Expression of antisense RNA to aldehyde dehydrogenase class-1 sensitizes tumor cells to 4-hydroperoxycyclophosphamide in vitro

Previous studies in this laboratory showed that the overexpression of human aldehyde dehydrogenase class-1 (ALDH-1) with a retroviral vector resulted in increased resistance to 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide. The present study examined the effect of AL DH-1 antisense RNA expression on ALDH-1 activity and sensitivity to 4-HC to xicity. Three different ALDH-1 cDNAs were synthesized that are either missi ng the N terminus (N), C terminus (C), or both (NC) and subcloned into the BamHI cloning site of pLXSN retroviral vector in the antisense (AS) orienta tion (AS-N, AS-C, and AS-NC, respectively). It was demonstrated that the ov erexpression of each of the AS constructs in K562 leukemic cells and A549 l ung cancer cells results in suppression of ALDH-1 mRNA and enzymatic activi ty. Furthermore, the AS-N and AS-NC were generally more effective than AS-C in reducing the ALDH-1 activity. Both K562 and A549 cells expressing the A LDH-1 AS became significantly more sensitive to 4-HC toxicity as demonstrat ed by clonogenic and liquid culture assays. The increase in 4-HC sensitivit y was in correlation with the degree of suppression of ALDH-1 activity. Mor eover, such increase in 4-HC sensitivity, especially with AS-N and AS-NC, w as to a similar degree seen with the use of diethylaminobenzaldehyde, a spe cific inhibitor of ALDH-1. These results indicate that ALDH-1 expression an d activity can be specifically and effectively suppressed by AS RNA and lea d to increased sensitivity to 4-HC.