Identification of a membrane-associated folate-binding protein in human leukemic CCRF-CEM cells with transport-related methotrexate resistance

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 microM 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/10(7) cells, but could be further increased to 30 pmol/10(7) 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 microM 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, car 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.