RETROVIRUS-MEDIATED GENE-TRANSFER OF RAT GLUTATHIONE-S-TRANSFERASE YCCONFERS ALKYLATING DRUG-RESISTANCE IN NIH 3T3 MOUSE FIBROBLASTS

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 alkyla tor resistance have implicated isoforms of glutathione S-transferase ( GST) but transfection experiments using cloned isoforms of GST have yi elded conflicting results. While there are several plausible explanati ons for these apparently contradictory findings, the issue that clonal variability might potentially confound the results of conventional tr ansfection experiments has been raised. To address this issue properly , we have studied rat GST-Yc expression and drug sensitivity to alkyla ting drugs in populations of mouse NIH 3T3 fibroblasts following eithe r 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 le vels of GST-Yc mRNA (Northern blot), increased levels of immunodetecta ble GST-Yc (Western blot), and, respectively, 1.4- and 1.9-fold increa ses in total GST activity and 6.1- and 8.3-fold increases in glutathio ne peroxidase activity (associated with the Yc subunit). Yc-transfecte d and Yc-transduced cell populations were, respectively, 5.8- (P < 0.0 01) 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-fo ld for chlorambucil and 4.6-6.1-fold for mechlorethamine (P < 0.05). I n contrast, these cells showed unaltered sensitivity to the antimetabo lite methotrexate, a nonalkylating drug. These results clearly demonst rate 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 possibili ty of using GST-Yc somatic gene transfer to confer protection to the h ematopoietic system in a gene therapy strategy applicable to cancer.