Grombacher T, Mitra S, Kaina B
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Germany.
Carcinogenesis. 1996 Nov;17(11):2329-36. doi: 10.1093/carcin/17.11.2329.
Repair of alkylated bases in DNA is performed by O6-methylguanine-DNA methyltransferase (MGMT) and a set of enzymes of the base excision repair pathway involving N-methylpurine-DNA glycosylase (MPG), apurinic endonuclease (APE), DNA polymerase beta (Pol beta) and DNA ligase. The level of expression of these enzymes may exert a profound effect on resistance of cells towards alkylating drugs. We have comparatively analyzed the expression of MGMT and the different base excision repair genes in rat hepatoma cells (line H4IIE) after exposure to alkylating agents, X-rays and the glucocorticoid hormone dexamethasone. Furthermore, the effect of these agents on the activity of the cloned human MGMT promoter was assayed. Exposure of cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or ionizing radiation increased MGMT mRNA levels up to 4.5-fold. Under the same conditions of treatment, exerting only a weak toxic effect, MPG and DNA ligase I mRNA levels were not enhanced, whereas the amounts of APE and Pol beta mRNA transiently increased by approximately 2-fold after X-ray and MNNG treatment, respectively. Dexamethasone induced both MGMT, APE and Pol beta mRNA and the induction paralleled the increase in mRNA of the glucocorticoid-dependent gene tyrosine aminotransferase. The observed increase in MGMT mRNA was due to promoter activation, which was shown in transient transfection assays with MGMT promoter-CAT reporter constructs in H4IIE cells. In these assays, the human MGMT promoter was found to be induced by methylating agents (MNNG and methyl methanesulfonate), ionizing radiation and dexamethasone. Weak induction of the promoter was observed after UV irradiation. Treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate was ineffective in promoter activation. The transfected MGMT promoter was not inducible by mutagens in HeLa S3 cells, which do not respond with induction of the endogenous MGMT gene. This is the first report showing hormone induction of a DNA repair gene (MGMT). The induction of MGMT and other genes encoding enzymes involved in DNA alkylation damage repair may be relevant in cancer therapy by causing resistance of tumor cells to alkylating drugs.
DNA中烷基化碱基的修复由O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）以及碱基切除修复途径中的一组酶来完成，这些酶包括N-甲基嘌呤-DNA糖基化酶（MPG）、脱嘌呤内切酶（APE）、DNA聚合酶β（Pol β）和DNA连接酶。这些酶的表达水平可能对细胞对烷基化药物的抗性产生深远影响。我们比较分析了大鼠肝癌细胞（H4IIE系）在暴露于烷基化剂、X射线和糖皮质激素地塞米松后MGMT以及不同碱基切除修复基因的表达。此外，还检测了这些试剂对克隆的人MGMT启动子活性的影响。细胞暴露于N-甲基-N'-硝基-N-亚硝基胍（MNNG）或电离辐射后，MGMT mRNA水平增加高达4.5倍。在相同的处理条件下，MPG和DNA连接酶I mRNA水平没有增强，仅产生微弱的毒性作用，而X射线和MNNG处理后，APE和Pol β mRNA的量分别短暂增加约2倍。地塞米松诱导了MGMT、APE和Pol β mRNA，且这种诱导与糖皮质激素依赖性基因酪氨酸转氨酶mRNA的增加平行。观察到的MGMT mRNA增加是由于启动子激活，这在H4IIE细胞中用MGMT启动子-CAT报告基因构建体进行的瞬时转染实验中得到了证实。在这些实验中，发现人MGMT启动子可被甲基化剂（MNNG和甲磺酸甲酯）、电离辐射和地塞米松诱导。紫外线照射后观察到启动子的弱诱导。用肿瘤启动子12-O-十四烷酰佛波醇-13-乙酸酯处理对启动子激活无效。转染的MGMT启动子在HeLa S3细胞中不能被诱变剂诱导，HeLa S3细胞对内源MGMT基因的诱导没有反应。这是首次报道激素诱导DNA修复基因（MGMT）。MGMT和其他编码参与DNA烷基化损伤修复的酶的基因的诱导可能与癌症治疗相关，因为这会导致肿瘤细胞对烷基化药物产生抗性。
