Breast cancer is the most frequent cancer in women and consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics. In this study, we aimed to identify DNA methylation based biomarkers to distinguish patients with locally advanced breast cancer who may benefit from neoadjuvant doxorubicin treatment.ABCB1, ATM, BRCA1, CDH3, CDKN2A, CXCR4, ESR1, FBXW7, FOXC1, GSTP1, IGF2, HMLH1, PPP2R2B, and PTEN) in 75 well-described pre-treatment samples from locally advanced breast cancer and correlated the results to the available clinical and molecular parameters. Six normal breast tissues were used as controls and 163 unselected breast cancer cases were used to validate associations with histopathological and clinical parameters.We investigated quantitatively the methylation patterns in the promoter regions of 14 genes (FOXC1 promoter. Absence of methylation at the ABCB1 promoter correlated with progressive disease during doxorubicin treatment. Most importantly, the DNA methylation status at the promoters of GSTP1, FOXC1 and ABCB1 correlated with survival, whereby the combination of methylated genes improved the subdivision with respect to the survival of the patients. In multivariate analysis GSTP1 and FOXC1 methylation status proved to be independent prognostic markers associated with survival.Aberrant methylation was detected in 9 out of the 14 genes including the discovery of methylation at the ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness. FOXC1 proved to be general prognostic factor, while ABCB1 and GSTP1 might be predictive factors for the response to and efficacy of doxorubicin treatment. Pharmacoepigenetic effects such as the reported associations in this study provide molecular explanations for differential responses to chemotherapy and it might prove valuable to take the methylation status of selected genes into account for patient management and treatment decisions.Quantitative DNA methylation profiling is a powerful tool to identify molecular changes associated with specific phenotypes. Methylation at the  Breast cancer, the most frequent cancer in women, consists of a heterogeneous collection of diseases with distinct histopathological, genetic and epigenetic characteristics . ConventWhile the contribution of genetic factors to breast carcinogenesis has long been recognized, it has become evident that epigenetic changes leading to transcriptional silencing of tumour suppressor genes are an at least equally contributing mechanism. In tumours a global loss of DNA methylation (hypomethylation) of the genome is observed at early stages of breast carcinogenesis which proceeds with increasing malignancy . The oveThe number of genes that has been identified to be aberrantly methylated in breast cancer is rapidly growing. Thus, high-throughput DNA methylation mapping technologies have the potential to identify distinct methylation signatures correlating with specific clinical stages and subtypes, but also to reveal the large heterogeneity of DNA methylation patterns within a tumour subgroup -9. ConsiGSTP1 promoter in breast cancers and to affect patient survival [ABCB1 [ATM [BRCA1 [CDH3 [CDKN2A [ESR1 [GSTP1 [IGF2 [HMLH1 [PPP2R2B [PTEN [CXCR4 [FOXC1 [FBXW7 [Recently, we reported the haplotype structure to influence the level of DNA methylation of the survival . Here wel [ABCB1 , ATM [12M [BRCA1 , CDH3 [1A1 [CDH3 , CDKN2A  [CDKN2A , ESR1 [12A [ESR1 , GSTP1 [1 [GSTP1 , IGF2 [1P1 [IGF2 , HMLH1 [2 [HMLH1 , PPP2R2B[PPP2R2B , PTEN [12B [PTEN ) or otheN [CXCR4 ), 2. gen4 [FOXC1 ) and 3. 1 [FBXW7 ). In totABCB1 (40 CpGs), ATM (56 CpGs), BRCA1 (46 CpGs), CDH3 (35 CpGs), CDKN2A (30 CpGs), CXCR4 (19 CpGs), ESR1 (50 CpGs) FBXW7 (31 CpGs), FOXC1 (14 CpGs), GSTP1 (21 CpGs), IGF2 (16 CpGs), MLH1 (24 CpGs), PPP2R2B (51 CpGs), and PTEN (39 CpGs). The six normal samples were unmethylated for all analyzed regions except for the highly methylated upstream region of BRCA1, the differentially methylated region of the imprinted IGF2 and the promoter region of ESR1 , FOXC1 (50%), PPP2R2B (56% and 65%), HMLH1 (14%), PTEN (22% and 76%) and GSTP1 (65% and 83%). All samples were unmethylated for the transcription start site of BRCA1, ATM, CDH3, CXCR4 and FBXW7. 10% of the samples exhibited a significant hypomethylation in the far upstream region of the BRCA1 CpG island. Some methylation was found around the transcription start site for ESR1 but also within the normal breast samples. None of the genes displayed an age-dependent variation of DNA methylation at the analyzed loci.We analyzed promoter methylation at 432 CpGs in 14 genes giving rise to 37.000 epigenotypes Figure . The ana1 Figure . Three aABCB1, FOXC1, GSTP1, PPP2R2B, PTEN promoters identifying thus strongly correlated methylation events on different chromosomes  showed abnormal methylation at one locus, 8 (11%) at two loci, 4 (5%) at three, 14 (19%) at four and five loci, respectively, 16 (21%) at six, 9 (12%) at seven, 5 (7%) at eight loci and two tumours (3%) displayed aberrant methylation at nine loci. On average, five loci were thus aberrantly methylated in any sample. Methylation events at the different loci were not randomly distributed and independent from each other Figure . As expes Figure . MethylaABCB1, FOXC1, PPP2R2B, and GSTP1 in both the basal-like and normal-like tumours, while IGF2, MLH1 and PTEN were hypomethylated in the basal-like tumours but not in the normal-like tumours. When analyzing the correlation between the expression level and the DNA methylation status of individual genes, genes with methylated promoters were almost exclusively not expressed, while unmethylated genes could be expressed as well as not be expressed weakening the correlation. The only significant correlation was obtained for GSTP1 . Because of their association with survival (see below) we analyzed the expression levels of GSTP1, ABCB1 and FOXC1 by qRT-PCR . Consequently a significant negative correlation between expression as measured by TaqMan and methylation was found for GSTP1 , while expression and methylation for FOXC1 and ABCB1 were not significantly correlated . Highly expressing genes were unmethylated for the respective promoter region of GSTP1 and FOXC1 and methylated promoters correlated with silenced expression. The weak correlation between expression and DNA methylation for FOXC1 was due to the fact that the gene was already silenced in most tumours independent of its methylation status. Four samples were methylated for ABCB1 but displayed high expression. This might be due to alternative usage of an upstream promoter [The observed methylation patterns were compared to the tumour subclasses as defined by microarray expression profiling . Basal-lpromoter  that is PPP2R2B in the pre-treatment sample was significantly associated with tumour grade (p = 0.019), whereby high-grade tumours were more frequently unmethylated than grade 1 and 2 tumours in the discovery cohort. The same was observed in the validation cohort of unselected breast cancers (p = 0.008). No association between methylation and tumour size was found. Estrogen receptor status positivity was associated with the presence and increased extent of methylation at the PPP2R2B promoter in both the discovery (p = 0.004) and the validation cohort (p = 0.006). Samples unmethylated for ABCB1 and those with increased levels of methylation in the differentially methylated region 2 of IGF2 had more often overexpression of the ERBB2 oncogene , previously analyzed by immunohistochemistry [Methylation was analyzed in the discovery and validation cohorts both as a categorical variable, i.e. the presence/absence of methylation at the respective promoter in association with the tumour characteristics, and as a quantitative variable investigating potential associations between the extent or the distribution of DNA methylation values and the analyzed clinical and molecular parameters  as well as in the validation cohort . Tumours unmethylated for the middle region of the ABCB1 CpG island were associated with mutations in the loop domains L2/L3 (p = 0.022), a region that has previously been shown to be associated with lack of response to doxorubicin based treatment. PPP2R2B did not show any differential degree of methylation in function of the type of TP53 mutation.We compared the observed DNA methylation profiles with the ABCB1, BRCA1, CDKN2A, FOXC1, GSTP1, IGF2, PPP2R2B and PTEN) within the discovery cohort were tested for association with survival by a logrank test. Breast cancer specific survival was significantly improved in patients with methylated promoters for ABCB1, GSTP1 and FOXC1 . Consistently, absence of methylation  in the patient cohort treated with doxorubicin. In the validation cohort treated with different regimens, a significant difference in survival between methylated and unmethylated samples was confirmed for FOXC1 (p = 0.024) with patients unmethylated for the promoter region having again worse survival. Methylation of GSTP1 did not condition improved survival in the validation cohort of patients (p = 0.331). Similarly, only a trend for improved survival was observed for the methylation status of ABCB1 (p = 0.070). The findings for GSTP1 and ABCB1 might indicate a treatment specific effect on survivalThe eight genes displaying variable DNA methylation patterns in a significant number of tumours (TP53 mutation status (p = 0.001), grade (p = 0.001) and the estrogen receptor status (p = 0.002) could slightly better differentiate two survival groups in the analyzed sample collection when compared to the methylation status of the single genes (ABCB1 (p = 0.004), GSTP1 (p = 0.004) and FOXC1 (p = 0.021)), while separation based on the progesterone receptor status and amplification of ERBB2 or TOP2A did not reach statistical significance. However, combination of two of the discovered DNA methylation markers further improved the distinction between doxorubicin treated patients having two, one or none of the genes methylated. No statistical difference on survival in function of the gene was found when comparing patients that had one of the two genes methylated and these were therefore combined for analysis. The best two-gene methylation pair comprised GSTP1 and FOXC1 (p = 8·10-5), followed by GSTP1 and ABCB1 (p = 0.001) and ABCB1 and FOXC1 (p = 0.01). Patients with all three genes methylated (n = 20) had an improved survival compared to patients with all three genes unmethylated . However, the separation lost its statistical significance when patients with mixed methylation patterns for all three genes were added to the analysis. We investigated if expression could be used as an alternative molecular measure to DNA methylation and divided samples in high versus low expression based on the mean expression values. The expression of GSTP1 was significantly associated with survival with patients with low levels of expression having as expected an improved survival (p = 0.048). FOXC1 (p = 0.247) and ABCB1 (p = 0.181) were not significant but again showed improved survival for low expressing patients. When combining DNA methylation and expression, patients with an unmethylated GSTP1 promoter and expressed gene had poorer survival compared to patients with a methylated promoter that did not express GSTP1 (p = 0.047). The same correlation was observed for FOXC1 (p = 0.045) and ABCB1 (p = 0.022).Survival analysis in the doxorubicin treated cohort based on the logrank test indicated that To identify significant parameters contributing to the observed differences in survival, Cox regression analysis was performed. The hazard ratio for each of the contributing factors was estimated separately  or modelled together .ABCB1, FOXC1 and GSTP1 as significant predictors of overall survival. Estrogen receptor status as well as TP53 status and grade were also significant predictors of survival in univariate analysis  and FOXC1  showed a higher risk of dying from breast cancer compared with patients methylated for the same genes  while increasing the HR for FOXC1 . The HR for the other parameters remained unchanged.Univariate analysis identified the methylation status of FOXC1 and GSTP1, stage, grade and estrogen receptor status  remained significant together with high grade (p = 0.002) and ER status (p = 0.001) (data not shown).In order to identify the model with the minimum number of covariates that fitted best the experimental data, we used the Akaike information criterion. The best model with a reduced number of covariates explaining survival included the methylation status of In the presented study we analyzed the methylation patterns in the promoter regions of fourteen genes in 75 pre-treatment samples from locally advanced breast cancers, six normal tissues and six widely used cell lines. Aberrant methylation events were detected in eleven out of the fourteen genes investigated. Discussion of the negative results can be found in the Additional File CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A) [LINE1/ALU) as a surrogate for genome-wide methylation levels, basal-like breast tumours are characterized by an overall loss of methylation . The observed hypomethylation in the far upstream region of BRCA1  . However . Howevestricted .ESR1), we studied its promoter methylation in normal samples as well as in a subset of the tumours. It has previously been shown that its degree of DNA methylation did not correlate well with hormone receptor status [Since the tumour sub-classification based on gene expression is driven to a significant extent by expression of the estrogen receptor (r status . Our datr status .PPP2R2B on 5q31-q32 encodes the regulatory subunit of the protein phosphates 2A complex (PP2A) and has been proposed as a tumour suppressor gene candidate due to its negative control of cell growth and the high frequency of LOH in breast cancers [TP53 mutation status is reported here for the first time and might provide an alternative molecular mechanism for gene inactivation, as also the LOH has previously been associated with TP53 mutations [Another studied gene, cancers . An assoutations . The preutations  was alsoABCB1 CpG island to be associated with ERBB2 amplification, TP53 mutation status, and response to doxorubicin treatment and overall survival in a doxorubicin-exposed cohort of primary breast cancers. Although the number of patients with progressive disease in the current study is limited and requires confirmation in other patient cohorts treated with anthracycline based treatment, there is good evidence that methylation of ABCB1 plays an important role in the response to doxorubicin. Lack of methylation in the central part of the ABCB1 CpG island was found to be associated with the TP53 mutation status and in particular with mutations in the L2/L3 DNA binding domain which have previously been associated with lack of response to treatment in the same patient cohort [TP53 and ABCB1 [ABCB1 has been shown to reduce intracellular doxorubicin concentration in cell cultures [in vivo studies has been conflicting [ABCB1 expression the mice leads to the development of doxorubicin resistance that might be reversed by ABCB1 inhibitors such as tariquidar [Our study is the first to show DNA methylation of the t cohort . This find ABCB1 . How muccultures  and re-ecultures . Althougflicting , a recenriquidar .TOP2A) gene significantly improves the outcome of anthracycline based adjuvant chemotherapy [TOP2A and ERBB2 are co-amplified in our dataset  warranting further investigation to explore the interaction between ABCB1 methylation status and TOP2A/ERBB2 amplification and how the combined effect of these proteins contributes to the drug resistance observed in anthracycline treatment regimens.A possible link between ERBB2 and ABCB1 expression has been observed in a multidrug resistant MCF-7 cell line . The ampotherapy ,38. InteIGF2 exerts its action on cellular growth through the insulin-like growth factor 1 receptor which interferes with anti-ERBB2 treatment through Akt signalling [IGF2 have been associated with overexpression of IGF2 [IGF2 is specifically observed in ERBB2 positive breast cancers providing a new potential mechanistic link between IGF1R expression and ERBB2 status via IGF2 methylation status.gnalling . In muri of IGF2 , which iGSTP1 and FOXC1 methylation status as independent prognostic markers for breast cancer survival in a uniform patient cohort receiving neoadjuvant doxorubicin monotherapy prior to surgery and five years of tamoxifen for all ER positive patients according to a clinical study protocol [FOXC1 methylation status might be a general prognostic marker as it is able to separate patients in good and poor survival groups in the doxorubicin treated as well as in validation cohort while GSTP1 and ABCB1 methylation status might be a predictive marker for doxorubicin monotherapy as the methylation status of these genes were not able to separate patients into survival groups in the validation cohort [GSTP1 methylation decreased when the operation status was taken into account indicating that those tumours that increased further or at least did not regress during neoadjuvant treatment were more often unmethylated for GSTP1 while FOXC1 hazard ratio increased as would be expected for a treatment independent effect. ABCB1 methylation status proved to be a marker for survival in the discovery cohort although it was not independent of other known markers in a multivariate model. The association with survival was less significant when the expression status instead of the DNA methylation status was analysed due to a strong correlation between DNA methylation and expression for GSTP1 only.We identify here the protocol . FOXC1 mn cohort . This isFOXC1, a member of the forkhead protein family, many members of which are involved in the development and progression of cancer [FOXC1 have recently been reported in a candidate re-sequencing approach of breast tumours [FOXC1 was found to be specifically hypomethylated and highly expressed in CD44+ compared to CD24+ stem cell progenitors, but no data correlating survival to the methylation patterns was presented [FOXC2 gene has been found to promote tumour metastasis and invasiveness [We confirm here a very recent report on the presence and extent of DNA methylation in the promoter of f cancer . Mutatio tumours  and FOXCresented . The oveglutathione-S-transferase 1 (GSTP1) is a well established biomarker for hormone dependent cancers. Low activity of GSTP1 resulting from promoter hypermethylation may increase the effective therapeutic dose of the pharmacological agent due to lower conjugation and inactivation of the drug leading to increased survival. In support of this hypothesis it has been shown that GSTP1 expression correlates with doxorubicin resistance in breast cancer cell lines [CpG hypermethylation of the promoter region of the ll lines . The obsll lines ,47.ABCB1 or GSTP1 promoter improved overall survival probably due to prolonged availability and activity of the drug in the cell while FOXC1 methylation might be a protective factor against tumour invasiveness.Methylation at the FOXC1 methylation status might be a widely applicable prognostic factor for breast cancer patients while the methylation status of ABCB1 and GSTP1 might be a predictive factor for doxorubicin and perhaps anthracycline treatment in general. However, further studies are necessary to confirm the predictive value of these markers requiring additional patient cohorts treated with a doxorubicin/anthracycline based monotherapy. Valuable time for treatment might be gained and the serious side-effects of a doxorubicin based regimen might be avoided taking the methylation status for treatment decisions into account. As the analyzed cohort consists of locally advanced primary tumours, it will be interesting to investigate the DNA methylation profiles also in T1/T2 and early stage breast cancer samples. Despite similar RNA expression profiles [The profiles , some biprofiles . Additio2) scheduled for 16 weeks. Patients with an operable tumour (n = 60) after neoadjuvant treatment had surgery followed by radiotherapy immediately after termination of the neoadjuvant chemotherapy. Eight patients had to be given radiotherapy prior to surgery due to local tumour extension, and seven patients were not eligible for surgery and were treated on an individual basis. Women with estrogen and/or progesterone positive tumours were all treated with tamoxifen (30 mg daily for 5 years). The main clinical characteristics of the analyzed 75 samples are given in Table Locally advanced breast cancer patients, admitted to the Haukeland University Hospital in Bergen (Norway) between 1991 and 1998 were part of a prospective study evaluating predictive factors for response to doxorubicin (n = 94). Tumour DNA was available in sufficient quantity to perform methylation analyses from 75 of the patients. Tissue was obtained by an incisional biopsy prior to therapy and was immediately snap-frozen (liquid nitrogen in the theater) as previously reported . DNA wasTP53 mutation status (n = 162) were available and used for validation. Follow-up/survival data was available for 87 of the patients. Tumour DNA extraction, bisulphite treatment and pyrosequencing analyses was performed using the same procedures as for the discovery dataset.163 random, unselected breast cancer samples were used for the validation of the observed associations with clinicopathological factors. Clinical and molecular parameters such as histological grade (n = 162), Estrogen receptor status (n = 158) and DNA samples from normal breast tissue were included as control samples for methylation analysis. Normal breast tissue (n = 6) was obtained from women who underwent a biopsy of the mammary gland because of mammographic abnormalities, but for which histology confirmed the presence of only normal tissue.The sample set was completed by immortalized human mammary epithelial cells (HMEC) and five widely used breast cancer cell lines .TP53 were analyzed in both the discovery and the validation cohorts by temporal temperature gradient electrophoresis (TTGE) followed by Sanger sequencing as previously described with primers covering regions (exons and introns) from exon 2-11 [Mutations in xon 2-11 ,49. 50 oxon 2-11 , and a s2, 100 μM dNTPs and 2.0 U HotStar Taq polymerase  in a 25 μl volume. The PCR program consisted of a denaturing step of 15 min at 95°C followed by 50 cycles of 30 s at 95°C, 30 s at the respective annealing temperature . TaqMan assays were all purchased from Applied Biosystems: Hs 00943351_g1 (GSTP1), Hs00184500_m1 (ABCB1) and Hs00559473_s1 (FOXC1). Human Breast Total RNA  was used to generate standard curves. PMM1 (Hs00963626_m1) was used as endogenous control and the relative gene expression levels were determined using the standard curve method and normalized to PMM1.50 of the tumours have previously been analyzed for gene expression using genome wide cDNA microarrays . For qua2 tests. Samples were scored as methylated when the methylation degree exceeded the average methylation degree of the normal samples by two times the standard deviation of the normal samples and had at least a methylation degree of 5% (detection limit of the technology). Odds ratio and 95% confidence intervals were calculated. Differences in the distribution of methylation were assessed by the non-parametric Mann-Whitney or the Kruskal-Wallis test. Correlation between the methylation status of the different genes was calculated by the non-parametric Kendall's tau test. Pearson's coefficients were used to study the correlation between methylation and expression levels. All calculations were performed using Statistical Package for Science version 15.0. The Cox proportional hazards model was used to evaluate the effect sizes (given as hazard ratios), 95% Confidence intervals (CI), regression coefficients and statistical significance of known clinicopathological features as well as the methylation status of selected genes. All covariates were treated as categorical variables. To investigate the relationship between multiple explanatory factors and survival, we used the Akaike information criterion (AIC) [TP53 mutation status as well as methylation of ABCB1, FOXC1 and GSTP1 respectively, were considered as covariates to the model. With L being the likelihood function of the model and k indicating the number of parameters of the model, the Akaike information criterion (AIC) is calculated by: AIC = -2log L+2k.Differences in the presence of methylation were determined by a two-sided Fisher's test and χon (AIC) . AIC evaThe authors declare that they have no competing interests.ED and JAR performed laboratory experiments and data analyses and participated in writing of the manuscript. HS was involved in the statistical analyses. PEL, SG and TA were responsible for the doxorubicin treated patient cohort and clinical database management. ALBD and VNK were responsible for the validation cohorts and IB for the control samples. IGG, ALBD, VNK and JT initiated and designed the study. JT wrote the manuscript and VNK, PEL and ALBD participated in writing the manuscript. All authors have read and approved the final manuscript.GSTP1, FOXC1 and ABCB1Correlation between DNA methylation and RNA expression for . Scatter plots showing the correlation between DNA methylation and RNA expression as measured by TaqMan for GSTP1, FOXC1 and ABCB1.Click here for fileAssociations of the presence/absence and degree of methylation and the clinical and molecular parameters. Associations of the presence/absence and degree of methylation and the clinical and molecular parameters of the samples by Fisher's exact test  or χ2 analysis  with odds ratio (OR) and 95% Confidence interval (CI) and their respective p-value in the validation cohort. Statistical significance of the differences in the distribution of the degree of methylation is assessed by the non-parametric Mann-Whitney and Kruskal-Wallis test. Samples are called methylated if the methylation degree exceeded 5% and the average methylation degree of the healthy tissue samples plus at least two times the standard deviation of the healthy tissues.Click here for fileBest models to fit the observed survival data. A) Best models with a varying number of covariates to fit the observed survival data. B) Multivariate analysis of the best model with the minimum number of covariates. The presented model is the only one where all covariates are significant in multivariate analysis.Click here for fileSupplementary discussion. Discussion of negative results.Click here for filePCR and pyrosequencing primers. Sequences of primers used for amplification and pyrosequencing reactions, Genbank accession numbers and nucleotides (Nt) corresponding to the amplified fragments as well as the annealing temperatures for the respective PCR amplifications. CpGs are numbered in the order of appearance from the 5' end of an amplification product. Y = pyrimidine.Click here for file


Hydralazine can substantially decrease blood flow and increase hypoxia in transplanted tumours. Previous indirect studies have suggested that hydralazine does not induce such effects in spontaneous tumours. We have now directly investigated the ability of hydralazine to increase hypoxia in both transplanted and spontaneous murine tumours by measuring tumour oxygen partial pressure (pO2) distributions using an Eppendorf oxygen electrode. Spontaneous tumours arose at different sites in CDF1 mice, while transplanted tumours were produced by implanting a C3H mouse mammary carcinoma on the backs of the same mouse strain. Measurements of pO2 were made in anaesthetised mice immediately before and 45 min after an intravenous injection of 5 mg kg-1 hydralazine. In the transplanted tumours hydralazine significantly decreased tumour oxygenation, such that the percentage of pO2 values < or = 5 mmHg increased from 45% to 87%, and median pO2 decreased from 5 to 3 mmHg. Similar significant changes were induced by hydralazine in the spontaneous tumours, the percentage of pO2 values < or = 5 mmHg increasing from 60% to 94% while the median pO2 values decreased from 8 to 2 mmHg. These results clearly show that there is no difference in the response of transplanted and spontaneous mouse tumours to hydralazine.


We analysed 26 metastases from 25 patients with sporadic cutaneous malignant melanoma for alterations in the CDKN2 gene by a combined polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP)/nucleotide sequencing approach. Eleven alterations  were concordantly and independently detected by both SSCP and nucleotide sequence analysis. Two of the exon 2 changes and the five changes in the non-coding exon 3 region are likely to represent natural polymorphism. Four (15%) of 26 metastases thus had CDKN2 mutations and belonged to 3 (12%) of 25 patients. Semi-quantitative PCR furthermore revealed no sign of homozygous deletions of the CDKN2 exon 2 region. The results support an involvement of the CDKN2 product in the development of a subgroup of sporadic melanomas and encourage the search for alterations in additional genes of the 9p21 region.


Epigenetic modifications associated with imprinting regulation can be compared to those associated with the more canonical developmental regulation, important for processes such as differentiation and tissue specificity. Here we test the hypothesis that these two mechanisms are associated with different histone modification enrichment patterns.The field of epigenetics is developing rapidly, however we are only beginning to comprehend the complexity of its influence on gene regulation. Using genomic imprinting as a model we examine epigenetic profiles associated with different forms of gene regulation. Imprinting refers to the expression of a gene from only one of the chromosome homologues in a parental-origin-specific manner. This is dependent on heritable germline epigenetic control at a Using high-throughput data extraction with subsequent analysis, we have found that particular histone modifications are more likely to be associated with either imprinting repression or developmental repression of imprinted genes. H3K9me3 and H4K20me3 are together enriched at imprinted genes with differentially methylated promoters and do not show a correlation with developmental regulation. H3K27me3 and H3K4me3, however, are more often associated with developmental regulation. We find that imprinted genes are subject to developmental regulation through bivalency with H3K4me3 and H3K27me3 enrichment on the same allele. Furthermore, a specific tri-mark signature comprising H3K4me3, H3K9me3 and H4K20me3 has been identified at all imprinting control regions.A large amount of data is produced from whole-genome expression and epigenetic profiling studies of cellular material. We have shown that such publicly available data can be mined and analysed in order to generate novel findings for categories of genes or regulatory elements. Comparing two types of gene regulation, imprinting and developmental, our results suggest that different histone modifications associate with these distinct processes. This form of analysis is therefore a useful tool to elucidate the complex epigenetic code associated with genome function and to determine the underlying features conferring epigenetic states. Epigenetic mechanisms play an important role in the control of gene expression. Modification to the packaging of DNA is believed to allow a more open or closed structure and influences association of the transcriptional machinery with the genetic material. The most characterised examples of epigenetic mechanisms to date in mammalian cells include DNA methylation of cytosine and post-translational modifications to the core histone proteins of the nucleosome (reviewed in ), thoughThe transcriptome of a cell is tightly regulated by epigenetic mechanisms to allow correct gene expression patterns at appropriate time points. The dynamic changes in gene expression required during the proliferation, differentiation and commitment of specific cell types are associated with specific epigenetic alterations. In order to simplify the description of this type of regulation we shall hereafter refer to this as developmental regulation.An additional mechanism of gene regulation is that of genomic imprinting, an epigenetic process affecting less than 1% of genes in the mammalian genome. An imprinted gene refers to a gene in which expression occurs solely or predominantly from only one of the parental chromosome homologues (reviewed in ). EitherBoth DNA methylation and post-translational histone modifications have been found to be enriched to a greater degree on one chromosome compared to its homologue at a number of imprinted loci in the mouse and human . DiffMany studies of specific loci have described differential enrichment of particular histone modifications between the two parental chromosomes at some ICRs and imprinted gene promoters or transcription start sites (TSSs). These marks include histone H3 acetylation, H4 acetylation, H3 dimethylation at lysine 4 (H3K4me2) and H3K4me3, which are found preferentially enriched on the unmethylated chromosome or normally active allele in comparison with its homologous counterpart, and H3K27me2, H3K27me3, H3K9me2 and H3K9me3 preferentially enriched on the methylated chromosome or inactive allele -42. H4K2Key to the work presented here, the active alleles of imprinted genes are also developmentally regulated. Imprinted genes that are developmentally expressed have only the 'normally active' allele expressed, whereas developmentally repressed imprinted genes have the 'normally active' allele repressed resulting in two repressed alleles. Very few studies have assessed the nature of epigenetic marks at developmentally repressed imprinted genes. We have used imprinted genes as a model system to compare epigenetic marks associated with the control of imprinting to those associated with developmental regulation to test the hypothesis that distinct epigenetic modifications are employed for these two mechanisms.An increasing number of groups are describing the epigenetic characteristics of various cell types across the whole genome in both mouse and human and these studies produce a large amount of publicly accessible data  and in more differentiated cell types . Secondly, 35% of imprinted genes are enriched with both H3K4me3 and H3K27me3 compared to 16% of all genes in the mouse genome . We discuss the H3K4me3/H3K9me3 profile below; whether the H3K4me3/H3K27me3 profile reflects developmental or imprinting regulation is addressed subsequently.Figure et al. [not to act as an ICR (Gnas Exon1A) does not exhibit this specific tri-mark profile. These findings imply that the presence of H3K4me3, H3K9me3 and H4K20me3 is a true epigenetic signature of ICRs.As H3K9me3 enrichment has previously been linked to H4K20me3 , we alsoet al.  using a n = 26) have a significantly different epigenetic profile for these four marks to imprinted genes without a promoter DMR (n = 22) in mouse ESCs .In order to assess the histone modification enrichment profiles at both germline and somatic DMRs we have compared imprinted genes with and without promoter DMRs for enrichment of H3K9me3, H4K20me3, H3K4me3 and H3K27me3 in mouse ESCs using the same source data as above , we find that all are germline DMRs rather than somatic DMRs indicating that the combined presence of H3K9me3 and H4K20me3 only occurs in the presence of H3K4me3 specifically at germline DMRs, defining the identified tri-mark profile.As seen from Figure et al. [Cd81, Osbpl5 and Tssc4 are biallelically expressed in ESCs [The 'normally active' allele of an imprinted gene can be developmentally regulated; we have therefore sorted mouse imprinted genes by expression status in ESCs using microarray data from Mikkelsen et al. . This mi in ESCs ). By intnot associated with developmental regulation, but rather with imprinting. Additional file If a particular modification is enriched to an equal degree at developmentally expressed and repressed imprinted genes, it is likely that this modification is without promoter DMRs that were assessed, none are enriched for H3K9me3 or H4K20me3 . Together, our results strongly suggest that H3K9me3 and H4K20me3 are not associated with developmental repression of imprinted genes in mouse ESCs, but rather with imprinting control.Histone modification enrichment at developmentally expressed or repressed imprinted genes can be assessed alongside the promoter DMR status. Of all developmentally repressed genes The results depicted in Additional file P < 0.01 for [P < 0.05 for [Our results in Figure 0.01 for  and 30% 0.05 for ; data no0.05 for . This maet al. [P < 0.0005). When comparing imprinted genes to all genes in the mouse genome enriched with both H3K4me3 and H3K27me3 a significant difference is observed for both NPCs and MEFs . A reduction in combined H3K4me3 and H3K27me3, but not complete removal, is observed between ESCs and the more differentiated cell types, suggestive of resolution of bivalency [41% in ESCs compared to 7% in NPCs  and 22% in MEFs   and in combination (H3K4me3 and H3K27me3) with regard to developmental expression status Figure . This imOf the few genes that possess both marks in NPCs and MEFs, we observe a somewhat higher number of developmentally repressed imprinted genes with coenrichment than expressed genes  across all cell types is significantly greater at developmentally expressed than repressed imprinted genes . For H3K27me3, enrichment is not observed at all imprinted genes that do not possess a promoter DMR, but rather at only 55% of these genes in ESCs . Furthermore, when assessing only imprinted genes that do not have a promoter DMR and are developmentally expressed in ESCs , H3K27me3 is enriched only 20% of the time ; H3K27me3 enrichment is found at 30% of developmentally expressed imprinted genes that do possess a promoter DMR (n = 10). This implies that H3K27me3 does not commonly repress the inactive allele in the absence of differential promoter methylation in mouse ESCs and that enrichment is not generally dependent on promoter DMR status. No developmentally expressed genes are enriched with H3K27me3 in NPCs; in MEFs an equal level of enrichment is observed at imprinted genes with promoter DMRs  and without promoter DMRs  of those that are developmentally expressed.The presence of H3K4me3 and H3K27me3 together at 25% and 20% of developmentally The comparative characterisation of histone modification profiles, DMR status and expression profiles for all confirmed imprinted genes provides a valuable resource for those interested in the role of these epigenetic marks in different classes of gene expression and repression and in the regulation of genomic imprinting in particular. Additionally, patterns of histone modification enrichment common to imprinted genes have been identified and highlight differences in the epigenetic profiles of these particular genes compared to other genes. In our analyses, we have been able to infer imprinted rather than developmental repression through the identification of modifications always associated with a germline DMR regardless of the developmental expression status of the gene. Histone modification enrichment profiles found to associate with the developmental expression state are acknowledged to be involved in the developmental regulation of imprinted genes.The repressive marks H3K9me3 and H4K20me3 are unlikely to play a prominent role in developmental repression of imprinted genes. This is apparent through the finding that H3K9me3 and H4K20me3 are not enriched at developmentally repressed genes any more often than at expressed genes in mouse ESCs . The presence of H3K27me3 at up to a quarter of developmentally expressed imprinted genes in both mouse ESCs and MEFs Figure  implies Some inactive imprinted alleles are associated with DNA methylation and others are not. As we have identified H3K27me3 to be involved in imprinting regulation at some genes, we investigated whether this histone mark might play a role in repressing the inactive allele in the absence of DNA methylation, as suggested by others . SeveralIgf2r/Airn imprinted locus. In mouse fibroblasts where Slc22a2 and Slc22a3 are developmentally  repressed, widespread H3K27me3 was enriched on both parental chromosomes [et al. [Grb10. They also established that H3K27me3 marks both chromosomes in neurons where this transcript is developmentally repressed. At the brain-type promoter of Grb10, Sanz et al. [The histone mark H3K27me3 is more often found at developmentally repressed than at expressed imprinted genes in mouse ESCs . Studieet al. [Our analyses show that all ICRs display enrichment of H3K9me3 and H4K20me3 in combination with H3K4me3, constituting an epigenetic signature. Previous reports have identified enrichment of H3K27me3 on the methylated chromosome of several ICRs ,25,35,41et al.  to identet al. ,35,36,38et al. ), which The analyses performed here provide insight into the association of histone modifications with different forms of genomic regulation. In all the cell types assessed, H3K27me3 is frequently found associated with developmental repression of imprinted genes, whereas H3K9me3 and H4K20me3 correlate with repression of the inactive allele of imprinted genes possessing promoter DMRs, rather than with spatiotemporally controlled developmental repression in ESCs. H3K4me3 is found associated with developmental regulation. We further propose that bivalent domains act to developmentally repress imprinted genes, as they do for other genes in the genome. In addition, we have identified an H3K4me3, H3K9me3 and H4K20me3 tri-mark signature at all ICRs without exception in mouse ESCs. Figure It is demonstrated here that high-throughput studies can be mined and analysed for smaller functional categories to generate novel insights into different forms of epigenetic regulation. Through genome-wide analysis, we have shown that alleles inactivated by genomic imprinting can be distinguished epigenetically from developmentally repressed alleles. These findings are consistent with published allele-specific data on individual imprinted loci  status: this worksheet details the promoter DMR status of imprinted genes used for analysis of histone modification profiles at genes with and without promoter DMRs, including references; Chromatin and expression states: this worksheet provides data extracted from six high-throughput studies for all available imprinted genes. Notably, not all imprinted genes were present in the source data files, most likely due to the high proportion of imprinted genes currently holding a predicted status in gene reference databases. If a gene was not present in one source data file the output is given as 'Gene not present'. Genes imprinted only in mice, or where imprinting status is not confirmed in humans, or where no orthologous human gene exists (*), are excluded from further human analyses. The same applies for human-specific imprinted genes (†), which are excluded from all mouse analyses. Isoform-dependent imprinted genes (‡) are not included in any further analyses. hESC, human embryonic stem cells; mESC, mouse embryonic stem cells; REH, human pro-B cells; mNPC, mouse neural progenitor cells; MEF, mouse embryonic fibroblasts; TSS, transcription start site; HCP, high CpG promoter; ICP, intermediate CpG promoter; LCP, low CpG promoter; MPSS, massively parallel signature sequencing.Click here for fileHistone modification enrichment at developmentally expressed and repressed imprinted genes in embryonic stem cells (ESCs). Enrichment of H3K4me3, H3K27me3, H3K9me3 and H4K20me3 at transcription start sites of developmentally expressed and repressed imprinted genes were assessed in mouse ESCs using high-throughput enrichment and expression source data from Mikkelsen et al. [P = 0.525).n et al. . The preClick here for fileAllele-specific histone modification enrichment at imprinted genes. In order to compare the results of our genome-wide analyses with allele-specific data, we have characterised previously published histone modification enrichment profiles involved in imprinting and developmental repression at imprinted genes. This data supports our findings and provides independent validation of our results.Click here for file


G21/NPRL2, RASSF1A, RASSF1C, SEMA3B, SEMA3F, RBSP3) were shown to inhibit tumour cell growth both in vitro and in vivo.We identified two 3p21.3 regions (LUCA and AP20) as most frequently affected in lung, breast and other carcinomas and reported their fine physical and gene maps. It is becoming increasingly clear that each of these two regions contains several TSGs. Until now TSGs which were isolated from AP20 and LUCA regions . Tumours grew in eight mice inoculated with HYAL1. Ectopic HYAL1 was deleted in all of them. HYAL2 was inoculated into 12 mice and only four tumours were obtained. In 3 of them the gene was deleted. In one tumour it was present but not expressed. As expected for tumour suppressor genes HYAL1 and HYAL2 were down-expressed in 15 fresh lung squamous cell carcinomas (100%) and clear cell RCC tumours (60–67%).The effect of expression in vivo without noticeable effect on growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Targeting the microenvironment of cancer cells is one of the most promising venues of cancer therapeutics. As major hyaluronidases in human cells, HYAL1 and HYAL2 may control intercellular interactions and microenvironment of tumour cells providing excellent targets for cancer treatment.The results suggest that the expression of either gene has led to inhibition of tumour growth  We have performed a deletion survey of 3p on more than 400 lung, renal, breast, cervical and ovarian carcinomas using a defined set of markers, combining conventional LOH (loss of heterozygosity) with quantitative real-time PCR (qPCR), comparative genomic and NotI microarrays hybridisations in vivo and in vitro. When the transfectants were passaged as tumours in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumours tested. In contrast, a non-functional mutant RB gene was maintained in all 10 tumours studied. In similar experiments with wt P53, the exogenous P53 gene was maintained and expressed in all 6 tumours tested, but in a mutated form. On the basis of these experiments we have developed the gene inactivation test (GIT) for a functional definition of TSG. It is based on the comparison of cell growth in vitro and tumour growth in vivo when the gene is/is not expressed. The main idea of the test is that a gene inhibiting growth of tumour cells should be inactivated in growing tumours by genetic or epigenetic mechanisms.The definition of a TSG is based on the demonstration of its regular inactivation by mutation or epigenetic silencing in tumour samples. It is also important to obtain supportive evidence from functional studies. We have previously found  non-random losses of human 3p21-p22 fragments from mouse-human microcell hybrids following progressive growth in SCID mice FUS1, SEMA3B, G21/NPRL2, RASSF1A, RASSF1C, RBSP3 (genes from AP20 and LUCA regions) and other TSGs inhibit tumour cell growth both in vitro and in vivoUsing GIT and growth analysis under cultural conditions we have shown that TCEA1, MLH1, RHOA, 3PK, PL6, 101F6, BLU, TGFBR2) did not show any effect in the tested cell lines However other genes from 3p21.3 (e.g. HYAL1 and HYAL2).Here we describe the functional analysis of two additional genes from the 3p21.3 LUCA region, hyaluronidases -1 and -2  is located in the LUCA region. It contains 6 exons producing a 2.6 kb mRNA (coding for 436 aa protein). It is well expressed in all analysed normal human tissues including lung and kidney. It was not expressed in 18 out of 20 lung cancer cell lines The HYAL2  contains 4 exons producing a 2 kb mRNA (that encode 473 aa). It is well expressed in all analysed human tissues including lung, kidney and many lung cancer cell lines. The protein is attached to the membrane by the glycosylphosphatidyl-inositol-anchor (GPI-anchor) HYAL2 protein was identified as a receptor for the sheep lung cancer retrovirus, JSRV, and a sequestration mechanism inactivating HYAL2 protein was demonstrated. The env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequesters the HYAL2 protein. The absence of HYAL2  leads to ligand-independent activation of the RON receptor tyrosine kinase and its downstream AKT and MAPK signaling pathways The These two genes contribute to intracellular and extracellular catabolism of hyaluronic acid (HA) in a CD44-dependent manner HYAL1 and HYAL2 can have a great importance not only for better understanding of HA catabolism and human carcinogenesis but could provide therapeutic targets for cancer treatment. In this study we have found that the expression of either gene suppressed tumour growth in vivo but not in vitro. These findings are consistent with earlier somatic hybrid studies by Henry Harris, George Klein and Francis Wiener, showing that somatic hybridisation of normal and malignant cells can suppress tumorigenicity in vivo but not cell growth in vitroin vivo suppressive effects have not been clarified, but at least part of them may have acted at the level of tumour-host interactions. The finding of a similar phenomenon with the two genes involved in the present study is a step towards the analysis of this important phenomenon.Thus analysis of HYAL1 and HYAL2. These genes are located in the minimally deleted region of LUCA , which were used in our growth suppression experiments for testing RASSF1A and G21/NPRL2HYAL1 and HYAL2 are well expressed in normal kidney and lung (see for example http://www.genecards.org/index.shtml) we have chosen KRC/Y and U2020 for our study.Expression of HYAL1 and HYAL2 were cloned into the episomal tetracycline-regulated pETE-Hyg vector HYAL1 and HYAL2 showed almost no inhibition of colony formation. When HYAL1 and HYAL2 were expressed the cloning efficiency was 77–100%, compared to the empty vector. As a positive control we used FUS1 gene, a strong TSG cloned in pETE FUS1 was less than 5% and for U2020 cells it was less than 25% compared to controls.HYAL1 and HYAL2 transgenes. The pETE-HYAL1 and pETE-HYAL2 vectors carrying wild type alleles of the tested genes were transfected into KRC/Y expressing tTA and clonal cell lines were selected. Expression of the transgenes was tested by Northern hybridization. Two of the best tetracycline regulated clones for each gene were then used in further experiments (see for example HYAL1 clones 1 and 4 were selected (HYAL1-KRC/Ycl.1 and HYAL1-KRC/Ycl.4) and for HYAL2 clones 13 and 14 (HYAL2-KRC/Ycl.13 and HYAL2-KRC/Ycl.14). Growth curves for HYAL1-KRC/Ycl.4 and HYAL2-KRC/Ycl.13 are shown in We performed also growth curve inhibition experiments using KRC/Y cells stably transformed with HYAL1 (HYAL1-U2020cl.4 and HYAL1-U2020cl.5) and HYAL2 (HYAL2-U2020cl.15 and HYAL2-U2020cl.18). All eight clones expressing either HYAL1 or HYAL2 in KRC/Y and U2020 cells were tested with CyQUANT NF Cell Proliferation Assay based on measurement of cellular DNA content via fluorescent dye binding  as described by Li et al., 1999 et al., 2002 in vivo. Clonal cell lines expressing transgenes were inoculated into SCID mice and the expression of the transgene was controlled by tetracycline administered ad libitum in the drinking water. In this setting TSGs suppressed tumour formation in SCID mice, unless they were eliminated or mutated. All grown tumours were analysed for the presence and expression of the transgene.We investigated HYAL1 and HYAL2 cell clones were inoculated into 22 SCID mice . For each SCID mouse, 5×106 cells were inoculated (one inoculation per mouse). In control mice, KRC/Y cells transfected with empty pETE vector were used (twelve SCID mice were injected). Half of the SCID mice were then given drinking water containing 1mg/ml tetracycline. Results of these experiments are shown in HYAL1 or HYAL2. All grown 12 tumours (T1–T12) were explanted and tested for the presence of pETE-HYAL1 and pETE-HYAL2 constructs by PCR. Transgenes were detected only in one tumour (HYAL2) and in eleven tumours it was deleted. In tumour T10 where the HYAL2 gene was present by PCR, there was no detectable expression by Northern (data not shown).KRC/Y derived HYAL1 and HYAL2 induce growth inhibition of tumours in SCID mice we tested expression of these genes in lung and renal tumours using qPCR (see As our data suggested that both qPCR see .HYAL1 was 7.8 (from 2.5 up to 77) and for HYAL2 8.8 (2.5–53) times . In seven cases expression of HYAL1 fell down more than 10-fold. For HYAL2 eight such cases were found. In six of fifteen lung biopsies we found more than 10-fold decrease of both HYAL1 and HYAL2 expression. In RCC samples expression of HYAL1 declined in 10 of 15 cases  and average decrease was 6.5 times (from 2 to 46). For HYAL2 reduced expression was detected in 9 of 15 samples  and average decrease was 4.9 times (2–11.4). Only in three RCC samples expression of HYAL1 decreased more than 10-fold and in one case we detected similar strong decline of HYAL2 expression. No statistically significant difference in cases with different tumour stages was observed both in SCC and RCC cases.Fifteen SCC and fifteen RCC tumours were studied. We assumed that expression fell down if it was at least 2-fold lower than in matched control samples. Expression of both genes was drastically decreased in all lung samples  did not show any effect in the tested cell lines. Six genes  had strong growth inhibitory activity, both in vitro and in vivo.In this work we describe functional characterization of NPRL2/G21 demonstrated growth inhibition in vitro in small cell lung cancer cell line U2020 and renal carcinoma cell line KRC/Y cells almost 90%. RASSF1A gene showed the same results. In SCID mice both genes either did not permit tumour formation or in growing tumours they were inactivated (for NPRL2 it was 10 mice and for RASSF1A it was 8 mice).For example, HYAL1 nor HYAL2 showed significant growth inhibition in colony formation and growth curve assays in vitro  even when the genes were repressed (water with tetracycline). However we have already shown that gene expression leakage in vivo is stronger than in vivo. Moreover it is known that tetracycline is a weaker inhibitor of expression compared to doxycycline in tTA system In contrast, neither itro see –3. CyQUAitro see  also demin vivo, most likely by influencing some interaction between the tumour cells and the host, but without directly affecting tumour cell growth in vitro. HYAL1 and HYAL2 thus differ in this aspect from other tumour suppressors like P53 or RASSF1A that inhibit growth both in vitro and in vivo. Since the products of both genes have a potential to influence intercellular interactions, their impairment may inhibit microenvironmental controls that normally protect the host from tumour growth.Thus, results clearly showed that the expression of either gene has led to the inhibition of tumour growth HYAL1 and HYAL2 are the major mammalian hyaluronidases in somatic tissues. They may act in concert to degrade high molecular weight hyaluronan  to the tetrasaccharide level HYAL2 hydrolyses high molecular weight hyaluronic acid  to produce an intermediate-sized product which is further hydrolysed by HYAL1 to give small oligosaccharides. Hyaluronan is claimed to be involved in tumour invasion and metastatic spread. The levels of HA surrounding tumour cells are often correlated with tumour aggressiveness and poor outcome Overproduction of HA enhances anchorage-independent tumour cell growth HYAL1 and HYAL2 in these studies, no frequent inactivating mutations were identified so far in these genes. Nevertheless it was reported that HYAL1 was inactivated in six of seven head and neck squamous cell carcinoma lines by illegitimate splicing HYAL2 promoter was methylated in more than 50% of non- small cell lung cancer (NSCLC) cases with decreased expression of the HYAL2 .In contrast to the tumour suppressor function of HYAL1 and HYAL2 was investigated in the study of Ji et al.HYAL1 and HYAL2 did not significantly inhibit tumour cells growth neither in vitro nor in vivo. However in this work it was demonstrated that HYAL2 inhibited experimental lung metastases in nu/nu mice. It is difficult to compare this work with our study. They used non-small cell lung cancer cell lines and recombinant adenoviral vectors. Completely different methods were used to measure suppressor activity. Despite these differences main results are similar to our work: HYAL1 and HYAL2 do not have strong growth inhibiting activity in vitro and HYAL2 displayed some tumour suppressor activity in vivo.Suppressor activity of HYAL1 and HYAL2 in SCID mice, we suggested that in primary renal and lung cancers expression of these genes might also be distorted. mRNA quantification was done for these genes in fifteen lung (SCC) and fifteen kidney (RCC) tumours using qPCR. In the majority of the tumour samples mRNA level of both genes was significantly decreased (HYAL1 and HYAL2 mRNA level was 0.67 (P = 0.005) in lung cancer samples . However in RCC samples it was only 0.4 . In any case, the results indicated that these genes may play important role in the development of lung and renal malignancies.Since we demonstrated tumour suppressor function of ecreased , sometimin vivo, without any inhibition of cell growth in vitro?What genes could be responsible for the suppression of tumour growth in vivo; products required for normal cellular responses to microenvironmental controls; or genes whose products inhibit angiogenesis There are at least three conceivable categories: genes encoding products required for responding to differentiation inducing signals Hyaluronidases are known to play an important role in tumour/host interactions. They may be examples of genes encoding interactive molecules that participate in microenvironmental growth control and carcinogenesis.There may be many other, as yet unidentified suppressor genes with similarly asymmetric inhibitory properties. The large numbers of LOHs that occur in the major human tumours and are not accounted for by known tumour suppressor genes, as well as the existence of “tumour suppressor gene clusters”, as documented on human chromosome 3p Paired tumour/normal samples were obtained from Blokhin Cancer Research Center, Russian Academy of Medical Sciences. Altogether fifteen specimens of lung squamous cell carcinoma (SCC) and fifteen clear cell renal carcinoma (RCC) cases and adjacent morphologically normal tissues  were obtained from patients after surgical resection of primary tumours and and stored in liquid nitrogen.. Top and bottom sections (3–5 µm thick) cut from frozen tumour tissues were examined histologically and only samples containing 70% or more tumour cells were used in the study. The samples were collected in accordance to the guidelines issued by the Ethic Committee of Blokhin Cancer Research Center, Russian Academy of Medical Sciences (Moscow). All patients gave written informed consent that is available upon request. The study was done in accordance with the principles outlined in the Declaration of Helsinki. All tumor specimens were characterized according to the International System of Clinico-Morphological Classification of Tumors (TNM).SCLC  cell line U2020 was described earlier HYAL1, HYAL2 and FUS1 genes was described previously FUS1mut containing Val33Met amino acid substitution was isolated from KRC/Y cell line transformed with wild type FUS1 (unpublished data). These genes were re-cloned into the pETE vector Molecular cloning of the human All molecular, cell biology and microbiology procedures were performed as described previously Construction of U2020 and KRC/Y cell lines producing tetracycline trans-activator tTA was described in HYAL1, HYAL2, FUS1 and FUS1mut genes were purified using R.E.A.L. Prep kit . Transfections were performed using Lipofectamine PLUS Reagent  according to the manufacturer's protocol.Plasmid DNAs containing After transfection, cells were selected with 200U/ml Hygromycin and 200ng/ml doxycycline for four weeks.6 cells were collected and total RNA was isolated with Trizol® reagent .PCR positive clones from each recombinant were grown in Iscove's cell culture medium supplemented with 10% heat-inactivated FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, 200 U/ml hygromycin and 200ng/ml doxycycline. Each clone was split into two parallel flasks, in one of them cells were grown without doxycycline. After one week, 10HYAL1 and HYAL2 probes were purified using electrophoresis and the Jetquick Gel Purification kit . The probes were labeled with α-P32 dCTP by random labeling.Northern blotting and hybridization were performed as described before in vivo and in vitro were done as described previously All growth inhibition experiments Briefly, U2020 or KRC/Y cells were transfected with plasmid DNA using Lipofectamine/Plus reagent . Transfected cells were stripped and plated on 100 mm cell culture plates. After selection with 400 µg/ml Hygromycin for 2 weeks, Giemsa-stained colonies were photographed and counted.6 cells were injected into 4-week old female SCID mice. Each mouse received only 1 injection. Twelve control mice were injected with empty pETE vector (six mice were drinking water with DOX and six without). Twenty two mice were used for the experiments  according to the manufacturer's protocol. Briefly, cells were plated at density of 100–500 cells per well in a 96-well plate . Number of cells in wells was counted every 24 hours: growth medium was removed, 50 µl of green-fluorescent CyQUANT GR dye (which exhibits strong fluorescence enhancement when bound to cellular nucleic acid) was added to the well and incubated for 30 min at 37°C. The fluorescence intensity of each sample was measured using a fluorescence microplate reader with excitation at 485nm and emission detection at 530nm. Plotted data points represent averages of triplicate samples, the plotted line is a linear regression fit of all data points. The assay is designed to produce a linear analytical response from at least 100–20,000 cells per well in most cell lines.GAPDH was assessed using qPCR . Ratio of mRNA level of target genes in all analysed tumours was measured relative to the reference “normal” samples .RNA isolated from primary tumour samples was reverse transcribed using the GeneAmp® RNA PCR Kit (Applied Biosystems) according to the manufacturer's protocol. The relative mRNA level of target genes and The primers and probes for transcripts were as follows:HYAL1:5′- TTTCTGCCCCTGGATGAGC-3′;Forward primer, 5′-CTCACCCAGAGCACCACTCC-3′;Reverse primer, 5′-FAM-CCCAGGCTGTGCTCCAGCTCA-[RTQ1]-3′;Probe, (amplicon size 80 bp);HYAL2:5′- CACCACAAGCACGGAGACCT-3′;Forward primer, 5′-CAGGCACTAGGCGGAAACTG-3′;Reverse primer, 5′-FAM-CCTTCCTGCATCTCAGCACCAACAG-[RTQ1]-3′;Probe (amplicon size 197 bp);GAPDH:5′- CGGAGTCAACGGATTTGGTC– 3′;Forward primer, 5′- TGGGTGGAATCATATTGGAACAT– 3′;Reverse primer, 5′-FAM-CCCTTCATTGACCTCAACTACATGGTTTACAT–[RTQ1]-3′;Probe, (amplicon size 141 bp);The optimal primer and probe concentrations for the target and control genes were as follows:HYAL1 primers, 300 nM, probe, 300 nM (for lung cancer cDNA samples); primers, 200 nM, probe, 200 nM (for RCC cDNA samples);HYAL2 primers, 300 nM, probe, 300 nM (for lung cancer cDNA samples); primers, 200 nM, probe, 200 nM (for RCC cDNA samples);GAPDH primers, 300 nM; probe, 150 nM.The thermocycler conditions were 10 min at 95°C, then 50 two-step cycles 15sec at 95°C and 60sec at 60° C. qPCR amplification was carried out in triplicate in 25-µl reaction volume using TaqMan Universal Master Mix (Applied Biosystems) and 10ng template cDNA. The sequences of the amplicons were verified by sequencing in 3730 DNA Analyzer automated sequencer (Applied Biosystems).T method was used as described previously The comparative CHYAL1 and HYAL2 and reference gene for the same SCC and RCC patients. The evaluation of statistical significance of mRNA level was tested for all studied cases. Nonparametric Spearmen's rank test was used to calculate the coefficient of correlation between the level of mRNA decrease for HYAL1 and HYAL2 genes. P-values <0.05 were considered statistically significant. All statistical procedures were performed using BioStat software Nonparametric Wilcoxon test was used to compare mRNA expression differences of


To date, BRCA1 and BRCA2 mutations in breast and/or ovarian patients have not been characterized in the Turkish population. We investigated the presence of BRCA mutations in 53 individuals with a personal and family history of breast and/or ovarian cancer, and 52 individuals with a personal history of breast cancer diagnosed below age 50 without additional family history. We have identified 11 mutations (nine BRCA1 and two BRCA2) using combined techniques involving protein truncation test, direct sequencing and heteroduplex analysis. We found eight out of 53 patients (15.1%) with a family history to carry BRCA gene mutations (seven BRCA1 and one BRCA2). Of these, four were found in 43 families presenting only breast cancer histories, and four were found in families presenting ovarian cancer with or without breast cancer. We also demonstrated two BRCA1 and one BRCA2 mutations in three out of 52 (5.8%) early-onset breast cancer cases without additional family history. Three of nine BRCA1 and both BRCA2 mutations detected in this study were not reported previously. These mutations may be specific to the Turkish population. The BRCA1 5382insC mutation, specific to Ashkenazi and Russian populations, was found twice in our study group, representing a possible founder mutation in the Turkish population. © 2000 Cancer Research Campaign


Hepatoblastoma is a malignant paediatric liver tumour. In order to approach the genetic background of this malignancy we have screened a panel of eighteen cases from Europe and Japan for chromosomal imbalances using comparative genomic hybridization (CGH). The most frequent losses included chromosomal regions 13q21–q22 (28%) and 9p22-pter (22%), while the most frequent gains occurred on 2q23–q24 (33%), 20q (28%) and 1q24–q25 (28%). A significant difference in CGH alterations between the tumours from patients of Caucasian and Japanese was revealed where loss of 13q was found only in the Japanese samples. In conclusion, the findings indicate several candidate regions for suppressor genes and oncogenes potentially involved in the hepatoblastomas of different ethnic origin. © 2000 Cancer Research Campaign


In contrast, the other methods revealed markedly lower efficiencies varying from 1% to 10%. The use of the HPPTP assay as a reliable processing procedure was validated by demonstrating a complete agreement in HPV detection and 93% agreement in HPV typing between 39 archival Pap-stained and paired fresh-frozen cervical smears. This method was applied to 40 archival smears from ten cervical cancer patients (selected from a group of 200 patients) which had a history of 3–6 smears with the first smear being Pap 1 or 2 taken at least 5 years before cancer was diagnosed. The average time period between the first Pap 1/2 smear that contained the same HPV type as in the corresponding carcinoma and diagnosis of cervical cancer was 12.0 ± 2.9 years. All subsequent smears were invariably positive for the same HPV type which was also found in the cervical cancer biopsy. In conclusion, the HPPTP assay provides a reliable and efficient means to extract DNA from Pap-stained archival cervical smears for the detection of HPV DNA by PCR and would be the method of choice for future HPV analysis of archival Pap-stained cervical smears. © 2000 Cancer Research CampaignThe efficacy of four methods to recover DNA from Papanicolaou (Pap)-stained archival cervical smears for optimal detection of human papillomavirus (HPV) DNA by GP5+/bioGP6+ polymerase chain reaction (PCR) was investigated. Two of the methods were based on proteinase K treatment and two based on treatment with guanidinium thiocyanate (GTC). The quality of the DNA as measured by PCR assays amplifying different sizes of the β-globin gene appeared to be superior for the GTC-based assays. Using competitive β-globin PCR assays, one of the GTC-based, assays, provisionally named High Pure PCR Template Preparation (HPPTP) assay, yielded by far the highest quantity of amplifiable DNA. It allowed the recovery of 2.2 × 10


Familial predisposition to basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) of the skin are apparent in the autosomal dominant syndromes naevoid basal cell carcinoma syndrome (NBCCS) and multiple self-healing squamous epitheliomata (MSSE) respectively. The gene responsible for NBCCS has been proposed to be a tumour-suppressor gene and is mapped to the same 2 Mb interval on 9q22.3 as the MSSE gene ESS1. In an attempt to further map the NBCCS gene, we have examined loss of heterozygosity (LOH) in 16 sporadic BCCs and two familial BCCs using microsatellite markers located within the candidate gene region. The overall frequency of LOH observed was 67% in the BCCs and partial or interstitial deletions were found in eight tumours, with the highest LOH frequency at markers D9S280, D9S287 and D9S180. To determine if the same genomic region also shows frequent LOH in tumours with a squamous phenotype, we have examined 11 SCCs, four actinic keratoses and 13 cases of Bowen's disease for LOH at 9q22.3. An overall LOH frequency of 50% was observed at D9S180, and occurred in all types of squamous tumours. In contrast, a much lower LOH frequency of only 6% was found at the D9S287 locus. Our observation of different patterns of LOH at 9q22.3 in sporadic BCCs and SCCs implies that more than one tumour-suppressor gene might be located in this genomic region.


The purpose of the present study was to explore the therapeutic potential of serial administration of shedding-inducing endotoxin in a mouse tumour bladder model. The studies were conducted with two variants derived from the MBT-2 tumour namely, T5 and T50, the latter being far more aggressive than the former. It was found that T5 tumours responded to intravesical lipopolysaccharides (LPS) instillation by a considerable reduction in their pace of growth (P < 0.0001) when treatment was initiated 3 days after tumour implantation, but not when started after 7 days. The T50 variant did not respond to LPS when treated 3 days after implantation, but a considerable reduction in rate of growth occurred when treatment was started after 1-2 days. Shedding induced by intravesically instilled LPS was found to retard considerably the progression rate of experimental bladder tumour.


Loss of multiple chromosomal regions is another genetic alteration frequently found in lung tumours. We have examined the association between p53 mutations, loss of heterozygosity (LOH) at frequently deleted loci in lung cancer, and tobacco exposure in 165 tumours from non-small cell lung cancer (NSCLC) patients. A highly significant association between p53 mutations and deletions on 3p, 5q, 9p, 11p and 17p was found. There was also a significant correlation between deletions at these loci. 86% of the tumours with concordant deletion in the 4 most involved loci  had p53 mutations as compared to only 8% of the tumours without deletions at the corresponding loci (P< 0.0001). Data were also examined in relation to smoking status of the patients and histology of the tumours. The frequency of deletions was significantly higher among smokers as compared to non-smokers. This difference was significant for the 3p21.3 (hMLH1 locus), 3p14.2 (FHIT locus), 5q11–13 (hMSH3 locus) and 9p21 (D9S157 locus). Tumours with deletions at the hMLH1 locus had higher levels of hydrophobic DNA adducts. Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and p53 mutations were significant and independent parameters for predicting LOH status at several loci. In the pathogenesis of NSCLC exposure to tobacco carcinogens in addition to clonal selection may be the driving force in these alterations. © 2001 Cancer Research Campaign http://www.bjcancer.comCarcinogenesis results from an accumulation of several genetic alterations. Mutations in the


Weekly intramuscular injections of 2.4million U Bicillin for 3 weeks was initiated as recommended by the Centers for Disease Control.Repeat sonogram 1 week after starting treatment revealed increased ascites and a new pericardialeffusion. Due to the worsening fetal condition, therapy was altered and the patient was admitted forIV penicillin. She received a continuous infusion of 18 million U penicillin G daily for 10 days. Serialsonograms showed improvement offetal ascites and pericardial effusion with 10 days of IV therapy,and complete resolution of hydrops was noted within 3 weeks. The fetus was born at term with nostigmata of congenital syphilis on newborn exam, and laboratory tests suggested adequate treatmentin utero.A 21 year old woman (G


BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions  were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations  were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations. © 2001 Cancer Research Campaignhttp://www.bjcancer.comMutations of the


Reconstitution of depleted thymicstroma with thymocytes showed evidence of defects in both developmentalcompartments.We have examined fetal thymic development in the trisomy 16 (Ts16) mouse, which isconsidered to be a model for human trisomy 21, or Down Syndrome. The Ts16 thymuscontains 10 to 20% of the number of lymphocytes found in a normal thymus at acomparable stage. Expression of thymocyte differentiation markers  is severely affected in Ts16 fetuses aged 14–18 gestational days.When thymuses from 14-day Ts16 mice were cultured


BRCA1 or BRCA2. In 104 families with three or more affected individuals (average 3.8) seeking counselling at family cancer clinics, mutation analysis was performed in the open reading frame of BRCA1 and BRCA2 by the protein truncation test and mutation-specific assays. In 31 of the 104 families tested, mutations were detected (30%). The majority of these mutations (25) occurred in BRCA1. Mutations were detected in 15 out of 25 families (60%) with both breast and ovarian cancer and in 16 out of 79 families (20%) with exclusively cases of breast cancer. Thus, an ovarian cancer case strongly predicted finding a mutation (P < 0.001). Within the group of small breast-cancer-only families, a bilateral breast cancer case or a unilateral breast cancer case diagnosed before age 40 independently predicted finding a BRCA1 or BRCA2 mutation . Therefore, even small breast/ovarian cancer families with at least one case of ovarian cancer, bilateral breast cancer, or a case of breast cancer diagnosed before age 40, should be referred for mutation screening. © 1999 Cancer Research CampaignFor families with a small number of cases of breast and/or ovarian cancer, limited data are available to predict the likelihood of genetic predisposition due to mutations in


TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM.In this study levels of prostaglandin E


These data provide a better understanding as the first step in the mechanism of action of the activin in the epithelial ovarian carcinoma. © 2000 Cancer Research CampaignIn this study, we have investigated the expression of inhibin subunits and activin receptors (ActRs) in normal and malignant ovarian cells. Each product of the inhibin subunits  and activin receptors (ActRs) amplified by reverse transcription polymerase chain reaction were detected as a single band in human granulosa cells, surface epithelial cells (OSE), and the ovarian cancer cell lines OVCAR 3 and SKOV 3. Western blot analysis was performed using polyclonal antibodies against ActR IIa or IIb peptides based on 13 COOH-terminal amino acids; cultured human granulosa cells were used as a positive control. Using ActR IIa antibody, one major band corresponding to approximately 80 kDa and one minor band corresponding to 105 kDa were observed in the samples. One single band at approximately 60 kDa was detected in OVCAR 3 and a 50 kDa band was detected with ActR IIb antibody in cultured granulosa cell, OSE and SKOV 3. Although no detectable change was induced in Smad 4 mRNA in OVCAR 3, Smad 2 mRNA levels were increased during 48 h treatment with activin A (50 ng ml


CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations  and 2 common polymorphisms (A148T and Nt500 G>C) were identified in the CDKN2A gene. In a family harbouring the R24P mutation, an intronic variant  of uncertain significance was detected in a non-carrier melanoma case. The overall incidence of CDKN2A mutations was 33.3%, but this percentage was higher in families with 3 or more melanoma cases (50%) than in those with only 2 affected relatives (25%). Noteworthy, functional analysis established that the novel mutated protein, while being impaired in cell growth and inhibition assays, retains some in vitro binding to CDK4/6. No variant in the p16-binding region of CDK4 was identified in our families. Our results, obtained in a heterogeneous group of families, support the view that inactivating mutations of CDKN2A contribute to melanoma susceptibility more than activating mutations of CDK4 and that other genetic factors must be responsible for melanoma clustering in a high proportion of families. In addition, they indicate the need for a combination of functional assays to determine the pathogenetic nature of new CDKN2A mutations. http://www.bjcancer.com © 2001 Cancer Research Campaignhttp://www.bjcancer.comPhysical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the


Fifty-seven patients with advanced prostate cancer resistant to first-line hormonal therapy were treated with estramustine and additionally randomized for treatment with clodronate or placebo. Clodronate treatment was started with 5 days intravenous administration (300 mg day[-1]) and followed by oral treatment (1.6 g day[-1]) for 12 months. Skeletal pain relief was only about 10% better in the clodronate than in the placebo group. The results do not support the superiority of combined intravenous and oral treatment with clodronate compared with oral administration only.


Estimates were made of the rates at which cancer cells were released directly into the renal vein in patients undergoing radical nephrectomy for primary renal cancer. Cancer cells were counted in blood samples taken from the renal vein using a density gradient centrifugation procedure, and identified using immunocytochemical techniques, on the basis of their cytoskeletal intermediate filament proteins. Cancer cells were released as single cells and multicell emboli in 8/10 patients, in numbers varying widely between 14-7509 emboli ml-1 of blood. Despite a calculated median input into the metastatic process of 3.7 x 10(7) cancer cells per day for at least 180 days, only 3/10 patients had extraperitoneal metastases prior to surgery and only 1 of the remaining disease-free patients subsequently developed distant metastases over a maximum 35 month period. These results are discussed in terms of primary tumour kinetics and metastatic inefficiency.


Despite infections by the dengue virus being a significant problem in tropical and sub-tropical countries, the mechanism by which the dengue virus enters into mammalian cells remains poorly described.A combination of biochemical inhibition, dominant negative transfection of Eps15 and siRNA mediated gene silencing was used to explore the entry mechanism of dengue into HepG2 cells.Results were consistent with entry via multiple pathways, specifically via clathrin coated pit mediated endocytosis and macropinocytosis, with clathrin mediated endocytosis being the predominant pathway.We propose that entry of the dengue virus to mammalian cells can occur by multiple pathways, and this opens the possibility of the virus being directed to multiple cellular compartments. This would have significant implications in understanding the interaction of the dengue virus with the host cell machinery. While most animal viruses enter into cells by receptor mediated endocytosis in clathrin coated pits ,2, evideHepG2 and Vero cells were maintained as previously described -13 Dengu# E6361).Confluent HepG2 cells were pretreated at 37°C with either 20 μM cytochalasin D or 30 μg/ml nystatin for 2 hr, or with 15 μg/ml chlorpromazine or 3 mM amiloride or 50 μM LY294002 or 0.2 μM wortmannin for 1.5 hr with 80% DMSO for 20 hours as a positive control. Cells were then trysinized and subsequently washed twice with cold-PBS. Cells were then washed with binding buffer  and resuspended in binding buffer. Annexin V-FITC  was added to the cell suspensions and samples were incubated in the dark for 15 min before analysis by flow cytometry  treatment . The inf2 for 2 days. Transfection efficiencies routinely exceeded 70% efficiency as determined by counting of multiple fields. Transfected cells were subsequently infected with dengue virus serotype 1, 2, 3 or 4 at an MOI of 20 for 1.5 hr followed by acid glycine (pH3) treatment to inactivate un-internalized viruses [2. A further set of cells were serum starved for 30 min and incubated with 5 μg transferrin conjugated with Alexa 594  at 37°C for 30 min follow by acid glycine treatment.Plasmid constructs of dominant negative (DIII and EH29) and control (D3Δ2) Eps15 were kindly provided by A. Benmerah . Transfections of HepG2 cells were undertaken using lipofectamine2000 . Briefly, 3 μg of the appropriate plasmid DNA was complexed with 4 μl of lipofectamine2000 for 30 min at room temperature and then added to HepG2 cells pre-grown to 70–80% confluency on glass coverslips. The cell/complex mix was incubated at 37°C, 10% CO viruses  and incuBoth dengue infected and transferrin treated transfected cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Transferrin control cells were directly mounted with Vectashield  while dengue virus infected cells were further permeabilized with 0.3% TritonX-100 and methanol. Nonspecific binding was blocked by incubation with 5% BSA for 1 hr at room temperature. Cells were incubated with an anti-dengue E protein monoclonal antibody, HB-114  at 4°C oNM_004859) and the green fluorescent protein  were determined using the on-line tool from Ambion, Austin, TX  and the selected sequences were subjected to siRNA template design to generate DNA oligonucleotide sequences for use with the Silencer™ siRNA Construction kit (Ambion). Six templates for siRNA generation were selected:Target sites on clathrin heavy chain  or U50974 (siGFP). All sequences were searched against the NCBI's database to confirm specificity. Sense and antisense DNA templates were chemically synthesized  and following the kit instructions based on 4 HepG2 cells was added and cell: complex mixtures incubated under standard conditions. Mock transfections (lipofectamine only) were performed in parallel. All transfections were undertaken in a final volume of 600 μl with siRNA at a final concentration at 50 nM. Transfections were harvested at 1 to 4 days post-transfection.HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum without antibiotics. Reverse transfections according to the manufacturers protocols were performed with Lipofectamine™RNAiMAX  by mixing the respective siRNA and 1.2 μl of Lipofectamine™RNAiMAX and adding to a single well of a 24 well plate. After 20 minutes of incubation at room temperature, a suspension of 5 × 1017 primer was used to synthesize first strand cDNA using ImpromII™ reverse transcriptase . The cDNA was then amplified in a multiplex reaction with 2 specific primer pairs for CHC  and GAPDH  as an internal control. Expected products were 343 bp (GAPDH) and 222 bp (CHC) Cycle conditions were 94°C for 3 minutes followed by 20 cycles of 94°C for 30 seconds, 58°C for 45 seconds and 72°C for 45 seconds followed by a final extension of 72°C for 7 minutes. PCR products were analyzed on 1.8% agarose gels containing ethidium bromide.Transfected cells from a single well of a 24-well plate were homogenized in 0.5 ml Trizol reagent  and RNA purified as recommended by the manufacturer. For the RT-PCR analysis, an oligo(dT)4) were grown on coverslips in single wells of a 24 well plate and transfected as above with either siRNAs as stated or mock transfected. At day 3 post transfection cells were infected with dengue virus serotype 2 MOI 20 for 2 hours followed by an acid glycine wash and subsequently incubated for 15 hours under standard conditions. Dengue virus E protein was detected as described above except that cells were also stained with DAPI. Parallel non-infected samples were incubated with transferrin as described above and were additionally stained with DAPI. Where biochemical inhibition was used in conjunction with siRNA silencing, samples were treated with 0.2 μM wortmannin for 30 minutes immediately preceding dengue virus infection.HepG2 cells . Working concentrations (the highest concentration without apparent cytotoxicity) were established as: cytochalasin D at 20 μM, amiloride at 3 mM, LY294002 at 50 μM, wortmannin at 0.2 μM, nystatin 20 μM at 30 μM and chlorpromazine at 15 μM (HepG2). The lack of cytotoxicity at these concentrations was confirmed by Annexin V staining and flow cytometry  significantly excluded the entry of transferrin Figure . HoweverNM_004859) were generated using in vitro transcription together with 1 siRNA targeted to the green fluorescent protein  for use as a control. To confirm all siRNAs were double-stranded, an aliquot of each siRNA was treated with RNaseIII which digests double-stranded RNA or RNaseA which digests single-stranded RNA. All siRNA constructs were confirmed to be off the appropriate size and to consist of dsRNA (data not shown).Given the significant entry of the dengue virus in the presence of over-expressing dominant negative mutants of Eps15, it is possible that either entry was occurring via multiple pathways, or the Eps15 mutants were not completely inhibiting clathrin mediated entry. To further explore this, RNA interference was used to down regulate the expression of clathrin heavy chain, an integral part of the clathrin vesicle . Five diTo optimize the silencing of the expression of the clathrin heavy chain, the 5 different siCHCs were transfected into HepG2 cells in parallel with transfections of siGFP and lipofectamine alone (mock). On days 1 to 4 days post-transfection, cells were harvested and RNA extracted. Multiplex RT-PCR was undertaken to detect messages from GAPDH and clathrin heavy chain (CHC) simultaneously and results analyzed by agarose gel electrophoresis. Experiments were undertaken independently in triplicate.Results showed a constant signal for GAPDH and the clathrin heavy chain (CHC) for mock and siGFP transfection Figure . MessageOptimal silencing of clathrin heavy chain expression was noted at day 3 post transfection with siRNA constructs siCHC3 and siCHC5. These two siRNAs were again transfected into HepG2 cells as above in parallel with transfections of siGFP and mock (transfection agent only) and on day 3 post transfection cells were infected with dengue virus serotype 2 and an MOI of 20 and cells allowed to grow for 15 hours (the time for one replication cycle of dengue serotype 2 minus two hours) under optimal conditions. At 15 hours cells were either analyzed by microscopy or by our adaptation of the standard plaque assay [Consistent with our results with transfections of dominant negative constructs of Eps15, a significant reduction of transferrin entry was seen with siCHC transfections, but not with mock or siGFP Figure . Dengue Results of dengue virus entry as seen by our adaptation of the standard plaque assay  were conTo determine whether the approximately 20% virus entry seen in cells in which clathrin mediated endocytosis has been inhibited is a result of background, or the result of dengue virus entry by macropinocytosis, we sought to simultaneously inhibit both pathways, clathrin mediated endocytosis through siRNA mediated RNA inhibition and macropinocytosis through biochemical inhibition using wortmannin.Cells were therefore transfected with siCHC5 to silence clathrin heavy chain or mock transfected and on day 3 post-transfection were either treated or not treated with wortmannin for 1 hour before being either incubated with transferrin or infected with dengue serotype 2 at an MOI of 20. Following acid glycine treatment of dengue infected cells, cells were incubated for 15 hours before being either examined by microscopy or the number of infected cells determined by our adaptation of the standard plaque assay [Results Figure  show thaResults from our adaptation of the plaque assay Figure , Panel BDespite flaviviral infections representing a significant world wide public health threat, little advance has been made in dissecting out the mechanism by which flaviviruses enter into either mammalian or insect cells. Studies on Japanese encephalitis virus and West Nile virus with either Vero (African Green monkey cells) or C6/36 cells have suggested that these two viruses enter by clathrin coated pit mediated endocytosis -10. WithRecently Chu et al.,  providedInterestingly Chu and colleagues also investigated the entry of West Nile Virus into the aedes albopictus cell line C6/36 using the same dominant negative mutant of Eps15  and simiMore recently Krishnan and colleagues have investigated the entry of the dengue virus into HeLa cells . This stOur data suggests that the remaining 20% virus entry observed is not the results of incomplete ablation of clathrin mediated endocytosis but represents virus entry by a viable, independent pathway, macropinocytosis. Entry of the dengue viruses  by multiple pathways as shown here raises some interesting questions, particularly with respect to the initial flavivirus: host cell interaction and may require a significant re-evaluation of our understanding of flavivirus entry into host cells.Consistently, inhibition of clathrin mediated endocytosis using dominant negative mutants of Eps 15 results in a reduction of dengue virus entry of approximately 80% as shown by this study and others -9. Our dThe authors declare that they have no competing interests.LS undertook the biochemical inhibition studies and Eps15 transfections and analyzed and interpreted the data. TS undertook the siRNA mediated silencing studies and analyzed and interpreted the data. DRS was responsible for design and implementation of the study as well as drafting the manuscript. All authors read and approved the final version.


A series of 23 children with primitive neuroectodermal tumours (PNET) were analysed with comparative genomic hybridization (CGH). Multiple chromosomal imbalances have been detected in 20 patients. The most frequently involved chromosome was chromosome 17, with a gain of 17q (11 cases) and loss of 17p (eight cases). Further recurrent copy number changes were detected. Extra copies of chromosome 7 were present in nine patients and gains of 1q were detected in six patients. A moderate genomic amplification was detected in one patient, involving two sites on 3p and the whole 12p. Losses were more frequent, and especially involved the chromosomes 11 (nine cases), 10q (eight cases), 8 (six cases), X (six patients) and 3 (five cases), and part of chromosome 9 (five cases). These recurrent chromosomal changes may highlight locations of novel genes with an important role in the development and/or progression of PNET. © 1999 Cancer Research Campaign