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Development of a detection and evaluation system for carcinogens with the use of cell lines deficient in DNA repair genes

利用DNA修复基因缺陷的细胞系开发致癌物检测和评估系统

基本信息

批准号：
12554035
负责人：
ITOH Riyoko
金额：
$ 8.77万
依托单位：
Fukuoka Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12554035/
关键词：
DNA repair enzyme O^6-methylguanine tumorigenesis mutation cell line gene deficient anticancer agent mouse アポトーシス発ガン

项目摘要

We have established a simple and accurate detection and evaluation system for carcinogens using cell lines from knockout mice, which are detective 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. Various cell lines were prepared by introduction of transforming vector, derived from SV40 virus, to primary cells isolated from lungs of mice with various genotypes, Mgmt^<-/->Mlh1^<+/+>, Mgmt^<-/->Mlh1^<-/->, Mgmt^<-/->Mlh1^<+/->, Mgmt^<+/+>Mlh1^<-/->. Mgmt^<-/->Mlh1^<+/+> cell line was hypersensitive to killing and mutation-inducing effects of methylnitrosourea (MNU), a simple alkylating agent. On the other hand, Mgmt^<-/->Mlh1^<-/-> cell line was as resistant to the killing effect of MNU as was wild cell line (Mgmt^<+/+>Mlh1^<+/+>), but MNU-induced mutant frequency of Mgmt^<-/->Mlh1^<-/-> cells was considerably higher than that of wild type cells. These results are consistent with killing and tumorigenic actions of MNU revealed with knockout mice. It is suggested that an increase in mutant frequency, caused by deficiency in DNA repair, would lead to tumor formation in organisms. To investigate whether this cell line system is useful for evaluation of therapeutic agents, we studied the killing and mutagenic effects of dacarbazine, which has alkylation potential. The results showed the same tendency as the case of MNU and effectiveness of this system is demonstrated. We are in progress to establish similar cell line systems for investigating spontaneous mutagenesis related to oxygen radicals, which can be produced through normal cellular metabolism.

我们已经建立了一个简单而准确的检测和评价系统的致癌物使用细胞系敲除小鼠，这是检测的Mgmt基因编码的DNA修复酶，O^6-甲基鸟嘌呤-DNA甲基转移酶，和/或Mlh 1基因编码的蛋白质参与错配修复。通过将衍生自SV 40病毒的转化载体引入分离自具有各种基因型Mgmt^<-/-> Mlh 1 ^<+/+>、Mgmt^<-/-> Mlh 1 ^<-/->、Mgmt ^<-/-> Mlh 1 ^<+/->、Mgmt^<+/+> Mlh 1 ^<-/->的小鼠肺的原代细胞中来制备各种细胞系。Mgmt^<-/-> Mlh ^<+/+>细胞系对甲基亚硝基脲（MNU）的杀伤和致突变作用高度敏感。另一方面，Mgmt^<-/-> Mlh 1 ^<-/->细胞系与野生型细胞系（Mgmt^<+/+> Mlh 1 ^<+/+>）一样对MNU的杀伤作用具有抗性，但Mgmt^<-/-> Mlh 1 ^<-/->细胞的MNU诱导突变频率显著高于野生型细胞。这些结果与用基因敲除小鼠揭示的MNU的杀伤和致瘤作用一致。这表明，DNA修复缺陷引起的突变频率增加会导致生物体中肿瘤的形成。为了研究该细胞系系统是否可用于评价治疗药物，我们研究了具有烷基化潜力的达卡巴嗪的杀伤和致突变作用。结果表明，与MNU的情况下，该系统的有效性证明了相同的趋势。我们正在建立类似的细胞系系统，用于研究与氧自由基相关的自发突变，氧自由基可以通过正常的细胞代谢产生。