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Membrane Transport of Natural Folates and Antifolate Compounds in Murine L1210 Leukemia Cells: Role of Carrier- and Receptor-mediated Transport Systems¹

Department of Oncology, [G. R. W., G. J., N. v. E., I. K.], Free University Hospital, P.O. Box 7057, 1007 MB, Amsterdam, Department of Internal Medicine, Netherlands Cancer Institute [J. H. S.], Amsterdam, and Laboratory of Medical Enzymology, Department of Haematology, University Hospital Utrecht, Utrecht [G. R.], The Netherlands; and Drug Development Section, Institute of Cancer Research, Sutton, Surrey, United Kingdom [A. L. J.]

L1210-B73 cells, variants of L1210 cells grown in medium containing nanomolar concentrations of folates, express a membrane associated folate binding protein (mFBP) in addition to the classical reduced folate/methotrexate carrier (RF/MTX-carrier) present in L1210 cells grown in standard high folate medium (G. Jansen et al., Cancer Res., 49: 1959–1963, 1989). In this study we used L1210-B73 and L1210 cells as a model system to study the affinity of the RF/MTX-carrier and the mFBP for the natural folate compounds folic acid and 5-formyltetrahydrofolate (5-CHO-THF), as well as a number of antifolate compounds. Furthermore we studied the contribution of the RF/MTX-carrier and the mFBP in membrane transport of these (anti)folates, and finally we analyzed the role of the mFBP and RF/MTX-carrier in the cytotoxic effects of the antifolates. The antifolates used were either inhibitors of dihydrofolate reductase, including methotrexate (MTX) and 10-ethyl-10-deazaaminopterine (10-EdAM), or two folate-based inhibitors of thymidylate synthase, N¹⁰-propargyl-5,8-dideazafolic acid (CB3717) and 2-deamino-2-methyl-N¹⁰-propargyl-5,8-dideazafolic acid (ICI-198,583).

The affinity of the RF/MTX-carrier for natural and antifolate compounds declined in the order 10-EdAM ICI-198,583 5-CHO-THF > MTX >> CB3717 >> folic acid. The mFBP exhibited a high binding affinity for CB3717 and ICI-198,583 but a poor binding affinity for MTX and 10-EdAM. Binding affinities of the mFBP decreased in the order CB3717 folic acid = ICI-198,583 5-CHO-THF >> MTX = 10-EdAM.

Over 24 h, at 25 nM, [³H]folic acid uptake in L1210-B73 cells was found to proceed for more than 98% via the mFBP. Uptake of [³H]-5-CHO-THF, at 50 nM extracellular concentration, occurred via both the mFBP (81%) and the RF/MTX-carrier (19%). With respect to antifolates, the mFBP in L1210-B73 cells contributed for less than 30% in the uptake of [³H]MTX but was the predominant route (92%) in the uptake of [³H]ICI-198,583.

Results from affinity and membrane transport observations were consistent with growth inhibition studies on L1210-B73 cells demonstrating that the mFBP played only a minor role in the cytotoxic effects of MTX or 10-EdAM. On the other hand, L1210-B73 cells were significantly more sensitive to CB3717 (220-fold) and ICI-198,583 (10-fold) than parental L1210 cells. The putative role of the mFBP in the uptake of CB3717 and ICI-198,583 in L1210-B73 cells was further supported by the fact that protection from growth inhibition could be achieved by folic acid rather than by 5-CHO-THF. In L1210 cells, expressing only the RF/MTX carrier, no protection with folic acid was observed.

These results suggest that multiple transport systems may play a role in the uptake of natural folates and nonclassical folate analogues with targets other than dihydrofolate reductase. The possible clinical relevance of these observations will be further discussed.

¹ This study was supported by the Dutch Cancer Society (Grant IKA 89-34). The authors are very grateful to ICI Pharmaceuticals, Alderly Park, Cheshire, United Kingdom, for their generous funding of the synthesis of the [³H]-ICI-198,583.

² To whom requests for reprints should be addressed.

Received 11/ 6/90. Accepted 8/ 8/91.

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