title: P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.
abstract: P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.
authors.affiliation: Department of Genetics, University of Illinois at Chicago 60607-7170, USA.
authors.forename: D
authors.initials: D
authors.lastname: de Graaf
authors.forename: R C
authors.initials: RC
authors.lastname: Sharma
authors.forename: E B
authors.initials: EB
authors.lastname: Mechetner
authors.forename: R T
authors.initials: RT
authors.lastname: Schimke
authors.forename: I B
authors.initials: IB
authors.lastname: Roninson
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000378:metabolism*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000911:Antibodies, Monoclonal; D001692:Biological Transport; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D018432:Drug Resistance, Multiple / Q000235:genetics; D005347:Fibroblasts; D005434:Flow Cytometry; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000633:toxicity*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism* / Q000633:toxicity*; D051379:Mice; D009363:Neoplasm Proteins; D010692:Phleomycins / Q000633:toxicity; D011743:Pyrimidines / Q000633:toxicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012190:Retroviridae; D014162:Transfection; D014747:Vinblastine / Q000633:toxicity
chemical_list: C116614:ABCG2 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D010692:Phleomycins; D011743:Pyrimidines; D011994:Recombinant Proteins; C489021:methotrexate transporter protein, mouse; D014747:Vinblastine; C024530:piritrexim; D008727:Methotrexate