Báo cáo khoa học: Perturbation of membrane microdomains in GLC4 multidrug-resistant lung cancer cells ) modification of ABCC1 (MRP1) localization and functionality docx

Perturbation of membrane microdomains in GLC4
multidrug-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:
(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 L

0
phase 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.
1470 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 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, V
a
,whichvaried
within the r ange 0.4–0.8 · 10
)18
molÆcell
)1
Æs
)1
.Concom-
itantly, the amount of cholesterol present exhibited some
variation within the r ange 1.3–2.0 · 10
)14
molÆcell
)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 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 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, V
MbCD
þ
,
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 V
MbCD
a
⁄ V
a
° as a function of [Chol]
MbCD
⁄ [Chol]°.
C. Marbeuf-Gueye et al. ABCC1 localization in light membrane microdomains
FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1471
V
a
° and V
MbCD
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 V
MbCD
a
⁄ V
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
V
GSH
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 (n ¼ 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, V
a
, of pump-
mediated efflux of drug was measured. Figure 4 shows
the plot of V
T
a
⁄ V
a
° as a function of Triton concentra-
tion, where V
T
a
and V
a
° 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. V
a
° 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 (V
a
). Cells, 10
6
⁄ mL,
were incubated for 15 min with MbCD at concentrations equal to
0m
M (a), 10 mM (b). Cells were then centrifuged as explained
under Experimental procedures, and incubated with 1 l
M PIRA.
The fluorescence intensity at 590 nm (k
ex

¼ 480 nm) was recorded
as a function of the time of incubation of cells with PIRA. The act-
ive efflux rate (V
a
) was determined from dF ⁄ dt after the addition of
glucose.
ABCC1 localization in light membrane microdomains C. Marbeuf-Gueye et al.
1472 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS
absence 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. V
T
a
⁄ V
a
° is

plotted as function of the Triton X-45 (d) or Triton X-165 (h)
added to the cells. V
T
a
and V
a
° 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.
C. Marbeuf-Gueye et al. ABCC1 localization in light membrane microdomains
FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1473
detergent-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.
ABCC1 localization in light membrane microdomains C. Marbeuf-Gueye et al.
1474 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 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 (HT29
col
). 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 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 reticu-
lum 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 L
0
phase coexists with the
L
C
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. 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 L
O
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 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
6±2lm 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,
C. Marbeuf-Gueye et al. ABCC1 localization in light membrane microdomains
FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1475
measured 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 37 °Cina
humidified incubator with 5% CO
2
. The resistant
GLC4 ⁄ ADR cells were cultured with 1.2 l m 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 (10
6
⁄ 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
)5
m with e
480
¼ 11 500 m
)1
Æcm
)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
(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, 1 mm CaCl
2
and 0.5 mm
MgCl
2
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 · 10
6
mL
)1
, were
washed once with NaCl ⁄ P
i
and suspended in 1 mL of reac-
tion buffer. The reaction buffer at pH 7.4 contained 0.1 m

NaCl ⁄ P
i
, 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 (k
ex
¼ 585 nm) was
monitored continuously when the following reactants were
added: 50 lm Amplex Red, 0.5 UÆmL
)1
HRP, and
0.1 UÆmL
)1
cholesterol oxidase (COase). The concentration
of cholesterol in the solution was proportional to the differ-
ence of the fluorescence signal DF ¼ F
COase
) F
HRP
, where
F
HRP
and F
COase
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
DF ¼ F
COase
) F
HRP
against 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 37 °C. 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
ABCC1 localization in light membrane microdomains C. Marbeuf-Gueye et al.
1476 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS
before [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 (k
ex
¼
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 · 10
6
⁄ 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 (C
e
) was equal to the cy-
tosolic free drug concentration (C
i
). 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 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 addi-
tion of glucose C
i
¼ C
e
, the passive influx and efflux were
equal, the net initial efflux represents the MRP
1
-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 10
6
cells ⁄ 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 · 10
6
)
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 addition 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 (10
6
⁄ mL) in the absence
or in the presence of the appropriate concentration of
MbCD. After specified time intervals, 2 mL aliquots con-
taining 2 · 10
6
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 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 · 10
6
,
were washed twice with NaCl ⁄ P
i
and suspended in this buf-
fer (2 · 10
8

cells ⁄ mL). They were then incubated for 2 h in
the absence or presence of 2.5 mm MbCD at 37 °C with
stirring. Cells were then again washed twice with NaCl ⁄ P
i
.
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 4 °C 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
C. Marbeuf-Gueye et al. ABCC1 localization in light membrane microdomains
FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1477
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
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 50 ° C for
MRP1 detection, or for 5 min 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 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 ⁄ P
i
overnight at 4 °C
and treated with 1 lgÆmL
)1
MRPm5 anti-MRP1 mouse
serum (Alexis Biochemical) overnight at 4 °C, 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 ⁄ P
i
,4lL 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 ⁄ P
i
. After being washed with
NaCl ⁄ P
i
, the membrane was incubated with HRP-conju-
gated CTB (dilution 1 : 5000, Sigma) in 0.1% Tween ⁄
NaCl ⁄ P
i
for 90 min, rinsed several times with NaCl ⁄ P
i
,
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 human
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
ABCC1 localization in light membrane microdomains C. Marbeuf-Gueye et al.
1478 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS
multidrug 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)-fluoroidarubicin, 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
C. Marbeuf-Gueye et al. ABCC1 localization in light membrane microdomains
FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS 1479
lipid micro-domains in the apical plasma membrane.
Nat Cell Biol 2, 582–592.
41 Gumbleton M, Hollins AJ, Omidi Y, Campbell L &
Taylor G (2003) Targeting caveolae for vesicular drug
transport. J Control Release 87, 139–151.
42 Schuck S, Honsho M, Ekroos K, Shevchenko A &
Simons K (2003) Resistance of cell membranes to differ-
ent detergents. Proc Natl Acad Sci USA 100, 5795–
5800.
43 Pike LJ, Han X, Chung K-N & Gross R (2002) Lipid

rafts are enriched in arachidonic acid and plasmeny-
lethanolamine and their composition is independent of
caveolin-1 expression: a quantitative electrospray ioniza-
tion ⁄ mass spectrometric analysis. Biochemistry 41,
2075–2088.
44 Janes PW, Ley SC & Magee AI (1999) Aggregation of
lipid rafts accompanies signaling via the T cell antigen
receptor. J Cell Biol 147, 447–461.
45 Ohvo H & Slotte P (1996) Cyclodextrin-mediated
removal of sterols from monolayers: effects of sterol
structure and phospholipids on desorption rate. Bio-
chemistry 35, 8018–8024.
46 Gimpl G, Burger K & Fahrenholz F (1997) Cholesterol
as modulator of receptor function. Biochemistry 36,
10959–10974.
47 Mouritsen OG & Zuckermann MJ (2004) What’s so
special about cholesterol? Lipids 39, 1101–1113.
48 Veatch SL & Keller SL (2003) Separation of liquid
phases in giant vesicles of ternary mixtures of phospho-
lipids and cholesterol. Biophys J 85, 3074–3093.
49 Reungpatthanaphong P, Marbeuf-Gueye C, Le Moyec
L, Salerno M & Garnier-Suillerot A (2004) Decrease of
P-glycoprotein activity in K562 ⁄ ADR cells by
MbetaCD and filipin and lack of effect induced by
cholesterol oxidase indicate that this transporter is not
located in rafts. J Bioenerg Biomembr 36, 533–543.
50 Borrel MN, Fiallo M, Veress I & Garnier-Suillerot A
(1995) The effect of crown ethers, tetraalkylammonium
salts, and polyoxyethylene amphiphiles on pirarubicin
incorporation in K562 resistant cells. Biochem Pharma-

col 50, 2069–2076.
51 Zijlstra JG, De Vries EGE & Mulder NH (1987) Multi-
factorial drug resistance in an adriamycin-resistant
human small cell lung carcinoma cell line. Cancer Res
47, 1780–1784.
52 Marbeuf-Gueye C, Broxterman H, Dubru F, Priebe W
& Garnier-Suillerot A (1998) Kinetics of anthracycline
efflux from multidrug resistance protein-expressing
cancer cells compared with P-glycoprotein-expressing
cancer cells. Mol Pharmacol 53, 141–147.
53 Amundson DM & Zhou M (1999) Fluorometric method
for the enzymatic determination of cholesterol. J Bio-
chem Biophys 38, 43–52.
54 Kimmich G, Randles J & Brand J (1975) Assay of pico-
mole amounts of ATP, ADP, and AMP using the luci-
ferase enzyme system. Anal Biochem 69, 187–192.
55 Salerno M & Garnier-Suillerot A (2001) Kinetics of
glutathione and daunorubicin efflux from multidrug
resistance protein overexpressing small-cell lung cancer
cells. Eur J Pharmacol 421, 1–9.
56 Fernandez-Checa JC & Kaplowitz N (1990) The use of
monochlorobimane to determine hepatic GSH levels
and synthesis. Anal Biochem 190
, 212–219.
57 Monneron A & d’Alayer J (1978) Isolation of plasma
and nuclear membranes of thymocytes. II. Biochemical
composition. J Cell Biol 77, 232–245.
ABCC1 localization in light membrane microdomains C. Marbeuf-Gueye et al.
1480 FEBS Journal 274 (2007) 1470–1480 ª 2007 The Authors Journal compilation ª 2007 FEBS