3T3 NIH murine fibroblasts and B78 murine melanoma cells expressing the Escherichia coli N3-methyladenine-DNA glycosylase I do not become resistant to alkylating agents.

Abstract

The role of alkylation of the N3 position of adenine in the cytotoxicity of alkylating agents in mammalian cells is still undefined. By co-transfecting NIH3T3 murine fibroblast and murine B78 H1 melanoma cells with pSG5tag and pSV2neo, we obtained clones expressing the mRNA of the bacterial tag gene coding for N3-methyladenine-DNA glycosylase I (Gly I), which specifically repairs N3-methyladenine. The levels of Gly I were 400 times higher in NIH3T3 pSG5tag (clone 3.9.4) and 12-33 times higher in B78 H1 tag clones (2A4, 2A6, 2C3 and 2D1) than in the respective control cells. The sensitivity to alkylating agents was evaluated in tag-expressing cells in comparison with pSG5, pSV2neo co-transfected control cells. As regards the cytotoxic activity of methylating agents (N-methylnitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine, dimethylsulfate and temozolomide) and other alkylators with different structure and different interactions with DNA such as CC-1065 and FCE-24517 (minor groove binders known to bind to N3 of adenine), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and cis-diamminedichloroplatinum II, cytotoxicity was the same for tag-expressing and non-expressing cells. These results suggest that the increased expression of N3-methyladenine-DNA glycosylase is not necessarily a crucial mechanism for the resistance of cells to alkylating agents.