DNA甲基化相关基因在子宫内膜癌细胞中的研究
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摘要
研究背景:
子宫内膜癌是女性生殖系统常见的恶性肿瘤之一,根据其病因和病理组织学特点可分为子宫内膜样癌和非子宫内膜样癌两种类型,又可称为Ⅰ型子宫内膜癌和Ⅱ型子宫内膜癌。Ⅰ型约占子宫内膜癌的80%——90%,该型多起源于增生期子宫内膜;与雌激素过度刺激有关;雌、孕激素受体表达常为阳性。近年来的研究显示,表观遗传信息的改变在此型内膜癌的发生、发展中起重要的作用。
表观遗传改变主要包括DNA甲基化的丢失、获得以及组蛋白修饰为特征的染色质结构的变化等。这些表观遗传的改变,特别是启动子的超甲基化引起的基因沉默,影响着肿瘤发展的各个阶段。通过基因启动子区及附近区域CpG岛胞嘧啶的甲基化可以在转录水平调节基因的表达,从而引起相应基因沉默,去甲基化又可恢复其表达。DNA甲基化在生理情况下就参与了控制基因的时空表达,在肿瘤发生时,癌细胞全基因组低甲基化是一个重要特征。肿瘤细胞基因组甲基化的程度与正常细胞相比仅为20——60%,同时伴有局部区域基因的高甲基化,包括肿瘤抑制基因、抑制肿瘤转移和浸润的基因、细胞周期调节基因、DNA修复基因、血管形成抑制基因等。DNA的甲基化对维持染色体的结构、X染色体的失活、基因印记以及许多人类基因病(如癌症,心血管疾病,糖尿病等)发生发展都起重要的作用。因此,基因启动子区异常甲基化作为一种高灵敏度的标志物在多种人类疾病发生发展机制的研究中受到越来越多的重视,是目前新的研究热点之一。
DNA甲基化是表遗传学上研究最深入的一种机制,是一种酶介导的化学修饰过程。DNA甲基化是指在DNA甲基转移酶(DNMT)的作用下,基因组CpG二核苷酸的胞嘧啶5′碳原子共价键结合一个甲基基团。DNA甲基化后核苷酸顺序未变,而基因表达受影响。在哺乳动物中,与甲基化有关的DNA甲基转移酶(DNMT)有三种:DNMT1、DNMT3A和DNMT3B。在发育过程中DNMT1主要作用是维持机体现存的DNA甲基化模式,而DNMT3A和DNMT3B主要是确立新的甲基化模式。三者间的关系目前还不太清楚,但编码3种酶的基因的mRNA和它们的表达蛋白在很多肿瘤细胞中都有过度表达,而且多种实验结果均提示三者可以分别通过直接或协同作用导致肿瘤细胞DNA异常甲基化。因此,它们在肿瘤细胞DNA甲基化的启动和维持中发挥着重要作用。有研究发现DNMT1和DNMT3B在子宫内膜癌中是过表达的,并且在高分化的内膜癌细胞系中的表达低于在低分化内膜癌细胞系中的表达水平,但其过表达的机制尚不清楚。
大约有5%的子宫内膜癌发生于具有较强遗传易感性妇女中,这种遗传易感性是由于和遗传性非息肉性样结直肠癌(hereditary nonpolyposis colorectalcancer,HNPCC)综合症相关的种系突变引起的,DNA修复基因的遗传性突变导致这些患者的肿瘤在整个基因组中出现大量的微卫星重复序列的突变,即微卫星不稳定性(microsatellite instability,MSI)。HNPCC表现为早发结肠癌的家族性聚集,一些其他类型癌症的发病率也有升高,其中妇女最显著的是子宫内膜癌。而这种微卫星不稳定性也出现于不带有DNA修复基因种系突变的某些散发性子宫内膜癌中,研究表明这些病例中错配修复基因功能的丢失是由于错配修复基因(如hMLH1)启动子甲基化引起的不表达造成的,同时hMLH1启动子甲基化也见于子宫内膜增生过长及邻近癌肿的正常子宫内膜,这提示错配修复基因的启动子甲基化是内膜癌发生过程中的早期事件。目前认为广泛的甲基化导致一些抑癌基因和DNA错配修复基因的降表达,可能是某些内膜癌尤其是Ⅰ型内膜癌的特征。DNA错配修复功能的丢失将加速和细胞恶性转化有关基因的微卫星序列的突变,从而加速肿瘤的恶性转化过程。而子宫内膜癌中错配修复基因(如hMLH1)启动子甲基化与DNMT3B是否有直接的关系尚不确定。
DNA甲基化引起大量基因的失活,从而在肿瘤发生中起重要作用,因此通过抑制甲基化可重新激活相应的基因,以达到治疗肿瘤的目的,这为子宫内膜癌的治疗开辟了一条新的道路。
第一部分
17-β雌二醇对子宫内膜癌细胞中DNA甲基转移酶1和DNA甲基转移酶3B的表达及活性的影响
目的:子宫内膜癌是女性生殖系统常见的恶性肿瘤之一,长期的雌激素过度刺激是子宫内膜癌发生的重要危险因素。近年来的研究显示,表观遗传的改变在此型内膜癌的发生、发展中起重要的作用。有学者发现DNMT1和DNMT3B在子宫内膜癌中是过表达的,并且在高分化的内膜癌细胞中的表达水平低于在低分化内膜癌细胞中的表达,但其过表达的机制尚不清楚。本研究利用人子宫内膜癌细胞系Ishikawa,探讨雌激素对Ishikawa细胞中DNMT1和DNMT3B表达及活性的影响。
方法:
1.Ishikawa细胞的培养:Ishikawa细胞在RPMI1640(添加10%FBS,2mM L-谷氨酸)培养基中贴壁生长。细胞在加药刺激前,在无酚红1640培养基中(添加5%碳吸附FBS,2mM L-谷氨酸)继续培养24h。
2.细胞周期的检测:实验分为五组:A对照组;B 10~(-6) M 17-β雌二醇组;C 10~(-8)M 17-β雌二醇组;D 10~(-10) M 17-β雌二醇组;E 10~(-12) M 17-β雌二醇组,加入不同浓度17-β雌二醇作用24h后,利用流式细胞术检测细胞周期的分布。
3.DNMT1和DNMT3B转录水平的检测:实验分为三组:a对照组;b 17-β雌二醇(10~(-8)M);c 17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M),加入药物作用24h后,Real-time PCR检测DNMT1和DNMT3BmRNA的表达。
4.DNMT1和DNMT3B蛋白水平的检测:实验分为三组:a对照组;b 17-β雌二醇(10~(-8)M);c 17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M),加入药物作用24h后,Western blot检测DNMT1和DNMT3B蛋白的表达。
5.17-β雌二醇作用后DNMT活性的检测:实验分为对照组和17-β雌二醇(10~(-8)M)组,药物作用24h后,检测DNA从头甲基化转移酶的活性。
结果:
1.不同浓度17-β雌二醇作用后对Ishikawa细胞增殖的影响:流式细胞术检测的细胞周期分布结果显示,与对照组相比,10~(-8)M 17-β雌二醇组S期所占的比值最大且与对照组相比较差异显著(P＜0.05),即此浓度对细胞的增殖活性最强,因此选10~(-8)M作为17-β雌二醇的作用浓度。
2.17-β雌二醇对Ishikawa细胞中DNMT1和DNMT3B转录水平的影响:与对照组和17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组相比,17-β雌二醇(10~(-8)M)组DNMT3BmRNA表达显著增加(P＜0.05);而17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组中DNMT3BmRNA表达较对照组虽有小幅度增加,但差异无显著性。ICI182780为雌激素受体拮抗剂,可拮抗雌激素的作用,加入ICI182780后DNMT3BmRNA表达水平较对照组并未见明显增加,提示DNMT3BmRNA表达的增加可能是雌激素受体途径介导的。为了进一步证明DNMT3BmRNA表达的增加不是雌激素刺激细胞增殖的结果,我们分别用GAPDH和PCNA标准化后结果仍然一致。然而,药物作用前后,DNMT1的表达未发现有明显改变。
3.17-β雌二醇对Ishikawa细胞中DNMT3B蛋白水平的影响:Western blot结果显示与对照组和17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组相比,17-β雌二醇(10~(-8)M)组DNMT3B蛋白表达显著增加(P＜0.05);而17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组中DNMT3B蛋白表达较对照组虽有小幅度增加,但差异无显著性。分别用β-actin和PCNA标准化后结果仍然一致。
4.对DNMT活性的检测:与对照组相比,17-β雌二醇(10~(-8)M)作用24h后,DNA甲基转移酶从头甲基化的活性显著提高(P＜0.05)。证明雌激素不但可以增加DNMT3B的表达,同时还使其活性提高。
结论:
1.雌激素可以显著增加子宫内膜癌细胞中DNMT3BmRNA和蛋白水平的表达,同时亦增加DNA甲基转移酶的活性,而雌激素对DNMT3B的上调有可能是通过雌激素受体途径实现的。
2.DNMT3B表达上调可导致肿瘤相关基因的异常甲基化,推测雌激素对DNMT3B的影响可能是参与子宫内膜恶性转化的机制之一。
第二部分
去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞凋亡的影响及hMLH1基因甲基化的相关研究
目的:检测去甲基化药物5-氮-2′-脱氧胞苷(5-aza-2'-deoxycytidine)对人子宫内膜癌细胞株Ishikawa、HHUA和KLE生物学行为的影响,并研究该药物对DNMT3B与hMLH1基因表达及hMLH1基因甲基化状态的影响,为子宫内膜癌的治疗提供新的作用靶点。
方法:
1.人子宫内膜癌细胞株Ishikawa、HHUA和KLE的培养:细胞在RPMI1640(添加10%FBS,2mM L-谷氨酸)培养基中贴壁生长。
2.细胞活力的检测:实验分为四组:A对照组;B0.1μM 5-氮-2′-脱氧胞苷组:C1μM 5-氮-2′-脱氧胞苷组和D5μM 5-氮-2′-脱氧胞苷组,各细胞株分组培养72h后,使用MTT方法检测细胞活力的变化。
3.观察去甲基化药物5-氮-2′-脱氧胞苷对细胞周期的影响。实验分为两组:A对照组;B1μM 5-氮-2′-脱氧胞苷组,药物作用72h后利用流式细胞术检测其对细胞周期的影响。
4.去甲基化药物5-氮-2′-脱氧胞苷对细胞凋亡的影响。实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后利用流式细胞术和TUNEL两种方法检测其对细胞凋亡的影响。
5.观察去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中hMLH1基因甲基化状态的影响。实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后Methylation-Specific PCR(MSP)检测子宫内膜癌细胞株中hMLH1基因的甲基化状态。
6.观察去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中DNMT3B与hMLH1mRNA及蛋白表达的影响。实验分为两组:A对照组:B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后用Real-time PCR和Western blot分别检测DNMT3B与hMLH1表达的变化。
结果:
1.细胞活力的检测:
MTT结果显示,去甲基化药物5-氮-2′-脱氧胞苷对细胞活力的抑制呈浓度依赖性,与正常对照相比,1μM 5-氮-2′-脱氧胞苷组可明显抑制细胞的增殖(P＜0.05)。5μM 5-氮-2′-脱氧胞苷组与对照组相比较亦可明显抑制细胞的增殖(P＜0.05),但与1μM 5-氮-2′-脱氧胞苷组比较并无显著性差异,因此确定1μM为去甲基化药物5-氮-2′-脱氧胞苷的作用浓度。观察1μM去甲基化药物5-氮-2′-脱氧胞苷作用72h后发现其对细胞活力的抑制呈时间依赖性。即去甲基化药物5-氮-2′-脱氧胞苷对细胞活力的抑制呈浓度及时间依赖性。
2.去甲基化药物5-氮-2′-脱氧胞苷对内膜癌细胞株细胞周期分布的影响:
实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后流式细胞术检测细胞周期的分布,发现细胞被阻断在G_2/M期。与对照组相比,药物组中细胞G_2/M期所占的比例增加了大约三倍。
3.去甲基化药物5-氮-2′-脱氧胞苷对细胞凋亡的影响:
利用流式细胞术和TUNEL两种方法检测发现5-氮-2′-脱氧胞苷可以显著的诱导细胞凋亡。利用Annexin-V流式细胞术可以检测早期凋亡,从而可以与坏死相鉴别,结果显示药物组早期凋亡的细胞百分比明显高于坏死的细胞,亦明显高于对照组,证明细胞活力的下降是凋亡所导致,而不是坏死。
4.去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中hMLH1甲基化状态的影响:
通过Methylation-Specific PCR对子宫内膜癌细胞株中hMLH1基因甲基化状态的检测发现去甲基化药物5-氮-2′-脱氧胞苷可以使hMLH1基因去甲基化从而激活hMLH1基因的表达。
5.去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中DNMT3B与hMLH1mRNA及蛋白表达的影响:
药物作用72h后利用Real-time PCR和Western blot检测DNMT3B与hMLH1表达水平的变化,发现去甲基化药物5-氮-2′-脱氧胞苷可以显著抑制DNMT3BmRNA及蛋白的表达,同时增加hMLH1mRNA及蛋白的表达。
结论:
1.去甲基化药物5-氮-2′-脱氧胞苷可以明显的抑制子宫内膜癌细胞的增殖,使其细胞周期阻断于G_2/M期,诱导细胞的凋亡,为子宫内膜癌的治疗提供一种新的候选药物。
2.初步印证所提出的设想:去甲基化药物5-氮-2′-脱氧胞苷通过抑制DNMT3B的表达使hMLH1去甲基化而被激活,使hMLH1的表达增加。
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 TypeⅠand TypeⅡ.Most patients present with TypeⅠwhich 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.
PARTⅠESTROGEN REGULATES DNA METHYLTRANSFERASE 3B EXPRESSION IN ISHIKAWA ENDOMETRIAL ADENOCACINOMA CELLS
Objective: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%CO_2 and 100% humidity.The entire medium was supplemented with 100μg/ml streptomycin and 100 unit/ml penicillin.To determine the effect of 17β-estradiol(E_2) 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 E_2 at various concentrations(10~(-6),10~(-8),10~(-10) and 10~(-12)M) 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 E_2(10~(-8) M) alone or E_2(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 E_2(10~(-8) M) alone or E_2(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 E_2:Ishikawa cells were cultured in the absence or in the presence of E_2 at the concentration of 10~(-8) M for 24 h.Then the DNMT activities of the cells were evaluated.
Results:
1.Effect of E_2 with different concentrations on the proliferation of Ishikawa cells: We treated the Ishikawa cells with E_2 at concentrations ranging from 10~(-6) M,10~(-8) M, 10~(-10) M to 10~(-12) M for 24 h and the effect of E_2 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 E_2.
2.Effect of E_2 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 E_2 treatment.We observed that E_2 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 E_2 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 E_2 treatment for 24 h in Ishikawa cells was observed.
3.Effect of E_2 on the protein expression of DNMT3B in Ishikawa cells:We assessed the regulation of DNMT3B in Ishikawa cells by E_2 using Western blotting analysis.Ishikawa cells were treated with E_2(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 E_2.Western blot analysis also revealed that the E_2-induced up-regulation of DNMT3B was suppressed by the addition of ICI182780.
4.E_2 increased de novo DNA methyltransferase activity:We sought to determine if E_2 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.E_2 treatment for 24 hours increased de novo DNA methylation in Ishikawa cells.These data suggest that E_2 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.
PARTⅡ5-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
Objective: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%CO_2 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 G_2/M phase of the cell cycle following treatment with 5-azadC,providing evidence of G_2/M arrest.By 72 h of treatment,approximately 3-fold of the cells arrested at G_2/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.
子宫内膜癌是女性生殖系统常见的恶性肿瘤之一,根据其病因和病理组织学特点可分为子宫内膜样癌和非子宫内膜样癌两种类型,又可称为Ⅰ型子宫内膜癌和Ⅱ型子宫内膜癌。Ⅰ型约占子宫内膜癌的80%——90%,该型多起源于增生期子宫内膜;与雌激素过度刺激有关;雌、孕激素受体表达常为阳性。近年来的研究显示,表观遗传信息的改变在此型内膜癌的发生、发展中起重要的作用。
表观遗传改变主要包括DNA甲基化的丢失、获得以及组蛋白修饰为特征的染色质结构的变化等。这些表观遗传的改变,特别是启动子的超甲基化引起的基因沉默,影响着肿瘤发展的各个阶段。通过基因启动子区及附近区域CpG岛胞嘧啶的甲基化可以在转录水平调节基因的表达,从而引起相应基因沉默,去甲基化又可恢复其表达。DNA甲基化在生理情况下就参与了控制基因的时空表达,在肿瘤发生时,癌细胞全基因组低甲基化是一个重要特征。肿瘤细胞基因组甲基化的程度与正常细胞相比仅为20——60%,同时伴有局部区域基因的高甲基化,包括肿瘤抑制基因、抑制肿瘤转移和浸润的基因、细胞周期调节基因、DNA修复基因、血管形成抑制基因等。DNA的甲基化对维持染色体的结构、X染色体的失活、基因印记以及许多人类基因病(如癌症,心血管疾病,糖尿病等)发生发展都起重要的作用。因此,基因启动子区异常甲基化作为一种高灵敏度的标志物在多种人类疾病发生发展机制的研究中受到越来越多的重视,是目前新的研究热点之一。
DNA甲基化是表遗传学上研究最深入的一种机制,是一种酶介导的化学修饰过程。DNA甲基化是指在DNA甲基转移酶(DNMT)的作用下,基因组CpG二核苷酸的胞嘧啶5′碳原子共价键结合一个甲基基团。DNA甲基化后核苷酸顺序未变,而基因表达受影响。在哺乳动物中,与甲基化有关的DNA甲基转移酶(DNMT)有三种:DNMT1、DNMT3A和DNMT3B。在发育过程中DNMT1主要作用是维持机体现存的DNA甲基化模式,而DNMT3A和DNMT3B主要是确立新的甲基化模式。三者间的关系目前还不太清楚,但编码3种酶的基因的mRNA和它们的表达蛋白在很多肿瘤细胞中都有过度表达,而且多种实验结果均提示三者可以分别通过直接或协同作用导致肿瘤细胞DNA异常甲基化。因此,它们在肿瘤细胞DNA甲基化的启动和维持中发挥着重要作用。有研究发现DNMT1和DNMT3B在子宫内膜癌中是过表达的,并且在高分化的内膜癌细胞系中的表达低于在低分化内膜癌细胞系中的表达水平,但其过表达的机制尚不清楚。
大约有5%的子宫内膜癌发生于具有较强遗传易感性妇女中,这种遗传易感性是由于和遗传性非息肉性样结直肠癌(hereditary nonpolyposis colorectalcancer,HNPCC)综合症相关的种系突变引起的,DNA修复基因的遗传性突变导致这些患者的肿瘤在整个基因组中出现大量的微卫星重复序列的突变,即微卫星不稳定性(microsatellite instability,MSI)。HNPCC表现为早发结肠癌的家族性聚集,一些其他类型癌症的发病率也有升高,其中妇女最显著的是子宫内膜癌。而这种微卫星不稳定性也出现于不带有DNA修复基因种系突变的某些散发性子宫内膜癌中,研究表明这些病例中错配修复基因功能的丢失是由于错配修复基因(如hMLH1)启动子甲基化引起的不表达造成的,同时hMLH1启动子甲基化也见于子宫内膜增生过长及邻近癌肿的正常子宫内膜,这提示错配修复基因的启动子甲基化是内膜癌发生过程中的早期事件。目前认为广泛的甲基化导致一些抑癌基因和DNA错配修复基因的降表达,可能是某些内膜癌尤其是Ⅰ型内膜癌的特征。DNA错配修复功能的丢失将加速和细胞恶性转化有关基因的微卫星序列的突变,从而加速肿瘤的恶性转化过程。而子宫内膜癌中错配修复基因(如hMLH1)启动子甲基化与DNMT3B是否有直接的关系尚不确定。
DNA甲基化引起大量基因的失活,从而在肿瘤发生中起重要作用,因此通过抑制甲基化可重新激活相应的基因,以达到治疗肿瘤的目的,这为子宫内膜癌的治疗开辟了一条新的道路。
第一部分
17-β雌二醇对子宫内膜癌细胞中DNA甲基转移酶1和DNA甲基转移酶3B的表达及活性的影响
目的:子宫内膜癌是女性生殖系统常见的恶性肿瘤之一,长期的雌激素过度刺激是子宫内膜癌发生的重要危险因素。近年来的研究显示,表观遗传的改变在此型内膜癌的发生、发展中起重要的作用。有学者发现DNMT1和DNMT3B在子宫内膜癌中是过表达的,并且在高分化的内膜癌细胞中的表达水平低于在低分化内膜癌细胞中的表达,但其过表达的机制尚不清楚。本研究利用人子宫内膜癌细胞系Ishikawa,探讨雌激素对Ishikawa细胞中DNMT1和DNMT3B表达及活性的影响。
方法:
1.Ishikawa细胞的培养:Ishikawa细胞在RPMI1640(添加10%FBS,2mM L-谷氨酸)培养基中贴壁生长。细胞在加药刺激前,在无酚红1640培养基中(添加5%碳吸附FBS,2mM L-谷氨酸)继续培养24h。
2.细胞周期的检测:实验分为五组:A对照组;B 10~(-6) M 17-β雌二醇组;C 10~(-8)M 17-β雌二醇组;D 10~(-10) M 17-β雌二醇组;E 10~(-12) M 17-β雌二醇组,加入不同浓度17-β雌二醇作用24h后,利用流式细胞术检测细胞周期的分布。
3.DNMT1和DNMT3B转录水平的检测:实验分为三组:a对照组;b 17-β雌二醇(10~(-8)M);c 17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M),加入药物作用24h后,Real-time PCR检测DNMT1和DNMT3BmRNA的表达。
4.DNMT1和DNMT3B蛋白水平的检测:实验分为三组:a对照组;b 17-β雌二醇(10~(-8)M);c 17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M),加入药物作用24h后,Western blot检测DNMT1和DNMT3B蛋白的表达。
5.17-β雌二醇作用后DNMT活性的检测:实验分为对照组和17-β雌二醇(10~(-8)M)组,药物作用24h后,检测DNA从头甲基化转移酶的活性。
结果:
1.不同浓度17-β雌二醇作用后对Ishikawa细胞增殖的影响:流式细胞术检测的细胞周期分布结果显示,与对照组相比,10~(-8)M 17-β雌二醇组S期所占的比值最大且与对照组相比较差异显著(P＜0.05),即此浓度对细胞的增殖活性最强,因此选10~(-8)M作为17-β雌二醇的作用浓度。
2.17-β雌二醇对Ishikawa细胞中DNMT1和DNMT3B转录水平的影响:与对照组和17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组相比,17-β雌二醇(10~(-8)M)组DNMT3BmRNA表达显著增加(P＜0.05);而17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组中DNMT3BmRNA表达较对照组虽有小幅度增加,但差异无显著性。ICI182780为雌激素受体拮抗剂,可拮抗雌激素的作用,加入ICI182780后DNMT3BmRNA表达水平较对照组并未见明显增加,提示DNMT3BmRNA表达的增加可能是雌激素受体途径介导的。为了进一步证明DNMT3BmRNA表达的增加不是雌激素刺激细胞增殖的结果,我们分别用GAPDH和PCNA标准化后结果仍然一致。然而,药物作用前后,DNMT1的表达未发现有明显改变。
3.17-β雌二醇对Ishikawa细胞中DNMT3B蛋白水平的影响:Western blot结果显示与对照组和17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组相比,17-β雌二醇(10~(-8)M)组DNMT3B蛋白表达显著增加(P＜0.05);而17-β雌二醇(10~(-8)M)+ICI182780(10~(-8)M)组中DNMT3B蛋白表达较对照组虽有小幅度增加,但差异无显著性。分别用β-actin和PCNA标准化后结果仍然一致。
4.对DNMT活性的检测:与对照组相比,17-β雌二醇(10~(-8)M)作用24h后,DNA甲基转移酶从头甲基化的活性显著提高(P＜0.05)。证明雌激素不但可以增加DNMT3B的表达,同时还使其活性提高。
结论:
1.雌激素可以显著增加子宫内膜癌细胞中DNMT3BmRNA和蛋白水平的表达,同时亦增加DNA甲基转移酶的活性,而雌激素对DNMT3B的上调有可能是通过雌激素受体途径实现的。
2.DNMT3B表达上调可导致肿瘤相关基因的异常甲基化,推测雌激素对DNMT3B的影响可能是参与子宫内膜恶性转化的机制之一。
第二部分
去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞凋亡的影响及hMLH1基因甲基化的相关研究
目的:检测去甲基化药物5-氮-2′-脱氧胞苷(5-aza-2'-deoxycytidine)对人子宫内膜癌细胞株Ishikawa、HHUA和KLE生物学行为的影响,并研究该药物对DNMT3B与hMLH1基因表达及hMLH1基因甲基化状态的影响,为子宫内膜癌的治疗提供新的作用靶点。
方法:
1.人子宫内膜癌细胞株Ishikawa、HHUA和KLE的培养:细胞在RPMI1640(添加10%FBS,2mM L-谷氨酸)培养基中贴壁生长。
2.细胞活力的检测:实验分为四组:A对照组;B0.1μM 5-氮-2′-脱氧胞苷组:C1μM 5-氮-2′-脱氧胞苷组和D5μM 5-氮-2′-脱氧胞苷组,各细胞株分组培养72h后,使用MTT方法检测细胞活力的变化。
3.观察去甲基化药物5-氮-2′-脱氧胞苷对细胞周期的影响。实验分为两组:A对照组;B1μM 5-氮-2′-脱氧胞苷组,药物作用72h后利用流式细胞术检测其对细胞周期的影响。
4.去甲基化药物5-氮-2′-脱氧胞苷对细胞凋亡的影响。实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后利用流式细胞术和TUNEL两种方法检测其对细胞凋亡的影响。
5.观察去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中hMLH1基因甲基化状态的影响。实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后Methylation-Specific PCR(MSP)检测子宫内膜癌细胞株中hMLH1基因的甲基化状态。
6.观察去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中DNMT3B与hMLH1mRNA及蛋白表达的影响。实验分为两组:A对照组:B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后用Real-time PCR和Western blot分别检测DNMT3B与hMLH1表达的变化。
结果:
1.细胞活力的检测:
MTT结果显示,去甲基化药物5-氮-2′-脱氧胞苷对细胞活力的抑制呈浓度依赖性,与正常对照相比,1μM 5-氮-2′-脱氧胞苷组可明显抑制细胞的增殖(P＜0.05)。5μM 5-氮-2′-脱氧胞苷组与对照组相比较亦可明显抑制细胞的增殖(P＜0.05),但与1μM 5-氮-2′-脱氧胞苷组比较并无显著性差异,因此确定1μM为去甲基化药物5-氮-2′-脱氧胞苷的作用浓度。观察1μM去甲基化药物5-氮-2′-脱氧胞苷作用72h后发现其对细胞活力的抑制呈时间依赖性。即去甲基化药物5-氮-2′-脱氧胞苷对细胞活力的抑制呈浓度及时间依赖性。
2.去甲基化药物5-氮-2′-脱氧胞苷对内膜癌细胞株细胞周期分布的影响:
实验分为两组:A对照组;B 1μM 5-氮-2′-脱氧胞苷组,药物作用72h后流式细胞术检测细胞周期的分布,发现细胞被阻断在G_2/M期。与对照组相比,药物组中细胞G_2/M期所占的比例增加了大约三倍。
3.去甲基化药物5-氮-2′-脱氧胞苷对细胞凋亡的影响:
利用流式细胞术和TUNEL两种方法检测发现5-氮-2′-脱氧胞苷可以显著的诱导细胞凋亡。利用Annexin-V流式细胞术可以检测早期凋亡,从而可以与坏死相鉴别,结果显示药物组早期凋亡的细胞百分比明显高于坏死的细胞,亦明显高于对照组,证明细胞活力的下降是凋亡所导致,而不是坏死。
4.去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中hMLH1甲基化状态的影响:
通过Methylation-Specific PCR对子宫内膜癌细胞株中hMLH1基因甲基化状态的检测发现去甲基化药物5-氮-2′-脱氧胞苷可以使hMLH1基因去甲基化从而激活hMLH1基因的表达。
5.去甲基化药物5-氮-2′-脱氧胞苷对子宫内膜癌细胞株中DNMT3B与hMLH1mRNA及蛋白表达的影响:
药物作用72h后利用Real-time PCR和Western blot检测DNMT3B与hMLH1表达水平的变化,发现去甲基化药物5-氮-2′-脱氧胞苷可以显著抑制DNMT3BmRNA及蛋白的表达,同时增加hMLH1mRNA及蛋白的表达。
结论:
1.去甲基化药物5-氮-2′-脱氧胞苷可以明显的抑制子宫内膜癌细胞的增殖,使其细胞周期阻断于G_2/M期,诱导细胞的凋亡,为子宫内膜癌的治疗提供一种新的候选药物。
2.初步印证所提出的设想:去甲基化药物5-氮-2′-脱氧胞苷通过抑制DNMT3B的表达使hMLH1去甲基化而被激活,使hMLH1的表达增加。
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 TypeⅠand TypeⅡ.Most patients present with TypeⅠwhich 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.
PARTⅠESTROGEN REGULATES DNA METHYLTRANSFERASE 3B EXPRESSION IN ISHIKAWA ENDOMETRIAL ADENOCACINOMA CELLS
Objective: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%CO_2 and 100% humidity.The entire medium was supplemented with 100μg/ml streptomycin and 100 unit/ml penicillin.To determine the effect of 17β-estradiol(E_2) 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 E_2 at various concentrations(10~(-6),10~(-8),10~(-10) and 10~(-12)M) 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 E_2(10~(-8) M) alone or E_2(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 E_2(10~(-8) M) alone or E_2(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 E_2:Ishikawa cells were cultured in the absence or in the presence of E_2 at the concentration of 10~(-8) M for 24 h.Then the DNMT activities of the cells were evaluated.
Results:
1.Effect of E_2 with different concentrations on the proliferation of Ishikawa cells: We treated the Ishikawa cells with E_2 at concentrations ranging from 10~(-6) M,10~(-8) M, 10~(-10) M to 10~(-12) M for 24 h and the effect of E_2 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 E_2.
2.Effect of E_2 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 E_2 treatment.We observed that E_2 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 E_2 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 E_2 treatment for 24 h in Ishikawa cells was observed.
3.Effect of E_2 on the protein expression of DNMT3B in Ishikawa cells:We assessed the regulation of DNMT3B in Ishikawa cells by E_2 using Western blotting analysis.Ishikawa cells were treated with E_2(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 E_2.Western blot analysis also revealed that the E_2-induced up-regulation of DNMT3B was suppressed by the addition of ICI182780.
4.E_2 increased de novo DNA methyltransferase activity:We sought to determine if E_2 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.E_2 treatment for 24 hours increased de novo DNA methylation in Ishikawa cells.These data suggest that E_2 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.
PARTⅡ5-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
Objective: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%CO_2 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 G_2/M phase of the cell cycle following treatment with 5-azadC,providing evidence of G_2/M arrest.By 72 h of treatment,approximately 3-fold of the cells arrested at G_2/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.
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1 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nature reviews Genetics 2002;3:415-428.
2 Ballestar E, Esteller M. The impact of chromatin in human cancer: linking DNA methylation to gene silencing. Carcinogenesis 2002;23:1103-1109.
3 El-Osta A. The rise and fall of genomic methylation in cancer. Leukemia 2004; 18:233-237.
4 Feinberg AP, Cui H, Ohlsson R. DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Seminars in cancer biology 2002;12:389-398.
5 Salvesen HB, MacDonald N, Ryan A, Jacobs IJ, Lynch ED, Akslen LA et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. International Joural of Cancer 2001 ;91:22-26.
6 Aebi S, Kurdihaidar B, Gordon R, Cenni B, Zheng H, Fink D et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Research 1996;56:3087-3090.
7 Sasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor B gene inactivation and CpG hypermethylation in human uterine endometrial cancer. Cancer Research 2001;61:97- 102.
8 Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293:1089-1093.
9 Bird AP. The relationship of DNA methylation to cancer. Cancer Surveys 1996; 28:87-101.
10 Warnecke PM, Bestor TH. Cytosine methylation and human cancer. Current opinion in oncology 2000;12:68- 73.
11 Jin F et al. Up-regulation of DNA methyltransferase 3B expression in endometrial cancers. Gynecologic oncology 2005;96:531-538.
12 Kallakury BV, Sheehan CE, Winn-Deen E, Oliver J, Fisher HA, Kaufman Jr. RP et al. Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas. Cancer 2001;92:2786- 2795.
13 Tsuda H, Yamamoto K, Inoue T, Uchiyama I, Umesaki N. The role of pl6-cyclind/CDK-pRb pathway in the tumorigenesis of endometrioid-type endometrial carcinoma. Br J Cancer 2000;82:675-682.
14 Gonzalez-Gomez P, Bello MJ, Lomas J, Arjona D, Alonso ME, Aminoso C et al. Aberrant methylation of multiple genes in neuroblastic tumours. Relationship with MYCN amplification and allelic status at 1p. European Joural of Cancer 2003;39:1478- 1485.
15 Ando T, Nishimura M, Oka Y. Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells. Leukemia 2000;14:1915-1920.
16 Wang H, Douglas W, Lia M, Edelmann W, Kucherlapati R, Podsypanina K et al. DNA mismatch repair deficiency accelerates endometrial tumorigenesis in PTEN heterozygous mice. The American journal of pathology 2002; 160:1481- 1486.
17 Shiozawa T, Itoh K, Horiuchi A, Konishi I, Fujii S, Nikaido T. Down-regulation of estrogen receptor by the methylation of the estrogen receptor gene in endometrial carcinoma. Anticancer Research 2002;22:139-143.
18 Risinger JI, Maxwell GL, Berchuck A, Barrett JC. Promoter hypermethylation as an epigenetic component in Type I and Type II endometrial cancers. Ann N Y Acad Sci 2003;983:208-212.
19 Whitcomb BP, Mutch DG, Herzog TJ, Rader JS, Gibb RK, Goodfellow PJ. Frequent HOXA11 and THBS2 promoter methylation, and a methylator phenotype in endometrial adenocarcinoma. Clinical Cancer Research 2003; 9:2277-2287.
20 Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A 1999;96:8681-8686.
1 Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16:6-21.
2 Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005; 74:481-514.
3 Leonhardt H, Page AW, Weier HU, Bestor TH. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 1992; 71:865-873.
4 Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99:247-257.
5 Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005; 6:597-610.
6 Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300: 455.
7 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415-428.
8 Lax SF. Molecular genetic pathways in various types of endometrial carcinoma: from a phenotypical to a molecular-based classification. Virchows Arch 2004; 444:213-223.
9 Kanaya T, et al. Frequent hypermethylation of MLH1 promoter in normal endometrium of patients with endometrial cancers. Oncogene 2003; 22:2352-2360.
10 Zhu WG, Otterson GA. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anti-Cancer Agents 2003; 3:187-199.
11 Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 2001; 134:573-586.
12 Christman JK. 5-azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002; 21:5483-5495.
13 Petti MC, et al. Pilot study of 5-aza-2_-deoxycytidine (Decitabine) in the treatment of poor prognosis acute myelogenous leukemia patients: preliminary results. Leukemia 1993; Suppl 1:36-41.
14 Kitagawa Y, et al. Demethylating reagent 5-azacytidine inhibits teloemerase activity in human prostate cancer cells through transcriptional repression of hTERT. Clin Cancer Res 2000; 6:2868-2875.
15 Greger V, Passarge E, Hopping W, Messmer E, Horsthemke B. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet 1989; 83:155-158.
16 Baylin SB, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001; 10:687-692.
17 Salvesen HB, et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. Int J Cancer 2001; 91:22-26.
18 Aebi S, et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 1996; 56:3087-3090.
19 Sasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor B gene inactivation and CpG hypermethylation in human uterine endometrial cancer. Cancer Res 2001; 61:97-102.
20 Gurin CC, Federici MG, Kang L, Boyd J. Causes and consequences of microsatellite instability in endometrial carcinoma. Cancer Res 1999; 59:462-466.
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5 Reik W,Dean W,Walter J.Epigenetic reprogramming in mammalian development.Science 2001;293:1089-1093.
6 Jin F,Dowdy SC,Xiong Y,et al.Up-regulation of DNA methyltransferase 3B expression in endometrial cancers.Gynecologic oncology 2005;96:531-538.
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1 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nature reviews Genetics 2002;3:415-428.
2 Ballestar E, Esteller M. The impact of chromatin in human cancer: linking DNA methylation to gene silencing. Carcinogenesis 2002;23:1103-1109.
3 El-Osta A. The rise and fall of genomic methylation in cancer. Leukemia 2004; 18:233-237.
4 Feinberg AP, Cui H, Ohlsson R. DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Seminars in cancer biology 2002;12:389-398.
5 Salvesen HB, MacDonald N, Ryan A, Jacobs IJ, Lynch ED, Akslen LA et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. International Joural of Cancer 2001 ;91:22-26.
6 Aebi S, Kurdihaidar B, Gordon R, Cenni B, Zheng H, Fink D et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Research 1996;56:3087-3090.
7 Sasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor B gene inactivation and CpG hypermethylation in human uterine endometrial cancer. Cancer Research 2001;61:97- 102.
8 Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293:1089-1093.
9 Bird AP. The relationship of DNA methylation to cancer. Cancer Surveys 1996; 28:87-101.
10 Warnecke PM, Bestor TH. Cytosine methylation and human cancer. Current opinion in oncology 2000;12:68- 73.
11 Jin F et al. Up-regulation of DNA methyltransferase 3B expression in endometrial cancers. Gynecologic oncology 2005;96:531-538.
12 Kallakury BV, Sheehan CE, Winn-Deen E, Oliver J, Fisher HA, Kaufman Jr. RP et al. Decreased expression of catenins (alpha and beta), p120 CTN, and E-cadherin cell adhesion proteins and E-cadherin gene promoter methylation in prostatic adenocarcinomas. Cancer 2001;92:2786- 2795.
13 Tsuda H, Yamamoto K, Inoue T, Uchiyama I, Umesaki N. The role of pl6-cyclind/CDK-pRb pathway in the tumorigenesis of endometrioid-type endometrial carcinoma. Br J Cancer 2000;82:675-682.
14 Gonzalez-Gomez P, Bello MJ, Lomas J, Arjona D, Alonso ME, Aminoso C et al. Aberrant methylation of multiple genes in neuroblastic tumours. Relationship with MYCN amplification and allelic status at 1p. European Joural of Cancer 2003;39:1478- 1485.
15 Ando T, Nishimura M, Oka Y. Decitabine (5-Aza-2'-deoxycytidine) decreased DNA methylation and expression of MDR-1 gene in K562/ADM cells. Leukemia 2000;14:1915-1920.
16 Wang H, Douglas W, Lia M, Edelmann W, Kucherlapati R, Podsypanina K et al. DNA mismatch repair deficiency accelerates endometrial tumorigenesis in PTEN heterozygous mice. The American journal of pathology 2002; 160:1481- 1486.
17 Shiozawa T, Itoh K, Horiuchi A, Konishi I, Fujii S, Nikaido T. Down-regulation of estrogen receptor by the methylation of the estrogen receptor gene in endometrial carcinoma. Anticancer Research 2002;22:139-143.
18 Risinger JI, Maxwell GL, Berchuck A, Barrett JC. Promoter hypermethylation as an epigenetic component in Type I and Type II endometrial cancers. Ann N Y Acad Sci 2003;983:208-212.
19 Whitcomb BP, Mutch DG, Herzog TJ, Rader JS, Gibb RK, Goodfellow PJ. Frequent HOXA11 and THBS2 promoter methylation, and a methylator phenotype in endometrial adenocarcinoma. Clinical Cancer Research 2003; 9:2277-2287.
20 Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A 1999;96:8681-8686.
1 Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16:6-21.
2 Goll MG, Bestor TH. Eukaryotic cytosine methyltransferases. Annu Rev Biochem 2005; 74:481-514.
3 Leonhardt H, Page AW, Weier HU, Bestor TH. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 1992; 71:865-873.
4 Okano M, Bell DW, Haber DA, Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999; 99:247-257.
5 Robertson KD. DNA methylation and human disease. Nat Rev Genet 2005; 6:597-610.
6 Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300: 455.
7 Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415-428.
8 Lax SF. Molecular genetic pathways in various types of endometrial carcinoma: from a phenotypical to a molecular-based classification. Virchows Arch 2004; 444:213-223.
9 Kanaya T, et al. Frequent hypermethylation of MLH1 promoter in normal endometrium of patients with endometrial cancers. Oncogene 2003; 22:2352-2360.
10 Zhu WG, Otterson GA. The interaction of histone deacetylase inhibitors and DNA methyltransferase inhibitors in the treatment of human cancer cells. Curr Med Chem Anti-Cancer Agents 2003; 3:187-199.
11 Santini V, Kantarjian HM, Issa JP. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 2001; 134:573-586.
12 Christman JK. 5-azacytidine and 5-aza-2'-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 2002; 21:5483-5495.
13 Petti MC, et al. Pilot study of 5-aza-2_-deoxycytidine (Decitabine) in the treatment of poor prognosis acute myelogenous leukemia patients: preliminary results. Leukemia 1993; Suppl 1:36-41.
14 Kitagawa Y, et al. Demethylating reagent 5-azacytidine inhibits teloemerase activity in human prostate cancer cells through transcriptional repression of hTERT. Clin Cancer Res 2000; 6:2868-2875.
15 Greger V, Passarge E, Hopping W, Messmer E, Horsthemke B. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum Genet 1989; 83:155-158.
16 Baylin SB, et al. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001; 10:687-692.
17 Salvesen HB, et al. PTEN methylation is associated with advanced stage and microsatellite instability in endometrial carcinoma. Int J Cancer 2001; 91:22-26.
18 Aebi S, et al. Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 1996; 56:3087-3090.
19 Sasaki M, Dharia A, Oh BR, Tanaka Y, Fujimoto S, Dahiya R. Progesterone receptor B gene inactivation and CpG hypermethylation in human uterine endometrial cancer. Cancer Res 2001; 61:97-102.
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