IN-VITRO SELECTION FOR K562 CELLS WITH HIGHER RETROVIRALLY MEDIATED COPY NUMBER OF ALDEHYDE DEHYDROGENASE CLASS-1 AND HIGHER RESISTANCE TO 4-HYDROPEROXYCYCLOPHOSPHAMIDE

Previously, we have reported the successful expression of human aldehy de dehydrogenase class-1 (ALDH-1) in K562 leukemia cells using a retro viral vector and demonstrated low expression that resulted in up to th reefold increase in resistance to 4-hydroperoxycyclophosphamide (4-HC) , an active derivative to cyclophosphamide. The purpose of this study was to investigate whether in vitro treatment with 4-HC will allow sel ection of K562 cells expressing higher levels of ALDH-1, and whether t hese selected cells are more resistant to 4-HC. Stably transfected or transduced K562 cells with retroviral pLXSN vector containing ALDH-1 c DNA (ALDH-1 cells) were treated repeatedly with 4-HC and then allowed to grow to confluence in liquid culture. Subsequently, the resistance to 4-HC of ALDH-1 cells treated once (ALDH-1+) or twice (ALDH-1++) wit h 4-HC was compared to ALDH-1 cells or wild-type K562 cells (WT cells) . The results show significant increase in 4-HC resistance of ALDH-1(2- to 16-fold, p < 0.005) over ALDH-1 or WT cells. No difference was detected between ALDH-1+ and ALDH-1++. In addition, higher ALDH-1 mRNA and enzyme activity were found in ALDH-1+ compared to ALDH-1 cells. S outhern analysis of DNA extracted from the different experimental grou ps demonstrated an eight-fold increase in ALDH-1 cDNA in ALDH-1+ versu s the ALDH-1 cells. This was confirmed by sequential FISH analysis usi ng biotin labeled pLXSN/ALDH-1 vector. Positive signals consistently l ocalized to the centromeric region of chromosome 9 and the long arm of chromosome 17 were demonstrated only in the ALDH-1+ cells and represe nted a fusion product of multiple copies of the pLXSN/ALDH-1 vector. I n summary, we have demonstrated that in vitro treatment with 4-HC resu lts in the selection of K562 cells with multiple copies of ALDH-1 gene that are clustered in two main integration sites. These cells demonst rate significantly higher resistance to 4-HC when compared to previous ly untreated cells. Such successful in vitro selection could have sign ificant implications for future cancer gene therapy protocols.