title: Kinetic analysis of fluorescein and dihydrofluorescein effluxes in tumour cells expressing the multidrug resistance protein, MRP1.
abstract: Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of two transporters the P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP1) which actively pump out multiple chemically unrelated substrates across the plasma membrane. A clear distinction in the mechanism of translocation of substrates by MRP1 or P-gp is indicated by the finding that, in most of cases, the MRP1-mediated transport of substrates is inhibited by depletion of intracellular glutathione (GSH), which has no effect on their P-gp-mediated transport. The aim of the present study was to quantitatively characterise the transport of anionic compounds dihydrofluorescein and fluorescein (FLU). We took advantage of the intrinsic fluorescence of FLU and performed a flow cytometric analysis of dye accumulation in the wild-type drug sensitive GLC4 that do not express MRP1 and its MDR subline which display high level of MRP1. The measurements were made in real time using intact cells. The kinetics parameters, k(a)=V(M)/K(m), which is a measure of the efficiency of the transporter-mediated efflux of a substrate, was very similar for the two FLU analogues. They were highly comparable with values for k(a) of other negatively charged substrates, such as GSH and calcein. The active efflux of both FLU derivatives was inhibited by GSH depletion.
authors.affiliation: Laboratoire de Physicochimie Biomoléculaire et Cellulaire (LPBC-CSSB) UMR CNRS 7033, Université Paris Nord, 74 rue Marcel Cachin, 93017 Bobigny, France.
authors.forename: Chantarawan
authors.initials: C
authors.lastname: Saengkhae
authors.forename: Chatchanok
authors.initials: C
authors.lastname: Loetchutinat
authors.forename: Arlette
authors.initials: A
authors.lastname: Garnier-Suillerot
mesh_terms: D001692:Biological Transport; D003498:Cyclic P-Oxides / Q000494:pharmacology; D005434:Flow Cytometry; D019793:Fluorescein / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D020024:Leukotriene Antagonists / Q000494:pharmacology; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D009539:Nicotinic Acids / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D003498:Cyclic P-Oxides; D019793:Fluorescein; D005452:Fluoresceins; D020024:Leukotriene Antagonists; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009539:Nicotinic Acids; D011422:Propionates; D011804:Quinolines; C110423:dihydrofluorescein; C059141:verlukast; C421109:multidrug resistance-associated protein 1