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Role of O⁶-Alkylguanine-DNA Alkyltransferase in Protecting against Cyclophosphamide-induced Toxicity and Mutagenicity¹

Section of Hematology/Oncology, Department of Medicine [Y. C., M. H. W., M. E. D.], Department of Radiation and Cellular Oncology [D. J. G.], and Cancer Research Center [M. E. D.], University of Chicago, Chicago, Illinois 60637, and Duke Comprehensive Cancer Center, Duke University, Durham, North Carolina 27710 [S. M. L.]

Cyclophosphamide is used to treat a wide range of human malignancies. However, it is also a known carcinogen associated with induction of therapy-related leukemia and bladder cancer. The DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT), protects cells from the toxic and mutagenic effects of O⁶-alkylating agents. We report here the contribution of AGT in protecting against the toxic and mutagenic effects of cyclophosphamide. CHO cells transduced with wild-type human AGT (CHO^AGT) and pcDNA3 (CHO^pcDNA3) were treated with activated cyclophosphamide derivatives, 4-hydroperoxycyclophosphamide (4-HC), 4-hydroperoxydidechlorocyclophosphamide (4-HDC), a progenitor of acrolein, and phosphoramide mustard (PM). The results show that CHO^AGT is 7- or 20-fold less sensitive to the toxic effects of 30 µM 4-HC or 300 µM 4-HDC, respectively, than CHO^pcDNA3 cells as measured by cell survival using a colony-forming assay. CHO^AGT cells treated with 20 µ M 4-HC or 200 µM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHO^pcDNA3 cells treated with the same dose of drugs. At 30 µM acrolein, the cell survival for CHO^AGT was 30% compared with 18.7% for CHO^pcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10⁶ cells in CHO^pcDNA3 compared with no mutants in CHO^AGT. In contrast, CHO^AGT and CHO^pcDNA3 cells treated with PM had similar survival curves and exhibited no difference in mutation frequency. The present study demonstrates that AGT plays an important role in protecting against the toxic and mutagenic effect of cyclophosphamide and suggests that acrolein, not PM, is responsible for generating the toxic and mutagenic lesion(s) protected by the AGT protein.

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