A germ line single nucleotide polymorphism (SNP) in the first intron of the gene encoding MDM2 at position 309, an important modulator of p53, has been described. BRCA1/2 mutation have been associated with increased rates of breast cancers with mutated P53. It was shown that the presence of MDM2 309 SNP correlated with younger cancer onset age in individuals with a p53 mutations. The differential effects of this SNP were also linked to estrogen receptor activation. Here we report on our study of 453 Ashkenazi breast cancer patients of whom 180 were positive for the known Ashkenazi BRCA1/2 mutations® statistical package , and JMP® software .DNA from breast cancer patients was obtained for analysis of one of the three common BRCA1/2 mutations and MDM2 SNP309. Data regarding cancer onset and death ages was obtained from our database and Statistical analysis was performed using the SPSSThe percentage of MDM2 SNP309 in control and BRCA 1/2 population which is similar to that reported for other Jewish Ashkenazi populations at 52.2% for the heterozygotes and 25.0% for MDM2SNP309G/G and 22.8% for MDM2SNP309T/T.There was not a statistical significant difference in median age of disease onset in the different MDM2 SNP309 subgroups of the BRCA1/2 carriers. When we further divided the group into under and above 51 years old ( presumed menopause age) in the BRCA1 positive subset we found that there were less patients of the MDM2SNP309 G/G versus the MDM2SNP309 T/T in the over 51 patient group (p = 0.049). This result has been obtained in a relatively small subgroup and is of borderline statistical significance. Interestingly, in the BRCA1/2 mutation carriers, we found a survival advantage for patients harboring the SNP309 G/G genotype (p = 0.0086) but not for the 272 patients not harbouring this mutations.MDM2SNP309G/G main effect on BRCA1/2 positive mutation carriers is linked to its effect on patients survival. Further research is needed in order to understand the reason for this difference. MDM2 regulates p53 by targeting its destruction through the ubiquitin pathway and also by directly blocking p53 transcriptional activity. A singlTwo recent articles have studied this subject again with somewhat conflicting results. Yarden et al , studiedSeveral studies have found negative correlations between MDM2 tumor expression and prognosis in various cancers including breast, ovarian and brain -9. Two sEstrogen seems also to influence the magnitude of the MDM2 SNP309 G/G effects on MDM2 expression. Estrogen receptor which has a DNA binding site close to the Sp1 site which is directly influences by MDM2 SNP309 G/G. Bond, Levine and colleagues have described a significant correlation between MDM2 SNP309 G/G and the prevalence of diffuse large cell lymphoma and soft tissue sarcoma, in women under the age of 51  . This fiWe have obtained DNA samples from a large group of breast cancer patients  for analysis of BRCA1/2 mutations and modifier genes. Here we describe the interactions we found out in this group between MDM2 SNP309 G/G, BRCA1/2 mutations and clinical findings including cancer onset age, survival, and the presence of other cancers.DNA was from breast cancer patients obtained for analysis of their BRCA status and modifier genes , and JMP® software . Assessment of the correlations between genetic carrier status and MDM2 was carried out using the Fisher Exact test or Chi-Squared test. A p value < 0.05 was considered significant. Similar methodology was used to assess the correlation between the frequency of other cancers and MDM2 SNP309 statusFollow up was calculated from the time of diagnosis to date of last follow-up. The rate of death was estimated using Kaplan-Meier methods. Statistical analysis was performed using the SPSSWe evaluated DNA samples from 180 Ashkenazi breast cancer BRCA1/2 positive . There was a distinct survival advantage in the BRCA1/2 mutation carrier group for the patients homozygous for G/G at the -309 position, compared to the patients harboring the SNP309 G/T or T/T Fig . Cox mulIn the current study we analyzed the relationship between MDM2 309 SNP status and cancer in a large group of Ashkenazi patients including a large group of BRCA1/2 mutation carriers Our results revealed high percentage of MDM2 309 SNP in this population of around a quarter of all women. This high prevalence of MDM2 309 SNP G/G in Ashkenazi women has been demonstrated by others before us. Yarden and colleagues recently presented data supporting the notion that the relative enrichment in MDM2 309 SNP G/G in Ashkenazi is a result of a selection process favoring this single nucleotide polymorphism. CurrentWe did not detect significant difference in cancer onset age in BRCA1/2 carriers harboring the different MDM2 SNP309 Fig  and 1d. The effect of the MDM2 SNP 309 G/G on cancer risk has been recently reviewed in a meta- analysis by Wilkeming and colleagues. These aWhen combining the BRCA1 and BRCA 2 mutation carriers we noted significantly better prognosis for the MDM2 SNP309 G/G carriers than for the other MDM2 SNP309 carriers (p = 0.0086). This are supposedly the tumors with higher levels of MDM2 expression. Cancers in BRCA1 and BRCA2 mutation carriers have substantial difference between them. HoweverSince MDM2 is an oncogene it seems counterintuitive that patients in which higher levels of MDM2 are expected will have longer survival. A possible explanation for such a counterintuitive result is based on our understanding of the role of p53 in carcinogensis. The presence MDM2 SNP309 G/G may lead to the overexpression of MDM2 and therefore cause downregulation of p53 in an effective manner and render mutational inactivation of p53 unnecessary. Thus tumors with MDM2 SNP309 G/G might still harbor a potentially active p53. Indeed, when Allazzouzi and colleagues studied p53 in colon cancer of SNP309 G/G carriers), they found a higher tumor prevalence of non dominant p53 mutations. When p5Therefore a possible scheme to explain a role for MDM2 SNP309 G/G in BRCA tumors could be that overexpression of MDM2 in BRCA1/2 tumors results in down-regulation of p53 and allows the development of BRCA1/2 tumors similar to the effect induced by a mutation in p53. Cells overexpressing MDM2 still contain p53 that in certain conditions. in which MDM2 is inactivated might be functional. As described MDM2 is inactivated by DNA damaging agents which includes chemotherapies. This hypothesis concurs with the hypothesis suggested by Mathoulin-Portie in their study of anthracyclin treated breast cancer women . This has been proposed as one of the main mechanisms of oncogenic transformation induced by this protein. MoreoveRecently two articles assessing the relationship between patients' survival and the MDM2 SNP309 were published. However, it is important to note that the patients in those studies suffered from a different disease than those in the current study and were treated with different therapies than those commonly used for treatments of breast cancer.Our data demonstrate that patients with BRCA1/2 breast cancers harboring any of the three MDM2 SNP309 had similar cancer onset ages. Only by studying the over 51 years old group of patients  we could detect a difference between the different MDM2 SNP309 genotypes – only in the BRCA1 mutation carriers. The relative percentage of the MDM2SNP309G/G carriers was reduced in this group of patients compared to the younger than 51 years old (p = 0.049). While this results concurs with those of Yarden and colleagues they were obtained from a small number of patients and thus need reaffirmation in larger studies.MDM2 SNP309G/G BRCA1/2 carriers had significantly longer survival compared to the combination of the other MDM2 SNP309 subgroups (p = 0.0086). This somewhat surprising result may be a result of differential sensitivity to adjuvant therapy in this subgroup. Further studies on MDM2 SNP309 may provide important insights as to factors effecting tumorigenesis and drug sensitivity in BRCA1/2 mutation carriers.The authors declare that they have no competing interests.Initiation of research HN, TP. Build up of database TP, TH, LK Collection of samples, DNA and statistical analysis – TH, SM. Discussion and Analysis of results HN, EP, TP, NS, LK.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/60/prepub


Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation. A Molecular Interaction Map (MIM) akin to a circuit diagram of an electric device can give a comprehensive view of cellular processes and help understand complex protein functions in cells. To this end, we generated a MIM focused on the p53-Mdm2-MdmX network proteins and performed computer simulations to help understand how Mdm2 and MdmX may regulate p53. Proper regulation of p53 is important for cell survival: elevated levels of p53 can lead to cell death, and decreased levels of p53 can lead to cancer. Mdm2 and MdmX are structurally homologous proteins that regulate p53. Mdm2 negatively regulates p53 by degradation, but MdmX regulation of p53 is not well understood. Recently, Mdm2 and MdmX have been recognized as potential cancer therapeutic targets. In an effort to better understand how MdmX can alter the p53 activity under various conditions, we used mathematical models based on the MIM network to generate hypotheses that can be tested by experiments. Our simulations suggest that MdmX may increase the dependency of p53 on Mdm2 or dampen p53 oscillations during DNA damage response. The transcription factor p53 is a tumor suppressor that causes cell cycle arrest or apoptosis in response to stress signals Mdm2 is a ubiquitin ligase that negatively regulates p53 by promoting ubiqutin-dependent p53 degradation In our model, MdmX is a key component. MdmX interacts with p53 or Mdm2 to form transcriptionally inactive p53∶MdmX or enzymatically inactive Mdm2∶MdmX. In order to model the interactions of MdmX with Mdm2 and p53, we selected a system of elementary processes that focused on the non-enzymatic interactions between MdmX and other molecules. Since the values of many kinetic constants for our model are unknown, we selected initial kinetic parameter sets to explore potential interesting behaviors. Then, we searched for regions of parameter space that showed biologically interesting behaviors and reproduced previously observed dynamic behaviors, including DNA damage induced oscillations To lay the foundation of a model, we first assembled the known molecular interactions among p53, Mdm2, and MdmX in the form of a heuristic MIM using the previously described notation http://discover.nci.nih.gov/mim)Four pathways of p53 regulation, functioning simultaneously, are highlighted in P53 activates  the transcription of target genes including Mdm2 and p21 (A1). Phosphorylation  and oligomerization  of p53 enhance the transactivation function (A3).p53 ubiquitination (A10) is mediated by the ring domain of p53-bound Mdm2 , and the ubiquitinated p53 undergoes degradation . Oligomerized p53 is more efficiently ubiquitinated and degraded (A13) than monomeric p53 because oligomerization of p53 enhances p53-Mdm2 binding. Ubiquitinated p53 can be deubiquitinated by Hausp (A12). Mdm2 binds to the transactivation domain (TAD) of p53 (A11), and MdmX competitively binds to the same site of p53 (A18). Binding between p53 and MdmX is stimulated by MdmX phosphorylation, mediated by Casein Kinase 1-alpha  . It is not known whether CK1-alpha is regulated by DNA damage. DNA damage-induced phosphorylations of p53 (A15) and Mdm2 (A17) coherently inhibit  the formation of p53∶Mdm2 heterodimer, thereby allowing the active forms of p53 to accumulate. P53 Ser20 phosphorylation attenuates the binding between p53 and MdmX (A21).Mdm2 is auto-ubiquitinated by the Mdm2 ring domain , and the ubiquitinated Mdm2 undergoes degradation . Mdm2 may have to form a homodimer to autoubiquitinate (A26), but it is not clear whether the autoubiquitination is in cis or trans. The Mdm2 autoubiqutination is enhanced (A27) by an interaction between an orphan receptor, TR3, and p53 (A28). Ubiquitinated Mdm2 can be deubiquitinated by Hausp (A29), and the deubiquitination is inhibited (A30) by DNA damage induced phosphorylation of Mdm2 at an unknown site (A31).The ring domains of MdmX and Mdm2 can bind each other to form an oligomer (A36), and MdmX is ubiquitinated by the ring domain of bound Mdm2 (A34). Ubiquitinated MdmX undergoes degradation . Ubiquitinated MdmX can be deubiquitinated by Hausp (A39). DNA damage induced phosphorylation controls the level of MdmX coherently via several pathways. DNA damage-induced MdmX phosphorylation (A38) enhances (A37) the binding between Mdm2 and MdmX (A36) which can result in the increased ubiquitination of MdmX (A35). MdmX phosphorylation (A38) inhibits (A40) the deubiquitination (A39) of MdmX by Hausp, which in turn increases ubiquitinated MdmX showing coherent regulation by DNA damage-induced kinases.Model simulations, based on the explicit MIM presented in In further simulations, an increased basal production rate constant (k6) of Mdm2 resulted in a decrease in p53 activity : negativWe hypothesized that the ability of MdmX to induce a sharp dependence of p53 activity on Mdm2 might be accounted for by the formation of MdmX∶p53 and Mdm2∶MdmX heterodimers. We tested this possibility in pre-equilibrated systems by examining the effects of heterodimer production rate constants  on the p53 responses to DNA damage. Transient increases of p53 activity   were obsPrevious studies indicated that the cellular response to DNA damage involves oscillatory behaviors at the molecular level The results showed that, in an oscillating system, MdmX (k15 = 10) tended to dampen or reduce the amplitude of oscillations  comparedNext we examined the effect of p53 level on the oscillatory behavior. p53 level was varied by changing the p53 basal production rate, k1. The results are shown in a bifurcation diagram . The oscThus, as expected, steady state of p53 activity (species 14) increases as the basal production rate constant (k1) of p53 increases , it begiAfter specific behaviors of interest were identified from the initial simulations, a wider range of kinetic parameter space was surveyed with the full model  using a In this search, we found regions of parameter space that exhibit oscillations and Nutlin responses that are consistent with experimental observations, and used the resulting parameter sets to simulate the effect of MdmX. These surveys were performed in two steps see . In the Nutlin is a drug that blocks p53 and Mdm2 interactions, and it was shown that Nutlin induces basal transcription activity of p53 without phosphorylation Sensitivity analyses were performed for two selected sets of kinetic parameters that were used for further simulations. The normalized local sensitivity of mRNA  during the first 180 min post DNA damage was considered as p53 activity and measured as output while basal production rate of Mdm2 (k6) was varied. Examples of the transient dynamic behavior observed when the production rates of Mdm2 and MdmX were varied are shown in  of MdmX ; for somover 50% . Such Mdover 50%  at some over 50% . As showover 50% .Late DNA damage response  with a full model  was examIn order to investigate the role of MdmX in the p53-dependent response to DNA damage, we prepared a heuristic MIM of the pathways leading from DNA damage to p53 activity , from whThe known pathways of p53 regulation are highlighted in four panels in Enhancement by p53 multimerization of the binding between p53 and Mdm2 [A13].Phosphorylation by Casein Kinase 1 alpha and its effect in binding between p53 and MdmX .Autoubiquitination of Mdm2  became a major reservoir during the pre-equilibration with low Mdm2 or when Mdm2∶MdmX (species 9) became a major reservoir during the pre-equilibration with high Mdm2 . Figure Unlike some previously reported switch-like behaviors arising from steady states Previous studies showed sustained oscillations of p53 during the long-term response to DNA damage Kinetic parameter space was explored in two stages. (1) During the first stage, two oscillatory regions, OSC1 and OSC2, were identified. The characteristic difference between the OSC1 and OSC2 clusters was the phosphorylation rate constant k3 in . Narrow The two groups of parameter sets derived from OSC1 or OSC2 underlie MdmX-sensitive and MdmX-insensitive models. It is likely that physiological condition will be explained better by one of those kinetic parameter sets. Experiments to test whether the over-expressed MdmX can in fact dampen DNA damage induced p53 oscillations will allow us to discriminate between the two models.Our theoretical study showed, with little prior knowledge, potentially interesting roles of MdmX in p53 regulation. Many of the kinetic constants used in the study were not biologically constrained, and some of the assumptions made in the models need to be biologically validated. The lack of prior knowledge demands rigorous experimental validation of the simulated results; however, it also allows the unbiased investigation of possible functions that are not intuitively obvious. The MdmX roles predicted in this study are not simply linear increases or decreases of p53 activity dependent upon the level of MdmX; the study suggests that manipulation of MdmX to alter p53 activity requires careful investigation of the dynamics of three proteins: MdmX, Mdm2, and p53. An experimental system with inducible MdmX and Mdm2 and a reporter protein to monitor p53 activity should allow us to test whether the switch-like behavior (increased dependency on Mdm2 by MdmX) or dampening of oscillations are observed. To observe the switch-like behavior, transient p53 activity has to be monitored, and it may require monitoring the p53 activity relative early  after DNA damage. To observe dampening of oscillations by MdmX, a long period of observation (>12 hr) will be necessary since the known peak-to-peak interval is approximately 6 hours. As shown in one example plot in the result section with the full model, the degree of level change in the switch-like effects by the increased MdmX was approximately 30%∼50% increase (low Mdm2) or decrease (high Mdm2) of p53 activity, and that level of change may require high sensitivity to measure p53 activity in experiments.We used highly simplified p53 models in this study because we can interpret the simulation results more easily. However, p53 is involved in many other regulations and feedback loops, and it is not obvious whether the conclusions will still hold in a larger p53 system. Although experimental verification will partially address the question, we could also proceed theoretically by introducing additional components successively to the current model and by surveying parameter space with an updated fitness function. The extension of the current model might include Wip1, which is known to trigger recurrent initiation of ATM activity in some cells One goal of this study was to understand how non-enzymatic interaction of MdmX with p53 and Mdm2 can regulate p53 during the response to DNA damage. The mechanism of p53 regulation by MdmX is poorly understood, at least in part because the role of Mdm2∶MdmX heterodimer in p53 ubiquitination and degradation is not clearly understood. It was previously speculated that p53∶MdmX heterodimer may serve as a reservoir to maintain a pool of p53 First, a heuristic molecular interaction map (MIM)  was consThe kinetic constants used for initial simulations were generated based on collected kinetic constants from previously published models As input, rate constants of phosphorylation (k3 = k8 = k17) and basal production rate of MdmX k15 in  and Mdm2We considered that DNA response may display bi-phasic responses: early and late responses. During initial simulations with the simple model , early rTo set realistic initial condition for early DNA damage response, cells without DNA damage were simulated by pre-equilibrating the model in the absence of DNA damage induced kinase activities (k3 = k8 = k17 = 0). For late DNA damage response, simulations were performed without pre-equilibration. DNA damage was simulated by setting all the rate constant of phosphorylation as a same positive value (k3 = k8 = k17>0) unless otherwise indicated. The rate constant of phosphorylation was kept constant during the simulation of DNA damage response because quite rapid saturation of ATM activity followed by a constant level of ATM activity up to 24 hr were observed in a previous study The bifurcation diagram shown in To search parameter space, we used a previously described algorithm The searches were done in a two step procedure. First, a subnetwork including a subset of interactions and kinetic constants was selected, based on their previously predicted roles in oscillatory behaviors The normalized local sensitivity of mRNA (species 15 in jLS) of the normalized local sensitivity was obtained by trapezoidal numerical integration The local sensitivity calculation was performed using the Simbiology toolbox in Matlab. The time integral  was measured because the full model include basal p53 activity (transcription activity by p53 monomer and promoter complex). Early DNA damage response was quantified by the maximum p53 activity (species 15) achieved during 0∼180 min after DNA damage; this period corresponds to the first rise in p53 activity after DNA damage The network was pre-equilibrated without Nutlin (k11>0) and without DNA damage (k3 = k8 = k17 = 0). For the simulation of Nutlin treatment, we simulated a network with k11 = 0, because Nutlin is known to affect Mdm2-p53 interaction only Some of example matlab scripts used for simulations were provided as supplemental information , S6, S7.Figure S1An informal diagram of (1.11 MB EPS)Click here for additional data file.Figure S2Regenerated example plots in (0.08 MB TIF)Click here for additional data file.Figure S3Effects of p53∶MdmX and Mdm2∶MdmX heterodimer reservoirs on p53 oscillatory behavior. The model is based on (0.64 MB TIF)Click here for additional data file.Figure S4MdmX-induced reduction of the amplitude of oscillating p53. This simulation included the p53-Mdm2 feedback loop (k33>0) with a time delay and without the TR3-mediated positive feedback loop (k14 = 0), and the model is based on (0.28 MB TIF)Click here for additional data file.Figure S5Comparison of experimental and simulated Nutlin responses. Simulations were performed based on the model in (1.35 MB EPS)Click here for additional data file.Figure S6Simulations were performed based on the model in (0.14 MB TIF)Click here for additional data file.Figure S7ijLS) of mRNA Click here for additional data file.Figure S8Region of switch-like behaviors as a function of association rate constants of p53∶MdmX and Mdm2∶MdmX. Simulations were performed based on the model in (0.26 MB TIF)Click here for additional data file.Figure S9Prediction of late response to DNA damage. Simulations were performed based on the model in (0.81 MB EPS)Click here for additional data file.Figure S10Distribution of each kinetic parameter in cluster OSC1 (A) and OSC2 (B) in (0.18 MB TIF)Click here for additional data file.Table S1Annotations of Mdm2-p53-MdmX MIM.(0.13 MB DOC)Click here for additional data file.Table S2Initial condition.(0.04 MB DOC)Click here for additional data file.Table S3Rank ordered time integral (LSj) of the normalized local sensitivity of the kinetic parameter sets from (A) (0.11 MB DOC)Click here for additional data file.Table S4Ordinary differential equations for (0.03 MB DOC)Click here for additional data file.Table S5Kinetic constants used in previous mathematical models.(0.04 MB DOC)Click here for additional data file.Table S6Assumptions used to generate an initial kinetic parameter set (column 3 in (0.03 MB DOC)Click here for additional data file.Text S1Matlab script: Parameter definition to be used to generate (1 KB TXT)Click here for additional data file.Text S2Matlab script: Function definition for a simple model.(2 KB TXT)Click here for additional data file.Text S3Matlab script: Simulate and plot (0.01 MB TXT)Click here for additional data file.Text S4Matlab script: Simulate and plot (0.01 MB TXT)Click here for additional data file.Text S5Matlab script: Function definition for the full model.(3 KB TXT)Click here for additional data file.Text S6Matlab script: Function required to generate (2 KB TXT)Click here for additional data file.Text S7Matlab script: Simulate and plot (3 KB TXT)Click here for additional data file.Text S8SBML format model. Define the full model  in SBML (0.04 MB SBML)Click here for additional data file.


Three out of four patients with primary (light chain) amyloid nephrotic syndrome treated with vincristine, doxorubicin and dexamethasone (VAD) induction obtained a partial response and are alive in continuing remission at 4.1, 6.5 and 9.3 years. These preliminary results are of considerable interest and suggest that prospective evaluation of this regimen is warranted in patients with this condition.


Mouse fibrosarcoma tumours were dissociated and divided into subpopulations of viable cells by centrifugation in linear density gradients of Renografin. Two of these subpopulations, designated Band 2 and Band 4, differed in their clonogenic ability in lung colony assay. The less dense Band 2 cells were significantly more clonogenic than the Band 4 cells (2.9 percent vs 1.4 percent respectively). Each band was further separated on the basis of cell size by centrifugal elutriation. Each size class of cells comprising Band 2 showed higher clonogenic ability than the corresponding size class in Band 4. Thus cell size differences were not responsible for the clonogenic differences between these bands. To determine whether cell-cycle distribution of the tumour cells was responsible for differences in cloning efficiency, flow microfluorometric and premature chromosome condensation methods were utilized. The unseparated and Band 4 populations showed a higher percentage of cells in S and G2 than did the Band 2 populations, but many of the S and G2 tumour cells showed extensive chromosome damage. From this study we conclude that the increased clonogenic ability of the lighter tumour cells is not due to differences in cell size or cell-cycle parameters.


Pancreas divisum has been postulated as a cause of acute pancreatitis and a chronic pain syndrome in a small subgroup of patients and can be treated with endoscopic dorsal pancreatic duct stent placementand minor papilla sphincterotomy. Twenty patients  were treated endoscopically. Dorsal duct stentswere placed in 19 patients with subsequent needle knife sphincterotomy of the minor papilla overthe stent. Clinical response was judged by comparison of symptoms . The symptom score improved from 9.3 to 5.1 in the pancreatitis groupand from 9.3 to 5.7 in the pain group. A good clinical response was observed in 3 of 7 patients in thepancreatitis group and in 6 of 11 in the pain group at a mean follow-up of 22 months. Complicationsof sphincterotomy were limited to pancreatitis in 6 patients (29%), 5 mild and 1 moderate accordingto published criteria. No patient required more than 4 days hospitalization. Two of 39 stents migratedinto the pancreas, and another stent fractured and remained lodged in the pancreas. Eight of 9 patientsevaluated demonstrated new morphologic duct changes on follow-up pancreatograms.Endoscopic stenting and sphincterotomy of the minor papilla are feasible and may be effective insome patients with pancreas divisum but carries a significant complication rate. The subjective improvementin patients with chronic pain warrants further controlled study.


Saccharomyces cerevisiae consists of 20 members; 18 genes encoding transporters  and two genes encoding sensors . The effect of oxygen provision on the expression of these genes was studied in glucose-limited chemostat cultivations . Transcript levels were measured from cells grown in five steady state oxygen levels , and from cells under conditions in which oxygen was introduced to anaerobic cultures or removed from cultures receiving oxygen.The gene family of hexose transporters in HXT gene family was distinct in cells grown under aerobic, hypoxic and anaerobic conditions. The transcription of HXT2, HXT4 and HXT5 was low when the oxygen concentration in the cultures was low, both under steady state and non-steady state conditions, whereas the expression of HXT6, HXT13 and HXT15/16 was higher in hypoxic than in fully aerobic or anaerobic conditions. None of the HXT genes showed higher transcript levels in strictly anaerobic conditions. Expression of HXT9, HXT14 and GAL2 was not detected under the culture conditions studied.The expression pattern of the When oxygen becomes limiting in a glucose-limited chemostat cultivation, the glucose uptake rate per cell increases. However, the expression of none of the hexose transporter encoding genes was increased in anaerobic conditions. It thus seems that the decrease in the moderately low affinity uptake and consequently the relative increase of high affinity uptake may itself allow the higher specific glucose consumption rate to occur in anaerobic compared to aerobic conditions. Saccharomyces cerevisiae contains the sugar transporter genes HXT1 to HXT17, GAL2 and the glucose sensor genes SNF3 and RGT2 -1 min-1 (vvm). For cultures which received less than 20.9% O2 in the gas stream, air was replaced with the equivalent volume of N2, so that total gas flow was maintained constant for all experiments. Cultures which were fed 2.8 or 20.9% O2 were subject to oscillations. To prevent these, approximately 5% of the total cell concentration in the bioreactor was added to the culture as cells in mid to late exponential phase at the time when continuous medium feed was started -1) and 20.9% (0.60 Cmol biomass [Cmol glucose]-1) O2 e]-1) O2  resulted2 approximately 25 h after O2 was provided. Fresh, exponentially growing cells were not added to prevent oscillations since the transcript levels in the added cells may have affected the overall results disproportionately.Oscillations occurred in cultures which were maintained in steady state anaerobic conditions and then provided with 20.9% Oet al. -1) so that the results for each probe in any analysis could be correlated to this internal standard, eliminating experimental variation in different hybridizations and samples. Probes were divided into 2 probe pools with 8 and 9 probes per pool. The identity of the probes was determined by the migration behaviour and the quantity by the peak area. Total polyA RNA was quantified from the cell lysates, after eluting the polyA RNA in dimethyl pyrocarbonate (DMPC) treated H2O, using the RiboGreen RNA quantification kit . Individual mRNA expression levels are given as the standardised  amount per total polyA RNA.Transcriptional analysis was performed with the TRanscript analysis with aid of Affinity Capture (TRAC) assay described by Rautio et al. . Total met al. [HXT1-5, HXT8-10, GAL2, RGT2 and SNF3 were designed to hybridise to the 3' end of the coding regions. The coding regions of HXT13 and HXT17 as well as HXT9 and HXT11 have sequence similarity of 97%. Only the 5' ends of the coding region of these genes differ enough to enable the design of specific probes. HXT6 and HXT7 are 99% identical within the coding region, but differences can be found in the 3' flank of the genes. The flanking region of HXT7 is AT rich which leads to a melting temperature of the probe (50°C) that is lower than that of the other probes (62–71°C). HXT15 and HXT16 are identical within their coding regions and within 1 kb in both directions from the coding region, except one nucleotide. Therefore only one probe was designed which detected both of these genes.The probes used in the TRAC analyses are listed in Table et al. . The proHXT3 promoter forward1 5'ACCGGTATATCAAATGGCGGTGTA 3', HXT3 promoter reverse 5'TCAGGCATGTTCATTACCTGAGAG 3', HXT6 promoter forward1 5'TGGCATCAAATTTGGGAA 3', HXT6 promoter reverse 5'GAGAGATGCTCCACAGGA 3', HXT13 promoter forward1 5'TGCTGCAATTTGCTATTT 3', HXT13 promoter reverse 5'CATCTCCATCGCTATCAA 3', HXT15 promoter forward1 5'CCATTTTTTCAGAATCCT 3', HXT15 promoter reverse 5'CTGTTTAGATTATCTGCA 3'. Three parallel PCR-reactions for each of the HXT promoters sequenced were carried out using Dynazyme Ext or Phusion high fidelity polymerases , and purified PCR-fragments were sequenced using the same oligonucleotides that were used in the PCR-reactions. In addition, the following oligonucleotides were used in the sequencing: HXT3 promoter forward2 5'GGAACATTCTAGCTCGTT 3', HXT6 promoter forward2 5'TACTTGGAAATTAATGTA 3', HXT13 promoter forward2 5'ATCATTTGTCGTGTTCCT 3' and HXT15 promoter forward2 5'CCAAATATCTTATACGTT 3'.The following oligonucleotides were used in PCR of promoter fragments from the genomic DNA of CEN.PK113-1A: Statistical analysis of the data was carried out using SPSS software . Analysis of variance (ANOVA) and Dunnett's T3 multiple comparison test were used for the comparative analysis of the data. All graphs were prepared using the R environment ,62.ER, LR and MP conceived the study. ER carried out the transcriptional, promoter and statistical analyses, participated in the fermentations and drafted the manuscript. AT and MGW carried out the fermentations and MGW revised the manuscript. LR supervised the work and revised the manuscript. All authors read and approved the final manuscript.


Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. Furthermore, naturally occurring insensitivity to IFN-α may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not been identified so far. To explore the molecular basis of IFN-α responsiveness, we analysed the expression pattern of about 7000 genes in IFN-α sensitive and resistant cell lines and we compared the transcription profiles of cells cultured in the presence or absence of the cytokine using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction upon IFN-α treatment by standard 3H-thymidine incorporation and flow-cytometry. The study of 4 sensitive and 2 resistant cell lines allowed the identification of 4 genes  preferentially transcribed in sensitive cells and 2 (SHB and PKC-ζ) preferentially expressed in resistant cells. IFN-α stimulation resulted in the expression of a panel of 19 known inducible genes in sensitive but not in resistant cells. Moreover a group of 30 novel IFN-α inducible genes was identified. These data may provide a useful basis to develop diagnostic tools to select potential IFN-α responders eligible for treatment, while avoiding unnecessary toxicity to non-responders. Furthermore, by extending the knowledge of the polymorphic effects of IFN-α on gene expression, they offer novel clues to the study of its pleiotropic toxicity. © 2001 Cancer Research Campaign


Fgf2 gene is expressed in the brain neuroepithelium during embryonic development and in astroglial cells throughout life. Previous knockout studies suggested that FGF2 plays a role in the proliferation of neural progenitors in the embryonic cerebral cortex. These studies exclusively used knockout alleles lacking the Fgf2 exon 1. However, the description of putative alternative exons located downstream from the canonical exon 1 raised the possibility that alternatively spliced transcripts may compensate for the lack of the canonical exon 1 in the Fgf2 -/- mice.The Fgf2 transcripts and contains essential heparin and receptor binding interfaces. The expression of Fgf2 exon 3 was prevented by inserting a transcriptional STOP cassette in the Fgf2 genomic locus. These mice demonstrate a phenotype in the adult neocortex characterized by decreased density and number of cortical excitatory neurons and astrocytes, which is virtually identical to that of the Fgf2 -/- mice lacking exon 1. In addition, we also show that the Fgf2 exon 3 knockout mice have decreased proliferation of precursors in the adult cerebral cortex, which had not been previously investigated in the other mutant lines.We generated and characterized a new line of Fgf2 knockout mice lacking the expression of exon 3, which is conserved in all Fgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis during embryonic development as well as in adulthood. Thus, FGF2 may be required for the maintenance of the pool of adult cortical progenitor cells.The results demonstrate that the phenotype of two completely different  Rather, the increase in total cell number can be attributed to an increase in the number of progenitor cells [FGF2, one of the most potent among the fibroblast growth factor ligands, is expressed in the neuroepithelium of the central nervous system ventricular zone (VZ) from early embryonic development, along with the transmembrane FGF receptors FGFR1-FGFR3. FGF2 promotes growth in the cerebral cortex by increasing the number of cortical progenitor cells in the VZ [The expression of FGF2 is steadily down-regulated during embryonic development -5-.Fgf2 null (Fgf2 -/-) mice demonstrated its unique role in regulating neurogenesis. Specifically, there is a 40% decrease in the number of neurons and astrocytes in the cerebral cortex of Fgf2 -/- mice [Fgf2 -/- mice [The analysis of -/- mice , as well-/- mice . This su-/- mice . Pyramid-/- mice . The dat-/- mice . In addi-/- mice .Fgf2 is encoded by three exons, exon 1, 2, and 3. In the original knockout studies, Fgf2 null mice were generated by deleting exon 1 and immediately upstream sequences in the Fgf2 gene locus [Fgf2 mRNA variants have been described that lack the canonical exon 1 and utilize alternative exons  that are located downstream from exon 1 and can be spliced to the second exon [Fgf2 -/- mice. To answer this question, we generated a new recombinant mouse model that lacks exon 3, an exon that is conserved among the Fgf2 transcript variants.ne locus -11-. Howond exon . These sFgf2 knockout mice was generated by inserting a stop codon in front of this exon. This stop sequence was flanked by Cre recognition sites, and as such it is amenable to being excised by Cre recombination, reconstituting the wild type gene. An important question to be addressed is whether these mice would show the same or more profound deficits in CNS development, as compared with the original exon 1 Fgf2 knockout line.Because exon 3 is over 5 kb long, the current strain of Fgf2 genomic sequence, respectively , the entire coding sequence for the βgeo gene  followed by a polyadenylation site and flanked by loxP sites. This cassette was inserted by homologous recombination into an intronic KpnI site, located approximately 1 kb upstream from the third exon of the Fgf2 gene   flanked ely Fig. . The SAβene Fig. . After eFgf2 alleles, the following primer pairs were used: upstream primer: 5'-TTGGTACCCTGGAATATTTTAGCCC-3'; downstream primer: 5'-AATAAGTAACCCAGAATATACTGG-3'. These are complementary to sequences in the intron 2 – 3 at the cassette insertion site   with an upstream primer in exon 2 and a downstream primer in exon 3  in 0.1 M Phosphate Buffered Solution (PBS), pH 7.4, followed by post-fixation overnight in 4% PFA and cryprotection in 20% sucrose. Thirty minutes prior to perfusion, the mice were injected with 100 mg/kg 2-bromodeoxururidine (BrdU). Brains were sectioned with a cryostat at 50μm thickness. For each antibody staining, a series of free-floating sagittal sections (1 out every 10) were blocked for one hour in a 0.1 M PBS/10% normal goat serum solution, pH 7.4, containing 0.2% Triton-X-100 and 0.1% Tween 20. Immunohistochemistry was performed with the following antibodies: neuronal-specific nuclear protein (NeuN) , glial fibrillary acidic protein (GFAP) , T-box brain 1 protein (TBR-1)  , parvalbVolume and total cell numbers were calculated as described  using thgeo gene, which was followed by a polyadenylation site  was inserted upstream of exon 3 in the Fgf2 gene by homologous recombination. In the modified gene locus, βgeo mRNA is expressed instead of exon 3, after which there is premature polyadenylation of the gene product. The truncated product is thought to be without function, since many of the receptor binding interfaces and most of the heparin binding sites are in exon 3 [Fgf2 gene can be reconstituted via Cre recombination, which should excise the SAβgeolox2DTA sequence.The SAβgeolox2DTA DNA cassette, containing a splice acceptor, preceding the entire coding sequence for the βn exon 3 . With thFgf2 exon 3-deficient mice were born at the normal Mendelian frequency and displayed no alteration of body size, fertility, or survival. To assess Fgf2 exon 3 expression, total RNA was extracted from the brain of 5-month-old Fgf2 exon 3 KO, wild type and heterozygous mice and reverse-transcribed into cDNA. RT-PCR analyses were carried out using an upstream primer corresponding to sequences in exon 2 and a downstream primer corresponding to sequences in exon 3 of the wild type Fgf2 gene. A specific 139-bp amplicon was obtained from Fgf2 wild type and heterozygous mice, but not from their KO littermates  homozygous for the wild type or the Fgf2 exon 3 KO allele. Cortical neurons and astroglial cells, identified by NeuN and GFAP immunostaining, respectively . To assess the number of actively proliferating cells, BrdU was administered 30 min before perfusion and the incorporation of the nucleotide in S-phase cells, visualized by immunostaining, was quantified by stereology in the whole cerebral cortex. Proliferating cells were decreased by 29% in the Fgf2 exon 3 KO mice as compared to their wild type littermates  is likely non-specific because were it to have resulted from read-through of the truncating stop codon, it would also appear at the 21 kD and 22 kD positions since all three isoforms are translated from the same transcripts, and it does not (0% of the intensity of the wildtype 21 kD and 22 kD bands).We produced mice with premature termination of the conserved Fgf2 exon 3 causes significant deficits in several major cortical cell populations. Overall, cortical NeuN+ neuron number was decreased by 23% and that of GFAP+ astrocytes by 34%; one subpopulation of cortical GABAergic interneurons, the PV+ interneurons, was not significantly affected, although we cannot exclude that a larger sample may reveal a deficit in this population, as well. This replicates the results obtained in our previous study of Fgf2 null mice [Fgf2 gene locus. In that study, NeuN+ cells were decreased by 24% and glial cells by 38%. Thus, the degree of deficit in these two cell populations in the two Fgf2 KO models is strikingly similar. The decrease in GFAP+ cells in the adult cortex is also in agreement with the results previously reported by another group [Fgf2 KO mice reported a minimal decrease or no significant change in total cortical volume. Given the decrease in neuron number, it is conceivable that some compensatory mechanism maintains normal cortical volume; an expansion of dendritic or synaptic neuropil is a possibility.This study shows that the lack of ull mice , which wer group . Also, iFgf2 KO mice that are slightly different from those described above [Fgf2 KO mice lacking exon 1. Possible reasons for this discrepancy include the genetic background (C57BL/6J/129/Sv versus Black Swiss/129/Sv in our mice) and the quantitative methods used for estimating cell numbers. We examined both total number and density of cells by stereology using the optical fractionator and unbiased sampling methods, whereas Dono et al. assessed cell density, presumably by counting cells in selected sections.Another group reported findings on the cortical phenotype of ed above . Dono etThe decrease in excitatory neurons expressing TBR-1 is less than what was previously found in the exon 1 KO mice using immunostaining for the neurotransmitter glutamate. However, TBR-1 + excitatory neurons represent only a subset of pyramidal cells, those predominantly found in layer 6 in adult mice. The data suggest that FGF2 may be relatively more important for the genesis of pyramidal cells in other cortical layers.Fgf2 KO study [In our prior exon1 KO study  the mutaKO study . This diFgf2 KO mouse lines, lacking exon 1 or exon 3, is remarkably similar. The combined results from these KO models clearly indicate that FGF2 plays a role in cortical cell genesis in embryos as well as in adulthood. This is shown by the similar phenotype in the two lines: a decrease in total cortical neuron number, due to a deficit in pyramidal cells as well as in astrocytes, as shown by NeuN, TBR-1 and GFAP immunostaining. Pyramidal neurons and astrocytes are progeny of radial glial cells, which are located in the VZ [Fgf2 exon 1 KO mice [Fgf2 KO mice are proliferating stem/progenitor cells in the adult subventricular zone [The implications of these results are manifold. First, the results demonstrate that the phenotype of two completely different n the VZ . This is KO mice . Anotherlar zone , which alar zone . It can The current results also show that FGF2 is required for normal cell proliferation in the mature cortex. In previous studies on exon 1 KO mice, proliferating cells were assessed only in embryos . The curFgf2 gene by inserting a transcriptional STOP cassette by homologous recombination in the Fgf2 genomic locus. Previous research has shown that there are three putative splice variants of exon 1: 1a, 1b, and 1c [Fgf2 KO models [Fgf2 KO mice. However, whether these FGF2 variants are actually transcribed into mRNA and protein is unclear. The generation of an exon 3 KO in the current study sought to verify the role of FGF2 in development because exon 3 is conserved in the all known FGF2 variants. The results clearly show that the phenotype of the Fgf2 exon 3 KO mice show no greater severity, and that the decrease in cell populations is very similar to that found in the exon 1 studies. Overall, the role of FGF2 in regulating cortical development is confirmed by removing the conserved exon 3. The relatively mild phenotype in this Fgf2 mutant may be explained by compensatory or redundant relationship with other FGF family genes. Furthermore, this line will allow for future studies in which the intact Fgf2 gene can be reconstituted by Cre recombination at selective stages in development or adulthood.This study illustrates a novel way of preventing the expression of a specific exon of the , and 1c . Exon 1aO models -11-. AltFgf2 exon 3 KO mice by molecular, immunohistochemical and stereological analyses and drafted the manuscript. YO participated in the molecular analyses for the characterization of the phenotype of Fgf2 exon 3 KO mice. DS generated the targeting vector for the Fgf2 exon 3 KO mice and carried out the screening of the targeted ES cells. TD participated in study design, helped with data analysis and helped to write the manuscript. LPS generated the knockout mice in the University of Cincinnati Gene-Targeted Mouse Service. HL carried out the Western blot assays. FMV conceived the study, participated in its design, helped with data analyses and interpretation and helped to write the manuscript.KC carried out the characterization of the brain phenotype of All authors read and approved the final manuscript.


DNA extracted from familial and sporadic colorectal neoplasms was compared with constitutional DNA using a range of hypervariable locus specific probes to assess the extent of allele loss during conversion to malignancy. Chromosome 5 allele loss was observed in 23% of carcinoma samples, as previously found by others. However, we have been able to show for the first time loss of the D5S43 locus on chromosome 5 in adenomas from three patients, two of whom had the precancerous condition adenomatous polyposis coli (APC). These results suggest significant genetic changes involving chromosome 5 are occurring in benign adenomas. Probes for chromosome 1 (loci D1S7 and D1S8) and for chromosome 7 (loci D7S21 and D7S22) revealed no notable alterations in the adenoma samples. Complete loss of alleles for loci on chromosome 7 was not observed in carcinomas but reduced intensity of one parental allele was found in three specimens one of which was known to have multiple copies of this chromosome. Results using probes for chromosome 1 suggest that deletion of the D1S7 or D1S8 loci is not a common event in colorectal carcinogenesis. Loss of chromosome 5 alleles in adenomas from APC patients provides evidence in support of Knudson's hypothesis.


We have used the polymerase chain reaction to detect DNA sequences related to human papillomavirus type 16, by simultaneous priming with oligonucleotides from the E6 and L1/L2 open reading frames of the HPV16 genome. The HPV16-related sequence is present at low levels in normal oral tissue, in addition to biopsies and cell cultures from patients with benign and malignant disease. Ultimate analysis of the amplified sequences from the E6(120bp) and L1/L2(173bp) regions of HPV16 was achieved by gel electrophoresis and comparative nucleotide sequencing. The oral carcinoma biopsies and tissue cultures contained DNA sequences which were identical to the E6 region of HPV16, but only rarely contained sequences closely related to the L1/L2 region. The PCR technology should permit the detection, identification and cloning of latent viruses from extremely small tissue biopsies.


N-acetyltransferases (NAT). This study examined the association of (NAT) 1 and 2 genotypes with the risk of smoking-related bladder cancer. A total of 74 pathologically confirmed bladder cancer patients and 184 controls were serially recruited from the National Taiwan University Hospital. History of cigarette smoking and other risk factors for bladder cancer was obtained through standardized questionnaire interview. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 and NAT2 by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Allele frequency distributions of NAT1 and NAT2 were similar between cases and controls. There was a significant dose–response relationship between the risk of bladder cancer and the quantity and duration of cigarette smoking. The biological gradients were significant among subjects carrying NAT1*10 allele or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele. The results are consistent with the hypothesis that NAT1 and NAT2 might modulate the susceptibility to bladder cancer associated with cigarette smoking. © 1999 Cancer Research CampaignAromatic amines from cigarette smoking or occupational exposure, recognized risk factors for bladder cancer, are metabolized by


Previous data have shown that this enzymatic complex is implicated in cell cycle progression and carcinogenesis. In yeast this enzyme acetylates peptides composed by methionine and aspartic acid or glutamic acid in their first two positions respectively and it has been shown the same specificity in human cells.Human NhNAA20 expression in hepatic cell lines using recombinant adenoviruses that express specific siRNAs against this gene and analyzed cell cycle progression and apoptosis induction after this treatment. Immunopurified hNatB enzymatic complexes from human cell lines were used for analyzing hNatB in vitro enzymatic activity using as substrate peptides predicted to be acetylated by NatB.We have silenced hNAA20 silencing in hepatic cell lines reduces cell proliferation in a p53 dependent and independent manner. At the same time this treatment sensitizes the cells to a proapototic stimulus. We have observed that the hNatB complex isolated from human cell lines can acetylate in vitro peptides that present an aspartic or glutamic acid in their second position as has been described in yeast.hNatB enzymatic complex is implicated in cell cycle progression but it exerts its effects through different mechanisms depending on the cellular characteristics. This is achievable because it can acetylate a great number of peptides composed by an aspartic or glutamic acid at their second residue and therefore it can regulate the activity of a great number of proteins. Although the two cotranslational events, cleavage of the initiator methionine and Nα-acetylation, are common and highly conserved from bacteria to higher eukaryotes, their function it is not well understood acetyl-CoA  for 2 hours at 37°C. The samples were centrifuged and the supernatant was incubated with SP-Sepharose (50% in 0.5 M acetic acid) for 10 minutes on a rotor before washing the SP-Sepharose three times with acetic acid 0.5 M and once with methanol. The radioactivity incorporated in the peptides was determined by scintillation counting.The NAT assay was performed basically as described previously ,16 but uHepG2 and Hep3B cells were seeded in 15 mm cover glasses and infected with siRNA expressing adenovirus pAdsiRNA2 or pAdsiRNA350 for 24, 48 and 72 hours, BrdU labeled for 1 hour and processed using the 5-bromo-2'-deoxyuridine Labeling and Detection Kit I  following the manufacturer's instructions.HepG2 and Hep3B cells were harvested after 72 hours of infection with the adenovirus pAdsiRNA2 or pAdsiRNA350 and processed using the CycleTEST PLUS DNA reagent kit (Becton Dickinson) following the manufacturer's instructions. Flow cytometric analysis of the different samples was performed using BD FACScalibur flow cytometer and DNA content was analyzed using the MODFIT software.HepG2 and Hep3B cells were incubated in 15 mm cover glasses and infected with adenovirus pAdsiRNA2 or pAdsiRNA350 for 48 hours prior adding the proteasome inhibitor MG132 (5 μM)  for an additional 8 hours. Cells were fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4). TUNEL assay was performed using the In Situ Cell Death Detection Kit (Roche Applied Science) according to the manufacturer's instructions. Cell apoptosis percentage was obtained as indicated in cell proliferation assay.hNAA20 shRNAs. As it is observed in Figure hNAA20 siRNA (pAdsiRNA2) is expressed. Previous data indicate that there is an inhibition of cell cycle progression when hNatB is inhibited. Therefore we measured cellular proliferation after hNAA20 knockdown and observed a clear inhibition in both cell lines . These differences are not restricted to hNAA20 inhibition in distinct cell lines because hNAA25 repression causes also cell growth arrest, but instead of upregulating p21(WAF/CIP1) expression, as hNAA20 knockdown, there is a clear p21(WAF/CIP1) reduction [The p53 tumour suppressor is a tightly regulated protein that acts by stopping cell-cycle progression or promoting apoptosis when cells encounter stress stimuli such as oncogene activation or DNA damage ,20. For eficient , induceseduction . But we hNAA20 knockdown in HepG2 and Hep3B is not associated with apoptosis induction (data not shown ) but it sensitizes the cells to the proapoptotic treatment with MG132 as it is presented in Figure hNAA20 siRNA, siRNA2. This effect correlates in HepG2 with a reduction of BCL2  [We have observed also that Hep3B cell line is more sensitive to MG132 treatment as Hep3B cells that express the unrelated siRNA present some sensitivity to MG132. This is Hep3B specific because a prolonged exposure of the cells to this proapoptotic stimulus induces an apoptotic cell death that it is not observed in Hela and HepG2 cells (data not shown). These findings are also reminiscent of the increased sensitivity to environmental stress when yeast strains were deleted of yNatB enzymatic complex (naa25-Δ) ,6.in vitro acetyltransferase assay using as substrate a peptide corresponding to human tropomyosin-1 amino terminus, that has been demonstrated to be a hNatB substrate [NAT enzymatic complexes are composed by a catalytic subunit and one or several accessory subunits. In the case of hNatA there are two possible catalytic subunits  and seveubstrate . We obsein vitro acetyltransferase activity we purified enzymatic complexes with the hNaa20p antibody from Hela cells as this has resulted the most efficient isolation method ; MT1A: Metallothionein-1A; RS28: 40S ribosomal protein S28; TNNC1: Troponin C, slow skeletal and cardiac muscles (TN-C); NAT2: N-acetyltransferase 2 (arylamine N-acetyltransferase); DCUP: Uroporphyrinogen decarboxylase; KAD1: Adenylate kinase isoenzyme 1; B3AT: Band 3 anion transport protein; CRBA1: Beta-crystallin A3; PTN1: Tyrosine-protein phosphatase non-receptor type 1; PPLA: Cardiac phospholamban.The authors declare that they have no competing interests.AA, CG, ML, EL, JP and RA designed research. AA carried out all the experiments concerning cell manipulation and analysis. CG and ML performed the in vitro acetylation assays. All authors designed research and analyzed data. RA coordinated the research and wrote the paper.


The purpose of this study was to determine the prevalence of BRCA1 mutations in Chinese breast cancer patients in Singapore. BRCA1 analysis was conducted in consecutive patients with breast cancer before the age of 40 years (76 women), or whose relatives had breast or ovarian cancer (16 women). Ten patients had both early onset breast cancer and affected relatives. Genomic DNA from peripheral mononuclear blood cells was studied by using the protein transcription–translation assay (exon 11) and single-strand conformational polymorphism, with subsequent DNA sequencing. All six disease-causing mutations occurred in women under 40 years (8.6%) with three occurring in patients under 35 years . Mis-sense mutations of unknown significance were found in three patients. Two of the ten women with affected relatives under 40 years had BRCA1 mutations. The prevalence of BRCA1 mutations in Chinese patients with early onset breast cancer is similar to that observed in Caucasian women. Most Chinese patients with affected relatives were not carriers of BRCA1 mutations. © 2000 Cancer Research Campaign


To understand genetic events which play a role in the development and/or progression of human endometrial cancer, we studied allelotypes on all autosomal chromosomes, as well as chromosome X, with 42 microsatellite markers and 56 endometrial cancers. The most frequent loss of heterozygosity (LOH) was observed on the long arm of chromosome 10 , which was commonly detected in grade 1 cancer. We constructed a detailed deletion map and defined two commonly deleted regions in 10q25-q26; one was the 8 cM region between D10S209 and D10S216, the other was the 12 cM region between D10S217 and D10S590. Replication errors at two or more loci were observed in 24 of 56 tumours (43%), suggesting that disruption of the DNA mismatch repair system also plays an important role in the course of endometrial carcinogenesis.


Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease.Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased.To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals. Down syndrome (DS) is a result of trisomy or partial trisomy of human chromosome 21 (HSA21) and has an incidence of 1 in approximately 1000 live births in utero precluding analysis of the postnatal consequences of trisomy We utilised a mouse model to investigate how trisomy 21 might affect gene regulatory networks and adult neurogenesis. Mouse chromosome 16 (MMU16) has significant homology with HSA21 A number of features analogous to DS have been demonfstrated in the partially trisomic mouse models and the extent and severity of the phenotypes observed mostly correlate with the size of the trisomic segment. Both Ts65Dn and Ts1Cje mice display craniofacial dysmorphology that directly parallels human DS and defective spatial learning and memory Proliferation defects have been demonstrated in the hippocampal dentate gyrus and neocortical germinal matrix of DS, Ts16 and Ts65Dn foetuses, postnatal Ts65Dn mice and in the hippocampal dentate gyrus and cerebellum of adult Ts65Dn mice Neurogenesis occurs prenatally and postnatally but also throughout adulthood, the latter in predominantly two regions of the brain, the subgranular zone of the dentate gyrus and the SVZ lining the lateral ventricles. Neural stem cells (NSCs) residing in neurogenic regions have the ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes in vivo and ex vivo experiments we show that Ts1Cje adult neurogenesis is reduced as a result of abnormal progenitor production and defects in cell lineage commitment during differentiation rather than defects in NSC development or homeostasis. Moreover, we identified specific regulators of cell cycle progression that are dysregulated in neural precursors isolated from the adult Ts1Cje SVZ. We hypothesise that these defects in adult neurogenesis confound pre-existing developmental defects and are likely to progressively contribute to problems in memory formation and possibly brain functions throughout adult life. We also expect that the defects will impair the ability of the DS brain to respond to neuronal loss and degeneration with repair.Based on a number of In order to identify genes dysregulated in adult Ts1Cje neurogenesis we performed a whole-genome expression analysis using GeneChip® Mouse Genome 430 2.0 Arrays  on 3 pairs of female Ts1Cje and disomic littermate control primary neural stem and progenitor cells after expansion for 7 days as neurosphere cultures. This data is freely accessible on the Gene Expression Omnibus website under the series accession number GSE17760.2 fold-changes (M) for trisomic versus disomic mice versus the average log2 expression (A). Each dot represents a single probe. Probes that were not expressed naturally did not show any change, i.e., the log2 fold-changes were around 0, but probes that were expressed at worthwhile levels  consistently showed fold changes of around 1.5  may be differentially expressed, although the fold changes were small so that few genes could be individually identified as differentially expressed at conventional significance levels. In order to uncover changes in groups of functionally interconnected genes we utilised two online analysis tools the Database for Innovation, Visualization and Integrated Discovery , Mcm7, Pbk, Phb, Prim1 and Suv39h1) by RT-qPCR in neurosphere cultures generated from 3 pairs of female Ts1Cje and disomic littermate controls independently of the original microarray analysis . Phb was shown to be slightly downregulated in the Ts1Cje neurospheres by RT-qPCR analysis but upregulated in the microarray analysis.Focusing on the IPA network with the largest number of differentially expressed probes  we analyanalysis . SimilarTo examine whether these small abnormalities in gene expression affect the Ts1Cje brain we examined the neurogenic lineage systematically from neural stem cells (NSCs) through neural progenitors, via neuroblasts, to neuronal differentiation and neurite outgrowth. In addition, we also examined neuroblast migration.Gene expression abnormalities in proliferation associated genes imply that there are defects in the proliferation of trisomic NSCs and/or progenitors. We used a double labelling assay with the thymidine analogues 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU) to investigate whether there is a reduction in the numbers of NSCs or a defect in the proliferation of NSCs in the adult Ts1Cje subventricular zone (SVZ). Thymidine analogues are incorporated into DNA during the DNA synthesis phase of the cell cycle and then diluted exponentially following cell division when half the analogue is distributed to the daughter cell. The thymidine analogue is undetectable by immunohistochemistry after 3 cell divisions.NSCs are relatively quiescent with a relatively long cell cycle length of ∼15 days and an S phase of ∼8 h P = 0.053; N = 4 pairs of male Ts1Cje and disomic controls; P = 0.97; N = 4 pairs of male Ts1Cje and disomic controls; data not shown). We therefore detected no difference in the number of NSCs in the adult Ts1Cje brain compared to their sex matched disomic littermate controls.IdU positive cells were counted in the subventricular neurogenic zone at 5 equally spaced rostrocaudal levels. We found no significant difference in the number of IdU long-term label retaining cells  and move as chains of migrating neuroblasts from the SVZ to the olfactory bulbs where they become periglomerular and granule cell interneurons The PSA-NCAM staining revealed that the Ts1Cje neuroblasts were reduced in number, but directional pattern of migration appeared normal and there was no obvious defects such as blockages or altered orientation. In the following experiment we examined their rate of migration using a medium-term thymidine analogue labelling assay.P = 0.056; N = 3 male and 1 female pairs of Ts1Cje and disomic littermates).Neuroblasts take between 2 and 6 days to migrate from the SVZ to the olfactory bulbs via the RMS Finally, we examined the ultimate product of SVZ neurogenesis, the differentiating neuron.in vitro. Following 7 days of primary culture, neurospheres were dissociated and plated onto poly-ornithine and laminin coated coverslips. Cell types were identified by immunofluorescence using antibodies against type III β-tubulin, glial fibrillary astrocyte protein (GFAP) and O4 to mark neurons, astrocytes and oligodendrocytes respectively. The number of neurons generated from Ts1Cje neurospheres was reduced with an average of 56% compared to sex matched disomic littermate controls  or the average length (P = 0.55) of primary neurite  or tertiary (P = 0.96) neurites . In this study we also observed dysregulated expression of genes involved in proliferation, particularly cell cycle control. We have shown a tendency for delayed neuroblast migration through the adult Ts1Cje SVZ. Importantly, we have uncovered severe defects in Ts1Cje neural differentiation, in particular a reduction in the production of neurons and a tendency for reduced side branching. Given that our work demonstrates neurogenesis defects exist during adulthood suggests that there is potential to slow aspects of the progression of cognitive decline and neurodegeneration in DS.We have used a combination of 2-phase of the cell cycle and Brca2 deficiency has been shown to impair cytokinesis and extend cell division 2/M phase Analysis of our microarray expression data from adult SVZ neurosphere cultures using both the Database for Innovation, Visualisation and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) identified dysregulation of genes involved in proliferation in Ts1Cje samples. The majority of proliferation associated genes showing disrupted expression were subtly downregulated and not from the Ts1Cje trisomic region, indicating that trisomy results in a large number of downstream gene expression abnormalities that combined would affect proliferation in adult neural progenitors. The largest IPA network of differentially expressed genes included a number of genes whose down regulation could delay cell cycle progression. The following discussion is focused around the genes from this network for which expression was most affected by trisomy. The helicase minichromosome maintenance proteins (MCMs) and Prim1  are involved in DNA replication and their reduced expression is likely to result in a delay or elongation of the S phase of the cell cycle 2 and M phases. Indeed, elongation of the G1 but particularly the G2 phase has been observed in trisomic neural precursors Ccnb1 and Skp2, two key regulators of the G2/M and G1/S transitions are decreased in neonatal (PN2) Ts65Dn cerebellar granule cell precursors 2/M phase may be a common theme in trisomic cell division. Notably, we found Ccnb1 to be downregulated in adult neurospheres. Furthermore, all probes located to the Skp loci on our microarray that were expressed in adult neurospheres were reduced in the Ts1Cje compared to disomic controls, with fold changes ranging from 0.64 to 0.87.Based on the changes in gene expression that we observed we would expect a delay in cell cycle progression in the S or Get al., (2009) used cells after at least four passages in culture, whilst we subjected cells to only one passage in culture before conducting the microarrays. We note that we have shown changes in gene expression in trisomic neural precursors even in different pathways to those observed by others despite some similarities in neural differentiation defects Rest regulated genes in the trisomic neural progenitor cells. This could be due to the differences in the source of cells investigated. We used primary neurospheres, whilst Bahn et al., (2002) analysed cells after more than 10 weeks of culture and Canzonetta et al., (2008) analysed cell lines following at least 3 passages. In this context it is important to note that striking differences have been observed between primary neuronal cells and cells cultured for extended periods such that primary cells are expected to reflect the situation in vivo more accurately A recent gene expression study on embryonic neural progenitors cultured as neurospheres from the E14.5 Ts1Cje neocortex also observed dysregulation of genes involved in proliferation, although changes in expression of individual genes differed from our study in vivo using a double thymidine analogue labelling assay. However, we observed a reduction in stem cell/progenitor derived primary neurospheres from the Ts1Cje SVZ and neuroblasts migrating through the RMS. A partial block in the S, G2 or M phases of the cell cycle could explain a low production of neural progenitors and neuroblasts. In concordance with previous findings and a reduction in the numbers of neural progenitors and neuroblasts the anterior-posterior length of the Ts1Cje olfactory bulbs were shorter than the disomic controls To our knowledge, no previous study has differentiated between the effects of trisomy 21 in humans, or 16 in mice on NSCs versus neural progenitor cells. We did not detect a difference between the number of adult Ts1Cje and disomic control NSCs 2 phase, which was also shown to be prolonged compared to the disomic In support of our findings others have reported similar defects in the production of neural progenitors, albeit the majority of studies have been focused on different areas of the brain, at different stages of development. A number of studies of Ts16, Ts65Dn and DS embryonic brain development have identified cell cycle and neurogenesis abnormalities in the neocortex and hippocampus Our neurosphere data imply that there is a major reduction in the capacity of Ts1Cje neural progenitors to differentiate into neurons by more than 50%. The number of neurons produced by the SVZ has not been assessed previously in models of DS. Defects in neural differentiation have been reported for other areas of trisomic brains and recently a greater number of GFAP-positive cells were observed prior to differentiation in neural progenitors isolated from the E14.5 Ts1Cje neocortex Dyrk1A), which is located on the Ts1Cje trisomic region. Indeed, the 3 Dyrk1A probes on our microarray were overexpressed at expected trisomic levels. Dyrk1A has been shown to have a highly dynamic expression pattern throughout mouse brain development suggestive of a key role in regulating the multiple events that occur during neuronal development 2-phases of the neuronal progenitor cell cycle combined with the altered levels of cell cycle regulators found in mice overexpressing Dyrk1A and the phosphorylation of cell cycle regulators by DYRK1A led to the suggestion that it is a regulator of neural progenitor proliferation KIP1, which has been shown to be involved in neuronal differentiation Dyrk1A have been generated and were shown to display hippocampal-dependent spatial learning and memory defects, developmental delay and motor deficits Dyrk1A in the striatum of a transgenic mouse model of Dyrk1A resulted in the restoration of motor coordination, attenuation of hyperactivity and improvement of sensorimotor gating We have demonstrated that the expression of a number of genes involved in control of the cell cycle are disrupted and that there are a number of abnormalities in adult Ts1Cje adult neurogenesis, however the question remains, which trisomic gene or genes are responsible? It is tempting to speculate that the abnormalities we have observed are due to the overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A gene  Neurogenic cells isolated from the adult Ts1Cje brain exhibited disrupted expression of genes encoding proteins involved in cell cycle progression. 2) Ts1Cje brains had normal numbers of long-term retaining NSCs. 3) In contrast, the numbers of self-renewing neural progenitor colonies, were reduced. 4) There was a reduction in the number of neuroblasts migrating through the Ts1Cje RMS compared to disomic controls. 5) Our data suggests that the speed of adult Ts1Cje neuroblasts migration was reduced. 6) The number of neurons produced by adult Ts1Cje neurogenic progenitors showed a striking reduction to less than one half whilst the numbers of astrocytes was increased. 7) Ts1Cje neurons showed a tendency for less elaborate neurites.All breeding and experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee .App  as described previously Sex matched disomic littermates were used as controls for all experiments. All mice were aged to 3 months (84 days). Ts1Cje and disomic mice were generated by mating Ts1Cje males  with C57BL/6 female mice for over 10 generations. Offspring were genotyped using multiplex PCR primers for neomycin and RNA was extracted from 3 Ts1Cje and 3 disomic female littermate control neurospheres (generated from adult subventricular zone (SVZ) cells) cultured for 7 days after isolation using a Qiagen RNeasy micro kit  according to the manufacturer's instructions with the DNase I digestion step. The quality and quantity of RNA was assessed using a 2100 Bioanalyzer . Each RNA sample was processed using the Two-Cycle Target Labelling Assay and hybridised onto an Affymetrix GeneChip® Mouse Genome 430 2.0 Arrays according to the manufacturer's instructions . Fluorescent signals were detected using a GeneChip® Scanner 3000 .t-statistics P-values were adjusted for multiple testing www.r-project.org) P-values was completed to estimate the true percentage of statistically significantly expressed genes http://www.ingenuity.com) were used to uncover changes in functionally interconnected genes from a list of the most differentially expressed genes . Primer and probe sequences are detailed in the 2O , on a LightCycler® 480 System . Conditions for the RT-qPCR were a pre-denaturing step of 95°C for 10 min, 45 cycles of; 95°C for 10 sec, 60°C for 30 sec, 72°C for 30 sec and finishing with a cooling step at 40°C for 1 min.RT-qPCR was used to validate the expression of 11 selected genes from the microarray analysis. RNA was extracted independently from the microarray samples from 3 female Ts1Cje and 3 disomic litermate control 7-day-old neurosphere culture pellets using a Qiagen RNeasy micro kit  according to the manufacturer's instructions with the DNase digestion step. RNA quality and quantity was assessed using a 2100 Bioanalyzer . First strand cDNA was synthesised using random hexamers and the SuperScript™III Reverse Transcriptase Kit  according to the manufacturer's instructions. Primers were designed and probes selected using ProbeFinder Version 2.34  for each signal was calculated based on the Second Derivative Maximum method. A set of serially diluted cDNAs synthesised from 3 µg of adult mouse brain RNA was used to construct a 3-data point standard curve for every PCR assay in each run. A PCR efficiency of between 90–110% and an R-squared value >0.98 were used to define successful assays. Relative quantification of target gene expression in Ts1Cje and disomic samples was carried out using the comparative Ct method. We performed intra-sample data normalization against 3 endogenous control reference genes 2+/Mg2+ free) with 200 U/ml penicillin, 200 µg/ml streptomycin for 30 min at 4°C and transferred into mouse tonicity phosphate-buffered saline (MTPBS) for 5 min at 37°C. The pancreatin/trypsin was inhibited briefly by 2% foetal bovine serum in MTPBS, and the tissue was mechanically dissociated. Cells were collected by centrifugation and cultured in neural stem cell (NSC) proliferation medium for 7 days: DMEM/F12, 5 mM HEPES, 13.4 mM NaHCO3, 0.6% D-glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, 25 µg/ml insulin, 60 µM putrescine dihydrocloride, 100 µg/ml apotransferrin, 30 nM sodium selenite, 20 nM progesterone, 10 ng/ml bovine recombinant fibroblast growth factor 2 (FGF2), 4 µg/ml porcine heparin sodium salt, 20 ng/ml murine epidermal growth factor (EGF) and 2 mg/ml bovine serum albumin fraction V. Under these conditions SVZ cells proliferate and form neurospheres. Neurospheres were collected by centrifugation and dissociated in 0.25 mg/ml trypsin, 10 µg/ml DNase I, 10 mM HEPES and 0.2 mg/ml EDTA in Ca2+/Mg2+ free HBSS at 37°C for 3 min. The trypsin was inhibited briefly by 140 µg/ml soybean trypsin inhibitor, in HEM with 10 µg/ml DNase I. 50,000 cells were plated onto poly-ornithine/laminin-coated 13 mm glass coverslips in differentiation medium  for 8 days.Cells from the SVZ were isolated and cultured as described previously For assessment of neuroblast migration along the rostral migratory stream (RMS) 4 pairs of Ts1Cje and disomic littermate controls  were injected with 5-bromo-2′deoxyuridine  twice daily 12 h apart for 4 days and left without treatment for 2 days. The migration front of BrdU labelled neuroblasts was identified on serial paraffin sections stained for BrdU. Adjacent sections were stained with cresyl violet to reveal histological morphology. To determine the distance migrated by the neuroblasts, the distance between the most rostral extent of the frontal cortex, as a reference point and the migration front was calculated based on the number of intervening sections and section thickness.To determine the number of NSCs and progenitors and to assess their proliferation 4 pairs of male Ts1Cje and disomic littermate controls were injected with 5-iodo-2′-deoxyuridine  twice daily 12 h apart for 7 days, left without treatment for 7 days and then injected with 5-chloro-2′-deoxyuridine  twice daily 12 h apart for 7 days and finally left without treatment for 7 days. The number of CldU positive, IdU positive and double positive cells were counted at five equally spaced rostrocaudal levels approximately 250 µm apart ranging from the most rostral section containing fimbria of the hippocampi to the section containing the fusion of the rhinal fissure. Stained sections were analysed microscopically and photographs were taken with a digital camera .To ensure that assays involving cell counts were accurate, the nuclei of cells throughout the SVZ from digital images at a magnification of 40× of 4 pairs of sex matched adult Ts1Cje and disomic littermates were compared. The longest diameter of 630 randomly selected nuclei from each genotype were measured. We found no significant difference in the mean length of the longest nuclei diameter between Ts1Cje and disomic controls (data not shown) and therefore numbers of labelled SVZ cell profiles could be compared For thymidine analogue incorporation studies, brains were perfusion-fixed in 4% PFA, postfixed in 4% PFA for 48 h, embedded in paraffin and coronally sectioned at 5 µm in preparation for staining.Immunohistochemistry was performed as described previously In Situ Apoptosis Detection Kit S7110 according to the manufacturer's instructions . The number of TUNEL positive cells was counted at 5 evenly spaced rostrocaudal intervals on sections adjacent to those assessed for thymidine analogue incorporation.Four pairs of sex matched  Ts1Cje and disomic littermate controls were assessed for the number of apoptotic cells in the SVZ using the ApopTag® Fluorescein 2O2 in methanol, and bound antibody was visualised using horseradish peroxidase-coupled streptavidin, diaminobenzidine and H2O2. Intervening wash steps were TBS with or without 0.5% Triton X-100. Photographs of stained tissue were taken using a low magnification stereomicroscope  and digital camera , PSA-NCAM-positive chains were traced and quantified using the NIH Image analysis software, ImageJ.Whole-mount immunohistochemistry was performed as described previously Stata®10  software and R language and environment were used to perform multifactorial ANOVAs and other statistical analyses (for further details see Text S1Supporting Text.(0.13 MB DOC)Click here for additional data file.Table S1DAVID data.(0.21 MB XLS)Click here for additional data file.Table S2Affymetrix and RT-qPCR data.(0.03 MB XLS)Click here for additional data file.


Mutations in the BRCA1/BRCA2 genes account for varying proportions of breast cancer families studied, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations in 17 families from Wales with two or more cases of breast cancer under age 50 and/or ovarian cancer. Eight out of 17 (47%) families had demonstrable mutations. Six out of 17 (35%) carried BRCA1 mutations and 2 out of 17 (12%) carried BRCA2 mutations. Two recurrent mutations in BRCA1 were identified, which appear to represent founder mutations in this population. These data support the existence of additional breast and ovarian cancer susceptibility genes.


A recent report suggests that the KLF6 gene encoding the Krüppel-like factor 6 protein is a frequently mutated, putative tumour suppressor gene in prostate cancer. The aims of the present study were to confirm these initial findings by determining the frequency of exon2 KLF6 mutations in a cohort of European prostate cancer patients, and to investigate whether there was evidence for mutational inactivation of both the KLF6 and TP53 tumour suppressor loci in some tumours. We examined 32 primary prostate tumours and three prostate tumour cell lines for mutations by PCR amplification and direct dideoxy sequencing (KLF6), and by oligonucleotide microarray (p53GeneChip™) analysis and dideoxy sequencing (TP53). Whereas TP53 mutations typical of prostate cancer were found at a frequency consistent with the literature, no KLF6 mutations were found in any of the tumour samples nor in the three prostate cancer cell lines. Amplification was performed in an MJB thermal cycler programmed for 40 cycles , and included an initial denaturation step at 95°C for 2 min, and a final elongation step of 5 min at 72°C. Polymerase chain reaction products were purified with Microcon 100 filters from Millipore  and used as template in dideoxy cycle sequencing reactions with fluorescent dye-labelled dideoxynucleotides and DNA polymerase from Applied Biosystems International . Four cycle sequencing reactions were performed, using primers KLF6Ex2FC, KLF6Ex2RC, KLF6Ex2R and KLF6Ex2FA  and used according to recommendations from the manufacturer. Chips were scanned with a Hewlett-Packard GeneArray scanner and signal intensities were evaluated with Affymetrix GeneChip software. The probe array methodology scans all coding exons and splice sites of the human p53 gene for point mutations with high efficiency and accuracy  suggest that KLF6 miscoding mutations in tumours of the prostate are uncommon. While the present report was under initial review, a new study on KLF6 alterations in prostate tumours appeared in the Additional studies are called for in order to clarify whether KLF6 is indeed a common mutational target in prostate carcinogenesis.


A number of randomized studies have been carried out in the UK and USA to determine the optimal radiotherapy dose schedule for advanced non-small-cell lung cancer (NSCLC). We have examined eight radiotherapy regimens from data taken from four randomized phase III studies carried out in the UK (1264 patients): 10 Gy single fraction; 17 Gy in two fractions over 8 days; 30 Gy in ten fractions over 14 days; 22.5 Gy in five fractions in 5 days; 27 Gy in six fractions over 11 days; 30 Gy in six fractions over 11 days; 36 Gy in 12 fractions over 16 days; and 39 Gy in 13 fractions over 17 days. We compared the clinical results in palliation, toxicity and survival with four regimens taken from one randomized study from the USA (365 patients): 40 Gy in 20 fractions over 4 weeks; 40 Gy 'split course' in ten fractions in 4 weeks; 50 Gy in 25 fractions over 5 weeks; and 60 Gy in 30 fractions over 6 weeks. Using the linear-quadratic (LQ) radiobiological model, we have calculated the radiobiological equivalent dose (BED) for acute-reacting tissues (BED10), late-reacting tissues (BED1.7) and tumour (BED25), and related the predicted response to the observed response in each tissue. There was a good correlation between the predicted response and the reported response in the case of late-reacting tissue toxicity and tumour response. The model confirmed that, in good performance status patients, a higher value for BED25 correlated with a higher degree of local control and survival and that radiotherapy regimens with a higher value for BED1.7 were associated with five cases of cord myelopathy, if the spinal cord was not shielded. In poor performance status patients the model suggested that the optimal regimen was a single fraction of 10 Gy because this resulted in an equivalent degree of symptom control as other regimens, needed only one hospital visit and was less likely to result in cord damage, thus, allowing for the possibility of retreatment at a later date.