title: Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.
abstract: We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.
authors.affiliation: Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Spinella
authors.forename: K E
authors.initials: KE
authors.lastname: Brigle
authors.forename: S J
authors.initials: SJ
authors.lastname: Freemantle
authors.forename: E E
authors.initials: EE
authors.lastname: Sierra
authors.forename: I D
authors.initials: ID
authors.lastname: Goldman
mesh_terms: D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000502:physiology*; D058973:Folate Receptors, GPI-Anchored; D007939:Leukemia L1210 / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D010453:Peptide Synthases / Q000378:metabolism; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; C492292:Slc19a2 protein, mouse; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D013762:Tetrahydrofolate Dehydrogenase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate