title: P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.
abstract: We have previously described a series of methotrexate (MTX)-selected CCRF-CEM sublines (CEM/MTX R1-3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P-glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P-glycoprotein-mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1-3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross-resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P-glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P-glycoprotein. Our data have implications for the P-glycoprotein-mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited.
authors.affiliation: Children's Cancer Research Institute, Sydney Children's Hospital, NSW, Australia.
authors.forename: A J
authors.initials: AJ
authors.lastname: Gifford
authors.forename: M
authors.initials: M
authors.lastname: Kavallaris
authors.forename: J
authors.initials: J
authors.lastname: Madafiglio
authors.forename: L H
authors.initials: LH
authors.lastname: Matherly
authors.forename: B W
authors.initials: BW
authors.lastname: Stewart
authors.forename: M
authors.initials: M
authors.lastname: Haber
authors.forename: M D
authors.initials: MD
authors.lastname: Norris
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D006801:Humans; D015458:Leukemia, T-Cell / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017354:Point Mutation; D058978:Reduced Folate Carrier Protein; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000964:Antimetabolites, Antineoplastic; D000972:Antineoplastic Agents, Phytogenic; D002352:Carrier Proteins; D004273:DNA, Neoplasm; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; C120043:SLC19A2 protein, human; D014750:Vincristine; D008727:Methotrexate