胶质瘤组织中DNA修复基因MGMT启动子区过甲基化研究

背景与目的：O^6-甲基鸟嘌呤-DNA甲基转移酶（O^6-methylguanine-DNA methyltransferase,MGMT）是肿瘤细胞产生亚硝脲药物抗药性的分子基础，启动子区过甲基化而导致MGMT基因的转灵失活，影响MGMT蛋白表达，本研究探索了胶质瘤组织中MGMT基因启动子区过甲基化状态及其与MGMT蛋白表达的关系。方法：采用甲基化特异性聚集合酶链反应分析胶质瘤组织中MGMT基因启动子区过甲基化状态，同时采用免疫组织化学法分析胶质瘤组织中MGMT蛋白表达情况。结果：在27例胶质瘤患者的肿瘤组织标本中，18例MGMT蛋白表达呈阳性的胶质瘤组织中7例MGMT基因启动甲基化，阳性率为38.9%；9例MGMT蛋白表达呈阴性的胶质瘤组织中7例MGMT基因启动子甲基化、阳性率为77.8%（P〈0.05）。结论：MGMT基因启动子区的甲基化状态与MGMT蛋白的表达相关，MGMT基因启动子过甲基化，MGMT蛋白表达较低；MGMT基因启动子去甲基化，MGMT蛋白表达较高。     

MGMT基因甲基化检测在神经胶质瘤治疗中的应用

摘 要：神经胶质瘤是一种常见的、复杂的恶性肿瘤,现有的治疗方法仍不能明显提高患者的生存期。近年来,越来越多的研究表明MGMT启动子甲基化状态与神经胶质瘤诊断、治疗和预后密切相关。编码修复O6-甲基鸟嘌呤的MGMT可以帮助肿瘤细胞逃避烷化剂的细胞毒作用,但MGMT启动子常会因为发生甲基化降低基因的表达,最终阻碍DNA的修复。研究已证实MGMT基因甲基化是一种可靠的胶质瘤预测标志物,且MGMT基因甲基化的胶质瘤患者对化、放疗更敏感,生存期更长。根据患者的MGMT 基因甲基化状态,制定及实施不同的治疗方案,实现个体化治疗或许可以提高化、放疗疗效和改善预后。因此,本文对MGMT基因甲基化检测在神经胶质瘤治疗中的作用以及目前常用的MGMT基因启动子甲基化检测方法的研究进展进行了综述。     

人原发胶质母细胞瘤组织中NF-κB、TP53及MGMT甲基化与MGMT蛋白表达的相关性研究

背景与目的：胶质瘤化疗敏感性与一些分子表达相关。本研究检测和分析原发性胶质母细胞瘤（glioblastoma multifolille,GBM）患者肿瘤组织中核因子κB（NF-κB）、突变型P53（TP53）及O^6-甲基鸟嘌呤-DNA甲基转移酶｜（O^6-methylguanine—DNA methyltransferase,MGMT）基因启动子区甲基化与MGMT蛋白表达的相关性．以探讨调控MGMT蛋白表达的作用机制。方法：采用甲基化特异性PCR方法（methylation specific PCR,MSP）检测我院收治的120例原发性GBM患者肿瘤组织标本MGMT基因启动子区甲基化：采用免疫组化方法检测NF—κB、TP53、MGMT蛋白表达情况。采用SPSS17．0软件及Spearman相关系数分析方法进行统计学分析。结果：免疫组化表明NF—κB、TP53表达与MGMT表达呈正相关（r=0．244,r=0．271,P均〈0．05）,NF—κB与TP53表达亦呈正相关r=-0．329,P〈0．05）。MSP结果显示MGMT基因启动子区甲基化率与MGMT蛋白表达强度无相关性。结论：转录因子NF—κB与TP53对原发性GBM肿瘤组织中MGMT蛋白表达可能存在正调控作用,而MGMT基因启动子区甲基化率与MGMT蛋白表达强度无相关性。     

甲基转移酶与肿瘤耐药预见性、个体化化疗的研究

如何预见和克服肿瘤细胞对化疗药物的耐药性是肿瘤化疗急需解决的问题。我们通过对中国人肿瘤细胞系、裸鼠移植瘤及肿瘤患者的组织分子水平的研究,探讨肿瘤细胞中O^6-甲基鸟嘌呤DNA甲基转移酶(O^6-methylguanine-DNA methyltraasferase,MGMT)活性和蛋白水平高低与细胞对烷化剂、特别是对亚硝脲化疗药物耐药的关系。研究证明,肿瘤细胞的MGMT蛋白水平高,对烷化剂耐药;MGMT蛋白水平低对烷化剂敏感。在研究工作基础上,我们提出了以分析MGMT蛋白为依据．合理使用烷化剂、特别是使用亚硝脲类药物的预见性、个体化治疗方案。并进一步采用制备MGMT单抗的技术途径,研制出MGMT免疫组化检测试剂盒。本文结合文献和我们的研究工作,对甲基转移酶与肿瘤耐药预见性、个体化化疗的研究,从理论依据到实验研究作一全面的介绍。     

胶质瘤患者外周血 DNA 修复基因甲基化与蛋白表达的相关性

目的：探讨胶质瘤患者O6-甲基鸟嘌呤-DNA-甲基转移酶（ O6-Methylguanine -DNA methyltransferase,MGMT）及人类错配修复基因 MSH2（Human mutS homolog2,hMSH2）蛋白表达与外周血相应基因启动子甲基化的相关性。方法分别采用免疫组化法及甲基化特异性PCR （ MSP ）检测275例胶质瘤患者肿瘤组织MGMT、hMSH2蛋白的表达及外周血中这两个基因启动子甲基化情况。结果脑胶质瘤患者肿瘤组织MGMT和hMSH2蛋白阴性表达率分别为47．2％和62．5％;基因启动子区甲基化阳性率分别为41．8％和22．4％。统计学分析显示外周血MGMT基因启动子甲基化与肿瘤组织蛋白阴性表达相关（P＜0．05）。 hMSH2基因启动子甲基化与肿瘤组织hMSH2蛋白表达不具有相关性（P＞0．05）。结论 MGMT基因甲基化是脑胶质瘤发生过程中常见的分子事件,可能与胶质瘤的发生有关;而hMSH2基因启动子甲基化可能并不是胶质瘤hMSH2蛋白失活的主要原因,可能存在其他重要因素影响其表达。     

胶质瘤MGMT启动子甲基化及其临床意义

目的 分析O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）基因启动子甲基化状态及其与胶质瘤临床病理特征、预后的关系。方法 收集2012年1月至2013年6月间行手术治疗的70例胶质瘤组织和14例非肿瘤患者正常脑组织，采用甲基化特异性 PCR法（MSP）检测MGMT甲基化水平，分析其与胶质瘤临床病理特征的关系。比较不同MGMT甲基化状态的高、低级别胶质瘤患者的总生存（OS），Cox比例风险回归模型分析影响低级别胶质瘤患者的OS的因素。结果 70例胶质瘤患者中48例（68.6％）MGMT基因启动子甲基化，而正常脑组织标本中仅2例（14.3％）MGMT甲基化，差异有统计学意义（P＜0.05）。MGMT甲基化与年龄、性别、肿瘤类型、KPS评分、p53和Ki-67表达无关（P＞0.05）；与病理分级有关（P＜0.05）。低级别脑胶质瘤患者中，MGMT甲基化患者中位OS为30个月，明显长于非甲基化者的11个月，差异具有统计学意义（P＜0.05）。单因素分析显示WHO病理分级、烷化剂化疗、MGMT甲基化与低级别脑胶质瘤患者OS有关（P＜0.05）。多因素分析WHOⅡ级、未接受烷化剂化疗、MGMT非甲基化是影响低级别胶质瘤患者OS的独立危险因素（P＜0.05）。结论 MGMT甲基化与胶质瘤的发生、发展有关，在判断胶质瘤恶性度、评估预后及指导临床治疗方面具有一定的价值。     

agat基因差异性表达及其甲基化在胃癌发生和治疗中的意义

O^6-烷基鸟嘌呤-DNA-烷基转移酶（AGAT）,由肿瘤抑制基因agat编码,能够修复甲基亚硝脲（MNU）等烷化剂造成的损伤,减少肿瘤的发生。然而,当agat基因因其启动子CpG岛发生异常甲基化导致其基因沉默（gene scilencing）时,agat不能正常表达,细胞中AGAT活性降低,DNA受到烷化剂的作用,发生G：C-A：C突变,进而引起肿瘤的发生、进展。另一方面,肿瘤细胞中AGAT活性较高时,将对同为重要化疗药物的烷化剂产生耐药性。综述近年来胃癌组织中agat基因异常甲基化及其在胃癌发生和治疗中的研究进展。     

食管胃交界部腺癌中MGMT基因启动子区甲基化状态对预后的影响北大核心CSCD

目的检测食管胃交界部腺癌组织中O6-甲基鸟嘌呤-DNA甲基转移酶(O^6-methylguanineDNA methyltransferase,MGMT)基因启动子区甲基化状态以及蛋白表达情况,评价其与临床参数及预后的关系。方法选取107例食管胃交界部腺癌患者,应用甲基化特异性聚合酶链反应(methylmion specific PCR,MSP)检测MGMT基因甲基化情况,免疫组织化学法检测MGMT蛋白表达情况,分析两者与临床特征及生存时间的关系。结果癌组织中MGMT基因启动子区甲基化率显著高于癌旁正常组织;癌组织中MGMT蛋白阳性率显著低于癌旁正常组织。Spearman等级相关分析表明MGMT基因启动子区甲基化与MGMT蛋白表达呈负相关。MGMT基因启动子甲基化及蛋白表达与淋巴结转移、pTNM分期之间具有相关性。多因素Cox回归分析,MGMT基因启动子区甲基化、MGMT蛋白表达及pTNM分期是影响患者生存的独立预后因素。结论 MGMT基因启动子甲基化、MGMT蛋白表达与pTNM分期是影响食管胃交界部腺癌患者预后的独立影响因素。     

DNA修复基因MGMT启动子区过甲基化与食管鳞状细胞癌

目的：O^6－甲基鸟嘌呤DNA甲基转移酶（MGMT）可以转移DNA加合物O^6－甲基鸟嘌呤中的甲基,从而修复DNA损伤,许多肿瘤中发现MGMT基因启动子过甲基化导致该基因失活,我们研究了MGMT基因启动子甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应及测序方法分析食管癌。癌旁组织和正常食管上皮中MGMT启动子甲基化状态。结果：在检测的199例食管癌组织中,46例（38．7％）有MGMT基因启动子过甲基化,相应癌旁组织22例中也有5例（22．7％）出现MGMT基因甲基化,而21例正常食管上皮均无此种改变。结论：MGMT基因启动子过甲基化是食管癌中常见的分子事件,可能发生在癌过程的早期阶段。     

脑胶质瘤O6-甲基鸟嘌呤-DNA甲基转移酶启动区甲基化及其表达异质性的研究

目的研究脑胶质瘤组织不同部位O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）启动区甲基化状态及其表达的差异性。方法在54例脑胶质瘤组织的中心和周边部位分别获取标本,共获得92份标本,采用巢式甲基化特异性PCR（MSP）进行MGMT启动区甲基化状态的检测,同时采用免疫组织化学方法进行MGMT蛋白表达的检测。结果 38例胶质瘤中获得了肿瘤两个不同部位MG-MT启动区甲基化的状态,其中24例胶质瘤MGMT启动区甲基化的状态是一致的,占总数的63.2%（24/38）。在29例胶质瘤患者中获得了肿瘤两个不同部位MGMT蛋白表达的情况,其中10例胶质瘤MGMT蛋白表达是一致的,占总数的34.5%（10/29）。两总体一致性概率间差值的95%置信区间为（5.6%和51.8%）,两者的差异有统计学意义。巢式MSP和免疫组化都获得检测结果的标本有77份,在61份MGMT启动区甲基化标本中,25份MGMT蛋白表达阴性,14份蛋白表达可疑阳性,18份蛋白表达阳性,4份蛋白表达强阳性。在16份MGMT启动区非甲基化标本中,2份MGMT蛋白表达阴性,2份蛋白表达可疑阳性,9份蛋白表达阳性,3份蛋白表达强阳性,MGMT启动区甲基化状态与蛋白表达呈负相关（r=-0.318,P=0.005）。结论脑胶质瘤MGMT蛋白表达在同一肿瘤的不同部位存在明显的异质性。虽然脑胶质瘤MGMT启动区甲基化的状态在同一肿瘤的不同部位存在一定的异质性,但是大多数脑胶质瘤MGMT启动区甲基化的状态在同一肿瘤的不同部位是一致的。脑胶质瘤MGMT启动区甲基化状态的一致性优于蛋白表达的一致性。脑胶质瘤MGMT启动区甲基化,MGMT蛋白表达较少,MGMT启动区非甲基化,MGMT蛋白表达较多。     

Correlation of Clinical Features and Methylation Status of MGMT Gene Promoter in Glioblastomas

In an effort to extend the potential relationship between the methylation status of MGMT promoter and response to CENU therapy, we examined the methylation status of MGMT promoter in 44 patients with glioblastomas. Tumor specimens were obtained during surgery before adjuvant treatment, frozen and stored at -80 degrees C until for DNA extraction process. DNA methylation patterns in the CpG island of the MGMT gene were determined in every tumor by methylation specific PCR (MSP). These results were then related to overall survival and response to alkylating agents using statistical analysis. Methylation of the MGMT promoter was detected in 68% of tumors, and 96.7% of methylated tumors exhibited also an unmethylated status. There was no relationship between the methylation status of the MGMT promoter and overall survival and response to alkylating agents. Our observations do not lead us to consider promoter methylation of MGMT gene as a prognostic factor of responsiveness to alkylating agents in glioblastomas.     

Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer

O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfite-sequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.     

食管癌中DNA修复基因MGMT启动子区CpG岛过甲基化研究

目的：探索O~0-甲基鸟嘌呤-DNA甲基转移酶(O~6-methylguanine-DNA methyltransferase,MGMT)基因启动子区过甲基化状态与食管癌的关系。方法：采用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)分析食管癌、癌旁和正常食管黏膜细胞中MGMT基因启动子区过甲基化状态。用免疫组织化学SP法检测上述组织中MGMT蛋白表达情况。结果：76例食管癌组织中有26例(34.2%)MGMT基因启动子过甲基化,相应的癌旁组织中有8例发生甲基化,而正常食管黏膜细胞中均无甲基化。所有甲基化的癌组织中均无MGMT蛋白表达,而所有非甲基化的癌组织、相应癌旁组织和8例正常组织中均有MGMT蛋白表达。结论：MGMT基因启动子区过甲基化而使其蛋白表达缺失,可能是食管癌发生的重要因素。     

TMS1/ASC基因CpG岛甲基化与膀胱移行细胞癌发生的关系

目的检测抑癌基因TMS1/ASC启动子区5′CpG岛甲基化状态及其在膀胱移行细胞癌（BTCC）中mRNA和蛋白表达水平。方法应用MSP技术检测膀胱移行细胞癌中TMS1/ASC基因启动子区甲基化状态,RT-PCR和Western blot法分别检测其mRNA和蛋白表达水平。结果TMS1/ASC基因在正常膀胱组织中未发生甲基化,而在癌组织中甲基化频率为46.9%（15/32）,并且随着肿瘤分级、分期的增加,其甲基化水平逐渐升高（χ^2=23.106,P〈0.05）。在15例启动子异常甲基化的BTCC标本中,14例同时伴有TMS1/ASC基因表达缺失或下调,两者存在明显的相关性（γ=0.5842,P〈0.05）。TMS1/ASC mRNA和蛋白表达在正常膀胱组织和BTCC组织中分别为81.3%（26/32）、18.8%（6/32）（P〈0.01）,不同病理分级、临床分期间差异有统计学意义（P〈0.05）。结论TMS1/ASC基因启动子区异常甲基化可能导致该基因转录表达失活,使其mRNA和蛋白表达减少,甚至缺失,这可能是膀胱癌发生、发展的原因之一。     

胃癌BRCA1基因甲基化与BRCA1 mRNA表达的关系研究

目的：探讨乳腺癌易感基因1（BRCA1）启动子CpG岛甲基化与BRCA1 mRNA表达的相关性及其意义。方法采用甲基化特异性PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1基因启动子CpG岛甲基化状态;采用逆转录PCR技术检测37例胃癌组织和对应癌旁组织及6例正常胃组织中BRCA1 mRNA 表达水平。结果胃癌组织中 BRCA1基因启动子 CpG 岛总甲基化率为48.6%（18/37）,显著高于癌旁组织[5.4%（2/37）]和正常胃组织[0.0%（0/6）],差异有统计学意义（χ2=17.541、5.021,P均〈0.05）。胃癌组织中BRCA1 mRNA阳性表达率为48.6%（18/37）,显著低于癌旁组织[94.6%（35/37）]和正常胃组织[100.0%（6/6）],差异有统计学意义（χ2=19.215、5.520,P均〈0.05）。BRCA1基因启动子CpG岛甲基化与BRCA1 mRNA表达成负相关（ r=-0.515,P〈0.05）。结论 BRCA1基因启动子CpG岛甲基化可能是导致BRCA1 mRNA失表达的主要原因之一。     

CpG island hypermethylation of the DNA repair enzyme methyltransferase predicts response to temozolomide in primary gliomas.

PURPOSE: The DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) inhibits the killing of tumor cells by alkylating agents, and its loss in cancer cells is associated with hypermethylation of the MGMT CpG island. Thus, methylation of MGMT has been correlated with the clinical response to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in primary gliomas. Here, we investigate whether the presence of MGMT methylation in gliomas is also a good predictor of response to another emergent alkylating agent, temozolomide. EXPERIMENTAL DESIGN: Using a methylation-specific PCR approach, we assessed the methylation status of the CpG island of MGMT in 92 glioma patients who received temozolomide as first-line chemotherapy or as treatment for relapses. RESULTS: Methylation of the MGMT promoter positively correlated with the clinical response in the glioma patients receiving temozolomide as first-line chemotherapy (n = 40). Eight of 12 patients with MGMT-methylated tumors (66.7%) had a partial or complete response, compared with 7 of 28 patients with unmethylated tumors (25.0%; P = 0.030). We also found a positive association between MGMT methylation and clinical response in those patients receiving BCNU (n = 35, P = 0.041) or procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (n = 17, P = 0.043) as first-line chemotherapy. Overall, if we analyze the clinical response of all of the first-line chemotherapy treatments with temozolomide, BCNU, and procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea as a group in relation to the MGMT methylation status, MGMT hypermethylation was strongly associated with the presence of partial or complete clinical response (P < 0.001). Finally, the MGMT methylation status determined in the initial glioma tumor did not correlate with the clinical response to temozolomide when this drug was administered as treatment for relapses (P = 0.729). CONCLUSIONS: MGMT methylation predicts the clinical response of primary gliomas to first-line chemotherapy with the alkylating agent temozolomide. These results may open up possibilities for more customized treatments of human brain tumors.     

DNA repair gene O6-methylguanine-DNA methyltransferase: promoter hypermethylation associated with decreased expression and G:C to A:T mutations of p53 in brain tumors

The DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) removes alkylating adducts from the O(6) position of guanine and protects cells from cytotoxic and mutagenic effects. Expression of MGMT is decreased in some cancers, which may be the result of methylation of CpG islands of both the promoter and coding regions of the gene. We studied the methylation status of the MGMT promoter in a very large collection of brain tumors (85) using methylation-specific polymerase chain reaction (PCR). Aberrant methylation occurred in 48% of 85 human brain tumor samples. Quantitative real-time PCR showed that expression of MGMT mRNA levels was significantly decreased (P < 0.001) in those brain tumors that had methylation of the promoter region of their MGMT gene. MGMT can prevent G to A mutations by removing alkyl groups from the O(6) position of guanine. We found a significantly increased frequency of G:C to A:T mutations of the p53 gene in brain tumors having a methylated MGMT promoter compared with those having an unmethylated MGMT promoter (P < 0.05), and all the non-CpG dinucleotide G:C to A:T mutations of p53 were in samples with a methylated MGMT promoter.     

O<Superscript>6</Superscript>-methylguanine DNA methyltransferase status determined by promoter methylation and immunohistochemistry in gliosarcoma and their clinical implications

O⁶-methylguanine-DNA methyltransferase (MGMT) is known as a DNA repair protein, and loss of function in MGMT is related to an increase in survival in patients with malignant gliomas treated with alkylating agents. In the present study, we determined the status of MGMT using methylation-specific polymerase chain reaction (PCR) and immunohistochemistry on paraffin-embedded specimens in 12 human gliosarcomas, and these results were then related to overall survival (OS) and response to alkylating agents. The MGMT promoter was methylated in six patients. Immunostaining of MGMT was positive in 58.3% of patients. MGMT methylation status was correlated with immunostaining results in five patients (41.7%). The median OS and progression-free survival (PFS) of the whole population were 13.4 months [95% confidence interval (CI), 12.3–14.5 months] and 8.3 months (95% CI, 7.4–9.2 months), respectively. In patients with methylated MGMT promoter, median OS was 15.0 months, compared with 11.3 months in the unmethylated group. Median PFS of gliosarcoma patients was 10.3 months for the methylated group, whereas it was 7.3 months for the unmethylated group. On multivariate analysis, patients with methylated MGMT promoter had better prognosis than patients with unmethylated MGMT promoter with respect to OS and PFS (P = 0.045 and 0.034, respectively). However, there was no statistical significance between MGMT protein expression and survival. The results show that a significant fraction of gliosarcomas have MGMT promoter methylation and protein expression, and suggest that patient survival is associated with MGMT methylation status.