Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells.
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 Mr 51,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and as a single charge species at isoelectric point = 5.8 in isoelectric focusing. Micromolar Km values were estimated with both propionaldehyde (Km = 5 microM) and 4-hydroxy cyclophosphamide (4-OH CPA) (Km = 4 microM) 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 (Ki = 6 microM) and 4-(diethylamino)benzaldehyde (Ki = 0.04 microM). 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.
Russo
J E
JE
Pharmacology Laboratory, Johns Hopkins Oncology Center, Baltimore, Maryland 21205.
Hilton
J
J
D000444:Aldehyde Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D003520:Cyclophosphamide / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004351:Drug Resistance; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology; D014410:Tumor Stem Cell Assay
D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase