Cyclophosphamide Resistant L1210 Cells Characterization of Cytosolic Aldehyde Dehydrogenase from Updated Version

The cytosolic aldehyde dehydrogenase (ALDH) isozyme from cyclophosphamide (f PA) resistant 1.1210 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, 51,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and as a single charge species at isoelectric point = 5.8 in isoelectric focusing. Micromolar Ä„, values were estimated with both propionaldehyde (A',,,= 5 /IM) and 4-hydroxy cyclophosphamide (4-OH (l'A) (A',„ = 4 /IM) as substrates, indicating that this isozyme is capable of oxidizing the acti vated cyclophosphamide intermediate 4-hydroxy CPA/aldophosphamide to carboxyphosphamide. This isozyme is also potently inhibited by disulfiram (A",= 6 MM)and 4-(diethylamino)benzaldehyde (A",= 0.04 /IM). 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 iso zyme in P388/CPA cells, mouse liver, mouse small intestine, and the 1C1C7 hepatoma cell line, whereas no ALDH is detected in sensitive LI 210 or P388 cells. Also, these antibodies show little cross-reactivity with the mitochondria! isozyme from mouse liver or 1(1(7 cells. From immunological and inhibitor characterization, the soluble ALDH isozyme in L1210/CPA cells appears identical to the normal mouse tissue isozyme.