Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line.
Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.
Schuetz
J D
JD
Department of Medicine, Virginia Commonwealth University, Medical College of Virginia, Richmond 23298.
Matherly
L H
LH
Westin
E H
EH
Goldman
I D
ID
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D058973:Folate Receptors, GPI-Anchored; D005784:Gene Amplification; D002955:Leucovorin / Q000494:pharmacology; D007939:Leukemia L1210 / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism / Q000494:pharmacology*; D051379:Mice; D010641:Phenotype; D011799:Quinazolines / Q000494:pharmacology; D011956:Receptors, Cell Surface; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism; D016597:Trimetrexate
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011799:Quinazolines; D011956:Receptors, Cell Surface; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D002955:Leucovorin; D016597:Trimetrexate; D008727:Methotrexate