Functional Activity of the Reduced Folate Carrier in KB, MA104, and IGROV-I Cells Expressing Folate-binding Protein1

The role of a membrane-associated folate binding protein (mFBP) in transport of folate analogues was investigated in three epithelial cell lines that were grown in high folate medium and folate-conditioned medium and express different levels of mFBP: human nasopharyngeal KB cells, monkey kidney MA 104 cells, and IGROV-I ovarian carcinoma cells. Folate analogues were selected for which mFBP exhibits a low affinity, i.e., methotrexate (MTX) and 10-ethyl-lO-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folie acid, i.e., N-(S[N-(3,4dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl(-A'-methylamino]-2theonyl)-L-glutamic acid (ZD1694), Nlo-propargyl-5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies with IGROV-I and MA104 cells demonstrated a lack of correlation between the affinity of mFBP for the antifolate drugs and their sensitivity profile; both cell lines were highly sensitive to growth inhibition by MTX, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were insensitive 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. This overall drug sensi tivity profile appeared to correlate with the differential efficiency of drug transport via the "classical" reduced folate/MTX carrier (RFC), rather than by mFBP. Characteristics that further supported functional RFC activity in KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prevented by the reduced folate leucovorin rather than by folie acid; (b) rates for uptake of | 'H 110-KdAM were 2-4-fold higher than for [3H]MTX at l UMextracellular concentra tions and coincided with the affinity of the RFC for these drugs, rather than those of the mFBP; (c) uptake of [3H]10-EdAM and [3H]leucovorin was markedly inhibited by leucovorin and 10-EdAM, respectively, or by an A'-hvdroxysuccinimide ester of MTX (irreversibly labeling RFC) but only to a minor extent by folie acid or an A'-hydroxysuccinimide ester of folie acid (irreversibly labeling mFBP); and, finally, (</1 labeling with an yV-hydroxysuccinimide ester of [3H]MTX identified a protein with a mo lecular weight within the range of that reported for the RFC in human leukemic cells. Altogether, these results indicate that both RFC and mFBP are coexpressed in three epithelial cell lines and that RFC is the preferential route of entry for antifolate compounds, even when mFBP is expressed to very high levels. INTRODUCTION For several decades, the RFC3 system has been implicated to be of physiological and pharmacological importance in controlling the upReceived 2/1/95; accepted 7/6/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely Io indicate this fact. ' Supported by Dutch Cancer Society Grants IKA 89-34 and NKI 92-43. ~ To whom requests for reprints should be addressed. Fellow of the Royal Netherlands Academy for Arts and Sciences. 3 The abbreviations used are: RFC, reduced folate/methotrexate carrier; 5-CH,-THF, 5-methyltetrahydrofolate; MTX, methotrexate; 10-EdAM, 10-ethyl-lO-deazaaminopterin; CB3717, A'")-propargyl-5,8-dideazafolic acid; ZD1694, A'-(5[A'-(3,4-dihydro-2-methyl-4oxoquinazoIin-6-ylmethyl(-W-methylamino]-2-theonyl)-L-glutamic acid; DDATHF, 5,10dideazatetrahydrofolic acid; DHFR, dihydrofolate reducÃ-ase;GARTF, glycinamide ribonucleotide transformylase; HBSS, HEPES-buffered saline solution; LF, low folate; HF, high folate (2.2 JAMfolie acid); leucovorin, L-5-formyltetrahydrofolate; mFBP, membraneassociated folate binding protein; TS, thymidylate synthase; IC50, 50% inhibitory con centration; NHS, A'-hydroxysuccinimide. take of folate cofactors (e.g., 5-CH3-THF) required for cell growth (1-4), or antifolate compounds (e.g., MTX) (1, 5-7) in neoplastic cells. Although A",,,values for RFC-mediated uptake of 5-CH3-THF or MTX are in the micromolar range, the influx capacity is amply sufficient to comply with the cellular folate requirements to sustain growth or to internalize cytotoxic concentrations of antifolate drugs. In recent years, however, mFBPs have received considerable atten tion as possible alternative folate transport systems (2-4, 8, 9). Documentation of mFBP biodistribution indicated variable expression levels in a wide variety of normal and malignant tissues and constitutively high expression levels in ovarian carcinoma (10-12). A functional role for mFBP in folate transport was illustrated by obser vations that transfection with cDNA encoding for mFBP allows cell growth at nanomolar concentrations of folate, both in vitro (13, 14) and in vivo (15). Besides uptake of natural reduced folate cofactors, it has been demonstrated that mFBP could have a functional role in mediating the uptake of antifolates in human nasopharyngeal KB cells (16,17), monkey kidney MA104 cells (18), MCF-7 breast cancer cells (19), and leukemia cells (20, 21). The mechanism of mFBP-mediated (anti)folate uptake is structur ally and functionally different from that of RFC-mediated uptake. The RFC is an integral membrane protein with a postulated channel function for (anti)folate compounds that is controlled by an aniÃ3n exchange mechanism as the driving force (1, 22). mFBP has a high affinity for folie acid and is linked to the plasma membrane via a glycosylphoshatidylinositol anchor (23). Two mechanisms have been suggested for mFBP-mediated (anti)folate uptake: one route is via the classical receptor-mediated endocytosis pathway; another route, iden tified in MA104 cells, includes a process termed potocytosis (24). Potocytosis comprises a series of events in which mFBP molecules are clustered in caveolae which can temporarily close from the exter nal environment, after which acidification of the lumen releases the ligand from the receptor. An important conceptional assumption in this model is that mFBP per se cannot translocate (anti)folates across the plasma membrane but requires a specific carrier protein. This carrier protein may have common features with RFC given the fact that its activity can be inhibited by probenecid (25). In this study we further delineated the role of RFC and mFBP in antifolate drug uptake as a function of cellular expression of mFBP: high expression levels in KB cells and moderate expression levels in IGROV-I and MA104 cells. The selected drugs are MTX and 10EdAM, both inhibitors of dihydrofolate reducÃ-ase, CB3717, and ZD1694, both inhibitors of TS and a glycinamide ribonucleotide formyltransferase inhibitor, DDATHF. These drugs are well charac terized for RFC-mediated transport, but exhibit different binding affinities for mFBP (20, 26). The drug sensitivity profile, transport studies with radiolabeled drugs, and affinity labeling studies indicate that regardless of the mFBP expression level, a carrier (RFC)-mediated transport route is functional in KB, IGROV-I, and MA104 cells, and appears to be the predominant route for antifolate uptake in these cells. Only in KB cells, but not MAI 04 and IGROV-I cells, was mFBP found to mediate the uptake of cytotoxic concentrations of CB3717, a compound that is poorly transported via RFC. 3795 on July 24, 2017. © 1995 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from REDUCED HOLATK/MTX CARRIER FUNCTION IN EPITHELIAL CELLS mFBP may contribute in the uptake of natural reduced folate cofactors, only at nanomolar extracellular concentrations and not at micromolar concentrations. MATERIALS AND METHODS