Resistance of the human O6-alkylguanine-DNA alkyltransferase containing arginine at codon 160 to inactivation by O6-benzylguanine.
Inactivation of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O6-benzylguanine is currently undergoing clinical trials for development as an agent to enhance chemotherapy. It has been reported recently that a polymorphism in the human O6-alkylguanine-DNA alkyltransferase gene exists, with about 15% of the population studied having arginine at codon 160 instead of glycine (Y. Imai et al., Carcinogenesis (Lond.), 16: 2441-2445, 1995). We have studied the effects of mutations of this glycine to arginine, tryptophan, or alanine on the interaction of human alkyltransferase with O6-benzylguanine using direct determination of the amount of activity remaining after incubation with various concentrations of the inhibitor and measurement of the rate of production of [8-3H]guanine from O6-benzyl[8-3H]guanine as assays. These mutations had little effect on the alkyltransferase activity in repairing O6-methylguanine in methylated DNA. Alteration of glycine 160 to tryptophan or alanine slightly increased the sensitivity to O6-benzylguanine (by up to 4-fold). However, alteration of glycine 160 to arginine drastically reduced the inactivation by O6-benzylguanine with at least a 20-fold increase in the ED50 value and a similar reduction in the production of guanine whether inactivation was carried out in the absence or presence of DNA. These results raise the possibility that a subpopulation of patients may be resistant to O6-benzylguanine and that higher doses or additional alkyltransferase inhibitors capable of inactivating this form of the alkyltransferase will be necessary.
Edara
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Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033-0850, USA.
Kanugula
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Goodtzova
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Pegg
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D001120:Arginine / Q000235:genetics; D003062:Codon / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D017354:Point Mutation / Q000235:genetics*
D003062:Codon; C064976:O(6)-benzylguanine; D006147:Guanine; D004247:DNA; D001120:Arginine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase