| Research Abstract |
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.
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