An enzyme O-6-methylguanine-DNA methyltransferase (MGMT) catalyzes transfer of a methyl group from O-6-methylguanine and O-4-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 MGMT(-/-) 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 G(2)-M arrest in the second cycle of cell proliferation.
