Inactivation of O⁶-Methylguanine-DNA Methyltransferase and Sensitization of Human Tumor Cells to Killing by Chloroethylnitrosourea by O⁶-Methylguanine as a Free Base

Abstract

Human fibroblasts and tumor cells with constitutive levels of the DNA repair protein O⁶-methylguanine-DNA methyltransferase were incubated with mm concentrations of the free base O⁶-methylguanine for up to 24 h. This treatment depleted the cells of their transferase activity, and sensitized the cells to killing by the antineoplastic drug 1-[2-chloroethyl]-1-nitrosourea. Cells constitutively lacking the methyltransferase were not sensitized to cell killing. Cell free extracts incubated with O⁶-methylguanine also lost methyltransferase activity. Other alkylpurines, such as O⁶-methylguanosine, S⁶-methylthioguanine, O⁶-ethylguanine, and 3-methyladenine, did not have this effect on extracts of human tumor cells, while O⁶-methylguanosine and O⁶-methylguanine inactivated purified methyltransferase from Escherichia coli. The data suggest that the free base O⁶-methylguanine is probably a substrate for the methyltransferase. Calculation of the second order rate constants for free base versus O⁶-methylguanine in DNA, and experiments in which the free base was mixed with DNA containing O⁶-methylguanine before reaction with methyltransferase, indicated that the base in DNA is about 4 × 10⁷ better as a substrate than is the free base. These results demonstrate that DNA repair capacity of tumor cells can be diminished without DNA damage, and suggest a method for increasing the efficiency of chemotherapy.