Maintenance of differential methotrexate toxicity between cells expressing drug-resistant and wild-type dihydrofolate reductase activities in the presence of nucleosides through nucleoside transport inhibition

Abstract

Methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR), has been used widely as a chemotherapeutic agent and as a selective agent for cells expressing drug-resistant DHFR activity. MTX deprives rapidly dividing cells of reduced folates that are necessary for thymidylate synthesis and de novo purine nucleotide synthesis. However, MTX toxicity can be circumvented by salvaging thymidine (TdR) and purine nucleosides. Here we have investigated conditions under which nucleoside transport inhibition can be used to maintain differential MTX toxicity between unmodified cells and cells expressing drug-resistant DHFR activity in the presence of exogenous nucleosides. PA317 cells (a 3T3 derivative cell line) were rescued from the toxicity of 0.1 microM MTX by 1.0 microM TdR in the presence of 100 microM inosine. The nucleoside transport inhibitor dipyridamole (DP) resensitized these cells to MTX, even in the presence of exogenous nucleosides. Furthermore, PA317 cells transduced with any of three retroviruses encoding drug-resistant DHFRs remained resistant to MTX over all concentrations tested (up to 10.0 microM) in the presence of DP. Similar results were obtained in transduced HuH7 and K562 cell lines, a human hepatoma and a human leukemia cell line, respectively. We conclude that nucleoside transport inhibition increases the toxicity and selectivity of MTX in cultured cells, and therefore is an effective way to maintain differential MTX toxicity between unmodified and DHFR-modified cells. Our results support the use of nucleoside transport inhibition in in vivo selection protocols involving the liver and hematopoietic systems.