From the Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom
Received for publication, February 7, 2003
, and in revised form, March 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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BCRP is a 655-amino acid, 72.1-kDa protein and is the second member of the G subfamily of ABC transporters. Members of the G subfamily are all half-transporters and include among others (i) the Drosophila white, brown, and scarlet proteins, which are involved in the transport of eye pigment (13); (ii) ABCG1, which is thought to be involved in the transport of cholesterol and phospholipids (14); and (iii) heterodimeric ABCG5/ABCG8, which has been implicated in the transport of cholesterol and plant sterols (15). In contrast to P-glycoprotein MDR1 and MRP1, which are full size transporters, BCRP most likely functions as a homodimer (16).
In normal tissue, high expression of the BCRP is found in stem cells (17), epithelial cells of small and large intestines, ducts and lobules of the breast, endothelial cells of veins and capillaries (18), and synchitiotrophoblastic cells of the placenta (19). The localization of BCRP suggests that it could have a potential role in protection against toxins. The recent observation in BCRP knock-out mice that BCRP protects against a chlorophyll-derived dietary phototoxin and protoporphyria is consistent with this notion (20).
Previously, we have characterized the molecular basis of the drug specificity of LmrA, a half-transporter homologue of human P-glycoprotein MDR1, in the Gram-positive bacterium Lactococcus lactis (21, 22). To allow a detailed comparison of BCRP and LmrA, human BCRP was functionally expressed in L. lactis using the nisin A-induced expression system that is used for the expression of LmrA. BCRP was active as an ATP-dependent multidrug transporter in L. lactis and was able to interact with sterols. We conclude that the substrate specificity of BCRP partly overlaps with that proposed for ABCG1 and ABCG5/ABCG8. Our observations may suggest a physiological role for BCRP in sterol metabolism in human in addition to its role in mediating resistance to xenobiotics and toxins arising from dietary intake and cellular metabolism.
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EXPERIMENTAL PROCEDURES |
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Genetic Manipulations
The human BCRP gene was amplified from pcDNA3-BCRP
(2) by PCR using the forward
primer 5'-GCCGCTAATACCATGGCTTCCAGTAATGTCG-3' to introduce an
NcoI site at the 5' end of BCRP and the reverse primer
5'-GCTTGGTACCGAGCTCTCTAGAAATTTAAGAATA-3' to introduce an
XbaI site at the 3' end of BCRP. The internal
NcoI sites in BCRP were removed through silent mutations by
PCR-based site-directed mutagenesis using two internal complementary primer
pairs: the forward primer
5'-CTGTAATCTGATTTATTACCGATGGGGATGTTACC-3' and the reverse primer
5'-GGTAACATCCCCATCGGTAATAAATCAGATAACAG-3' for C1440G and the
forward primer 5'-GCTTATTCAGCCAGTTCGATGGCACTGGCCATAGCAGCAGG-3' and
the reverse primer 5'-CTGCTGCTATGGCCAGTGCCATCGAACTGGCTGAATAAGC-3'
for C1566G. The final PCR product was digested with NcoI and
XbaI and cloned into the lactococcal pNZ8048 expression vector
(23), giving pNZ-BCRP. All
PCR-amplified DNA fragments were sequenced to ensure that only the intended
changes had been introduced.
Preparation of Inside-out Membrane Vesicles
For the isolation of BCRP-containing and control inside-out membrane
vesicles, L. lactis NZ9000 cells harboring pNZ-BCRP or pNZ8048,
respectively, were grown at 30 °C to an A660 of
0.3. At this density, 0.4% (v/v) of the supernatant of the
nisin-producing L. lactis strain NZ9700 (containing about 10 ng/ml of
nisin A) was added to the culture to induce transcription of the BCRP
gene under control of the nisA promoter. Following the incubation at
30 °C for 2 h, the cells were harvested by centrifugation at an
A660 of
0.70.9 and washed either with 100
mM KPi (pH 7.0) for transport assays or 50 mM Tris-HCl
(pH 7.4) for ATPase assays. The cell pellet was resuspended to an
A660 of 5 in KPi or Tris buffer supplemented with Complete
protease inhibitor mixture (Roche Applied Science). Lysozyme was then added to
a final concentration of 2 mg/ml, and the suspension was incubated at 30
°C for 30 min to digest the cell wall. The cells were lysed by three
passages through a Basic Z cell disruptor (Constant Systems, Northants, UK) at
20,000 p.s.i. Subsequently, DNase (10 µg/ml), RNase (2 µg/ml), 10
mM MgSO4, and 15 mM K-EDTA (pH 7.0) were
added, and the suspension was further incubated for 30 min at 30 °C.
Unbroken cells and debris were removed by centrifugation at 13,000 x
g for 15 min at 4 °C. Inside-out membrane vesicles were collected
by centrifugation at 125,000 x g for 40 min at 4 °C and
resuspended in either 50 mM KPi (pH 7.0) or 50 mM Tris
(pH 7.4) supplemented with 10% glycerol. The membrane vesicles were stored in
100-µl aliquots in liquid nitrogen.
Immunoblotting
Inside-out membrane vesicles were subjected to 10% SDS-PAGE. The proteins
were electroblotted to Hybond-P membrane (Amersham Biosciences) and were
detected in the presence of a 1:1000 dilution of the monoclonal anti-BCRP
antibody BXP-21 in accordance with the manufacturer's recommendations
(Signet). Detection of the primary antibody was performed using the ECL system
(Amersham Biosciences) as suggested by the manufacturer.
Cell Cytotoxicity Assays
L. lactis NZ9000 cells containing pNZ8048 or pNZ-BCRP were grown
at 30 °Ctoan A660 of about 0.3 in a 96-well plate.
Subsequently, protein expression was induced by the addition of nisin A as
described under "Preparation of Inside-out Membrane Vesicles."
Cytotoxic drugs were added to the cell suspensions at a concentration ranging
from 0 to 200 µM. The A660 of the cultures
were measured every 10 min for 6 h in a VersaMax plate reader (Molecular
Devices, CA). The relative growth rates were determined, and the
IC50 values were calculated. The data were obtained in duplicate
from three independent experiments.
Transport Assays
Ethidium Bromide TransportL. lactis NZ9000 cells containing
pNZ8048 or pNZ-BCRP were grown at 30 °C to an A660 of
about 0.3. Subsequently, protein expression was induced in the presence of
nisin A as described under "Preparation of Inside-out Membrane
Vesicles." The cells were washed three times in 50 mM KPi (pH
7.0) containing 5 mM MgSO4. To deprive cells of
metabolic energy, the cell suspensions were incubated for 30 min at 30 °C
in the presence of 0.5 mM dinitrophenol and washed three times in
50 mM KPi (pH 7.0) containing 5 mM MgSO4. The
cell pellet was resuspended to an A660 of 0.5 in 2 ml of
the KPi buffer and incubated for 5 min at 30 °C in the presence of 25
mM glucose with or without the inhibitor FTC (0.5
µM). Ethidium bromide was added to a final concentration as
indicated in the legend to Fig.
2, and its fluorescence was followed at 30 °C in a
Perkin-Elmer LS 55B fluorimeter using excitation and emission wavelengths of
500 and 580 nm, respectively, and slit widths of 2.5 nm each.
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Hoechst 33342 TransportInside-out membrane vesicles (1 mg
of total membrane protein) were diluted in 2 ml of 50 mM KPi (pH
7.4) supplemented with 2 mM MgSO4, 0.1 mg/ml creatine
kinase, and 5 mM phosphocreatine. For experiments with the
inhibitor, 0.5 µM FTC was added to the buffer. After 1 min
of incubation at 30 °C, 1 µM Hoechst 33342 (Molecular
Probes, Leiden, The Netherlands) was added, and the binding of the dye to the
membrane vesicles was followed by fluorimetry (excitation and emission
wavelength of 355 and 457 nm, respectively, and slit widths of 2.5 nm) until a
steady state was reached. Subsequently, ATP (or AMP-PNP in the control
experiments) was added to a final concentration of 5 mM, and the
fluorescence intensity was followed until a new steady state was reached. For
the substrate competition studies, estradiol was added to the cuvette at
concentrations as indicated in Fig.
6A prior to the addition of Hoechst 33342.
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[3H]Estradiol Uptake in Whole Cells
BCRP-expressing and control L. lactis cells were generated as
described under "Preparation of Inside-out Membrane Vesicles,"
washed three times in 50 mM KPi (pH 7.0) containing 5 mM
MgSO4, resuspended in KPi buffer supplemented with 1 mg bovine
serum albumin/ml to an A660 of 0.5, and kept on ice until
use. Cell suspensions (100-µl aliquots) were preincubated at 30 °C for
5 min in the presence of [2,4,6,7-3H]estradiol (87 Ci/mmol)
(Amersham Biosciences) at a final concentration of 500 nM.
Metabolic energy for active transport of estradiol was then generated in the
cells through the addition of 25 mM glucose. Following the
incubation at the times indicated in Fig.
6B, the cell suspensions were mixed with 3 ml of ice-cold
20 mM Tris-HCl (pH 7.4) containing 5 mM MgSO4
and rapidly filtered over Whatman GF/G glass fiber filters that were
pre-equilibrated overnight at 20 °C in Tris buffer. The filters were
washed twice with ice-cold Tris buffer, and radioactivity retained on the
filters was measured by liquid scintillation counting. All of the data were
corrected by subtracting nonspecific binding of [3H]estradiol to
the filters, which was usually less than 510% of the total
radioactivity.
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RESULTS |
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Heterologously Expressed BCRP Is Active as a Multidrug TransporterThe drug resistance of L. lactis cells harboring pNZ-BCRP was compared with that of cells harboring the pNZ8048 control vector. The growth rate of the two strains in liquid culture containing 40 pg/ml nisin A was determined at increasing concentrations of ethidium bromide, Hoechst 33342, rhodamine 123, or tetramethylrosamine. The concentrations of drugs necessary to reduce the growth rate of cells by 50% (IC50) are listed in Table I. BCRP expression in L. lactis significantly increased the drug resistance of the organism.
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To test whether drug extrusion from the cell is the underlying mechanism of drug resistance in L. lactis expressing BCRP, ethidium bromide uptake in cells was measured by monitoring the fluorescence of the intracellular ethidium-polynucleotide complex. In the presence of glucose, the uptake of ethidium bromide in cells expressing BCRP was significantly lower than that observed in control cells without BCRP (Fig. 2). This difference in ethidium accumulation between the two cell types reflected a higher ethidium efflux rate in BCRP-expressing cells. Upon the addition of the BCRP-specific inhibitor FTC, the ethidium accumulation in cells expressing BCRP was similar to that observed in control cells in the presence or absence of FTC, pointing to the inhibition of BCRP activity under these conditions.
The fluorescent lipophilic dye Hoechst 33342 is transported by BCRP expressed in mammalian cells (19, 25). To further analyze the activity of BCRP in L. lactis, the transport of Hoechst 33342 was studied in L. lactis-derived inside-out membrane vesicles in which the nucleotide-binding domain of BCRP was exposed on the outside surface of the membrane. The addition of Hoechst 33342 to the inside-out membrane vesicles resulted in a rapid increase in fluorescence up to a steady state level because of the partitioning of the dye in the hydrophobic environment of the phospholipid bilayer (Fig. 3). The subsequent addition of MgATP resulted in a rapid quenching of the Hoechst 33342 fluorescence in membrane vesicles containing BCRP (Fig. 3A) but not in control membrane vesicles without BCRP (Fig 3B). FTC strongly inhibited the ATP-dependent quenching of Hoechst 33342 fluorescence in membrane vesicles containing BCRP. In contrast, no significant changes in the steady state level of Hoechst 33342 fluorescence were observed in BCRP-containing and control membrane vesicles in the presence of AMP-PNP alone or AMP-PNP plus FTC (Fig. 3). These observations point to the BCRP-dependent transport of Hoechst 33342 from the phospholipid bilayer into the aqueous lumen of the membrane vesicles.
Functional studies in membrane vesicles of L. lactis also aimed at the ATPase activity of BCRP. In contrast to control inside-out membrane vesicles, BCRP-containing inside-out membrane vesicles displayed a significant amount of orthovanadate-sensitive ATPase activity that was stimulated up to 5-fold in the presence of daunomycin (Fig. 4A), a substrate of BCRP (2). The concentration of daunomycin required for half-maximal stimulation (SC50) of the vanadate-sensitive ATPase activity was about 20 µM. FTC significantly reduced the daunomycin-stimulated vanadate-sensitive ATPase activity, with an IC50 concentration below 2.5 µM (Fig. 4B). These data strongly suggest that the drug-stimulated vanadate-sensitive ATPase activity is associated with BCRP activity.
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Taken together, the observations of (i) multidrug resistance and reduced ethidium bromide accumulation in cells expressing BCRP, (ii) the ATP-dependent Hoechst 33342 transport and the drug-stimulated vanadate-sensitive ATPase activity in inside-out membrane vesicles containing BCRP, and (iii) the inhibition of these drug transport and ATPase activities by the BCRP inhibitor FTC demonstrate the functional expression of human BCRP in L. lactis.
Interaction of BCRP with SterolsAlthough BCRP (ABCG2) was originally identified as a multidrug transporter, other members in the ABCG subfamily (e.g. ABCG1 and ABCG5/G8) have been implicated in the transport of sterols. To explore the interaction of BCRP with sterols, the effect of sterols on the BCRP-associated ATPase activity was examined. Surprisingly, the sterols estradiol and cholesterol both stimulated the BCRP-associated ATPase activity about 4-fold, with SC50 values of about 10 and 8 µM, respectively (Fig. 5A). In addition, the BCRP-associated ATPase activity was stimulated 4-fold by the natural steroid progesterone and 7-fold by testosterone at SC50 values of 5 and 15 µM, respectively, (Fig. 5B). Finally, the estrogen receptor modulator tamoxifen (26) stimulated the BCRP-associated ATPase activity almost 3-fold with an SC50 of about 50 µM (Fig. 5C). None of these sterols significantly affected the low level of vanadate-sensitive ATPase activity observed in control membrane vesicles lacking BCRP (Fig. 5).
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The ability of BCRP expressed in L. lactis to interact with estradiol was further analyzed in Hoechst 33342 transport assays in which estradiol was included as a competing substrate. As shown in Fig. 6A, the presence of estradiol significantly inhibited the BCRP-mediated transport of Hoechst 33342 in inside-out membrane vesicles. The degree of inhibition by estradiol was proportional to the concentration of estradiol used, suggesting that estradiol is a potential transport substrate for BCRP. Estradiol did not affect the fluorescence of Hoechst 33342 in control membrane vesicles without BCRP (data not shown). The ability of BCRP to transport estradiol was directly assessed by measuring the uptake of [3H]estradiol in L. lactis cells. In the presence of glucose, BCRP-expressing cells exhibited a 4-fold lower uptake of [3H]estradiol than the control cells or BCRP-expressing cells in the absence of glucose (Fig. 6B). These results demonstrate the BCRP-mediated transport of estradiol in L. lactis. Interestingly, the amount of cell-associated estradiol was not reduced in glucose-energized L. lactis cells expressing LmrA, suggesting the lack of a significant LmrA-mediated transport of estradiol under the experimental conditions (Fig. 6B).
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DISCUSSION |
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To study sterol transport by human BCRP, we have expressed the protein in L. lactis, a bacterium that is devoid of mammalian sterols but that synthesizes hopanoids to regulate membrane fluidity (36). This property gives L. lactis an advantage over insect cells and mammalian cells where the sterol content of the plasma membranes can vary between 5 and 25%, which may hinder direct measurements of sterol transport and sterol-stimulated ATPase activities in the experimental setting. In addition, the expression of BCRP in L. lactis allows a comparison of its substrate specificity with that of the ABC half-transporter LmrA, a lactococcal homologue of P-glycoprotein MDR1. Three lines of experimental evidence suggested that BCRP was functionally expressed in L. lactis. Firstly, BCRP expression conferred multidrug resistance on cells. Secondly, BCRP expression enhanced the efflux of ethidium in cells and the transport of Hoechst 33342 in inside-out membrane vesicles. Both activities were inhibited in the presence of the BCRP-specific modulator FTC. Finally, BCRP-containing inside-out membrane vesicles displayed a vanadate and FTC-sensitive ATPase activity, which was stimulated by drugs (such as daunomycin) that are transported by BCRP. The observation of a BCRP-associated ATPase activity in inside-out membrane vesicles of L. lactis is consistent with published studies in which a drug-stimulated vanadate-sensitive ATPase activity in isolated membranes of insect cells (37) and mammalian cells (38) was shown to be due to the presence of BCRP.
The BCRP-associated ATPase activity in inside-out membrane vesicles of L. lactis was also significantly stimulated in the presence of sterols, including estradiol, cholesterol, progesterone, testosterone, and tamoxifen. It has been shown for P-glycoprotein MDR1 that the lipid environment can significantly influence the characteristics of purified and functionally reconstituted protein (39). Hence, the stimulation of the BCRP-associated ATPase activity in inside-out membrane vesicles of L. lactis by mammalian sterols could reflect a requirement of BCRP for the presence of these sterols in its lipid environment. However, the observations of (i) the efflux of [3H]estradiol in BCRP-expressing L. lactis cells but not LmrA-expressing cells (Fig. 6B) and (ii) the inhibition of BCRP-mediated Hoechst 33342 transport by estradiol (Fig. 6A) at concentrations that stimulate the BCRP-associated ATPase activity (Fig. 5A) imply competition between sterols and drugs for binding to common binding sites in BCRP. These data would also argue against indirect mechanisms of coupling between the estradiol-induced stimulation of BCRP-associated ATPase activity and [3H]estradiol translocation, in which (i) [3H]estradiol transport would represent a secondary flux associated with the active translocation of an endogenous compound (e.g. lipid) by BCRP and (ii) simultaneously, estradiol would interact with an allosteric binding site, rather than a transport site, to enhance the BCRP-associated ATPase activity.
Interestingly, as the concentration of daunomycin or sterols increased beyond that which stimulated the BCRP-associated ATPase maximally, the ATPase activity then decreased (Figs. 4 and 5) similar to observations for the P-glycoprotein MDR1 ATPase (34, 39). The biphasic pattern of stimulation and inhibition of the drug/sterol stimulated BCRP-associated ATPase activity may depend on the saturation state of BCRP transport sites, with enhanced binding of drug/sterol to transport sites at the inside surface of the membrane at low substrate concentrations and reduced dissociation of these substrates from release sites at the outside surface of the membrane at high substrate concentrations. Alternatively, the inhibition of the BCRP-associated ATPase activity at high drug/sterol concentrations may reflect changes in the lipid environment, i.e. in lateral pressure, that are less optimal for BCRP activity.
Altogether the data presented in this paper suggest that human BCRP is able to interact with sterols and that BCRP may play a role in the transport of sterols, steroids, and estrogen receptor antagonists used in the treatment of breast tumors, in addition to its ability to transport chemotherapeutic drugs and cellular toxins.
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FOOTNOTES |
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Recipient of a Commonwealth Scholarship.
To whom correspondence should be addressed. Fax: 44-1223-334040; E-mail:
hwv20{at}cam.ac.uk.
1 The abbreviations used are: ABC, ATP-binding cassette; BCRP, breast cancer
resistance protein; FTC, fumitremorgin C; IC50, drug concentration
required for half-maximal inhibition of the cellular growth rate; MDR,
multidrug resistance; AMP-PNP, adenosine
5'-(,
-imido)triphosphate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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