Expression of an E.coli O6-alkylguanine DNA alkyltransferase gene in Chinese hamster cells protects against N-methyl and N-ethylnitrosourea induced reverse mutation at the hypoxanthine phosphoribosyl transferase locus

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Abstract
The spontaneous hypoxanthine phosphoribosyl transferase deficient (HPRT− ) mutants of V79 cells (TG11 and TG15) were transfected with a retrovirus-based plasmid containing a truncated form of the Escherichia coli gene which codes for O6-alkylguanine (O6-AG) DNA alkyltransferase (ATase). The resultant cell lines TG11SB5 and TG15SB7 were G418 resistant and expressed high levels of O6-AG ATase activity. The frequency of revertants induced by equitoxic doses of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) was 10- to 50-fold higher in TG11 than in TG15. In TG11SB5 and TG15SB7 induced revertant frequencies were reduced relative to TG11 and TG15 by factors of 6–8 and 1.5–3.0, respectively, immediately after treatment. On delayed plating the frequency of MNU-induced revertant colonies decreased at a rate inversely proportional to dose in both TG11 and TG11SB5. In contrast, after exposure of TG11SB5 to ENU (50 or 75 μg/ml) initial reversion frequencies were low compared with TG11, but then rose to a plateau frequency by 24 h, which was maintained for up to 72 h. The frequency of reversion observed, the degree of protection afforded by the E.coli O6-AG ATase and the kinetics of expression of revertants were thus cell line specific suggesting that DNA sequence specific alkylation and/or preferential repair may be responsible. The initial protection against mutagenesis is consistent with the hypothesis that MNU- and ENU-induced reversion is the result of miscoding opposite O6-AG or O4-alkylthymine residues. Expression of O6-AG ATase activity was variable when cells were continually cultured over long periods despite the presence of the selective antibiotic G418.

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