Analysis of time-lapse cinematographic film permitted the construction of pedigrees from 88 well oxygenated cells of a mouse osteosarcoma (MOS). These cells have been chronically treated with various concentrations of the hypoxic cell sensitizer misonidazole (MIS) over periods of up to 96 h. At concentrations of 0.5 and 7 mM there is a 2--3 h increase in cell-cycle time. Concentrations of 2 mM show an intermitotic time delay of 7.6--10.3 h. At 4 mM cells divided only once. With increasing drug concentration there was an increase in the number of abnormal mitoses. These results were compared with cloning efficiency (PE) experiments. PE at 0.5 mM is 80%, at 1 mM 40 and at 2 mM is reduced to 4%. Cells treated with 2mM MIS over a period of 28.6 h resume their normal cycle when the drug is washed from the culture. This may indicate that DNA is not a major target for MIS. It is concluded that this hypoxic cell sensitizer is also toxic for MOS cells in well oxygenated conditions.


Cyclin D1 is a cell cycle regulator essential for G1 phase progression and is frequently overexpressed in several human tumour types as a consequence of gene amplification or chromosomal rearrangements. We analysed the expression of cyclin D1 in 75 patients with transitional cell carcinoma (TCC) to investigate the possible relationship between its expression and clinical outcome as well as histopathological findings using the immunohistochemical method. We observed strong staining  for cyclin D1 in 19 cases (25.3%) and weak staining  in 19 cases (25.3%). Overexpression of cyclin D1 was not associated with tumour invasion. No significant association was found between overexpression of cyclin D1 and tumour grade (P > 0.05). We assessed the differences of disease-free interval in superficial tumours and actuarial survival probability in invasive tumours according to the status of cyclin D1 expression. Tumours with (++) staining for cyclin D1 recurred much more rapidly than (-) and/or (+) staining tumours . However, overexpression of cyclin D1 was not associated with a shortened overall survival of patients with invasive tumours (P < 0.1). These results suggest that genetic alteration of cyclin D1 appears to be an early event in the tumorigenesis of bladder TCC and is associated with early recurrence in superficial tumours.


Changes which lead to excessive cyclin production or to loss of cell cycle inhibition by proteins such as p16/MTS1 may release breast tumour cells from the constraints of cell division. In order to establish the frequency of MTS1/p16 gene alteration and its relation with genetic damage to the p53 and cyclin D1 genes, we have studied these gene abnormalities in 164 human primary breast cancers and in six breast cancer cell lines. Two breast cancer cell lines and one primary tumour showed a homozygous deletion of exon 2 of the MTS1 gene. Using single-strand conformation polymorphism and subsequent sequencing analysis, one tumour showed an alteration at codon 67 . Another tumour showed a mutation at codon 98 (without amino acid change) with an additional polymorphism at codon 140. This polymorphism was also found in 13 other tumour samples, but has no effect on (disease-free) survival. From these data we conclude that the occurrence of CDKN2 (p16/MTS1) mutation in primary breast cancer is a rare event and is not likely to be involved in human breast tumour carcinogenesis and progression.


Fifteen patients (34.9%) achieved partial remission  for a median duration of 6 months; the median survival of these patients has not yet been reached. A further 15 had stable disease for a median of 4 months and a median survival of 7 months. The 10 patients (23.2%) who experienced disease progression had a median survival of 4 months. Three patients are not evaluable. The 1-year actuarial survival rate is 31.1%. The treatment was well tolerated: only 35% of the patients had grade 3 or 4 granulocytopenia on day 14, none experienced episodes of neutropenic fever, and there was no evidence of severe haematological toxicity upon recycling. Only 9% of the patients suffered from gastrointestinal toxicity (grade 3); increased but reversible transaminase levels were observed in 11.6%. In conclusion, the results of this phase II study show that the combination of gemcitabine and vinorelbine is active and well tolerated in NSCLC, and thus encourage its use in elderly or unfit patients. © 2000 Cancer Research CampaignCisplatin-based combinations are efficacious in increasing the overall survival of patients with non-small cell lung cancer (NSCLC), but their toxicity makes them unsuitable for elderly and unfit patients. The primary objective of this non-randomized phase II study was to evaluate the feasibility and activity of the gemcitabine plus vinorelbine combination in previously untreated elderly and/or unfit patients with measurable stage III or IV NSCLC. Forty-three patients aged ≥ 65 years or with contraindications against cisplatin treatment  received intravenous (i.v.) gemcitabine 1000 mg m


H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the These results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells. IGF2) gene plays a role in the pathogenesis of the overgrowth disorder Beckwith-Wiedemann syndrome , the growth-restriction condition Silver-Russell syndrome , as well as various human cancers including Wilms tumour, rhabdomyosarcoma, hepatoblastoma, colorectal and breast carcinomas Aberrant imprinting of the Insulin-like growth factor 2  H19 DMR) is located 5′ of the H19 promoter is methylated on the paternal chromosome and is known as the imprinting control region IC1 because it functions as an insulator mediated by the CCCTC binding factor (CTCF) Igf2 allele and in down-regulation of H19Igf2 exists such that deletion or mutation within the IC1 also results in methylation changes in DMRs within the Igf2 gene Igf2 DMRs in a mutually exclusive and parent of origin specific manner influenced by CTCF binding and DNA methylation IGF2 DMRs in mouse and human. Specifically, IGF2 DMR0, located 5′ to the main IGF2 promoters is methylated on the maternal allele only in placentas in the mouse IGF2 allele was inferred using cell lines derived from a BWS patient with paternal uniparental disomy at 11p15.5 loci (UPD) IGF2 DMR0 has been carried out in many cancer studies following the demonstration that hypomethylation of this DMR is associated with loss of imprinting in Wilms tumour IGF2 DMR0 plays a role in reactivation of the silent maternal allele Mouse models have been used to demonstrate that the imprinted expression of the closely linked IGF2 and biallelic silencing of H19. Another group of BWS patients has normal IC1 methylation but abnormal methylation at IC2, a maternally methylated imprinting control region within the KCNQ1 and KCNQ1OT1 gene cluster which seems to operate independent of IGF2 and H19IGF2 and biallelic activation of H19The molecular basis of BWS is heterogeneous with 5% of patients exhibiting gain of methylation at IC1 with biallelic activation of IGF2 DMR0 region in BWS and SRS patients and it is not known to what extent the proposed silencing function of this DMR contributes to the aetiology of BWS and SRS. We therefore examined parent of origin methylation at the DMR0 in various subtypes of BWS and SRS patients and also in a cohort of Wilms tumour samples. Our results indicate that the active paternal IGF2 allele is methylated at the DMR0 in cis with H19 methylation in normal individuals and that in SRS and BWS the DMR0 and IC1 have similar methylation abnormalities. In contrast, in Wilms tumour samples DMR0 methylation is negatively correlated with IC1 methylation. These results imply IGF2 imprinting defects in congenital growth disorders and Wilms tumours arise through different epigenetic mechanisms.To date there have been no reports on the methylation status of the trans or that methylation at the DMR0 is on the paternal allele.We examined the methylation status of DMR0 in peripheral blood leukocyte DNA derived from BWS patients with IC1 hypermethylation (n =  7), IC2 hypomethylation (n = 37) or normal methylation at both IC1 and IC2 (n = 27) and SRS patients with IC1 hypomethylation (n =  2), using bisulphite and pyrosequencing analysis. The region analysed is shown in IGF2 DMR0 in some of our BWS and SRS patients and 6 control individuals in order to confirm our findings. The patients with maternal duplication had reduced methylation levels (44%), while all the paternal UPD and the paternal duplication patients had hypermethylation , cis with methylation at the IC1  or the paternal allele fails to become methylated and is silenced (SRS). These allele-specific methylation changes have the effect of an imprint switch so that both alleles look like either a paternal allele (BWS) or a maternal allele (SRS). Pyrosequencing results were verified by bisulphite sequencing  and two SRS patients with maternal 11p15.5 duplications. We also determined directly the parental origin of methylation at the  the IC1 . These rquencing .We analysed DMR0 methylation in two Wilms tumour patients that had arisen in individuals affected by BWS and a series of non-syndromic Wilms tumour patients. One of the BWS patients had constitutional hypermethylation at IC1, the other had paternal UPD. The non-syndromic Wilms tumour patients comprised of individuals that had tumour-specific IC1 hypermethylation (n = 10), 11p15.5 LOH with loss of the maternal allele (n = 13) or normal methylation (n = 10) at the IC1. Remarkably, all tumours with hypermethylation at IC1 and most of those with LOH, including those arisen in individuals affected by BWS, showed reduced methylation at DMR0 , while those with normal IC1 methylation had normal DMR0 methylation  . Indeed,IGF2 gene in Wilms tumour samples with and without loss of imprinting MspI/ HpaII restriction analysis , while others have reported exceptions in ovarian and bladder cancer During tumorigenesis global hypomethylation occurs in repeat rich regions, LINE and SINE elements, while CpG islands associated with promoters are subject to hypermethylation IGF2-H19 locus and associated with imprinting loss in BWS, SRS and Wilms Tumour. In the growth disorders, germline allele-specific methylation changes have the effect of an imprint switch so that both alleles look like either a paternal allele (BWS) or a maternal allele (SRS) while in cancer both parental marks are lost and reprogrammed. Our findings demonstrate that loss of IGF2 imprinting is a complex phenomenon that occurs with different mechanisms in human disease, probably reflecting different molecular causes and responses.In summary, we show three different reprogramming events occurring at the http://www.geneclinics.org). The study also involved 40 Wilms Tumour patients enrolled by Paediatric Oncology Units affiliated to Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). All tumours were histologically diagnosed.85 BWS and 3 SRS cases were recruited from different Italian Pediatric Departments and were clinically diagnosed according to the criteria described in the literature  which included six CpGs, three of which were previously reported to be hypomethylated in colorectal patients with LOI at IGF25′TGAGGATGGGTTTTTGTTTGGTAT3′ and biotinylated reverse primer 5′TCCTCAATCCACCCAAAATAATAT3′. 10 uL of the biotinylated PCR product was used for each sequencing assay using the following sequencing primers 5′GGGGTGGAGGGTGTA 3′ and 5′-AAAAGTTATTGGATATATAGT 3′. Pyrosequencing was carried on PSQ HS 96 System and PyroMark MD System using Pyro Gold Reagent kits . Allele-specific pyrosequencing was carried out by replacing the above biotinylated primer with allele-specific biotinylated primers, 5′CCCAAAATAATATCTATAAAAAAAAAATTCAC3′, or 5′CCCAAAATAATATCTATAAAAAAAAAATTCAT3′ which recognized the G or A allele of the rs3741210 polymorphism on the reverse strand after bisulphite conversion.We designed a standard pyrosequencing assay for Methylation was quantified using Pyro Q-CpG Software  that calculates the ratio of converted C's (T's) to unconverted C's at each CpG and expresses this as a percentage methylation. Average methylation across the DMR0 for all 6 CpGs were calculated. Median and IQR methylation for different categories of patients were analysed using Prism Graphpad software.We verified allele-specific methylation by conventional bisulphite sequencing of cloned PCR products in all our informative cases.Figure S1Bisulphite sequencing analyses of IGF2 DMR0 Methylation in normal, congenital growth disorders and Wilms Tumour. IGF2 DMR0 Methylation in normal, congenital growth disorders and Wilms Tumour. Methylation of 6 CpGs was determined by bisulphite genomic sequencing on DNA extracted from peripheral blood leukocytes  or tumour tissue (Wilms Tumour) of individuals informative for the rs3741210 polymorphism. Filled circles represent methylated CpGs and open circles unmethylated CpGs. The maternal (MAT) and paternal (PAT) alleles of the IGF2 DMR0 region are indicated.(1.33 MB TIF)Click here for additional data file.


Telomerase is a specialized ribonucleoprotein polymerase that directs the synthesis of telomerase repeats at chromosome ends. Accumulating evidence has indicated that telomerase is stringently repressed in normal human somatic tissues but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity plays a role in carcinogenesis and immortalization. In this work, the status of telomerase activity during the development of human thyroid cancer was determined using telomeric repeat amplification protocol (TRAP) in 14 nodular hyperplasia, 14 adenomas, 23 papillary carcinomas and 11 follicular carcinomas. Positive telomerase activity was detected in 2 of 14 nodular hyperplasias (14%), 4 of 14 adenomas (29%), 12 of 23 papillary carcinomas (52%) and 10 of 11 follicular carcinomas (91%). The cancers that are negative for telomerase activity are mostly in early stage (stage I or II). These results suggest that telomerase reactivation plays a role during the development of thyroid cancer.


Experimental evidence for this mechanism in urinary bladder, ureter, gut, lung, uterus, tooth-pulp and tongue is reviewed. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by agents that interfere with mechanosensory transduction in the organs considered, including P2X A schem3 homomeric and P2X2/3 heteromeric receptors on subepithelial sensory nerves initiating impulses in sensory pathways to pain centres in the central nervous system (CNS) )3 and/or P2X2/3 receptors on subepithelial sensory nerve terminals to relay nociceptive messages via sensory ganglia and spinal cord to pain centres in the CNS.Compelling evidence has been presented for the role of purinergic mechanosensory transduction where ATP, released from epithelial cells lining the bladder, ureter and gut during distension, acts on P2X3 and P2X2/3 receptors are being explored to treat visceral pain and the possibilities for development of agents that inhibit ATP transport from epithelial cells or enhance ATP breakdown after its release are discussed.Antagonists to P2XThe author declares that he has no competing interests.


The parental conflict hypothesis predicts that the mother inhibits embryo growth counteracting growth enhancement by the father. In plants the DNA methyltransferase MET1 is a central regulator of parentally imprinted genes that affect seed growth. However the relation between the role of MET1 in imprinting and its control of seed size has remained unclear. Here we combine cytological, genetic and statistical analyses to study the effect of MET1 on seed growth. We show that the loss of MET1 during male gametogenesis causes a reduction of seed size, presumably linked to silencing of the paternal allele of growth enhancers in the endosperm, which nurtures the embryo. However, we find no evidence for a similar role of MET1 during female gametogenesis. Rather, the reduction of MET1 dosage in the maternal somatic tissues causes seed size increase. MET1 inhibits seed growth by restricting cell division and elongation in the maternal integuments that surround the seed. Our data demonstrate new controls of seed growth linked to the mode of reproduction typical of flowering plants. We conclude that the regulation of embryo growth by MET1 results from a combination of predominant maternal controls, and that DNA methylation maintained by MET1 does not orchestrate a parental conflict. In flowering plants, meiosis is followed by the production of haploid structures, the male pollen and the female embryo sac, each containing two gametes. After double-fertilization, the female gametes, the egg cell and central cell, respectively give rise to the embryo and its nurturing annex, the endosperm. The embryo and the endosperm develop within the maternally derived seed integuments. Seed size is controlled primarily by interactions between the endosperm and integuments MET1, using a dominant antisense construct, MET1a/sMET1a/s causes an increase of seed size whereas paternal inheritance has an opposite effect. MET1 is a key player in the control of parental genomic imprinting, which restricts gene expression from one of the two parental alleles Arabidopsis, it was proposed that MET1 controls the expression of two pools of imprinted genes: maternally expressed inhibitors and paternally expressed enhancers of endosperm growth Arabidopsis two imprinted genes dependent on MET1 have been identified FWA and FERTILIZATION INDEPENDENT SEED 2 (FIS2) in the male gametes FIS2 and FWA are expressed in the female central cell FIS2 and FWA are expressed in the endosperm from their maternal allele, while MET1 maintains silencing on the paternal allele FIS2 and FWA as imprinted genes.The parental contributions to seed size were identified in crosses involving diploid and tetraploid plants. Tetraploid mothers produced smaller seeds when crossed to diploid fathers, however tetraploid fathers crossed to diploid mothers produced larger seeds MET1a/s were mediated by removal of silencing of the paternal allele of endosperm growth inhibitors, thus causing seed size increase and vice versa MET1a/s has a dominant effect, which does not allow distinguishing whether seed size variations in wild type (wt)×MET1a/s crosses originated from the loss of MET1 in the previous parental generation (sporophyte) or in the haploid generation producing the gametes (gametophyte). In addition, MET1a/s lines accumulate epimutations met1-6met1 during male gametogenesis reduces seed size. This result was also in agreement with the demonstration of a gametophytic effect of met1-3 on the silencing of the paternal alleles of the imprinted genes FIS2 and FWAmet1-6 on seed size remained unclear met1-6 loss of function on the diploid parental sporophytic generation was not tested explicitly. To address these concerns, we restricted our analysis to homozygous and heterozygous mutants derived from a self-fertilized heterozygous met1-3/+ mother and compared the effects on seed development of met1-3 loss of function during male gametogenesis, female gametogenesis and the parental diploid generation.It was expected that the contrasting effects of met1-3 causes a loss of DNA methylation in first generation homozygous plants met1 function is caused by a T-DNA insert linked to a gene conferring resistance to the herbicide BASTA. To confirm specific parental contributions of met1-3 to seed size, we analyzed digital images of seeds from crosses that varied MET1 genotype and parent of transmission   , Table 1met1-3, seeds from wt×met1-3/+ crosses were visually sorted according to their size relative to a wild type control, and BASTA resistance associated to met1-3 was tested. Two populations of seeds were distinguished. All smallest seeds were resistant to BASTA (n = 323) while all largest seeds were sensitive to BASTA (n = 336). The 1∶1 proportion supported the predicted association of the paternal effect of met1-3 to gametogenesis (p = 0.6126 χ2). As we did not analyze the entire population we may have missed a complex genetic component regulating seed size. To ensure that abnormally small seeds or seed lethality were not missing from our bulked seed population, we analyzed all seeds from single crosses between wild-type mothers and pollen from met1-3/+ plants  demonstrating that paternal inheritance of met1-3 causes seed size reduction as a result of the loss of MET1 activity during male gametogenesis. The loss of MET1 during male gametogenesis may allow paternal expression of imprinted growth inhibitors and cause a decrease of endosperm and seed size. Loss-of-function paternal effects are uncommon and until now have only been linked to defects in fertilization in DrosophilaC.elegansArabidopsismet1-3 causes a paternal effect associated with defects after fertilization and thus representing a distinct class of paternal effect mutations.To confirm the link between the small seeds and paternal inheritance of + plants , Table 2MET1a/s pollen caused a decrease of seed size, a symmetrical increase of seed size was observed in seeds from the reciprocal crosses MET1a/s×wt. met1-3 from met1-3/+ mothers would increase size in 50% of the seeds. Crosses between ovules from met1-3/+ plants and wild-type pollen did exhibit increased seed size relative to wild type controls . This is contrary to the expected consequence of a maternal gametophytic effect of met1-3/+, which should produce a greater proportion BASTA resistance among the largest seeds in a population derived from met1-3/+×wt crosses. To confirm this finding we compared BASTA resistance and seed size in an entire population of seeds from met1-3/+×wt crosses from a single plant. We observed that larger seeds did not always inherit the met1-3 allele and the means of size measurements did not differ between seed genotypes  was supplied by J. Finnegan met1-3 (Col) was supplied by J. Paszkowsky and contains a TDNA insert conferring resistance to BASTA met1-3 line was maintained as heterozygous by repeated backcrosses to wild-type plants in order to avoid accumulation of epigenetic defects. Once allowed to self, the resulting segregating homozygous plants were used for emasculation for crosses to wild-type plants and for observation of autonomous development.The wild-type control lines C24 and Col were supplied by the ABRC stock center. The line Plants were grown at 22 C and 60% hygrometry in short days (16 h night) for three weeks followed by long days (8 h night) in Conviron Growth chambers.Developing seeds were cleared with Hoyer's medium and observed with DIC optics with a Leica microscope (DM600). Images were recorded with a Snapshot camera and processed with Metamorph for morphometric measurements. For confocal microscopy, material was prepared and observed as described previously met1 we performed a series of four experiments. We produced crosses between wild type and met1-3/+ plants grown in the same conditions and obtained two populations of 900 seeds with inheritance of met1 from the mother or from the father. We visually separated seeds according to size categories in each population and tested BASTA resistance in a subset representing the largest or smallest seeds. In a second series of crosses we produced crosses between wild type emasculated plants and wild-type or met1-3/+ plants or met1-3/met1-3 plants grown together. The seeds obtained were imaged and seed size was measured as detailed below and the data are reported in In order to evaluate the relationship between seed size and parental inheritance of 2 test (χ2). Finally, we used the Kalmagorov-Smirnoff Normality test (K-S) to determine whether seed size phenotypes fit a normal distribution based on comparison to a generated ideal normal distribution of similar mean and standard deviation. Calculations were performed using StatView 5.0.1 , except for χ2, which was calculated on Excel×(Microsoft). p-values provided in the text are followed by the abbreviation of the test used.To determine seed area and height, digital images of seeds on a white background were thresholded in Adobe Photoshop CS2  and at 6 DAP  in wild-type seeds , in seeds resulting from crosses between wild-type ovules and pollen from met1-3/+ plants . Scale bars represent 20 µm  and 50 µm . Cytological observations were performed to establish the origin of the reduction of seed size caused by paternal inheritance of met1. The final seed size depends both on the extent of cell proliferation in the embryo and on the degree of endosperm growth during the early phase of seed development 1 to 4 Days After Pollination (DAP). Until the late heart stage we did not observe any reduction of cell proliferation in the embryo of seeds, which inherit met1 paternally or maternally. Patterns of embryo development did not show obvious modifications and met1/+ embryos were viable. In contrast seeds which inherited met1 paternally showed a reduction of endosperm size as early as the beginning of the embryo globular stage. Early endosperm development is characterized by a series of nuclei divisions, not followed by cell divisions leading to a syncytium. The frequency and number of syncytial divisions were not altered by paternal inheritance of met1 and both smaller met1/+ seeds and larger wild-type seeds contained approximately 100 nuclei as expected at 3 DAP. The difference in size between the two populations of seeds increased during development leading to two easily distinguishable classes.(0.49 MB DOC)Click here for additional data file.Figure S2Parental effect of met1-3/+ ovules crossed to wild-type pollen on seed size during seed development, correlated with BASTA resistance (R) or sensitivity (S).(1.73 MB PDF)Click here for additional data file.Figure S3Autonomous development of fruit and seed in MET1a/s. (a) Increase of fruit elongation in MET1a/s plants in absence of fertilization 7 Days After Emasculation (DAE). (b) Autonomous development of ovules in met1a/s silique in absence of fertilization (7 DAE). (c) Cleared autonomous seed from MET1a/s plant 7 DAE show remnants of the central nucleus and the egg cell. (d) Wild-type seed (7 DAE). Bars represent 0.8 mm (a), 0.5 mm (b), and 40 µm .(0.94 MB PDF)Click here for additional data file.


Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte hypertrophy. However, experimental perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. Thus, it has not been possible to conclude whether the effects on chondrocyte differentiation are cell autonomous or whether they are mediated by Dlx expression in adjacent tissues, notably the perichondrium. To address this question we first engineered transgenic mice in which Dlx5 expression was specifically restricted to immature and differentiating chondrocytes and not the perichondrium. Col2a1-Dlx5 transgenic embryos and neonates displayed accelerated chondrocyte hypertrophy and mineralization throughout the endochondral skeleton. Furthermore, this transgene specifically rescued defects of chondrocyte differentiation characteristic of the Dlx5/6 null phenotype. Based on these results, we conclude that the role of Dlx5 in the hypertrophic pathway is cell autonomous. We further conclude that Dlx5 and Dlx6 are functionally equivalent in the endochondral skeleton, in that the requirement for Dlx5 and Dlx6 function during chondrocyte hypertrophy can be satisfied with Dlx5 alone.The axial and appendicular skeleton of vertebrates develops by endochondral ossification, in which skeletogenic tissue is initially cartilaginous and the differentiation of chondrocytes via the hypertrophic pathway precedes the differentiation of osteoblasts and the deposition of a definitive bone matrix. Results from both loss-of-function and misexpression studies have implicated the related homeobox genes  The adult vertebrate skeleton appears to be a rather uniform bony tissue. This apparent uniformity belies its embryonic origins, as a mosaic structure originating from diverse progenitors that arise in different germ layers, and its development via two distinct ontological processes: intramembranous and endochondral ossification Dlx homeobox genes encode nuclear transcription factors Dlx5 and Dlx6 are expressed in all anlagen of the endochondral skeleton. Their expression has been noted in precartilaginous limb bud mesenchyme Sox9Dlx5 and Dlx6 are expressed in the post-mitotic prehypertrophic and hypertrophic zones but not in immature chondroblasts in the resting or proliferating zones Dlx5 and Dlx6 are also expressed in the perichondrium/periosteum in the long bones as well as ribs and vertebrae −/−Dlx5 and doubly deficient −/−Dlx5/6 mice have revealed requirements for Dlx5 and Dlx6 during chondrogenesis Dlx5 or Dlx6 alone in chicken limb bud micromass cultures stimulated chondrogenesis Dlx5 in vivo reduced proliferation of epiphysial chondrocytes and resulted in precocious chondrocyte hypertrophy Dlx5 and Dlx6 as partially redundant positive regulators of chondrocyte differentiation. However, to date, perturbations of Dlx expression have either not been cell type specific or have been done in the context of endogenous Dlx5 expression. This general concern is particularly germane when seeking to elucidate a cell-autonomous function for Dlx5 in chondrocyte hypertrophy given endogenous Dlx5 expression in both the differentiating chondrocytes and in the perichondrium, the site of synthesis of secreted factors that regulate this process in the chondrogenic core. Here, we first describe a transgenic line of mice in which exogenous Dlx5 expression is targeted to immature chondrocytes using regulatory elements from the Col2a1 gene. Tissue-specific misexpression of Dlx5 accelerated chondrocyte hypertrophy and promoted precocious ossification in the endochondral skeleton of these transgenic mice. Visualization of transgene expression in the absence of endogenous Dlx5 expression  indicated that transgene expression was limited to the chondrogenic core and not the perichondrium. The subsequent rescue of endochondral ossification defects in −/−; Col2a1-Dlx5Dlx5/6 mice therefore establishes a cell-autonomous function for Dlx5 during chondrocyte hypertrophy and, furthermore, demonstrates functional equivalence of Dlx5 and Dlx6 in the endochondral skeleton.Dlx5 and its cis-linked paralogue Dlx6 function as positive regulators of both chondrogenesis and chondrocyte hypertrophy in the endochondral skeleton Dlx5 during chondrocyte hypertrophy in vivo, we generated transgenic mice in which a Dlx5 cDNA was expressed under control of the promoter and intron 1 enhancer of the Col2a1 gene  had a variable number of copies of the transgene, as measured by semi-quantitative PCR from genomic DNA  or P1 after staining cartilage and mineralizing bone with alcian blue and alizarin red respectively. All three dead founders, or viable neonates bearing the t19Col2a1-Dlx5 allele, examined this way had a common phenotype of hypermineralization of the endochondral skeleton  at two developmental stages and is shown, along with an explanation of the scoring system, in t1/+Col2a1-Dlx5 founder, this hypermineralization was so excessive that the supraoccipital and exoccipital bones were completely fused, as were the first two cervical vertebrae and there was hypermineralization in the chondrocranium surrounding the interparietal bone  showed precocious ossification between these centers in the form of narrow bridges of mineralized tissue that joined the ossified centrum and neural arch. This precocious ossification was further advanced in t19/t19Col2a1-Dlx5 homozygotes (n = 9) such that the majority of centra were completely fused to the adjacent neural arches , lateral , and ventral . Embryonic vertebrae have discrete ossification centres in the centrum and the neural arches, which normally fuse post-natally. The vertebrae of P1 l arches . The oveenotypes , resultienotypes .Col2a1-Dlx5 neonates were mildly affected. t19/+Col2a1-Dlx5 limbs were indistinguishable from wild type in both overall size and in the extent of mineralization. The limbs of homozygous t19/t19Col2a1-Dlx5 neonates averaged slightly smaller than wild type littermates  in which the long bones were shorter and thicker than non-transgenic controls, particularly in the hindlimb, and the relative extent of mineralization was more advanced in the femur compared to wild type controls . Thereafter, the initiation of mineralization in the centrum at a specific axial level in wild type embryos was somewhat variable such that, by E15.5, the number of vertebral centra in which mineralization had commenced was quite disparate among littermates of the same genotype, varying from zero to twelve . However, no significant difference in the extent of ossification in the long bones of the limbs was apparent up to P0 . Thus, expression of Dlx5 in somitic cartilage did not appear to affect the initial timing of mineralization, but rather contributed to a dose-dependent acceleration of ossification once initiated.We next asked whether the timing of onset of mineralization was affected in o twelve . While t however . We also however . By E17.l arches . In contly fused . We alsot19/+Col2a1-Dlx5 transgenic neonates confirmed that mineralization had occurred throughout the vertebrae, whereas the ossification centres of the centrum and neural arches of wild type pups were separated by blocks of cartilaginous tissue that contained both radially flattened and hypertrophic chondrocytes  and hypertrophic (Col10a1) markers was expanded in the limbs and vertebrae of t19/+Col2a1-Dlx5 embryos compared with wild type littermates  with heterozygous +/−Dlx5/6 mice (C57Bl/6 x DBA background) to specifically reconstitute Dlx5 function in the cartilage of otherwise Dlx5/6 null embryos. −/−Dlx5/6; t19/+Col2a1-Dlx5 neonates showed a rudiment of a mineralized element of varying size, located dorsal to the exoccipital bones, which was interpreted to be supraoccipital in identity  and was rescued to wild type levels in hemizygous Col2a1-Dlx5 embryos while complete rescue of the supraoccipital bones required homozygosity of the transgene in a Dlx5/6 null background. Nonetheless, it is quite likely that some functions of the Dlx5-Dlx6 locus will depend on diverged protein functions; some genes behave differently following loss of Dlx6 versus Dlx5 in the mandibular arch, for example Dlx5, Runx2 (encoding two isoforms that differ at their amino-termini) is a multifunctional regulator of both chondrocyte and osteoblast differentiation Dlx5, Runx2 is expressed in cartilaginous condensations and later, during long bone growth, both genes are expressed in prehypertrophic and hypertrophic chondrocytes. In addition, Dlx5 and Runx2 are expressed in the perichondrium flanking the prehypertrophic and hypertrophic zones and, in both cases, perichondrial expression extends over the proliferating zone . In common with Runx2 then, Dlx5 accelerates chondrocyte differentiation but, in contrast, while Runx2 is capable of initiating chondrocyte hypertrophy de novo, Dlx5 is unable to induce ectopic chondrocyte hypertrophy in tissues where it would not normally occur, nor advance the initial timing of mineralization. Thus, it is likely that Dlx5 requires the synthesis of other rate-limiting critical cofactors for its hypertrophic function. This is an interesting situation given that Dlx5 appears to be a direct upstream regulator of Runx2 during osteoblast differentiation Runx2 in chondroblasts. Indeed, the signals and upstream transcriptional regulators of Runx2 in chondrocytes are not yet known.Like ing zone . As thisDlx5 expression is not without precedent; non-uniform effects of Col2a1-driven transgenes in the endochondral skeleton have been a hallmark of other studies too. For example, the supraoccipital bone and vertebral skeleton was more sensitive to the effects of a dominant negative PTHrP receptor than the limbs, which only exhibited an effect at the highest number of transgene copies and then only in discrete elements Dlx5 and other regulators of cartilage development no doubt reflects differences in the molecular milieu of anatomically distinct chondrocytes and further underscores the mosaic nature of the skeleton.The differential sensitivity of the appendicular and axial skeleton to a given level of ectopic Prior institutional approval was received for the animal work described in this study from the University of Guelph Animal Care Committee, in accordance with Canadian Council on Animal Care guidelines.Dlx5 open reading frame was amplified as a 5′ Flag-tagged HindIII-NcoI fragment, and shuttled into a modified pSlax13bpA) sequence from p3000i3020Col2α1Dlx5 as an NcoI-XbaI fragment, and the FlagDlx5-bpA cassette was cloned back into p3000i3020Col2α1 as a HindIII-SalI fragment, replacing the βgeo-bpA sequences. The construct was digested with NotI and SalI and the linear transgene cassette was microinjected into the pronuclei of fertilized CD-1 oocytes. Genotyping of transgene bearing mice was done with forward primer 5′-AACAGTTCCCCGAAAGAGGT-3′ and reverse primer 5′-GAGCGCTTTGCCATAAGAAG-3′, which amplified a 1040 bp fragment. The t19Col2a1-Dlx5 allele was maintained in a hemizygous state on a CD-1 background; hemizygous animals were occasionally bred to generate homozygous embryos or neonates. Offspring from t19/+Col2a1-Dlx5 x +/−Dlx5/6 crosses were backcrossed to Dlx5/6 heterozygotes to generate transgene-positive, Dlx5/6 null mice. A 438 bp genomic Ihh fragment was amplified with primers: 5-CACTTGTGGTGGAGGATGTG-3′ and 5′-TACCACACGCTTGTCAGCTC-3′. Dlx5/6 heterozygotes were genotyped with the lacZ primer pair: 5′-GCGTTACCCAACTTAATCG-3′ and 5′-TGTGAGCGAGTAACAACC-3′FlagDlx5 and β-actin mRNA was determined on total RNA prepared from E16.5 embryos using the Trizol Reagent (Invitrogen). 1 µg of RNA was reverse transcribed  and PCR amplified  with standard protocols. Primer sequences used for RT-PCR were: FlagDlx5 (For 5′-GACTACAAGGACGACGATGAC-3′ and Rev 5′-GAGCGCTTTGCCATAAGAAG-3′) and β -actin .The murine 2O/glycerol to 100% glycerol.Alcian Blue and Alizarin Red staining was used to visualize cartilaginous and mineralised skeletal tissues respectively of embryos and neonates. Briefly, eviscerated and skinned (>E16.5) bodies were fixed in 95% ethanol over several days. Embryos were stained in 80% ethanol, 20% acetic acid, 0.01% Alcian blue 8GX for 2–4 days, cleared overnight in 1% KOH, stained 1–2 days in 0.001% Alizarin Red S in 1% KOH, then rinsed in 1% KOH, and a graded series of HTissues were fixed overnight in 4% paraformaldehyde then dehydrated and embedded in paraffin using standard techniques. 7 µm sections were stained with hematoxylin and eosin using a standard protocol.in situ hybridisation was done as described previously Dlx5, a 0.87 kb BamHI – HindIII fragment corresponding to the full length open reading frame Col2a1, a 0.4 kb cDNA corresponding to nt 1–402 of Genbank X57982; Ihh, a 1.8 kb partial cDNA EcoRI fragment Col10a1, a 0.86 kb ApaI - SalI fragment containing nt 1351–2215 of Genbank X65121.Whole mount and cryosection Images were taken using a MicroPublisher colour digital camera on a Leica MZ12.5 Stereomicroscope with Qcapture software (QImaging) or on a Leica DMRA2 upright microscope with Openlab software (Improvision) and processed using Adobe Photoshop.


Forty fixed tissue sections from patients with small cell lung carcinoma (SCCL) have been stained with a panel of 10 monoclonal antibodies using a peroxidase anti-peroxidase method and the incidence of staining has been compared to patient characteristics at presentation and to survival. An inverse association between HMFG2 staining and survival was found with median survival in HMFG2 negative patients 13 months compared to 8 months for HMFG2 positive patients. No such association was found with the other antibodies and no association was found between staining and disease extent or primary versus secondary deposits with this panel of antibodies. Epidermal growth factor receptor was detected in 3/38 presentation biopsies and in these 3 patients mean survival was only 5 months. Further prospective study of HMFG2 as a prognostic indicator in SCCL is suggested.


Rotavirus G10P[11] strains have long been associated with asymptomatic neonatal infections in some parts of India. We have previously reported G10P[11] strains associated with both asymptomatic infections and severe gastrointestinal disease in neonates from Vellore in southern India, with >90% partial nucleotide and amino acid identity to the VP4, VP6, VP7 and NSP4 genes of the exclusively asymptomatic G10P[11] strain I321.In this study, the whole genome of a G10P[11] isolate (N155) from a neonate with severe gastrointestinal disease was characterized to determine whether there were significant differences in its genetic makeup in comparison to G10P[11] strain I321 and to establish the origin of the G10P[11] strains in Vellore.PCR amplification and complete genome sequencing was carried out for all 11 gene segments of symptomatic G10P[11] rotavirus isolate N155. Nucleotide and amino acid sequence similarity with I321, other human and bovine strains for each gene segment were determined. The origin of each gene was determined based on the degree of identity to bovine or human rotavirus strains.N155 was found to be a reassortant between human and bovine rotaviruses. With the exception of NSP2, gene sequences of strain N155 showed >90% identity to published sequences of I321. Gene segments encoding NSP1, 2 and 3 were of human rotavirus origin for both strains; however, phylogenetic analysis of NSP2 sequences indicated that the human parental strain that led to the origin of these bovine-human reassortant strains was different. There were no significant differences between NSP2 sequences of strains from symptomatic and asymptomatic neonates in the same setting.The study shows that the difference in clinical presentations in neonates may not be due to the limited variability in the genome sequence of G10P[11] strains and that G10P[11] strains in different parts of India could have evolved through reassortment of different parental strains. An abdominal radiograph revealed dilated bowel loops and NEC. The baby was given intravenous fluids and kept nil oral for three days after which time feeds were initiated and well tolerated. The sample was screened for other enteropathogens, including Norovirus genogroups I and II, enteric adenovirus, 3.3http://frodo.wi.mit.edu/) and BioEdit softwares (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) based on corresponding genes sequences available in GenBank. Primer pairs were designed to amplify short fragments of each gene ranging from 387 to 1355 bp, with each fragment overlapping the next one by about 100 bp. RT-PCR was carried out with an initial denaturation at 95 °C for 5 min and 35 cycles of amplification using 5U of Taq polymerase (Invitrogen). High-fidelity Taq polymerase (Invitrogen) was used for the amplification of three genes  whose amplicon sizes were >1000 bp. The final sequence was determined by assembling consensus genome sequences from all fragments for the gene. The primer sequences used for sequencing the beginning and end of each gene in this study are given in RNA extraction and random priming reverse transcription for cDNA synthesis was carried out as described previously. Primers for the amplification of individual genes were designed using primer3 (3.44-TTCCTTATGCAGCTGATCACTCTGTGTCA-C6-NH2-3′) to the 3′ ends of both strands of the viral dsRNA using T4 RNA Ligase . One step RT-PCR using Superscript III RT-PCR kit  was carried out with a primer complementary to the modified amino-linked oligonucleotide (5′-TGACACAGAGTGATCAGC-3′) and a gene specific internal primer.The 5′ and 3′ sequences of the 11 gene segments were determined by using the single-primer amplification method.3.5PCR amplicons were purified using PCR cleanup filter plates  and sequenced with ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit . Sequences were resolved in the ABI PRISM 310 Genetic Analyzer . Deduced amino acid (aa) sequences were derived using the Expert Protein Analysis System (ExPASy) proteomics server of the Swiss Institute of Bioinformatics (SIB). The nucleotide (nt) and deduced amino acid sequences of all genes were compared with sequences available in the NCBI  GenBank database using BLAST . Multiple alignments and phylogenetic analysis of nt and deduced aa sequences were performed using Clustal W and neighbor-joining algorithms of the BioEdit (version 7.0.5.3) or Bionumerics  software packages. Dendrograms were confirmed by neighbor-joining and maximum parsimony methods. Genetic lineages were confirmed by bootstrap values of >95% using 1000 pseudoreplicate runs.3.6EU200793 to EU200803.The nucleotide sequences of the 11 gene segments of N155 have been deposited in GenBank under the accession numbers 44.1A total of 18,242 nucleotides encoding 5781 amino acids of the eleven segments of the G10P[11] strain N155 were sequenced. The complete coding sequences (cds) were determined for all genes except VP6 and NSP3, in which the first two and three amino acids, respectively, of the open reading frames could not be ascertained.N155 sequences were compared with published sequences of corresponding genes, with origin determined based on the degree of identity to bovine or human rotavirus strains. The closest sequence match and similarity with I321, human and bovine strains for each gene of N155 is shown in 4.2The VP1, VP2 and VP3 genes of N155 were of bovine origin, with 93%, 94% and 92% identity, respectively, with bovine strains NCDV or UK. The VP6 sequence of strain N155 clustered with SGI rotavirus strains and was more closely related to I321 (97% identity at the nucleotide (nt) level) and VP6 SGI sequences derived from bovine strains than SGI sequences of common human rotavirus strains . On tran4.3The gene encoding NSP1 of N155 was 97% identical to the corresponding sequence of I321, and likely of human origin . Human rThe NSP2 encoding gene of N155 was closely related to sequences from human rotaviruses showing >95% nt and 98% aa identity with recent human isolates from Vellore and Bangladesh of common genotypes (G1P[8] and G9P[8]), and >92% with human prototype strain Wa. Only 80.9% nt identity was seen with I321 and 82.8–89.1% with bovine strains . PhylogeIn order to assess the role of this difference in virulence, partial nucleotide sequence analysis of the region encoding aa 15–291 was carried out from 5 additional G10P[11] strains (2 symptomatic and 3 from asymptomatic infections). No significant differences were seen among NSP2 sequences of these isolates . TerminaThe NSP3 gene of N155 was similar to I321 (>95% identity at the nt level) and was highly related to human rotaviruses . Two disThe NSP4 sequence of N155 showed 95% identity with I321 and other G10P[11] strains isolated in Vellore , and werGene segment 11 of strain N155 showed 95% identity at the nt level to bovine strain VMRI .5Sequence analysis of the complete genome of strain N155 isolated from a neonate with symptomatic rotavirus infection confirms that G10P[11] strains found in humans in Vellore are bovine–human reassortant strains. Eight out of 11 gene segments are likely to have originated from bovine strains. With the exception of NSP2, a high degree of identity was seen between N155 and I321 in six of seven genes for which sequence data was available for both strains. Sequence data on I321 for genes encoding VP1, VP2, VP3 and NSP5 were not available for comparison although these have been reported to be of bovine origin through whole genome hybridization studies.This study showed that the NSP2 gene of both I321 and N155 originated from human rotavirus strains, but the strains belonged to two distinct genetic lineages with <80% identity between them. These findings would suggest that independent reassortment events between human and bovine strains led to the emergence of G10P[11] strains in Vellore and Bangalore. Both strains have three genes of human rotavirus origin, but the gene encoding NSP2 clearly originated from different human parental strains. These differences however do not explain the differences in virulence, as shown through more extensive analysis of NSP2 sequences from additional G10P[11] strains from symptomatic and asymptomatic infections.Early studies on I321 had revealed strong cross hybridization of NSP1, NSP2 and NSP3 genes with human rotavirus strain Wa.Based on the whole genome characterization of G10P[11], the 3 genes of human rotavirus origin—NSP1, NSP2 and NSP3 are probably essential for efficient replication of a bovine strain in a human host. The role of these genes in host restriction has previously been discussed in other studies but the data are inconclusive.Complete genome sequences are available from few strains throughout the worldNone.


BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in  BRCA2 has been linked to the development of prostate cancer, although the precise role that BRCA2 dysfunction plays in the development of prostate cancer is unclear. To address this, we have generated an animal model in which the mouse Brca2 gene is specifically deleted in the adult prostate. These mice develop precancerous prostate lesions, which progress in severity and incidence with the loss-of-function of an additional tumour suppressor, Trp53. Importantly, blocking male steroidal hormone production by castration leads to partial regression of the prostate lesions, however cells continue to proliferate after androgen withdrawal. This suggests human BRCA2 mutant prostate tumours, like the majority of prostate cancers, will respond to hormone therapy, but will relapse, as frequently occurs in this disease. In summary, our model suggests that BRCA2 acts as a tumour suppressor in the prostate and provides a pre-invasive model to test novel therapeutics.In Western countries, prostate cancer is the most common male cancer and the second biggest cause of cancer-related deaths in men. Men with a familial history of either breast or ovarian cancer have an elevated predisposition to prostate cancer, suggesting there is a genetic element to this disease. Indeed, the inheritance of a mutated form of the breast cancer susceptibility gene  Prostate cancer is the most common cancer in men in developed countries, with a rising incidence of the disease. However, the etiology of this malignancy is still unclear. Prostate cancer progresses through a pathologically defined series of steps involving increasing grades of PIN, invasive adenocarcinoma and metastatic cancer BRCA2 predispose to both breast and ovarian cancer making it a good candidate gene for prostate cancer etiology. There is an increased risk of prostate cancer in individuals carrying a mutation in BRCA2, particularly early-onset disease BRCA2 mutation that rose to 7.33 in men under 65 years of age BRCA2 carriers account for between 0.8–2% of prostate cancer cases, compared with the prevalence of 0.1% BRCA2 mutations in the general population BRCA2 mutation carriers have been associated with aggressive prostate cancer Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility BRCA2 mutations frequently demonstrating loss-of-heterozygosity with loss of the wild-type allele. BRCA2 plays an important role in the repair of DNA double-strand breaks (DSB) through homologous recombination (HR) BRCA2-deficient cells form chromosomal aberrations spontaneously in culture and are more sensitive to certain DNA damaging agents BRCA2 is thought to principally lead to tumour progression by the failure to repair DNA by HR, leading to genomic instability.BRCA2 is thought to act as a tumour suppressor, with tumours arising from in vivo tumour suppressor role for Brca2 in the mammary gland and have demonstrated a synergistic tumour suppressor activity with Trp53. However, Brca2 heterozygous animals do not show a predisposition to tumour formation and Brca2 null mice result in embryonic lethality Cre-LoxP system has been used to conditionally delete Brca2 in a tissue-specific manner. Deletion of Brca2 from the mouse mammary epithelium either fails to produce mammary-gland tumours or results in mammary-gland tumour formation with long latency (1.4–1.6 years) Brca2 mutant mice that were Trp53 heterozygous Brca2 and Trp53 developed mammary tumours with high penetrance at 6 months Mouse models have shown a direct Brca2 in prostate cancer we have used a prostate–specific Cre line and a conditional Brca2 allele to delete Brca2 in adult mouse prostate epithelia. We show that loss of Brca2 in the prostate results in focal hyperplasia and low-grade (LG) PIN. Mice with conditional deletion of Brca2 and Trp53 have a high incidence of high-grade (HG) PIN, which contain cells with elevated DNA damage. PIN lesions in Brca2;Trp53 homozygous mutant prostates persist and continue to proliferate after androgen depletion. This work confirms the role of Brca2 as a tumour suppressor in the prostate and provides a model to test potential therapeutics in Brca2-deficient prostate neoplasia.To understand the role of Brca2 in the prostate we deleted Brca2 from the adult mouse prostate epithelia. To achieve this we mated mice carrying a Brca2 allele that has exon 11 flanked by loxP sites (F/FBrca2) to transgenic mice carrying Cre recombinase under the control of a prostate-specific composite rat probasin promoter, PBCre4Cre line has been used successfully to delete tumour suppressor genes and activate oncogenes to drive prostate neoplasia and tumour progression Brca2 conditional allele results in the loss of a Rad51-interacting domain, and consequently, homozygous germline deletion leads to embryonic lethality To investigate the role of F/FBrca2), Brca2 heterozygous  and Brca2 mutant  animals were generated and analysed for tumour progression at 6 months, 10–14 months and 15–20 months of age. None of the F/+;PBCre4Brca2 prostates had any observable morphological differences compared to control prostates at any time point analysed   . LG PIN  0.0001) . These fTP53 is frequently mutated in BRCA2 cancers and studies in the mouse have shown a genetic interaction between Brca2 and Trp53Brca2 and Trp53 cooperate in the prostate we deleted both of these genes in the prostate epithelia using the PBCre4 transgene. Cohorts of male control , Brca2 homozygous and Trp53 heterozygous  and Brca2 and Trp53 double homozygous  animals were generated and analysed for tumour progression.The tumour suppressor Brca2 mutants, deletion of Brca2 and Trp53 resulted in the formation of HG PIN lesions. At 10–14 months, F/F;Trp53F/+;PBCre4Brca2 animals had focal LG PIN and hyperplasia  . Hyperplasia and LG PIN lesions were similar to those found in F/F;PBCre4Brca2 mutants. Frequently multiple ducts of each lobe had HG PIN, which were present in proximal and distal regions of the prostate and consisted of many atypical cells filling the lumen. Atypical cells were unorganised with poor orientation, severe nuclear pleomorphism and abnormal nuclear to cytoplasm ratios  . In thesm ratios . Mitoticm ratios . In somem ratios . These ad tissue .Brca2 and Brca2;Trp53 mutant prostates. An early response to DNA damage is the phosphorylation of histone H2AX (γH2AX) Brca2 mutant and Brca2;Trp53 mutant prostates contained cells that were positive for γH2AX, which were not present in control prostates  . Notably, there was a 20 fold increase in apoptotic cells in areas of HG PIN in Brca2;Trp53 homozygous mutant prostates (2% TUNEL positive cells vs 0.1% in control) (Deletion of control) . A 4 folcontrol) . An antiF/F;PBCre4Brca2 mutant prostates (2.2% Ki-67 positive cells vs 0.4% in control) , and dramatically increased by 30 fold in F/F;Trp53F/F;PBCre4Brca2 HG PIN lesions (12% Ki-67 positive cells vs 0.4% in control) (Ki-67 is a marker of proliferating cells and a prognostic indicator in prostate cancer control) . Levels control) .F/F;PBCre4Brca2 and F/F;Trp53F/+;PBCre4Brca2 mutant prostates had increased AR expression in the cytoplasm and nucleus of epithelial cells that correlated with regions of LG PIN  expressed in the luminal epithelium usually increase in the nucleus during human prostate carcinoma progression f LG PIN . The leval cells .Brca2;Trp53 homozygous mutant HG PIN lesions frequently contained large groups of p63-expressing cells, a marker of basal cells  cells are observed in human prostates al cells . Insteadal cells . Brca2;Tal cells . These ral cells  [36].Brca2 and Trp53 to androgen ablation, we surgically castrated animals at 16 months when HG PIN lesions have already formed and analysed them 4 days post-castration. Castration of control animals resulted in normal prostate regression, with a reduction in lumen size   . HoweverF/F;Trp53F/F;PBCre4Brca2 animals resulted in AR expression in a diffuse pattern throughout most prostate luminal and stromal cells   .Brca2;Trp53 mutants that persisted after castration, compared to control castrated animals (5.5% Ki-67 positive cells vs 0.3% in castrated control)  .BRCA2 mutations have implicated this gene in prostate cancer etiology, but its function in this malignancy is unclear. We have undertaken a genetic analysis of Brca2 function in the adult mouse prostate to define its role in prostate cancer and to create an in vivo model of Brca2-dependent prostate disease progression. Our study has demonstrated that loss of Brca2 in the mouse prostate epithelium results in hyperplasia and LG PIN. These lesions have an increase in the number of cells with DNA damage and apoptotic cells, which could be the result of the impairment of DNA repair pathways. This demonstrates not only that Brca2 can play a role in the initiation of prostate neoplasia but also that other factors are required for prostate tumour progression.Studies on human carriers of deleterious Brca2 and Trp53 in mouse prostate epithelia resulted in a shorter latency and increased frequency of prostate neoplasia compared to deletion of Brca2 alone  mice and F/FTrp53 (targeting exons 2–10) mice ARR2PBCre transgenic mice, PBCre4Histological phenotype of samples was assessed on haematoxylin and eosin stained sections. Serial sections were then stained for immunohistochemical analysis. Histological assessment was based on published guidelines and assisted by a pathologist Ki-67 or TUNEL staining was performed by immunohistochemistry on sections and cells stained with nuclear brown DAB chromogen were counted as positive. Cells from at least 4 high power fields were counted per animal, which totalled more than 900 cells per animal. Five animals of each genotype were analysed. Randomly selected fields were counted for control analysis and sections corresponding to histologically identified areas of hyperplasia and PIN were counted for mutant animals. All values are significant with p<0.05 using Student t-test unless otherwise stated.Antibody stains were done on paraffin sections as previously described Figure S1Brca2 and Trp53 are deleted in mutant adult prostates. PCR analysis of Cre, Brca2, and Trp53 on dissected anterior prostate (AP), lateral prostate (LP), dorsal prostate (DP), and ventral prostate (VP) tissue from a F/F;Trp53F/FBrca2 (control) F/F;Trp53F/F;PBCre4and Brca2 (mutant) animal. Wild type (WT) and water (H20) are controls.(0.07 MB DOC)Click here for additional data file.


Kip1p27 gene has been identified as inductor of cell cycle arrest at the G1 checkpoint to prevent entry of somatic cells into the S phase of the cell cycle when substantial DNA damage has occurred. It has been suggested that decreased expression of the p27Kip1 protein may contribute to the development of human malignancies due to loss of critical antiproliferative mechanisms. In the present study, 95 specimens (T1–T4) from 95 randomly selected patients undergoing radical prostatectomy at the Urological Department of Hannover University (82 patients) as well as in the Josef Hospital Regensburg (13 patients) between 1981 and 1992 for whom tissue blocks for immunohistochemical investigation were available, were investigated for different biological and clinical characteristics as possible predictors for recurrence-free and long-term survival: age, depth of tumour infiltration, histological grade, lymph node status, as well as decreased expression of the p27Kip1 protein. After a median follow-up up of 56 months (24–151 months), seven of 21 (33%) patients (Group 1) with loss of p27Kip1 protein expression or a relative amount of <10% of positively stained tumour cells developed recurrent disease in contrast to 17 of 74 (23%) patients (Group 2) with retained p27Kip1 protein expression (≥10% of positively stained tumour cells). The median recurrence-free survival was 14 months (5–40 months) for patients from Group 1 and 31 months (7–133 months) for Group 2 patients (P = 0.02). In multivariate analysis, loss of p27Kip1 protein expression was identified as the only independent prognostic parameter for recurrence-free survival. In contrast, neither the univariate nor the multivariate analysis showed a correlation between loss of p27Kip1 protein expression and the long-term survival of the patients. Prospective studies are urgently needed to confirm the independent prognostic value of decreased p27Kip1 protein expression together with overexpression of the p53 tumour suppressor protein in patients with localized prostate cancer. The availability of more refined prognostically important biological variables in addition to established prognostic factors like tumour stage or Gleason score might help decision making in patients at high risk for the development of local recurrence or systemic tumour progression. © 1999 Cancer Research CampaignThe


BRCA2 in sporadic breast and ovarian carcinomas are exceedingly rare. This led to the suggestion that large genomic rearrangements could be involved. We performed Southern blots in genomic DNA from 130 primary breast cancers and 83 cancer cell lines  and found no genomic rearrangements. These results suggest that a gene other than BRCA2 is the target of the frequent 13q12.3 allelic deletions in human cancers. © 2001 Cancer Research Campaign http://www.bjcancer.comMutations of


To the Editor: We read the article by Pelat et al. (The weekly queries from January 2004 through February 2009 were downloaded from Google Insights for Search (gripe (Spanish for influenza) showed a maximum correlation (ρ = 0.70) 2 weeks before the declared ILI (DILI). When excluding the terms for aviar (avian) and vacuna (vaccine), the correlation peak (ρ = 0.81) was likewise observable 2 weeks before the DILI. The maximum correlation observed for symptom queries was for tos (Spanish for cough) 2 weeks before the DILI (ρ = 0.74); for conditions associated with influenza the correlation was for neumonía  2 weeks after the DILI (ρ = 0.84). The queries for varicela (Spanish for chickenpox) showed a maximum correlation (ρ = 0.96) 1 week after the declared illness, as observed by Pelat et al (The queries for In conclusion, our study points out the utility of Internet queries for the surveillance of ILI and chickenpox in Spain. In the case of ILI, this information can be used as an early warning tool used complementarily to standard surveillance systems. More detailed studies are necessary regarding the usefulness and limitations of this tool in Spain, as well as in other contexts.


BRCA1 and BRCA2 has promoted the investigation of various polymorphisms in the p53 gene as possible risk modifiers in BRCA1/2 mutation carriers. Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, and it has been recently reported that a p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at the onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population. In this study, we have evaluated this association in a series of 2932 BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2.The close functional relationship between p53 and the breast cancer susceptibility genes  Specifically, two polymorphisms in p53, c.97-147ins16bp and p.Arg72Pro have been analysed as putative breast cancer susceptibility variants, although not all studies have yielded consistent results (p53 and the breast cancer susceptibility genes BRCA1 and BRCA2 (BRCA1/2 mutation carriers (p53 haplotype combining the absence of the 16-bp insertion and the presence of proline at codon 72 (No Ins-72Pro) was associated with an earlier age at onset of the first primary tumour in BRCA2 mutation carriers in the Spanish population (BRCA1/2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2 (CIMBA) . In addition, individuals homozygous for Ins16bp were directly sequenced. The concordance rate was 100% in both instances; accordingly, the GCHBOC mutation carriers were included in all subsequent analyses. Call rates ranged between 92 and 100% across studies.Genotypes for the two polymorphisms – Ins16bp and Arg72Pro – were determined for each sample using previously described methodology (BRCA1 and BRCA2 mutation carriers separately (Haplotypes were imputed using the R-package ‘hapassoc’ . Associaparately  and all BRCA1 and BRCA2 mutation carriers. No evidence of association was found for any of the genotypes or haplotypes analysed with either breast or ovarian cancer risk, including the No Ins-72Pro haplotype, previously reported to be associated with an increased risk to develop a first primary tumour before 35 years of age in BRCA2 mutation carriers (Genotype distributions and frequencies for the Ins16bp and Arg72Pro polymorphisms are shown in To confirm that this negative result was not due to the different analytic approach performed in this study, we carried out a logistic regression analysis, as was done in the original study (p53 with an increased cancer risk in BRCA2 mutation carriers (In summary, the previously reported association of the No Ins-72Pro haplotype in


E2f4 null mice. Thus, the regulatory mechanisms maintaining quiescence are robust even to complete loss of conserved transcription factor binding events.Maintaining quiescent cells in G0 phase is achieved in part through the multiprotein subunit complex known as DREAM, and in human cell lines the transcription factor E2F4 directs this complex to its cell cycle targets. We found that E2F4 binds a highly overlapping set of human genes among three diverse primary tissues and an asynchronous cell line, which suggests that tissue-specific binding partners and chromatin structure have minimal influence on E2F4 targeting. To investigate the conservation of these transcription factor binding events, we identified the mouse genes bound by E2f4 in seven primary mouse tissues and a cell line. E2f4 bound a set of mouse genes that was common among mouse tissues, but largely distinct from the genes bound in human. The evolutionarily conserved set of E2F4 bound genes is highly enriched for functionally relevant regulatory interactions important for maintaining cellular quiescence. In contrast, we found minimal mRNA expression perturbations in this core set of E2f4 bound genes in the liver, kidney, and testes of  Quiescence of cellular proliferation is crucial for mammalian tissue homeostasis, and aberrant activation of cell cycle programs can lead to cancer Removal of key E2F components of the multisubunit complexes that control the cell cycle can cause aberrant activation of cellular proliferation in specific tissues during development and in adulthood reviewed in . For insE2f4 null mice to determine the transcriptional importance of E2f4 binding in primary mouse tissues.Despite wide fluctuations and evolutionary turnover of transcription factor binding events between mouse and human We identified the proximal promoter regions that E2F4 occupies in three primary human tissues  directly isolated from donor organs, and an asynchronous human cell line (HepG2), using chromatin immunoprecipitation and promoter microarrays representing 13,000 regions in the human genome On our array platform, we found that E2F4 binds approximately 500 to 700 human genes, depending on the tissue. Among all three quiescent primary human tissues and the proliferating HepG2 carcinoma line, we observed overlap greater than 70% and as high as 84% . This ovA substantial majority of genes bound by E2F4 are bound in most tissues; we identified a core set of approximately 450 genes common to all human tissues in our study . These i−4, a core set of approximately 450 genes are bound in common by E2f4 in all mouse tissues  may vary between mouse and human; a variation that may have functional implications for E2f4's role in mouse Second, a feature of the approximately fifty genes where E2F4 binding is conserved is their remarkable enrichment in cell cycle, proliferation, and DNA repair functions. This result is consistent with the hypothesis that when a transcription factor binding event is conserved, this conservation is a good indictor of a functional regulatory connection E2f4 null tissues is nevertheless surprising, despite the largely normal physiology of these mice in adulthood. It has been known since the report of viable E2f4 null mice that the function of most tissues, even those directly impacted during development by absence of E2f4, recovers. Our findings reveal that this recovery extends to the level of gene expression; it appears that the absence of E2f4 can have profound, yet remarkably transient, implications for tissue-specific transcriptional programs. This recovery almost certainly depends on the overlapping roles that other members of the E2f family can play. For instance, it has been previously shown that E2F5 can largely compensate for the absence of E2F4 in vivo We expected that removal of E2f4, as a key member of the DREAM complex would have profound implications for the correct gene expression of the genes bound by E2f4 in vivo. Instead, however, we found that developmental recovery and survival to adulthood corresponds with almost completely normal gene expression in multiple tissues, relative to their phenotypically normal heterozygous littermates. Given the substantial developmental defects described above, the subtle and limited changes in the gene expression profiles of Our results also provide direct support to the hypothesis that transcriptional binding is often neutral in nature We have used comparative genomics approaches to explore the evolutionarily conserved regulatory pathways that E2F4, a key member of the DREAM complex, uses to maintain control of gene expression programs. We find that that most genes bound by E2F4 are common to numerous primary tissues within a species. This suggests that the regulatory architecture controlling quiescence may be similar among tissues that have remarkably different capacities for re-entry into the cell cycle. Understanding this architecture will require further studies to explore how E2f4 acts within specific cell types during development. The striking and specific conservation of E2F4 binding between mouse and human at cell cycle and proliferation genes suggests that only the targets crucial for the function of the DREAM complex are under selective pressure, yet our discovery that complete removal of E2f4 has at best modest effects on gene expression programs dramatically underscores the well-known redundancy within cell cycle regulation. Our study demonstrates the power of using the conservation of transcription factor binding at orthologous mouse-human genes as a tool to identify regulatory connections that appear to be under evolutionary pressure.All chemicals were purchased from Sigma-Aldrich, and used as received unless otherwise noted. E2F4 antisera were obtained from Santa Cruz Biotechnology  and used as described in prior studies Mice used in chromatin immunoprecipitation experiments were F1 males from a B6/C57 male cross with an A/J female (Jackson Laboratories). Islets suitable for ChIP studies were isolated by standard techniques and hand picking at Joslin Diabetes Center on mixed gender mice of the same genetic background (B6/C57xA/J). Other tissues were harvested using standard techniques, soaked or perfused with 1% formaldehyde, and homogenized in neutralization buffer followed by ChIP. For expression studies, E2f4 −/− homozygous and E2f4 +/− heterozygous knock-out mice were derived in a B6/C57 and 129S2/SvPas cross background Primary human hepatocytes were obtained from the Liver Tissue Procurement and Distribution Program (NIDDK contract number N01DK92310) at the University of Pittsburgh. Human pancreatic islets and pancreatic acinar tissues were the kind gifts of Gordon Weir, Abdulkadir Omer (Joslin Diabetes Center) and Nicolas Benshoff (University Minnesota) .Mouse tissues were harvested from two E2f4 −/− and two E2f4 +/− heterozygous littermates limmaaffyaffyPLM package The The procedure for chromatin immunoprecipitation has been reported previously limma package. Quality of the arrays was assessed using array images of the background and foreground intensities and also of the red/green (Cy5/Cy3) ratios to check for spatial artifacts. MA-plots (log-fold change against average log-intensity for each gene) −4). The set of commonly bound genes was defined using the less stringent value of 10−3, to capture binding events falling just under the more stringent threshold in one or two tissues. To find orthologous mouse and human genes on the promoter arrays, gene symbols and accession numbers were linked to the Ensembl IDs for each species to match the two gene lists. The data in −4. The comparison with Litovchick ChIP data was done by comparing their published binding results to the cut-offs described in The statistical analysis was performed using the We determined the enrichment of functional categories among the bound gene sets using the GOstat tool Given the sequences of the promoters on the ChIP array, a Perl script was used to automate BLAST searches of alignment across the genome and screen the BLAST output and BioPerl was used to extract the genomic coordinates of the promoters. To interrogate for the presence of the E2F4 motif, we used the one-kilobase E2F4 bound regions plus an additional 300 bases upstream and downstream to account for the resolution of the ChIP technique limma package within R was used in the genomic microarray data analysis. Quality assessment included boxplots and images of the background and foreground intensities for both red (Cy5) and green (Cy3) channels, as well as MA-plots. All the arrays used in the analysis exhibited good quality. Expression red/green log-ratios were median-normalized, after background subtraction, within each array. E2F4 binding events were empirically assigned as minimum contiguous regions comprising, at least, one probe for which the red/green ratio was greater than 5, or two adjacent probes with ratios greater than 2.5 and a combined (summed) ratio greater than 6.5, or three adjacent probes with ratios greater than 2 and a combined (summed) ratio greater than 9. A Perl script was used to compute the genomic base pair distance between each binding event and the closest transcription start site. Transcriptomic annotation relied on tables downloaded from the UCSC Genome Browser server. The smoothed histograms in density function (default parameters) to the distance distributions and plotting the respective outcomes.Human E2F4 ChIPs in primary human liver were hybridized in duplicate to human whole chromosome 21 microarrays; mouse E2f4 ChIPs in primary mouse liver were hybridized in duplicate to mouse whole chromosome 16 microarrays . The Figure S1(0.05 MB PDF)Click here for additional data file.Figure S2(0.06 MB PDF)Click here for additional data file.Figure S3(0.09 MB XLS)Click here for additional data file.Figure S4(0.17 MB PDF)Click here for additional data file.Figure S5(0.13 MB PDF)Click here for additional data file.Figure S6(0.08 MB PDF)Click here for additional data file.Figure S7(0.14 MB XLS)Click here for additional data file.Figure S8(0.08 MB XLS)Click here for additional data file.


The response of multicellular tumour spheroids of the EMT6 cell line to combinations of hyperthermia and Bleomycin (BLM) or Adriamycin (ADM) has been investigated. Using this model system, we have demonstrated enhanced BLM cytotoxicity at 43 degrees C and also heat-induced drug tolerance to BLM at 43 degrees C. ADM cytotoxicity was not significantly increased after 43 degrees C x 1 h but after 6 h at 42 degrees C greatly enhanced cell-killing was evident. These results are discussed in relation to our previous data for EMT6 cells growing either as monolayer cultures in vitro or as solid tumours in mice.