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Characteristics of Transport of 4-Amino Antifolates and Folate Compounds by Two Lines of L5178Y Lymphoblasts, One with Impaired Transport of Methotrexate¹

Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom [B. T. H.], and Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298 [B. D. B., J. C. W., I. D. G.]

In a line of L5178Y lymphoblasts resistant to methotrexate (MTX) by virtue of a permeability defect, there is a marked decrease in the membrane transport of MTX, aminopterin, and 5-methyltetrahydrofolate. Influx is depressed by 93, 92, and 96%, respectively, as compared to MTX-sensitive cells when the extracellular levels are 1 µM. This is compatible with a common defect in a high-affinity transport carrier mechanism shared by these three folate compounds. Conversely, however, adsorption of MTX to the surface of resistant cells is slightly increased. Uptake of ³H following exposure of these cells to [³H]folic acid is comparable in both resistant and sensitive lines, supporting the concept that influx of folic acid is mediated largely by a process independent of the MTX-tetrahydrofolate cofactor carrier. In addition to the quantitative differences in the rates of transport of 4-amino antifolates and 5-methyltetrahydrofolate, there are important differences in the characteristics of transport of these substances in these two cell lines. The Q_27–37°'s for MTX influx in MTX-sensitive and -resistant cells are 6.4 and 1.4, respectively. While influx is markedly inhibited by p-chloromercuriphenylsulfonic acid and 5-formyltetrahydrofolate in MTX-sensitive cells, only a small component of influx in resistant cells (25%) is inhibited by these agents. Transstimulation of MTX influx can be demonstrated in both cell lines but to a much lesser extent in resistant L5178Y cells. Sodium azide stimulates MTX influx in sensitive cells but has no effect on influx in resistant cells. However, this metabolic poison augments net uptake of MTX in both cell lines. Net accumulation of exchangeable MTX is markedly reduced in resistant cells, but differences in influx and net transport between resistant and sensitive cells decrease as extracellular MTX is increased, indicating the utility of high extracellular MTX levels in generating free intracellular drug as an approach to overcoming the permeability defect. These data suggest that the bulk of transport of antifolates and 5-methyltetrahydrofolate in resistant cells occurs by a process distinct from the MTX-tetrahydrofolate cofactor carrier system. The MTX-tetrahydrofolate cofactor transport carriers in MTX-resistant cells have undergone a marked reduction in affinity for their substrate, or else the major portion of these carriers have been deleted or are not operational. The observation that azide enhances net MTX uptake in resistant as well as sensitive L5178Y cells along with findings in other studies that the net uptake of folic acid is enhanced by azide raises the possibility that the effects of metabolic inhibitors on net transport of MTX and folic acid may be mediated, at least in part, by inhibition of a common exit process.

¹ Supported in part by USPHS Grant CA-16906 from the National Cancer Institute.

² These studies were initiated during a sabbatical period spent at the Medical College of Virginia, Richmond, Va.

³ To whom requests for reprints should be addressed.

Received 8/18/78. Accepted 3/23/79.