The oxazaphosphorine antitumor agents cyclophosphamide and ifosfamide are the most widely used alkylating agents used in chemotherapy. Although they have similar structures, there is a substantial body of clinical evidence to suggest that there are important differences between these two agents which need to be elucidated both to better understand their mechanism of action and to determine if unique resistance mechanisms operate in normal and neoplastic tissue. The cytotoxicity of many alkylating agents is reduced or eliminated by their conjugation with glutathione. We propose to investigate the role of specific human glutathione transferases in the conjugation of the nitrogen mustards released from cyclophosphamide and ifosfamide and to measure the appearance of interstrand cross-links in cells exposed to activated cyclophosphamide and activated ifosfamide and their respective mustards, phosphoramide mustard and ifosfamide mustard. Additional studies will investigate the role of 4-glutathionyl adducts of cyclophosphamide and ifosfamide. These recently reported adducts occur at a point in the metabolism of the oxazaphosphorines where they could influence the overall cytotoxicity of the parent compounds and the extent of their appearance may be closely related to the intracellular glutathione content. Studies of the enzymic and non-enzymic formation of glutathione adducts will employ radiolabelled alkylating agents and analysis of the hydrolytic and conjugation products by HPLC. Similar methods will be used to investigate the role of specific transferase isozymes in the conjugation of busulfan, an alkylating agent widely used in combination with cyclophosphamide in preparative regimens for bone marrow transplantation. Some aspects of this proposal will utilize NMR methods to follow the formation and transformation of adducts, including those at the 4-position of the oxazaphosphorine ring. A series of experiments using flow cytometric methods will investigate the distribution of factors related to alkylating agent resistance in discrete populations of normal and neoplastic human marrow. The concentration of glutathione and the levels of glutathione transferases and O6-alkyl transferase will be measured in hematopoietic cell populations.
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