Abstract
Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer alkylating agents. Overexpression of class 1 ALDH (ALDH-1) has been observed in cells selected for survival in the presence of OAPs. We used transfection to induce de novo expression of human ALDH-1 in V79/SD1 Chinese hamster cells to clearly quantitate the role of hALDH-1 expression in OAP resistance. Messenger RNA levels correlated well with hALDH-1 protein levels and enzyme activities (1.5-13.6 milliunits/mg with propionaldehyde/NAD+ substrate, compared to < 1 milliunit/mg in controls) in individual clonal transfectant lines, and slot blot analysis confirmed the presence of the transfected cDNA. Expressed ALDH activity was closely correlated (r = 0.99) with resistance to mafosfamide, up to 21-fold relative to controls. Transfectants were cross-resistant to other OAPs but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. Resistance was completely reversed by pretreatment with 25 microM diethylaminobenzaldehyde, a potent ALDH inhibitor. Alkaline elution studies showed that expression of ALDH-1 reduced the number of DNA cross-links commensurate with mafosfamide resistance, and this reduction in cross-links was fully reversed by the inhibitor. Thus, overexpression of human class 1 ALDH alone is sufficient to confer OAP-specific drug resistance.
Highlights
Human class 1 aldehyde dehydrogenase can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer alkylating agents
1 The abbreviations used are: ALDH, aldehyde dehydrogenase; hALDH-3, human class 3 aldehyde dehydrogenase; hALDH-1, human class 1 aldehyde dehydrogenase; CPA, cyclophosphamide; 4-OH-CPA, 4-hydroxy-cyclophosphamide; CBP, carboxyphosphamide; 4-hp-CPA, 4-hydroperoxy-cyclophosphamide; 4-hp-IF, 4-hydroperoxyifosfamide; MAF, mafosfamide; PM, phosphoramide mustard; ALDO, aldophosphamide; OAP, oxazaphosphorine; DEAB, diethylaminobenzaldehyde; enzymes is presumed to function as an important component of cellular defenses against toxic aldehydes (1–3)
In order to quantitatively examine the effect of variable ALDH expression on OAP-specific resistance and to clearly establish that expression of ALDH-1 alone is sufficient to cause resistance, we have developed transgenic cell lines that express a broad range of activities of either class 1 or class 3 ALDH, via transfection with mammalian expression vector constructs that contain the respective cDNAs encoding each isozyme
Summary
Western Blot Analysis—Cytosolic protein (100 g/lane) was prepared as for enzyme activity assay, electrophoresed on a 14% SDS-PAGE gel, transferred by semidry electroblotting at 200 mA for 1 h onto a nitrocellulose membrane, and probed as described previously (24) with a 1:2000 dilution of anti-rat class 1 ALDH antisera, generously provided by Dr Ronald Lindahl (University of South Dakota). This antisera was cross-reactive with the human ALDH-1 homolog. Treated with 75 M DEAB for 10 min prior to and during a 30-min exposure to 100 M MAF
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