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 μM 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 μM. In contrast, at least 100 μM 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 μM 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(methylsulfonyl)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. (.