Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation

Abstract

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea, Carmustine] is a nitrosourea that crosslinks DNA and is useful in cancer chemotherapy. Tumor cells resistant to BCNU produce high levels of O⁶-alkylguanine-DNA-alkltransferase (AT), a protein that removes the O⁶-guanine adduct formed by BCNU prior to crosslinking. By the transfection of a human cosmid library into the Chinese hamster ovary cell line AA8, several transgenic cell lines which express the AT gene have been constructed. These ‘BR’ cells were isolated on the basis of their resistance to G-418 and BCNU. Like human mer⁺ strains, BR cells (relative to the parental AA8 cells) are ˜500 times more resistant to the cytotoxic effects of 80 μM BCNU. Treatment with exogenous O⁶-methylguanine (O⁶MG), which depletes cellular AT, abolishes their BCNU resistance. Also consistent with the mer⁺ phenotype, BR cells are resistant to the mutagenic and killing activity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Treatment with exogenous O⁶MG, while reversing the resistance to MNNG mutation, does not reverse the resistance to MNNG killing. Unexpectedly, BR cells also exhibit resistance to killing by dimethylsulfate (DMS). The BR cells are not, however, detectably resistant to UV light. These results suggest that AT activity in mammalian cells is dosely linked to the activity of other DNA repair pathways.