Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells ) modification of ABCC1 (MRP1) localization and functionality ´ Carole Marbeuf-Gueye, Verene Stierle, Paiwan Sudwan, Milena Salerno and Arlette Garnier-Suillerot ´ ´ Laboratoire Biophysique Moleculaire, Cellulaire et Tissulaire (BioMoCeTi), UMR CNRS 7033, Universite Paris 13 et Paris 6, Bobigny, France Keywords ABCC1 functionality; ABCC1 localization; membrane cholesterol level; multidrug resistance; raft Correspondence M Salerno, Laboratoire BioMoCeTi, CNRS UMR 7033, UFR SMBH, 74 rue Marcel Cachin, 93017 Bobigny, Cedex France Fax: +33 48 38 88 88 Tel: +33 48 38 77 48 E-mail: m.salerno@smbh.univ-paris13.fr (Received 20 November 2006, revised 20 December 2006, accepted 10 January 2007) doi:10.1111/j.1742-4658.2007.05688.x The multidrug resistance-associated protein transporter ABCC1 (MRP1) is an integral plasma membrane protein involved in the multidrug resistance phenotype It actively expels a number of cytotoxic molecules from cells To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we used multidrug-resistant GLC4 ⁄ ADR cells, which overexpress MRP1 Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed A detergent-free methodology was used to separate ‘light’ and ‘heavy’ plasma membrane fractions Our data show that MRP1 was exclusively found in ‘light’ fractions known as L0 phase membrane microdomains, together with  23% of gangliosides GM1 and 40% of caveolin-1 Depletion of the membrane cholesterol level to 40% by treatment with the cholesterol-chelating agent methyl-b-cyclodextrin did not modify MRP1 activity, as evidenced either by the rate of efflux of pirarubicin or that of glutathione Further cholesterol depletion below 40% yielded both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality Taken together, these data suggest that MRP1 funtionality depends on its localization in cholesterol-rich membrane microdomains A major obstacle to the success of chemotherapy is multidrug resistance (MDR) [1] The classical MDR phenotype is characterized by cross-resistance to a wide variety of structurally unrelated chemotherapeutic agents of natural origin after exposure to only one The roles, in vitro, of ABCB1 (P-glycoprotein, P-gp) and ABCC1 (multidrug resistance-associated protein, MRP1) in MDR are uncontested [2–5] Both proteins are members of the ATP-binding-cassette (ABC) superfamily of transport proteins, which lower the intracellular drug content through active drug efflux The well-documented membrane protein sensitivity to a lipid environment has led to the hypothesis of functional cross-talk between membrane proteins and membrane microdomains such as rafts and caveolae, inspiring numerous studies over the past 10 years [6] Both types of microdomain, rich in cholesterol and glycosphingolipids, are supposed to share similar physicochemical properties, in particular a Lo phase, less fluid than a liquid crystal phase [7] Raft domains, and by extension caveolae, are generally isolated from intact cells on the basis of their insolubility in cold, Abbreviations ABC, ATP-binding cassette; COase, cholesterol oxidase; CTB, cholera toxin B; GSH, glutathione; GST, glutathione S-transferase; HRP, horseradish peroxidase; MbCD, methyl-b-cyclodextrin; MCB, monochlorobimane; MDR, multidrug resistance; MRP1, multidrug resistanceassociated protein; P-gp, p-glycoprotein; PIRA, pirarubicin 1470 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS C Marbeuf-Gueye et al nonionic detergents such as Triton X-100, Brij 96 or 98, Tween-20 or Chaps octylglucoside In fact, the widely different sensitivities of membrane proteins to extraction by various detergents is well documented, and, barring possible artefacts, suggests that lipid rafts represent a heterogeneous collection of domains showing differences in both lipid and protein content [8,9] Other protocols without detergent have also been used, and present the advantage of not making a preselection of the microdomain protein or lipidic components by the detergent [10] Recent studies of the colocalization of ABC transporters such as P-gp in raft or caveolae microdomains have been interpreted in different ways [11] Similar studies on MRP1 are scarce, and have only used methods with the detergents Lubrol or Triton X-100 [12–15] Hinrichs et al [12] found that MRP1 was predominantly located in the Lubrol-based fraction of detergent-insoluble membrane domains in the colchicine-selected cell line HT29-col Although there was a cocalization of caveolin-1 and MRP1 in the low-density fraction, they proposed a different localization within the microdomains, on the basis of immunoprecipitation experiments with sequential Triton X-100 and Lubrol extraction The aim of this work was to determine the localization of MRP1 in the plasma membrane and to determine whether the modification of its localization would modify its functional properties Therefore, using a detergent-free method to isolate the low-density microdomains of the MDR lung cancer cell line GLC4 ⁄ ADR, we studied the effect of cholesterol depletion by MbCD (a) on the localization of MRP1 and (b) on the functionality of MRP1, i.e its ability to pump out an anthracycline derivative, pirarubicin (PIRA) on the one hand and reduced glutathione (GSH) on the other hand Our data show that MRP1 is totally localized in the low-density membrane fraction, together with some GM1 and caveolin-1 The reduction of cholesterol to 40% did not affect the anthracycline transport by MRP1 However, when more cholesterol was removed, we observed a shift of MRP1 from the low-density to the high-density fraction membrane, paralleled by a decrease of its functionality Results The GLC4 small lung cancer cell line, and its multidrug-resistant counterpart GLC4 ⁄ ADR, were used to study the influence of cholesterol on the activity of MRP1 in its membrane microenvironment The amount of intracellular nonesterified cholesterol was determined, together with the rate of MRP1- ABCC1 localization in light membrane microdomains mediated efflux of the anthracycline derivative, PIRA, and of GSH Fifteen independent experiments were performed on 15 different days During that time, the resistance factor varied slightly, yielding a rate of MRP1-mediated efflux of PIRA, Va, which varied within the range 0.4–0.8 · 10)18 molỈcell)1Ỉs)1 Concomitantly, the amount of cholesterol present exhibited some variation within the range 1.3–2.0 · 10)14 molỈcell)1 For this reason, the data are presented in term of ratios between the rate of PIRA efflux in the presence of modulator to that in the absence of modulator; the same holds for the rate of GSH efflux and for the cholesterol concentration In non-MbCD-treated cells, no correlation was found between the membrane cholesterol content and the MRP1-mediated efflux of PIRA Effect of MbCD on cellular cholesterol content Cells were incubated with 15 mm MbCD for various times ranging from 30 s to 20 min, and the cholesterol content was determined As can be seen in Fig 1, cellular cholesterol depletion was fast, 50% depletion being observed after less than min, and 80% depletion being observed after 10 of incubation; this was not modified by a longer time of incubation Effect of cholesterol depletion on the rate of MRP1-mediated efflux of PIRA Cells were incubated for 10 with various MbCD concentrations, ranging from to 12 mm This incubation yielded an increase in the cell membrane permeability to PIRA In other words, the rate of passive uptake of the drug in the presence of MbCD, V MbCD , ỵ is higher than the rate, V+, in its absence; for instance, a h incubation of cells with 15 mm MbCD yielded a three-fold increase of the rate of PIRA passive uptake In order to determine an eventual impact of MbCD on the rate of MRP1-mediated efflux of PIRA, experiments were performed with energy-depleted resistant cells, which were incubated with MbCD as previously described At steady state, the incorporation of PIRA was the same as in sensitive cells [16–20] At this stage, the addition of mm glucose yielded an increase in the fluorescent signal due to the MRP1-mediated efflux of the drug only, and as there was no concentration gradient across the plasma membrane, the effect of MbCD on passive PIRA diffusion did not need to be taken into account (Fig 2) The cholesterol content of the cells was also determined on the same samples Figure shows the plots of V MbCD ⁄ Va° as a function of [Chol]MbCD ⁄ [Chol]° a FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1471 ABCC1 localization in light membrane microdomains C Marbeuf-Gueye et al Fig Time course of cholesterol depletion in GLC4 ⁄ ADR cells by incubation with MbCD Cells (106 ⁄ mL) were incubated in Hepes buffer in the presence of 15 mM MbCD for various times, ranging from to 30 Data points are from a representative experiment (n ¼ 3) Fig Variation of the MRP1-mediated efflux of PIRA as a function of the fraction of cholesterol present in the cells V MbCD ⁄ Va° is plota ted as a function of [Chol]MbCD ⁄ [Chol]° Cholesterol depletion was obtained by 10 of incubation of cells with different MbCD concentrations Va° and V MbCD are the rates of MRP1-mediated efflux a of PIRA, before and after, respectively, treatment with MbCD [Chol]° and [Chol]MbCD are the cellular cholesterol contents before and after treatment with MbCD They are the mean ± SE of five independent experiments performed on different days cholesterol depletion to 40% of the initial content did not give rise to modification of the ratio V MbCD ⁄ Va° a that is characteristic of the MRP1-mediated efflux of PIRA However, further depletion yielded a decrease of the ratio Effect of cholesterol depletion on the rate of MRP1-mediated efflux of GSH Fig Incorporation of PIRA in energy-depleted GLC4 ⁄ ADR cells and determination of the active efflux rate (Va) Cells, 106 ⁄ mL, were incubated for 15 with MbCD at concentrations equal to mM (a), 10 mM (b) Cells were then centrifuged as explained under Experimental procedures, and incubated with lM PIRA The fluorescence intensity at 590 nm (kex ¼ 480 nm) was recorded as a function of the time of incubation of cells with PIRA The active efflux rate (Va) was determined from dF ⁄ dt after the addition of glucose Va° and V MbCD are the rates of MRP1-mediated efflux a of PIRA before and after treatment with MbCD, respectively [Chol]° and [Chol]MbCD are the cellular cholesterol contents before and after treatment with MbCD They are the mean of five independent experiments performed on different days As can be seen, 1472 VGSH was determined after incubation of cells with MbCD for Measurement was also performed after incubation of cells for 30 with mm MbCD; in both cases, no modification of the rate of MRP1mediated efflux of GSH was observed Effect of Triton derivatives on the MRP1-mediated efflux of PIRA Triton X-45 (n ¼ 5) and Triton X-165 (n ¼ 16) were used at nonpermeabilizing concentrations The active efflux was measured: energy-depleted cells were incubated with lm PIRA in the presence of various concentration of Triton, either X-45 or X-165 At steady state, glucose was added and the rate, Va, of pumpmediated efflux of drug was measured Figure shows the plot of V T ⁄ Va° as a function of Triton concentraa tion, where V T and Va° are the rates of efflux in the a FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS C Marbeuf-Gueye et al ABCC1 localization in light membrane microdomains A Fig Effect of Triton on MRP1-mediated efflux of PIRA V T ⁄ Va° is a plotted as function of the Triton X-45 (d) or Triton X-165 (h) added to the cells V T and Va° are the rates of MRP1-mediated a efflux of PIRA in the presence or in the absence, respectively, of Triton absence and presence of Triton, respectively Fifty per cent inhibition of PIRA efflux was observed with 16 ± lm Triton, either X-45 or X-165 Similar experiments were also performed with Triton X-100 (data not shown), and strictly analogous data were obtained MRP1, GM1 and caveolin-1 distribution within membrane fractions In order to determine the effect of MbCD on the integrity of rafts and the localization of MRP1, GM1 and caveolin-1, GLC4 ⁄ ADR cells were treated or not treated with MbCD Low-density membrane fractions were then isolated, and the distributions of MRP1, GM1 and caveolin-1 were assessed by western blotting and dot blotting of the analytical density gradients (Figs and 6) Fraction represents the top of the gradient, and fraction 11 is the bottom of the gradient Fractions and and fractions and contain the 10% ⁄ 22% and 22% ⁄ 35% sucrose interface, respectively In untreated cells, 100% of MRP1 was found in fraction 4, 40% of GM1 was found in fraction and  10–15% in fractions 5–9, fractions and corresponding to the cytoplasm and intracellular membranes, and caveolin-1 was present in all the fractions but was slightly more abundant in fraction than in the others After mild treatment with MbCD, the amount of MRP1 present in fraction decreased to 70%, with a shift of 30% to fraction 6; GM1 present in fractions decreased to  25%, whereas in fraction it increased to 20%, and it remained equal to 15% in fractions B β C Fig Detection of MRP1 in GLC4 ⁄ ADR cell lysates GLC4 ⁄ ADR cells were lysed by sonication, before (empty square) or after (full circle) treatment with MbCD The lysate was separated by density centrifugation, and collected from the top in mL fractions Fraction is from the top of the gradient (A) MRP1 expression was analyzed with IMAGE J v1.30 software In the absence of treatment with MbCD, fraction contains MRP1 (100%) After treatment with MbCD, MRP1 is found in fraction (70%) and in fraction (30%) Immunodetection of MRP1 in the absence (B) and in the presence (C) of treatment with MbCD S and R stand for GLC4 (sensitive) and GLC4 ⁄ ADR (resistant) cells 6–9 The amount of caveolin-1 present in fraction also decreased slightly Discussion Membrane lipids not form a homogeneous phase consisting of glycerophospholipids and cholesterol, but a mosaic of domains with unique biochemical compositions Among these domains, those containing sphingolipids and cholesterol, referred to as lipid rafts, have received much attention in the past few years [21] Tight interactions between the sterol and the sphingolipids result in the formation of domains that are resistant to solubilization in detergents at low temperature [22–24] and are destabilized by cholesterol-depleting and sphingolipid-depleting agents Caveolae can be considered as a functional specialized raft, because they contain several specific lipids and proteins typical of FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1473 ABCC1 localization in light membrane microdomains C Marbeuf-Gueye et al A B Fig Detection of GM1 and caveolin-1 in GLC4 ⁄ ADR cell lysates GLC4 ⁄ ADR cells were lysed by sonication, before (empty square) or after (full circles) treatment with MbCD The lysate was separated by density centrifugation, and collected from the top in mL fractions Fraction is from the top of the gradient (A) GM1 content and (B) caveolin-1 expression were analyzed with IMAGE J v1.30 software detergent-resistant-membrane components They are typical flask-shaped invaginations of the plasma membrane that originate from the presence of caveolin family proteins Among the three isoforms, caveolin-1, caveolin-2 and caveolin-3, caveolin-1 is the predominant isoform responsible for this structure and is used as a biochemical marker of caveolae [25,26] It now seems clear that caveolae are stable membrane domains that are kept in place by the actin cytoskeleton Rather than having a specific function, caveolae might be considered to be multifunctional organelles with a physiologic role that varies according to cell type and cellular needs [25,26] Importantly, the various possible functions of caveolae not seem to be of vital importance for the 1474 organism, as reflected by the relatively weak phenotype seen in the knockout mice Caveolae have recently been shown to be involved in MDR However, reports on the effect of caveolins on the development of MDR are controversial: Caveolin-1 expression has been shown to be upregulated in MDR phenotypes in a number of human cell lines [27,28], but expression of caveolin-1 and caveolin-2 has not been detected in several MDR cell lines that express high levels of P-gp [29–31], suggesting that caveolin-1 expression is not associated with that of P-gp protein or MDR1 genes [32] Increased levels of glucosylceramide have been observed in many MDR tumor cells [33–35] These conflicting data can be explained by the use of different methods to isolate rafts and ⁄ or caveolae Actually, most biochemical purifications of lipid rafts are based on an operational definition, namely that they are insoluble in Triton X-100 and have low buoyant density A simplified definition of rafts is the 1% Triton X-100-insoluble material that floats at the interface of a 5% ⁄ 30% sucrose step gradient [36] Thus, Triton X-100-resistant lipid rafts are distinguished from bulk plasma membrane because they are enriched in cholesterol and sphingolipids, but are relatively depleted in glycerolphospholipids Subsequent to the identification of low-density detergent-resistant domains in Triton X-100 cell extracts, a variety of other detergents, including Lubrol WX, Lubrol PX, Brij 58, Brij 96, Brij 98, Nonidet P40, Chaps, and octylglucoside, have been employed at different concentrations to prepare detergent-resistant membrane domains [37–41] Unsurprisingly, use of these different detergents in the preparation of rafts yielded membrane domains with different lipid compositions from those of standard, Triton X-100-resistant membranes [42] It is not clear whether this heterogeneity preexisted in rafts or was induced by the application of the detergent However, nondetergent methods that not involve the solubilization properties of membranes can be used to isolate rafts These methods largely obviate problems such as membrane mixing and the selective extraction of lipids In addition, these preparations seem to retain a greater fraction of inner-leafletmembrane lipids [43] than detergent-extracted rafts do, and may therefore yield domains in which the coupling between raft leaflets is maintained [44,45] For these reasons, rafts prepared by nondetergent methods seem more likely to reproduce the in vivo composition of these microdomains accurately Several studies have been performed to determine whether P-gp is localized with raft or caveolae; FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS C Marbeuf-Gueye et al however, very few studies have been done with MRP1 [12–14] In this work, after cholesterol extraction from the membrane, we measured both the functionality of MRP1 and its localization in the plasma membrane We report that membrane cholesterol is a central element in the control of both MRP1 functionality and localization in the GLC4 ⁄ ADR cell line In this study we used a nondetergent method to isolate rafts In this isolation procedure, the light and heavy fractions were found to be derived from the plasma membrane, whereas the extra-heavy third fraction originated mainly from intracellular components We examined the distribution of signaling molecules as well as plasma membrane markers in each fraction Our data show that MRP1 is exclusively localized in light membrane fraction (Fig 5) Hinrichs et al also found, with the detergent-based method, that MRP1 was localized in microdomains in human colon carcinoma cells (HT29col) Nevertheless, MRP1 localization in microdomains was partial [12] Caveolin-1, the marker of caveolin, and GM1, the marker of rafts, are also found in microdomain fraction It should be emphazised that it is difficult to distinguish between caveolar and noncaveolar rafts, given the cofractionation properties that they have in common Caveolin-1 is present not only in light fraction 4, but in all the membrane cell fractions (Fig 6) This is not surprising, as it is now clear that multiple locations for caveolin-1 exist throughout the cell, and caveolin-1 has been reported to be present in the plasma membrane and in a number of other cellular sites, including mitochondria, the endoplasmic reticulum lumen, and secretory vesicles [26] GM1 distribution was similar to that of caveolin-1 in ‘light’ and ‘heavy’ membranes In a first set of experiments, in order to determine whether cholesterol affects MRP1 function, we used MbCD to extract cholesterol from the lipid phase of intact living cells MbCD is a highly hydrophilic cyclic oligosaccharide that specifically binds sterol, rather than other membrane lipids, to form water-soluble complexes [45], without causing further membrane perturbation by insertion [46] Depletion of the membrane cholesterol level down to 40% by treatment with the cholesterol-chelating agent MbCD did not modify MRP1 activity, as followed either by the rate of efflux of PIRA or that of GSH However, further cholesterol depletion below 40% yields both a partial shift of MRP1 to the high-density fraction and a decrease of its functionality (Figs and 5) Indeed, membrane models made of binary or ternary cholesterol lipid mixtures can exhibit a bell-shaped phase diagram where the cholesterol-rich L0 phase coexists with the ABCC1 localization in light membrane microdomains LC phase [47,48] For these reasons, it is not surprising that MRP1 funtionality is not affected by cholesterol depletion down to 40%, as the microdomains are probably still present At this stage, it is interesting to compare the present data with those that we have previously obtained with K562 ⁄ ADR cells overexpressing P-gp The transport functionality vs the cholesterol content, obtained after different MbCD treatments, shows different profiles for P-gp and MRP1 (Fig and [49]) In K562 ⁄ ADR cells, which not express caveolin-1 protein and therefore not possess caveolae, the progressive removal of cholesterol with MbCD yields a progressive inhibition of P-gp functionality, whereas MRP1 functionality does not depend on the cholesterol content being reduced to 40% Given that in K562 ⁄ ADR cells, P-gp is not localized in the microdomain, whereas we found that MRP1 is exclusively localized in the microdomain, these different profiles could be explained by the fact that cholesterol could be more easily removed from nonmicrodomain regions than from the LO phase in the microdomain, where it is more tightly packed Now let us compare the impact of the membrane fluidity on P-gp and MRP1 transport activity We have checked (a) that the passive influx of PIRA was the same in both sensitive and energy-depleted cells, meaning that there is no variation of passive influx during the acquisition of MDR in the two cell lines [20], and (b) that the initial rate of passive PIRA uptake was not modified by the addition of fluidizing agents such as Triton derivatives at the low concentrations used in this study [50] The effect of Triton derivatives on P-gp and MRP1 functionality was measured in K562 ⁄ ADR and GLC4 ⁄ ADR cells, respectively, It appears that ± lm Triton X-45 yields 50% of P-gp functionality inhibition [50], whereas a concentration of 16 ± lm was required to inhibit 50% of MRP1 functionality, corroborating the observation that as MRP1 is localized in a more tightly packed fraction than P-gp, more Triton is required to modify the fluidity of the membrane around the transporter Second, the whole membrane fluidity of these two cell lines can be compared using the rate of passive influx of various molecules through the plasma membrane We have previously measured the rate of passive influx of anthracycline derivatives in these two cell lines [20], and we have observed that, systematically, for a given molecule the rate of its influx in GLC4 ⁄ ADR cells was 3–4 times higher than in K562 ⁄ ADR cells, showing that, as a whole, the GLC4 ⁄ ADR membrane is more fluid than the K562 ⁄ ADR membrane In summary, our results show that MRP1 is localized in microdomains and that its functionality, FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1475 ABCC1 localization in light membrane microdomains C Marbeuf-Gueye et al measured as its ability to pump out PIRA, is conserved when it is localized in rafts only In addition, cholesterol perturbation is more difficult to achieve in rafts than in the other membrane regions Altogether, these data suggest that MRP1-related functions can be regulated by the microdomain membrane Experimental procedures Cell lines and culture GLC4 cells and MRP1-overexpressing GLC4 ⁄ ADR cells [51] were cultured in RPMI 1640 (Sigma Chemical Co., St Louis, MO) medium supplemented with 10% fetal bovine serum (Gibco Cergy Pointoise, France) at 37 °C in a humidified incubator with 5% CO2 The resistant GLC4 ⁄ ADR cells were cultured with 1.2 lm doxorubicin until 1–4 weeks before the experiments Cell cultures used for experiments were split : day before use in order to ensure logarithmic growth Cells (106 ⁄ mL; mL per cuvette) were energy-depleted by preincubation for 30 in Hepes buffer with sodium azide but without glucose [52] We have previously checked that no P-gp was expressed in the resistant cells and that anthracycline efflux is due only to MRP1 [52] Drugs and chemicals Doxorubicin and PIRA were kindly provided by Pharmacia-Upjohn (St Quentin Yualines, France) Concentrations were determined by diluting working solutions to approximately 10)5 m with e480 ẳ 11 500 m)1ặcm)1 Working solutions were prepared just before use MbCD (mean degree of substitution: 10.5–14.7), horseradish peroxidase (HRP), GSH, dithiothreitol and compounds of the polyoxyethylene series were purchased from Sigma and were dissolved in water Trade names of polyoxyethylene (where n is the number of ethylene oxide units) are as follows: Triton X-45 (n ¼ 5), Triton X-100 (n ¼ 9.6) and Triton X-165 (n ¼ 165) 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) and monochlorobimane (MCB) were supplied by Molecular Probes (Eugene, OR) Before the experiments, the cells were counted, centrifuged at 5000 g for 30 s and resuspended in Hepes buffer solutions containing 20 mm Hepes plus 132 mm NaCl, 3.5 mm KCl, mm CaCl2 and 0.5 mm MgCl2 at pH 7.3, with or without mm glucose Other chemicals were of the highest grade available Deionized double-distilled water was used throughout the experiments MRPm5 anti-MRP1 mouse serum was purchased from Alexis Biochemical (San Diego, CA, USA), anti-caveolin-1 (N-20 sc 894) rabbit serum was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA) Poly(vinylidene difluoride) membrane was purchased from Hybond-P, Amersham Pharmacia Biotech (Orsay, France) 1476 Determination of the nonesterified cholesterol content of GLC4 cells The nonesterified cholesterol assay was adapted from a spectrofluorometric method used in the kit assay from Molecular Probes [53] Briefly, cells, 2.5 · 106 mL)1, were washed once with NaCl ⁄ Pi and suspended in mL of reaction buffer The reaction buffer at pH 7.4 contained 0.1 m NaCl ⁄ Pi, 0.05 m NaCl, and 0.1% Triton X-100 Samples were incubated at 37 °C for 15 min, and this was followed by sonication on ice (three times for 30 s) and then one additional hour of incubation at 37 °C under continuous stirring Unless indicated otherwise, 160 lL of this sample was added to the reaction buffer (total volume 1.6 mL), and the fluorescence signal at 560 nm (kex ¼ 585 nm) was monitored continuously when the following reactants were added: 50 lm Amplex Red, 0.5 mL)1 HRP, and 0.1 mL)1 cholesterol oxidase (COase) The concentration of cholesterol in the solution was proportional to the difference of the fluorescence signal DF ¼ FCOase ) FHRP, where FHRP and FCOase are the fluorescence signal intensities before and after the addition of HRP and COase, respectively Because the interaction of the cell suspension with HRP yielded a small change in the fluorescence signal (not shown), a control standard was systematically carried out in the presence of 160 lL of sonicated cell suspension to which 0–10 lm cholesterol solution was added The curve DF ¼ FCOase ) FHRP against the exogenous cholesterol concentration was linear within the 0–5 lm range Cholesterol titration was not affected by the presence of MbCD at the concentrations used in this study Treatment of cells with MbCD The methylated derivatives of b-cylodextrin are known to preferentially trap membrane cholesterol in comparison to other cyclodextrins, which also show affinity for phospholipids and proteins [45] We therefore used a methylated b-cylodextrin (MbCD) with a substitution degree of 10.5– 14.7 to study the effect of cholesterol depletion on GLC4 ⁄ ADR and GLC4 cells Cells were grown as described The standard culture medium was replaced with Hepes buffer, to which 2–20 mm MbCD had been added Unless stated otherwise, cells were then incubated for 10 at 37 °C Because MbCD interacted with anthracycline and modified the fluorescence signal, cells were washed with Hepes buffer, and the transport activity was then measured as described below Cellular anthracycline accumulation The rationale and validation of our experimental set-up for measuring the kinetics of the transport of anthracyclines in tumor cells has been extensively described and discussed FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS C Marbeuf-Gueye et al before [16–19] It is based on the continuous spectrofluorometric monitoring (Perkin Elmer LS50B spectrofluorometer Perkin Elmer, Courtabouef, France) of the decrease in the fluorescence signal of anthracycline at 590 nm (kex ¼ 480 nm) after incubation with the cells in a cm quartz cuvette (Fig 2) The decrease in fluorescence occurring during incubation with cells is due to the quenching of fluorescence after intercalation of anthracycline between the base pairs of DNA We have previously shown that this methodology allows the accurate measurement of the free cytosolic concentration of anthracyclines under steady-state conditions, their initial rates of uptake, and the kinetics of active efflux [16–20] Determination of the MRP1-mediated efflux of PIRA Cells (1 · 106 ⁄ mL; mL per cuvette) were preincubated for 30 in Hepes buffer with sodium azide, but without glucose (energy-deprived cells) Depletion of ATP in these cells was 90%, as checked with the luciferin–luciferase test [54] The cells remained viable throughout the experiment, as checked with Trypan blue and calcein vital stain (not shown) After addition of PIRA, the decrease in the signal was monitored until steady state was reached As the pH of the medium was chosen to equal the intracellular pH, at steady state, the extracellular free drug concentration (Ce) was equal to the cytosolic free drug concentration (Ci) Glucose was then added, which led to the restoration of control ATP levels within and to an increase in the fluorescence signal due to the efflux of PIRA ATP-dependent PIRA efflux was determined from the slope of the tangent of the curve F ¼ f(t), where F is the fluorescence intensity at the time of addition of glucose (Fig 2) As under these conditions, at the moment of addition of glucose Ci ¼ Ce, the passive influx and efflux were equal, the net initial efflux represents the MRP1-mediated active efflux only [52] GSH measurements In order to quantify free GSH, either inside the cells or released in the extracellular medium, an enzymatic essay was used [55] MCB, itself nonfluorescent, is conjugated to GSH by glutathione S-transferase (GST) to yield a fluorescent adduct [56] We have used this property to develop a very rapid and sensitive fluorometric method for GSH measurement [55] Briefly, a 10 mm stock solution of MCB was prepared in ethanol, and aliquots were stored at ) 80 °C in the dark The nonenzymatic reaction that occurred between GSH and MCB was very slow However, when GST was added, the increase of the fluorescent signal characteristic of MCB–GSH derivative formation was very fast The initial rate of MCB–GSH formation was determined as the increase in the fluorescent signal between 100 and 150 s after the addition of GST to MCB plus GSH ABCC1 localization in light membrane microdomains The MCB and GST concentrations were kept constant at 100 lm and 0.5 mL)1, respectively The fluorescence signal recorded over a short time (50 s), which was used as a measure of the initial rate of MCB–GSH formation, is directly proportional to the concentration of GSH at least within the range 0–20 lm (this corresponded to the concentrations expected when 106 cells ⁄ mL were lysed, the intracellular GSH concentrations being within the 0–20 mm range) We have checked that oxidized glutathione did not give rise to any modification of the fluorescence signal For the intracellular GSH determination, cells (2 · 106) suspended in mL of buffer were disrupted by sonication on ice (3 · 10 s, power 2) The rate of MCB–GSH formation was followed after addition of MCB 100 lm and GST 0.5 mL)1, as described above For the determination of GSH released by the cells, they were resuspended in Hepes buffer (106 ⁄ mL) in the absence or in the presence of the appropriate concentration of MbCD After specified time intervals, mL aliquots containing · 106 cells were centrifuged at 5000 g for 30 s and washed twice, and the GSH in the extracellular medium, and therefore released from the cells, and the GSH present in the pellet were determined The extracellular concentration of GSH was not affected by 250 lm acivicin, indicating negligible activity of c-glutamyltransferase in the membrane of GLC4 ⁄ ADR cells Isolation of ‘light’ and ‘heavy’ membrane fractions We used a detergent-free procedure for purification of membrane domains ‘Light’ and ‘heavy’ fractions were isolated according to previous methods [10,45,57], with the following modifications GLC4 ⁄ ADR cells, 10–15 · 106, were washed twice with NaCl ⁄ Pi and suspended in this buffer (2 · 108 cells ⁄ mL) They were then incubated for h in the absence or presence of 2.5 mm MbCD at 37 °C with stirring Cells were then again washed twice with NaCl ⁄ Pi The pellet was suspended in 500 lL of sodium carbonate buffer at pH 9–10, and sonicated in a cold bath three times 30 s (50 Hz, 117 V, and 80 W) (Vibra cell; Sonics & Materials Inc., Danbury, CT) The lysate was mixed with 80% sucrose to yield mL of 40% sucrose solution This mixture was transferred to the bottom of the ultracentrifugation tube (Beckman Instruments, Palo Alto, CA) and was overlaid with mL of 35% sucrose, mL of 22% sucrose, and mL of 10% sucrose solution The ultracentrifugation was performed at 160 000 g for 14 h at °C with an SW41 swinging rotor Light-scattering bands confined at the 10–22% sucrose and 22–35% sucrose interfaces, respectively, were observed Eleven mL fractions were collected by suction with a syringe from the top to the bottom The velocity of aspiration was kept low in order to avoid disturbance of the sucrose layers The first fraction was called F1, and the last fraction was called F11 The total protein FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1477 ABCC1 localization in light membrane microdomains C Marbeuf-Gueye et al concentration was measured in each fraction using the Bradford reagent (Bio-Rad, Mames La Coquette, France) Western blotting measurement of MRP1 and caveolin-1 expression References Equal volumes (12 lL) of membrane fractions were mixed with concentrated SDS reducing buffer (final concentrations are 0.75% SDS, 45 mm Tris, pH 6.8, 75 mm dithiothreitol) The samples were then incubated for h at 50 °C for MRP1 detection, or for at 95 °C for caveolin-1 detection Protein samples were separated on 7.5% (MRP1) or 12% (caveolin) SDS ⁄ PAGE, and then transferred to poly(vinylidene difluoride) membrane for h and 20 for MRP1 in transfer buffer (25 mm Tris-base, 192 mm glycine, 0.1% SDS) and for h and 45 for caveolin-1 in transfer buffer (25 mm Tris-base, 192 mm glycine, 0.1% SDS, 10% methanol) The membrane was blocked with 5% nonfat dry milk in 0.1% Tween ⁄ NaCl ⁄ Pi overnight at °C and treated with lgỈmL)1 MRPm5 anti-MRP1 mouse serum (Alexis Biochemical) overnight at °C, or with lgỈmL)1 anti-caveolin-1 (N-20 sc 894) rabbit serum (Santa Cruz) for h at room temperature Detection by HRP-linked was performed according to the manufacturer’s protocol (ECL plus kit with mouse IgG, HRP-linked whole antibody; Amersham Pharmacia Biotech) MRP1 and caveolin expression were evaluated after densitometric scanning of film and analysis with image j 1.30 software Localization of GM1 ganglioside GM1 is commonly found in high concentrations in rafts [57], and can be labeled using fluorescein isothiocyanateconjugated cholera toxin B (CTB) CTB [45] binds to the glycosphingolipid GM1, and we used it as a marker for raft localization in ‘light’ and ‘heavy’ plasma membrane fractions We employed a CTB–HRP conjugate for simple, rapid detection of ganglioside GM1 Briefly, after activation of a PVDF membrane with methanol and washing with water and 0.1% Tween ⁄ NaCl ⁄ Pi, lL of each gradient was dotted onto the wet membrane The air-dried membrane was reactivated and blocked with 5% nonfat dry milk in 0.1% Tween ⁄ NaCl ⁄ Pi After being washed with NaCl ⁄ Pi, the membrane was incubated with HRP-conjugated CTB (dilution : 5000, Sigma) in 0.1% Tween ⁄ NaCl ⁄ Pi for 90 min, rinsed several times with NaCl ⁄ Pi, and then detected by enhanced chemiluminescence 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GM1 and caveolin-1 distribution within membrane fractions In order to determine the effect of MbCD on the integrity of rafts and the localization of MRP1, GM1 and caveolin-1, GLC4 ⁄ ADR cells. .. MRP1 in the absence (B) and in the presence (C) of treatment with MbCD S and R stand for GLC4 (sensitive) and GLC4 ⁄ ADR (resistant) cells 6–9 The amount of caveolin-1 present in fraction also decreased... resistance factor, kinetics of uptake, and kinetics of the Pglycoprotein-mediated efflux of doxorubicin, daunorubicin, 8-(S)-fluoroidarubicin, and idarubicin in multidrugresistant K562 cells Mol Pharmacol