Interaction of N-hydroxy(sulfo) succinimide active esters with the reduced folate/methotrexate transport system from human leukemic CCRF-CEM cells

G Jansen, G R Westerhof, G Rijksen, J H Schornagel

Research output: Contribution to journalArticle*Academicpeer-review

Abstract

The membrane impermeant protein cross-linker 3,3'-dithiobissulfosuccinimidyl propionate (DTSSP) is a well-known inhibitor of human erythrocyte band 3-mediated inorganic anion transport. We observed that DTSSP is also a potent inhibitor of reduced folate/methotrexate transport in human CCRF-CEM leukemia cells. An interaction of DTSSP with the reduced folate/MTX is substantiated by findings that: (a) like MTX transport itself, the concentration of DTSSP required for half-maximal inhibition of [3H]methotrexate transport varied substantially with the anionic composition of the external medium. In a saline buffer and an anion-deficient buffer the I50 values were 7 and 1 microM, respectively; (b) saturation of the carrier with 1-5 microM methotrexate completely protected the transport system from interaction by DTSSP; (c) methotrexate transport activity in DTSSP-treated cells could be restored after cleavage of the disulfide bond in DTSSP under mild reducing conditions; and (d) pretreatment of cells with DTSSP reduced the incorporation of [3H]methotrexate after labeling with an N-hydroxysuccinimide ester of [3H]methotrexate (NHS-MTX), another potent inhibitor of methotrexate transport. Comparison of DTSSP- and NHS-MTX-induced inhibition of methotrexate transport showed that DTSSP inhibition, in contrast to NHS-MTX inhibition, was (a) less potent, (b) dependent on buffer conditions, (c) reversible by reducing agents, and (d) required only a very low molar ratio of methotrexate over DTSSP to afford maximal protection.

Original languageEnglish
Pages (from-to)266-70
Number of pages5
JournalBiochimica et biophysica acta. Reviews on cancer
Volume985
Issue number3
Publication statusPublished - 3 Nov 1989

Keywords

  • Biological Transport/drug effects
  • Cell Membrane/metabolism
  • Cross-Linking Reagents
  • Dithiothreitol/pharmacology
  • Folic Acid/pharmacokinetics
  • Humans
  • Leukemia/metabolism
  • Methotrexate/antagonists & inhibitors
  • Succinimides/pharmacology
  • Tumor Cells, Cultured/metabolism

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