Cai Y, Wu M H, Ludeman S M, Grdina D J, Dolan M E
Department of Medicine, University of Chicago, Illinois 60637, USA.
Cancer Res. 1999 Jul 1;59(13):3059-63.
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 (CHOpcDNA3) 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 microM 4-HC or 300 microM 4-HDC, respectively, than CHOpcDNA3 cells as measured by cell survival using a colony-forming assay. CHO(AGT) cells treated with 20 microM 4-HC or 200 microM 4-HDC produced 4- or 7-fold lower mutation frequency as measured at the HPRT locus than CHOpcDNA3 cells treated with the same dose of drugs. At 30 microM acrolein, the cell survival for CHO(AGT) was 30% compared with 18.7% for CHOpcDNA3. The mutation frequency of acrolein at the same dose was 57 mutants/10(6) cells in CHOpcDNA3 compared with no mutants in CHO(AGT). In contrast, CHO(AGT) and CHOpcDNA3 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.
环磷酰胺用于治疗多种人类恶性肿瘤。然而，它也是一种已知的致癌物，与治疗相关白血病和膀胱癌的诱发有关。DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（AGT）可保护细胞免受O6-烷基化剂的毒性和诱变作用。我们在此报告AGT在抵御环磷酰胺的毒性和诱变作用方面的贡献。用野生型人AGT（CHO(AGT)）和pcDNA3（CHOpcDNA3）转导的CHO细胞用活化的环磷酰胺衍生物、4-氢过氧环磷酰胺（4-HC）、4-氢过氧二氯环磷酰胺（4-HDC，丙烯醛的前体）和磷酰胺芥（PM）进行处理。结果表明，通过集落形成试验测量细胞存活率，CHO(AGT)对30 microM 4-HC或300 microM 4-HDC的毒性作用的敏感性分别比CHOpcDNA3细胞低7倍或20倍。用20 microM 4-HC或200 microM 4-HDC处理的CHO(AGT)细胞在HPRT位点测得的突变频率比用相同剂量药物处理的CHOpcDNA3细胞低4倍或7倍。在30 microM丙烯醛作用下，CHO(AGT)的细胞存活率为30%，而CHOpcDNA3为18.7%。相同剂量下丙烯醛在CHOpcDNA3中的突变频率为57个突变体/10(6)细胞，而CHO(AGT)中无突变体。相比之下，用PM处理的CHO(AGT)和CHOpcDNA3细胞具有相似的存活曲线，且在突变频率上无差异。本研究表明AGT在抵御环磷酰胺的毒性和诱变作用中起重要作用，并表明丙烯醛而非PM是产生由AGT蛋白保护的毒性和诱变损伤的原因。
