Abstract

The potential role of the polyglutamate derivatives of methotrexate in the cytotoxicity of methotrexate has been examined in H-35 hepatoma cells in culture. Pulse doses of methotrexate result in the accumulation of a nonexchangeable fraction of methotrexate that is toxic to the cells and consists almost entirely of polyglutamates. The toxicity of the polyglutamates appears to correlate with their effects on de novo thymidine synthesis, which was measured in intact cells by the release of tritium from [5-³H]deoxyuridine. Evidence for this comes from the observation that the dose-inhibition response following pulses of methotrexate is nearly identical for cell growth and tritium release. Protection studies demonstrated that both thymidine and hypoxanthine are needed to prevent methotrexate toxicity, offering evidence that inhibition of cell growth is due to a depletion of reduced folate coenzymes. Cells treated with methotrexate were analyzed for the composition of the species bound to the target enzyme dihydrofolate reductase (EC 1.5.1.3). With 0.03 and 10 µM methotrexate in the medium the dihydrofolate reductase bound material consisted of 82 and 95% polyglutamates, respectively, compared with 78 and 88% in the total cell pool. These results demonstrate that the polyglutamates have at least an equivalent affinity for the enzyme in the intact cell when compared to methotrexate and, as such, can be chiefly responsible for the toxicity of methotrexate in those cells that have sufficient capacity to convert methotrexate to its γ-linked glutamate derivatives.