The overall goal is to define the relative contributions of four key metabolic enzymes in determining the efficacy, toxicity and cellular resistance mechanisms the determine the therapeutic outcome of treatment with cyclophosphamide (CPA) and it analogs. The investigato'rs previous studies have shown that overexpression in V79 of aldehyde dehydrogenase (ALDH) -1 or -3 leads to resistance to CPA analogs by decreasing DNA interstrand crosslinks; due to oxidation of the intermediate aldophosphamide (ALDO) to the inactive carboxyphosphamide (CBP). Purified ALDH-3 does not catalyze the reaction, thus in Specific Aim 1 reconstitution studies will be carried out to identify cytosolic cofactors. There is a higher (6-10 fold) resistance to mafosfamide (MAF) than to 4-hydroxycyclophosphamide which is presumably due to the thiol moiety MESNA that is released from this analog. MAF depletes glutathione (GSH) more slowly. ALDH resistance-mediated resistance may synergize with intra- or extracellular GSH or other thiols.
Specific Aim 2 will define the extent and mechanism of this interaction. The relative efficacy of ALDH for detoxification of CPA activated intra- versus extracellularly is unknown. The investigator will use activation-competent, ALDH-transfected cells to test the hypothesis that the ALDH pathway will confer higher resistance at equitoxic doses under conditions of continuous endogenous activation of CPA by Cyt P450 than when ALDO is presented as a bolus (Aim 3), perhaps due in part to effects of GSH maintenance. Fourth, a novel activation pathway implicated in CPA toxicity to lung and bladder, involving co-oxidation by cyclooxyenases (COX-1 or COX-2) will be studied using transfected cell lines already expressing these isozymes. The cells will be examined first to determine if they activate CPA in an arachidonatedependent manner. If so, they will be used as recipients for transfection with the ALDH expression vectors. Cytotoxicity studies and metabolite and GSH analysis will be used to compare the role of COX and ALDH expression in these cells to see if differences exist between the different activation pathways +/- detoxification by ALDH (Aim 4) These mechanistic studies in genetically defined models will provide new insights to the dynamics of different CPA metabolism pathways.
| Townsend, A J; Leone-Kabler, S; Haynes, R L et al. (2001) Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells. Chem Biol Interact 130-132:261-73 |
| Haynes, R L; Brune, B; Townsend, A J (2001) Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation. Free Radic Biol Med 30:884-94 |
| Haynes, R L; Szweda, L; Pickin, K et al. (2000) Structure-activity relationships for growth inhibition and induction of apoptosis by 4-hydroxy-2-nonenal in raw 264.7 cells. Mol Pharmacol 58:788-94 |
| Bunting, K D; Townsend, A J (1998) Dependence of aldehyde dehydrogenase-mediated oxazaphosphorine resistance on soluble thiols: importance of thiol interactions with the secondary metabolite acrolein. Biochem Pharmacol 56:31-9 |
