Depletion of mammalian O6-alkylguanine-DNA alkyltransferase activity by O6-benzylguanine provides a means to evaluate the role of this protein in protection against carcinogenic and therapeutic alkylating agents.
O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 microM O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferase activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 microM. In contrast, at least 100 microM O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 microM O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methysulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be valuable to increase the chemotherapeutic effectiveness of chloroethylating agents.
Dolan
M E
ME
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, Hershey 17033.
Moschel
R C
RC
Pegg
A E
AE
D000970:Antineoplastic Agents / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008130:Lomustine / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008698:Mesylates / Q000494:pharmacology; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured / Q000166:cytology* / Q000187:drug effects
D000970:Antineoplastic Agents; D008698:Mesylates; C064976:O(6)-benzylguanine; C041189:clomesone; D006147:Guanine; D008130:Lomustine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase