Molecular Pharmacology, Vol 12, 711-719, Copyright © 1976 by the American Society for Pharmacology and Experimental Therapeutics

Mechanism of Action of Methotrexate

IV. Free Intracellular Methotrexate Required to Suppress Dihydrofolate Reduction to Tetrahydrofolate by Ehrlich Ascites Tumor Cells in Vitro

¹ Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298

Incubation of Ehrlich ascites tumor cells in vitro with [³H]dihydrofolate results in its rapid conversion to [³H]tetrahydrofolate and other radiolabeled compounds, so that dihydrofolate cannot be detected in the cell. When cells are exposed to 10µM methotrexate for 30 min, a condition sufficient to generate intracellular methotrexate in excess of the tightly bound fraction within the cell (exchangeable methotrexate), following which exchangeable intracellular methotrexate is eliminated, conversion of dihydrofolate to tetrahydrofolate is negligibly affected. Even when cells are maintained in a medium containing 0.05 µM methotrexate, following exposure to 10 µM methotrexate, there is virtually no inhibition of dihydrofolate metabolism. However, as the extracellular methotrexate level is increased to 0.1, 1.0, and 10 µM in the second incubation, there is progressive inhibition of tetrahydrofolate synthesis along with a progressive increase in the level of dihydrofolate within the cells, complete at the highest methotrexate concentration employed. Fifty percent inhibition of the cell tetrahydrofolate level requires an extracellular methotrexate concentration of approximately 0.2 µM, comparable to an exchangeable intracellular level of about 0.15 µM. In contrast to the results with dihydrofolate, after treatment of cells with 10 µM methotrexate for 30 min followed by removal of exchangeable drug, there is marked (approximately 85%) inhibition of folic acid reduction to tetrahydrofolate. Residual reduction of folic acid is completely eliminated when the cells are continuously exposed to 10 µM methotrexate. The requirement for free intracellular methotrexate to abolish tetrahydrofolate synthesis appears to be the basis for the observations from this laboratory that free intracellular methotrexate is necessary to suppress [³H]deoxyuridine incorporation into DNA and [¹⁴C]formate incorporation into RNA, DNA, and protein. The demonstration in other studies that mammalian cells have a limited capacity to accumulate free intracellular methotrexate supports the concept that this intracellular component of the drug may be an important determinant of cytotoxicity.

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