CEM/MTX cells, a subline of CCRF-CEM cells resistant to methotrexate (MTX) by virtue of impaired transport by the reduced folate/methotrexate transport system, were grown in media containing folate levels in the physiological range rather than in standard media supplemented with high folate concentrations. Over a 7-month period folic acid concentrations were gradually lowered from 2 µm to 2 nm without subsequent changes in the transport-defective phenotype. In these cells we observed the up regulation of a membrane-associated folate-binding protein with high affinities for folic acid and reduced folates, but poor affinities for the folate antagonists MTX and 10-ethyl-10-deazaaminopterin. The binding capacity for [3H]folic acid was 12.5 pmol/107 cells, but could be further increased to 30 pmol/107 cells following cell transfer to completely folate-deficient medium for 3 days, except that in the latter situation cell growth stopped. The affinities of the folate-binding protein for 5-methyltetrahydrofolate, folinic acid, and MTX were 0.33, 0.11, and 0.009, respectively, relative to that of folic acid. Growth of CEM/MTX cells was promoted by nanomolar concentrations of either folic acid (median effective concentration, 0.35 nm) or folinic acid (median effective concentration, 0.75 nm), suggesting that the folate-binding protein is not only involved in folate binding, but also in the uptake of folates. The insensitivity of CEM/MTX cells to MTX was correlated with the poor affinity of the folate-binding protein for MTX, compared to folic acid. MTX was only growth inhibitory when added at concentrations at least 30-fold exceeding those of folic acid in the culture medium. On the other hand, CEM/MTX cells grown at 2 µm or 2 nm folic acid were equally sensitive to the lipophilic antifolate trimetrexate.
Despite the low affinity for MTX, the folate-binding protein could be specifically labeled by an N-hydroxysuccinimide ester of [3H]MTX and appeared to have a molecular weight of 44,000 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that an alternative folate uptake system, a folate-binding protein, can play an important role in transport-related methotrexate resistance. Moreover, since all these effects were observed for CEM/MTX cells grown at folate levels in the physiological range, it is conceivable that this mechanism of methotrexate resistance can also be of significance in leukemic cells in vivo.
This study was supported by Grant UUKC 8516 of the Netherlands Cancer Foundation.