Point mutations in human O6-alkylguanine-DNA alkyltransferase prevent the sensitization by O6-benzylguanine to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea

Abstract

Chinese hamster ovary (CHO) cells lack O6-alkylguanine-DNA alkyltransferase (AGT) activity and are sensitive to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea (BCNU). Transfection of these cells with a plasmid leading to the production of wild-type human AGT rendered them resistant to BCNU but this resistance could be overcome by treatment with O6-benzylguanine, an AGT inhibitor. Transfection with plasmids expressing mutants of the AGT in which either proline140 is converted to alanine or glycine156 is converted to alanine also gave rise to CHO cells resistant to BCNU, but these mutations rendered the expressed AGT less sensitive to O6-benzylguanine, and O6-benzylguanine was therefore much less effective in restoring sensitivity to BCNU. The G156A mutation provided the greater amount of resistance to O6-benzylguanine, and the CHO cells expressing this mutant AGT were not effectively killed by the O6-benzylguanine plus BCNU combination. CHO cells expressing the mutant AGTs were also much less sensitive than those expressing the control protein with respect to loss of AGT activity and enhancement of killing by BCNU in response to the more potent AGT inhibitor, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine. Although these results raise the possibility that resistance to therapy with O6-benzylguanine and chloroethylating agents may arise by the selection for tumor cells expressing a mutated AGT, they also provide a means by which the therapeutic effectiveness of agents forming O6-alkylguanine adducts such as BCNU might be enhanced. Expression of the G156A mutant AGT in hematopoietic progenitor cells by gene therapy techniques could be used to increase their AGT activity and provide a form that was resistant to O6-benzylguanine. Such resistance would provide a way to select for cells expressing the inserted gene and would provide an increase in the therapeutic index for treatment of tumors which would have an AGT activity sensitive to O6-benzylguanine.