Takagi Yasumitsu, Takahashi Masayuki, Sanada Masayuki, Ito Riyoko, Yamaizumi Masaru, Sekiguchi Mutsuo
Frontier Research Center, Fukuoka Dental College, Fukuoka 814-0193, Japan.
DNA Repair (Amst). 2003 Oct 7;2(10):1135-46. doi: 10.1016/s1568-7864(03)00134-4.
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.
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作用，我们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/或编码参与错配修复的蛋白质的Mlh1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，而通过在Mlh1基因中引入额外突变可克服MNU的这种作用。Mgmt(-/-)Mlh1(-/-)细胞比野生型细胞对MNU更具抗性。当引入带有强启动子的人Mgmt cDNA序列时，野生型细胞获得了与Mgmt(-/-)Mlh1(-/-)细胞相同的高水平MNU抗性。虽然在这种甲基转移酶过度表达的野生型细胞中未观察到MNU诱导的突变频率明显增加，但Mgmt(-/-)Mlh1(-/-)细胞经MNU处理后突变频率提高了10倍。携带约正常水平一半MLH1蛋白的Mgmt(-/-)Mlh1(+/-)细胞显示出自发突变频率正常，但对烷基化致癌物的诱变作用仍高度敏感。在细胞存活试验中也观察到了Mlh1突变的这种单倍剂量不足特征；Mgmt(-/-)Mlh1(+/-)细胞对MNU的抗性与Mgmt(-/-)Mlh1(-/-)细胞相同。在用MNU处理后，Mgmt(-/-)Mlh1(+/+)细胞中诱导了半胱天冬酶-3，而在Mgmt(-/-)Mlh1(+/-)细胞或Mgmt(-/-)Mlh1(-/-)细胞中未发生诱导。MLH1蛋白的细胞含量似乎对于确定受损细胞是进入死亡途径还是诱变途径至关重要。Mlh1杂合细胞的单倍剂量不足表型可能是由于MLH1同源物在异二聚体形成中的竞争所致。
