Leukocyte O6-alkylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O6-benzylguanine.
O6-Alkylguanine-DNA alkyltransferase (AGT) is the key DNA repair protein responsible for resistance to chloroethylating and methylating agents that attack at the O6 position of guanine. O6-Benzylguanine (BG), a potent inhibitor of AGT, has recently entered clinical trials. A number of point mutations and at least one human polymorphism within AGT are associated with AGT resistance to inactivation by BG. In this study, we evaluated AGT inhibition 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/microg DNA and was similar in Japanese and Caucasian donors. Depletion of AGT by BG was uniform in all donors with mean ED50s of 037 microM BG in Caucasians and 0.36 microM BG in Japanese. To determine whether the gly160arg AGT polymorphism described in the Japanese population, and recently shown to be BG resistant, could be detected by this assay, we mixed purified gly160arg AGT protein with blood mononuclear cell extract and measured in vitro BG inactivation. The ED50 for the mixture of the gly160arg AGT and mononuclear cell extract was 9 microM BG. On the basis of results in 56 donors, we conclude that BG-resistant AGT, defined as an ED50 in mononuclear cells of >1 microM BG, is present in 0 of 56 donors, (95% confidence interval, 0-6%), suggesting that polymorphisms producing AGT-resistant BG are unusual in humans.
Gerson
S L
SL
Division of Hematology/Oncology and the Ireland Cancer Center at Case Western Reserve University and University Hospital of Cleveland, Ohio 44106, USA. slg5@po.cwru.edu
Schupp
J
J
Liu
L
L
Pegg
A E
AE
Srinivasen
S
S
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000201:enzymology
D004791:Enzyme Inhibitors; C064976:O(6)-benzylguanine; D006147:Guanine; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase