Loss of Multidrug Resistance Protein 1 Expression and Folate Efflux Activity Results in a Highly Concentrative Folate Transport in Human Leukemia Cells

Abstract

Folate cofactors serve as one-carbon donors in the de novo biosynthesis of purines and thymidylate (1). As such, normal and neoplastic dividing cells have an absolute folate requirement for DNA replication (1). Disruption of folate biosynthesis with folic acid antagonists (i.e. antifolates) is the pharmacological basis for the antitumor activity of methotrexate (MTX) 1 and various antifolates (2). Because mammalian cells are devoid of folate biosynthesis, they rely on folate vitamin uptake from exogenous sources. Membrane transport of folates and MTX is mediated by several systems (3, 4): (a) the reduced folate carrier (RFC) is the major uptake route that functions as a bi-directional anion exchanger (5, 6) taking up folates through an antiport exchange mechanism with intracellular organic phosphates (7); (b) folate receptors mediate the unidirectional uptake of folate cofactors into mammalian cells via an endocytotic process (8); and (c) an apparently independent transport system with optimal folate uptake activity at low pH (9 -11).Apart from RFC, efflux of folates and MTX (12, 13) is mediated by multidrug resistance proteins (MRP) MRP1-4 (14 -19), which belong to the ATP-binding cassette superfamily (20,21). Members of the MRP family, currently comprising nine genes (i.e. MRP1-9), function as ATP-driven efflux transporters of various natural product anions and acidic charged drug conjugates (14, 15). Mammalian cells transfected with MRP1-4 accumulate decreased levels of MTX and consequently display resistance to this drug, particularly upon short term drug exposure (16 -19). Membrane vesicles isolated from MRP1-and MRP2-transfected cells exhibit ATP-dependent transport of MTX (16). Detailed kinetic analysis of folic acid, leucovorin (LCV; 5-formyl-tetrahydrofolate) and MTX transport into MRP1-and MRP3-rich membrane vesicles reveals K m values in the low millimolar range (22). Hence, the free intracellular level of folates and antifolates including MTX is determined by the net activities of these influx (i.e. RFC) and efflux (RFC and MRP) transport pathways.CEM-7A is a human leukemia CCRF-CEM subline previously established by gradual deprivation of LCV from the growth medium (23), resulting in RFC gene amplification (24) and carrier overexpression (23,24). Consequently, CEM-7A cells displayed a marked increase in the influx of MTX and LCV accompanied by a comparable increase in the steady-state transmembrane gradient of MTX (23). Surprisingly, however, there was no increase in the efflux rate constant for MTX (23). This is in contrast with previous studies (25) 1 The abbreviations used are: MTX, methotrexate; MRP, multidrug resistance protein; RFC, reduced folate carrier; LCV, Leucovorin (5-formyl-tetrahydrofolate); TMQ, trimetrexate; BCRP, breast cancer resistance protein; Pgp, P-glycoprotein; NHS-MTX, N-hydroxysuccinimide ester of MTX; AM, acetoxymethyl ester; HBS, Hepes-buffered saline.

Sulfasalazine is a potent inhibitor of the reduced folate carrier: Implications for combination therapies with methotrexate in rheumatoid arthritis

“…Although the objective of this study was not to elucidate a mechanistic basis for the efficacy of the triple DMARD therapy, recent studies by our group (47) did indicate that CEM/SSZ cells displayed enhanced sensitivity to both chloroquine and dexamethasone. In the case of chloroquine, this may be related to downregulation in the expression of MRP-1 (see Figure 4), which, apart from a physiologic role of MRP-1 in folate homeostasis (53)(54)(55), is best known as a drug efflux pump that can export various drugs (52,66), including chloroquine (67,68). Down-regulation of MRP-1 may therefore lead to increased intracellular retention of chloroquine, resulting in enhanced efficacy.…”

“…At the level of the cell membrane, cellular folate homeostasis is regulated by at least 2 groups of transporters. The RFC mediates folate transport inward (29,32), while members of the MRP family (52), in particular MRP-1, control cellular export of folates (53)(54)(55). In order to assess the effects of long-term exposure to SSZ, we analyzed RFC protein expression and transport activity and MRP-1 protein expression in CEM/SSZ cells.…”

“…Interestingly, MRP-1 levels were markedly decreased in CEM/SSZ cells compared with CEM cells (Figure 4). It is conceivable that this down-regulation of MRP-1 is a response to minimize export of folates after inhibition of RFC-mediated uptake of natural folates by SSZ (53,55).…”

Expression of resistance markers to methotrexate predicts clinical improvement in patients with rheumatoid arthritis

“…24 25 MRP function was measured by flow cytometry of total mononuclear blood cells using a calcein acetoxymethylester/ probenecid assay with a final probenecid concentration of 1 mM. [26][27][28][29][30][31] In brief, cells were preincubated with warm RPMI 1640 medium after several washing steps to remove any remaining serum components, followed by a 15 minute incubation with calcein acetoxymethylester. After 90 minutes' incubation in medium with or without probenecid as modulator of MRP activity, fluorescence was measured with a FACScan cytometer (Becton Dickinson, Franklin Lakes, New Jersey, USA).…”

The Mechanism of Carrier-Mediated Transport of Folates in BeWo Cells: The Involvement of Heme Carrier Protein 1 in Placental Folate Transport

“…Moreover, folates are also substrates for ATP-binding cassette transporters, MRPs (multidrug resistance-associated proteins), and BCRP (breast cancer resistance protein). [14][15][16] Hence it is thought that these transporters contribute greatly to folate kinetics. Although there have been reports on folate transport in the placenta, 17,18) the mechanism of folate transport in the placenta has not been elucidated in detail.…”