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Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: role of carrier- and receptor-mediated transport systems. 1991

G R Westerhof, and G Jansen, and N van Emmerik, and I Kathmann, and G Rijksen, and A L Jackman, and J H Schornagel
Department of Oncology, Free University Hospital, Amsterdam, The Netherlands.

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-deazaaminopterin (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 greater than or equal to ICI-198,583 greater than or equal to 5-CHO-THF greater than MTX much greater than CB3717 much greater than 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 greater than or equal to folic acid = ICI-198,583 greater than or equal to 5-CHO-THF much greater than 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.(ABSTRACT TRUNCATED AT 400 WORDS)

UI MeSH Term Description Entries
D007939 Leukemia L1210 An experimental LYMPHOCYTIC LEUKEMIA of mice. Leukemia L 1210,L 1210, Leukemia,L1210, Leukemia
D008727 Methotrexate An antineoplastic antimetabolite with immunosuppressant properties. It is an inhibitor of TETRAHYDROFOLATE DEHYDROGENASE and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA. Amethopterin,Methotrexate Hydrate,Methotrexate Sodium,Methotrexate, (D)-Isomer,Methotrexate, (DL)-Isomer,Methotrexate, Dicesium Salt,Methotrexate, Disodium Salt,Methotrexate, Sodium Salt,Mexate,Dicesium Salt Methotrexate,Hydrate, Methotrexate,Sodium, Methotrexate
D011956 Receptors, Cell Surface Cell surface proteins that bind signalling molecules external to the cell with high affinity and convert this extracellular event into one or more intracellular signals that alter the behavior of the target cell (From Alberts, Molecular Biology of the Cell, 2nd ed, pp693-5). Cell surface receptors, unlike enzymes, do not chemically alter their ligands. Cell Surface Receptor,Cell Surface Receptors,Hormone Receptors, Cell Surface,Receptors, Endogenous Substances,Cell Surface Hormone Receptors,Endogenous Substances Receptors,Receptor, Cell Surface,Surface Receptor, Cell
D002352 Carrier Proteins Proteins that bind or transport specific substances in the blood, within the cell, or across cell membranes. Binding Proteins,Carrier Protein,Transport Protein,Transport Proteins,Binding Protein,Protein, Carrier,Proteins, Carrier
D002462 Cell Membrane The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells. Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D005492 Folic Acid A member of the vitamin B family that stimulates the hematopoietic system. It is present in the liver and kidney and is found in mushrooms, spinach, yeast, green leaves, and grasses (POACEAE). Folic acid is used in the treatment and prevention of folate deficiencies and megaloblastic anemia. Pteroylglutamic Acid,Vitamin M,Folacin,Folate,Folic Acid, (D)-Isomer,Folic Acid, (DL)-Isomer,Folic Acid, Calcium Salt (1:1),Folic Acid, Monopotassium Salt,Folic Acid, Monosodium Salt,Folic Acid, Potassium Salt,Folic Acid, Sodium Salt,Folvite,Vitamin B9,B9, Vitamin
D005575 Formyltetrahydrofolates Tetrahydrofolates which are substituted by a formyl group at either the nitrogen atom in the 5 position or the nitrogen atom in the 10 position. N(5)-Formyltetrahydrofolate is leucovorin (citrovorum factor) while N(10)-formyltetrahydrofolate is an active coenzyme which functions as a carrier of the formyl group in a number of enzymatic reactions. Formyltetrahydrofolic Acids,N(10)-Formyltetrahydrofolate
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013762 Tetrahydrofolate Dehydrogenase An enzyme of the oxidoreductase class that catalyzes the reaction 7,8-dihyrofolate and NADPH to yield 5,6,7,8-tetrahydrofolate and NADPH+, producing reduced folate for amino acid metabolism, purine ring synthesis, and the formation of deoxythymidine monophosphate. Methotrexate and other folic acid antagonists used as chemotherapeutic drugs act by inhibiting this enzyme. (Dorland, 27th ed) EC 1.5.1.3. Dihydrofolate Dehydrogenase,Dihydrofolate Reductase,Folic Acid Reductase,Acid Reductase, Folic,Dehydrogenase, Dihydrofolate,Dehydrogenase, Tetrahydrofolate,Reductase, Dihydrofolate,Reductase, Folic Acid
D013940 Thymidylate Synthase An enzyme of the transferase class that catalyzes the reaction 5,10-methylenetetrahydrofolate and dUMP to dihydrofolate and dTMP in the synthesis of thymidine triphosphate. (From Dorland, 27th ed) EC 2.1.1.45. Thymidylate Synthetase,Synthase, Thymidylate,Synthetase, Thymidylate

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