Summary Decreased accumulation of the fluorescent dye BCECF [2', 7'-bis-(2-carboxyethyl)-5-(6)-carboxyfluorescein] characterized murine and human multidrug-resistant cell lines overexpressing the multidrug resistance protein (MRP). Indomethacin (10 gM), a known cyclooxygenase and glutathione-S-transferase inhibitor as well as a modulator of anion transport, increased accumulation and blocked efflux of BCECF in MRP-expressing murine and human cells. The drug did not affect P-glycoprotein (P-gp)-mediated export of rhodamine 123. The indomethacin effect on BCECF efflux was not reversed by the addition of exogenous prostaglandins, suggesting that the drug acts by a mechanism other than decreasing prostaglandin synthesis. Indomethacin also increased multidrug susceptibility of both murine and human cell lines overexpressing MRP, but not those displaying P-gp-associated resistance. In addition, indomethacin modulated the decreased vincristine accumulation in cells expressing MRP, but not in those expressing P-gp. These data suggest that indomethacin is a specific inhibitor of MRP, possibly functioning by inhibition of glutathione-S-transferase or, alternatively, by direct competition with the drug at the transport site.Keywords: BCECF; chemosensitizer; modulator; transport inhibitors Multidrug resistance in mammalian cells has been associated with altered drug transport. Active efflux or altered intracellular sequestration has been attributed to membrane-associated P-glycoprotein (Pgp) or the multidrug resistance protein (MRP) (Inaba et al, 1979; Zamon et al, 1994). We have previously demonstrated that murine and human cell lines that overexpressed MRP also showed altered accumulation and increased efflux of the free acid form of the fluorescent dye BCECF (Draper et al, 1996). BCECF is loaded into the cells as the membrane-permeable acetoxymethyl (AM) ester, BCECF-AM. This lipophilic ester derivative can cross cell membranes, entering the cytoplasm and various organelles, where the AM ester is hydrolysed by intracellular esterases to release BCECF free acid, which is normally retained by the cell. We used the altered BCECF accumulation phenotype of cell lines overexpressing MRP to look for drugs that could modulate MRP function. One of the drugs examined first was indomethacin as it has previously been reported to modulate efflux of BCECF from cultured epithelial cell monolayers, although the mechanism is unknown (Collington et al., 1992). We report here on the ability of indomethacin, a member of the non-steroidal family of anti-inflammatory drugs (NSAID), to specifically modulate MRP-mediated resistance.

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

SummaryThe relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines.Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmu) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3.

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