Molecular Pharmacology, Vol 10, 257-274, Copyright © 1974 by the American Society for Pharmacology and Experimental Therapeutics

The Mechanism of Action of Methotrexate

I. Interaction with a Low-Affinity Intracellular Site Required for Maximum Inhibition of Deoxyribonucleic Acid Synthesis in L-Cell Mouse Fibroblasts

¹ Departments of Medcine and Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514

L-cell mouse fibroblasts were loaded with methotrexate (MTX) in vitro to a level which exceeded the capacity of high-affinity intracellular binding sites, following which MTX in excess of the tightly bound fraction was eliminated from the intracellular and extracellular compartments. Although binding of MTX was irreversible over the short interval of these experiments and the medium was free of folates and serum, cells continued to incorporate deoxyuridine into DNA at a rate which was depressed by only 27%. Upon further exposure of cells to MTX there was increased inhibition of deoxyuridine incorporation into DNA, which was a hyperbolic function of the extracellular and intracellular MTX concentrations, with 50% inhibition at 0.2µM and 0.2-0.4 µM, respectively. The net cellular uptake of radioactivity after exposure of cells to deoxyuridine was characterized by an initial rapid uptake of label, following which the net cellular uptake slowed to approximately the rate of incorporation of label into cellular constituents which do not penetrate the cell membrane. The net cellular uptake of label over 5 min was not decreased by a reduction of temperature from 37° to 23.5°. A 5-min exposure of cells to MTX at 37° markedly inhibited net cellular uptake of radioactivity, but this process was unaffected by MTX at 23.5° (when influx of MTX was markedly reduced) unless the cells had first been loaded with MTX at 37°. MTX inhibited net cellular uptake of radioactivity under conditions in which incorporation of deoxyuridine into DNA was already negligible, and inhibited incorporation of label into the trichloracetic acid supernatant fraction. MTX was not metabolized by L-cells. MTX did not accelerate the initial rate of efflux of a rapid-exit component of radioactivity from cells loaded with N⁵-methyl[¹⁴C]tetrahydrofolate, but quickly displaced (in less than 17 min) a small fraction of a slow-exit component. However, even after exposure of cells to 12µM MTX (sufficient for complete suppression of deoxyuridine incorporation into DNA) for 30 min (an interval which should be sufficient to eliminate displaceable endogenous tetrahydrofolates for this MTX level), and under conditions in which the medium should have been cleared of displaced folates, exchangeable intracellular MTX in the range of 0.2-0.4 µM still produced marked inhibition of deoxyuridine incorporation into DNA in comparison to cells in which all exchangeable intracellular MTX was eliminated. These studies suggest that in addition to tight binding to dihydrofolate reductase, MTX inhibits a lower-affinity receptor site necessary for the maintenance of deoxyuridine metabolism.

Note:
ACKNOWLEDGMENT The excellent technical assistance of Mrs. Sharon Loftfield is acknowledged.