Abstract
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.
Footnotes
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Send reprint requests to: Jan S. Moreb, M.D., Division of Hematology/Oncology, Department of Medicine, University of Florida College of Medicine, P.O. Box 100277, Gainesville, FL 32610-0277. E-mail: morebjs{at}medicine.ufl.edu
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↵1 This study was supported by Grant R29-CA59684 awarded by the National Cancer Institute (to J.S.M.).
- Abbreviations:
- ALDH-1
- aldehyde dehydrogenase class-1
- 4-HC
- 4-hydroperoxycyclophosphamide
- AS
- antisense
- NSCLC
- nonsmall cell lung cancer
- FBS
- fetal bovine serum
- bp
- base pair
- WT
- wild type
- ECL
- enhanced chemiluminescence
- dCTP
- deoxy cytidine 5′-triphosphate
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- DEAB
- diethylaminobenzaldehyde
- CP
- cyclophosphamide
- CYP
- cytochrome P450
- Received November 1, 1999.
- Accepted January 7, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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