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Characterization of Cytosolic Aldehyde Dehydrogenase from Cyclophosphamide Resistant L1210 Cells¹

Pharmacology Laboratory, The Johns Hopkins Oncology Center [J. E. R., J. H.], and Department of Pharmacology and Molecular Science [J. E. R.], The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

The cytosolic aldehyde dehydrogenase (ALDH) isozyme from cyclophosphamide (CPA) resistant L1210 cells (L1210/CPA) was purified to apparent homogeneity using ternary enzyme complex-dye ligand chromatography. The purified isozyme migrates as a single band at M_r 51,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and as a single charge species at isoelectric point = 5.8 in isoelectric focusing. Micromolar K_m values were estimated with both propionaldehyde (K_m = 5 µM) and 4-hydroxy cyclophosphamide (4-OH CPA) (K_m = 4 µM) as substrates, indicating that this isozyme is capable of oxidizing the activated cyclophosphamide intermediate 4-hydroxy CPA/aldophosphamide to carboxyphosphamide. This isozyme is also potently inhibited by disulfiram (K_i = 6 µM) and 4-(diethylamino)benzaldehyde (K_i = 0.04 µM). Both of these inhibitors are capable of sensitizing L1210/CPA cells to activated CPA in clonogenic survival assays. Thus, the increased levels of only the cytosolic ALDH isoform in L1210/CPA cells appear to be the single phenotypic difference necessary for conferring resistance to CPA. Monospecific antibodies to the L1210/CPA isozyme have been used in Western blot analysis to detect nanogram levels of ALDH in cell and tissue extracts. These antibodies cross-react with the cytosolic isozyme in P388/CPA cells, mouse liver, mouse small intestine, and the 1C1C7 hepatoma cell line, whereas no ALDH is detected in sensitive L1210 or P388 cells. Also, these antibodies show little cross-reactivity with the mitochondrial isozyme from mouse liver or 1C1C7 cells. From immunological and inhibitor characterization, the soluble ALDH isozyme in L1210/CPA cells appears identical to the normal mouse tissue isozyme.

¹ This research was supported by USPHS Grants CA-36966 and CA-16783 awarded by the National Cancer Institute, Department of Health and Human Services.

² Supported by a National Science Foundation Graduate Fellowship. To whom requests for reprints should be addressed, at Pharmacology Laboratory 1-120, The Johns Hopkins Oncology Center, The Johns Hopkins School of Medicine, 600 N. Wolfe St., Baltimore, MD 21205.

Received 12/21/87. Revised 3/ 3/88. Accepted 3/ 8/88.

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