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.