Inactivation of O6-methylguanine-DNA methyltransferase and sensitization of human tumor cells to killing by chloroethylnitrosourea by O6-methylguanine as a free base.
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 10(7) 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.
D002470:Cell Survival / Q000187:drug effects; D004260:DNA Repair; D005038:Ethylnitrosourea / Q000031:analogs & derivatives* / Q000494:pharmacology; D005347:Fibroblasts / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D009369:Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013379:Substrate Specificity
C017139:1-(2-chloroethyl)-1-nitrosourea; D006147:Guanine; C008449:O-(6)-methylguanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D005038:Ethylnitrosourea