COEXPRESSION OF THE HERPES-SIMPLEX VIRUS THYMIDINE KINASE GENE POTENTIATES METHOTREXATE RESISTANCE CONFERRED BY TRANSFER OF A MUTATED DIHYDROFOLATE-REDUCTASE GENE

We have previously shown that transfer of a mutated dihydrofolate redu ctase (DHFR) confers resistance to methotrexate (MTX) to infected cell s. We report herein the construction of a retrovirus vector, DC/SV6S31 tk, which carries the herpes simplex virus thymidine kinase gene (HSVt k) as well as-the mutated Serine 31 DHFR (S31) cDNA. 3T3 cells infecte d with DG/SV6S31tk are more resistant to MTX than cells infected with DC/SV6S31, which carries the S31 and NEOr gene. In DC/SV6S31tk-infecte d cells, a fraction of cells (20-40%) were more resistant to MTX compa red with DC/SV6S31-infected cells, and these cells survived a 5-day ex posure to 200 mu M of NeMTX. The mechanism of this augmented resistanc e is attributed to the salvage of thymidine by HSVtk, as the augmentat ion is reversed when dialyzed serum is used for cytotoxicity assays. T he cells that survive high-dose MTX selection have high levels of expr ession of S31 DHFR and HSVtk, although copy numbers of he proviral seg uences do not increase significantly. Transduction of cells with the D C/SV6S31tk vector also sensitizes cells to ganciclovir (GCV). Co-expre ssion of a metabolically related gene in a retroviral vector to potent iate the resistance imparted by a drug resistance gene may be useful f or gene therapy for cancer patients.