Effect of O-6-benzylguanine on alkylating agent-induced toxicity and mutagenicity in Chinese hamster ovary cells expressing wild-type and mutant O-6-alkylguanine-DNA alkyltransferases

The DNA repair protein O-6-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating a gents by removing lesions from the O-6 position of guanine, O-6-Benzylguani ne (BG) is a potent inactivator of AGT, resulting in an increase in the sen sitivity of cells to the toxic effects of chemotherapeutic alkylating agent s. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-ty pe AGT (CHOWTAGT) and a mutant GGT [P138 M/V139I/P140K (CHOMIK)] known to b e resistant to BG were treated with BG and various alkylating agents. BG tr eatment alone dramatically decreased AGT activity in CHOWTAGT cells but res ulted in no depletion in AGT activity in CHOMIK cells. In the absence of AG T, these cells are highly sensitive to the toxic and mutagenic effects of t emozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHOWTAGT cells ar e resistant to temozolomide and BCNU, and treatment with BG resulted in a s ignificantly higher cell killing and mutation frequency, CHOMIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG, Both cell killing and mutation frequency of 4-hydroperoxycyclophospham ide (4-HC) in CHO, CHOWTAGT and CHOMIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PIM) and acrolein, BG had no effect on 4-hydropernxydidechlorocyclophosphamide ( which generates acrolein and a nonalkylating form of PM) in CHO cells and C HOMIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite o f 4-HC, Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O-6 alkylating agen ts and that a mutant AGT resistant to inactivation by BG effectively preven ts PC-enhanced toxicity and mutagenicity induced by these agents. Expressio n of the AGT protein contributes to resistance of 4-HC, BG also enhances th e toxicity of 4-HC and PM by a mechanism that may not involve the AGT repai r protein.