Role of codon 160 in the sensitivity of human O-6-alkylguanine-DNA a alkyltransferase to O-6-benzylguanine

O-6-Alkylguanine DNA alkyltransferase (AGT) is a DNA repair protein that pr ovides protection from alkylating agents such as dacarbazine, temozolomide, and 1,3-bis- (2-chloroethyl)-1-nitrosourea (BCNU), which are used for canc er chemotherapy. O-6-Benzylguanine (BG) is an inhibitor of AGT that sensiti zes tumors to these agents. BG is currently in clinical trials. It is possi ble that the presence of resistant farms of AGT may limit the effectiveness of this strategy. Previous studies have shown that the AGT mutant G160R, w hich may occur naturally as a result of a polymorphism in the AGT gene, is resistant to BG, whereas the mutants G160W and G160A are actually more sens itive to the inhibitor. To examine other mutations at this site, a random s equence was placed at codon 160 in the AGT cDNA, and a plasmid library was constructed to express these sequences in Escherichia coli. After selection with BG and N-methyl N (')-nitro-N-nitrosoguanidine, BG-resistant mutants were obtained and analyzed. Eleven different amino acid substitutions were found to impart BG resistance by this assay. The most resistant mutants con tained histidine or arginine, which had EC50 values of 12 and 4.7 mu M, res pectively, compared with the wild-type EC50 of 0.08 mu M, but nine other al terations led to at least a 10-fold rise in the EC50 value. Three additiona l mutations at codon 160 were constructed by site-directed mutagenesis, and these led to 6- to 11-fold increases in resistance to BG. Comparisons of t he properties of mutants G160R and G160E showed that the presence of DNA en hanced the reaction with BG much more strongly when an acidic residue was p resent at this position. This may account for the lack of selection of the G160E mutation even though it did impart resistance to BG. These results in dicate that many alterations of AGT at position 160 can lead to significant resistance to BG. (C) 1999 Elsevier Science Inc.