O~6-甲基鸟嘌呤-DNA甲基转移酶在恶性脑胶质瘤组织中的表达及意义

目的探讨O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)在恶性脑胶质瘤组织中的表达及其与预后的关系。方法选取2011年1月至2016年1月间中山市陈星海医院收治的经手术切除的64例恶性脑胶质瘤(Ⅲ、Ⅳ级)患者,采用免疫组化法检测肿瘤组织中MGMT蛋白的表达,评定治疗效果,追踪生存时间(OS),评价MGMT与生存时间之间的关系。结果 MGMT阳性表达率为62.5%,MGMT阴性表达率为37.5%。WHOⅢ级恶性胶质瘤组织中MGMT阳性表达率与Ⅳ级比较,差异无统计学意义(P>0.05);但Ⅳ级恶性胶质瘤MGMT(++)表达率为60.0%,明显高于Ⅲ级的48.3%,差异有统计学意义(P<0.05)。MGMT阴性患者总有效率(ORR)为62.5%,高于阳性患者的17.5%;与MGMT(-)比较,MGMT(+)和MGMT(++)平均OS均降低,差异均有统计学意义(均P<0.05)。MGMT阳性患者最长无进展生存时间(PFS)为32个月,MGMT(-)最长PFS为40个月。结论 MGMT表达与脑胶质瘤恶性程度有关,表达强度越高,恶性程度越高,检测MGMT可一定程度预知脑胶质瘤治疗的有效率及预后。     

不同级别胶质瘤中p53蛋白与O~6-甲基鸟嘌呤-DNA甲基转移酶表达的相关性

目的探讨不同级别胶质瘤中p53蛋白与O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)表达的相关性。方法选取2013年1月至2013年12月间行神经外科手术切除的98例脑胶质瘤组织作为研究对象,采用免疫组织化学的方法分析不同级别胶质瘤组织中p53蛋白与MGMT表达的关系。结果 MGMT在不同级别胶质瘤组织间表达无显著差异,在表达强度上差异有统计学意义(P<0.05)。p53在不同级别胶质瘤组织中的表达差异有统计学意义,在表达强度上并无明显特征。MGMT与p53在表达强度上呈负相关。结论 p53与MGMT的表达在不同级别的胶质瘤中均有一定的关系,且p53表达增强和MGMT表达减弱均对胶质瘤的发生、发展及其恶性程度产生一定的影响。     

脑肿瘤组织中O6-甲基鸟嘌呤-DNA甲基转移酶表达的研究

探讨Ｏ６┐甲基鸟嘌呤┐ＤＮＡ甲基转移酶（ＭＧＭＴ）表达与脑肿瘤对氯乙基亚硝脲（ＣＥＮＵｓ）耐药的关系。方法采用Ｗｅｓｔｅｒｎｂｌｏｔ方法检测５９例不同类型脑肿瘤组织ＭＧＭＴ蛋白表达水平,对其中３０例胶质瘤同时采用ＲＴ┐ＰＣＲ检测其ＭＧＭＴｍＲＮＡ表达水平,ＭＧＭＴｍＲＮＡ表达水平与ＭＧＭＴ活性进行相关分析。结果ＭＧＭＴｍＲＮＡ表达水平与ＭＧＭＴ活性正相关（ｒ＝０．９１１,Ｐ＜０．０１）,非胶质瘤及转移癌ＭＧＭＴ蛋白表达阳性率明显较胶质瘤高,分别为９０％,７８％,７％（Ｐ＜０．０５）;不同个体胶质瘤ＭＧＭＴｍＲＮＡ表达有较大异质性,范围为０～１．２９,平均为０．５±０．３７,Ｍｅｒ－率为３７％;高、低级别星形细胞瘤ＭＧＭＴｍＲＮＡ表达无差异（０．４７±０．３６和０．４４±０．３４）;髓母细胞瘤有较高的ＭＧＭＴｍＲＮＡ表达水平（０．８０）。结论胶质瘤ＭＧＭＴ总体水平较低,为此ＣＥＮＵｓ作为化疗首选药提供了新的理论依据;但半数以上胶质瘤为Ｍｅｒ＋表型,提示对ＣＥＮＵｓ耐药,因而化疗前检测Ｍｅｒ表型以筛选Ｍｅｒ－个体进行化疗将进一步提高疗效。     

潮汕地区食管癌患者O6-甲基鸟嘌呤-DNA甲基转移酶基因多态性的分析

背景与目的初步研究中国食管癌高发区之一潮汕地区食管癌患者和正常人群O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因编码84、143及160位密码子的单核苷酸多态性(Single nucleotide polymorphism,SNP)。材料与方法应用双色荧光杂交微阵列技术分别检测潮汕地区100例食管鳞状细胞癌患者及65例健康对照外周血MGMT基因84位密码子(C2740214T)单核苷酸多态性;用同样的方法检测76例食管鳞状细胞癌患者和50例健康对照MGMT基因143位密码子(A2798995G)、160位密码子(G2799046A)单核苷酸多态性。结果基因型分布符合Hardy-Weinberg定律。统计分析显示食管鳞状细胞癌组与对照组84位密码子2740214C和2740214T等位基因频率差异无统计学意义(P=0.402),2740214CT基因型在病例组和对照组中所占的比例分别为16%和12.3%,2740214TT基因型在病例组中所占的比例是2%。食管鳞状细胞癌组变异基因型拥有者(CT TT)高于对照组,但差异无统计学意义(P=0.327),2740214TT基因型仅见于个别肿瘤患者,而在正常对照中未发现。143位密码子A2798995G位点仅在对照组中发现一例杂合子,其余均为野生型纯合子,病例组与对照组基因型和等位基因频率差异均无统计学意义。160位密码子G2799046A位点检测在所有的病人和对照中均为野生型纯合子。结论潮汕地区食管鳞状细胞癌患者和正常人群中存在MGMT基因多态性,84位密码子C2740214T位点的突变型纯合子仅见于肿瘤患者,可能与食管鳞状细胞癌有关。     

O6-甲基鸟嘌呤-DNA甲基转移酶基因多态性与肿瘤易感性的关系

目的:探讨中国南方汉族人群O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因5个外显子的多态性与肿瘤易感性的关系.方法:采用聚合酶链式反应(PCR)-单链构象多态性(SSCP)-DNA直接测序法对100例正常人和88例肿瘤患者外周血白细胞进行MGMT基因多态性研究.结果:正常人群和肿瘤患者第3外显子第84位密码子的第1个碱基C被T所取代,导致错义突变(CTT→TTT),氨基酸由亮氨酸(Leu)变成了苯丙氨酸(Phe);第4外显子第94位密码子的第3个碱基C被T所取代,导致同义突变(TTC→TTT,均编码Phe).这2个位点的多态性在2组人群中的检出率差异不存在统计学意义(P＞0.05).同时在肿瘤患者中还检测到MGMT基因第5外显子第200位密码子的第2个碱基G被C取代,即GGC→GCC,所编码的氨基酸由甘氨酸变为缬氨酸,这一多态不存在于正常对照中.结论:MGMT基因第3、4、5外显子上存在着多态性,其中第3、4外显子的多态改变与肿瘤形成的关系较弱,而第5外显子的多态仅见于肿瘤患者,可能与肿瘤的形成有关.     

O^6-甲基鸟嘌呤-DNA甲基转移酶在大肠癌中的表达及意义

目的：检测大肠癌组织中O^6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的表达情况及其在肿瘤发生发展中的作用和临床意义。方法：采用SABC免疫组化法检测79例大肠癌标本中MGMT的表达情况。结果：大肠癌中MGMT阳性率为53．16％（42／79），其阳性表达与性别、癌细胞浸润深度无关（P〉0．05），与患者年龄、淋巴结转移情况有关（P〈0．05）。结论：检测MGMT的表达情况对于大肠癌恶性程度的判断及临床化疗方案的制定具有指导作用。     

O~6-甲基鸟嘌呤-DNA甲基转移酶甲基化状态和同期放化治疗与中国香港胶质母细胞瘤患者生存的相关性研究

背景与目的:胶质母细胞瘤(GBM)是一种恶性程度最高的星形细胞瘤,其主要的治疗方法是外科手术和放射疗法,2005年批准使用替莫唑胺,化学疗法的效果才得以确定。本文比较中国香港原发性GBM患者接受同期放化治疗或单纯放疗的生存时间,探讨GBM肿瘤组织中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)的甲基化状态在预后评价的价值。方法:回顾性分析2005年3月至2007年6月期间35例经手术后病理确诊并接受过同期放化治疗或单纯放疗的中国香港GBM患者的资料,从石蜡包埋的GBM肿瘤组织中分离出基因组DNA,采用甲基化特异性聚合酶链反应方法(MSP)检测基因MGMT甲基化状态。采用Kaplan Meier方法计算总生存时间(OS)和无进展生存时间(PFS),比较同期放化治疗或单纯放疗和MGMT甲基化或非甲基化状态对生存时间的影响。结果:35例患者中,男性27例,女性8例,平均年龄50.4岁。35例患者的中位PFS和中位OS分别是4.7个月(3.1~6.2个月)和11.7个月(6.5~16.6个月),其中18例仅接受单纯放疗患者的中位PFS和中位OS分别是4.2个月(3.4~5.0个月)和5.8个月(2.0~9.6个月),17例接受同期放化治疗患者的中位PFS和中位OS分别是6.0个月(2.0~10个月)和13.2个月(8.1~18.3个月),P值>0.05。15例(43%)肿瘤组织中存在MGMT甲基化,MGMT甲基化和非甲基化患者的中位OS分别是16.9个月(12.7~21.1个月)和10个月(5.8~14.1个月),P值>0.05。结论:尽管差异无统计学意义,但与单纯放疗比较,接受了同期放化治疗的GBM患者显示出了更长的总体生存趋势,MGMT甲基化可能是GBM的明显预后较好的因素。     

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。     

人O6-烷基鸟嘌呤-DNA烷基转移酶突变体对O6-苄基鸟嘌呤耐药性的研究

目的　探讨人O6 烷基鸟嘌呤 DNA烷基转移酶 (O6 alkylguanine DNAalkyltransferase ,O6 AGT)中单个氨基酸的改变对O6 苄基鸟嘌呤 (O6 benzylguanine ,O6 BG)抑制作用敏感性的影响。方法应用定点诱导突变的方法 ,将人AGT的第 15 6位点的甘氨酸突变为丙氨酸 ,第 140位点的脯氨酸突变为丙氨酸 ,并检测了重组蛋白的活性。结果　点突变G15 6A和P140A的重组蛋白的AGT活性分别为(2 44± 90 )fmol/mg和 (2 31± 48)fmol/mg蛋白 ,与野生型AGT活性 (2 2 3± 35 )fmol/mg蛋白相近 ,并且对O6 BG具有耐药性。O6 BG对突变后的G15 6A AGT和P140A AGT的IC50 分别为 7.2 0 μg/ml和 0 .92 μg/ml,而对野生型AGT的IC50 为 0 .0 6 8μg/ml,耐药性分别提高了 10 5 .8及 13.5倍。 结论　应用G15 6A和P140A基因转导 ,可克服BG与烷化剂联合化疗的骨髓抑制作用     

O6-甲基鸟嘌呤-DNA甲基转移酶在大肠癌中的表达及意义

目的:检测大肠癌组织中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)的表达情况及其在肿瘤发生发展中的作用和临床意义.方法:采用SABC免疫组化法检测79例大肠癌标本中MGMT的表达情况.结果:大肠癌中MGMT阳性率为53.16%(42/79),其阳性表达与性别、癌细胞浸润深度无关(P>0.05),与患者年龄、淋巴结转移情况有关(P＜0.05).结论:检测MGMT的表达情况对于大肠癌恶性程度的判断及临床化疗方案的制定具有指导作用.     

Predictive impact of MGMT promoter methylation in glioblastoma of the elderly

O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation identifies a subpopulation of glioblastoma patients with more favorable prognosis and predicts a benefit from alkylating agent chemotherapy (CT). Little is known about its prevalence and clinical significance in older glioblastoma patients. We studied 233 glioblastoma patients aged 70 years or more (144 males, 89 females, median age: 74 years, range: 70.0-86.6 years), who were prospectively enrolled in the German Glioma Network, for MGMT promoter methylation by methylation-specific PCR (MSP) in all patients and DNA pyrosequencing in 166 patients. MGMT data were correlated with patient outcome. Median progression-free survival (PFS) was 4.8 months (95% CI: 4.3-5.3) and median overall survival (OS) was 7.7 months (95% CI: 6.3-9.0). MGMT promoter methylation was detected by MSP in 134 patients (57.5%). For the whole cohort, PFS was 5.2 versus 4.7 months (p = 0.207) and OS was 8.4 versus 6.4 months (p = 0.031) in patients with versus without MGMT promoter methylation. Patients with MGMT methylated tumors had longer PFS when treated with radiotherapy (RT) plus CT or CT alone compared to patients treated with RT alone. Patients with MGMT unmethylated tumors appeared to derive no survival benefit from CT, regardless of whether given at diagnosis together with RT or as a salvage treatment. Patients treated with RT plus CT or CT alone demonstrated longer OS when pyrosequencing revealed >25% MGMT methylated alleles. Taken together, MGMT promoter methylation may be a useful biomarker to stratify elderly glioblastoma patients for treatment with versus without alkylating agent CT.     

选择性化疗方案治疗恶性胶质瘤的疗效和生存情况分析

背景与目的：O^6-甲基鸟嘌呤-DNA甲基转移酶（O^6-methylguanine-DNA methyhransferase,MGMT）表达与胶质瘤患者的化疗耐药相关。本研究总结MGMT高表达和低表达恶性胶质瘤患者的化疗方案、近期疗效和生存情况．分析选择性化疗是否对MGMT高表达患者有益。方法：自2000年8月至2006年1月,中山大学肿瘤防治中心神经肿瘤科收治的经手术后病理确诊的成人恶性脑胶质瘤患者57例．所有病例化疗前均有可评价病灶。化疗前用免疫组化方法检测肿瘤组织MGMT表达情况．对MGMT高表达者．尽量避免使用亚硝脲类或替莫唑胺单药化疗,采用不含亚硝脲类或替莫唑胺方案,或由替莫唑胺（temozolomide,TMZ）和顺铂组成联合化疗方案;或亚硝脲类药物、替莫唑胺分别与其他细胞毒药物组成联合化疗方案（VM-26、DDP、CBP、IFO、VP16）;对MGMT低表达者,不限制亚硝脲类药物或替莫唑胺的应用。结果：35例患者MGMT高表达,22例MGMT低表达,MGMT低表达组的客观有效率（objective response,OR）和疾病控制率（response rate,RR）高于MGMT高表达组（40．9％：22．9％和72．7％：60.0%．但差异无统计学意义（P〉0．05）。57例中位随访时间11．7个月（0．7～53．4）。MGMT低表达组和高表达组中位无疾病进展时间（Drogressive-free survival,PFS）分别是8．5个月（95％CI 4．8—19．3）和6．7（95％CI 3．7—9．3）,中位生存时间（overail survival,OS）分别是20．3（95％CI 14．3～）和16．105％CI 11．1～26．2）,中位PFS和0S在MGMT低表达组和高表达组差异无统计学意义（P〉0．05）。结论：在化疗前检测恶性胶质瘤MGMT表达情况,对MGMT高表达患者选用有助于克服耐药的化疗方案进行选择性化疗。可使MGMT高表达患者的近期疗效（客观有效率和疾病控制率）和生存时间（无疾病进展生存和总生存）达到MGMT低表达患者水平。     

DNA甲基化与胶质瘤

表遗传学修饰的DNA甲基化在胶质瘤的发生发展中起重要作用。大量肿瘤抑制基因、细胞周期调控基因、DNA损伤修复基因、肿瘤侵袭相关基因等启动子区域CpG岛甲基化,与胶质瘤的发生发展密切相关。不同基因的甲基化发生频率与胶质瘤的组织类型和病理分级有关。某些基因的DNA甲基化可以为胶质瘤的早期诊断和预后评价提供分子生物学标记物,同时由于DNA甲基化具有可逆转性,也可为胶质瘤的基因治疗提供新的治疗靶点。     

MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系

背景与目的:目前的研究已经证实DNA修复酶——6-氧-甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine-DNAmethyltransferase,MGMT)在脑胶质瘤组织中的表达与肿瘤的耐药性有一定的关系,并且能够影响肿瘤的化疗效果。本研究通过分析MGMT在脑胶质瘤组织中的表达及其与患者生存期的关系,为基于耐药机制上的脑胶质瘤分子分类提供参考资料。方法:用组织芯片技术和免疫组织化学方法检测311例脑胶质瘤石蜡标本中MGMT的表达情况,并对所有患者进行手术后的5年随访。结果:MGMT表达阳性者126例,占40.51%(126/311)。其中,星形细胞瘤中阳性率为50.41%(61/121),少枝胶质细胞瘤中为25.71%(18/70),少枝星形细胞瘤中为28.13%(18/64),胶质母细胞瘤中为51.79%(29/56);在Ⅰ～Ⅱ级胶质瘤中,MGMT表达的阳性率为36.56%(68/186),而在Ⅲ～Ⅳ级胶质瘤中为46.40%(58/125),经χ2检验分析,两者之间有显著性差异(P<0.001);将MGMT的表达与患者生存期的关系绘制成Kaplan-Meier生存曲线,并进行log-rank分析,MGMT表达阳性者与阴性者之间的差异有显著性(P<0.05)。结论:MGMT在脑胶质瘤的异常表达与肿瘤的组织类型、病理级别有关,MGMT表达阳性患者的生存期明显低于表达阴性的患者。     

O(6) -methylguanine-DNA methyltransferase (MGMT) promoter methylation and low MGMT-encoded protein expression as prognostic markers in glioblastoma patients treated with biodegradable carmustine wafer implants after initial surgery followed by radiotherapy with concomitant and adjuvant temozolomide

BACKGROUND:

O⁶‐methylguanine‐DNA methyltransferase (MGMT) promoter methylation status was proposed as a prognostic biomarker for patients with glioblastoma. However, the prognostic impact of MGMT in patients with newly diagnosed glioblastoma who receive carmustine‐releasing wafers (Gliadel) along with temozolomide (TMZ) is still unknown.

METHODS:

MGMT promoter methylation status and protein expression were analyzed in formalin‐fixed, paraffin‐embedded tumor specimens obtained from 111 French patients with newly diagnosed glioblastoma. Patients received the Gliadel wafers followed by radiotherapy plus concomitant and adjuvant TMZ chemotherapy while they were enrolled in a French multicenter prospective study.

RESULTS:

For the whole cohort, the median overall survival (OS) was 17.5 months, and the progression‐free survival was 10.3 months. Patients with tumors that harbored MGMT methylation had a significantly longer OS compared with patients who had wild‐type MGMT (21.7 months vs 15.1 months; P = .025). Similarly, patients who had low MGMT protein expression (≤15%) had a significantly improved OS compared with patients who had high MGMT expression (27.0 months vs 15.1 months; P = .021). The extent of resection was the strongest clinical predictor of outcome. In multivariate Cox models that were adjusted for sex, performance status, and extent of surgery, both MGMT methylation and protein expression were identified as independent prognosticators, and the finding was validated internally using a bootstrap resampling technique. Discrepancies were identified between protein expression and MGMT methylation status, thus suggesting that the 2 assays probably assess different biologic features.

CONCLUSIONS:

MGMT promoter methylation status and low MGMT expression both were identified as positive prognosticators in patients with newly diagnosed glioblastoma who underwent surgical resection and received Gliadel wafer implants followed by adjuvant radiotherapy and concomitant oral TMZ chemotherapy (the Stupp protocol). Cancer 2012. © 2012 American Cancer Society.     

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUNDO⁶-methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the mispairing base O⁶-methyl-guanine induced by environmental and experimental carcinogens. It can transfer the alkyl group to a cysteine residue in its active site and became inactive. The chemical carcinogen N-nitroso compounds (NOCs) can directly bind to the DNA and induce the O⁶-methylguanine adducts, which is an important cause of gene mutation and tumorigenesis. However, the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis, especially in the initiation phase, remains largely unclear.AIMTo investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODSWe established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitroso-urea (MNU) treatment. Cell proliferation, colony formation, soft agar, cell migration, and xenograft assays were used to verify the malignant phenotype. By using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis, we detected the MGMT expression in malignant transformed cells. We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry. MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR. The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays. RESULTSWe observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment. Moreover, we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells. Inhibition of the MGMT expression by O⁶-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes. Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU. Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues, but downregulated in the gastric cancer tissues.CONCLUSIONOur finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation. The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis, which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.     

Contribution of O6-methylguanine-DNA methyltransferase to monofunctional alkylating-agent resistance in human brain tumor—derived cell lines

The DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and gliomaderived cell lines differ in sensitivity to the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD₁₀). By using the substrate analogue inhibitor O⁶-benzylguanine (O⁶-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD₁₀ among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD₁₀, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O⁶-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial. © 1995 Wiley-Liss, Inc.     

Expression of human O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells and restoration of cellular resistance to certain N-nitroso compounds.

We have constructed a plasmid in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA is driven by the Rous sarcoma virus promoter sequence. Transfection of this plasmid into Chinese hamster ovary (CHO) cells results in expression of MGMT and in cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(2-chloroethyl)-1-nitrosourea (CNU), but not to N-nitroso-N-ethylurea. The specific activity of MGMT in transfected CHO cells correlated well with their resistance to MNNG and CNU. Southern analysis showed that the plasmid had been integrated into the CHO cell genome. Western analysis of extracts from transfected CHO cells using an antibody against a peptide corresponding to the carboxyl-terminal end of the human MGMT protein demonstrated a single band with a molecular size of 24-25 kDa; no such band was observed in extracts from wild-type CHO cells. These transfected cells may therefore be used to study the role of MGMT in the repair of alkylating DNA lesions and to determine its importance in carcinogenesis as well as in chemotherapy.