TRANSDUCTION OF NIH 3T3 CELLS WITH A RETROVIRUS CARRYING BOTH HUMAN MDR1 AND GLUTATHIONE-S-TRANSFERASE-PI PRODUCES BROAD-RANGE MULTIDRUG-RESISTANCE

In these experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate tra nscription units. We previously described the pHaMSV vector, which inc ludes the human MDR1 gene as a selectable marker and chemoprotective g ene, plus an internal SV40 promoter for expressing a second heterologo us gene along with MDR1 [M. E. Metz, D. M. Best, and S. E. Kane. Virol ogy, 208: 634-643, 1995]. To test the ability of this vector to delive r two therapeutic genes simultaneously, the cDNA for human glutathione S-transferase pi (GST pi, 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 pi ecotropic retrovirus, which was produced at a titer of 2 x 10(6) colony-forming units/ml, was used to transduce NIH 3T3 cells. After initial selection in 60 ng/ml colchicine, a populati on of transduced cells was exposed to stepwise increasing colchicine c oncentrations to select for amplified expression of MDR1. As MDR1 expr ession increased, the expression of GST increased in concert, as demon strated by Northern analysis, Western analysis, and measurements of gl utathione 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 tr ansduced cells growing in 60 ng/ml colchicine. Northern hybridizations demonstrated a 3-5-fold increase in both the full-length retroviral m essage encoding MDR1 and the subgenomic mRNA encoding GST pi. after am plification of resistance from 60 to 1280 ng/ml colchicine. The cytoto xic effects of several xenobiotics were evaluated in NIH 3T3 cells tra nsfected with MDR1 (3T3.MDR) or transduced with the MDR1-GST pi retrov irus (3T3.GST640 or 3T3.GST1280) to evaluate the ability of our vector to produce a spectrum of drug resistances specific for the genes expr essed, 3T3.MDR and 3T3.GST1280 cells expressing equivalent levels of M DR1 had identical levels of resistance to doxorubicin or colchicine. T hese results suggest that GST pi; expression did not contribute to dox orubicin resistance in this model system. However, 3T3.GST640 cells we re about 4-fold resistant to ethacrynic acid and 1-chloro-2,4-dinitrob enzene compared to cells expressing MDR1 alone, consistent with the ab ility of GST pi to conjugate both of these cytotoxins. Increases in dr ug resistance paralleled increases in gene-specific mRNA and recombina nt protein levels in all cases. Thus, our studies suggest that the amp lifiable coexpression of MDR1 plus a second potentially therapeutic ge ne of interest is a feasible strategy for the delivery of multiple dru g resistance genes to normal cells for protection against the toxic si de effects of combination chemotherapy.