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.
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 micrograms/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.
Fox
M
M
Department of Biochemical Genetics, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK.
Margison
G P
GP
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005038:Ethylnitrosourea / Q000494:pharmacology*; D005796:Genes / Q000187:drug effects; D005798:Genes, Bacterial; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007700:Kinetics; D008770:Methylnitrosourea / Q000494:pharmacology*; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase; D014162:Transfection
D008770:Methylnitrosourea; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D005038:Ethylnitrosourea