Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: role of carrier- and receptor-mediated transport systems.
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)
Westerhof
G R
GR
Department of Oncology, Free University Hospital, Amsterdam, The Netherlands.
Jansen
G
G
van Emmerik
N
N
Kathmann
I
I
Rijksen
G
G
Jackman
A L
AL
Schornagel
J H
JH
D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002462:Cell Membrane; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005575:Formyltetrahydrofolates / Q000378:metabolism; D007939:Leukemia L1210 / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005575:Formyltetrahydrofolates; D011956:Receptors, Cell Surface; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; C059966:ICI 198583; D008727:Methotrexate