Osteosarcoma cells, resistant to methotrexate due to nucleoside and nucleobase salvage, are sensitive to nucleoside analogs

Purpose: To test a novel strategy for overcoming intrinsic resistance to methotrexate (MTX) in osteosarcoma (OS) due to nucleoside and nucleobase salvage (NS). Methods: Four OS cell lines, found to be highly resistant to MTX, were tested to determine the dominant mechanism of resistance. Sensitivity to MTX was tested in the presence of dialyzed serum or the transport inhibitor dipyridamole (DP) to confirm the contribution of NS to MTX resistance. We then investigated whether increased NS activity could be exploited using cytotoxic nucleoside analogs. Results: Like other cell types, OS cells are capable of circumventing inhibition of de novo nucleotide synthesis by relying on NS. MTX, at concentrations as high as 1 mM did not inhibit cell growth in culture medium supplemented with undialyzed serum. In contrast, when NS was inhibited by DP or in medium depleted of nucleosides and nucleobases, sensitivity to MTX was seen at nanomolar concentrations. In medium with dialyzed serum, thymidine and hypoxanthine provided dose-dependent protection from MTX toxicity at concentrations similar to those seen in human plasma. No evidence of other significant mechanisms of resistance were found. All four cell lines were sensitive to 3-day exposures to cytarabine (IC50 0.22 to 2.88 muM) and vidarabine (IC50 0.09 to 0.95 muM). Conclusions: Salvage of de novo nucleotide synthesis inhibition by extracellular thymidine and hypoxanthine, at physiologically relevant concentrations, contributes to resistance to MTX in OS. However, this same process may impart a collateral sensitivity to nucleoside analogs. These findings support clinical trials for patients with OS using nucleoside analogs, either alone or in combination.