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Membrane Transport of Methotrexate in Human Lymphoblastoid Cells

Medicine Branch, Clinical Pharmacology Section, National Cancer Institute, NIH, Bethesda, Maryland 20014

The mechanism of transport of the antifolate, methotrexate (MTX), was investigated in the CEM cell line, a human lymphoblastoid cell line grown in tissue culture. The time course of uptake revealed a rapid initial phase at extracellular MTX concentrations of 1 and 100 µM with steady-state intracellular levels reached by 60 min. Two distinct routes of MTX entry were observed. A "low-dose" route was operative at extracellular MTX concentrations of 20 µM or less. This route was saturable, had a K_m of 5.94 µM, was temperature dependent, and was not inhibited by metabolic posions. The "high-dose" component of transport was observed at MTX concentrations in excess of 20 µM; it was saturable, had a K_m of 125.7 µM, and was neither temperature nor energy dependent. The low-dose route was inhibited by the structural analogs, folic or folinic acid, but not at equimolar concentrations, while the high-dose route was not inhibited at folic or folinic acid concentrations in excess of the MTX concentration.

The data suggest that the low-dose component is carrier mediated; however, a Q₁₀ of 2.0 and a distribution ratio in excess of 1.0 provide the only evidence that the low-dose component is energy dependent. Analyses of the high-dose route could not discriminate between carrier-mediated transport and passive diffusion since a Q₁₀ was not demonstrable and the K_m was in excess of the highest MTX concentration studied. This was further substantiated by the inability of structural analogs to effect the influx velocity of MTX in this concentration range. The elucidation of a second route of MTX transport at high dose concentrations in human neoplastic cells may explain the apparent efficacy of high-dose MTX in MTX-"resistant" tumors.

¹ To whom requests for reprints should be addressed, at Medicine Branch, National Cancer Institute, Bldg. 10, Room 12N226, 9000 Rockville Pike, Bethesda, Md. 20014.

Received 9/ 6/77. Accepted 12/ 8/77.

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