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O6-Methylguanine-DNA methyltransferase (MGMT) is responsible for removal of O6-alkylguanine from DNA induced by alkylating mutagens/carcinogens. To analyze the involvement of O6-alkylguanine in the generation and MGMT in avoidance of various genotoxic effects of alkylating agents, we transfected Chinese hamster ovary (CHO) cells that lack MGMT activity with human MGMT cDNA cloned into a mammalian expression vector (pSV2MGMT). A high proportion (60-80%) of transfectants selected for a cotransfected neo gene survived treatment with high doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-hydroxyethyl-N-chloroethylnitrosourea (HeCNU). Parallel transfections with an expression vector containing the bacterial ada gene (pSV2ada) showed the human MGMT to be more effective than the ada expression vector in mediating alkylation resistance. Various clonal CHO cell lines have been established stably transfected with the human MGMT cDNA. The transfectants expressed human MGMT at levels ranging from 8600 to 210,000 molecules per cell. The high MGMT expressors became strongly resistant to the killing effects of MNNG, HeCNU, N-methyl-N-nitrosourea (MNU) and, to a significant lesser degree, methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). No killing resistance was observed to N-ethyl-N-nitrosourea (ENU), though the MGMT and ada transfectants showed reduction in mutation frequency induced by this agent. Protection from mutation induction by MGMT (and ada) expression was also demonstrated for MNNG. The transfectants were also protected from the sister chromatid exchange (SCE) inducing and, to a lesser degree, clastogenic effect of MNNG and MNU, and slightly to EMS and MMS. Again no protection was observed towards ENU. Correlations between MGMT activity and resistance to a given end point suggest that, for MNNG, O6-methylguanine is the preponderant toxic, mutagenic and SCE inducing lesion. About 90% of MNNG (and MNU) induced SCEs and nearly all of the MNNG-induced gene mutations seem to be due to this adduct. For alkylation-induced chromosomal aberrations, however, and for cell killing and SCEs induced by MMS, EMS and ENU, other lesions than O6-alkylguanine appear to be of major importance. The data strongly support the view that O6-methylguanine is a genotoxic lesion and MGMT a function decisively involved in avoidance of genotoxic effects in cells exposed to MNNG and related compounds. They indicate also that it is important to take into account the property and mode of action of any given alkylating agent in assessing the protective role of MGMT against alkylation-induced genotoxicity.

We have previously identified four human astrocytoma cell strains as defective in the repair of N-methyl-N' -nitro-N-nitrosoguanidine (MNNG) damaged adenovirus 5. We now show that two of these strains (the only two tested), in comparison to other tumor strains or normal human skin fibroblasts, are very sensitive to MNNG-produced killing as measured by colony forming ability, but are normally sensitive to ultraviolet light. Further, such repair deficient cells may be cultured from tumors of the colon, lung, skin, and neck. The phenotype of deficient repair of MNNG-treated adenovirus 5 has now been found in a subgroup of 9 of the 39 human tumor strains tested. We propose to call this phenotype the Mer(-) phenotype. None of the 22 strains of normal human skin fibroblasts tested showed deficient repair of MNNG damage. MNNG treatment (80 microM) causes a decrease in semi-conservative DNA synthesis from which Mer(-) tumor cells do not recover, but from which cells capable of normal repair of MNNG damage (Mer(+)) do. Somewhat paradoxically, Mer(-) cells show more MNNG-stimulated DNA synthesis ('repair synthesis') than do Mer(+) cells. Besides being deficient in the repair of MNNG-damaged adenoviruses Mer(-) cells also have difficulty in repairing viruses damaged either by other N-alkyl-N'-nitro-N-nitrosoguanidines, or by N-methyl- or N-ethyl-N-nitrosoureas.