Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase

Abstract

We have constructed a retroviral bicistronic vector, MFG/GID, that transduces the expression of both the A3 isoform of the rat glutathione S-transferase (GST A3), and the tyr-22 variant of the human dihydrofolate reductase (DHFR(L22Y)). Transduction of murine 3T3 fibroblasts with this vector increased their in vitro resistance to chlorambucil (1.8-fold) and trimetrexate (TMTX) (748-fold). TMTX selection of a mixed population of 20% GID-transduced NIH 3T3 cells and 80% control cells resulted in a marked increase in the GST peroxidase activity associated with the GST A3 isoform (17.7-fold). MFG/GID-transduced primary clonogenic murine hematopoietic progenitor cells were likewise more resistant to TMTX and chlorambucil than control beta-gal-transduced cells. Selecting GID-transduced hematopoietic cells with a combination of TMTX and a nucleoside transport inhibitor resulted in a marked increase in resistance upon re-exposure to TMTX (99% survival). Similarly, GID-transduced hematopoietic cells selected with TMTX were more resistant to chlorambucil, with 40% survival at a drug concentration that killed practically all control cells. These results suggest that antifolate-mediated selection of MFG/GID-transduced hematopoietic cells could be used as a mean to enrich the population of transduced cells prior to or following transplantation, thus potentially conferring in vivo chemoprotection to nitrogen mustards and antifolates.