Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.
Transfection and expression of human O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against the genotoxic effects of alkylating agents.
复制标题
DOI：
10.1093/carcin/12.10.1857
复制
发表时间：
1991-10
期刊：
影响因子：
4.7
通讯作者：
Bernd Kaina;Gerhard Fritz;Sankar Mitra;T. Coquerelle
中科院分区：
文献类型：
--
作者：
Bernd Kaina;Gerhard Fritz;Sankar Mitra;T. Coquerelle
O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）负责去除由烷基化诱变剂/致癌剂诱导的DNA中的O 6-烷基鸟嘌呤。为了分析参与的O 6-烷基鸟嘌呤的产生和MGMT在避免各种遗传毒性效应的烷化剂，我们转染中国仓鼠卵巢（CHO）细胞，缺乏MGMT活性与人MGMT cDNA克隆到哺乳动物表达载体（pSV 2 MGMT）。选择用于共转染neo基因的高比例（60-80%）转染子在高剂量N-甲基-N '-硝基-N-亚硝基胍（MNNG）和N-羟乙基-N-氯乙基亚硝基脲（HeCNU）处理后存活。用含有细菌ADA基因的表达载体（pSV 2ada）平行转染显示人MGMT在介导烷基化抗性方面比ADA表达载体更有效。已经建立了用人MGMT cDNA稳定转染的各种克隆CHO细胞系。转染子表达的人MGMT的水平范围为8600至210，000分子/细胞。高MGMT表达者变得对MNNG、HeCNU、N-甲基-N-亚硝基脲（MNU）的杀伤作用具有强烈抗性，并且在显著较低的程度上对甲磺酸甲酯（MMS）和甲磺酸乙酯（EMS）具有抗性。没有观察到对N-乙基-N-亚硝基脲（ENU）的杀伤抗性，尽管MGMT和ada转染子显示由该药剂诱导的突变频率降低。也证明了MNNG对MGMT（和ada）表达诱导突变的保护作用。转染子也受到保护，从姐妹染色单体交换（SCE）诱导，并在较小程度上，染色体断裂的影响MNNG和MNU，并轻微EMS和MMS。同样没有观察到对ENU的保护。MGMT活性和对给定终点的抗性之间的相关性表明，对于MNNG，O 6-甲基鸟嘌呤是优势毒性、致突变性和SCE诱导损伤。大约90%的MNNG（和MNU）诱导的SCE和几乎所有的MNNG诱导的基因突变似乎是由于这种加合物。然而，对于烷基化诱导的染色体畸变，以及MMS、EMS和ENU诱导的细胞杀伤和SCE，O 6-烷基鸟嘌呤以外的其他病变似乎具有重大意义。这些数据强烈支持这样的观点，即O 6-甲基鸟嘌呤是一种遗传毒性病变，MGMT是一种功能，决定性地参与避免细胞暴露于MNNG和相关化合物的遗传毒性作用。它们还表明，在评估MGMT对烷基化诱导的遗传毒性的保护作用时，考虑任何给定烷化剂的性质和作用模式是重要的。
O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.