Interaction of N-hydroxy(sulfo) succinimide active esters with the reduced folate/methotrexate transport system from human leukemic CCRF-CEM cells.
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.
Jansen
G
G
Department of Internal Medicine, University Hospital, Utrecht, The Netherlands.
Westerhof
G R
GR
Rijksen
G
G
Schornagel
J H
JH
D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004229:Dithiothreitol / Q000494:pharmacology; D005492:Folic Acid / Q000493:pharmacokinetics*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D008727:Methotrexate / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*; D013388:Succinimides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003432:Cross-Linking Reagents; D013388:Succinimides; D005492:Folic Acid; C011240:dithiobis(succinimidylpropionate); D004229:Dithiothreitol; D008727:Methotrexate