Receptor-mediated folate accumulation is regulated by the cellular folate content.

Abstract

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro [3',5',7,9-3H4] Folates are a class of pteridine compounds that are essential for normal growth and maturation. Reduced folic acid coenzymes are involved in one-carbon transfer reactions such as those necessary for the biosynthesis of methionine, serine, deoxythymidylic acid, and purines. Although these folatemediated reactions have been studied extensively and many of the key enzymes that catalyze these reactions have been purified and characterized (1), little is known about the overall mechanism ofcellular folate homeostasis. The normal plasma folate concentration in humans is 10-20 nM. However, tissue concentrations are 3 orders of magnitude higher (2-30 nmol/g of wet weight). Even when the plasma concentration of folate is experimentally increased 20-to 40-fold above normal, the steady-state tissue concentration remains constant (2). Therefore, most likely folate enters cells by a high-affinity uptake process that can be regulated.To better understand folate metabolism, many investigators have utilized tissue culture systems. The generally accepted model is that folate uptake is mediated by a membrane carrier that either facilitates the diffusion of folate across the membrane or actively transports the molecule into the cell (3). Another possibility is that folate is internalized by high-affinity receptors on the cell surface by a process that is similar to the receptor-mediated endocytosis of macromolecules. In the latter model, as in the case of vitamin MATERIALS AND METHODSBuffers and Tissue Culture Medium. Dulbecco's phosphate-buffered saline (PBS) was obtained from Hazelton Laboratories (Denver, PA). Medium 199 (M199) with Earle's salts and folic acid was purchased from GIBCO and supplemented with glutamine (GIBCO). RPMI 1640 medium without folic acid was formulated in our laboratory. Organic components were purchased from Sigma; the inorganic salts were obtained from Fisher Scientific. The RPMI medium was supplemented with adenine sulfate (10 mg/liter), thymine (0.3 mg/liter), xanthine (0.3 mg/liter), and hypoxanthine (0.3 mg/liter) (Sigma), and it was buffered with Hepes (25 mM, Sigma) and sodium bicarbonate (2 g/liter). All tissue culture medium contained fetal bovine serum (Hazelton Laboratories). To prepare folate-deficient medium, the serum was treated with activated charcoal (Norit A, Fisher Scientific) as detailed elsewhere (8) 5983The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Relationship between folate-binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines

“…Because these culture conditions ensured adequate delivery of folate to all cell lines used, the differential cisplatin responsiveness cannot be attributed to different availability of endogenous folates but instead might reflect differences in the capacity to use them. FBP expression levels have been reported to be inversely regulated by the extra-and intracellular folate concentration (Kamen et al, 1986;Kane et al, 1988) but, in our hands, only one (L-SKOV3) out of five ovarian carcinoma cell lines tested showed FBP up-modulation when adapted at physiological folate concentrations (20 nM) . In the same study, all the cell lines showed a marked decrease in endogenous folates compared with cells grown in standard medium (2.3 ,UM), and the amount of total folate in the cells was in general directly proportional to the level of membrane FBP expression, consistent with the concept that intracellular folate content is maintained primarily by FBP in low-folate cultured cells (Matsue et al, 1992 (Table).…”

Dynamics of antifolate transport via the reduced folate carrier and the membrane folate receptor in murine leukaemia cells in vitro and in vivo

“…Then cells were centrifuged and the cell pellet was analyzed for radioactivity. The total folic acid binding capacity was determined after a 20 min acidic treatment with HBSS buVer (pH 3.5) to remove surface bound folates [29]. After centrifugation, cells were resuspended in HBSS pH 7.4 and incubated with [ 3 H]-folic acid as described above.…”

“…Membrane folate receptors are membrane-associated folate binding proteins that constitute a second group of folate transporters and are characterized by high binding aYnity for folic acid (K d : 0.1-1 nM) and 5-methyltetrahydrofolate (K d : 3 nM), but markedly lower aYnities for other reduced folates and MTX [26,29,44,64].…”

“…Two mechanisms of internalization of folates by MFR have been proposed: classical endocytosis [47] and a process called potocytosis which involves the sequestration of MFRs in speciWc membrane vesicles (caveolae) in which, upon acidiWcation, the (anti) folates dissociate from the receptors and are transported into the cytosol by a putative folate carrier [2]. Although the exact role and the relative importance of the RFC in mediating antifolate antitumor activity in vivo has been fairly well established [5,29,31,50], the role of MFR is still unclear [12,67]. However, it may be anticipated that optimal MFR-mediated transport could be revealed under conditions of low extracellular folates when there is less receptor occupancy and competition with circulating reduced folates [15,61,68].…”

Loss of heterozygosity at the 5,10-methylenetetrahydrofolate reductase locus in human ovarian carcinomas

“…It is well known that in several cases FBP expression is physiologically regulated by the intracellular folate content (Kamen and Capdevila, 1986;Kane et al, 1988;Matsue et al, 1992;Miotti et al, 1995), most probably by the 5-methyltetrahydrofolate (5-CH3-H4 folate) content, since this coenzyme is the natural ligand of FBP and represents the predominant circulatory form (>90% of the overall seric folate, under physiological conditions) (Antony, 1992). Intracellular 5-CH3-H4 folate shortage may be consequent on limited bioavailability of extracellular folates or on biochemical defects in the intracellular regeneration or cellular retention of this folate form.…”