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

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 I 50 values were 7 and 1 μM, respectively; (b) saturation of the carrier with 1–5 μM 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.