Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis
Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis
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DOI：
10.1016/s1568-7864(03)00134-4
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发表时间：
2003-10-07
期刊：
影响因子：
3.8
通讯作者：
Sekiguchi, M
中科院分区：
文献类型：
--
作者：
Takagi, Y;Takahashi, M;Sekiguchi, M
为了研究错配修复系统在烷基化诱导的细胞凋亡和诱变中的参与，从基因靶向小鼠中建立了编码DNA修复酶、O-6-甲基鸟嘌呤-DNA甲基转移酶的Mgmt基因和/或编码错配修复中所涉及的蛋白质的Mlh 1基因缺陷的细胞系。Mgmt（-/-）细胞对N-甲基-N-亚硝基脲（MNU）的杀伤作用高度敏感，通过在Mlh 1基因中引入额外的突变来克服MNU的这种作用。Mgmt（-/-）Mlh 1（-/-）细胞比野生型细胞对MNU更具抗性。当引入具有强启动子的人Mgmt cDNA序列时，野生型细胞获得了与Mgmt（-/-）Mlh 1（-/-）细胞相同的高水平的MNU抗性。虽然在这种甲基转移酶过量产生的野生型细胞中没有观察到MNU诱导的突变频率的明显增加，但Mgmt（-/-）Mlh 1（-/-）细胞的突变频率在用MNU处理后变得高10倍。携带大约一半正常水平MLH 1蛋白的Mgmt（-/-）Mlh 1（+/-）细胞显示出正常水平的自发突变频率，但仍对烷化剂的致突变作用具有高度反应性。在细胞存活试验中也观察到Mlh 1突变的这种单倍不足特征; Mgmt（-/-）Mlh 1（+/+）细胞与Mgmt（-/-）Mlh 1（-/-）细胞一样对MNU具有抗性。MNU处理后，Mgmt（-/-）Mlh 1（+/+）细胞中caspase-3被诱导，而Mgmt（-/-）Mlh 1（+/-）细胞和Mgmt（-/-）Mlh 1（-/-）细胞中没有发生诱导。MLH 1蛋白的细胞内容物似乎对于确定受损细胞是否进入死亡或突变诱导途径至关重要。Mlh 1-杂合细胞的单倍不足表型可以通过MLH 1同源物之间异二聚体形成的竞争来解释。(C)2003 Elsevier B. V.保留所有权利。
To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O-6-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt(-/-) cells are hypersensitive to the killing effect of N-methyl-N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt(-/-)Mlh1(-/-) cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt(-/-)Mlh1(-/-) cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt(-/-)Mlh1(-/-) cells became 10-fold higher after being treated with MNU. Mgmt(-/-)Mlh1(+/-) cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt(-/-)Mlh1(+/+) cells were as resistant to MNU as were Mgmt(-/-)Mlh1(-/-) cells. While caspase-3 was induced in Mgmt(-/-)Mlh1(+/+) cells after treatment with MNU, no induction occurred in Mgmt(-/-)Mlh1(+/-) cells or in Mgmt(-/-)Mlh1(-/-) cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues. (C) 2003 Elsevier B.V. All rights reserved.