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 ALDH-1 antisense RNA expression on ALDH-1 activity and sensitivity to 4-HC toxicity. Three different ALDH-1 cDNAs were synthesized that are either missing 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) orientation (AS-N, AS-C, and AS-NC, respectively). It was demonstrated that the overexpression of each of the AS constructs in K562 leukemic cells and A549 lung cancer cells results in suppression of ALDH-1 mRNA and enzymatic activity. 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 ALDH-1 AS became significantly more sensitive to 4-HC toxicity as demonstrated by clonogenic and liquid culture assays. The increase in 4-HC sensitivity was in correlation with the degree of suppression of ALDH-1 activity. Moreover, such increase in 4-HC sensitivity, especially with AS-N and AS-NC, was to a similar degree seen with the use of diethylaminobenzaldehyde, a specific inhibitor of ALDH-1. These results indicate that ALDH-1 expression and activity can be specifically and effectively suppressed by AS RNA and lead to increased sensitivity to 4-HC.
Moreb
J S
JS
Division of Hematology/Oncology, Department of Medicine, University of Florida, Gainesville, Florida32610-0277, USA. morebjs@medicine.ufl.edu
Maccow
C
C
Schweder
M
M
Hecomovich
J
J
D000444:Aldehyde Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000096:biosynthesis; D005822:Genetic Vectors; D006801:Humans; D007942:Leukemia, Experimental / Q000378:metabolism; D008175:Lung Neoplasms / Q000378:metabolism; D016372:RNA, Antisense / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D016372:RNA, Antisense; D012333:RNA, Messenger; D003520:Cyclophosphamide; D000444:Aldehyde Dehydrogenase; C011272:perfosfamide