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 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 G2-M arrest in the second cycle of cell proliferation.