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Specificity of Bischloroethylnitrosourea-induced Mutation in a Chinese Hamster Ovary Cell Line Transformed to Express Human O⁶-Alkylguanine-DNA Alkyltransferase¹

Environmental Health Sciences. Case Western Reserve University [D. T. M., S. L. G., M. L. V., W. D. S.], Departments of Medicine [S. L. G., L. L. D., M. L. V., W. D. S.], and Ireland Cancer Center of University Hospitals [S. L. G., L. L. D., M. L. V., W. D. S.], Cleveland, Ohio 44106

The human O⁶-alkylguanine-DNA alkyltransferase complementary DNA was transfected into the alkyltransferase-deficient Chinese hamster ovary cell line, D422, in an effort to dissect the underlying mechanisms of bischloroethylnitrosourea (BCNU)-induced mutations. The alkyltransferase-transformed cell line exhibited 100-fold protection against BCNU-induced toxicity and an overall decrease in mutation frequency to 25% of that observed in the parental cell line at the hemizygous adenine phosphoribosyl transferase gene target. The frequency of the predominant mutation in the parental cell line, the G:CT:A transversion, was reduced from 16 x 10^-6 to 0.7 x 10^-6 in the O⁶-alkyltransferase-transformed cell line. Likewise, the G:CA:T transitions, the second most common BCNU-induced mutation in the parental cell line, was reduced in frequency from 5.2 x 10^-6 to 0.9 x 10^-6 in the alkyltransferase-transformed Chinese hamster ovary cells. These findings suggest that both the G:CT:A transversions and G:CA:T transitions were O⁶-alkylguanine-mediated mutations. In the alkyltransferase-transformed Chinese hamster ovary cell line, T:AG:C transversions, comprising 45% (23 of 51) of the recovered mutations, emerged as the most common base substitution. In summation, in the absence of alkyltransferase-dependent DNA repair, mutations resulting from O⁶-alkylation of guanine underlie both the cytotoxic and mutagenic activity of BCNU. In cells expressing high levels of alkyltransferase activity, the cytotoxic and mutagenic actions of BCNU are greatly reduced and mutations resulting from A:T base pair modifications appear to be the major genotoxic lesions induced by the drug.

¹ This work was supported in part by National Institute for Environmental Health Sciences Research Grant R01 ESO5540 (W. D. S.); a Research Grant from the Cuyahoga County Unit, Ohio Division of the American Cancer Society (M. L. V.); a Research Grant from Glaxo, Inc. (W. D. S., M. L. V.); a Cancer Center Core grant to The Ireland Cancer Center (P30CA43703); Grants P01CA51183 (S. L. G.) and K08ES00244 (L. L. D.) from the NIH; and Grants CN-34 (S. L. G.) and RA322 (L. L. D.) from the American Cancer Society. S. L. G. is a Mallinckrodt Foundation Scholar.

² To whom requests for reprints should be addressed, at Case Western Reserve University, Department of Medicine, Hematology/Oncology, UCRC Bldg. 2, Suite 200, 11001 Cedar Rd. Cleveland, OH 44106.

³ Present address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.

⁴ Present address: Department of Pathology and Laboratory Medicine, Brown University School of Medicine, Providence, RI 03912.

Received 7/16/92. Accepted 12/16/92.

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