ALKYLATION-INDUCED APOPTOSIS OF EMBRYONIC STEM-CELLS IN WHICH THE GENE FOR DNA-REPAIR, METHYLTRANSFERASE, HAD BEEN DISRUPTED BY GENE TARGETING

An enzyme O-6-methylguanine-DNA methyltransferase (MGMT) catalyzes tra nsfer of a methyl group from O-6-methylguanine and O-4-methylthymine o f alkylated DNA to its own molecule, thereby repairing the pre-mutagen ic lesions in a single step reaction, Making use of gene targeting, we developed mouse embryonic stem (ES) cell lines deficient in the methy ltransferase. Quantitative immunoblot analysis and enzyme assay reveal ed that MGMT(-/-) cells, in which both alleles were disrupted, contain ed no methyltransferase protein while cells with one intact allele (MG MT(+/-)) contained about half the amount of protein carried by the par ental MGMT(+/+) cells. MGMT(-/-) cells have an extremely high degree o f sensitivity to simple alkylating agents, N-methyl-N'-nitro-N-nitroso guanidine (MNNG) and N-methyl-N-nitrosourea (MNU), whereas MGMT(+/-) c ells are slightly more sensitive to these agents, as compared with fin dings from normal cells, A high frequency of mutation was induced in M GMT(-/-) cells on exposure to a relatively low dose of MNNG, Electroph oretic analyses of the DNAs as well as fluorochrome staining of the ce lls revealed that MGMT(-/-) cells treated with MNNG undergo apoptotic death, which occurs after G(2)-M arrest in the second cycle of cell pr oliferation.