Active Efflux of the Free Acid form of the Fluorescent Dye 2′,7′‐Bis(2‐Carboxyethyl)‐5(6)‐Carboxyfluorescein in Multidrug‐Resistance‐Protein‐Overexpressing Murine and Human Leukemia Cells

Abstract

Murine and human cell lines overexpressing the multidrug-resistance protein (MRP) showed a marked decreased accumulation of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In contrast, less altered accumulation was seen in the P-glycoprotein(P-gp)-overexpressing cell lines. The decreased drug accumulation was reversed by the energy inhibitors sodium azideI2-deoxyglucose and by the vinca alkaloid, vincristine, but not by the chemotherapeutic agents, etoposide and adriamycin. Decreased accumulation was linked to active efflux of the hydrophilic free acid form of BCECF from the MRP-overexpressing cell lines, indicating that dye extrusion occurs after the dye ester has been converted to the free acid form in the cytoplasm. The finding suggests that MRP mediates removal of substrates from a cytoplasmic location. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreased the vincristine and etoposide resistance displayed by the MRP-expressing murine cell lines, but did not affect the accumulation of BCECF. Thus, while glutathione may be involved in MRP-mediated resistance to some chemotherapeutic agents, it is not necessary for efflux of substrates such as BCECF.Keywords: 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; P-glycoprotein; multidrug-resistance protein; glutathione; murine.Two membrane-associated transport proteins have been identified that mediate resistance to natural product anticancer drugs : the 170-kDa P-glycoprotein (P-gp) [l] and the more recently identified 190-kDa multidrug-resistance protein (MRP) [2]. While both proteins belong to the ATP-binding-cassette transporter superfamily, they share overall less than 15 % amino acid identity [ 3 ] . In addition they also display a different pattern of drug resistance, with MRP displaying an apparent preference for more hydrophilic xenobiotics, as supported by the findings that MRP confers resistance to antimonial and arsenical oxyanions [3].Recently there has been much discussion concerning the finding that overexpression of MRP results in transport of negatively charged molecules [leukotriene C, (LTC,), S-dinitrophenylglutathione (Dnp-GS)] [4-71 as well as transport of the positively charged and neutral drugs daunorubicin (DNR) and VP-16 [S, 91. The transport of these neutral and positively charged molecules appears to be dependent upon the intracellular glutathione (GSH) levels, since incubation of cells with

Inflammatory Cytokines, but Not Bile Acids, Regulate Expression of Murine Hepatic Anion Transporters in Endotoxemia

“…The nonfluorescent ester 5-carboxyfluorescein diacetate 5-CFDA (Sigma-Aldrich) is rapidly and passively taken up into cells where it is converted by intracellular esterases to the fluorescent moiety 5-CF, which is a specific substrate of the Mrp transporters (Draper et al, 1997). Briefly, cells were grown in sixwell culture dishes (Costar, Cambridge, MA) to confluence.…”

Localization of a Substrate Specificity Domain in the Multidrug Resistance Protein

“…This is supported by experiments in which Pgp-overexpressing cell lines have been shown to efflux the hydrophobic esters of the fluorescent dyes calcein AM and 2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein AM (BCECF AM) but not their free acids, which are formed following cleavage by cytosolic esterases (41). In similar studies, MRP was able to transport both free calcein and calcein AM as well as BCECF AM, suggesting that the protein can recognize substrates in the cytoplasm as well as the plasma membrane (42)(43)(44)(45)(46). Thus initial interactions with MRP/mrp may occur via two different routes in which drugs and other hydrophobic substrates bind in the lipid environment of the plasma membrane, whereas the primary interaction of hydrophilic organic anions may be with the cytoplasmic loops of the protein.…”

Role of MRP1 in multidrug resistance in acute myeloid leukemia

“…58,59 Thus, while glutathione may be involved in MRP1-mediated resistance to some chemotherapeutic agents, it is not necessary for the efflux of substrates such as these two compounds. 59,60 Another substrate, DiOC2(3), which is a substrate of Pgp, is not a substrate of MRP1. 57 Among some of these compounds are substrates of both MRP1 and Pgp, such as calcein-AM, Rh123, DNR.…”