POINT MUTATIONS IN HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE PREVENT THE SENSITIZATION BY O-6-BENZYLGUANINE TO KILLING BY N,N'-BIS(2-CHLOROETHYL)-N-NITROSOUREA

Chinese hamster ovary (CHO) cells lack O-6-alkylguanine-DNA alkyltrans ferase (AGT) activity and are sensitive to killing by N,N'-bis(2-chlor oethyl)-N-nitrosourea (BCNU). Transfection of these cells with a plasm id leading to the production of mild-type human AGT rendered them resi stant to BCNU but this resistance could be overcome by treatment with O-6-benzylguanine, an AGT inhibitor, Transfection with plasmids expres sing mutants of the AGT in which either proline(140) is converted to a lanine or glycine(156) is converted to alanine also gave rise to CHO c ells resistant to BCNU, but these mutations rendered the expressed AGT less sensitive to O-6-benzylguanine, and O-6-benzylguanine was theref ore much less effective in restoring sensitivity to BCNU. The G156A mu tation provided the greater amount of resistance to O-6-benzylguanine, and the CHO cells expressing this mutant AGT were not effectively kil led by the O-6-benzylguanine plus BCNU combination, CHO cells expressi ng the mutant AGTs were also much less sensitive than those expressing the control protein with respect to loss of AGT activity and enhancem ent of killing by BCNU in response to the more potent AGT inhibitor, 2 ,4-diamino-6-benzgloxy-5-nitrosopyrimidine. Although these results rai se the possibility that resistance to therapy with O-6-benzylguanine a nd chloroethylating agents may arise by the selection for tumor cells expressing a mutated AGT, they also provide a means by which the thera peutic effectiveness of agents forming O-6-alkylguanine adducts such a s BCNU might be enhanced, Expression of the G156A mutant AGT in hemato poietic progenitor cells by gene therapy techniques could be used to i ncrease their AGT activity and provide a form that was resistant to O- 6-benzylguanine. Such resistance would provide a way to select for cel ls expressing the inserted gene and would provide an increase in the t herapeutic index for treatment of tumors which would have an AGT activ ity sensitive to O-6-benzylguanine.