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Effect of O⁶-Benzylguanine on Alkylating Agent-induced Toxicity and Mutagenicity in Chinese Hamster Ovary Cells Expressing Wild-Type and Mutant O⁶-Alkylguanine-DNA Alkyltransferases¹

Section of Hematology-Oncology, Department of Medicine [Y. C., M. H. W., M. E. D.] and Cancer Research Center [M. E. D.], University of Chicago, Chicago, Illinois 60637; Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [M. X-W., A. E. P.]; and Duke Comprehensive Cancer Center, Duke University, Durham, North Carolina 27710 [S. M. L.]

The DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (AGT) has been shown to protect cells from the toxic and mutagenic effect of alkylating agents by removing lesions from the O⁶ position of guanine. O⁶-Benzylguanine (BG) is a potent inactivator of AGT, resulting in an increase in the sensitivity of cells to the toxic effects of chemotherapeutic alkylating agents. Chinese hamster ovary (CHO) cells and CHO cells transfected with wild-type AGT (CHO^WTAGT) and a mutant AGT [P138 M/V139I/P140K (CHO^MIK)] known to be resistant to BG were treated with BG and various alkylating agents. BG treatment alone dramatically decreased AGT activity in CHO^WTAGT cells but resulted in no depletion in AGT activity in CHO^MIK cells. In the absence of AGT, these cells are highly sensitive to the toxic and mutagenic effects of temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), and no further sensitization occurs in the presence of BG. In contrast, CHO^WTAGT cells are resistant to temozolomide and BCNU, and treatment with BG resulted in a significantly higher cell killing and mutation frequency. CHO^MIK cells were completely resistant to temozolomide or BCNU in the presence and absence of BG. Both cell killing and mutation frequency of 4-hydroperoxycyclophosphamide (4-HC) in CHO, CHO^WTAGT, and CHO^MIK cells were increased in the presence of BG. 4-HC generates two active metabolites, phosphoramide mustard (PM) and acrolein. BG had no effect on 4-hydroperoxydidechlorocyclophosphamide (which generates acrolein and a nonalkylating form of PM) in CHO cells and CHO^MIK cells, but enhancement of toxicity was observed with PM in both these cell lines. Therefore, we attribute the enhancement to the PM metabolite of 4-HC. Our results demonstrate that wild-type AGT plays an important role in protecting against the toxic and mutagenic effect of O⁶ alkylating agents and that a mutant AGT resistant to inactivation by BG effectively prevents BG-enhanced toxicity and mutagenicity induced by these agents. Expression of the AGT protein contributes to resistance of 4-HC. BG also enhances the toxicity of 4-HC and PM by a mechanism that may not involve the AGT repair protein.

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