INDUCTION OF METHOTREXATE RESISTANCE BY RETROVIRAL-MEDIATED TRANSFER OF A MUTANT DIHYDROFOLATE REDUCTASE GENE

Methotrexate (MTX), a folate analog which inhibits the enzyme dihydrofolate reductase (DHFR), is an effective antineoplastic drug. However, MTX-induced myelosuppression limits the effectiveness of this agent. Selective induction of MTX resistance in bone marrow stem cells, prior to treatment with MTX, might prevent this toxicity and improve the therapeutic index of the drug. In these studies drug resistance was transferred to mouse and human bone marrow stem cells by retroviral expression vectors containing coding sequences of a mutant DHFR with a decreased affinity for MTX. Three retroviral expression vectors were analyzed. The CIS DR vector contained the mutant DHFR gene inserted into the replication-defective amphotropic 4070 virus, Cistor. The other vectors contained the mutant DHFR inserted into either the env region (SDHT1) or gag-pol region (SDHT2) of a replication-defective spleen focus-forming virus. All three constructs induced approximately a 200-fold resistance to MTX when transfected into NIH3T3 cells. Amphotropic infectious retroviruses were obtained by transfecting the mutant DHFR vectors into a packaging cell line, which supplied the gag, pol, and env proteins for virus production. Virus titers of 4.5 x 10('3) colony-forming units (CFU)/ml (CIS DR), 1.5 x 10('4) CFU/ml (SDHT2), and 5 x 10('5) CFU/ml (SDHT1) were measured by the transfer of MTX resistance to NIH3T3 cells. Modest increases in virus titers could be achieved by either selecting the virus-producing cells in stepwise increasing concentrations of MTX or by introducing the vector into the packaging cell line via infection rather than transfection. The amphotropic SDHT1 virus efficiently induced MTX resistance in cells of several species, including mouse NIH3T3 cells (5 x 10('5) CFU/ml), monkey CV1 cells (4 x 10('3) CFU/ml), and human MCF-7 cells (6 x 10('4) CFU/ml). When cocultured with SDHT1 virus-producing cells, both mouse and human bone marrow cells could be infected and rendered resistant to MTX. Approximately 30-35% of the mouse progenitor cell colonies (CFU/C) and 4-10% of the human CFU/C formed colonies in soft agar in the presence of MTX. Mouse cytotoxic T lymphocytes (generated in a mixed lymphocyte reaction) and mouse helper T lymphocytes can also be made resistant to MTX. After coculture with irradiated virus-producing cells, T lymphocytes proliferated in response to antigen and incorporated ('3)H -deoxyuridine into DNA in the presence of MTX. Thus, this retroviral expression vector can infect both bone marrow stem cells and T lymphocytes and render them resistant to the cytotoxic effects of MTX.