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.
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.