RESISTANCE OF THE HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE CONTAINING ARGININE AT CODON-160 TO INACTIVATION BY O-6-BENZYLGUANINE

Inactivation of O-6-alkylguanine-DNA alkyltransferase by O-6-benzylgua nine renders tumor cells more sensitive to killing by methylating and chloroethylating agents, and O-6-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 O-6-alk ylguanine-DNA alkyltransferase gene exists, with about 15% of the popu lation studied having arginine at codon 160 instead of glycine (Y. Ima i al at, Carcinogenesis (Lond.), 16: 2441-2415, 1995). We have studied the effects of mutations of this glycine to arginine, tryptophan, or alanine on the interaction of human alkyltransferase with O-6-benzylgu anine using direct determination of the amount of activity remaining a fter incubation with various concentrations of the inhibitor and measu rement of the rate of production of [8-H-3]guanine from O-6-benzyl[8-H -3]guanine as assays. These mutations had little effect on the alkyltr ansferase activity in repairing O-6-methylguanine in methylated DNA. A lteration of glycine 160 to tryptophan or alanine slightly increased t he sensitivity to O-6-benzylguanine (by up to ii-fold), However, alter ation of glycine 160 to arginine drastically reduced the inactivation by O-6-benzylguanine with at least a 20-fold increase in the ED(50) va lue and a similar reduction in the production of guanine whether inact ivation was carried out in the absence or presence of DNA. These resul ts raise the possibility that a subpopulation of patients may be resis tant to O-6-benzylguanine and that higher doses or additional alkyltra nsferase inhibitors capable of inactivating this form of the alkyltran sferase will be necessary.