OVEREXPRESSION OF THE HUMAN ALDEHYDE DEHYDROGENASE CLASS-I RESULTS ININCREASED RESISTANCE TO 4-HYDROPEROXYCYCLOPHOSPHAMIDE

A correlation between overexpression of aldehyde dehydrogenase and res istance to oxazaphosphorines, widely used anticancer agents, has been shown. To investigate the direct role of the human aldehyde dehydrogen ase class 1 (ALDH-1) in the resistance to one of these agents, 4-hydro peroxycyclophosphamide (4-HC), an active metabolite of cyclophosphamid e, neomycin-selectable plasmid or retroviral constructs harboring the wild-type ALDH-1 complementary DNA in the sense orientation were trans fected into K562 leukemic cell lines. Polymerase chain reaction (PCR) analysis confirmed the presence of vector DNA in the stably transfecte d K562 cells. Reverse transcriptase PCR and Northern and Western blot analysis showed expression of ALDH-1 mRNA and protein in the cells tra nsfected with ALDH-1 in the sense orientation but not in cells transfe cted with vector alone. The activity of the expressed ALDH-1 was demon strated using spectrophotometric assay. Stably transfected K562 cells were subjected to various doses of 4-HC, and cell viability was assaye d using clonogenic cell culture in semisolid medium. Results demonstra te that K562 cells transfected with ALDH-1 in the sense orientation di splay increased resistance to 4-HC compared with wild-type or vector-t ransfected K562 cells. Furthermore, the addition of diethylaminobenzal dehyde, a specific inhibitor for ALDH-1, restored the sensitivity of t he ALDH-l-expressing K562 cells to 4-HC. Thus, the data pinpoint the d irect role of ALDH-1 in the protection against 4-HC cytotoxicity.