FUNCTIONAL-ACTIVITY OF THE REDUCED FOLATE CARRIER IN KB, MA104, AND IGROV-I CELLS EXPRESSING FOLATE-BINDING PROTEIN

The role of a membrane-associated folate binding protein (mFBP) in tra nsport of folate analogues was investigated in three epithelial cell l ines that were grown in high folate medium and folate-conditioned medi um and express different levels of mFBP: human nasopharyngeal KB cells , monkey kidney MA104 cells, and IGROV-I ovarian carcinoma cells. Fola te analogues were selected for which mFBP exhibits a low affinity, i.e ., methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folic acid, i.e., n-6-ylmeth yl(-N-methylamino]-2-theonyl)-L-glutamic acid (ZD1694), N-10-propargyl -5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies wit h IGROV-I and MA104 cells demonstrated a lack of correlation between t he affinity of mFBP for the antifolate drugs and their sensitivity pro file; both cell lines were highly sensitive to growth inhibition by MT X, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were inse nsitive for CB3717. The same drug sensitivity profile was observed for KB cells, with the exception that these cells were also sensitive to growth inhibition by CB3717 but only in folate-conditioned medium. Thi s overall drug sensitivity profile appeared to correlate with the diff erential efficiency of drug transport via the ''classical'' reduced fo late/MTX carrier (RFC), rather than by mFBP. Characteristics that furt her supported functional RFC activity In KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prev ented by the reduced folate leucovorin rather than by folic acid; (b) rates for uptake of [H-3]10-EdAM were 2-4-fold higher than for [H-3]MT X at 1 mu m extracellular concentrations and coincided,vith the affini ty of the RFC for these drugs, rather than those of the mFBP; (c) upta ke of [H-3]10-EdAM and [H-3]leucovorin was markedly inhibited by leuco vorin and 10-EdAM, respectively, or by an N-hydroxysuccinimide ester o f MTX (irreversibly labeling RFC) but only to a minor extent by folic acid or an N-hydroxysuccinimide ester of folic acid (irreversibly labe ling mFBP); and, finally, (d) labeling with an N-hydroxysuccinimide es ter of [H-3]MTX identified a protein with a molecular weight within th e range of that reported for the RFC in human leukemic cells. Altogeth er, these results indicate that both RFC and mFBP are coexpressed in t hree epithelial cell lines and that RFC is the preferential route of e ntry for antifolate compounds, even when mFBP is expressed to very hig h levels.