title: Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin.
abstract: To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.
authors.affiliation: Department of Laboratory Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193.
authors.forename: Satomi
authors.initials: S
authors.lastname: Asai
authors.forename: Hayato
authors.initials: H
authors.lastname: Miyachi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Takemura
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Ando
mesh_terms: D019943:Amino Acid Substitution; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005472:Fluorouracil / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D007424:Intracellular Fluid / Q000737:chemistry; D002955:Leucovorin / Q000008:administration & dosage / Q000494:pharmacology*; D015458:Leukemia, T-Cell / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D020125:Mutation, Missense; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D012641:Selection, Genetic; D017384:Sequence Deletion; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D016597:Trimetrexate / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D003062:Codon; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; C120043:SLC19A2 protein, human; D013762:Tetrahydrofolate Dehydrogenase; D002955:Leucovorin; D005472:Fluorouracil; D016597:Trimetrexate; D008727:Methotrexate