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Retrovirus-mediated Gene Transfer of Rat Glutathione S-Transferase Yc Confers Alkylating Drug Resistance in NIH 3T3 Mouse Fibroblasts¹

Department of Medicine, Divisions of Hematology and Experimental Medicine, and Department of Oncology, Montreal General Hospital and Research Institute, Montreal H3G 1A4 [M. G., S. L., H. A., D. C.]; Experimental Pharmacology Program, Lady Davis Institute for Medical Research and Sir Mortimer B. Davis Jewish General Hospital, Montreal H3T 1E2 [R. L. S., G. B.]; and McGill University, Montreal H3A 2T5 [M. G., S. L., H. A., R. L. S., G. B., D. C.], Canada

A major limitation to successful cancer treatment is the existence of drug resistance. While several mechanisms of drug resistance have now been well characterized, mechanisms of resistance to alkylating drugs have remained less well defined. Several experimental models of alkylator resistance have implicated isoforms of glutathione S-transferase (GST) but transfection experiments using cloned isoforms of GST have yielded conflicting results. While there are several plausible explanations for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional transfection experiments has been raised. To address this issue properly, we have studied rat GST-Yc expression and drug sensitivity to alkylating drugs in populations of mouse NIH 3T3 fibroblasts following either transfection or transduction with an N2-based retrovirus vector. In comparison with cells treated with an antisense vector, Yc-transfected and Yc-transduced populations of NIH 3T3 cells expressed increased levels of GST-Yc mRNA (Northern blot), increased levels of immunodetectable GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increases in total GST activity and 6.1- and 8.3-fold increases in glutathione peroxidase activity (associated with the Yc subunit). Yc-transfected and Yc-transduced cell populations were, respectively, 5.8- (P < 0.001) and 2.4-fold (P < 0.05) resistant to chlorambucil and 10.8- (P < 0.01) and 5.4-fold (P < 0.001) resistant to mechlorethamine. The range of resistance of clonal isolates from either population was 1.8–6.0-fold for chlorambucil and 4.6–6.1-fold for mechlorethamine (P < 0.05). In contrast, these cells showed unaltered sensitivity to the antimetabolite methotrexate, a nonalkylating drug. These results clearly demonstrate that the rat GTS-Yc is able to confer alkylating drug resistance in mouse fibroblasts. The ability to confer alkylating drug resistance following retrovirus-mediated gene transfer also raises the possibility of using GST-Yc somatic gene transfer to confer protection to the hematopoietic system in a gene therapy strategy applicable to cancer.

¹ This work was supported by grants from the National Cancer Institute of Canada (G. B.), the Cancer Research Society, Inc. (D. C.), and the Medical Research Council of Canada (D. C.); and by personal career awards from the Fonds de la Recherche en Santé du Québec (G. B., D. C.). R. L. S. was the recipient of a fellowship from the Research Cancer Society, Inc.

² To whom requests for reprints should be addressed, at Division of Hematology, Montreal General Hospital, 1650 Cedar Avenue, Room A7132, Montreal, Quebec H3G 1A4, Canada.

Received 1/18/94. Accepted 6/16/94.

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