients
with metastatic melanoma with the BRAF V600E mutation.
METHODS: We conducted a phase 3 randomized clinical trial comparing vemurafenib
with dacarbazine in 675 patients with previously untreated, metastatic melanoma
with the BRAF V600E mutation. Patients were randomly assigned to receive either
vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter
of body-surface area intravenously every 3 weeks). Coprimary end points were
rates of overall and progression-free survival. Secondary end points included
the response rate, response duration, and safety. A final analysis was planned
after 196 deaths and an interim analysis after 98 deaths.
RESULTS: At 6 months, overall survival was 84% (95% confidence interval [CI], 78
to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine
group. In the interim analysis for overall survival and final analysis for
progression-free survival, vemurafenib was associated with a relative reduction
of 63% in the risk of death and of 74% in the risk of either death or disease
progression, as compared with dacarbazine (P<0.001 for both comparisons). After
review of the interim analysis by an independent data and safety monitoring
board, crossover from dacarbazine to vemurafenib was recommended. Response rates
were 48% for vemurafenib and 5% for dacarbazine. Common adverse events
associated with vemurafenib were arthralgia, rash, fatigue, alopecia,
keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and
diarrhea; 38% of patients required dose modification because of toxic effects.
CONCLUSIONS: Vemurafenib produced improved rates of overall and progression-free
survival in patients with previously untreated melanoma with the BRAF V600E
mutation. (Funded by Hoffmann-La Roche; BRIM-3 ClinicalTrials.gov number,
NCT01006980.). BACKGROUND: The sustained clinical activity of the BRAF inhibitor vemurafenib
(PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited
primarily by the development of acquired resistance leading to tumor
progression. Clinical trials are in progress using MEK inhibitors following
disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT
pathway can also induce resistance to the inhibitors of MAPK pathway.
METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity to vemurafenib or the MEK
inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell
lines exploring differences in activation-associated phosphorylation levels of
major signaling molecules, leading to the testing of co-inhibition of the
AKT/mTOR pathway genetically and pharmacologically. There was a high degree of
cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant
cell lines that acquired a secondary mutation in NRAS. In other cell lines,
acquired resistance to both drugs was associated with persistence or increase in
activity of AKT pathway. siRNA-mediated gene silencing and combination therapy
with an AKT inhibitor or rapamycin partially or completely reversed the
resistance.
CONCLUSIONS/SIGNIFICANCE: Primary and acquired resistance to vemurafenib in
these in vitro models results in frequent cross resistance to MEK inhibitors,
except when the resistance is the result of a secondary NRAS mutation.
Resistance to BRAF or MEK inhibitors is associated with the induction or
persistence of activity within the AKT pathway in the presence of these drugs.
This resistance can be potentially reversed by the combination of a RAF or MEK
inhibitor with an AKT or mTOR inhibitor. These combinations should be available
for clinical testing in patients progressing on BRAF inhibitors. Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a
small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in
BRAFV600 mutant melanoma, creating the need for a well-validated companion
diagnostic to select patients for treatment. We describe analytic performance
characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to
select patients for the pivotal vemurafenib trials. This real-time polymerase
chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA
from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed
using blends of cell lines or tumor DNA, and tumor specimens with low levels of
mutant alleles, as determined by 454 sequencing (a quantitative next-generation
pyrosequencing method). A >96% hit rate was obtained across all specimen types
with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from
one 5-μm section. The cobas test showed a higher sensitivity and specificity
than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross
reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by
2 operators, using different instruments and reagent lots, yielded correct calls
in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified
that gave invalid test results. A simple 1:2 dilution resulted in a valid test
result of 76% in such cases. The cobas test is a reproducible assay that detects
some non-V600E mutations and is more accurate than direct sequencing in
detecting BRAFV600E. CONTEXT: A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF
V600 Mutation Test) was recently approved by the US Food and Drug Administration
to select patients with BRAF-mutant metastatic melanoma for treatment with the
BRAF inhibitor vemurafenib.
OBJECTIVES: (1) To compare the analytic performance of the cobas test to Sanger
sequencing by using screening specimens from phase II and phase III trials of
vemurafenib, and (2) to assess the reproducibility of the cobas test at
different testing sites.
DESIGN: Specimens from 477 patients were used to determine positive and negative
percent agreements between the cobas test and Sanger sequencing for detecting
V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel
pyrosequencing method (454) to resolve discordances between polymerase chain
reaction and Sanger results. Reproducibility of the cobas test was assessed at 3
sites by using 3 reagent lots and an 8-member panel of melanoma samples.
RESULTS: A valid cobas result was obtained for all eligible patients. Sanger
sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433
specimens, positive percent agreement was 96.4% (215 of 223) and negative
percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger
V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by
454. The cobas test detected 70% of V600K mutations. In the reproducibility
study, a correct interpretation was made for 100% of wild-type specimens and
specimens with greater than 5% mutant alleles; V600E mutations were detected in
90% of specimens with less than 5% mutant alleles.
CONCLUSIONS: The cobas test (1) had a lower assay failure rate than that of
Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most
V600K mutations, and (4) was highly reproducible. Prognosis for advanced and metastatic melanoma is poor, with a 5-year survival
of 78, 59 and 40% for patients with stage IIIA, IIIB and IIIC, respectively, and
a 1-year survival of 62% for M1a, 53% for M1b and 33% for M1c. The
unsatisfactory results of actual standard therapies for metastatic melanoma
highlight the need for effective new therapeutic strategies. Several drugs,
including BRAF, KIT and MEK inhibitors, are currently being evaluated after
promising data from Phase I and Phase II studies; Vemurafenib, a BRAF-inhibitor
agent, has been approved by the Food and Drug Administration (FDA) for the
treatment of patients with unresectable or metastatic melanoma with the BRAF
V600E mutation after a significant impact on both progression-free and overall
survival was demonstrated compared with dacarbazine in a Phase III trial.
Ipilimumab, an immunotherapeutic drug, has proven to be capable of inducing
long-lasting responses and was approved for patients with advanced melanoma in
first- and second-line treatment by the FDA and in second-line treatment by the
European Medicines Agency. Furthermore, a significant survival benefit of the
combination of ipilimumab with dacarbazine compared with dacarbazine alone for
first-line treatment was reported. In the near future, patients with BRAF
mutations could have the chance to benefit from treatment with BRAF inhibitors;
patients harboring BRAF or NRAS mutations could be treated with MEK inhibitors;
finally, the subgroup of patients with acral, mucosal or chronic sun-damaged
melanoma harboring a KIT mutation could benefit from KIT inhibitors. Ipilimumab
could become a standard treatment for metastatic melanoma, both as a single
agent and in combination; its efficacy has been proven, and researchers should
now address their efforts to understanding the predictive variables of response
to treatment. BACKGROUND: Approximately 50% of melanomas harbor activating (V600) mutations in
the serine-threonine protein kinase B-RAF (BRAF). The oral BRAF inhibitor
vemurafenib (PLX4032) frequently produced tumor regressions in patients with
BRAF V600-mutant metastatic melanoma in a phase 1 trial and improved overall
survival in a phase 3 trial.
METHODS: We designed a multicenter phase 2 trial of vemurafenib in patients with
previously treated BRAF V600-mutant metastatic melanoma to investigate the
efficacy of vemurafenib with respect to overall response rate (percentage of
treated patients with a tumor response), duration of response, and overall
survival. The primary end point was the overall response rate as ascertained by
the independent review committee; overall survival was a secondary end point.
RESULTS: A total of 132 patients had a median follow-up of 12.9 months (range,
0.6 to 20.1). The confirmed overall response rate was 53% (95% confidence
interval [CI], 44 to 62; 6% with a complete response and 47% with a partial
response), the median duration of response was 6.7 months (95% CI, 5.6 to 8.6),
and the median progression-free survival was 6.8 months (95% CI, 5.6 to 8.1).
Primary progression was observed in only 14% of patients. Some patients had a
response after receiving vemurafenib for more than 6 months. The median overall
survival was 15.9 months (95% CI, 11.6 to 18.3). The most common adverse events
were grade 1 or 2 arthralgia, rash, photosensitivity, fatigue, and alopecia.
Cutaneous squamous-cell carcinomas (the majority, keratoacanthoma type) were
diagnosed in 26% of patients.
CONCLUSIONS: Vemurafenib induces clinical responses in more than half of
patients with previously treated BRAF V600-mutant metastatic melanoma. In this
study with a long follow-up, the median overall survival was approximately 16
months. (Funded by Hoffmann-La Roche; ClinicalTrials.gov number, NCT00949702.). BACKGROUND: The development of keratoacanthomas (KAs) and well-differentiated
squamous cell carcinomas (SCCs) is a known adverse effect of novel BRAF
inhibitors such as vemurafenib. With multiple such neoplasms often arising after
BRAF inhibitor therapy, surgical excision is often impractical.
OBSERVATIONS: We describe a patient with stage IV melanoma who received the BRAF
inhibitor vemurafenib (recently approved by the US Food and Drug Administration)
as part of a clinical trial and developed numerous diffuse, pathology-proven KAs
and SCCs. The high number of lesions across a broad area precluded surgical
treatment; instead, a noninvasive field approach using photodynamic therapy
(PDT) was initiated. Compared with untreated tumors, most lesions demonstrated
significant clinical regression following successive cycles of PDT.
CONCLUSIONS: Given vemurafenib's recent approval by the US Food and Drug
Administration, we provide a timely case report on the effective use of PDT in
the treatment of BRAF inhibitor-associated KAs and SCCs. Although further
studies are needed to better understand the biological processes of these
secondary neoplasms, our observation provides an alternative noninvasive
solution for improving the quality of life for patients receiving BRAF inhibitor
therapy. INTRODUCTION: Leptomeningeal metastases are occurring at higher frequency in
cancer patients. The prognosis of leptomeningeal metastases is poor and standard
treatment, which includes radiotherapy and chemotherapy, is mostly ineffective.
Melanoma represents one of the tumors with the highest incidence of
leptomeningeal metastases. For such a disease, the BRAF inhibitors have recently
been demonstrated to be effective on melanoma brain metastases harboring the
V600EBRAF mutation.
CASE PRESENTATION: We report a case of a 39-year-old Italian woman with advanced
melanoma with brain, lung and peritoneum metastases harboring the V600EBRAF
mutation. In August 2010 she was enrolled into the BRIM3 trial and after the
randomization process she received dacarbazine. After two cycles, there was
evidence of disease progression in her peritoneum and lung. For this reason, she
was enrolled into another clinical trial with the GSK2118436 BRAF inhibitor,
dabrafenib, as a second line of therapy. She had a partial response that was
maintained until 13 weeks of treatment. In January 2011 she developed symptoms
typical for brain metastases and received a diagnosis of leptomeningeal
involvement of melanoma cells after an examination of her cerebral spinal fluid;
magnetic resoce imaging was negative for meningitis or brain metastases.
Analysis of her cerebral spinal fluid sample confirmed that the melanoma cells
still carried the V600EBRAF mutation. After a few days, our patient went into a
coma and died.
CONCLUSION: Starting with a clinical case, we discuss the pathogenesis of
leptomeningeal metastases and whether the leptomeninges may represent a
sanctuary where melanoma cells may generate resistance and/or BRAF inhibitors
cannot reach an adequate concentration for significant activity. We assess
whether treatment with BRAF inhibitors in melanoma patients should be
interrupted as soon as disease progression appears or continued beyond
progression, through the administration of additional compounds. BACKGROUND: Resistance to therapy with BRAF kinase inhibitors is associated with
reactivation of the mitogen-activated protein kinase (MAPK) pathway. To address
this problem, we conducted a phase 1 and 2 trial of combined treatment with
dabrafenib, a selective BRAF inhibitor, and trametinib, a selective MAPK kinase
(MEK) inhibitor.
METHODS: In this open-label study involving 247 patients with metastatic
melanoma and BRAF V600 mutations, we evaluated the pharmacokinetic activity and
safety of oral dabrafenib (75 or 150 mg twice daily) and trametinib (1, 1.5, or
2 mg daily) in 85 patients and then randomly assigned 162 patients to receive
combination therapy with dabrafenib (150 mg) plus trametinib (1 or 2 mg) or
dabrafenib monotherapy. The primary end points were the incidence of cutaneous
squamous-cell carcinoma, survival free of melanoma progression, and response.
Secondary end points were overall survival and pharmacokinetic activity.
RESULTS: Dose-limiting toxic effects were infrequently observed in patients
receiving combination therapy with 150 mg of dabrafenib and 2 mg of trametinib
(combination 150/2). Cutaneous squamous-cell carcinoma was seen in 7% of
patients receiving combination 150/2 and in 19% receiving monotherapy (P=0.09),
whereas pyrexia was more common in the combination 150/2 group than in the
monotherapy group (71% vs. 26%). Median progression-free survival in the
combination 150/2 group was 9.4 months, as compared with 5.8 months in the
monotherapy group (hazard ratio for progression or death, 0.39; 95% confidence
interval, 0.25 to 0.62; P<0.001). The rate of complete or partial response with
combination 150/2 therapy was 76%, as compared with 54% with monotherapy
(P=0.03).
CONCLUSIONS: Dabrafenib and trametinib were safely combined at full monotherapy
doses. The rate of pyrexia was increased with combination therapy, whereas the
rate of proliferative skin lesions was nonsignificantly reduced.
Progression-free survival was significantly improved. (Funded by
GlaxoSmithKline; ClinicalTrials.gov number, NCT01072175.). INTRODUCTION: Metastatic melanoma is an aggressive disease resistant to
chemotherapy. Recent clinical trials have reported improved survival for two
novel agents; ipilimumab, a humanized, IgG1 monoclonal antibody that blocks
cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and vemurafenib , a BRAF
(v-raf murine sarcoma viral oncogene homolog B1) inhibitor targeting an
activating mutation in the serine-threonine-protein kinase BRAF gene.
AREAS COVERED: The authors reviewed preclinical and clinical data examining the
safety of vemurafenib in melanoma. MEDLINE and EMBASE were searched using the
medical subject heading 'vemurafenib' and the following text terms: melanoma,
BRAF inhibition, vemurafenib. This review provides the reader with an overview
of current data examining the efficacy and safety of vemurafenib in metastatic
melanoma.
EXPERT OPINION: Vemurafenib is an oral agent licensed for patients with BRAF
V600E mutation-positive inoperable and metastatic melanoma. The most common
adverse effects observed in Phase III clinical trials were dermatological
events, arthralgia and fatigue. Specific dermatological toxicities included
development of cutaneous squamous cell cancers and keratoacanthomas.
Prolongation of the QT interval was also reported. Regular dermatological
assessments and electrocardiograms are recommended. Ongoing trials are examining
vemurafenib in both the adjuvant setting and metastatic setting in combination
with ipilimumab and MEK inhibitors (mitogen-activated protein
kinase/extracellular signal-regulated kinase). Understanding and overcoming
mechanisms of resistance to BRAF inhibitors is the focus of ongoing research. Vemurafenib is the first molecularly targeted therapy to be licensed in the US
and Europe for treatment of advanced melanoma. Its mechanism of action involves
selective inhibition of the mutated BRAF V600E kinase that leads to reduced
signalling through the aberrant mitogen-activated protein kinase (MAPK) pathway.
Its efficacy is restricted to melanomas carrying the BRAF V600E mutation, which
is seen in approximately 50% of all melanomas. In a randomized phase III trial,
it was superior to dacarbazine in first-line treatment of advanced melanoma,
with an overall response rate (ORR) of 48% (95% CI 42, 45), an estimated 6-month
progression-free survival (PFS) of 5.3 versus 1.6 months (hazard ratio [HR]
0.26; 95% CI 0.20, 0.33; p < 0.001) and a statistically superior 12-month
overall survival (OS) rate of 55% versus 43% (HR 0.62 [95% CI 0.49, 0.77]).
Vemurafenib is generally well tolerated, but its use can be associated with
development of cutaneous neoplasms such as squamous cell carcinoma (SCC) and
keratoacanthoma (KA). These lesions can be excised safely without the need for
withholding the drug or reducing its dose. Mechanisms of resistance to
vemurafenib do not involve development of secondary mutations in the BRAF kinase
domain, but may be related to BRAF V600E over-amplification, bypassing
mechanisms via upregulation and overexpression of other components in the MAPK
signalling cascade or activation of alternative pathways with potential to
enhance cell growth, proliferation and survival. Clinical trials to test the
efficacy of vemurafenib in combination with immunomodulatory agents, such as
ipilimumab, and MAPK kinase (MEK) inhibitors, such as GDC-0973, in the treatment
of advanced melanoma are currently underway. Also under investigation is the use
of vemurafenib in other solid tumours with BRAF mutations, such as papillary
thyroid cancer. The purpose of this study is to review the development of BRAF inhibitors, with
emphasis on the trials conducted with dabrafenib (GSK2118436) and the evolving
role of dabrafenib in treatment for melanoma patients. Fifty percent of
cutaneous melanomas have mutations in BRAF, resulting in elevated activity of
the mitogen-activated protein kinase signaling pathway. Dabrafenib inhibits the
mutant BRAF (BRAF(mut)) protein in melanomas with BRAF(V600E) and BRAF(V600K)
genotypes. BRAF(V600E) metastatic melanoma patients who receive dabrafenib
treatment exhibit high clinical response rates and compared with dacarbazine
chemotherapy, progression-free survival. Efficacy has also been demonstrated in
BRAF(V600K) patients and in those with brain metastases. Dabrafenib has a
generally mild and manageable toxicity profile. Cutaneous squamous cell
carcinomas and pyrexia are the most significant adverse effects. Dabrafenib
appears similar to vemurafenib with regard to efficacy but it is associated with
less toxicity. It is expected that new combinations of targeted drugs, such as
the combination of dabrafenib and trametinib (GSK1120212, a MEK inhibitor), will
provide higher response rates and more durable clinical benefit than dabrafenib
monotherapy. BACKGROUND: To examine the association between level and patterns of baseline
intra-tumoural BRAF(V600E) protein expression and clinical outcome of
BRAF(V600E) melanoma patients treated with selective BRAF inhibitors.
METHODS: Fifty-eight BRAF(V600E) metastatic melanoma patients treated with
dabrafenib or vemurafenib on clinical trials had pre-treatment tumour
BRAF(V600E) protein expression immunohistochemically (IHC) assessed using the
BRAF V600E mutant-specific antibody VE1. Sections were examined for staining
intensity (score 1-3) and percentage of immunoreactive tumour cells, and from
this an immunoreactive score (IRS) was derived (intensity × per cent
positive/10). The presence of intra-tumoural heterogeneity for BRAF(V600E)
protein expression was also assessed. BRAF(V600E) expression was correlated with
RECIST response, time to best response (TTBR), progression-free survival (PFS)
and overall survival (OS).
RESULTS: Expression was generally high (median IRS 28 (range 5-30)) and
homogeneous (78%). Expression of mutated protein BRAF(V600E) as measured by
intensity, per cent immunoreactive cells, or IRS did not correlate with RECIST
response, TTBR, PFS or OS, including on multivariate analysis. Heterogeneity of
staining was seen in 22% of cases and did not correlate with outcome.
CONCLUSION: In the current study population, IHC-measured pre-treatment
BRAF(V600E) protein expression does not predict response or outcome to BRAF
inhibitor therapy in BRAF(V600E) metastatic melanoma patients. INTRODUCTION: The development of selective BRAF inhibitors in patients with
metastatic BRAF V600 mutant melanoma has proven to be an effective therapeutic
strategy. While vemurafenib was the first approved BRAF inhibitor for this
indication, another selective BRAF inhibitor, dabrafenib, has demonstrated
efficacy in patients with BRAF mutant melanoma, including those with active
brain metastases and other maligcies.
AREAS COVERED: This review covers the current role of BRAF inhibitors in
patients with metastatic melanoma and the clinical development of dabrafenib.
The pharmacological, safety and efficacy data are discussed from the Phases I,
II and III studies of dabrafenib. In addition, the results of the Phase II study
of dabrafenib in melanoma patients with active brain metastases (BREAK-MB) and
the Phase I/II study of dabrafenib/trametinib are examined.
EXPERT OPINION: While dabrafenib has demonstrated comparable efficacy to
vemurafenib in BRAF V600E mutant melanoma patients, the BREAK-MB and
dabrafenib/trametinib studies have taken BRAF inhibitor strategies further with
evidence of disease activity in patients with metastatic melanoma brain
metastases and potential abrogation of BRAF inhibitor resistance. The incidence of melanoma is increasing steadily both in Poland and worldwide.
Until 2010 three drugs were approved for the treatment of metastatic melanoma -
dacarbazine (DTIC) in Europe and USA, fotemustine in Europe and interleukin-2
(IL-2) in USA. Approval of ipilimumab and vemurafenib in Europe and USA has
changed the standard of care, while the next candidates such as dabrafenib and
trametinib have improved survival in phase III studies in metastatic melanoma
patients. An encouraging treatment strategy is the combination of dabrafenib and
trametinib, evaluated in a phase I/II study with an ongoing phase III trial.
Another promising new immune modulating monoclonal antibody (mAb) is anti-PD1
(BMS-936558), tested in an early phase trial in monotherapy or in combination
with a multipeptide vaccine in metastatic melanoma patients. Ipilimumab or BRAF
inhibitors (vemurafenib, dabrafenib) seem to be active in patients with brain
metastases. Intensive research of melanoma vaccines is currently being carried
out in a number of countries worldwide. However, no vaccine in the treatment of
melanoma has been approved by regulatory authorities so far. Lack of effective
therapy in patients with high-risk resected melanoma led to a number of clinical
studies of adjuvant treatment. Interferon-α (INF-α) therapy in this setting is
still controversial. A dendritic cell-based vaccine in a randomized phase II
trial showed a survival benefit over the control group in patients with
high-risk resected melanoma. Promising results of long-term survival of advanced
resected melanoma patients in a phase II study evaluating the genetically
modified tumour vaccine (GMTV) AGI-101 were reported. This review provides an
update on clinical strategies used or tested in patients with metastatic
melanoma. Cutaneous squamous cell carcinoma (cSCC) is a concerning toxicity with BRAF
inhibitors in the treatment for melanoma. While the two drugs shown to improve
survival, vemurafenib, and dabrafenib, have similar efficacy, the reported rates
of cSCC are quite different. Drawing upon preclinical and clinical trial data,
this article discusses the potential factors behind the different cSCC
incidences reported with the two BRAF inhibitors and provides a strategic
approach to understand this issue further. PURPOSE: Dabrafenib (GSK2118436) is a potent inhibitor of mutated BRAF kinase.
Our multicenter, single-arm, phase II study assessed the safety and clinical
activity of dabrafenib in BRAF(V600E/K) mutation-positive metastatic melanoma
(mut(+) MM).
PATIENTS AND METHODS: Histologically confirmed patients with stage IV
BRAF(V600E/K) mut(+) MM received oral dabrafenib 150 mg twice daily until
disease progression, death, or unacceptable adverse events (AEs). The primary
end point was investigator-assessed overall response rate in BRAF(V600E) mut(+)
MM patients. Secondary end points included progression-free survival (PFS) and
overall survival (OS). Exploratory objectives included the comparison of BRAF
mutation status between tumor-specific circulating cell-free DNA (cfDNA) and
tumor tissue, and the evaluation of cfDNA as a predictor of clinical outcome.
RESULTS: Seventy-six patients with BRAF(V600E) and 16 patients with BRAF(V600K)
mut(+) MM were enrolled onto the study. In the BRAF(V600E) group, 45 patients
(59%) had a confirmed response (95% CI, 48.2 to 70.3), including five patients
(7%) with complete responses. Two patients (13%) with BRAF(V600K) mut(+) MM had
a confirmed partial response (95% CI, 0 to 28.7). In the BRAF(V600E) and
BRAF(V600K) groups, median PFS was 6.3 months and 4.5 months, and median OS was
13.1 months and 12.9 months, respectively. The most common AEs were arthralgia
(33%), hyperkeratosis (27%), and pyrexia (24%). Overall, 25 patients (27%)
experienced a serious AE and nine patients (10%) had squamous cell carcinoma.
Baseline cfDNA levels predicted response rate and PFS in BRAF(V600E) mut(+) MM
patients.
CONCLUSION: Dabrafenib was well tolerated and clinically active in patients with
BRAF(V600E/K) mut(+) MM. cfDNA may be a useful prognostic and response marker in
future studies. The mitogen-activated protein kinase (MAPK) pathway is particularly important
for the survival and proliferation of melanoma cells. Somatic mutations in BRAF
and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors
vemurafenib and dabrafenib have emerged as promising agents for the treatment of
melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors
induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells,
rapid emergence of acquired resistance and secondary skin tumors as well as
presence of few effective treatment options for melanoma bearing wild-type BRAF
(including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate
that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF
wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi
and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated
melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF
dimerization but inhibits the kinase activity of the RAF dimer, probably because
of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent
antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated
melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation
or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632
and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic
antiproliferative effects on these cells. Our findings characterize the unique
features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further
investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas
refractory to BRAF inhibitors. OBJECTIVE: To summarize the clinical development of dabrafenib and to highlight
the clinically relevant distinct characteristics of dabrafenib in contrast to
vemurafenib.
DATA SOURCE: An English-language literature search of MEDLINE/PubMed (1966-June
2013), using the keywords GSK2118436, dabrafenib, vemurafenib, selective BRAF
inhibitor, and advanced melanoma, was conducted. Data were also obtained from
package inserts, meeting abstracts, and clinical registries.
STUDY SELECTION AND DATA EXTRACTION: All relevant published articles on
dabrafenib and vemurafenib were reviewed. Clinical trial registries and meeting
abstracts were used for information about ongoing studies.
DATA SYNTHESIS: BRAF(V600E) mutation confers constitutive BRAK kinase activation
in melanoma cells, promoting tumor growth. This discovery led to the development
of BRAF kinase inhibitors like vemurafenib and dabrafenib. Dabrafenib has been
approved to treat patients with BRAF(V600E)-positive unresectable or metastatic
melanoma based on its clinical benefit demonstrated in a randomized phase III
study. It has also been shown to be safe and effective in patients with BRAF
mutant advanced melanoma involving the brain. Dabrafenib is well tolerated, with
the most common adverse effects being hyperkeratosis, headache, pyrexia, and
arthralgia. Currently, there is no evidence to suggest that one BRAF inhibitor
is superior to the other. With similar efficacy, therapy selection will likely
be influenced by differential tolerability and cost.
CONCLUSIONS: Dabrafenib joins vemurafenib to confirm the superior clinical
outcome of the BRAF inhibitors when compared with dacarbazine in patients with
BRAF(V600E)-positive advanced melanoma. Active research is ongoing to expand its
utility into the adjuvant setting and to circumvent rapid emergence of drug
resistance. Melanoma is the most aggressive form of skin cancer. The treatment of patients
with advanced melanoma is rapidly evolving due to an improved understanding of
molecular drivers of this disease. Somatic mutations in BRAF are the most common
genetic alteration found in these tumors. Recently, two different
mutant-selective small molecule inhibitors of BRAF, vemurafenib and dabrafenib,
have gained regulatory approval based on positive results in randomized phase
III trials. While the development of these agents represents a landmark in the
treatment of melanoma, the benefit of these agents is limited by the frequent
and rapid onset of resistance. The identification of several molecular
mechanisms of resistance to BRAF inhibitors is rapidly leading to the clinical
testing of combinatorial strategies to improve the clinical benefit of these
agents. These mechanisms, and the lessons learned from the initial testing of
the BRAF inhibitors, provide multiple insights that may facilitate the
development of targeted therapies against other oncogenic mutations in melanoma,
as well as in other cancers. BACKGROUND & AIM: Brain metastases are frequent in patients with metastatic
melanoma, indicating poor prognosis. We investigated the BRAF kinase inhibitor
vemurafenib in patients with advanced melanoma with symptomatic brain
metastases.
METHODS: This open-label trial assessed vemurafenib (960mg twice a day) in
patients with BRAF(V600) mutation-positive metastatic melanoma with
non-resectable, previously treated brain metastases. The primary end-point was
safety. Secondary end-points included best overall response rate, and
progression-free and overall survival.
RESULTS: Twenty-four patients received vemurafenib for a median treatment
duration of 3.8 (0.1-11.3) months. The majority of discontinuations were due to
disease progression (n=22). Twenty-three of 24 patients reported at least one
adverse event (AE). Grade 3 AEs were reported in four (17%; 95% confidence
interval [CI], 4.7-37.4%) patients and included cutaneous squamous cell
carcinoma in four patients. Median progression-free survival was 3.9 (95% CI,
3.0-5.5) months, and median survival was 5.3 (95% CI, 3.9-6.6) months. An
overall partial response (PR) at both intracranial and extracranial sites was
achieved in 10 of 24 (42%; 95% CI, 22.1-63.4) evaluable patients, with stable
disease in nine (38%; 95% CI, 18.8-59.4) patients. Of 19 patients with
measurable intracranial disease, seven (37%) achieved >30% intracranial tumour
regression, and three (16%; 95% CI, 3.4-39.6%) achieved a confirmed PR. Other
signs of improvement included reduced need for corticosteroids and enhanced
performance status.
CONCLUSIONS: Vemurafenib can be safely used in patients with advanced
symptomatic melanoma that has metastasised to the brain and can result in
meaningful tumour regression. Activating BRAF mutations, leading to constitutive activation of the MAPK
signaling pathway, are common in a variety of human cancers. Several small
molecule BRAF inhibitors have been developed during the last years and shown
promising results in clinical trials, especially for metastatic melanoma, while
they have been less effective in colon cancer. Two inhibitors, vemurafenib and
dabrafenib, have been approved for treatment of melanoma. Unfortunately, in most
patients who initially respond the tumors eventually develop acquired resistance
to the BRAF inhibitors. So far, a number of resistance mechanisms have been
identified, including secondary NRAS mutations and BRAF alternative splicing,
leading to reactivation of the MAPK pathway. Other alterations, both upstream
and downstream of BRAF can have the same effect, and activation of alternative
pathways can also play a role in resistance to BRAF inhibitors. In addition,
intra-tumor heterogeneity with the presence of clones of tumor cells lacking
BRAF mutations needs to be considered, since wildtype BRAF can be activated by
inhibitors designed to target mutated BRAF. Combination of the BRAF inhibitor
dabrafenib with the MEK inhibitor trametinib has significantly prolonged
progression free survival compared to dabrafenib alone in metastatic melanoma.
Combination treatments of BRAF inhibitors with other agents may not only
circumvent or delay resistance, but may also lead to fewer side effects, such as
development of secondary squamous tumors. Several clinical trials are underway
for many different BRAF mutation positive cancers with BRAF inhibitors alone or
in combination with other small molecule inhibitors, immunotherapies or
conventional chemotherapy. BRAF V600 mutation has been reported in more than 50% of melanoma cases and its
presence predicts clinical activity of BRAF inhibitors (iBRAF). We evaluated the
role of MIA, S100 and LDH to monitor iBRAF efficiency in advanced melanoma
patients presenting BRAF V600 mutations. This was a prospective study of
melanoma patients harboring the BRAF V600 mutation and treated with iBRAF within
a clinical trial (dabrafenib) or as part of an expanded access program
(vemurafenib). MIA, S100 and LDH were analyzed in serum at baseline, and every
4-6 weeks during treatment. Eighteen patients with melanoma stages IIIc-IV were
enrolled with 88.8% of response rate to iBRAF. Baseline concentrations of all
the tumor markers correlated with tumor burden. MIA and S100 concentrations
decreased significantly one month after the beginning of treatment and, upon
progression, their concentrations increased significantly above the minimum
levels previously achieved. MIA levels lower than 9 μg/L one month after the
beginning of treatment and S100 concentrations lower than 0.1 μg/L at the moment
of best response were associated with improved progression-free survival. In
conclusion, MIA and S100 are useful to monitor response in melanoma patients
treated with iBRAF. Dabrafenib is a BRAF kinase inhibitor indicated for the treatment of BRAF V600E
mutation-positive melanoma. The population pharmacokinetics of dabrafenib,
including changes over time and relevant covariates, were characterized based on
results from four clinical studies using a nonlinear mixed effects model with a
full covariate approach. Steady-state exposures of dabrafenib metabolites
(hydroxy-, carboxy-, and desmethyl-dabrafenib) were characterized separately.
The pharmacokinetics of dabrafenib were adequately described by non-inducible
and inducible apparent clearance that increased with dose and time. Total
steady-state clearance (CL/F) at 150 mg BID dose was 34.3 L/h. Based on the
induction half-life (67 hours), steady state should be achieved within 14 days
of dosing. Capsule shell was the most significant covariate (55%) while sex and
weight had only a small impact on exposure (<20%). The AUC ratio
(hypromellose:gelatin capsule) is predicted to be 1.80 and 1.42 following single
and repeat dosing, respectively. Age, renal (mild and moderate), and hepatic
(mild) impairment were not significant covariates. Steady-state pre-dose
concentration (%CV) of dabrafenib and of hydroxy-, carboxy-, and
desmethyl-dabrafenib at 150 mg BID were 46.6 ng/mL (83.5%), 69.3 ng/mL (64.1%),
3608 ng/mL (14.7%), and 291 ng/mL (17.2%), respectively. Capsule shell,
concomitant medications, older age, and weight were predictors of metabolite
exposure. The clinical development of selective BRAF inhibitors for metastatic BRAF V600
mutant melanoma patients has been a major breakthrough in targeted therapeutics.
Objective response rates of approximately 50% have been observed in the Phase
III studies of the BRAF inhibitors vemurafenib and dabrafenib. The side effects
can be relatively common, including proliferative skin toxicities. The latter
range from hyperkeratosis and keratoacanthomas (KAs) to squamous cell carcinomas
(SCCs) and new primary melanomas. In addition, case reports on the emergence of
gastric/colonic polyps and RAS mutant maligcies have been described during
BRAF inhibitor therapy. These events have been attributed to paradoxical
activation of the MAPK pathway in BRAF wild-type cells exposed to selective BRAF
inhibitors in addition to increased RAS activity. Combined BRAF and MEK
inhibition appears to improve clinical outcomes and reduce cutaneous
proliferation events as fewer KAs and SCCs have been observed with combination
therapy. Next-generation pan-RAF inhibitors ('paradox breakers') and ERK
inhibitors may further enhance clinical activity in metastatic BRAF-mutant
melanoma patients and mitigate this paradoxical oncogenesis. Further
investigation into the potential long-term effects of selective BRAF inhibitors
is warranted as expanded use of these agents is expected in patients with
BRAF-mutant melanoma and other maligcies. We studied the efficacy, tolerability and clinical courses of dabrafenib in
patients with metastatic melanoma who were ineligible for enrolment into a
clinical trial. Between July 2011 and May 2013, patients with unresectable stage
III or stage IV, V600-mutated metastatic melanoma who were not eligible for
inclusion into clinical trials were offered treatment with dabrafenib through a
named patient programme. Routine efficacy and toxicity data were collected
throughout treatment and studied retrospectively. The endpoints were
progression-free survival (PFS), overall survival and best overall response.
Thirty-one patients commenced dabrafenib therapy including six individuals who
had progressed on a prior BRAF-inhibitor treatment. The majority of patients had
cerebral metastases (n=17) and/or a poor performance status [Eastern Cooperative
Oncology Group (ECOG)≥2, n=11]. Median overall survival was 5.6 months (range
0.1-22 months). Median PFS was 3.3 months (range 0.1-21) and was similar despite
performance status. One patient had a complete response and eight showed partial
responses to treatment. Patients with cerebral metastases (n=17) had a median
PFS of 4.6 months. Five patients (16%) had dose-limiting toxicities. Despite
several poor prognostic features, dabrafenib is a safe and effective treatment
in the community setting, with occasional impressive outcomes. INTRODUCTION: Approximately 50% of patients with cutaneous melanoma have an
activating mutation in BRAF kinase, leading to constitutive activation of the
mitogen-activated protein kinase pathway and unregulated cell growth. Selective
inhibitors of the mutated BRAF kinase produce response rates of approximately
50% with median progression-free survival of 6 - 7 months. BRAF-blocking
therapies work rapidly, with responses seen within 2 weeks after therapy
initiation, and they are associated with generally mild toxicities. The BRAF
inhibitor dabrafenib recently was approved for use in patients with BRAF
V600-mutated metastatic melanoma.
AREAS COVERED: This article discusses the mechanisms of action and
pharmacokinetic and pharmacodynamic changes as well as clinical efficacy and
safety of dabrafenib for treatment of patients with advanced melanoma including
unresectable stage IIIc and stage IV patients who harbor a BRAF V600 mutation.
Clinical trial data are reviewed, and efficacy of dabrafenib in patients with
brain metastases and in combination with the MEK inhibitor trametinib is
discussed.
EXPERT OPINION: Despite rapid and significant tumor reduction in a majority of
patients with BRAF-mutant metastatic melanoma who are treated with dabrafenib,
this drug's use as a single agent is limited because of its relatively short
duration of response. Various combinations with drug(s) inhibiting other target
kinases and/or with immunomodulating agent(s) will likely be the standard in the
near future. Recent breakthroughs in the treatment of advanced melanoma are based on
scientific advances in understanding oncogenic signaling and the immunobiology
of this cancer. Targeted therapy can successfully block oncogenic signaling in
BRAF(V600)-mutant melanoma with high initial clinical responses, but relapse
rates are also high. Activation of an immune response by releasing inhibitory
check points can induce durable responses in a subset of patients with melanoma.
These advances have driven interest in combining both modes of therapy with the
goal of achieving high response rates with prolonged duration. Combining BRAF
inhibitors and immunotherapy can specifically target the BRAF(V600) driver
mutation in the tumor cells and potentially sensitize the immune system to
target tumors. However, it is becoming evident that the effects of paradoxical
mitogen-activated protein kinase pathway activation by BRAF inhibitors in
non-BRAF-mutant cells needs to be taken into account, which may be implicated in
the problems encountered in the first clinical trial testing a combination of
the BRAF inhibitor vemurafenib with ipilimumab (anti-CTLA4), with significant
liver toxicities. Here, we present the concept and potential mechanisms of
combinatorial activity of targeted therapy and immunotherapy, review the
literature for evidence to support the combination, and discuss the potential
challenges and future directions for rational conduct of clinical trials. BACKGROUND: Addition of a MEK inhibitor to a BRAF inhibitor enhances tumour
growth inhibition, delays acquired resistance, and abrogates paradoxical
activation of the MAPK pathway in preclinical models of BRAF-mutated melanoma.
We assessed the safety and efficacy of combined BRAF inhibition with vemurafenib
and MEK inhibition with cobimetinib in patients with advanced BRAF-mutated
melanoma.
METHODS: We undertook a phase 1b study in patients with advanced
BRAF(V600)-mutated melanoma. We included individuals who had either recently
progressed on vemurafenib or never received a BRAF inhibitor. In the
dose-escalation phase of our study, patients received vemurafenib 720 mg or 960
mg twice a day continuously and cobimetinib 60 mg, 80 mg, or 100 mg once a day
for either 14 days on and 14 days off (14/14), 21 days on and 7 days off (21/7),
or continuously (28/0). The primary endpoint was safety of the drug combination
and to identify dose-limiting toxic effects and the maximum tolerated dose.
Efficacy was a key secondary endpoint. All patients treated with vemurafenib and
cobimetinib were included in safety and efficacy analyses (intention-to-treat).
The study completed accrual and all analyses are final. This study is registered
with ClinicalTrials.gov, number NCT01271803.
FINDINGS: 129 patients were treated at ten dosing regimens combining vemurafenib
and cobimetinib: 66 had recently progressed on vemurafenib and 63 had never
received a BRAF inhibitor. Dose-limiting toxic effects arose in four patients.
One patient on a schedule of vemurafenib 960 mg twice a day and cobimetinib 80
mg once a day 14/14 had grade 3 fatigue for more than 7 days; one patient on a
schedule of vemurafenib 960 mg twice a day and cobimetinib 60 mg once a day 21/7
had a grade 3 prolongation of QTc; and two patients on a schedule of vemurafenib
960 mg twice a day and cobimetinib 60 mg 28/0 had dose-limiting toxic
effects-one developed grade 3 stomatitis and fatigue and one developed
arthralgia and myalgia. The maximum tolerated dose was established as
vemurafenib 960 mg twice a day in combination with cobimetinib 60 mg 21/7.
Across all dosing regimens, the most common adverse events were diarrhoea (83
patients, 64%), non-acneiform rash (77 patients, 60%), liver enzyme
abnormalities (64 patients, 50%), fatigue (62 patients, 48%), nausea (58
patients, 45%), and photosensitivity (52 patients, 40%). Most adverse events
were mild-to-moderate in severity. The most common grade 3 or 4 adverse events
were cutaneous squamous-cell carcinoma (12 patients, 9%; all grade 3), raised
amounts of alkaline phosphatase (11 patients, 9%]), and anaemia (nine patients,
7%). Confirmed objective responses were recorded in ten (15%) of 66 patients who
had recently progressed on vemurafenib, with a median progression-free survival
of 2·8 months (95% CI 2·6-3·4). Confirmed objective responses were noted in 55
(87%) of 63 patients who had never received a BRAF inhibitor, including six
(10%) who had a complete response; median progression-free survival was 13·7
months (95% CI 10·1-17·5).
INTERPRETATION: The combination of vemurafenib and cobimetinib was safe and
tolerable when administered at the respective maximum tolerated doses. The
combination has promising antitumour activity and further clinical development
is warranted in patients with advanced BRAF(V600)-mutated melanoma, particularly
in those who have never received a BRAF inhibitor; confirmatory clinical testing
is ongoing.
FUNDING: F Hoffmann-La Roche/Genentech. BACKGROUND: BRAF inhibitor-based therapies have been shown to induce cutaneous
toxicities, with onset generally in the first 8-26 weeks of therapy.
OBJECTIVES: To determine whether cutaneous toxicities persist in patients who
have remained on BRAF inhibitor-based therapies for longer than 52 weeks, and
therefore whether ongoing dermatology assessment is required.
METHODS: All patients treated with the BRAF inhibitors vemurafenib or dabrafenib
or combination BRAF inhibitor and mitogen-activated protein kinase kinase (MEK)
inhibitor therapy at Westmead Hospital, Sydney, Australia underwent regular
dermatological assessments for the duration of therapy. All patients enrolled in
a clinical trial, and 18% of patients in the compassionate access scheme
underwent a baseline assessment prior to commencement of therapy and every
4-8 weeks thereafter. Patients' adverse events were recorded in a specific
database.
RESULTS: Patients continued to develop cutaneous adverse events after 52 weeks
of continuous therapy. Patients on single-agent BRAF inhibitor therapy suffered
from Grover disease (45%), plantar hyperkeratosis (45%), verrucal keratosis
(18%) and even cutaneous squamous cell carcinoma (16%). The most frequent
adverse event seen in patients in the combination BRAF and MEK inhibitor group
was an acneiform eruption (40%).
CONCLUSIONS: Patients on BRAF inhibitor-based therapies need to continue to have
regular dermatological follow-up independent of the duration of their therapy. OBJECTIVE: To examine the current controversies and discuss consensus
recommendations regarding treatment sequencing and the role of BRAF inhibitor at
disease progression.
DATA SOURCE: An English-language literature search of MEDLINE/PubMed (1966-May
2014), using the keywords advanced melanoma, ipilimumab, cytotoxic T-lymphocyte
antigen 4, dabrafenib, vemurafenib, BRAF inhibitor, trametinib, MEK inhibitor,
and treatment sequencing was conducted. Data were also obtained from package
inserts, meeting abstracts, and clinical registries.
STUDY SELECTION AND DATA EXTRACTION: All relevant published articles and
abstracts on ipilimumab, vemurafenib, dabrafenib, and trametinib were reviewed.
Clinical trial registries and meeting abstracts were used for ongoing studies.
DATA SYNTHESIS: The availability of new agents has made therapy selection more
complex. Immunotherapy supporters reason that immunotherapy offers the best
chance for long-term benefit and does not compromise the antitumor activity of
subsequent BRAF inhibitors. Targeted therapy advocates rely on the high
probability and rapid onset of response to BRAF inhibitors. Currently, there is
insufficient evidence regarding the role of BRAF inhibitor at disease
progression.
CONCLUSIONS: Therapy should be individualized based on patient- and
disease-specific factors. Immunotherapy represents the best option for durable
remission; however, targeted therapy is more appropriate for patients who are
symptomatic or have rapidly growing tumors. The novel therapies have also
demonstrated meaningful intracranial activity; thus, the presence of brain
metastases should be taken into consideration in selecting therapy. Limited data
exist about the continuation of BRAF inhibitors after therapeutic failure.
Active research is ongoing to define the best option for patients with BRAF
inhibitor refractory disease. OBJECTIVE: To evaluate the cost effectiveness of dabrafenib versus dacarbazine
and vemurafenib as first-line treatments in patients with BRAF V600
mutation-positive unresectable or metastatic melanoma from a Canadian healthcare
system perspective.
METHODS: A partitioned-survival analysis model with three mutually exclusive
health states (pre-progression, post-progression, and dead) was used. The
proportion of patients in each state was calculated using survival distributions
for progression-free and overall survival derived from pivotal trials of
dabrafenib and vemurafenib. For each treatment, expected progression-free,
post-progression, overall, and quality-adjusted life-years (QALYs), and costs
were calculated. Costs were based on list prices, a clinician survey, and
published sources. A 5-year time horizon was used in the base case. Costs (in
2012 Canadian dollars [CA$]) and QALYs were discounted at 5% annually.
Deterministic and probabilistic sensitivity analyses were conducted.
RESULTS: Dabrafenib was estimated to yield 0.2055 more QALYs at higher cost than
dacarbazine. The incremental cost-effectiveness ratio was CA$363,136/QALY. In
probabilistic sensitivity analyses, at a threshold of CA$200,000/QALY, there was
an 8.2% probability that dabrafenib is cost effective versus dacarbazine. In
deterministic sensitivity analyses, cost effectiveness was sensitive to survival
distributions, utilities, and time horizon, with the hazard ratio for overall
survival for dabrafenib versus dacarbazine being the most sensitive parameter.
Assuming a class effect for efficacy of BRAF inhibitors, dabrafenib was domit
versus vemurafenib (less costly, equally effective), reflecting its assumed
lower daily cost. Assuming no class effect, dabrafenib yielded 0.0486 more QALYs
than vemurafenib.
CONCLUSIONS: At a threshold of CA$200,000/QALY, dabrafenib is unlikely to be
cost effective compared with dacarbazine. It is not possible to make reliable
conclusions regarding the relative cost effectiveness of dabrafenib versus
vemurafenib based on available information. |
which are the risk factors for sudden cardiac death in patients with hypertrophic cardiomyopathy? | The following risk factors for sudden cardiac death in patients with hypertrophic cardiomyopathy have been identified: 1) previous cardiac arrest; 2) sustained ventricular tachycardia; 3) family history of sudden cardiac death; 4) high-risk genetic mutations; 5) unexplained syncope; 6) non-sustained ventricular tachycardia; 7) hypotensive response to exercise; 8) marked left ventricular hypertrophy; 9) J-wave on ECG and 10) myocardial fibrosis using late gadolinium enhancement | Patients with hypertrophic cardiomyopathy are at increased risk for sudden
death. Recent studies have improved our ability to risk-stratify such patients
and have elucidated several potential mechanisms of sudden death and syncope.
Certain noninvasive tests, such as signal-averaged electrocardiography and
measurements of cardiac autonomic function and QT/QT dispersion, are often
abnormal in hypertrophic cardiomyopathy, but are not useful for risk
stratification. Myocardial ischemia determined by exercise thallium
scintigraphy, however, identifies young patients with hypertrophic
cardiomyopathy who are at high risk for cardiac arrest and syncope. Nonsustained
ventricular tachycardia on ambulatory Holter monitoring in the absence of
symptoms of impaired consciousness is associated with a benign prognosis and is
not predictive of sudden death. Conversely, ventricular tachycardia induced at
electrophysiologic study identifies adult patients with hypertrophic
cardiomyopathy who subsequently experience sudden death. Finally,
characterization of the natural history of the genetic defects will increasingly
become an integral part of risk evaluation in hypertrophic cardiomyopathy. Patients with hypertrophic cardiomyopathy (HC) die suddenly. Proposed risk
factors for sudden cardiac death (SCD) in HC are youth, a family history of SCD,
syncope, and ventricular tachycardia. Hemodynamic variables have not
convincingly proved to be risk factors for SCD. Therefore, this study was
designed to examine predictors of SCD in a large number of patients with HC
during long-term follow-up periods. The relation of studied variables (clinical,
electrocardiographic, echocardiographic, hemodynamic, and exercise test
findings) to SCD in 309 patients with HC who were initially diagnosed during
1971 through 1994 (mean follow-up 9.4 years) was examined by multivariate
analysis. SCD occurred in 28 patients. Independent predictors of SCD were a
smaller difference between peak and rest systolic blood pressure during exercise
testing (p=0.006), and higher left ventricular outflow tract pressure gradient
at rest (p=0.003). Exercise-related SCD occurred in 8 patients and
exercise-unrelated SCD in 20 patients (mean age 28 vs 47 years, p <0.05). Thus,
patients of exercise-related SCD were younger and had smaller increases in
systolic blood pressure during exercise testing, whereas patients with
exercise-unrelated SCD were older and had higher left ventricular outflow tract
pressure gradient. Hypertrophic cardiomyopathy (HCM) is a myocardial disease with variable
phenotpye and genotype. To demonstrate that the mutation Arg719Trp in the
cardiac beta-myosin heavy chain (beta MHC) gene is a high risk factor for sudden
death and can be associated with an unusual apical non-obstructive HCM, we
report the case of a 6 1/2 year old boy, who suffered cardiac arrest. The
proband had a de novo mutation of the beta MHC gene (Arg719Trp) on the paternal
beta MHC allele and a second maternally transmitted mutation (Met349Thr), as was
shown previously (Jeschke et al. 1998 (11)). Here we report the clinical
phenotype of the proband and of his relatives in detail. The proband had a
marked apical and midventricular hypertrophy of the left and right ventricle
without obstruction. There was an abnormal relaxation of both ventricles. Holter
monitoring detected no arrhythmia. Ventricular fibrillation was inducible only
by aggressive programmed stimulation. The boy died 3 1/2 years later after
another cardiac arrest due to arrhythmia. Five carriers of the Met349Thr
mutation in the family were asymptomatic and had no echocardiographic changes in
the heart, suggesting a neutral inherited polymorphism or a recessive mutation.
It is concluded that there is an association of the mutation Arg719Trp in the
beta-myosin heavy chain with sudden cardiac death in a young child. Disease
history in conjunction with the genetic analysis suggests that the implantation
of a defibrillator converter would have been a beneficial and probably life
saving measure. BACKGROUND: Hypertrophic cardiomyopathy is a genetic disease inherited as an
autosomal domit trait associated with risk of sudden death. The majority of
cases of sudden death occur in young adults with no or few symptoms, which
underlines the importance of risk stratification as a basis for selecting a
therapeutic strategy. Implantable cardioverter-defibrillators are indicated in
patients resuscitated following cardiac arrest, and those with sustained
ventricular tachycardia or two or more risk factors identified in non-invasive
tests.
AIM: The aim of this study was to determine the number of appropriate therapies
(anti-tachycardia pacing and defibrillation) and the risk factors, or
association of risk factors, that predict therapies in patients with
hypertrophic cardiomyopathy and an implantable cardioverter-defibrillator.
METHODS: We studied 17 consecutive patients with hypertrophic cardiomyopathy and
cardioverter-defibrillators implanted between December 1992 and June 2003. The
following risk factors were analyzed: 1) previous cardiac arrest or sustained
ventricular tachycardia; 2) family history of sudden cardiac death; 3) high-risk
genetic mutations; 4) syncope; 5) non-sustained ventricular tachycardia; 6)
hypotensive response to exercise; and 7) marked left ventricular hypertrophy.
Appropriate therapies were determined and the predictive value of the different
sudden death risk stratification parameters was analyzed.
RESULTS: During a mean follow-up of 40 +/- 29 months, 7 patients (41%) received
a total of 293 appropriate therapies. Of the 9 patients with previous cardiac
arrest or ventricular tachycardia, 4 received appropriate therapies. In the
remaining 8 patients, with implantable cardioverter-defibrillators for primary
prevention, 3 received appropriate therapies. Family history of sudden death was
associated with a positive predictive value of 25% for appropriate therapies,
40% for syncope and 50% for non-sustained ventricular tachycardia. The presence
of any two risk factors was associated with a positive predictive value of 33%
and the presence of three factors with 100%.
CONCLUSION: In this group of patients, considered to be at high risk for sudden
cardiac death, a considerable percentage had ventricular tachycardias that were
correctly identified and treated by the implantable cardioverter-defibrillator.
The percentage of patients with appropriate therapies was slightly higher in the
group who had a cardioverter-defibrillator for secondary prevention of sudden
death (aborted sudden death or sustained ventricular tachycardia). In patients
with an implantable cardioverter-defibrillator for primary prevention,
non-sustained ventricular tachycardia was the risk factor with the highest
predictive value. An association of risk factors was also predictive of
arrhythmic events. CONTEXT: Recently, the implantable cardioverter-defibrillator (ICD) has been
promoted for prevention of sudden death in hypertrophic cardiomyopathy (HCM).
However, the effectiveness and appropriate selection of patients for this
therapy is incompletely resolved.
OBJECTIVE: To study the relationship between clinical risk profile and incidence
and efficacy of ICD intervention in HCM.
DESIGN, SETTING, AND PATIENTS: Multicenter registry study of ICDs implanted
between 1986 and 2003 in 506 unrelated patients with HCM. Patients were judged
to be at high risk for sudden death; had received ICDs; underwent evaluation at
42 referral and nonreferral institutions in the United States, Europe, and
Australia; and had a mean follow-up of 3.7 (SD, 2.8) years. Measured risk
factors for sudden death included family history of sudden death, massive left
ventricular hypertrophy, nonsustained ventricular tachycardia on Holter
monitoring, and unexplained prior syncope.
MAIN OUTCOME MEASURE: Appropriate ICD intervention terminating ventricular
tachycardia or fibrillation.
RESULTS: The 506 patients were predominately young (mean age, 42 [SD, 17] years)
at implantation, and most (439 [87%]) had no or only mildly limiting symptoms.
ICD interventions appropriately terminated ventricular tachycardia/fibrillation
in 103 patients (20%). Intervention rates were 10.6% per year for secondary
prevention after cardiac arrest (5-year cumulative probability, 39% [SD, 5%]),
and 3.6% per year for primary prevention (5-year probability, 17% [SD, 2%]).
Time to first appropriate discharge was up to 10 years, with a 27% (SD, 7%)
probability 5 years or more after implantation. For primary prevention, 18 of
the 51 patients with appropriate ICD interventions (35%) had undergone
implantation for only a single risk factor; likelihood of appropriate discharge
was similar in patients with 1, 2, or 3 or more risk markers (3.83, 2.65, and
4.82 per 100 person-years, respectively; P = .77). The single sudden death due
to an arrhythmia (in the absence of advanced heart failure) resulted from ICD
malfunction. ICD complications included inappropriate shocks in 136 patients
(27%).
CONCLUSIONS: In a high-risk HCM cohort, ICD interventions for life-threatening
ventricular tachyarrhythmias were frequent and highly effective in restoring
normal rhythm. An important proportion of ICD discharges occurred in primary
prevention patients who had undergone implantation for a single risk factor.
Therefore, a single marker of high risk for sudden death may be sufficient to
justify consideration for prophylactic defibrillator implantation in selected
patients with HCM. Hypertrophic cardiomyopathy (HCM) is the most common hereditary cardiac
condition and the leading cause of sudden cardiac death (SCD) in young adults.
Given that SCD can be the first and most devastating clinical expression of HCM,
identifying individuals at high risk is paramount. Determining an individual's
risk for HCM-related SCD requires a thorough understanding of the recognized
risk factors, of which there are seven established or 'major' and five
'possible'. Major risk factors can be identified by thoroughly reviewing a
patient's personal medical history and noninvasive cardiovascular testing. The
presence of major risk factors identify patients who are at high enough risk of
SCD to warrant consideration of an implantable cardioverter-defibrillator;
whereas the absence of any major risk factors provides considerable reassurance
to both patient and physician. The risk of HCM-related SCD in patients with no
major risk factors is, however, not zero. Possible risk factors gain importance
in the presence of an isolated major risk factor. Here, we provide a
contemporary review of established and possible risk factors for HCM-related
SCD. We also examine microvolt T-wave alters and cardiovascular MRI as
emerging risk stratification tools that could further hone our ability to
accurately identify the high-risk patient. Sudden cardiac death is the most devastating manifestation of hypertrophic
cardiomyopathy (HCM) and often occurs in young and previously asymptomatic
patients. Therefore, risk stratification for sudden death has a major role in
the management of HCM and has acquired even greater relevance since the
implantable cardioverter-defibrillator (ICD) has proved to be highly effective
in preventing sudden death in this disease. The ICD is definitely indicated for
secondary prevention of sudden death in patients with HCM who have survived a
cardiac arrest with documented ventricular fibrillation, or experienced one or
more episodes of sustained ventricular tachycardia. However, uncertainties
persist regarding the precise selection of patients for primary prophylactic ICD
implantation. A number of risk markers are used to assess the magnitude of risk,
including family history of premature sudden death; extreme left ventricular
(LV) hypertrophy (> 30 mm) in young patients; nonsustained ventricular
tachycardia on Holter electrocardiographic recording; unexplained (not neurally
mediated) syncope, particularly in young patients; and blood pressure decrease
or inadequate increase during upright exercise. Multiple risk factors convey a
definite increase in risk. However, a single risk factor such as family history
of multiple sudden deaths, massive LV hypertrophy in a young patient, or
frequent and/or prolonged runs of nonsustained ventricular tachycardia on
Holter, may also justify consideration of a prophylactic ICD. BACKGROUND: The appearance of a discrete upward deflection of the ST segment
termed "the ST hump sign" (STHS) during exercise testing has been associated
with resting hypertension and exaggerated blood pressure response to exercise.
OBJECTIVE: We investigated the prevalence and clinical significance of this sign
in a population of patients with hypertrophic cardiomyopathy.
METHODS: Eighty-one patients with hypertrophic cardiomyopathy (HCM) who
underwent cardiopulmonary exercise testing were followed in a retrospective
cohort study for a mean period of 5.3 years.
RESULTS: The appearance of the STHS at the peak of exercise testing was observed
in 42 patients (52%), particularly in the inferior and the lateral leads.
Patients with the STHS had higher fractional shortening and maximum left
ventricular wall thickness and exhibited more frequently outflow tract gradient
>30 mmHg at rest. Furthermore, the presence of STHS was a strong independent
predictor of the risk of sudden cardiac death (SCD), as the latter occurred in
eight of the patients with this sign (8/42, 19%) and in none of the patients
without it (0/39, 0%) (P < 0.001).
CONCLUSION: The appearance of a "hump" at the ST segment during exercise testing
appears to be a risk factor for SCD in patients with HCM. However, further
studies are necessary to validate this finding in larger populations and to
elucidate the mechanism of the appearance of the "hump." The effect of left ventricular outflow tract obstruction (LVOTO) at rest on the
incidence of sudden death (SD) in patients with hypertrophic cardiomyopathy is
rather conflicting. The aim of this study was the evaluation of LVOTO at rest as
a new potential risk factor for SD in hypertrophic cardiomyopathy. A total of
166 patients (112 men, 51.8 +/- 15.6 years) were studied; 50 patients (30.1%)
had peak instantaneous LVOTO gradients of > or = 30 mm Hg at rest. During the
follow-up period (median 32.4 months, range 1 to 209), 13 patients either died
suddenly, or had cardiac arrest, documented sustained ventricular tachycardia,
or implantable cardioverter defibrillator discharge. The cumulative event-free
survival rate was 92% in patients with LVOTO, and 92% in patients without
obstruction (p = NS). LVOTO at rest was associated with a particularly low
positive predictive value for SD (8%), although a high negative predictive value
(92%) was recorded. Patients having syncope or presenting with a maximum wall
thickness > or =3 cm in echocardiography were more sensitive to SD emergence
because they had a 13.07 (95% confidence interval 4.00 to 46.95, p <0.0001) and
a 10.07 (95% confidence interval 2.92 to 34.79, p = 0.003) greater relative
risk, respectively. In conclusion, our cohort study results do not support LVOTO
as an independent risk factor for SD in patients with hypertrophic
cardiomyopathy. BACKGROUND: Myocardial late gadolinium enhancement (LGE) on contrast-enhanced
magnetic resoce imaging (CE-MRI) of patients with hypertrophic cardiomyopathy
(HCM) has been suggested to represent intramyocardial fibrosis and, as such, an
adverse prognostic risk factor. We evaluated the characteristics of LGE on
CE-MRI and explored whether LGE among patients with HCM was associated with
genetic testing, severe symptoms, ventricular arrhythmias, or sudden cardiac
death (SCD).
METHODS AND RESULTS: Four hundred twenty-four patients with HCM (age=55+/-16
years [range 2 to 90], 41% females), without a history of septal
ablation/myectomy, underwent CE-MRI (GE 1.5 Tesla). We evaluated the relation
between LGE and HCM genes status, severity of symptoms, and the degree of
ventricular ectopy on Holter ECG. Subsequent SCD and appropriate implanted
cardioverter defibrillator (ICD) therapies were recorded during a mean follow-up
of 43+/-14 months (range 16 to 94). Two hundred thirty-nine patients (56%) had
LGE on CE-MRI, ranging from 0.4% to 65% of the left ventricle. Gene-positive
patients were more likely to have LGE (P<0.001). The frequencies of New York
Heart Association class >or=3 dyspnea and angina class >or=3 were similar in
patients with and without LGE (125 of 239 [52%] versus 94 of 185 [51%] and 24 of
239 [10%] versus 18 of 185 [10%], respectively, P=NS). LGE-positive patients
were more likely to have episodes of nonsustained ventricular tachycardia (34 of
126 [27%] versus 8 of 94 [8.5%], P<0.001), had more episodes of nonsustained
ventricular tachycardia per patient (4.5+/-12 versus 1.1+/-0.3, P=0.04), and had
higher frequency of ventricular extrasystoles/24 hours (700+/-2080 versus
103+/-460, P=0.003). During follow-up, SCD occurred in 4 patients, and
additional 4 patients received appropriate ICD discharges. All 8 patients were
LGE positive (event rate of 0.94%/y, P=0.01 versus LGE negative). Two additional
heart failure-related deaths were recorded among LGE-positive patients.
Univariate associates of SCD or appropriate ICD discharge were positive LGE
(P=0.002) and presence of nonsustained ventricular tachycardia (P=0.04). The
association of LGE with events remained significant after controlling for other
risk factors.
CONCLUSIONS: In patients with HCM, presence of LGE on CE-MRI was common and more
prevalent among gene-positive patients. LGE was not associated with severe
symptoms. However, LGE was strongly associated with surrogates of arrhythmia and
remained a significant associate of subsequent SCD and/or ICD discharge after
controlling for other variables. If replicated, LGE may be considered an
important risk factor for sudden death in patients with HCM. We performed a systematic literature review of recommended 'major' and
'possible' clinical risk markers for sudden cardiac death (SCD) in hypertrophic
cardiomyopathy (HCM). We searched the Medline, Embase and Cochrane databases for
articles published between 1971 and 2007. We included English language reports
on HCM patients containing follow-up data on the endpoint (sudden) cardiac death
using survival analysis. Analysis was undertaken using the quality of reporting
of meta-analyses (QUORUM) statement checklist. The quality was checked using a
quality assessment form from the Cochrane Collaboration. Thirty studies met
inclusion criteria and passed quality assessment. The use of the six major risk
factors (previous cardiac arrest or sustained ventricular tachycardia,
non-sustained ventricular tachycardia, extreme left ventricular hypertrophy,
unexplained syncope, abnormal blood pressure response, and family history of
sudden death) in risk stratification for SCD as recommended by international
guidelines was supported by the literature. In addition, left ventricular
outflow tract obstruction seems associated with a higher risk of SCD. Our
systematic review provides sound evidence for the use of the six major risk
factors for SCD in the risk stratification of HCM patients. Left ventricular
outflow tract obstruction could be included in the overall risk profile of
patients with a marked left ventricular outflow gradient under basal conditions. INTRODUCTION: Although implantable cardioverter-defibrillators (ICDs) are used
in sudden cardiac death (SCD) prevention in high-risk patients with hypertrophic
cardiomyopathy (HCM), long-term results as well as precise risk stratification
are discussed in a limited number of reports. The aim of the study was to assess
the incidence of ICD intervention in HCM patients with relation to clinical risk
profile.
METHODS AND RESULTS: We studied 104 consecutive patients with HCM implanted in a
single center. The mean age of study population was 35.6 (SD, 16.2) years with
the average follow-up of 4.6 (SD, 2.6) years. ICD was implanted for secondary (n
= 26) and primary (n = 78) prevention of SCD. In the secondary prevention group,
14 patients (53.8%) experienced at least 1 appropriate device intervention
(7.9%/year). In the primary prevention (PP) group appropriate ICD discharges
occurred in 13 patients (16.7%) and intervention rate was 4.0%/year.
Nonsustained VT was the only predictive risk factor (RF) for an appropriate ICD
intervention in the PP (positive predictive value 22%, negative predictive value
96%). No significant difference was observed in the incidence of appropriate ICD
discharges between PP patients with 1, 2, or more RF. Complications of the
treatment included: inappropriate shocks (33.7%), lead dysfunction (12.5%), and
infections: 4.8% of patients. Four patients died during follow-up.
CONCLUSION: ICD therapy is effective in SCD prevention in patients with HCM,
although the complication rate is significant. Nonsustained ventricular
tachycardia seems to be the most predictive RF for appropriate device
discharges. Number of RF did not impact the incidence of appropriate ICD
interventions. Cardiac magnetic resoce with late gadolinium enhancement (LGE) is a
well-established method for in vivo detection of myocardial scarring. Several
recent studies have investigated the prognostic value of LGE in patients with
hypertrophic cardiomyopathy (HC). We discuss the prevalence and patterns of
scarring in HC and its pathophysiologic significance, with focus on ventricular
arrhythmias and sudden cardiac death. The available evidence that myocardial
scar demonstrated by LGE is a good independent predictor of cardiac mortality in
HC is summed up. Recommendations of current guidelines for prevention of sudden
cardiac death in HC are discussed with regard to recent results, and the
significance of LGE as an emerging risk factor is pointed out. In conclusion, it
is demonstrated that LGE has incremental value in addition to clinical risk
factors for risk stratification and management of patients with HC. OBJECTIVES: The aim of this study was to determine the efficacy of implantable
cardioverter-defibrillators (ICDs) in children and adolescents with hypertrophic
cardiomyopathy (HCM).
BACKGROUND: HCM is the most common cause of sudden death in the young. The
availability of ICDs over the past decade for HCM has demonstrated the potential
for sudden death prevention, predomitly in adult patients.
METHODS: A multicenter international registry of ICDs implanted (1987 to 2011)
in 224 unrelated children and adolescents with HCM judged at high risk for
sudden death was assembled. Patients received ICDs for primary (n = 188) or
secondary (n = 36) prevention after undergoing evaluation at 22 referral and
nonreferral institutions in the United States, Canada, Europe, and Australia.
RESULTS: Defibrillators were activated appropriately to terminate ventricular
tachycardia or ventricular fibrillation in 43 of 224 patients (19%) over a mean
of 4.3 ± 3.3 years. ICD intervention rates were 4.5% per year overall, 14.0% per
year for secondary prevention after cardiac arrest, and 3.1% per year for
primary prevention on the basis of risk factors (5-year cumulative probability
17%). The mean time from implantation to first appropriate discharge was 2.9 ±
2.7 years (range to 8.6 years). The primary prevention discharge rate
terminating ventricular tachycardia or ventricular fibrillation was the same in
patients who underwent implantation for 1, 2, or ≥3 risk factors (12 of 88
[14%], 10 of 71 [14%], and 4 of 29 [14%], respectively, p = 1.00). Extreme left
ventricular hypertrophy was the most common risk factor present (alone or in
combination with other markers) in patients experiencing primary prevention
interventions (17 of 26 [65%]). ICD-related complications, particularly
inappropriate shocks and lead malfunction, occurred in 91 patients (41%) at 17 ±
5 years of age.
CONCLUSIONS: In a high-risk pediatric HCM cohort, ICD interventions terminating
life-threatening ventricular tachyarrhythmias were frequent. Extreme left
ventricular hypertrophy was most frequently associated with appropriate
interventions. The rate of device complications adds a measure of complexity to
ICD decisions in this age group. AIMS: A retrospective, case-control study to investigate the J wave, a J-point
elevation on resting 12-lead electrocardiograms, as a risk factor for sudden
cardiac arrest (SCA) in patients diagnosed with hypertrophic cardiomyopathy
(HCM).
METHODS: Patients with HCM and age- and sex-matched healthy control subjects
were recruited, and 12-lead electrocardiograms were performed. The prevalence
and related characteristics of J waves were assessed. Patients were followed-up
for a mean of 47.9 months.
RESULTS: Patients with HCM (n = 345) had significantly higher prevalence of J
waves than healthy controls (n = 690; 11.6% and 7.1%, respectively). During
follow-up, 14 patients with HCM experienced SCA (SCA subgroup). The prevalence
of J waves was significantly higher in the SCA subgroup than in the non-SCA
subgroup (42.9% and 10.3%, respectively). There were no significant differences
between the SCA and non-SCA subgroups in J wave morphology.
CONCLUSION: The J wave may be a risk factor for SCA in patients with HCM. BACKGROUND: Cardiac magnetic resoce (CMR) is used in the diagnosis and risk
stratification of hypertrophic cardiomyopathy (HCM) and can detect myocardial
replacement fibrosis (anindependent predictor of adverse cardiac outcomes) using
late gadolinium enhancement (LGE).
METHODS: We retrospectively analysed CMR studies carried out over a 2 year
period identifying those which were diagnostic of HCM. 117 cases were analysed.
Mean age of subjects was 53 years and 78 (67%) were male. Mean ejection fraction
(EF) was 68.3% with a mean left ventricular (LV) mass index of 89.4 g/m².
Hypertrophy was predomitly asymmetric in 94 (80%).
RESULTS: All subjects received gadolinium and 80 (68%) had evidence of LGE. LVEF
was lower (67 vs. 71%; p = 0.015) and LV mass index higher (94 vs. 81 g/m²; p =
0.007) in the LGE group. The proportion of patients with at least 1 clinical
risk factor for sudden cardiac death (SCD) was similar in groups with and
without LGE (48% vs. 32%; p = 0.160). In this study, a significant proportion
(62%) of patients without clinical risk factors for SCD were found to have LGE
on CMR. These patients would not currently be considered for therapy with an
implantable cardiac defibrillator.
CONCLUSIONS: 1. Patients with HCM are at increased risk of SCD, but identifying
patients who may benefit from implantable defibrillators is difficult. 2. LGE is
associated with adverse cardiovascular outcomes in HCM, but is present in a
large proportion of patients. 3. Many patients without clinical risk factors for
SCD have LGE and would not currently be considered for an implantable cardiac
device. |
What is known about the economic cost of urinary incontinence? | The estimated total economic cost in treating overactive bladder was 117 billion Korean Won (KRW, the currency of South Koea) in 2006 and 145 billion KRW in 2007. The estimated total cost in treating stress urinary incontinence was 122 billion KRW in 2006 and 59 billion KRW in 2007.
The estimated total economic cost of OAB was 12.02 billion dollars in 2000, with 9.17 and 2.85 billion dollars incurred in the community and institutions, respectively. Community female and male OAB costs totaled 7.37 and 1.79 billion dollars, respectively. The estimated total cost was sensitive to the estimated prevalence of OAB; therefore, we calculated the average cost per community-dwelling person with OAB, which was 267 dollars per year.
An estimated 1835628 community-dwelling women over the age of 18 years had urinary incontinence in 1998. The total annual cost of this urinary incontinence is estimated at $710.44 million, or $387 per incontinent woman, comprising $338.47 million in treatment costs and $371.97 million in personal costs. An estimated 60% of women with incontinence in 1998 were aged 40 years or over. Assuming the prevalence of incontinence remains constant and, allowing for inflation, we project that the total annual cost in 20 years' time will be $1267.85 million, 93% ($1.18 billion) of which will constitute costs associated with women aged over 40 years.
For individuals 65 years of age and older these costs are substantial, increasing from $8.2 billion (1984 dollars) to $16.4 billion (1993 dollars). The 1995 societal cost of incontinence for individuals aged 65 years and older was $26.3 billion, or $3565 per individual with urinary incontinence. | Urinary incontinence imposes a significant ficial burden on individuals,
their families, and healthcare organizations. For individuals 65 years of age
and older these costs are substantial, increasing from $8.2 billion (1984
dollars) to $16.4 billion (1993 dollars). Both of these cost-of-illness
estimates, however, relied on data and factors that have changed over time. This
study updates these cost estimates. The 1995 societal cost of incontinence for
individuals aged 65 years and older was $26.3 billion, or $3565 per individual
with urinary incontinence. Limitations, implications, and directions for future
research are also discussed. Overactive bladder (OAB) is a highly prevalent condition among older patients,
and its presence is associated with the use of substantial healthcare resources
and economic costs. Within the next 30 years, it is expected that the demand for
services related to OAB will increase dramatically. Treatment of OAB is
challenging and depends on several factors, including the age of the patient,
cognitive functioning, and the degree of mobility. Pharmacotherapy, such as the
use of tolterodine and oxybutynin, is a viable option for the treatment of OAB,
and muscarinic antagonists are commonly used. The efficacy of an agent may
differ in older patients compared with younger ones. In addition, certain side
effects can be particularly troublesome in the geriatric population. A
retrospective analysis of a large managed care database showed an age-related
increase in the number of women seeking care for OAB. Caring for incontinent
patients in the long-term care setting was shown to result in substantial
additional costs, which were higher in those with more frequent incontinent
episodes. Prompted voiding may be effective in reducing the number of
incontinent episodes for those in institutionalized care; however, this practice
is labor intensive and generally is only effective in 40% of cases. Moreover,
assistance with prompted voiding must be maintained continuously. Future
research should focus on defining the most cost-effective methods of treating
OAB in the long-term care setting. PURPOSE: We quantified and described the economic burden of overactive bladder
and stress urinary incontinence in Korea. We calculated direct costs by
identifying public and private data sources that contain population-based data
on resource utilization by patients with stress urinary incontinence and
overactive bladder.
METHODS: For estimating indirect costs (productivity loss), the human capital
approach was applied. Data were collected from several institutes, including the
Health Insurance Review Agency.
RESULTS: The estimated total economic cost in treating overactive bladder was
117 billion Korean Won (KRW, the currency of South Koea) in 2006 and 145 billion
KRW in 2007. The estimated total cost in treating stress urinary incontinence
was 122 billion KRW in 2006 and 59 billion KRW in 2007.
CONCLUSIONS: By quantifying the total economic costs of overactive bladder and
stress urinary incontinence, this study provides an important perspective in
Korea. Because the average age of the Korean population is rapidly increasing,
this study provides important information on the direct and indirect costs of
overactive bladder and stress urinary incontinence for an aging society. BACKGROUND: Patients with neurologic diseases often have neurogenic detrusor
overactivity (NDO), which can result in a loss of voluntary bladder control and
uncontrollable urinary incontinence (UI).The impact of UI due to NDO on
patients' lives has not been well studied. The objective of this review was to
assess the health-related quality of life (HRQoL) and economic burden in
patients with urgency UI due to NDO in select countries in North America, the
European Union, Asia, and Australia.
METHODS: Systematic literature searches and reviews of articles published in
English (January 2000 to February 2011) were conducted using MEDLINE®, EMBASE®,
and the Cochrane Library. Studies assessing the impact of UI on HRQoL of
patients with an underlying neurologic condition of interest (i.e., multiple
sclerosis, spinal cord injury, Parkinson's disease, stroke, or spina bifida)
were included. Economic studies in urgency UI also were included.
RESULTS: Of 876 citations generated in the initial search, a total of 27
articles were deemed relevant: 16 articles presented HRQoL data and 11 articles
presented information on the economic burden of UI. Humanistic studies used a
range of HRQoL instruments to measure HRQoL burden, and the economic studies
included different cost components to quantify the economic burden, making
meaningful comparisons challenging. Despite this heterogeneity, the literature
suggests that HRQoL in patients with UI due to NDO is worse than patients with
UI in general or those with the same underlying neurologic condition without UI.
In addition, urgency UI also results in substantial economic costs.
CONCLUSIONS: Incontinent patients with underlying neurologic conditions have
impaired HRQoL as well as substantial economic burden attributable to UI due to
NDO. There is a need for urgency UI treatments that improve HRQoL of these
patients and alleviate the economic burden of this condition. Overactive bladder (OAB), often accompanied by urinary incontinence, is most
prevalent among the elderly, but also affects many middle-aged men and women in
the US. OAB may severely impair quality of life, and its overall economic costs
to society are substantial. Although antimuscarinic agents relieve OAB symptoms
effectively, treatment persistence generally is low. This has been attributed in
part to the occurrence of dry mouth and other anticholinergic adverse events.
High plasma concentrations of N-desethyloxybutynin (DEO), an active metabolite
of oxybutynin, have been identified as the major cause of anticholinergic
adverse effects associated with oral oxybutynin. Transdermal formulations of
oxybutynin generate much lower DEO plasma concentrations compared with oral
formulations. In a placebo-controlled US Phase III study in patients with OAB,
the recently approved oxybutynin topical gel (OTG) was efficacious and well
tolerated. Dry mouth occurred in 6.9% of patients treated with OTG and 2.8% of
patients on placebo. Incidences of other anticholinergic events were low and
similar for OTG and placebo. OTG rarely caused application site skin reactions.
OTG provides significant benefits to patients with OAB, particularly those who
are sensitive to anticholinergic adverse effects. |
Which are the coactivators of the Yes-associated protein (yap)? | The Yap protein forms complex with Tead (TEA domain) transcription factors. | The T-box transcription factor TBX5 plays essential roles in cardiac and limb
development. Various mutations in the TBX5 gene have been identified in patients
with Holt-Oram syndrome, which is characterized by congenital defects in the
heart and upper extremities. In this study, we identified a WW-domain-containing
transcriptional regulator TAZ as a potent TBX5 coactivator. TAZ directly
associates with TBX5 and markedly stimulates TBX5-dependent promoters by
interacting with the histone acetyltransferases p300 and PCAF. YAP, a
TAZ-related protein with conserved functional domains, also stimulates
TBX5-dependent transcription, possibly by forming a heterodimer with TAZ. TBX5
lacks a PY motif, which mediates the association of other proteins with TAZ, and
interacts with TAZ through multiple domains including its carboxyl-terminal
structure. Truncation mutants of TBX5 identified in patients with Holt-Oram
syndrome were markedly impaired in their ability to associate with and be
stimulated by TAZ. These findings reveal key roles for TAZ and YAP in the
control of TBX5-dependent transcription and suggest the involvement of these
coactivators in cardiac and limb development. The structure of the complex between the transcription cofactor Vgll1 and the
transcription factor TEAD4, the mammalian equivalent of the Drosophila Vestigial
and Scalloped, respectively, is determined in this study. Remarkably, Vgll1
interacts with TEAD in a manner similar to the transcription coactivators, as
well as oncogenes YAP and TAZ, despite having a varied primary sequence.
Vgll1-TEAD complex upregulates the expression of IGFBP-5, a
proliferation-promoting gene, and facilitates anchorage-independent cell
proliferation. The YAP/TAZ-TEAD complex also upregulates several other
proliferation-promoting genes and also promotes anchorage-independent cell
proliferation. Given its structural and functional similarity to YAP/TAZ, Vgll1
has the potential to promote cancer progression. TEAD proteins are transcription factors that are crucial for development, but
also play a role in cancers. Several developmentally and pathologically
important genes are upregulated by TEADs. TEADs have a TEA domain that enables
them to bind specific DNA elements and a transactivation domain that enables
them to interact with coactivators. TEADs on their own are unable to activate
transcription and they require the help of coactivators. Several
TEAD-interacting coactivators are known and they can be classified into three
groups: (1) YAP and its paralog TAZ; (2) Vgll proteins; and (3) p160s.
Accordingly, these coactivators also play a role in development and cancers.
Recent studies have shown that TEADs and their coactivators aid in the
progression of various cancers, including the difficult to treat glioblastoma,
liver and ovarian cancers. They facilitate cancer progression through expression
of proliferation promoting genes such as c-myc, survivin, Axl, CTGF and Cyr61.
There is also a good correlation between high TEAD or its coactivator expression
and poor prognosis in various cancers. Given the fact that TEADs and their
coactivators need to work together for a functional outcome, disrupting the
interaction between them appears to be a viable option for cancer therapy.
Structures of TEAD-coactivator complexes have been elucidated and will
facilitate drug design and development. The discovery of the Hippo pathway can be traced back to two areas of research.
Genetic screens in fruit flies led to the identification of the Hippo pathway
kinases and scaffolding proteins that function together to suppress cell
proliferation and tumor growth. Independent research, often in the context of
muscle biology, described Tead (TEA domain) transcription factors, which bind
CATTCC DNA motifs to regulate gene expression. These two research areas were
joined by the finding that the Hippo pathway regulates the activity of Tead
transcription factors mainly through phosphorylation of the transcriptional
coactivators Yap and Taz, which bind to and activate Teads. Additionally, many
other signal transduction proteins crosstalk to members of the Hippo pathway
forming a Hippo signal transduction network. We discuss evidence that the Hippo
signal transduction network plays important roles in myogenesis, regeneration,
muscular dystrophy, and rhabdomyosarcoma in skeletal muscle, as well as in
myogenesis, organ size control, and regeneration of the heart. Understanding the
role of Hippo kinases in skeletal and heart muscle physiology could have
important implications for translational research. |
How many genes outside of the MHC locus have been genetically associated to Rheumatoid Arthritis through GWAS? | Large genome-wide association studies (GWAS) have identified more than 30 loci involved in RA pathogenesis. To date, over 30 non-MHC RA-associated loci have been identified in humans, and over 100 arthritis-associated loci have been identified in rodent models of RA. The most relevant non-HLA gene single nucleotide polymorphisms (SNPs) associated with RA include PTPN22, IL23R, TRAF1, CTLA4, IRF5, STAT4, CCR6, PADI4. Previous studies demonstrate that 6 of the established non-HLA CD and RA risk loci (out of 26 loci for each disease) are shared between both diseases. | A major goal of current human genome-wide studies is to identify the genetic
basis of complex disorders. However, the availability of an unbiased, reliable,
cost efficient and comprehensive methodology to analyze the entire genome for
complex disease association is still largely lacking or problematic. Therefore,
we have developed a practical and efficient strategy for whole genome
association studies of complex diseases by charting the human genome at 100 kb
intervals using a collection of 27,039 microsatellites and the DNA pooling
method in three successive genomic screens of independent case-control
populations. The final step in our methodology consists of fine mapping of the
candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs)
analysis. This approach was validated upon application to rheumatoid arthritis,
a destructive joint disease affecting up to 1% of the population. A total of 47
candidate regions were identified. The top seven loci, withstanding the most
stringent statistical tests, were dissected down to individual genes and/or SNPs
on four chromosomes, including the previously known 6p21.3-encoded Major
Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based
genome-wide association analysis complemented by end stage SNP typing provides a
new tool for genetic dissection of multifactorial pathologies including common
diseases. To identify new genetic risk factors for rheumatoid arthritis, we conducted a
genome-wide association study meta-analysis of 5,539 autoantibody-positive
individuals with rheumatoid arthritis (cases) and 20,169 controls of European
descent, followed by replication in an independent set of 6,768 rheumatoid
arthritis cases and 8,806 controls. Of 34 SNPs selected for replication, 7 new
rheumatoid arthritis risk alleles were identified at genome-wide significance (P
< 5 x 10(-8)) in an analysis of all 41,282 samples. The associated SNPs are near
genes of known immune function, including IL6ST, SPRED2, RBPJ, CCR6, IRF5 and
PXK. We also refined associations at two established rheumatoid arthritis risk
loci (IL2RA and CCL21) and confirmed the association at AFF3. These new
associations bring the total number of confirmed rheumatoid arthritis risk loci
to 31 among individuals of European ancestry. An additional 11 SNPs replicated
at P < 0.05, many of which are validated autoimmune risk alleles, suggesting
that most represent genuine rheumatoid arthritis risk alleles. Epidemiology and candidate gene studies indicate a shared genetic basis for
celiac disease (CD) and rheumatoid arthritis (RA), but the extent of this
sharing has not been systematically explored. Previous studies demonstrate that
6 of the established non-HLA CD and RA risk loci (out of 26 loci for each
disease) are shared between both diseases. We hypothesized that there are
additional shared risk alleles and that combining genome-wide association study
(GWAS) data from each disease would increase power to identify these shared risk
alleles. We performed a meta-analysis of two published GWAS on CD (4,533 cases
and 10,750 controls) and RA (5,539 cases and 17,231 controls). After genotyping
the top associated SNPs in 2,169 CD cases and 2,255 controls, and 2,845 RA cases
and 4,944 controls, 8 additional SNPs demonstrated P<5 × 10(-8) in a combined
analysis of all 50,266 samples, including four SNPs that have not been
previously confirmed in either disease: rs10892279 near the DDX6 gene
(P(combined) = 1.2 × 10(-12)), rs864537 near CD247 (P(combined) = 2.2 ×
10(-11)), rs2298428 near UBE2L3 (P(combined) = 2.5 × 10(-10)), and rs11203203
near UBASH3A (P(combined) = 1.1 × 10(-8)). We also confirmed that 4 gene loci
previously established in either CD or RA are associated with the other
autoimmune disease at combined P<5 × 10(-8) (SH2B3, 8q24, STAT4, and TRAF1-C5).
From the 14 shared gene loci, 7 SNPs showed a genome-wide significant effect on
expression of one or more transcripts in the linkage disequilibrium (LD) block
around the SNP. These associations implicate antigen presentation and T-cell
activation as a shared mechanism of disease pathogenesis and underscore the
utility of cross-disease meta-analysis for identification of genetic risk
factors with pleiotropic effects between two clinically distinct diseases. PURPOSE OF REVIEW: To review recent progress in the genetics of rheumatoid
arthritis (RA) and discuss the implications for understanding the pathogenesis
of the disease as well as clinical application.
RECENT FINDINGS: Protection against anticitrullinated protein antibody (ACPA)
positive RA was shown to be associated wit DRB1*1301. Genome-wide association
studies (GWASs) added about 10 new loci to the list of already more than 20 loci
associated with RA, so the list is now over 30. Typing for the known risk loci
is not helpful for prediction of the risk for RA. It is remarkable how few
functional studies have been published.
SUMMARY: Known genetic factors explain 50-60% of the genetic variance for
susceptibility to ACPA-positive and 30-50% for ACPA-negative RA. Searching for
the remaining missing or hidden heritability is in all probability not going to
yield much for prediction and/or targeted intervention. Therefore, I conclude
that if you want to find more genes you should have a lot of patience, time and
money, stop with convential GWAS and invest in large-scale sequencing of
selected patients and controls. I have a better suggestion, however: use the
information that is already available to perform functional studies in order to
understand the mechanism of the known associations! Recent results from genetic and treatment studies have shed new light on chronic
inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). In
particular, genome-wide association studies (GWAS) have provided supportive
evidence that RA is a disease with a strong genetic background. Interestingly, a
series of candidate genes have been identified outside of the classical major
histocompatibility (MHC) locus, which had long been regarded as the major
contributor to the pathogenesis of this disease. Among these genes, PTPN22 plays
an outstanding role. CD40, STAT4, PRM1, and TNFAIP3 also seem to be of
relevance. Interestingly, there is a significant overlap between RA
susceptibility genes and those of other autoimmune diseases such as systemic
lupus erythematosus (SLE) and type 1 diabetes, which suggests common pathogenic
mechanisms. Genetic analyses may not only provide new insights into the
pathogenesis of RA, but may also open new avenues for therapeutic approaches,
because overactive immune-signaling pathways might specifically be addressed by
biologic therapies. However, the predictive value of many of the recent findings
of large-scale genetic analyses in identifying new genetic polymorphisms remains
low. We describe the current knowledge about the role of non-MHC genes in the
pathogenesis of rheumatoid arthritis. Rheumatoid arthritis (RA) is a polygenic autoimmune disease primarily affecting
the synovial joints. Numerous animal models show similarities to RA in humans;
some of them not only mimic the clinical phenotypes but also demonstrate the
involvement of homologous genomic regions in RA. This paper compares
corresponding non-MHC genomic regions identified in rodent and human genome-wide
association studies (GWAS). To date, over 30 non-MHC RA-associated loci have
been identified in humans, and over 100 arthritis-associated loci have been
identified in rodent models of RA. The genomic regions associated with the
disease are designated by the name(s) of the gene having the most frequent and
consistent RA-associated SNPs or a function suggesting their involvement in
inflammatory or autoimmune processes. Animal studies on rats and mice
preferentially have used single sequence length polymorphism (SSLP) markers to
identify disease-associated qualitative and quantitative trait loci (QTLs) in
the genome of F2 hybrids of arthritis-susceptible and arthritis-resistant rodent
strains. Mouse GWAS appear to be far ahead of rat studies, and significantly
more mouse QTLs correspond to human RA risk alleles. OBJECTIVE: Candidate gene association studies and genome-wide association
studies (GWAs) have identified a large number of single nucleotide polymorphisms
(SNPs) loci affecting susceptibility to rheumatoid arthritis (RA). However, for
the same locus, some studies have yielded inconsistent results. To assess all
the available evidence for association, we performed a meta-analysis on
previously published case-control studies investigating the association between
SNPs and RA.
METHODS: Two hundred and sixteen studies, involving 125 SNPs, were reviewed. For
each SNP, three genetic models were considered: the allele, domit and
recessive effects models. For each model, the effect summary odds ratio (OR) and
95% CIs were calculated. Cochran's Q-statistics were used to assess
heterogeneity. If the heterogeneity was high, a random effects model was used
for meta-analysis, otherwise a fixed effects model was used.
RESULTS: The meta-analysis results showed that: (1) 30, 28 and 26 SNPs were
significantly associated with RA (P<0.01) for the allele, domit, and
recessive models, respectively. (2) rs2476601 (PTPN22) showed the strongest
association for all the three models: OR = 1.605, 95% CI: 1.540-1.672,
P<1.00E-15 for the T-allele; OR = 1.638, 95% CI: 1.565-1.714, P<1.00E-15 for the
T/T+T/C genotype and OR = 2.544, 95% CI: 2.173-2.978, P<1.00E-15 for the T/T
genotype. (3) Only 23 (18.4%), 13 (10.4%) and 15 (12.0%) SNPs had high
heterogeneity (P<0.01) for the three models, respectively. (4) For some of the
SNPs, there was no publication bias according to Funnel plots and Egger's
regression tests (P<0.01). For the other SNPs, the associations were tested in
only a few studies, and may have been subject to publication bias. More studies
on these loci are required.
CONCLUSION: Our meta-analysis provides a comprehensive evaluation of the RA
association studies from the past two decades. The detailed meta-analysis
results are available at:
http://210.46.85.180/DRAP/index.php/Metaanalysis/index. The "Bermuda triangle" of genetics, environment and autoimmunity is involved in
the pathogenesis of rheumatoid arthritis (RA). Various aspects of genetic
contribution to the etiology, pathogenesis and outcome of RA are discussed in
this review. The heritability of RA has been estimated to be about 60 %, while
the contribution of HLA to heritability has been estimated to be 11-37 %. Apart
from known shared epitope (SE) alleles, such as HLA-DRB1*01 and DRB1*04, other
HLA alleles, such as HLA-DRB1*13 and DRB1*15 have been linked to RA
susceptibility. A novel SE classification divides SE alleles into S1, S2, S3P
and S3D groups, where primarily S2 and S3P groups have been associated with
predisposition to seropositive RA. The most relevant non-HLA gene single
nucleotide polymorphisms (SNPs) associated with RA include PTPN22, IL23R, TRAF1,
CTLA4, IRF5, STAT4, CCR6, PADI4. Large genome-wide association studies (GWAS)
have identified more than 30 loci involved in RA pathogenesis. HLA and some
non-HLA genes may differentiate between anti-citrullinated protein antibody
(ACPA) seropositive and seronegative RA. Genetic susceptibility has also been
associated with environmental factors, primarily smoking. Some GWAS studies
carried out in rodent models of arthritis have confirmed the role of human
genes. For example, in the collagen-induced (CIA) and proteoglycan-induced
arthritis (PgIA) models, two important loci - Pgia26/Cia5 and Pgia2/Cia2/Cia3,
corresponding the human PTPN22/CD2 and TRAF1/C5 loci, respectively - have been
identified. Finally, pharmacogenomics identified SNPs or multiple genetic
signatures that may be associated with responses to traditional
disease-modifying drugs and biologics. Investigators have made key advances in rheumatoid arthritis (RA) genetics in
the past 10 years. Although genetic studies have had limited influence on
clinical practice and drug discovery, they are currently generating testable
hypotheses to explain disease pathogenesis. Firstly, we review here the major
advances in identifying RA genetic susceptibility markers both within and
outside of the MHC. Understanding how genetic variants translate into pathogenic
mechanisms and ultimately into phenotypes remains a mystery for most of the
polymorphisms that confer susceptibility to RA, but functional data are
emerging. Interplay between environmental and genetic factors is poorly
understood and in need of further investigation. Secondly, we review current
knowledge of the role of epigenetics in RA susceptibility. Differences in the
epigenome could represent one of the ways in which environmental exposures
translate into phenotypic outcomes. The best understood epigenetic phenomena
include post-translational histone modifications and DNA methylation events,
both of which have critical roles in gene regulation. Epigenetic studies in RA
represent a new area of research with the potential to answer unsolved
questions. |
Are optogenetics tools used in the study and treatment of epilepsy? | Using optogenetics tools it is possible to begin to address some of the fundamental unanswered questions in epilepsy, to dissect epileptic neuronal circuits and to develop new intervention strategies. | The optogenetic approach to gain control over neuronal excitability both in
vitro and in vivo has emerged as a fascinating scientific tool to explore
neuronal networks, but it also opens possibilities for developing novel
treatment strategies for neurologic conditions. We have explored whether such an
optogenetic approach using the light-driven halorhodopsin chloride pump from
Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to
hyperpolarize central neurons, may inhibit excessive hyperexcitability and
epileptiform activity. We show that a lentiviral vector containing the NpHR gene
under the calcium/calmodulin-dependent protein kinase IIalpha promoter
transduces principal cells of the hippocampus and cortex and hyperpolarizes
these cells, preventing generation of action potentials and epileptiform
activity during optical stimulation. This study proves a principle, that
selective hyperpolarization of principal cortical neurons by NpHR is sufficient
to curtail paroxysmal activity in transduced neurons and can inhibit stimulation
train-induced bursting in hippocampal organotypic slice cultures, which
represents a model tissue of pharmacoresistant epilepsy. This study demonstrates
that the optogenetic approach may prove useful for controlling epileptiform
activity and opens a future perspective to develop it into a strategy to treat
epilepsy. Optogenetic tools comprise a variety of different light-sensitive proteins from
single-cell organisms that can be expressed in mammalian neurons and effectively
control their excitability. Two main classes of optogenetic tools allow to
either depolarize or hyperpolarize, and respectively generate or inhibit action
potentials in selective populations of neurons. This opens unprecedented
possibilities for delineating the role of certain neuronal populations in brain
processing and diseases. Moreover, optogenetics may be considered for developing
potential treatment strategies for brain diseases, particularly for excitability
disorders such as epilepsy. Expression of the inhibitory halorhodopsin NpHR in
hippocampal principal cells has been recently used as a tool to effectively
control chemically and electrically induced epileptiform activity in slice
preparations, and to reduce in vivo spiking induced by tetanus toxin injection
in the motor cortex. In this review we give a comprehensive summary of what has
been achieved so far in the field of epilepsy using optogenetics, and discuss
some of the possible strategies that could be envisaged in the future. We also
point out some of the challenges and pitfalls in relation to possible outcomes
of using optogenetics for controlling network excitability, and associated brain
diseases. This article is part of the Special Issue entitled 'New Targets and
Approaches to the Treatment of Epilepsy'. Epilepsy is characterised by the propensity of the brain to generate spontaneous
recurrent bursts of excessive neuronal activity, seizures. GABA-mediated
inhibition is critical for restraining neuronal excitation in the brain, and
therefore potentiation of GABAergic neurotransmission is commonly used to
prevent seizures. However, data obtained in animal models of epilepsy and from
human epileptic tissue suggest that GABA-mediated signalling contributes to
interictal and ictal activity. Prolonged activation of GABA(A) receptors during
epileptiform bursts may even initiate a shift in GABAergic neurotransmission
from inhibitory to excitatory and so have a proconvulsant action. Direct
targeting of the membrane mechanisms that reduce spiking in glutamatergic
neurons may better control neuronal excitability in epileptic tissue.
Manipulation of brain pH may be a promising approach and recent advances in gene
therapy and optogenetics seem likely to provide further routes to effective
therapeutic intervention. Cerebrocortical injuries such as stroke are a major source of disability.
Maladaptive consequences can result from post-injury local reorganization of
cortical circuits. For example, epilepsy is a common sequela of cortical stroke,
but the mechanisms responsible for seizures following cortical injuries remain
unknown. In addition to local reorganization, long-range, extra-cortical
connections might be critical for seizure maintece. In rats, we found that
the thalamus, a structure that is remote from, but connected to, the injured
cortex, was required to maintain cortical seizures. Thalamocortical neurons
connected to the injured epileptic cortex underwent changes in HCN channel
expression and became hyperexcitable. Targeting these neurons with a closed-loop
optogenetic strategy revealed that reducing their activity in real-time was
sufficient to immediately interrupt electrographic and behavioral seizures. This
approach is of therapeutic interest for intractable epilepsy, as it spares
cortical function between seizures, in contrast with existing treatments, such
as surgical lesioning or drugs. Temporal lobe epilepsy is the most common type of epilepsy in adults, is often
medically refractory, and due to broad actions and long-time scales, current
systemic treatments have major negative side-effects. However, temporal lobe
seizures tend to arise from discrete regions before overt clinical behaviour,
making temporally and spatially specific treatment theoretically possible. Here
we report the arrest of spontaneous seizures using a real-time, closed-loop,
response system and in vivo optogenetics in a mouse model of temporal lobe
epilepsy. Either optogenetic inhibition of excitatory principal cells, or
activation of a subpopulation of GABAergic cells representing <5% of hippocampal
neurons, stops seizures rapidly upon light application. These results
demonstrate that spontaneous temporal lobe seizures can be detected and
terminated by modulating specific cell populations in a spatially restricted
manner. A clinical approach built on these principles may overcome many of the
side-effects of currently available treatment options. PURPOSE OF REVIEW: Tremendous advances have occurred in recent years in
elucidating basic mechanisms of epilepsy at the level of ion channels and
neurotransmitters. Epilepsy, however, is ultimately a disease of functionally
and/or structurally aberrant connections between neurons and groups of neurons
at the systems level. Recent advances in neuroimaging and electrophysiology now
make it possible to investigate structural and functional connectivity of the
entire brain, and these techniques are currently being used to investigate
diseases that manifest as global disturbances of brain function. Epilepsy is
such a disease, and our understanding of the mechanisms underlying the
development of epilepsy and the generation of epileptic seizures will
undoubtedly benefit from research utilizing these connectomic approaches.
RECENT FINDINGS: MRI using diffusion tensor imaging provides structural
information, whereas functional MRI and electroencephalography provide
functional information about connectivity at the whole brain level.
Optogenetics, tracers, electrophysiological approaches, and calcium imaging
provide connectivity information at the level of local circuits. These
approaches are revealing important neuronal network disturbances underlying
epileptic abnormalities.
SUMMARY: An understanding of the fundamental mechanisms underlying the
development of epilepsy and the generation of epileptic seizures will require
delineation of the aberrant functional and structural connections of the whole
brain. The field of connectomics now provides approaches to accomplish this. Epilepsy is a devastating disease, currently treated with medications, surgery
or electrical stimulation. None of these approaches is totally effective and our
ability to control seizures remains limited and complicated by frequent side
effects. The emerging revolutionary technique of optogenetics enables
manipulation of the activity of specific neuronal populations in vivo with
exquisite spatiotemporal resolution using light. We used optogenetic approaches
to test the role of hippocampal excitatory neurons in the lithium-pilocarpine
model of acute elicited seizures in awake behaving rats. Hippocampal pyramidal
neurons were transduced in vivo with a virus carrying an enhanced halorhodopsin
(eNpHR), a yellow light activated chloride pump, and acute seizure progression
was then monitored behaviorally and electrophysiologically in the presence and
absence of illumination delivered via an optical fiber. Inhibition of those
neurons with illumination prior to seizure onset significantly delayed
electrographic and behavioral initiation of status epilepticus, and altered the
dynamics of ictal activity development. These results reveal an essential role
of hippocampal excitatory neurons in this model of ictogenesis and illustrate
the power of optogenetic approaches for elucidation of seizure mechanisms. This
early success in controlling seizures also suggests future therapeutic avenues. The complexity of the brain, in which different neuronal cell types are
interspersed and complexly interconnected, has posed a major obstacle in
identifying pathophysiological mechanisms underlying prevalent neurological
disorders. This is largely based in the inability of classical experimental
approaches to target defined neural populations at sufficient temporal and
spatial resolution. As a consequence, effective clinical therapies for prevalent
neurological disorders are largely lacking. Recently developed optogenetic
probes are genetically expressed photosensitive ion channels and pumps that in
principal overcome these limitations. Optogenetic probes allow millisecond
resolution functional control over selected optogenetically transduced neuronal
populations targeted based on promoter activity. This optical cell control
scheme has already been applied to answer fundamental questions pertaining to
neurological disorders by allowing researchers to experimentally intercept, or
induce, pathophysiological neuronal signaling activity in a highly controlled
manner. Offering high temporal resolution control over neural activity at high
cellular specificity, optogenetic tools constitute a game changer in research
aiming at understanding pathophysiological signaling mechanisms in neurological
disorders and in developing therapeutic strategies to correct these. In this
regard, recent experimental work has provided new insights in underlying
mechanisms, as well as preliminary proof-of-principle for optogenetic therapies,
of several neurological disorders, including Parkinson's disease, epilepsy and
progressive blindness. This review synthesizes experimental work where
optogenetic tools have been applied to explore pathologic neural network
activity in models of neurological disorders. Axonal sprouting of excitatory neurons is frequently observed in temporal lobe
epilepsy, but the extent to which inhibitory interneurons undergo similar axonal
reorganization remains unclear. The goal of this study was to determine whether
somatostatin (SOM)-expressing neurons in stratum (s.) oriens of the hippocampus
exhibit axonal sprouting beyond their normal territory and innervate granule
cells of the dentate gyrus in a pilocarpine model of epilepsy. To obtain
selective labeling of SOM-expressing neurons in s. oriens, a Cre
recombinase-dependent construct for channelrhodopsin2 fused to enhanced yellow
fluorescent protein (ChR2-eYFP) was virally delivered to this region in SOM-Cre
mice. In control mice, labeled axons were restricted primarily to s.
lacunosum-moleculare. However, in pilocarpine-treated animals, a rich plexus of
ChR2-eYFP-labeled fibers and boutons extended into the dentate molecular layer.
Electron microscopy with immunogold labeling demonstrated labeled axon terminals
that formed symmetric synapses on dendritic profiles in this region, consistent
with innervation of granule cells. Patterned illumination of ChR2-labeled fibers
in s. lacunosum-moleculare of CA1 and the dentate molecular layer elicited
GABAergic inhibitory responses in dentate granule cells in pilocarpine-treated
mice but not in controls. Similar optical stimulation in the dentate hilus
evoked no significant responses in granule cells of either group of mice. These
findings indicate that under pathological conditions, SOM/GABAergic neurons can
undergo substantial axonal reorganization beyond their normal territory and
establish aberrant synaptic connections. Such reorganized circuitry could
contribute to functional deficits in inhibition in epilepsy, despite the
presence of numerous GABAergic terminals in the region. Optogenetic techniques provide powerful tools for bidirectional control of
neuronal activity and investigating alterations occurring in excitability
disorders, such as epilepsy. In particular, the possibility to specifically
activate by light-determined interneuron populations expressing
channelrhodopsin-2 provides an unprecedented opportunity of exploring their
contribution to physiological and pathological network activity. There are
several subclasses of interneurons in cortical areas with different functional
connectivity to the principal neurons (e.g., targeting their perisomatic or
dendritic compartments). Therefore, one could optogenetically activate specific
or a mixed population of interneurons and dissect their selective or concerted
inhibitory action on principal cells. We chose to explore a conceptually novel
strategy involving simultaneous activation of mixed populations of interneurons
by optogenetics and study their impact on ongoing epileptiform activity in mouse
acute hippocampal slices. Here we demonstrate that such approach results in a
brief initial action potential discharge in CA3 pyramidal neurons, followed by
prolonged suppression of ongoing epileptiform activity during light exposure.
Such sequence of events was caused by massive light-induced release of GABA from
ChR2-expressing interneurons. The inhibition of epileptiform activity was less
pronounced if only parvalbumin- or somatostatin-expressing interneurons were
activated by light. Our data suggest that global optogenetic activation of mixed
interneuron populations is a more effective approach for development of novel
therapeutic strategies for epilepsy, but the initial action potential generation
in principal neurons needs to be taken in consideration. We have reviewed some of the important studies published within the last 18
months that have advanced our understanding of the epilepsies, their aetiology
and treatment. Clinical studies have revealed new insights into old themes
including seizure prediction, mortality in epilepsy, febrile seizures and the
pathophysiology of focal cortical dysplasias. The rapid advances in genetics and
particularly whole exome sequencing have had an impact on our understanding of
epileptic encephalopathies, and the aetiology of hippocampal sclerosis.
Experimental research techniques such as viral vector gene delivery,
optogenetics and cell based transplantation techniques have set the framework
for novel approaches to the treatment of pharmacoresistant epilepsy. These few
examples are indicative of the great strides that have recently been made in
epilepsy research. New genetic investigation techniques, including next-generation sequencing,
epigenetic profiling, cell lineage mapping, targeted genetic manipulation of
specific neuronal cell types, stem cell reprogramming, and optogenetic
manipulations within epileptic networks are progressively unraveling the
mysteries of epileptogenesis and ictogenesis. These techniques have opened new
avenues to discover the molecular basis of epileptogenesis and to study the
physiologic effects of mutations in epilepsy-associated genes on a multilayer
level, from cells to circuits. This manuscript reviews recently published
applications of these new genetic technologies in the study of epilepsy, as well
as work presented by the authors at the genetic session of the XII Workshop on
the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation
sequencing is providing investigators with an unbiased means to assess the
molecular causes of sporadic forms of epilepsy and has revealed the complexity
and genetic heterogeneity of sporadic epilepsy disorders. To assess the
functional impact of mutations in these newly identified genes on specific
neuronal cell types during brain development, new modeling strategies in
animals, including conditional genetics in mice and in utero knock-down
approaches, are enabling functional validation with exquisite cell-type and
temporal specificity. In addition, optogenetics, using cell-type-specific Cre
recombinase driver lines, is enabling investigators to dissect networks involved
in epilepsy. In addition, genetically encoded cell-type labeling is providing
new means to assess the role of the nonneuronal components of epileptic networks
such as glial cells. Furthermore, beyond its role in revealing coding variants
involved in epileptogenesis, next-generation sequencing can be used to assess
the epigenetic modifications that lead to sustained network hyperexcitability in
epilepsy, including methylation changes in gene promoters and noncoding
ribonucleic acid (RNA) involved in modifying gene expression following seizures.
In addition, genetically based bioluminescent reporters are providing new
opportunities to assess neuronal activity and neurotransmitter levels both in
vitro and in vivo in the context of epilepsy. Finally, genetically rederived
neurons generated from patient induced pluripotent stem cells and genetically
modified zebrafish have become high-throughput means to investigate disease
mechanisms and potential new therapies. Genetics has changed the field of
epilepsy research considerably, and is paving the way for better diagnosis and
therapies for patients with epilepsy. BACKGROUND: Electrical high frequency stimulation (HFS) has been shown to
suppress seizures. However, the mechanisms of seizure suppression remain unclear
and techniques for blocking specific neuronal populations are required.
OBJECTIVE: The goal is to study the optical HFS protocol on seizures as well as
the underlying mechanisms relevant to the HFS-mediated seizure suppression by
using optogenetic methodology.
METHODS: Thy1-ChR2 transgenic mice were used in both vivo and in vitro
experiments. Optical stimulation with pulse trains at 20 and 50 Hz was applied
on the focus to determine its effects on in vivo seizure activity induced by
4-AP and recorded in the bilateral and ipsilateral-temporal hippocampal CA3
regions. In vitro methodology was then used to study the mechanisms of the in
vivo suppression.
RESULTS: Optical HFS was able to generate 82.4% seizure suppression at 50 Hz
with light power of 6.1 mW and 80.2% seizure suppression at 20 Hz with light
power of 2.0 mW. The suppression percentage increased by increasing the light
power and saturated when the power reached above-mentioned values. In vitro
experimental results indicate that seizure suppression was mediated by
activation of GABA receptors. Seizure suppression effect decreased with
continued application but the suppression effect could be restored by
intermittent stimulation.
CONCLUSIONS: This study shows that optical stimulation at high frequency
targeting an excitatory opsin has potential therapeutic application for fast
control of an epileptic focus. Furthermore, electrophysiological observations of
extracellular and intracellular signals revealed that GABAergic
neurotransmission activated by optical stimulation was responsible for the
suppression. Optogenetics is a novel technology that combines optics and genetics by optical
control of microbial opsins, targeted to living cell membranes. The versatility
and the electrophysiologic characteristics of the light-sensitive ion-channels
channelrhodopsin-2 (ChR2), halorhodopsin (NpHR), and the light-sensitive proton
pump archaerhodopsin-3 (Arch) make these optogenetic tools potent candidates in
controlling neuronal firing in models of epilepsy and in providing insights into
the physiology and pathology of neuronal network organization and
synchronization. Opsins allow selective activation of excitatory neurons and
inhibitory interneurons, or subclasses of interneurons, to study their activity
patterns in distinct brain-states in vivo and to dissect their role in
generation of synchrony and seizures. The influence of gliotransmission on
epileptic network function is another topic of great interest that can be
further explored by using light-activated Gq protein-coupled opsins for
selective activation of astrocytes. The ever-growing optogenetic toolbox can
also be combined with emerging techniques that have greatly expanded our ability
to record specific subtypes of cortical and hippocampal neurons in awake
behaving animals such as juxtacellular recording and two-photon guided
whole-cell recording, to identify the specific subtypes of neurons that are
altered in epileptic networks. Finally, optogenetic tools allow rapid and
reversible suppression of epileptic electroencephalography (EEG) activity upon
photoactivation. This review outlines the most recent advances achieved with
optogenetic techniques in the field of epilepsy by summarizing the presentations
contributed to the 13th ILAE WONOEP meeting held in the Laurentian Mountains,
Quebec, in June 2013. Epilepsy is a neurological disorder that affects around 1% of the population
worldwide. The two main therapies, pharmacology and the electrical stimulation,
both have some shortcomings. For instance, pharmacological therapy is frequently
accompanied by side effects, and current anticonvulsive drugs fail to be
effective to around a third of patients. These patients could suffer
astrocyte-related epilepsy, as increasing evidence indicates that dysfunctions
of astrocytes can result in epilepsy. However, epilepsy drugs that affect
astrocytes are not available currently. Although electrical stimulation has
benefited many patients, the electrode stimulates unselective neurons or
circuits. All these need to develop new strategies for improving the life of the
patients. As channelrhodopsins (ChRs) were discovered, a novel method referred
to as "optogenetics" was developed. It has advantages over electrical
stimulation of being less-invasiveness and allowing spatiotemporally
stimulation. Recently, a number of experiments have explored the treatments for
epilepsy with optogenetic control of neurons. Here, we discuss the possibility
that an optogenetic approach could be used to control the release of
gliotransmitters and improve astrocyte function such as glutamate and K(+)
uptake, and thereby offer a potential strategy to investigate and treat
astrocyte-related epilepsy. Current treatment options for epilepsy are inadequate, as too many patients
suffer from uncontrolled seizures and from negative side effects of treatment.
In addition to these clinical challenges, our scientific understanding of
epilepsy is incomplete. Optogenetic and designer receptor technologies provide
unprecedented and much needed specificity, allowing for spatial, temporal and
cell type-selective modulation of neuronal circuits. Using such tools, it is now
possible to begin to address some of the fundamental uswered questions in
epilepsy, to dissect epileptic neuronal circuits and to develop new intervention
strategies. Such specificity of intervention also has the potential for direct
therapeutic benefits, allowing healthy tissue and network functions to continue
unaffected. In this Perspective, we discuss promising uses of these technologies
for the study of seizures and epilepsy, as well as potential use of these
strategies for clinical therapies. |
In which phase of cell cycle does stress-induced transcription-associated mutagenesis (TAM) occur? | Factors involved in RNA polymerase (RNAP) processivity or transcriptional derepression, such as Mfd (transcription coupling repair factor), contribute to the generation of stress-induced mutations. Under stress, transcription-associated mutagenesis is increased. Stress-induced transcription-associated mutations are acquired by nondividing cells, during stationary phase, and are not observed under conditions of exponential growth. | Previously, using a chromosomal reversion assay system, we established that an
adaptive mutagenic process occurs in nongrowing Bacillus subtilis cells under
stress, and we demonstrated that multiple mechanisms are involved in generating
these mutations (41, 43). In an attempt to delineate how these mutations are
generated, we began an investigation into whether or not transcription and
transcription-associated proteins influence adaptive mutagenesis. In B.
subtilis, the Mfd protein (transcription repair coupling factor) facilitates
removal of RNA polymerase stalled at transcriptional blockages and recruitment
of repair proteins to DNA lesions on the transcribed strand. Here we demonstrate
that the loss of Mfd has a depressive effect on stationary-phase mutagenesis. An
association between Mfd mutagenesis and aspects of transcription is discussed. BACKGROUND: During transcription, the nontranscribed DNA strand becomes
single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases
in the ssDNA are less protected from mutagens and hence experience more
mutations than do paired bases. These mutations are called
transcription-associated mutations. Transcription-associated mutagenesis is
increased under stress and depends on the DNA sequence. Therefore, selection
might significantly influence protein-coding sequences in terms of the
transcription-associated mutability per transcription event under stress to
improve the survival of Escherichia coli.
METHODOLOGY/PRINCIPAL FINDINGS: The mutability index (MI) was developed by
Wright et al. to estimate the relative transcription-associated mutability of
bases per transcription event. Using the most stable fold of each ssDNA that
have an average length n, MI was defined as (the number of folds in which the
base is unpaired)/nx(highest -DeltaG of all n folds in which the base is
unpaired), where DeltaG is the free energy. The MI values show a significant
correlation with mutation data under stress but not with spontaneous mutations
in E. coli. Protein sequence diversity is preferred under stress but not under
favorable conditions. Therefore, we evaluated the selection pressure on MI in
terms of the protein sequence diversity for all the protein-coding sequences in
E. coli. The distributions of the MI values were lower at bases that could be
substituted with each of the other three bases without affecting the amino acid
sequence than at bases that could not be so substituted. Start codons had lower
distributions of MI values than did nonstart codons.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that the majority of
protein-coding sequences have evolved to promote protein sequence diversity and
to reduce gene knockout under stress. Consequently, transcription-associated
mutagenesis increases protein sequence diversity more effectively than does
random mutagenesis under stress. Nonrandom transcription-associated mutagenesis
under stress should improve the survival of E. coli. Scientists have been aware for many years of genetic programs that get activated
under stress and produce genetic variants in cells that escape non-proliferating
conditions. These programs are well conserved in all organisms and expand our
view of evolution. They mediate genome instability, create diversity in antibody
formation, expand metabolism and increase fitness of pathogens within host
environments. Error-prone DNA replication and repair are genetic
variability-causing agents that get stimulated by the onset of cellular
stresses. Embedded in these programs is the ability to limit mutagenesis to
defined genomic regions and times, ensuring integrity of most of the genome.
Recent evidence suggests that factors involved in RNA polymerase (RNAP)
processivity or transcriptional derepression contribute to the generation of
stress-induced mutations. In Bacillus subtilis, transcription-associated
mutagenesis has been shown to be independent of recombination-dependent repair
and, in some cases, of the Y DNA polymerases. Central to stationary-phase
mutagenesis in B. subtilis is the requirement for Mfd, transcription coupling
repair factor, which suggests a novel mechanism from those described in other
model systems. |
Which proteins are related to the loss of cell-cell adhesion during EMT (epithelial-mesenchymal transition)? | Transcriptional and post-transcriptional regulatory mechanisms mediated by several inducers of EMT, in particular the ZEB and Snail factors, downregulate the expression and/or functional organization of core polarity complexes. Functional loss of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration during embryonic development and tumour invasion. Recently, we found that aPKC can also phosphorylate Par6 to drive EMT and increase the migratory potential of non-small cell lung cancer cells. We propose that the regulation of EMT by SIRT1 involves modulation of, and cooperation with, the EMT inducing transcription factor ZEB1. Knockdown of Numb by shRNA in MDCK cells led to a lateral to apical translocation of E-cadherin and beta-catenin, active F-actin polymerization, mis-localization of Par3 and aPKC, a decrease in cell-cell adhesion and an increase in cell migration and proliferation. Growth factors such as TGFb and EGF have also been shown to be related to EMT. | We have previously demonstrated that, following acquisition of endocrine
resistance, breast cancer cells display an altered growth rate together with
increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive
interactions can promote an aggressive phenotype, we investigated the integrity
of this protein complex in our breast cancer model of tamoxifen resistance. In
culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies
with loss of cell-cell junctions and demonstrated altered morphology
characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT).
Neutralising E-cadherin function promoted the invasion and inhibited the
aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on
the behaviour of TamR cells. Additionally, TamR cells had increased levels of
tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated
beta-catenin was decreased. These cells also displayed loss of association
between beta-catenin and E-cadherin, increased cytoplasmic and nuclear
beta-catenin and elevated transcription of beta-catenin target genes known to be
involved in tumour progression and EMT. Inhibition of EGFR kinase activity in
TamR cells reduced beta-catenin tyrosine phosphorylation, increased
beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such
treated cells, the association of beta-catenin with Lef-1 and the transcription
of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest
that homotypic adhesion in tamoxifen-resistant breast cancer cells is
dysfunctional due to EGFR-driven modulation of the phosphorylation status of
beta-catenin and may contribute to an enhanced aggressive phenotype and
transition towards a mesenchymal phenotype in vitro. The epithelial-to-mesenchymal transition (EMT) is a crucial process in tumour
progression providing tumour cells with the ability to escape from the primary
tumour, to migrate to distant regions and to invade tissues. EMT requires a loss
of cell-cell adhesion and apical-basal polarity, as well as the acquisition of a
fibroblastoid motile phenotype. Several transcription factors have emerged in
recent years that induce EMT, with important implications for tumour
progression. However, their effects on cell polarity remain unclear. Here, we
have re-examined the data available related to the effect of EMT related
transcription factors on epithelial cell plasticity, focusing on their impact on
cell polarity. Transcriptional and post-transcriptional regulatory mechanisms
mediated by several inducers of EMT, in particular the ZEB and Snail factors,
downregulate the expression and/or functional organization of core polarity
complexes. We also summarize data on the expression of cell polarity genes in
human tumours and analyse genetic interactions that highlight the existence of
complex regulatory networks converging on the regulation of cell polarity by EMT
inducers in human breast carcinomas. These recent observations provide new
insights into the relationship between alterations in cell polarity components
and EMT in cancer, opening new avenues for their potential use as therapeutic
targets to prevent tumour progression. Functional loss of the cell-cell adhesion molecule E-cadherin is an essential
event for epithelial-mesenchymal transition (EMT), a process that allows cell
migration during embryonic development and tumour invasion. In most carcinomas,
transcriptional repression has emerged as the main mechanism responsible for
E-cadherin downregulation. Here, we report the identification of class I bHLH
factor E2-2 (TCF4/ITF2) as a new EMT regulator. Both isoforms of E2-2 (E2-2A and
E2-2B) induce a full EMT when overexpressed in MDCK cells but without affecting
the tumorigenic properties of parental cells, in contrast to other EMT inducers,
such as Snail1 or class I bHLH E47. E-cadherin repression mediated by E2-2 is
indirect and independent of proximal E-boxes of the promoter. Knockdown studies
indicate that E2-2 expression is dispensable for maintece of the EMT driven
by Snail1 and E47. Comparative gene-profiling analysis reveals that E2-2 factors
induce similar, yet distinct, genetic programs to that induced by E47 in MDCK
cells. These results, together with the embryonic expression pattern of Tcf4 and
E2A (which encodes E12/E47), support a distinct role for E2-2 and suggest an
interesting interplay between E-cadherin repressors in the regulation of
physiological and pathological EMT processes. BACKGROUND: A feature of epithelial to mesenchymal transition (EMT) relevant to
tumour dissemination is the reorganization of actin cytoskeleton/focal contacts,
influencing cellular ECM adherence and motility. This is coupled with the
transcriptional repression of E-cadherin, often mediated by Snail1, Snail2 and
Zeb1/deltaEF1. These genes, overexpressed in breast carcinomas, are known
targets of growth factor-initiated pathways, however it is less clear how
alterations in ECM attachment cross-modulate to regulate these pathways. EGF
induces EMT in the breast cancer cell line PMC42-LA and the kinase inhibitor
staurosporine (ST) induces EMT in embryonic neural epithelial cells, with
F-actin de-bundling and disruption of cell-cell adhesion, via inhibition of
aPKC.
METHODS: PMC42-LA cells were treated for 72 h with 10 ng/ml EGF, 40 nM ST, or
both, and assessed for expression of E-cadherin repressor genes (Snail1, Snail2,
Zeb1/deltaEF1) and EMT-related genes by QRT-PCR, multiplex tandem PCR (MT-PCR)
and immunofluorescence +/- cycloheximide. Actin and focal contacts (paxillin)
were visualized by confocal microscopy. A public database of human breast
cancers was assessed for expression of Snail1 and Snail2 in relation to outcome.
RESULTS: When PMC42-LA were treated with EGF, Snail2 was the principal
E-cadherin repressor induced. With ST or ST+EGF this shifted to Snail1, with
more extreme EMT and Zeb1/deltaEF1 induction seen with ST+EGF. ST reduced stress
fibres and focal contact size rapidly and independently of gene transcription.
Gene expression analysis by MT-PCR indicated that ST repressed many genes which
were induced by EGF (EGFR, CAV1, CTGF, CYR61, CD44, S100A4) and induced genes
which alter the actin cytoskeleton (NLF1, NLF2, EPHB4). Examination of the
public database of breast cancers revealed tumours exhibiting higher Snail1
expression have an increased risk of disease-recurrence. This was not seen for
Snail2, and Zeb1/deltaEF1 showed a reverse correlation with lower expression
values being predictive of increased risk.
CONCLUSION: ST in combination with EGF directed a greater EMT via actin
depolymerisation and focal contact size reduction, resulting in a loosening of
cell-ECM attachment along with Snail1-Zeb1/deltaEF1 induction. This appeared
fundamentally different to the EGF-induced EMT, highlighting the multiple
pathways which can regulate EMT. Our findings add support for a functional role
for Snail1 in invasive breast cancer. The conserved polarity proteins Par6 and aPKC regulate cell polarization
processes. However, increasing evidence also suggests that they play a role in
oncogenic progression. During tumor progression, epithelial to mesenchymal
transition (EMT) delineates an evolutionary conserved process that converts
stationary epithelial cells into mesenchymal cells, which have an acquired
ability for independent migration and invasion. In addition to signaling
pathways that alter genetic programes that trigger the loss of cell-cell
adhesion, alternative pathways can alter cell plasticity to regulate cell-cell
cohesion and increase invasive potential. One such pathway involves TGFβ-induced
phosphorylation of Par6. In epithelial cells, Par6 phosphorylation results in
the dissolution of junctional complexes, cytoskeletal remodelling, and increased
metastatic potential. Recently, we found that aPKC can also phosphorylate Par6
to drive EMT and increase the migratory potential of non-small cell lung cancer
cells. This result has implications with respect to homeostatic and
developmental processes involving polarization, and also with respect to cancer
progression-particularly since aPKC has been reported to be an oncogenic
regulator in various tumor cells. |
Is micro RNA 1 (miR-1) implicated in cardiac arrhythmias? | Yes. miR-1 overexpression may contribute to the increased susceptibility of the heart to AVB, which provides us novel insights into the molecular mechanisms underlying ischemic cardiac arrhythmias. As miR-1 has been shown in animal models and clinical studies to contribute to arrhythmogenesis by regulating pacemaker channel genes, our finding of miR-1 up-regulation in patients with myocardial infarction indicates that it might be responsible for the higher risk for arrhythmias in these patients. | AIMS: The present study was designed to investigate whether the beneficial
effects of beta-blocker propranolol are related to regulation of microRNA miR-1.
METHODS AND RESULTS: We demonstrated that propranolol reduced the incidence of
arrhythmias in a rat model of myocardial infarction by coronary artery
occlusion. Overexpression of miR-1 was observed in ischaemic myocardium and
strikingly, administration of propranolol reversed the up-regulation of miR-1
nearly back to the control level. In agreement with its miR-1-reducing effect,
propranolol relieved myocardial injuries during ischaemia, restored the membrane
depolarization and cardiac conduction slowing, by rescuing the expression of
inward rectifying K(+) channel subunit Kir2.1 and gap junction channel connexin
43. Our results further revealed that the beta-adrenoceptor-cAMP-Protein Kinase
A (PKA) signalling pathway contributed to the expression of miR-1, and serum
response factor (SRF), which is known as one of the transcriptional enhancers of
miR-1, was up-regulated in ischaemic myocardium. Moreover, propranolol inhibited
the beta-adrenoceptor-cAMP-PKA signalling pathway and suppressed SRF expression.
CONCLUSION: We conclude that the beta-adrenergic pathway can stimulate
expression of arrhythmogenic miR-1, contributing to ischaemic arrhythmogenesis,
and beta-blockers produce their beneficial effects partially by down-regulating
miR-1, which might be a novel strategy for ischaemic cardioprotection. BACKGROUND: MicroRNAs (miRNAs/miRs) are small conserved RNA molecules of 22
nucleotides that negatively modulate gene expression primarily through base
paring to the 3' untranslated region of target messenger RNAs. The
muscle-specific miR-1 has been implicated in cardiac hypertrophy, heart
development, cardiac stem cell differentiation, and arrhythmias through
targeting of regulatory proteins. In this study, we investigated the molecular
mechanisms through which miR-1 intervenes in regulation of muscle cell growth
and differentiation.
METHODS AND RESULTS: On the basis of bioinformatics tools, biochemical assays,
and in vivo models, we demonstrate that (1) insulin-like growth factor-1 (IGF-1)
and IGF-1 receptor are targets of miR-1; (2) miR-1 and IGF-1 protein levels are
correlated inversely in models of cardiac hypertrophy and failure as well as in
the C2C12 skeletal muscle cell model of differentiation; (3) the activation
state of the IGF-1 signal transduction cascade reciprocally regulates miR-1
expression through the Foxo3a transcription factor; and (4) miR-1 expression
correlates inversely with cardiac mass and thickness in myocardial biopsies of
acromegalic patients, in which IGF-1 is overproduced after aberrant synthesis of
growth hormone.
CONCLUSIONS: Our results reveal a critical role of miR-1 in mediating the
effects of the IGF-1 pathway and demonstrate a feedback loop between miR-1
expression and the IGF-1 signal transduction cascade. BACKGROUND: Atrial fibrillation (AF) is associated with increased
inward-rectifier current activity that may stabilize atrial rotors maintaining
the arrhythmia. Left atrial (LA) structures are important for AF maintece,
but previous studies have mostly evaluated changes in the right atrium.
MicroRNA-1 (miR-1) reciprocally regulates inwardly rectifying potassium channel
(Kir)2.1 expression in coronary disease, contributing to arrhythmogenesis.
OBJECTIVES: This study sought to evaluate changes in miR-1 and Kir2 subunit
expression in relation to I(K1) alterations in LA of patients with persistent
AF.
METHODS: Atrial tissue was obtained from 62 patients (31 with AF) undergoing
mitral valve repair or bypass grafting. Currents were recorded from isolated
cells. Proteins were quantified from immunoblots. mRNA and miR-1 levels were
measured with real-time polymerase chain reaction. Immunohistochemistry was
applied to localize connexin (Cx) 43.
RESULTS: I(K1) density was increased in LA cells from patients with AF (at -100
mV: -5.9 +/- 1.3 vs. -2.7 +/- 0.7 sinus rhythm, P <.05). There was a
corresponding increase in Kir2.1 protein expression, but no change in other Kir
or Cx proteins. Expression of inhibitory miR-1 was reduced by approximately 86%
in tissue samples of AF patients. Kir2.1 mRNA was significantly increased. No
change in Cx43 localization occurred. Ex vivo tachystimulation of human atrial
slices up-regulated Kir2.1 and down-regulated miR-1, suggesting a primary role
of atrial rate in miR-1 down-regulation and I(K1) up-regulation.
CONCLUSION: miR-1 levels are greatly reduced in human AF, possibly contributing
to up-regulation of Kir2.1 subunits, leading to increased I(K1). Because
up-regulation of inward-rectifier currents is important for AF maintece,
these results provide potential new insights into molecular mechanisms of AF
with potential therapeutic implications. MicroRNAs (miRNAs) are increasingly reported to have important roles in diverse
biological and pathological processes. Changes in abundance of muscle-specific
microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia
and heart failure. However, the specific molecular targets and cellular
mechanisms involved in the miR-1 function in the heart are only beginning to
emerge. In this study, we investigated miR-1 expression and its potential role
in the mouse model of viral myocarditis (VMC). The expression levels of miR-1
and its target gene Connexin 43 (Cx43) were measured by real-time PCR and
western blotting, respectively. The miR-1 expression levels were significantly
increased in cardiac myocytes from VMC mice in comparison with control samples
(relative expression: 10 ± 2.5 vs. 31 ± 7.6, P < 0.05). Among the target genes
of miR-1, the expression Cx43 protein was significantly reduced in such mice
while there was no significant difference in the its mRNA levels. Our results
revealed an inverse correlation between miR-1 levels and Cx43 protein expression
in VMC samples. Using a bioinformatics-based approach, we found two identical
potential binding sites were found in mouse miR-1 and Cx43 3'- untranslated
region, this confirms a possible regulatory role of miR-1. In cultured, miRNA
transfected myocardial cells, we show overexpression of miR-1 accompanied by a
decrease in Cx43 protein's expression. There was only a slight (not
statistically significant) drop in Cx43 mRNA levels. Our results indicate that
miR-1 is involved in VMC via post-transcriptional repression of Cx43, and might
constitute potentially valuable data for the development of a new approach in
the treatment of this disease. The present study was designed to investigate whether microRNAs (miRNAs) are
involved in atrioventricular block (AVB) in the setting of myocardial ischemia
(MI). A cardiac-specific miR-1 transgenic (Tg) mouse model was successfully
established for the first time in this study using microinjection. miR-1 level
was measured by real-time qRT-PCR. Whole-cell patch clamp was employed to record
L-type calcium current (I Ca,L) and inward rectifier K(+) current (I K1).
Expression of connexin 43 (Cx43) protein was determined by western blot
analysis. Alternations of [Ca(2+)]i was detected by laser scanning confocal
microscopy in ventricular myocytes. The incidence of AVB was higher in miR-1 Tg
mice than that in wild-type (WT) mice. The normalized peak current amplitude of
I Ca,L was lower in ventricular myocytes from miR-1 Tg mice as compared with WT
mice. Similarly, the current density of I K1 was decreased in miR-1 Tg mice than
that in WT mice. Compared with WT mice, miR-1 Tg mice exhibited a significant
decrease of the systolic [Ca(2+)]i in ventricular myocytes but a prominent
increase of the resting [Ca(2+)]i. Moreover, Cx43 protein was downregulated in
miR-1 Tg mice compared to that in WT mice. Administration of LNA-modified
antimiR-1 reversed all the above changes. miR-1 overexpression may contribute to
the increased susceptibility of the heart to AVB, which provides us novel
insights into the molecular mechanisms underlying ischemic cardiac arrhythmias. Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction
of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein
connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy
leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether
miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy.
Thus, in this study we investigated whether a reduction in the extent of cardiac
hypertrophy could limit the pathological electrical remodeling of Cx43 and the
onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical
constriction of the ascending aorta to induce pathologic left ventricular
hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg
valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12
weeks. Sham surgery was performed for control groups. Programmed ventricular
stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When
compared to sham controls, rats from LVH group showed a significant decrease of
miR-1 and an increase of Cx43 expression and its ERK1/2-dependent
phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL
administration to rats with aortic banding significantly reduced cardiac
hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and
phosphorylation. Gain- and loss-of-function experiments in neonatal
cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1.
Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and
increased Cx43 expression and phosphorylation compared to un-stimulated NCMs.
Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector
intra-myocardial injection reduced Cx43 expression and phosphorylation in mice
with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression
and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of
pressure overload-induced myocyte hypertrophy reduces the risk of
life-threatening VT by normalizing miR-1 expression levels with the consequent
stabilization of Cx43 expression and activity within the gap junction. |
Are there Conserved Noncoding Elements (CNEs) in invertebrate genomes? | Yes. | We have identified Conserved Non-coding Elements (CNEs) in the regulatory region
of Caenorhabditis elegans and Caenorhabditis briggsae mab-9, a T-box gene known
to be important for cell fate specification in the developing C. elegans
hindgut. Two adjacent CNEs (a region 78 bp in length) are both necessary and
sufficient to drive reporter gene expression in posterior hypodermal cells. The
failure of a genomic mab-9::gfp construct lacking this region to express in
posterior hypodermis correlates with the inability of this construct to
completely rescue the mab-9 mutant phenotype. Transgenic males carrying this
construct in a mab-9 mutant background exhibit tail abnormalities including
morphogenetic defects, altered tail autofluorescence and abnormal lectin-binding
properties. Hermaphrodites display reduced susceptibility to the C. elegans
pathogen Microbacterium nematophilum. This comparative genomics approach has
therefore revealed a previously unknown role for mab-9 in hypodermal function
and we suggest that MAB-9 is required for the secretion and/or modification of
posterior cuticle. In addition to protein coding sequence, the organism genome contains a
significant amount of regulatory DNA. Comparative genomics reveals that the
organism genomes of vertebrates, tunicate, cephalochordate, flies, and nematodes
contain cis-regulatory elements with highly conserved non-coding elements
(CNEs). CNEs that cluster around trans-dev genes are part of core gene
regulatory networks (GRNs), and usually, they can act as transcriptional
enhancers. In this review, we described the identification of CNEs and
summarized their key properties across the metazoans, and then discussed the
evolution of CNEs after large-scale genome duplication events and the role of
CNEs in the evolution of ani-mal body plan. Urochordates are the closest relatives of vertebrates and at the larval stage,
possess a characteristic bilateral chordate body plan. In vertebrates, the genes
that orchestrate embryonic patterning are in part regulated by highly conserved
non-coding elements (CNEs), yet these elements have not been identified in
urochordate genomes. Consequently the evolution of the cis-regulatory code for
urochordate development remains largely uncharacterised. Here, we use
genome-wide comparisons between C. intestinalis and C. savignyi to identify
putative urochordate cis-regulatory sequences. Ciona conserved non-coding
elements (ciCNEs) are associated with largely the same key regulatory genes as
vertebrate CNEs. Furthermore, some of the tested ciCNEs are able to activate
reporter gene expression in both zebrafish and Ciona embryos, in a pattern that
at least partially overlaps that of the gene they associate with, despite the
absence of sequence identity. We also show that the ability of a ciCNE to
up-regulate gene expression in vertebrate embryos can in some cases be localised
to short sub-sequences, suggesting that functional cross-talk may be defined by
small regions of ancestral regulatory logic, although functional sub-sequences
may also be dispersed across the whole element. We conclude that the structure
and organisation of cis-regulatory modules is very different between vertebrates
and urochordates, reflecting their separate evolutionary histories. However,
functional cross-talk still exists because the same repertoire of transcription
factors has likely guided their parallel evolution, exploiting similar sets of
binding sites but in different combinations. |
Which tumor suppressor is referred to as "the guardian of the genome"? | The major tumour suppressor protein, p53, is one of the most well-studied proteins in cell biology. It plays a crucial role in regulating the transcription of numerous genes responsible for cells cycle arrest, DNA repair, angiogenesis, cell senescence, or apoptosis in response to various stress signals, and is considered one of the most important players in the development of cancer. p53 contributes to the maintenance of genomic stability. Thus, p53 has been described as "the guardian of the genome". | The tumor suppressor p53 is a multifunctional protein whose main duty is to
preserve the integrity of the genome. This function of wild-type p53 as
"guardian of the genome" is achieved at different levels, as a cell cycle
checkpoint protein, halting the cell cycle upon DNA damage, and via a direct
involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis.
Mutations in the p53 gene occur in about 50% of all human tumors and eliminate
the tumor suppressor functions of p53. However, many mutant p53 proteins have
not simply lost tumor suppressor functions but have gained oncogenic properties
which contribute to the progression of tumor cells to a more maligt
phenotype. The molecular basis for this gain of function of mutant p53 is still
unknown. However, mutant (mut) p53 specifically binds to nuclear matrix
attachment region (MAR) DNA elements. MAR elements constitute important higher
order regulatory elements of chromatin structure and function. By binding to
these elements, mut p53 could modulate important cellular processes, like gene
expression, replication, and recombination, resulting in phenotypic alterations
of the tumor cells. Mut p53 thus could be the first representative of a new
class of oncogenes, which exert their functions via long-range alterations or
perturbation of chromatin structure and function. The tumor-suppressor gene p53 acts as "the guardian of the genome", sensing DNA
damage and initiating protective responses. To examine the hypothesis that p53
abnormality leads to increased genomic alterations in primary tumor cells, our
study utilized 51 primary tumors of cervical carcinoma and 10 microsatellite
markers. These markers were mapped to the short arms of chromosomes 3 and 5,
covering the regions 3p13-25 and 5p15.1-15.3. Genomic deletion on 3p and 5p was
correlated with genetic or epigenetic p53 inactivation pathways, including p53
mutation, genetic deletion of p53 and cervical infection with human
papillomavirus. The proportion of abnormal p53 was found to be significantly
higher in the cases exhibiting loss of heterozygosity (LOH) on 5p (p < 0.001),
supporting the hypothesis of the presence of a p53-dependent pathway to cervical
tumorigenesis. In contrast, however, LOH on 3p was found to be independent of
p53 inactivation. A common deletion region, 3p22-24, was identified in 44% of
informative cases, and genomic loss at this specific region was correlated with
early tumorigenic onset and poor grade of tumor differentiation. Diversity
within the patterns of genomic alteration in the same form of cancer suggests
different sets of risk/tumorigenic profiles, molecular pathogenesis, as well as
prognosis and outcome. The p53 guardian of the genome is inactivated in the majority of cancers, mostly
through missense mutations that cause single residue changes in the DNA binding
core domain of the protein. Not only do such mutations result in the abrogation
of wild-type p53 activity, but the expressed p53 mutant proteins also tend to
gain oncogenic functions, such as interference with wild-type p53-independent
apoptosis. Because p53 mutants are highly expressed in cancer cells and not in
normal cells, their reactivation to wild-type p53 function may eliminate the
cancer by apoptosis or another p53-dependent mechanism. Several studies that
embarked on this quest for reactivation have succeeded in restoring wildtype p53
activity to several p53 mutants. However, mutants with more extensive structural
changes in the DNA binding core domain may be refractory to reactivation to the
wild-type p53 phenotype. Therefore, understanding the structure and functions of
oncogenic p53 mutants may lead to more potent reactivation modalities or to the
ability to eliminate mutant p53 gain of function. The PTEN tumor suppressor protein inhibits phosphatidylinositol 3-kinase
(PI3K)/Akt signaling that promotes translocation of Mdm2 into the nucleus. When
restricted to the cytoplasm, Mdm2 is degraded. The ability of PTEN to inhibit
the nuclear entry of Mdm2 increases the cellular content and transactivation of
the p53 tumor suppressor protein. Retroviral transduction of PTEN into U87MG
(PTEN null) glioblastoma cells increases p53 activity and expression of p53
target genes and induces cell cycle arrest. U87MG/PTEN glioblastoma cells are
more sensitive than U87MG/PTEN null cells to death induced by etoposide, a
chemotherapeutic agent that induces DNA damage. Previously, tumor suppressor
proteins have been supposed to act individually to suppress cancers. Our results
establish a direct connection between the activities of two major tumor
suppressors and show that they act together to respond to stresses and
maligcies. PTEN protects p53 from survival signals, permitting p53 to
function as a guardian of the genome. By virtue of its capacity to protect p53,
PTEN can sensitize tumor cells to chemotherapy that relies on p53 activity. p53
induces PTEN gene expression, and here it is shown that PTEN protects p53,
indicating that a positive feedback loop may amplify the cellular response to
stress, damage, and cancer. The tumor-suppressor p53 is a multifunctional protein mainly responsible for
maintaining genomic integrity. p53 induces its tumor-suppressor activity by
either causing cell-cycle arrest (G(1)/S or G(2)/M) or inducing cells to undergo
apoptosis. This function of wild-type p53 as "guardian of the genome" is
presumably achieved by forming molecular complexes with different DNA targets as
well as by interacting with a number of cellular proteins, e.g., Mdm2, Gadd45,
p21, 14-3-3sigma, Bax and Apaf-1. Upon activation, p53 activates p21, which in
turn controls the cell cycle by regulating G(1) or G(2) checkpoints. Here, we
report SMAR1 as one such p53-interacting protein that is involved in delaying
tumor progression in vivo as well as in regulating the cell cycle. SMAR1 is a
newly identified MARBP involved in chromatin-mediated gene regulation. The SMAR1
gene encodes at least 2 alternatively spliced variants: SMAR1(L) (the
full-length form) and SMAR1(S) (the shorter form). We report that expression of
SMAR1(S), but not of SMAR1(L), mRNA was decreased in most of the human cell
lines examined, suggesting selective silencing of SMAR1(S). Overexpression of
SMAR1(S) in mouse melanoma cells (B16F1) and their subsequent injection in
C57BL/6 mice delays tumor growth. Exogenous SMAR1(S) causes significant
retardation of B16F1 cells in the G(2)/M phase of the cell cycle compared to
SMAR1(L). SMAR1(S) activates p53-mediated reporter gene expression in mouse
melanoma cells, breast cancer cells (MCF-7) and p53 null cells (K562), followed
by activation of its downstream effector, p21. We further demonstrate that SMAR1
physically interacts and colocalizes with p53. These data together suggest that
SMAR1 is the only known MARBP that delays tumor progression via direct
activation and interaction with tumor-suppressor p53. PURPOSE: An extensive body of literature regarding p53 has accumulated during
the last 2 decades. The cellular mechanisms of p53 are complex yet well-defined,
whereas its clinical usefulness in the management of bladder cancer remains
controversial. We outline the basic constitutive functions of p53 and summarize
its current role in the management of transitional cell carcinoma of the
bladder.
MATERIALS AND METHODS: We conducted a MEDLINE based literature review concerning
the fundamental mechanisms of p53 and its role in the management of bladder
cancer.
RESULTS: The p53 gene is a tumor suppressor gene that acts as "guardian of the
genome." Many diverse cellular events, including DNA damage and hypoxia,
activate the p53 gene. The p53 protein functions as a transcription factor,
regulating downstream genes involved in cell cycle arrest, DNA repair and
programmed cell death. Loss of p53 function confers genomic instability,
impaired apoptosis and diminished cell cycle restraint. Therefore, p53 mutations
select for certain critical features of maligcy. Alteration of P53 is the
most common mutation in human cancer. Roughly half of all human maligcies,
including many urological cancers, exhibit p53 mutations. In bladder cancer p53
mutations have been associated with higher tumor grade and advanced stage, as
well as progression of superficial disease to muscle invasion. Moreover, p53
nuclear over expression appears to be an independent predictor of disease
progression and decreased survival after cystectomy.
CONCLUSIONS: The importance of p53 mutation in tumor cell biology is
irrefutable. Wild-type p53 mediates imperative functions such as regulation of
the cell cycle and programmed cell death. Deficiency of p53 function by mutation
or inactivation abrogates normal cell cycle checkpoints and apoptosis,
generating a favorable milieu for genomic instability and carcinogenesis.
However, despite the manifest importance of p53 in human maligcy, its current
role in the management of bladder cancer appears somewhat limited. A multitude
of retrospective studies have associated p53 mutations with adverse outcomes in
superficial and muscle invasive disease. Nonetheless, randomized prospective
studies are needed to determine the potential clinical implications of p53 in
bladder cancer. Although p53 is clearly involved in the salvage pathway to DNA damage, its
frequent mutations do not explain the efficacy of radiotherapy and chemotherapy.
Indeed, around 50% of all human cancers show mutations in p53, and a further
fraction show a functional inactivation of the protein. Nevertheless, patients
seem to respond to therapy that would otherwise require a functional p53. At
least in part, these responses could be explained by the pathway mediated by
p73. This mechanism is parallel to, but independent of the p53 pathway. Several
pieces of evidence show a significant interaction between these two proteins.
Therefore, while p53 can be rightly defined as the guardian of the genome, we
could think of p73 as the "assistant" guardian of the genome! The process of maligt transformation universally entails genetic damage and
oncogenic signaling, two stresses that are signaled to p53 through different
genetic pathways. Based on this, it is possible to distinguish two jobs for p53:
"guardian of the genome" that consists in sensing and reacting to DNA damage
through the ATM/ATR and Chk1/Chk2 kinases, and "policeman of the oncogenes"
that, correspondingly, consists in responding to oncogenic signaling through the
p53-stabilizing protein ARF. Contrary to expectation, recent genetic evidence in
mice indicates that the response of p53 to DNA damage has little or no impact on
cancer protection. In contrast, ARF-dependent activation of p53 is critical for
p53-mediated tumor suppression. Here, we discuss the mechanistic implications of
these observations and their relevance for cancer therapy. The p53 tumor suppressor protein has long been recognized as the central factor
protecting humans from cancer. It has been famously dubbed "the guardian of the
genome" due to its ability to respond to genotoxic stress, such as DNA damage
and other stress signals, and to protect the genome by inducing a variety of
biological responses including DNA repair, cell cycle arrest, and apoptosis.
However, the tumor suppressive effects of p53 go far beyond its roles in
mediating these three processes. There is growing evidence that p53 also exerts
its effects on multiple aspects of tumor formation, including suppression of
metastasis and, as summarized in this review, inhibition of new blood vessel
development (angiogenesis). The p53 protein has been shown to limit angiogenesis
by at least three mechanisms: (1) interfering with central regulators of hypoxia
that mediate angiogenesis, (2) inhibiting production of proangiogenic factors,
and (3) directly increasing the production of endogenous angiogenesis
inhibitors. The combination of these effects allows p53 to efficiently shut down
the angiogenic potential of cancer cells. Inactivation of p53, which occurs in
approximately half of all tumors, reverses these effects; as a consequence,
tumors carrying p53 mutations appear more vascularized and are often more
aggressive and correlate with poor prognosis for treatment. Thus, the loss of
functional p53 during tumorigenesis likely represents an essential step in the
switch to an angiogenic phenotype that is displayed by aggressive tumors. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor
suppressor is a phosphatase that antagonizes the phosphoinositol-3-kinase/AKT
signaling pathway and suppresses cell survival as well as cell proliferation.
PTEN is the second most frequently mutated gene in human cancer after p53.
Germline mutations of PTEN have been found in cancer susceptibility syndromes,
such as Cowden syndrome, in which over 80% of patients have mutations of PTEN.
Homozygous deletion of Pten causes embryonic lethality, suggesting that PTEN is
essential for embryonic development. Mice heterozygous for Pten develop
spontaneous tumors in a variety of organs comparable with the spectrum of its
mutations in human cancer. The mechanisms of PTEN functions in tumor suppression
are currently under intense investigation. Recent studies demonstrate that PTEN
plays an essential role in the maintece of chromosomal stability and that
loss of PTEN leads to massive alterations of chromosomes. The tumor suppressor
p53 is known as a guardian of the genome that mediates the cellular response to
environmental stress, leading to cell cycle arrest or cell death. Through
completely different mechanisms, PTEN also protects the genome from instability.
Thus, we propose that PTEN is a new guardian of the genome. In this review, we
will discuss new discoveries on the role of PTEN in tumor suppression and
explore mechanisms by which PTEN maintains genomic stability. The p53 gene has been referred to as 'the guardian of the genome' because it
controls apoptosis and cell cycle arrest. The purpose of this study was to
evaluate the association of p53 codon 72 genetic polymorphism and the p53
immunohistochemistry with Helicobacter pylori-associated gastroduodenal
diseases, including gastric cancer. This study included 1,852 subjects: controls
and patients with gastric cancer, dysplasia, benign gastric ulcers, and duodenal
ulcers (DU). Biallelic polymorphism was genotyped by restriction fragment length
polymorphism. Immunohistochemical analysis for the detection of mutant type p53
expression was performed. The frequency of the Pro/Pro allele of the p53 codon
72 was higher in the patients with H. pylori-positive dysplasia than in controls
(OR: 2.3, 95% CI: 1.3-4.3), but it was less frequent among patients with a H.
pylori-positive DU (OR: 0.5, 95% CI: 0.3-0.8). However, there was no significant
association with gastric cancer, including the location, stage, or histological
type of gastric cancer. Expression of a mutant type of p53 protein was detected
in 6.3% of dysplastic tissues and 26.5% of cancerous tissues compared 0% in the
controls. Positive expression was higher in the intestinal type of cancer
(34.9%) than in the diffuse type (15.0%; P = 0.001). These results suggest that
genetic polymorphism of p53 codon 72 played a role in the determination of H.
pylori-associated gastroduodenal diseases, but p53 immunostaining did not
correlate with those of the p53 genetic polymorphism analysis. The tumor suppressor p53 is often referred to as "the guardian of the genome"
because of its central role in the cellular response to oncogenic stress and
prevention of tumor development. Mutations of p53 in acute myeloid leukemia
(AML) are rare but resistance to chemotherapy has been reported because of the
deregulation of the p53 signaling and differentiation pathways. It is known that
the interaction of the vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25D)
with its functional vitamin D receptor leads to differentiation, G(1) arrest,
and increased cell survival in p53-null AML cells. However, there are no reports
on the effect of 1,25D in leukemia cells expressing wild-type p53. Here, we
examine vitamin D signaling in AML cells MOLM-13 and OCI-AML3 expressing
wild-type p53 in the presence and absence of the MDM2 antagonist nutlin-3. We
find that 1,25D alone induces monocytic differentiation in these cell lines
similar to that seen in p53-null AML cells, suggesting that the presence of
wild-type p53 is compatible with activation of vitamin D signaling. Combination
of nutlin-3a with 1,25D accelerated programmed cell death, likely because of
enhanced nutlin-induced upregulation of the proapoptotic PIG-6 protein and
downregulation of antiapoptotic BCL-2, MDMX, human kinase suppressor of Ras 2,
and phosphorylated extracellular signal-regulated kinase 2. Tumor development in the skin may be a multistep process where multiple genetic
alterations occur successively. The p53 gene is involved in genome stability and
thus is referred to as "the guardian of the genome." To better understand the
antigenotoxic effects of p53 in ultraviolet light B (UVB)-induced mutagenesis,
mutations were measured in the epidermis of UVB-irradiated p53(+/+) and p53(-/-)
gpt delta mice. In the mouse model, point mutations and deletions are separately
identified by the gpt and Spi(-) assays, respectively. The mice were exposed to
UVB at single doses of 0.5, 1.0, or 2.0 kJ/m(2) . The mutant frequencies (MFs)
were determined 4 weeks after the irradiation. All doses of UVB irradiation
enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no
significant differences in gpt MFs and the mutation spectra between p53(+/+) and
p53(-/-) mice. The predomit mutations induced by UVB irradiation were G:C to
A:T transitions at dipyrimidines. In contrast, in unirradiated p53(-/-) mice,
the frequencies of Spi(-) large deletions of more than 1 kb and complex-type
deletions with rearrangements were significantly higher than those of the Spi(-)
large deletions in p53(+/+) counterparts. The specific Spi(-) mutation frequency
of more than 1 kb deletions and complex types increased in a dose-dependent
manner in the p53(+/+) mice. However, no increase of such large deletions was
observed in irradiated p53(-/-) mice. These results suggest that the
antigenotoxic effects of p53 may be specific to deletions and complex-type
mutations induced by double-strand breaks in DNA. The tumor suppressor p53 homologues, TA-p73, and p63 have been shown to function
as tumor suppressors. However, how they function as tumor suppressors remains
elusive. Here, I propose a number of tumor suppressor pathways that illustrate
how the TA-p73 and p63 could function as negative regulators of invasion,
metastasis, and cancer stem cells (CSCs) proliferation. Furthermore, I provide
molecular insights into how TA-p73 and p63 could function as tumor suppressors.
Remarkably, the guardians--p53, p73, and p63--of the genome are in control of
most of the known tumor suppressor miRNAs, tumor suppressor genes, and
metastasis suppressors by suppressing c-myc through
miR-145/let-7/miR-34/TRIM32/PTEN/FBXW7. In particular, p53 and TA-p73/p63 appear
to upregulate the expression of (1) tumor suppressor miRNAs, such as let-7,
miR-34, miR-15/16a, miR-145, miR-29, miR-26, miR-30, and miR-146a; (2) tumor
suppressor genes, such as PTEN, RBs, CDKN1a/b/c, and CDKN2a/b/c/d; (3)
metastasis suppressors, such as Raf kinase inhibitory protein, CycG2, and DEC2,
and thereby they enlarge their tumor suppressor network to inhibit
tumorigenesis, invasion, angiogenesis, migration, metastasis, and CSCs
proliferation. The classical functions of p53 protein are those related to its role on DNA
damage, cell growth arrest, senescence and apoptosis. For this reason it is
called 'the guardian of the genome' and is considered one of the most important
players in the development of cancer. However, more recently it has been show
that p53 is not only involved in cancer, but also in ageing. p53 is stimulated
by stress, which in turn results in the activation of a wide range of
transcriptional targets. Low-intensity stress will activate p53 in a manner
which results in antioxidant response, thus protecting against ageing because of
its antioxidant function. On the contrary, high-intensity activation of p53 will
result in an increase of oxidative stress by activation of p53-mediated
pro-oxidant targets, thus increasing the rate of ageing, but protecting against
cancer. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in the cell
cycle, ensuring correct chromosome segregation and normal progression through
mitosis. Aurora B is overexpressed in many types of human cancers, which has
made it an attractive target for cancer therapies. Tumor suppressor p53 is a
genome guardian and important negative regulator of the cell cycle. Whether
Aurora B and p53 are coordinately regulated during the cell cycle is not known.
We report that Aurora B directly interacts with p53 at different subcellular
localizations and during different phases of the cell cycle (for instance, at
the nucleus in interphase and the centromeres in prometaphase of mitosis). We
show that Aurora B phosphorylates p53 at S183, T211, and S215 to accelerate the
degradation of p53 through the polyubiquitination-proteasome pathway, thus
functionally suppressing the expression of p53 target genes involved in cell
cycle inhibition and apoptosis (e.g., p21 and PUMA). Pharmacologic inhibition of
Aurora B in cancer cells with WT p53 increased p53 protein level and expression
of p53 target genes to inhibit tumor growth. Together, these results define a
mechanism of p53 inactivation during the cell cycle and imply that oncogenic
hyperactivation or overexpression of Aurora B may compromise the tumor
suppressor function of p53. We have elucidated the antineoplastic mechanism for
Aurora B kinase inhibitors in cancer cells with WT p53. Tumor suppressor p53, known as 'the guardian of the genome', has the ability to
prevent the emergence of transformed cells by the induction of cell cycle arrest
and apoptosis. Otherwise, there were researches about the function of p53, such
as NDA repair, regulating metabolism and maternal reproduction in recent years.
Furthermore, there was a new function for p53 in antiviral apoptosis mentioned
in the research, Integration of interferon-alpha/beta signaling to p53 responses
in tumour suppression and antiviral defense. In order to define the antiviral
function of p53, many target genes has been defined, such as IRF9, IRF5, ISG15
and TLR3. All of these implied there must be a complex mechanism for role of p53
in antiviral innate immunity, adaptive immunity and inflammation. The tumor suppressor protein p53 is often referred to as the guardian of the
genome. In the past, controversial findings have been presented for the role of
the C-terminal regulatory domain (RD) of p53 as both a negative regulator and a
positive regulator of p53 activity. However, the underlying mechanism remained
enigmatic. To understand the function of the RD and of a domit
phosphorylation site within the RD, we analyzed p53 variants in vivo and in
vitro. Our experiments revealed, surprisingly, that the p53 RD of one subunit
interacts with the DNA binding domain of an adjacent subunit in the tetramer.
This leads to the formation of intersubunit contacts that stabilize the
tetrameric state of p53 and enhance its transcriptional activity in a
cooperative manner. These effects are further modulated by phosphorylation of a
conserved serine within the RD. The tumor suppressor protein p53 has been described "as the guardian of the
genome" for its crucial role in regulating the transcription of numerous genes
responsible for cells cycle arrest, senescence, or apoptosis in response to
various stress signals. Although p53 promotes longevity by decreasing the risk
of cancer through activation of apoptosis or cellular senescence, several
findings suggest that an increase of its activity may have deleterious effects
leading to selected aspects of the aging phenotype and neurodegenerative
diseases. There is the link between p53 and oxidative stress, the latter a
crucial factor that contributes to neurodegenerative processes like Alzheimer
disease (AD). In the present study, using a proteomics approach, we analyzed the
impact of lack of p53 on the expression of several brain mitochondrial proteins
involved in different pathways, and how lack of p53 may present a target to
restore neuronal impairments. Our investigation on isolated brain mitochondria
from p53((-/-)) mice also provides a better understanding of the
p53-mitochondria relationship and its involvement in the development of many
diseases. The TP53 gene, first described in 1979, was identified as a tumor suppressor
gene in 1989, when it became clear that its product, the p53 nuclear
phosphoprotein, was frequently inactivated in many different forms of cancers.
Nicknamed "guardian of the genome", TP53 occupies a central node in stress
response networks. The p53 protein has a key role as transcription factor in
limiting oncogenesis through several growth suppressive functions, such as
initiating apoptosis, senescence, or cell cycle arrest. The p53 protein is
directly inactivated in about 50% of all tumors as a result of somatic gene
mutations or deletions, and over 80% of tumors demonstrate dysfunctional p53
signaling. Beyond the undeniable importance of p53 as a tumor suppressor, an
increasing number of new functions for p53 have been reported, including its
ability to regulate energy metabolism, to control autophagy, and to participate
in various aspects of differentiation and development. Recently, studies on
genetic variations in TP53 among different populations have led to the notion
that the p53 protein might play an important role in regulating fertility. This
review summarizes current knowledge on the basic functions of different genes of
the TP53 family and TP53 pathway with respect to fertility. We also provide
original analyses based on genomic and genotype databases, providing further
insights into the possible roles of the TP53 pathway in human reproduction. The major tumour suppressor protein, p53, is one of the most well-studied
proteins in cell biology. Often referred to as the Guardian of the Genome, the
list of known functions of p53 include regulatory roles in cell cycle arrest,
apoptosis, angiogenesis, DNA repair and cell senescence. More recently, p53 has
been implicated as a key molecular player regulating substrate metabolism and
exercise-induced mitochondrial biogenesis in skeletal muscle. In this context,
the study of p53 therefore has obvious implications for both human health and
performance, given that impaired mitochondrial content and function is
associated with the pathology of many metabolic disorders such as ageing, type 2
diabetes, obesity and cancer, as well as reduced exercise performance. Studies
on p53 knockout (KO) mice collectively demonstrate that ablation of p53 content
reduces intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial yield,
reduces cytochrome c oxidase (COX) activity and peroxisome
proliferator-activated receptor gamma co-activator 1-α protein content whilst
also reducing mitochondrial respiration and increasing reactive oxygen species
production during state 3 respiration in IMF mitochondria. Additionally, p53 KO
mice exhibit marked reductions in exercise capacity (in the magnitude of 50 %)
during fatiguing swimming, treadmill running and electrical stimulation
protocols. p53 may regulate contractile-induced increases in mitochondrial
content via modulating mitochondrial transcription factor A (Tfam) content
and/or activity, given that p53 KO mice display reduced skeletal muscle
mitochondrial DNA, Tfam messenger RNA and protein levels. Furthermore, upon
muscle contraction, p53 is phosphorylated on serine 15 and subsequently
translocates to the mitochondria where it forms a complex with Tfam to modulate
expression of mitochondrial-encoded subunits of the COX complex. In human
skeletal muscle, the exercise-induced phosphorylation of p53(Ser15) is enhanced
in conditions of reduced carbohydrate availability in association with enhanced
upstream signalling through 5'adenosine monophosphate-activated protein kinase
but not p38 mitogen-activated protein kinase. In this way, undertaking regular
exercise in carbohydrate restricted states may therefore be a practical approach
to achieve the physiological benefits of consistent p53 signalling. Although our
knowledge of p53 in exercise metabolism has advanced considerably, much of our
current understanding of p53 regulation and associated targets is derived from
various non-muscle cells and tissues. As such, many fundamental questions remain
uswered in contracting skeletal muscle. Detailed studies concerning the
time-course of p53 activation (including additional post-translational
modifications and subsequent subcellular translocation), as well as the effects
of exercise modality (endurance versus resistance), intensity, duration, fibre
type, age, training status and nutrient availability, must now be performed so
that we can optimise exercise prescription guidelines to strategically target
p53 signalling. The emerging role of p53 in skeletal muscle metabolism therefore
represents a novel and exciting research area for exercise and muscle
physiologists. During the past ten years, microRNAs (miRNAs) have been shown to play a more
significant role in the formation and progression of cancer diseases than
previously thought. With an increase in reports about the dysregulation of
miRNAs in diverse tumor types, it becomes more obvious that classic
tumor-suppressive molecules enter deep into the world of miRNAs. Recently, it
has been demonstrated that a typical tumor suppressor p53, known as the guardian
of the genome, regulates some kinds of miRNAs to contribute to tumor suppression
by the induction of cell-cycle arrest and apoptosis. Meanwhile, miRNAs
directly/indirectly control the expression level and activity of p53 to
fine-tune its functions or to render p53 inactive, indicating that the interplay
between p53 and miRNA is overly complicated. The findings, along with current
studies, will underline the continuing importance of understanding this
interlocking control system for future therapeutic strategies in cancer
treatment and prevention. |
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