Reversibility of High-Affinity Binding of Methotrexate in L1210 Murine Leukemia Cells

The intracellular binding of methotrexate to high-affinity site(s) was examined in L1210 murine leukemia cells in vitro. Following exposure of cells to an extracellular [³H]methotrexate concentration of 2 μm, a peak intracellular level of 10.2 nmol methotrexate per g dry weight was achieved after 20 min. Resuspension of cells in a drugfree medium led to achievement of a steady-state intracellular level of 6.3 nmol/g, dry weight, a level that was constant for up to 120 min. Sephadex G-15 chromatography confirmed that this residual intracellular [³H]methotrexate coeluted with dihydrofolate reductase (assayed at pH 7.8), was nondialyzable, and reverted to a late-migrating peak after boiling. These findings are all consistent with the hypothesis that residual [³H]-methotrexate persisted in a tightly bound complex with dihydrofolate reductase. The reversibility of [³H]methotrexate binding in this complex was examined by resuspending cells after achieving a steady-state intracellular level of 6.3 nmol/g, dry weight, in unlabeled methotrexate at concentrations of 1 to 100 μM for up to 60 min. Rapid loss of [³H]methotrexate from the bound complex occurred at extracellular methotrexate concentrations of 10 μM or greater with release of 90% of intracellular [³H]-methotrexate within 60 min. The rate of loss of labeled intracellular drug was a linear first-order function of time at or above concentrations of 10 μM unlabeled methotrexate. An “off” rate constant of-3.8*10^-2/min was calculated; this off rate constant was greater than predicted from experiments with purified dihydrofolate reductase from these cells (k_off=-1.4*10^-3). These experiments demonstrate the reversibility of methotrexate binding to high-affinity intracellular sites in vitro.