Leukocyte O-6-akylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O-6-benzylguanine

O-6-Alkylguanine-DNA alkyltransferase (AGT) is the key DNA repair protein r esponsible for resistance to chloroethylating and methylating agents that a ttack at the O-6 position of guanine. O-6-Benzylguanine (BG), a potent inhi bitor of AGT, has recently entered clinical trials. A number of point mutat ions and at least one human polymorphism within AGT are associated with AGT resistance to inactivation by BG, In this study, we evaluated AGT inhibiti on by BG in an in vitro assay of peripheral blood mononuclear cell AGT from 56 normal donors, 42 Caucasians, and 14 Japanese. AGT activity ranged from 2.7 to 21.9 fmol/pg DNA and was similar in Japanese and Caucasian donors. Depletion of AGT by BG was uniform in all donors with mean ED(50)s of 0.37 mu M BG in Caucasians and 0.36 mu M BG in Japanese. To determine whether th e gly160arg AGT polymorphism described in the Japanese population, and rece nt;ly shown to be BG resistant, could be detected by this assay, we mixed p urified gly160arg AGT protein with blood mononuclear cell extract and measu red in vitro BG inactivation, The ED50 for the mixture of the gly160arg AGT and mononuclear cell extract was 9 mu M BG. On the basis of results in 55 donors, we conclude that BG-resistant AGT, defined as an ED50 in mononuclea r cells of >1 mu M BG, is present in 0 of 56 donors, (95% confidence interv al, 0-6%), suggesting that polymorphisms producing AGT-resistant BG are unu sual in humans.