The Study Of Hypermethylation Related Genes In Endometrial Cancer Cells

Background:Endometrial cancer is a common malignancy of the female genital tract.According to their etiological and pathological features,endometrial cancers are divided into endometrioid and non-endometrioid histologic subtypes,also referred to Typeâ… and Typeâ…¡.Most patients present with Typeâ… which frequently expresses estrogen and progesterone receptors.This type of tumor often arises in a background of hyperplasia,and is associated with unopposed estrogen stimulation.Recent studies have indicated that epigenetic alterations may play a significant role in this type of cancer.Methylation on the 5'carbon position of pyrimidine ring of deoxycytidines located within CpG dinucleotides represents the major epigenetic modification of the human genome.DNA methylation is an essential epigenetic modification required for normal mammalian development,gene regulation,genomic imprinting,and chromatin structure.Aberrant DNA methylation pattern of CpG islands located in promoter regions is related to transcriptional regulation.It has been well elucidated that silencing of tumor suppressor genes and DNA mismatch repair genes are associated with alterations in regional methylation patterns in many human malignant cells.Altered DNA methylation in the form of global hypomethylation and regional hypermethylation is one of the most consistent epigenetic changes in cancer.The silencing of the tumor suppressor gene by DNA methylation may participate in the pathogenesis of cancer.DNA methylation is known to be mediated by a family of proteins called DNA methyltransferases(DNMTs),including DNMT1,DNMT3A and DNMT3B.Although the three DNMTs partially cooperate to establish and maintain genomic methylation pattems,they also have distinctive functions.DNMT1 is considered as the major maintenance methyltransferase that has a preference for hemi-methylated DNA and may be responsible for the symmetrical methylation of nascent DNA strands during DNA replication.However,DNMT3A and DNMT3B are thought to function as de novo methyltransferases with higher activity on unmethylated substrates,suggesting that they may be responsible for the aberrant methylation in cancer cells.It has been reported that DNMT1 and DNMT3B were up-regulated in endometrial cancers as compared to normal endometrium controls,but the mechanism was unclear.Microsatellite instability(MSI) was first detected in tumors arising in individuals with hereditary nonpolyposis colorectal cancer(HNPCC).Since endometrial carcinoma is the second most common tumor in women with HNPCC,MSI studies were performed,and MSI was detected in approximately 25%of sporadic endometrial cancers.Unlike the familial cases in which the affected member carries a germline mutation in one of the DNA mismatch repair genes,hMLH1 promoter hypermethylation is the predominant cause of MSI in sporadic cases.In addition,loss of hMLH1 expression due to methylation of its promoter was observed in normal-appearing endometrial glands adjacent to endometrial carcinomas.These findings strongly suggest that MMR deficiency is crucial in early stages of endometrial carcinogenesis.Epigenetic factors such as DNA methylation was known to contribute to the malignant transformation of cells by silencing critical genes.Drugs that inhibit DNA methyltransferases were shown to have the potential to reactivate silenced genes and induce differentiation or apoptosis of malignant cells. PARTâ… ESTROGEN REGULATES DNA METHYLTRANSFERASE 3B EXPRESSION IN ISHIKAWA ENDOMETRIAL ADENOCACINOMA CELLSObjective:It is well-known that exposure to unopposed estrogen is considered as an important risk factor for endometrial cancer.Recent studies have shown that over-expression of DNA methyltransferases(DNMTs) are involved in the development of endometrial cancer.Therefore,the present study was undertaken to elucidate the impact of estrogen on the expression of DNMTs in endometrial cancer.Methods:1.Cell culture:Ishikawa cell line(a well-differentiated endometrial adenocarcinoma cell line which expresses estrogen receptors) was maintained in RPMI1640 medium containing 5%heat inactivated fetal bovine serum(FBS) and 2 mM L-glutamine in culture flasks with 37℃in an atmosphere containing 5%CO₂ and 100% humidity.The entire medium was supplemented with 100μg/ml streptomycin and 100 unit/ml penicillin.To determine the effect of 17β-estradiol(E₂) on DNMT1 and DNMT3B expression,the cells were maintained for 24 h in phenol red-free 1640 medium supplemented with 5%charcoal-dextran-stripped calf serum before the application of treatment.2.Analysis of cell cycle distribution:Ishikawa cells were cultured in the absence or in the presence of E₂ at various concentrations(10^-6,10^-8,10^-10 and 10^-12M) for 24 h.Then the cells were analyzed by Flow cytometry analysis.3.Detection of the mRNA levels of DNMT1 and DNMT3B.The cells were grown and treated with E₂(10^-8 M) alone or E₂(10^-8 M) with addition of ICI182780(10^-8 M) respectively for 24 h.The mRNA levels of DNMT1 and DNMT3B were measured by real-time PCR.4.Detection of the protein expression levels of DNMT1 and DNMT3B:The cells were grown and treated with E₂(10^-8 M) alone or E₂(10^-8 M) with addition of ICI182780(10^-8 M) respectively for 24 h.The protein expression levels of DNMT1 and DNMT3B were measured by Western blot analysis.5.Measurement of DNMT activities after treatment with E₂:Ishikawa cells were cultured in the absence or in the presence of E₂ at the concentration of 10^-8 M for 24 h.Then the DNMT activities of the cells were evaluated.Results:1.Effect of E₂ with different concentrations on the proliferation of Ishikawa cells: We treated the Ishikawa cells with E₂ at concentrations ranging from 10^-6 M,10^-8 M, 10^-10 M to 10^-12 M for 24 h and the effect of E₂ with different concentrations on the cell cycle distribution of Ishikawa cells were confirmed by flow cytometry analysis.The maximum stimulation of cell proliferation was detected at the concentration of 10^-8 M of E₂.2.Effect of E₂ on the mRNA level of DNMT1 and DNMT3B:We examined the steady state levels of DNMT1 and DNMT3B mRNA using real-time PCR in Ishikawa cells following E₂ treatment.We observed that E₂ profoundly elevated transcription of DNMT3B in Ishikawa cells.It is noteworthy that throughout these experiments,the stimulant effect on DNMT3B mRNA was unchanged when the results were standardized against PCNA,a control for cell cycle alteration.Significant inhibition in DNMT3B mRNA was observed using both E₂ and anti-estrogen ICI182780.These results suggest the presence of estrogen-mediated transcriptional activation of DNMT3B.In contrast,no significant alteration in DNMT1 mRNA with E₂ treatment for 24 h in Ishikawa cells was observed.3.Effect of E₂ on the protein expression of DNMT3B in Ishikawa cells:We assessed the regulation of DNMT3B in Ishikawa cells by E₂ using Western blotting analysis.Ishikawa cells were treated with E₂(10^-8 M) alone or in combination with ICI182780(10^-8 M) for 24 h.An increase in DNMT3B expression occurred in Ishikawa cells with the treatment of E₂.Western blot analysis also revealed that the E₂-induced up-regulation of DNMT3B was suppressed by the addition of ICI182780.4.E₂ increased de novo DNA methyltransferase activity:We sought to determine if E₂ treatment could alter DNA methylation capacities by the standard in vitro assay using synthetic,unmethylated,CpG-rich oligonucleotide substrates and radioactive S-adenosyl-L-methionine.E₂ treatment for 24 hours increased de novo DNA methylation in Ishikawa cells.These data suggest that E₂ increased de novo DNA methyltransferase activities.Conclusion:Our study suggests that estrogen up-regulating the expression of DNMT3B in an ER-dependent pathway may be a possible mechanism for estrogen facilitates the malignant transformation of endometrial cancer cells. PARTâ…¡5-AZA-2'-DEOXYCYTIDINE IS A POTENT INHIBITOR OF DNA METHYLTRANSFERASE 3B AND INDUCES APOPTOSIS IN HUMAN ENDOMETRIAL CANCER CELL LINES WITH THE UP-REGULATION OF HMLH1Objective:Our study was to evaluate the effects of 5-aza-2'-deoxycytidine (5-azadC) on cell growth inhibition,cell cycle arrest,apoptosis as well as the expression levels of hMLH1 and DNMT3B in human endometrial cancer cell lines.Methods:1.Cell culture:The human endometrial cancer cell lines Ishikawa,HHUA and KLE were maintained in RPMI 1640 medium supplemented with 10%heat-inactivated fetal calf serum and 2 mM L-glutamine at 37℃in a 5%CO₂ incubator.The cells were treated with a freshly prepared solution of 5-aza-2'-deoxycytidine.2.Cell viability:Cells were plated in 96-well plates,allowed to grow overnight and treated with 0μM,0.1μM,1μM and 5μM 5-azadC for 72 h(fresh drug was added every 24 h).Cell viability was assessed by a colorimetric assay using MTT.3.Effect of 5-azadC on cell cycle distribution:Cells were cultured in the presence of 5-azadC at the concentration of 1μM for 72 h.Cell cycle profiles were determined by analyzing DNA content using propidium iodide(PI) staining and flow cytometry.4.Effect of 5-azadC on cell apoptosis:Cells were cultured in the presence of 5-azadC at the concentration of 1μM for 72 h.To understand and confirm the nature of cell death,we used the Annexin-V flow cytometry analysis and TUNEL assay.5.Effect of 5-azadC on the methylation status of hMLH1 in human endometrial cancer cell lines:Cells were cultured in the presence of 5-azadC at the concentration of 1μM for 72 h.The methylation status of the hMLH1 gene was monitored by methylation-specific PCR.6.Effect of 5-azadC on hMLH1 and DNMT3B expression:Cells were treated with 1μM 5-azadC for 72 h,and then were harvested for real-time PCR and western blotting analysis.Results:1.Cell viability:The dose of 5-azadC required to inhibit cell growth was evaluated in Ishikawa,HHUA and KLE cell lines.Cells were treated with 5-azadC(0μM,0.1μM,1μM and 5μM),and cell morphology and viability were monitored for 72 h using the MTT assay.A dose-dependent inhibition of cell viability was observed upon treatment with 5-azadC.Furthermore,there was no significant difference between 1μM group and 5μM group,so we determined 1μM as the treatment dose.2.Effect of 5-azadC on cell cycle distribution:Cell cycle distribution analysis showed an increase,within 72 h,in the number of cells in the G₂/M phase of the cell cycle following treatment with 5-azadC,providing evidence of G₂/M arrest.By 72 h of treatment,approximately 3-fold of the cells arrested at G₂/M phase compared with the control group.3.5-azadC treatment induced apoptosis in human endometrial cancer cell lines:To understand and confirm the nature of cell death,we used the Annexin-V flow cytometry analysis and TUNEL assay.Numerous TUNEL-positive cells with apoptotic characteristics,based on the rounded,shrunken shape of the nucleus and on intense staining of FITC-conjugated dUTP,appeared in the 5-aza-CR-treated cultures.A time-dependent increase of apoptotic cells occurred in 5-azadC-treated cell cultures.Drug exposure also caused a strong increase of Annexin-V staining,a typical feature of early apoptosis.The proportion of apoptotic cells at 72 h post-treatment was significantly higher than that of necrotic cells,indicating that apoptosis rather than necrosis is the mechanism of 5-azadC-induced cell death in Ishikawa cells.5-azadC treatment induced the same apoptotic effect in HHUA and KLE cell lines either.4.Effect of 5-azadC on hMLH1 gene methylation:Considering the fact that hemimethylation of the hMLH1 promoter is observed in endometrial cancer cell lines (presence of both methylated and unmethylated bands in the untreated control),we assessed the effect of 5-aza-CR on the methylation status of the hMLH1 gene promoter.Following treatment with 5-aza-CR(1μM) for 72 h,the methylated band almost completely disappeared.Corresponding to the disappearance of the methylation-specific band was the up-regulation of hMLH1 mRNA and protein expression in Ishikawa cells.The same results were detected in HHUA and KLE cell lines.5.Effect of 5-azadC on hMLH1 and DNMT3B expression:Cells were treated with 1μM 5-azadC for 72 h,and then were harvested for real-time PCR and western blotting analysis.We observed that the demethylating agent 5-azadC significantly elevated the transcription level of hMLH 1 in Ishikawa cells.Western blotting analysis confirmed the induction of hMLH1 expression in Ishikawa cells.At 72 h post-treatment,a significant activation of hMLH1 expression occurred,a time when apoptosis was extensive.Furthermore,the demethylating agent 5-azadC resulted in the reduction of DNMT3B in Ishikawa cells.Aider 72 h treatment,an increase of hMLH1 and a decrease of DNMT3B were also revealed in HHUA and KLE cell lines,suggesting that a correlation between hMLH1 and DNMT3B,and 5-aza-2' -deoxycytidine is a potent inhibitor of DNA methyltransferase 3B and induces apoptosis in human endometrial cancer cell lines with the up-regulation of hMLH1.Conclusion:Our results suggested that 5-aza-2'-deoxycytidine is a potent inhibitor of DNA methyltransferase 3B and induces apoptosis in Ishikawa cells with the up-regulation of hMLH1.