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 aldehyde dehydrogenase class-1 (ALDH-1) in K562 leukemia cells using a retroviral vector and demonstrated low expression that resulted in up to threefold 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 selection of K562 cells expressing higher levels of ALDH-1, and whether these selected cells are more resistant to 4-HC. Stably transfected or transduced K562 cells with retroviral pLXSN vector containing ALDH-1 cDNA (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++) with 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. Southern analysis of DNA extracted from the different experimental groups demonstrated an eight-fold increase in ALDH-1 cDNA in ALDH-1+ versus the ALDH-1 cells. This was confirmed by sequential FISH analysis using biotin labeled pLXSN/ALDH-1 vector. Positive signals consistently localized to the centromeric region of chromosome 9 and the long arm of chromosome 17 were demonstrated only in the ALDH-1+ cells and represented a fusion product of multiple copies of the pLXSN/ALDH-1 vector. In summary, we have demonstrated that in vitro treatment with 4-HC results in the selection of K562 cells with multiple copies of ALDH-1 gene that are clustered in two main integration sites. These cells demonstrate significantly higher resistance to 4-HC when compared to previously untreated cells. Such successful in vitro selection could have significant implications for future cancer gene therapy protocols.