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

Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892 [D. B. Y., S. H-C.], and Laboratory of Molecular Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [M. A. B., R. S. D.]

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 x 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.

¹ Present address: Applied Genetics, Inc., 205 Buffalo Ave., Freeport, NY 11520.

² Present address: Laboratory of Molecular Immunology, National Institute of Dental Research, Building 30, Room 306, Bethesda, MD 20892.

³ To whom requests for reprints should be addressed.

Received 5/ 6/85. Revised 11/21/85. Accepted 12/31/85.