Alkylation-induced apoptosis of embryonic stem cells in which the gene for DNA-repair, methyltransferase, had been disrupted by gene targeting

An enzyme O⁶-methylguanine-DNA methyltransferase (MGMT) catalyzes transfer of a methyl group from O⁶-methylguanine and O⁴-methylthymine of alkylated DNA to its own molecule, thereby repairing the pre-mutagenic lesions in a single step reaction. Making use of gene targeting, we developed mouse embryonic stem (ES) cell lines deficient in the methyltransferase. Quantitative immunoblot analysis and enzyme assay revealed that MGMT(-/-)cells, in which both alleles were disrupted, contained no methyltransferase protein while cells with one intact allele (MGMT(+/-)) contained about half the amount of protein carried by the parental MGMT(+/+) cells. MGMT(-/-) cells have an extremely high degree of sensitivity to simple alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU), whereas MGMT(+/-) cells are slightly more sensitive to these agents, as compared with findings from normal cells. A high frequency of mutation was induced in MGM(-/-) cells on exposure to a relatively low dose of MNNG. Electrophoretic analyses of the DNAs as well as fluorochrome staining of the cells revealed that MGMT(-/-) cells treated with MNNG undergo apoptotic death, which occurs after G₂-M arrest in the second cycle of cell proliferation.