Upon altering the plasma membrane cholesterol content of these cells, membrane localization and the activity of MRP1 were analyzed.. Therefore, using a detergent-free method to isolate t
Trang 1multidrug-resistant lung cancer cells ) modification of
ABCC1 (MRP1) localization and functionality
Carole Marbeuf-Gueye, Ve´rene Stierle, Paiwan Sudwan, Milena Salerno and
Arlette Garnier-Suillerot
Laboratoire Biophysique Mole´culaire, Cellulaire et Tissulaire (BioMoCeTi), UMR CNRS 7033, Universite´ Paris 13 et Paris 6, Bobigny, France
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)
super-family of transport proteins, which lower the
intracel-lular 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,
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 1 48 38 88 88
Tel: +33 1 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 L0phase membrane microdomains, together with
23% of gangliosides GM1 and 40% of caveolin-1 Depletion of the mem-brane 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 local-ization in cholesterol-rich membrane microdomains
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 resistance-associated protein; P-gp, p-glycoprotein; PIRA, pirarubicin.
Trang 2nonionic 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
show-ing 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
preselec-tion of the microdomain protein or lipidic components
by the detergent [10]
Recent studies of the colocalization of ABC
transport-ers 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
caveo-lin-1 and MRP1 in the low-density fraction, they
pro-posed 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
localiza-tion of MRP1 in the plasma membrane and to
deter-mine whether the modification of its localization
would modify its functional properties Therefore,
using a detergent-free method to isolate the
low-den-sity 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
frac-tion, 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
multi-drug-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-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)18molÆcell)1Æs)1 Concom-itantly, the amount of cholesterol present exhibited some variation within the range 1.3–2.0· 10)14molÆcell)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 1 min, and 80% deple-tion being observed after 10 min of incubadeple-tion; 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 min with various MbCD concentrations, ranging from 0 to 12 mm This incuba-tion yielded an increase in the cell membrane permeab-ility to PIRA In other words, the rate of passive uptake of the drug in the presence of MbCD, VMbCDþ ,
is higher than the rate, V+, in its absence; for instance,
a 1 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, experi-ments 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 5 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 deter-mined on the same samples Figure 3 shows the plots
of VMbCD
a ⁄ Va as a function of [Chol]MbCD⁄ [Chol]
Trang 3Va and VMbCD
a are the rates of MRP1-mediated efflux
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
experi-ments performed on different days As can be seen,
cholesterol depletion to 40% of the initial content did not give rise to modification of the ratio VMbCD
a ⁄ Va 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
VGSH was determined after incubation of cells with MbCD for 5 min Measurement was also performed after incubation of cells for 30 min with 5 mm MbCD;
in both cases, no modification of the rate of MRP1-mediated efflux of GSH was observed
Effect of Triton derivatives on the MRP1-mediated efflux of PIRA Triton X-45 (n¼ 5) and Triton X-165 (n ¼ 16) were used at nonpermeabilizing concentrations The active efflux was measured: energy-depleted cells were incuba-ted with 1 lm PIRA in the presence of various concen-tration of Triton, either X-45 or X-165 At steady state, glucose was added and the rate, Va, of pump-mediated efflux of drug was measured Figure 4 shows the plot of VT
a⁄ Va as a function of Triton concentra-tion, where VT
a and Va are the rates of efflux in the
Fig 3 Variation of the MRP1-mediated efflux of PIRA as a function
of the fraction of cholesterol present in the cells V MbCD
a ⁄ V a is plot-ted as a function of [Chol] MbCD ⁄ [Chol] Cholesterol depletion was obtained by 10 min of incubation of cells with different MbCD con-centrations Va and V MbCD
a are the rates of MRP1-mediated efflux
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.
Fig 1 Time course of cholesterol depletion in GLC4 ⁄ ADR cells by
incubation with MbCD Cells (10 6 ⁄ mL) were incubated in Hepes
buffer in the presence of 15 m M MbCD for various times, ranging
from 0 to 30 min Data points are from a representative experiment
(n ¼ 3).
Fig 2 Incorporation of PIRA in energy-depleted GLC4 ⁄ ADR cells
and determination of the active efflux rate (Va) Cells, 10 6 ⁄ mL,
were incubated for 15 min with MbCD at concentrations equal to
0 m M (a), 10 m M (b) Cells were then centrifuged as explained
under Experimental procedures, and incubated with 1 l M PIRA.
The fluorescence intensity at 590 nm (kex¼ 480 nm) was recorded
as a function of the time of incubation of cells with PIRA The
act-ive efflux rate (Va) was determined from dF⁄ dt after the addition of
glucose.
Trang 4absence and presence of Triton, respectively Fifty per
cent inhibition of PIRA efflux was observed with
16 ± 2 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 5 and 6) Fraction 1 represents the top of the
gradient, and fraction 11 is the bottom of the gradient
Fractions 3 and 4 and fractions 6 and 7 contain the
10%⁄ 22% and 22% ⁄ 35% sucrose interface,
respect-ively In untreated cells, 100% of MRP1 was found in
fraction 4, 40% of GM1 was found in fraction 4 and
10–15% in fractions 5–9, fractions 8 and 9
corres-ponding to the cytoplasm and intracellular membranes,
and caveolin-1 was present in all the fractions but was
slightly more abundant in fraction 4 than in the others
After mild treatment with MbCD, the amount of
MRP1 present in fraction 4 decreased to 70%, with a
shift of 30% to fraction 6; GM1 present in fractions 4
decreased to 25%, whereas in fraction 6 it increased
to 20%, and it remained equal to 15% in fractions
6–9 The amount of caveolin-1 present in fraction 4 also decreased slightly
Discussion
Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids and cholesterol, but
a mosaic of domains with unique biochemical composi-tions Among these domains, those containing sphingol-ipids 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 consid-ered as a functional specialized raft, because they con-tain several specific lipids and proteins typical of
Fig 4 Effect of Triton on MRP1-mediated efflux of PIRA VT⁄ V a is
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
efflux of PIRA in the presence or in the absence, respectively, of
Triton.
B
C
A
β
Fig 5 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 1 mL fractions Frac-tion 1 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 4 contains MRP1 (100%) After treatment with MbCD, MRP1 is found in fraction 4 (70%) and in fraction 6 (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.
Trang 5detergent-resistant-membrane components They are
typical flask-shaped invaginations of the plasma
mem-brane that originate from the presence of caveolin
fam-ily 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 do not seem to be of vital importance for the
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: Caveo-lin-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 buoy-ant density A simplified definition of rafts is the 1% Triton X-100-insoluble material that floats at the inter-face 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 con-centrations to prepare detergent-resistant membrane domains [37–41] Unsurprisingly, use of these different detergents in the preparation of rafts yielded mem-brane domains with different lipid compositions from those of standard, Triton X-100-resistant membranes [42] It is not clear whether this heterogeneity pre-existed in rafts or was induced by the application of the detergent
However, nondetergent methods that do not involve the solubilization properties of membranes can be used
to isolate rafts These methods largely obviate prob-lems such as membrane mixing and the selective extraction of lipids In addition, these preparations seem to retain a greater fraction of inner-leaflet-membrane 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;
A
B
Fig 6 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
separ-ated by density centrifugation, and collected from the top in 1 mL
fractions Fraction 1 is from the top of the gradient (A) GM1
con-tent and (B) caveolin-1 expression were analyzed with IMAGE J v1.30
software.
Trang 6however, 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
ele-ment in the control of both MRP1 functionality and
localization in the GLC4⁄ ADR cell line
In this study we used a nondetergent method to
iso-late rafts In this isolation procedure, the light and
heavy fractions were found to be derived from the
plasma membrane, whereas the extra-heavy third
frac-tion 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 4 (Fig 5) Hinrichs et al also
found, with the detergent-based method, that MRP1
was localized in microdomains in human colon
carci-noma 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
frac-tion 4 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
frac-tion 4, but in all the membrane cell fracfrac-tions (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
reticu-lum reticu-lumen, and secretory vesicles [26] GM1
distribu-tion 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
per-turbation 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 3 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
LCphase [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 3 and [49]) In K562⁄ ADR cells, which do not express caveolin-1 protein and therefore do not possess caveolae, the progressive removal of cholesterol with MbCD yields a progressive inhibition of P-gp functionality, whereas MRP1 func-tionality 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 micro-domain, 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, mean-ing that there is no variation of passive influx durmean-ing 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
6 ± 2 lm Triton X-45 yields 50% of P-gp function-ality inhibition [50], whereas a concentration of
16 ± 2 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 fluid-ity 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,
Trang 7measured as its ability to pump out PIRA, is
con-served 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 37C 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 1 : 2 1 day before use in order to
ensure logarithmic growth Cells (106⁄ mL; 2 mL per
cu-vette) were energy-depleted by preincubation for 30 min 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
Pharma-cia-Upjohn (St Quentin Yualines, France) Concentrations
were determined by diluting working solutions to
approxi-mately 10)5mwith e480¼ 11 500 m)1Æcm)1 Working
solu-tions 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
(n¼ 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, 1 mm CaCl2 and 0.5 mm
MgCl2 at pH 7.3, with or without 5 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)
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· 106mL)1, were washed once with NaCl⁄ Piand suspended in 1 mL of reac-tion buffer The reacreac-tion 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 37C for 15 min, and this was followed
by sonication on ice (three times for 30 s) and then one additional hour of incubation at 37C 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 (kex¼ 585 nm) was monitored continuously when the following reactants were added: 50 lm Amplex Red, 0.5 UÆmL)1 HRP, and 0.1 UÆmL)1cholesterol oxidase (COase) The concentration
of cholesterol in the solution was proportional to the differ-ence of the fluorescdiffer-ence signal DF¼ FCOase) FHRP, where
FHRP and FCOase are the fluorescence signal intensities before and after the addition of HRP and COase, respect-ively 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
DF¼ FCOase) FHRPagainst the exogenous cholesterol con-centration 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 phospho-lipids 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 des-cribed The standard culture medium was replaced with He-pes buffer, to which 2–20 mm MbCD had been added Unless stated otherwise, cells were then incubated for
10 min at 37C Because MbCD interacted with anthra-cycline 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
Trang 8before [16–19] It is based on the continuous
spectrofluoro-metric monitoring (Perkin Elmer LS50B spectrofluorometer
Perkin Elmer, Courtabouef, France) of the decrease in the
fluorescence signal of anthracycline at 590 nm (kex¼
480 nm) after incubation with the cells in a 1 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; 2 mL per cuvette) were preincubated for
30 min in Hepes buffer with sodium azide, but without
glu-cose (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
monit-ored 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
cy-tosolic free drug concentration (Ci) Glucose was then added,
which led to the restoration of control ATP levels within
2 min 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 F¼ 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
addi-tion of glucose Ci¼ 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
fluores-cent 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
deter-mined as the increase in the fluorescent signal between 100
and 150 s after the addition of GST to MCB plus GSH
The MCB and GST concentrations were kept constant at
100 lm and 0.5 UÆmL)1, respectively The fluorescence sig-nal 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 concen-trations expected when 106cells⁄ mL were lysed, the intra-cellular 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 2 mL of buffer were disrupted by sonication
on ice (3· 10 s, power 2) The rate of MCB–GSH forma-tion was followed after addiforma-tion of MCB 100 lm and GST 0.5 UÆmL)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, 2 mL aliquots con-taining 2· 106cells 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 concentra-tion 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 iso-lated according to previous methods [10,45,57], with the following modifications GLC4⁄ ADR cells, 10–15 · 106
, were washed twice with NaCl⁄ Piand suspended in this buf-fer (2· 108cells⁄ mL) They were then incubated for 2 h in the absence or presence of 2.5 mm MbCD at 37C 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 & Mate-rials Inc., Danbury, CT) The lysate was mixed with 80% sucrose to yield 2 mL of 40% sucrose solution This mix-ture was transferred to the bottom of the ultracentrifuga-tion tube (Beckman Instruments, Palo Alto, CA) and was overlaid with 3 mL of 35% sucrose, 3 mL of 22% sucrose, and 3 mL of 10% sucrose solution The ultracentrifugation was performed at 160 000 g for 14 h at 4C with an SW41 swinging rotor Light-scattering bands confined at the 10–22% sucrose and 22–35% sucrose interfaces, respect-ively, were observed Eleven 1 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 distur-bance of the sucrose layers The first fraction was called F1, and the last fraction was called F11 The total protein
Trang 9concentration was measured in each fraction using the
Bradford reagent (Bio-Rad, Mames La Coquette, France)
Western blotting measurement of MRP1
and caveolin-1 expression
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 1 h at 50C for
MRP1 detection, or for 5 min at 95C 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 2 h and 20 min
for MRP1 in transfer buffer (25 mm Tris-base, 192 mm
gly-cine, 0.1% SDS) and for 1 h and 45 min 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 ⁄ Piovernight at 4C
and treated with 1 lgÆmL)1 MRPm5 anti-MRP1 mouse
serum (Alexis Biochemical) overnight at 4C, or with
1 lgÆmL)1 anti-caveolin-1 (N-20 sc 894) rabbit serum
(Santa Cruz) for 2 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
cave-olin 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
isothiocyanate-conjugated 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
frac-tions 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, 4 lL of each gradient
was dotted onto the wet membrane The air-dried
mem-brane 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-conju-gated CTB (dilution 1 : 5000, Sigma) in 0.1% Tween⁄
NaCl⁄ Pi for 90 min, rinsed several times with NaCl⁄ Pi,
and then detected by enhanced chemiluminescence
(Amer-sham Pharmacia Biotech) and analyzed with image j 1.30
software
Acknowledgements
We thank Professor Laurence Le Moyec and
Dr Catherine Herve´ du Penhoat for critical review
of the manuscript This work was supported by grants from the Centre National de la Recherche Scientifique and l’Universite Paris XIII
References
1 Leslie EM, Deeley RG & Cole SP (2005) Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense Toxicol Appl Pharmacol 204, 216–237
2 Cole SP & Deeley RG (1998) Multidrug resistance mediated by the ATP-binding cassette transporter pro-tein MRP Bioessays 20, 931–940
3 Nielsen D & Skovsgaard T (1992) P-glycoprotein as multidrug transporter: a critical review of current multi-drug resistant cell lines Biochim Biophys Acta 1139, 169–183
4 Fojo AT, Shen DW, Mickley LA, Pastan I & Gottes-man MM (1987) Intrinsic drug resistance in huGottes-man kidney cancer is associated with expression of a human multidrug-resistance gene J Clin Oncol 5, 1922–1927
5 Leier I, Jedlitschky G, Buchholz U, Cole SP, Deele RG
& Keppler D (1994) The MRP gene encodes an ATP-dependent export pump for leukotriene C4 and structu-rally related conjugates J Biol Chem 269, 27807–27810
6 Pike LJ (2004) Lipid rafts: heterogeneity on the high seas Biochem J 378, 281–292
7 Pike LJ (2003) Lipid rafts: bringing order to chaos
J Lipid Res 44, 655–667
8 Pizzo P & Viola A (2003) Lymphocyte lipid rafts: struc-ture and function Curr Opin Immunol 15, 255–260
9 Radeva G, Perabo J & Sharom FJ (2005) P-Glycopro-tein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains FEBS J 272, 4924–4937
10 MacDonald JL & Pike LJ (2005) A simplified method for the preparation of detergent-free lipid rafts J Biol Chem 280, 26796–26804
11 Orlowski S, Martin S & Escargueil A (2006) P-glycopro-tein and ‘lipid rafts’: some ambiguous mutual relation-ships (floating on them, building them or meeting them
by chance?) Cell Mol Life Sci 63, 1038–1059
12 Hinrichs JWJ, Klappe K, Hummel I & Kok JW (2004) ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resis-tant cancer cells J Biol Chem 279, 5734–5738
13 Hinrichs JWJ, Klappe K, van Riezen M & Kok JW (2005) Drug resistance-associated changes in sphingo-lipids and ABC transporters occur in different regions
of membrane domains J Lipid Res 46, 2367–2376
14 Klappe K, Hinrichs JWJ, Kroesen B-J, Sietsma H & Kok JW (2004) MRP1 and glucosylceramide are coordi-nately over expressed and enriched in rafts during
Trang 10multidrug resistance acquisition in colon cancer cells.
Int J Cancer 110, 511–522
15 Omidi Y, Campbell L, Barar J, Connell D, Akhtar S &
Gumbleton M (2003) Evaluation of the immortalised
mouse brain capillary endothelial cell line, b End3, as
an in vitro blood–brain barrier model for drug uptake
and transport studies Brain Res 990, 95–112
16 Fre´zard F & Garnier-Suillerot A (1991) Determination
of the osmotic active drug concentration in the
cyto-plasm of anthracycline-resistant and -sensitive K562
cells Biochim Biophys Acta 1091, 29–35
17 Fre´zard F & Garnier-Suillerot A (1991) Comparison of
the membrane transport of anthracycline derivatives in
drug-resistant and drug-sensitive K562 cells Eur J
Bio-chem 196, 483–491
18 Mankhetkorn S, Dubru F, Hesschenbrouck J, Fiallo M
& Garnier-Suillerot A (1996) Relation among the
resis-tance factor, kinetics of uptake, and kinetics of the
P-glycoprotein-mediated efflux of doxorubicin,
daunorubi-cin, 8-(S)-fluoroidarubidaunorubi-cin, and idarubicin in
multidrug-resistant K562 cells Mol Pharmacol 49, 532–539
19 Borrel MN, Pereira E, Fiallo M & Garnier-Suillerot A
(1994) Mobile ionophores are a novel class of
P-glyco-protein inhibitors The effects of ionophores on
4¢-O-tetrahydropyranyl-adriamycin incorporation in K562
drug-resistant cells Eur J Biochem 233, 125–133
20 Marbeuf-Gueye C, Ettori D, Priebe W, Kozlowski H &
Garnier-Suillerot A (1999) Correlation between the
kinetics of anthracycline uptake and the resistance
fac-tor in cancer cells expressing the multidrug resistance
protein or the P-glycoprotein Bochim Biophys Acta
1450, 374–384
21 Fantini J, Garmy N, Mahfoud R & Yahi N (2002)
Lipid rafts: structure, function and role in HIV,
Alzhei-mers and prion diseases Expert Rev Mol Med 2002,
1–22
22 Brown DA & London E (2000) Structure and function
of sphingolipid- and cholesterol-rich membrane rafts
J Biol Chem 275, 17221–17224
23 Edidin M (2003) The state of lipid rafts: from model
membranes to cells Annu Rev Biophys Biomol Struct 32,
257–283
24 Shogomori H & Brown DA (2003) Use of detergents to
study membrane rafts: the good, the bad, and the ugly
Biol Chem 384, 1259–1263
25 Van Deurs B, Roepstorff K, Hommelgaard AM &
Sandvig A (2003) Caveolae: anchored, multifunctional
platforms in the lipid ocean Trends Cell Biol 13,
92–100
26 Quest AFG, Leyton L & Parraga M (2004) Caveolins,
caveolae, and lipid rafts in cellular transport, signaling,
and disease Biochem Cell Biol 82, 129–144
27 Lavie Y, Fiucci G & Liscovitch M (1998) Up-regulation
of caveolae and caveolar constituents in
multidrug-resis-tant cancer cells J Biol Chem 273, 32380–32383
28 Pang A, Au WY & Kwong YL (2004) Caveolin-1 gene
is coordinately regulated with the multidrug resistance 1 gene in normal and leukemic bone marrow Leuk Res
28, 973–977
29 Haber M, Burkhart CA, Regi DL, Madafiglio J, Norris
MD & Horwitz SB (1995) Altered expression of M beta
2, the class II beta-tubulin isotype, in a murine J774.2 cell line with a high level of taxol resistance J Biol Chem 270, 31269–31275
30 Greenberger LM, Lothstein L, Williams SS & Horwitz
SB (1988) Distinct P-glycoprotein precursors are over-produced in independently isolated drug-resistant cell lines Proc Natl Acad Sci USA 85, 3762–3766
31 Parolini I, Sargiacomo M, Galbiati F, Rizzo G, Grignani F, Engelman JA, Okamoto T, Ikezu T, Scherer PE, Mora R et al (1999) Expression of caveo-lin-1 is required for the transport of caveolin-2 to the plasma membrane Retention of caveolin-2 at the level
of the golgi complex J Biol Chem 274, 25718–25725
32 Davidson B, Goldberg I, Givant-Horwitz V, Nesland
JM, Berner A, Bryne M, Risberg B, Koplovic J, Kris-tensen GB, Trope CG et al (2002) Caveolin-1 expres-sion in ovarian carcinoma is MDR1 independent Am J Clin Pathol 117, 225–234
33 Lavie Y, Cao H, Bursten SL, Giuliano AE & Cabot
MC (1996) Accumulation of glucosylceramides in multi-drug-resistant cancer cells J Biol Chem 271, 19530– 19536
34 Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu
CM & Muller M (2000) Differential expression of sphingolipids in MRP1 overexpressing HT29 cells Int J Cancer 87, 172–178
35 Veldman RJ, Klappe K, Hinrichs J, Hummel L, van der Schaaf G, Sietama H & Kok JW (2002) Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynth-esis in the Golgi apparatus FASEB J 16, 582–592
36 Hope HR & Pike LJ (1996) Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insolu-ble lipid domains Mol Biol Cell 7, 843–851
37 Madore N, Smith KL, Graham CH, Jen A, Brady K, Hall S & Morris R (1999) Functionally different GPI proteins are organized in different domains on the neuronal surface EMBO J 18, 6917–6926
38 Drevot P, Langlet C, Guo X-J, Bernard A-M, Colard
O, Chauvin J-P, Lasserre R & He H-T (2002) TCR signal initiation machinery is pre-assembled and acti-vated in a subset of membrane rafts EMBO J 21, 1899–1908
39 Ilangumaran S, Arni S, van Echten-Deckert G, Borisch
B & Hoessli DC (1999) Microdomain-dependent regula-tion of Lck and Fyn protein-tyrosine kinases in T lym-phocyte plasma membranes Mol Biol Cell 10, 891–905
40 Roper K, Corbeil D & Huttner WB (2000) Retention of prominin in microvilli reveals distinct cholesterol-based