Mammalian cells expressing Escherichia coli O-6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes

The effect of expression of the DNA repair protein, O-6-alkylguanine-DNA al kyltransferase, on the growth inhibitory effects of the dibromoalkanes (DBA ) dibromomethane (DBM) and dibromoethane (DBE) was determined in Chinese ha mster lung fibroblasts transfected with and expressing high levels of the E scherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E, coli ada gene encoding either O-6-alkylguanine (O-6-alkG) or alkylphosphotriester(alkPT) ATase activitie s. The functional activity of the ATase in these cells was demonstrated by in vitro assay of cell extracts using H-3-methylated DNA as a substrate, an d by the protection they provided against the growth inhibitory effects of methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl -N-nitrosourea (MNU) and the chloroethylating agent 1,3-bis(2-chloroethyl)- 1-nitrosourea (BCNU). However, cells expressing the full length or the O-6- alkG: ATase region, but not the alkPT ATase region, of Ada were found to be more sensitive to the growth inhibitory effects of the DBA; Ogt expression sensitized cells to DBM but not significantly to DBE. Addition of DBA to c ell extracts depleted O-6-alkG ATase activity on the methylated DNA substra te, but had no effect on alkPT ATase activity. This suggests that ATase-med iated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthion ine sulfoximine in attempts to augment or deplete cellular levels of GSH ha d no marked effect on the ATase-mediated sensitization to DBA. This suggest s that rather than GSH-mediated DNA damage, the effect may be mediated by a DNA adduct caused by the oxidative metabolic pathway. These observations i ndicate that expression of ATase may have a detrimental effect on cellular sensitivity to environmentally relevant alkylating agents.