Abstract
Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents (eg. cyclophosphamide). We previously demonstrated that expression of transfected rat ALDH-3 can confer OAP-specific resistance in human MCF-7 cells (Bunting, K. D., Lindahl, R., and Townsend, A. J. (1994) J. Biol. Chem. 269, 23197-23203). However, the aldophosphamide intermediate inactivated by human class 1 ALDH (hALDH-1) has not proven to be a good substrate for the purified hALDH-3. We have examined the ability of transfected human or rat ALDH-3 to confer OAP resistance in V79/SDl cells. Clones expressing elevated human (386-5938 milliunits/mg) or rat (4-597 milliunits/mg, benzaldehyde/NADP+ substrate) ALDH-3 activity were 1.3- to 12-fold resistant to mafosfamide relative to control cells (<1 milliunit/mg). Resistance was correlated with hALDH-3 activity, and was reversed by pretreatment with the ALDH inhibitor diethylaminobenzaldehyde. Transfectants were cross-resistant to 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. DNA interstrand cross-links were reduced commensurately with the fold resistance to mafosfamide in the highest activity clone. A key finding was the detection of a metabolite, most likely carboxyphosphamide, that is formed only by cytosols from cells expressing either class 3 or class 1 ALDH.
Highlights
Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents
Transfection of Rat or Human Class 3 ALDH Expression Constructs—Class 3 ALDH-expressing cell lines were generated by calcium phosphate transfection of rat or human class 3 ALDH expression vector constructs into V79 cells
The ALDH-1-mediated resistance was accompanied by increased formation of carboxyphosphamide [1], the inactivated product of ALDH oxidation of aldophosphamide, and decreased DNA cross-linking was found [24]
Summary
Materials—Sources of chemical and pharmacologic materials are described in our companion study that reports resistance conferred by hALDH-1 [28]. DNA sequence analysis of approximately the first 150 base pair indicated a match of the sequence with that previously reported by Hsu et al [20] This cDNA was subcloned into the XhoI site of the ⌬pCEP4⌬ mammalian expression vector, a derivative of the pCEP4 vector (Invitrogen) that was modified to prevent episomal replication and favor host integration [28]. Aliquots (500 l) of this activation premix were adjusted to contain 1 ϫ ALDH assay buffer, 5 mM mercaptoethanol, 100 l of cell cytosols (12,000 ϫ g supernatant, approximately 5 mg/ml), and either 1 mM NADϩ (for class 1 and control extracts) or 1 mM NADPϩ (for class 3 extracts) in a final volume of 1.0 ml, and the incubation was continued for an additional hour at 25 °C. After analysis on the PhosphorImager, regions in each lane corresponding to the carboxyphosphamide band were scraped from the dried plate, and total counts/min were determined by scintillation counting
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