In VitroSelection for K562 Cells with Higher Retrovirally Mediated Copy Number of Aldehyde Dehydrogenase Class-1 and Higher Resistance to 4-Hydroperoxycyclophosphamide

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Abstract
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. Transfer of drug resistance genes into hematopoietic progenitors is an important developing field in cancer gene therapy. However, the main problems remain achieving high transfer efficiency as well as high functional expression of the gene of interest. Previously, we have reported the successful expression of low levels of ALDH-1 in K562 leukemic cells with up to three-fold increase in resistance to 4-HC. In this study, we used 4-HC treatment to select for K562 cells with the highest pLXSN/ALDH-1 copy number and ALDH-1 expression. We showed that cells surviving one treatment of 4-HC (ALDH-1+) indeed have significantly higher ALDH-1 expression and enzyme activity when compared to untreated ALDH-1 or WT K562 cells. Furthermore, Southern analysis confirmed that the in vitro treatment with 4-HC resulted in the selection of cells with higher copy numbers of ALDH-1 cDNA. FISH analysis using biotin-labeled pLXSN/ALDH-1 vector showed positive signal only in the ALDH-1+ cells. Interestingly, two integration sites of pLXSN/ALDH-1 fusion product were detected. These results may have significant clinical implications, especially in the design of clinical gene therapy protocols. In the setting of autologous stem cell transplantation, the in vitro selection of hematopoietic stem cells with the highest levels of ALDH-1 may allow these cells to compete better in the repopulation of the hematopoietic system and thus allow the use of high doses of cyclophosphamide repeatedly after autologous transplantation.

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