Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

J. D. Schuetz, L. H. Matherly, E. H. Westin, I. David Goldman

Research output: Contribution to journalArticle

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Abstract

Properties of the methorexate (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 V(max) without a change in K(m). From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx V(max). 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.

Original languageEnglish (US)
Pages (from-to)9840-9847
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number20
StatePublished - 1988
Externally publishedYes

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Leukemia L1210
Carrier transport
Methotrexate
Cells
Cell Line
Defects
Tetrahydrofolate Dehydrogenase
Leucovorin
Cell membranes
Labeling
Esters
Genes
Molecular Weight
Molecular weight
Binding Sites
Cell Membrane
Derivatives
Membranes
Recovery
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line. / Schuetz, J. D.; Matherly, L. H.; Westin, E. H.; Goldman, I. David.

In: Journal of Biological Chemistry, Vol. 263, No. 20, 1988, p. 9840-9847.

Research output: Contribution to journalArticle

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