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Transduction of NIH 3T3 Cells with a Retrovirus Carrying Both Human MDR1 and Glutathione S-Transferase Produces Broad-Range Multidrug Resistance¹

Departments of Medical Oncology and Therapeutics Research [J. H. D., L. M., K. A. W.] and Cell and Tumor Biology [M. Z. M., S. E. K.], City of Hope National Medical Center, Duarte, California 91010; Department of Biochemistry, Hokkaido University School of Medicine, N15, W7, Sapporo 060, Japan [M. S.]; and Department of Biochemistry, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun 350-04, Saitama, Japan [M. M.]

In these experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units. We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous gene along with MDR1 [M. E. Metz, D. M. Best, and S. E. Kane. Virology, 208: 634–643, 1995]. To test the ability of this vector to deliver two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase (GST , the most abundant member of the glutathione S-transferase family in human tumor cells) was inserted into pHaMASV, and this plasmid was transfected into ecotropic packaging cells. The resulting pHaMASV.GST ecotropic retrovirus, which was produced at a titer of 2 x 10⁶ colony-forming units/ml, was used to transduce NIH 3T3 cells. After initial selection in 60 ng/ml colchicine, a population of transduced cells was exposed to stepwise increasing colchicine concentrations to select for amplified expression of MDR1. As MDR1 expression increased, the expression of GST increased in concert, as demonstrated by Northern analysis, Western analysis, and measurements of glutathione S-transferase activity. Transduced cells growing in 1280 ng/ml colchicine had about 3-fold higher total glutathione S-transferase activity than nontransduced cells and 2.5-fold higher activity than transduced cells growing in 60 ng/ml colchicine. Northern hybridizations demonstrated a 3–5-fold increase in both the full-length retroviral message encoding MDR1 and the subgenomic mRNA encoding GST after amplification of resistance from 60 to 1280 ng/ml colchicine. The cytotoxic effects of several xenobiotics were evaluated in NIH 3T3 cells transfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST retrovirus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expressed. 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of MDR1 had identical levels of resistance to doxorubicin or colchicine. These results suggest that GST expression did not contribute to doxorubicin resistance in this model system. However, 3T3.GST640 cells were about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrobenzene compared to cells expressing MDR1 alone, consistent with the ability of GST to conjugate both of these cytotoxins. Increases in drug resistance paralleled increases in gene-specific mRNA and recombinant protein levels in all cases. Thus, our studies suggest that the amplifiable coexpression of MDR1 plus a second potentially therapeutic gene of interest is a feasible strategy for the delivery of multiple drug resistance genes to normal cells for protection against the toxic side effects of combination chemotherapy.

¹ Supported by National Cancer Institute Grants CA59308 and CA33572.

² To whom requests for reprints should be addressed, at Department of Cell and Tumor Biology, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010.

Received 5/27/94. Accepted 7/13/95.

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