Mammalian cells expressing Escherichia coli O6-alkylguanine-DNA alkyltransferases are hypersensitive to dibromoalkanes.
The effect of expression of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, on the growth inhibitory effects of the dibromoalkanes (DBA) dibromomethane (DBM) and dibromoethane (DBE) was determined in Chinese hamster lung fibroblasts transfected with and expressing high levels of the Escherichia coli alkyltransferase (ATase) genes. These included the ogt gene and complete or truncated versions of the E. coli ada gene encoding either O6-alkylguanine (O6-alkG) or alkylphosphotriester (alkPT) ATase activities. The functional activity of the ATase in these cells was demonstrated by in vitro assay of cell extracts using 3H-methylated DNA as a substrate, and 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 O6-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 cell extracts depleted O6-alkG ATase activity on the methylated DNA substrate, but had no effect on alkPT ATase activity. This suggests that ATase-mediated sensitization of the intact cells may be related to the inactivation of the ATase protein. Addition to the cell culture medium of GSH or buthionine sulfoximine in attempts to augment or deplete cellular levels of GSH had no marked effect on the ATase-mediated sensitization to DBA. This suggests that rather than GSH-mediated DNA damage, the effect may be mediated by a DNA adduct caused by the oxidative metabolic pathway. These observations indicate that expression of ATase may have a detrimental effect on cellular sensitivity to environmentally relevant alkylating agents.
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CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester M20 4BX, U.K.
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D000818:Animals; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D004926:Escherichia coli; D015946:Ethylene Dibromide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D006842:Hydrocarbons, Brominated / Q000494:pharmacology; D008168:Lung / Q000166:cytology / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis
D006842:Hydrocarbons, Brominated; D011994:Recombinant Proteins; D015946:Ethylene Dibromide; D019328:Buthionine Sulfoximine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005978:Glutathione; C027947:methylene bromide