title: Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells.
abstract: We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.
authors.affiliation: Department of Biology, The Technion, Haifa 32000, Israel. assaraf@technion.ac.il
authors.forename: Yehuda G
authors.initials: YG
authors.lastname: Assaraf
authors.forename: Lilah
authors.initials: L
authors.lastname: Rothem
authors.forename: Jan Hendrik
authors.initials: JH
authors.lastname: Hooijberg
authors.forename: Michal
authors.initials: M
authors.lastname: Stark
authors.forename: Ilan
authors.initials: I
authors.lastname: Ifergan
authors.forename: Ietje
authors.initials: I
authors.lastname: Kathmann
authors.forename: Ben A C
authors.initials: BA
authors.lastname: Dijkmans
authors.forename: Godefridus J
authors.initials: GJ
authors.lastname: Peters
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D001692:Biological Transport; D015153:Blotting, Western; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D005492:Folic Acid / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D002955:Leucovorin / Q000494:pharmacology; D008727:Methotrexate / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009154:Mutation; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D005492:Folic Acid; D002955:Leucovorin; C007740:fluorexon; D008727:Methotrexate