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Vol. 55, Issue 5, 929-937, May 1999
Deutsches Krebsforschungszentrum, Heidelberg, Germany
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Summary |
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 Top
 Summary
 Introduction
Experimental Procedures
Results
Discussion
References
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The multidrug resistance protein MRP1 functions as an ATP-dependent
conjugate export pump and confers multidrug resistance. We cloned MRP2
(symbol ABCC2), a MRP family member localized to the apical membrane of
polarized cells. Stable expression of MRP2 in transfected human
embryonic kidney (HEK-293) and Madin-Darby canine kidney (MDCK) cells
was enhanced by inhibitors of histone deacetylase. In polarized MDCK
cells, both rat and human MRP2 were sorted to the apical plasma
membrane. An antibody raised against the amino terminus of rat MRP2
recognized the recombinant protein on the apical surface of
nonpermeabilized cells, providing direct evidence for the extracellular
localization of the amino terminus of MRP2. ATP-dependent transport by
recombinant human and rat MRP2 was measured with membrane vesicles from
stably transfected cells. The Km value of
human MRP2 was 1.0 ± 0.1 µM for leukotriene C4 and
7.2 ± 0.7 µM for 17
-glucuronosyl estradiol; the
Km values of human MRP1 were 0.1 ± 0.02 µM for leukotriene C4 and 1.5 ± 0.3 µM for
17-glucoronosyl estradiol. Thus, the
conjugate-transporting ATPases MRP2 and MRP1 differ not only by
their domain-specific localization but also by their kinetic
properties. Drug resistance conferred by recombinant MRP2 was studied
in MDCK and HEK-293 cells using cell viability assays. Expression of
human and rat MRP2 enhanced the resistance of MDCK cells to etoposide
5.0-fold and 3.8-fold and to vincristine 2.3- and 6.0-fold,
respectively. Buthionine sulfoximine reduced resistance to these drugs.
Human MRP2 overexpressed in HEK-293 cells enhanced the resistance to etoposide (4-fold), cisplatin (10-fold), doxorubicin (7.8-fold), and
epirubicin (5-fold). These results demonstrate that MRP2 confers resistance to cytotoxic drugs.
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Introduction |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Membrane
proteins mediating the ATP-dependent transport of conjugates of
lipophilic compounds with glutathione, glucuronate, or sulfate have
been recognized as members of the multidrug resistance protein (MRP)
family. This family of conjugate-transporting ATPases includes MRP
(Cole et al., 1992
), also known as MRP1, and the canalicular isoform of
MRP (Büchler et al., 1996), also known as canalicular
multispecific organic anion transporter (Paulusma et al., 1996;
Taniguchi et al., 1996; Ito et al., 1997) or apical MRP, now widely
termed MRP2; as well as related ATP-binding cassette transporters from
yeast, plants, and Caenorhabditis elegans (reviewed by
Keppler and König, 1997). Substrate-dependent ATPase activity of
the purified protein has been demonstrated for MRP1 (Chang et al.,
1997). ATP-dependent transport of conjugates and other amphiphilic
anions into inside-out-oriented membrane vesicles from
MRP1-overexpressing drug-selected and MRP1-transfected cells has been
established (Jedlitschky et al., 1994, 1996; Leier et al., 1994;
Müller et al., 1994; Loe et al., 1996).
The apical isoform of MRP was originally recognized in the rat
hepatocyte canalicular membrane by immunofluorescence microscopy and by
cloning of a novel 347-bp cDNA fragment that was distinct from rat MRP
(Mrp1); this cDNA fragment was not expressed in the liver from mutant
rats that lack ATP-dependent transport of conjugates across the
canalicular membrane (Mayer et al., 1995). Subsequent cloning of the
full-length cDNA revealed rat apical MRP (Mrp2; Paulusma et al., 1996;
Büchler et al., 1996; Ito et al., 1997; Madon et al., 1997). The
human ortholog, MRP2, was cloned from liver tissue (Büchler et
al., 1996; Paulusma et al., 1997) and from drug-resistant tumor cells
(Taniguchi et al., 1996). The amino acid identity between human MRP1
and MRP2 is 49% (Keppler and König, 1997). MRP1 and
MRP2 have been localized to chromosomes 16p13.1 (Cole et
al., 1992) and 10q24 (Taniguchi et al., 1996), respectively. The
absence of MRP2 from the human hepatocyte canalicular membrane has been
recognized as the cause of the Dubin-Johnson syndrome (Kartenbeck et
al., 1996; Keppler and Kartenbeck, 1996), and several mutations in the
MRP2 gene were reported in this hereditary disorder
(Paulusma et al., 1997; Wada et al., 1998). The predominant localization of rat and human MRP2 in the hepatocyte canalicular membrane has been consistent with a hepatic canalicular transporter. However, the additional localization of this transport protein to the
apical membrane of kidney proximal tubules (Schaub et al., 1997)
indicates that the characteristic expression and sorting of MRP2 is to
the apical domain of polarized cells.
The permanent expression of human MRP2 in stably transfected cell lines
enables studies on the sorting of this integral membrane protein, and
membrane vesicles prepared from MRP2-transfected cells
provide an important tool for establishing the substrate specificity of
this ATP-dependent transporter, both with respect to physiological
endogenous substrates and with respect to anticancer drugs and their
derivatives. Previously, some information on the substrate specificity
of rat Mrp2 has been gathered from determinations of ATP-dependent
transport into inside-out hepatocyte canalicular membrane vesicles from
normal rats in comparison with membrane vesicles from mutant rats
selectively lacking Mrp2 (Ishikawa et al., 1990; Oude Elferink et al.,
1995; Jedlitschky et al., 1997). Direct information on the substrate
specificity of recombinant rat Mrp2 has been obtained recently after
transient expression in COS7 cells and Xenopus laevis
oocytes (Madon et al., 1997) and in transfected NIH3T3 cells (Ito et
al., 1998). Evers et al. (1998) most recently showed that membrane
vesicles from MRP2-transfected cells derived from
Madin-Darby canine kidney (MDCK) cells exhibit ATP-dependent
transport of the glutathione S-conjugates of
2,4-dinitrophenol and ethacrynic acid and that recombinant human MRP2
expressed in these polarized cells is sorted to the apical membrane.
Furthermore, recombinant rabbit Mrp2 expressed in insect cells was
recognized as an ATP-dependent transporter for leukotriene
C4 (LTC4) and 17-glucuronosyl estradiol (van Aubel et al. 1998). However, a kinetic characterization allowing for the direct comparison of Km values between recombinant human MRP1
and MRP2 has been lacking, and, importantly, it remained an open
question whether MRP2 confers multidrug resistance. Our results
demonstrate for the first time that recombinant MRP2 confers resistance
to etoposide, vincristine, cisplatin, doxorubicin, and epirubicin. As a
prerequisite for these studies, we describe that inhibitors of histone
deacetylase, such as butyrate and trichostatin A, induce a manifold
overexpression of recombinant MRP2.
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Experimental Procedures |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Materials.
[14,15,19,20-3H]LTC4 (6.1 TBq/mmol) and 17-D-glucuronosyl
[6,7-3H]estradiol (2 TBq/mmol) were obtained
from DuPont/New England Nuclear (Boston, MA). Unlabeled
LTC4 was from Cascade Biochem Ltd. (Reading,
Berkshire, UK), and unlabeled 17-D-glucuronosyl estradiol was obtained from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose filters (pore size 0.2 µm) were obtained from
Schleicher & Schüll (Dassel, Germany). Cell culture media and
supplements were obtained from Sigma-Aldrich Chemie (Deisenhofen,
Germany). G418 (Geneticin) was purchased from Calbiochem (Bad
Soden, Germany). Sodium butyrate was obtained from Merck-Schuchardt
(Hohenbrunn, Germany). Trichostatin A
(4,6-dimethyl-7-[p-dimethylaminophenyl]-7-oxohepta-2,4-dienohydroxamic acid) and buthionine sulfoximine (BSO) were purchased from
Sigma-Aldrich Chemie. LY335979
(4-(1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl)-
-[(5-quinolinyloxy)methyl]-1-piperazineethanol; Dantzig et al., 1996) was kindly provided by Drs. J. J. Starling and A. Dantzig from the Eli Lilly Research Laboratories (Indianapolis, IN).
Antibodies.
EAG5 is a polyclonal antibody raised in rabbits
against the carboxy-terminal sequence of human MRP2 (Büchler et
al., 1996; Jedlitschky et al., 1997). The polyclonal antibody EAG15 was
raised against the corresponding sequence of rat Mrp2 (Büchler et
al., 1996). MDK is a polyclonal antibody raised in rabbits against the
amino-terminal sequence of rat Mrp2 (residues 1-25) coupled to keyhole
limpet hemocyanin (Büchler et al., 1996) via the carboxyl end of
the peptide. QCRL-1, an MRP1-specific monoclonal antibody, was kindly
provided by Drs. R. G. Deeley and S. P. C. Cole
(Queen's University, Kingston, Ontario, Canada). Monoclonal antibody
C219 against MDR P-glycoproteins was purchased from Centocor
(Malvern, PA).
Cloning of Human MRP2 and Vector Constructions.
Using 5 µg of poly(A)+-enriched RNA (mRNA) and the Zap
cDNA synthesis kit (Stratagene, Heidelberg, Germany), a unidirectional human liver cDNA library was constructed according to the
manufacturer's instructions. The screening and plaque purification was
performed as described (Büchler et al., 1996) with a fragment of
human MRP2 as probe. The hybridization yielded several
MRP2 clones, the longest being 2.3 kb (pMRP2.3'). A
reverse transcription-polymerase chain reaction (PCR) strategy was used
to obtain the 5'-end of MRP2 cDNA. Human liver total RNA
(5 µg) was reverse-transcribed in 50 µl of transcription buffer
[50 mM Tris (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM
dithiothreitol, 1 mM 2'-deoxynucleoside 5'-triphosphate, 40 U of
RNasin] in the presence of 50 pmol hcrev3200 (5'-ATT TGA TGC
ATG GAC GA-3'; bases 3162-3146) with 50 U of StrataScript reverse
transcriptase at 37°C for 1 h. The resulting single-stranded cDNAs were purified by centrifugation through Microcon-100 (Millipore, Eschborn, Germany) and used for the subsequent PCR. PCR was performed in a total volume of 50 µl of PCR buffer (provided by
manufacturer) containing 2.5 U TaqPlus DNA polymerase
(Stratagene), and a 0.25-mM concentration of each sense and antisense
primer. For the amplification of the 5'-fragment of human
MRP2, the following primer pair was used: the sense
primer hcfor5', 5'-ATA GAA GAG TCT TCG TTC-3' (bases 
37 to 20); and
the antisense primer revdeg-I, 5'-TTT GTC CTT TCA CTA GTT C-3' (bases
2848-2830). The 5'-proximal sequence information needed for the design
of the sense primer hcfor5' was obtained from the expressed
sequence tag (EST) library by searching for putative human
MRP2 sequences using the homologous 5'-proximal sequence of
rat mrp2 (Büchler et al., 1996). A clone (no.
124379; GenBank/EBI Data Bank no. R02250) was found to share an 80.7%
identity with the rat mrp2 sequence. With the assumption that this sequence was the 5' part of human MRP2, we
designed the primer hcfor5' in front of the ATG start codon. The PCR
was run at a denaturing temperature of 94°C for 1 min, at an
annealing temperature of 50°C for 1 min, and at an elongation
temperature of 72°C for 3 min, for a total of 35 cycles. The reaction
was completed by a 10-min incubation at 72°C. The amplified fragment was cloned into the vector pCR2.1 (Invitrogen, NV Leek, the
Netherlands). The resulting clone pMRP2.5' and the clone pMRP2.3' were
sequenced by the dideoxynucleotide chain termination method of Sanger
et al. (1980) using the T7 sequencing kit of Pharmacia Biotech
(Freiburg, Germany) and [-35S]dATP (DuPont/NEN). For
the construction of the full-length MRP2 cDNA, the clone
pMRP2.3' was digested with NotI and SpeI
and ligated with the 5' 2900-bp fragment obtained by digesting the
clone pMRP2.5' with NotI and SpeI. This
full-length human MRP2 cDNA is available under the
GenBank/EBI Data Bank accession no. X96395.
37 to 4861) was subcloned between the NotI and
ApaI restriction sites of the mammalian expression vector
pcDNA3.1(+) (Invitrogen). The 4.9-kb (bases 49 to 4839) cDNA fragment
of rat mrp2 (Büchler et al., 1996) was inserted into
the mammalian expression vector pcDNA3 (Invitrogen) between the
restriction sites EcoRI and XhoI. Successful
subcloning was verified by restriction map analysis and DNA sequencing.
Both human MRP2 and rat mrp2 were, thus, under the control of the cytomegalovirus promoter and enhancer elements.
Cell Culture and Transfection.
MDCK II cells were obtained
from Dr. K. Simons (European Molecular Biology Laboratory, Heidelberg,
Germany). HEK-293 cells were obtained from the American Type Culture
Collection (Manassas, VA). MDCK II and HEK-293 cells were cultured in
minimum essential medium containing 5 and 10% fetal bovine serum,
respectively, supplemented with L-glutamine (2 mM),
penicillin (100 U/ml), and streptomycin (100 µg/ml; 0.17 mM). The
parental vector pcDNA3.1 or the constructs described above were
transfected into the cells by electroporation (280 V; 1050 µF;
automatic pulse length). After transfection, cells were selected with
600 µg/ml (0.8 mM) G418 for 2 to 3 weeks. G418-resistant clones were
screened for human MRP2 or rat Mrp2 expression by immunoblot analysis
and immunofluorescence microscopy. Expression of MRP2 and Mrp2 in
positive clones was further enhanced by culturing the cells with sodium
butyrate or trichostatin A, both of which are inhibitors of histone
deacetylase and known to activate viral promoter and enhancer elements
(Chen et al., 1997).
Preparation of Membrane Vesicles and Crude Membrane
Fractions.
Inside-out membrane vesicles from transfected HEK-293
and MDCK II cells were prepared as described previously (Keppler et al., 1998). Membrane vesicles were frozen and stored in liquid nitrogen. Crude membrane fractions were prepared as follows. Cells were
disrupted by sonication in hypotonic buffer (1 mM EDTA, 5 mM
sodium/potassium phosphate; pH 7.0). After centrifugation
(100,000g; 4°C; 45 min) pellets were resuspended in Tris
buffer (50 mM; pH 7.4). Crude membrane fractions were frozen and stored
at 20°C. All membranes were prepared in the presence of proteinase
inhibitors (0.3 µM aprotinin, 1.0 µM leupeptin, 0.1 mM
phenylmethylsulfonyl fluoride, and 1.0 µM pepstatin).
Immunoblot Analysis. Membrane fractions were diluted with sample buffer and incubated at 37°C for 30 min before separation on 5% stacking and 7.5% resolving polyacrylamide gels. Immunoblotting was performed with a tank blotting system from Bio-Rad (Munich, Germany) and enhanced chemiluminescence detection (Amersham-Buchler, Braunschweig, Germany). Primary antibodies were diluted in TTBS [10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20] to the following final concentrations: EAG5, 1:5,000; EAG15, 1:10,000. The secondary antibody was a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) used at a 1:2000 dilution.
Northern Blot Analysis.
Total RNA was isolated from
transfected HEK-293 cells with a RNA-Clean kit (Angewandte
Gentechnologie Systeme, Heidelberg, Germany). Total RNA samples
(20 µg each) were separated by formaldehyde-agarose gel
electrophoresis and transferred onto Duralon UV membranes (Stratagene)
as described previously (Büchler et al., 1996). Sample loading
and efficiency of transfer were determined by staining of the membranes
with ethidium bromide before prehybridization. A 347-bp (Mayer et al.,
1995) and a 585-bp (Büchler et al., 1996) cDNA probe were used to
detect rat mrp2 and human MRP2, respectively. Autoradiographs were exposed for 2 days.
Vesicle Transport Studies.
Transport of 100 nM
[3H]LTC4 (0.17 TBq/mmol)
or 1.5 µM 17-glucuronosyl [3H]estradiol
(0.20 TBq/mmol) into membrane vesicles was measured by the rapid
filtration method (Keppler et al., 1998). Briefly, membrane vesicles
(20 µg of protein) were incubated in the presence of 4 mM ATP, 10 mM
creatine phosphate, 100 µg/ml creatine kinase, and labeled substrate
in an incubation buffer (250 mM sucrose, 10 mM Tris/HCl; pH 7.4) at
37°C. The final volume was 55 µl. Aliquots (15 µl) were taken at
the indicated time points, diluted in 1 ml of ice-cold incubation
buffer, and immediately filtered through presoaked nitrocellulose
membrane (0.2-µm pore size). Filters were rinsed twice with 5 ml of
incubation buffer, dissolved in liquid scintillation fluid, and
counted for radioactivity. In control experiments, ATP was replaced by
an equal concentration of 5'-AMP. ATP-dependent transport was
calculated by subtracting values obtained in the presence of 5'-AMP
from those in the presence of ATP. For determination of kinetic
constants, initial transport rates were measured at five different
substrate concentrations (25-1000 nM for LTC4
and 0.5-8 µM for 17-glucuronosyl estradiol). The concentrations
of the labeled substrate were kept constant and varying concentrations
of unlabeled substrate were added. Km
values are determined as the substrate concentration at half-maximal velocity of transport under the experimental conditions described above. Similar results were obtained by direct curve-fitting to the
Michaelis-Menten equation and by the use of double-reciprocal plots
according to Lineweaver and Burk (1934).
Immunofluorescence and Confocal Laser Scanning Microscopy.
For immunolocalization of MRP2, transfected MDCK cells were grown on
Transwell membrane inserts (pore size 3 µm; Costar, Cambridge, MA)
for 7 days. Butyrate, at a final concentration of 2 mM, was added to
the culture medium 24 h before use of the cells. Cells were fixed
with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton
X-100 in PBS containing 5% fetal bovine serum. Membranes were
incubated with polyclonal antibody EAG5 or EAG15 (both diluted 1:25
with PBS) at room temperature for 30 min. After three washes with PBS,
membranes were reincubated with Cy2-conjugated goat anti-rabbit IgG
(Dianova, Hamburg, Germany; dilution 1:200 in PBS). Nuclei were stained
with 0.2 µg/ml propidium iodide added to the solution of the
secondary antibody. Confocal laser scanning fluorescence microscopy was
performed under conditions described recently (Mayer et al., 1995;
Büchler et al., 1996) with an LSM 410 apparatus (Carl Zeiss,
Jena, Germany). For localization of the amino terminus of rat Mrp2,
nonpermeabilized cells were used for immunofluorescence studies.
Briefly, MDCK transfectants grown on glass coverslips were either first
fixed with 4% paraformaldehyde/PBS and then incubated with the MDK
antibody (1:25) at room temperature for 30 min, or first incubated with
MDK antibody (1:25) at 4°C for 1 h and then fixed with 4%
paraformaldehyde/PBS. After permeabilization with 1% Triton X-100,
coverslips were reincubated with Cy2-conjugated goat anti-rabbit IgG
(1:200) at room temperature for 30 min.
Cytotoxicity Assays.
Sensitivity to cytotoxic drugs was
determined using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell
viability assay according to the method of Mosmann (1983). Briefly,
MDCK or HEK-293 cells were seeded in 96-well plates at a density of
5 × 104 and 1.5 × 104 cells per well, respectively. The histone
deacetylase inhibitor trichostatin A (125 nM) was added to enhance the
expression of recombinant MRP2 in MDCK cells. Drugs were added 24 h after seeding. After 3 days of incubation under normal culture
conditions, MTT was added at a final concentration of 250 µg/ml (0.6 mM). In the case of vincristine, minimum essential medium without
riboflavin was used to avoid radical-mediated degradation of
vincristine (Granzow et al., 1995). The IC50
value was defined as the drug concentration required to reduce
cell survival, as determined by the relative absorbance of reduced MTT,
to 50%. Relative resistance factors (RRs) were calculated by dividing
the IC50 value of cells transfected with MRP2
expression vectors by the IC50 of cells transfected with the control vector. The effect of MDR1
P-glycoprotein modulator LY335979 (Dantzig et al., 1996), at
a concentration of 0.25 µM, and the glutathione synthesis inhibitor
BSO (20 µM) on the sensitivity of transfectants to the cytostatic
agents was studied by including them in the cell viability assays.
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Results |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Cloning of Human MRP2.
Human
MRP2 cDNA was obtained after screening a 
-ZAP cDNA
library combined with a reverse transcription-PCR amplification of the
5' part. The full-length cDNA of human MRP2 contains a single open reading frame of 4635 bp that encodes a predicted protein
of 1545 amino acids. The deduced amino acid sequence shares a 78%
identity with rat Mrp2 (Büchler et al., 1996; Paulusma et al.,
1996; Ito et al., 1997) and an 82% identity with rabbit Mrp2 (van
Aubel et al., 1998). The molecular mass of unglycosylated human MRP2 is
174,090 Da. After an alignment of the three currently known orthologs
of MRP2, 13 transmembrane segments were predicted by a transmembrane
topology analysis program (TMAP) (Persson and Argos, 1994). Four
transmembrane segments were predicted between both nucleotide-binding
domains and nine transmembrane segments between the amino terminus and
the first nucleotide-binding domain. This topology prediction suggests
an extracellular localization of the amino terminus. A similar topology
with 13 transmembrane segments was predicted by the TMAP program
(Persson and Argos, 1994) when human MRP1 and mouse Mrp1 were included
in the alignment, together with human MRP2, rabbit Mrp2, and rat Mrp2.
However, epitope insertion studies favor six transmembrane segments
between both nucleotide-binding domains of MRP1 (Kast and Gros, 1998).
Expression of MRP2 in HEK and MDCK Cells.
HEK-293 and MDCK II
cells were stably transfected with the vector constructs containing
human MRP2 or rat mrp2, as well as the parental
vector pcDNA3.1(+). G418-resistant clones were screened for MRP2 and
Mrp2 expression. The expression level of MRP2 and Mrp2 in the positive
clones was markedly enhanced by culturing the transfected cells with
sodium butyrate for 24 h. As shown in Fig.
1A, Mrp2 expression in HEK-Mrp2 cells
(HEK-293 cells stably transfected with rat mrp2) was
increased by sodium butyrate in a concentration-dependent manner. No
cytotoxicity of butyrate was observed at the optimal concentration of 5 mM. The maximal effect of butyrate was achieved at a concentration of
10 mM in MDCK transfectants. However, concentrations above 2 mM caused visible cytotoxicity in this cell line. Therefore, HEK-293
transfectants and MDCK transfectants were cultured with 5 mM and 2 mM
butyrate, respectively, for 24 h before use in immunofluorescence
studies. The expression of both MRP2 and Mrp2 was stable when G418 was omitted from the culture medium.
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; Jedlitschky et al., 1997; Evers et
al., 1998). Deglycosylation indicated that the MRP2 overexpressed in
HEK-293 cells was glycosylated to the same extent as in human liver
(data not shown). The expression of MRP2 was much higher in HEK-293
transfectants than in MDCK transfectants as determined by immunoblot
analysis. It is noticeable that endogenous MRP2 was not detectable in
HEK-Co cells transfected with the vector alone (Fig. 1B). Moreover,
MRP1 was not detected in HEK-293 cells by immunoblot analysis using the
monoclonal antibody QCRL-1 directed against MRP1 (not shown).
The expression of MRP2 and mrp2 in HEK-293
transfectants was further verified by Northern blotting analysis
performed on 20 µg of total RNA. Only one mRNA species with a length
of about 5.3 kb was detected in HEK-MRP2 cells using the 585-bp cDNA
fragment (Büchler et al., 1996) as probe for MRP2
(Fig. 2A). In HEK-Co cells, no signal was
detected with this cDNA probe. A 5.3-kb mRNA was also found in HEK-Mrp2
cells by using the 347-bp cDNA fragment (Mayer et al., 1995) as a probe
for mrp2 (Fig. 2B).
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Localization of MRP2 in Transfectants.
Indirect
immunofluorescence with HEK-MRP2 cells with the antibody EAG5 indicated
that MRP2 was localized to a major portion to intracellular membranes
(not shown). However, in polarized MDCK transfectants, the localization
of recombinant MRP2 was different. Immunofluorescence and confocal
laser scanning microscopy using the antibodies EAG5 or EAG15 revealed a
homogenous plasma membrane staining in MDCK transfectants expressing
MRP2 or Mrp2 (Fig. 3, A and C). Vertical
sections showed intense green fluorescence for MRP2 and Mrp2 on the
apical membrane (Fig. 3, B and D). Under the same conditions, no
specific staining was observed in MDCK cells transfected only with the
parental vector pcDNA3.1(+).
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Transport Studies with Membrane Vesicles.
Membrane vesicles
were prepared from HEK-293 and MDCK II transfectants cultured for
24 h in the presence of 5 mM and 10 mM butyrate, respectively.
These relatively high concentrations of butyrate induced maximum levels
of MRP2 in these cells. Because of the higher expression level of human
MRP2 and rat Mrp2 in HEK-293 transfectants as compared with MDCK
transfectants, membrane vesicles from the former were preferred for
studies on transport function. These MRP2-containing membrane vesicles
of HEK-293 transfectants, which are derived, in part, from
intracellular membrane vesicles, were most useful for transport
studies. [3H]LTC4 was
transported ATP-dependently into membrane vesicles from HEK-MRP2 cells
at a rate of 48 ± 9 pmol · min
1
· mg protein1 at 100 nM
LTC4 (mean ± S.D.; n = 5;
Fig. 4). In membrane vesicles from rat
mrp2-transfected HEK-293 and MDCK cells, transport rates at
this concentration of
[3H]LTC4 were 31 ± 3 and 24 ± 2 pmol · min1 · mg
protein1, respectively. For the determination
of kinetic constants, human MRP2-mediated transport was calculated by
subtracting ATP-dependent transport into membrane vesicles from HEK-Co
cells from those measured with membrane vesicles from HEK-MRP2 cells.
An apparent Km of 1.0 ± 0.1 µM
(n = 4) was determined for human MRP2 by
double-reciprocal plots (Table 1).
ATP-dependent transport of
[3H]LTC4 was also
detectable in membrane vesicles from HEK-Co cells (Fig. 4B), but the
transport rate (11 ± 4 pmol · min1 · mg protein1; n = 3) at 100 nM
LTC4 was only 22% of that measured with membrane vesicles from HEK-MRP2. The endogenous transport system has a Km of 0.28 ± 0.09 µM
(n = 4). Membrane vesicles from HEK-MRP2 were also
active in the ATP-dependent transport of 17-glucuronosyl [3H]estradiol (Fig.
5). An apparent
Km of 7.2 ± 0.7 µM
(n = 4) was obtained (Table 1). The transport of
17-glucuronosyl [3H]estradiol into membrane
vesicles from HEK-Co cells was below 10% of that measured with
membrane vesicles from HEK-MRP2 cells (Fig. 5B). The
Km value for 17-glucuronosyl
[3H]estradiol in these control transfectants
was 2.8 ± 0.3 µM (n = 4). The kinetic constants
were also determined for rat Mrp2 and compared with those for human
MRP2 and with the constants determined earlier for human MRP1 (Table
1).
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Drug Resistance Conferred by MRP2.
MDCK-transfectants were
studied at confluency for MTT assays when a polarized monolayer had
been formed, which is a prerequisite for the apical localization of
MRP2. Under this condition, both rat and human MRP2 conferred
significant resistance to etoposide and vincristine (Fig.
6; Table
2). It was reported that MDCK cells
express endogenous MDR1 P-glycoprotein (Horio et al., 1989). Our
immunoblot analysis with the monoclonal antibody C219 showed that the
expression level of a 170-kDa protein is similar in all transfectants
(not shown). The interference of MDR1 P-glycoprotein with the
cytotoxicity assays was, therefore, suppressed by the specific MDR1
modulator LY335979 (Dantzig et al., 1996; Shepard et al., 1998).
LY335979, at a concentration of 0.25 µM, sensitized all
MDCK-transfectants to etoposide and vincristine without lowering the
relative resistance conferred by MRP2 (Fig. 6A; Table 2), indicating
that resistance was actually conferred by MRP2 transfected into these
cells. The effect of 20 µM BSO, an inhibitor of glutathione synthesis
that has been shown to inhibit MRP1-mediated multidrug resistance
(Zaman et al., 1995), was also determined. This concentration of BSO
did not inhibit MRP1- or MRP2-mediated ATP-dependent transport of
LTC4. Coincubation with 20 µM BSO in the
cytotoxicity assays reduced the level of etoposide resistance conferred
by MRP2 from 5-fold to 2-fold (Fig. 6; Table 2). Human recombinant MRP2
conferred low-level resistance to epirubicin (1.9-fold;
P < .01) and to doxorubicin (1.5-fold;
P < .01) in these confluent MDCK cells. In these
cells, MRP2-mediated resistance to cisplatin could not be detected.
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Summary
Introduction
Experimental Procedures
Results
Discussion
References
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Human MRP2 and its orthologs from rat and rabbit were cloned and
sequenced recently (Mayer et al., 1995; Büchler et al., 1996;
Paulusma et al., 1996, 1997; Taniguchi et al., 1996; Ito et al., 1997;
Madon et al., 1997; van Aubel et al., 1998; Evers et al., 1998). The
function, particularly of rat Mrp2, as a conjugate-transporting ATPase
has been deduced predominantly from studies with hepatocyte canalicular
membrane vesicles from normal and Mrp2-deficient animals (Ishikawa et
al., 1990; Oude Elferink et al., 1995; Jedlitschky et al., 1997).
Stable transfection of MRP2 and its expression at a high level are
prerequisites for a detailed functional characterization of this
transport protein, which plays an important role in hepatobiliary and
renal excretion (Keppler and König, 1997). Stable transfectants expressing human MRP2 (Figs. 1-5) are also of considerable interest for studies on multidrug resistance conferred by this isoform of MRP1.
In this study, we established transfected HEK-293 and MDCK cell lines
permanently expressing recombinant human MRP2 or rat Mrp2 (Figs. 1 and
2). Expression of recombinant MRP2, driven by the cytomegalovirus
promoter, was markedly enhanced by the addition of inhibitors of
histone deacetylase, such as butyrate and trichostatin A, to the cell
culture (Fig. 1A).
Our previous immunofluorescence studies have demonstrated that human
and rat MRP2 are localized under physiological conditions to the apical
membrane domain of polarized cells, including hepatocytes and proximal
tubule epithelia of the kidney (Büchler et al., 1996; Keppler and
Kartenbeck, 1996; Schaub et al., 1997). In contrast, the major portion
of recombinant rat and human MRP2 expressed in this study in HEK-293
cells was not sorted to the plasma membrane but retained in a fully
glycosylated and functionally active form on intracellular membranes
(Fig. 1B; Table 1). HEK-293 cells were reported to be epithelial in
nature (Chan et al., 1997); however, these cells did not form a
distinct polarized monolayer under our culture conditions. It is
unclear whether the incomplete sorting of rat and human MRP2 to the
plasma membrane of HEK-293 cells is due to missing proteins required
for sorting or a result of an incomplete polarity of the HEK-293 cells
in culture. Importantly, the intracellular membranes containing
recombinant MRP2 formed vesicles with a high specific activity of
ATP-dependent transport of labeled substrates (Table 1; Figs. 4 and 5).
However, extensive sorting of human and rat MRP2 to the apical plasma
membrane domain was observed in polarized MDCK cells stably transfected
with the respective vector constructs (Fig. 3). This observation is
consistent with a recent study on MRP2 in this cell line (Evers et al.,
1998). Cultures of MDCK cells were shown to form polarized epithelial monolayers with the apical membrane oriented to the medium (Richardson et al., 1981). The apical localization of recombinant rat and human
MRP2 demonstrated by confocal laser scanning microscopy (Fig. 3)
differs from the sorting and localization of recombinant human MRP1 to
the lateral membrane of transfected kidney cells (Evers et al., 1996).
It will be of interest to define the amino acid sequences determining
the differential sorting of MRP1 and its isoform, MRP2, in polarized cells.
The membrane topology of rat Mrp2, as predicted by the TMAP program
(Persson and Argos, 1994), indicated an extracellular localization of
the amino terminus (Büchler et al., 1996; Keppler and
König, 1997). An extracellular amino terminus of MRP1 was recognized recently on the basis of site-directed mutagenesis studies
(Hipfner et al., 1997; Kast and Gros, 1997). In the present study, we
used a polyclonal antibody raised against the amino-terminal 25 amino
acids of rat Mrp2 to test the predicted membrane topology in
nonpermeabilized MDCK cells expressing recombinant Mrp2 (Fig. 3E).
Staining of the apical membrane was only observed with the MDK antibody
directed against the amino terminus and not with the EAG15 antibody
directed against the carboxyl terminus of Mrp2 (Fig. 3G). This result
provides direct evidence for the extracellular localization of the
amino terminus of Mrp2. Together with the recent analysis of the
topology of MRP1 (Hipfner et al., 1997; Kast and Gros, 1997), these
analyses suggest that an extracellular amino terminus may be a common
feature of the members of the MRP family.
The substrate specificity of human MRP1 and MRP2 has been considered to
be quite similar (Keppler et al., 1998), although some kinetic
differences were detected recently (Jedlitschky et al., 1997). The
availability of membrane vesicles rich in recombinant human MRP2 (Fig.
1B) now has enabled a comparison with recombinant human MRP1 with
respect to the substrate specificity and to the kinetic properties of
ATP-dependent transport (Table 1). We studied two established
endogenous substrates for ATP-dependent transport by members of the MRP
family, the glutathione S-conjugate,
LTC4, and the glucuronate conjugate,
17-glucuronosyl estradiol. With both substrates, a high maximal
velocity of transport into membrane vesicles from
MRP2-transfected HEK-293 cells relative to the membrane vesicles from MRP1-transfected HeLa cells was observed. As
indicated by the higher
Vmax/Km ratio
in the same membrane vesicle preparation, LTC4
was a better substrate than 17-glucuronosyl estradiol, both for MRP2
and for MRP1 (Table 1). The concentration of LTC4
required for half-maximal transport velocity was 10-fold higher for
recombinant human MRP2 than for MRP1. The concentration of
17-glucuronosyl estradiol at half-maximal transport velocity was
4.8-fold higher for recombinant MRP2 than for MRP1. The kinetic
constants for recombinant rat Mrp2 were not significantly different
from those for human MRP2 (Table 1) although both transporter orthologs share only 78% identical amino acids (Keppler and König, 1997). The Km values of rat and human MRP2 for
LTC4 (Table 1) were also very similar to the
Km in rat hepatocyte canalicular membrane vesicles (i.e., 1.3 µM) when the same methodology for measurement of
ATP-dependent membrane transport was used (Leier et al., 1994; Jedlitschky et al., 1996). This indicates that differences in membrane
composition between the HEK-293 cells and rat hepatocytes did not
result in a detectable effect on the kinetic properties of Mrp2. We
conclude that the intracellular membrane vesicles from our stably
transfected HEK-293 cells are well suited to characterize the
functional properties of rat and human MRP2, although they lack the
complete sorting of MRP2 to the apical membrane domain that is observed
in the transfected MDCK cells.
MRP1 has been shown to confer multidrug resistance in transfected cell
lines (Grant et al., 1994; Zaman et al., 1995). MRP2 differs markedly
from MRP1 by its apical localization and its expression in polarized
cells (Büchler et al., 1996; Evers et al., 1996, 1998; Keppler
and Kartenbeck, 1996; Schaub et al., 1997), but not so much in its
substrate specificity (Table 1). This suggests that drug conjugates
(Jedlitschky et al., 1996) and drug complexes with glutathione (Loe et
al., 1996) may be substrates for both MRP isoforms. It is in line with
this view that a reduction of the MRP2 protein by expression of an
antisense construct increased the sensitivity of hepatic cancer cells
to several anticancer agents and increased the cellular content of reduced glutathione (Koike et al., 1997). The results of cytotoxicity assays shown in this work provide the first direct evidence for the
ability of MRP2 to confer drug resistance. Despite the relatively low
sensitivity of MDCK control cells to etoposide
(IC50 = 163 µM) and vincristine
(IC50 = 8.2 µM), transfection with human or rat
MRP2 genes conferred additional resistance to these
cytotoxic drugs (Fig. 6; Table 2). Immunoblot analysis and
sensitization of the cells using the MDR1 modulator LY335979 indicated
that the endogenous canine MDR1 P-glycoprotein in MDCK cells is one of
the reasons for the low sensitivity of these cells to these cytotoxic
agents. The resistance factor conferred by rat or human MRP2 was not
significantly affected by LY335979 (Table 2). Sensitization of
MRP2-transfected MDCK cells by BSO indicates that the drug resistance conferred by MRP2 is related to the intracellular GSH level.
Vincristine has been shown to be a substrate for MRP1 only in the
presence of GSH (Loe et al., 1996). However, it is unknown at present
whether etoposide and vincristine are transported by MRP2 as a GSH complex.
Resistance of tumor cells to cisplatin has been associated with MRP2
overexpression (Taniguchi et al., 1996; Koike et al., 1997; Kool et
al., 1997). In our present study, we provide direct evidence that MRP2
overexpression can confer resistance to cisplatin in transfected
HEK-293 cells (Table 3). Interestingly, coincubation with 50 µM BSO
in the cytotoxicity assays reduced resistance to cisplatin from
10.0-fold to 2.9-fold (data not shown). As the major proportion of
recombinant MRP2 is localized to intracellular membranes in HEK-293
cells, rather than to the plasma membrane, one may consider the
possibility that cisplatin or its glutathione conjugate is first
sequestered in intracellular vesicles and subsequently released into
the extracellular space. Alternatively or additionally, the fraction of
recombinant MRP2 in the plasma membrane is sufficient to pump out the
cisplatin glutathione conjugate. These possibilities are currently
under investigation in our laboratory.
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Acknowledgments |
|---|
We thank Dr. Wolfgang Hagmann and Dr. H.-R. Rackwitz for their
support in the generation of the MDK antibody, Dr. Roger Deeley and Dr.
Susan Cole for providing their MRP1-transfected HeLa cells (Grant et
al., 1994), and Johanna Hummel-Eisenbeiss for her contributions to the
measurements of ATP-dependent transport.
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Footnotes |
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Received September 25, 1998; Accepted February 19, 1999
This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Grants SFB 352/B3 and SFB 601/A2); the Forschungsschwerpunkt Transplantation, Heidelberg; and the Tumorzentrum, Heidelberg/Mannheim.
1 The nucleotide sequences reported in this paper have the GenBank/EBI Data Bank accession numbers X96395 (human MRP2), X96393 (rat Mrp2), and X90643 (347 bp fragment of rat Mrp2).
Send reprint requests to: Dr. Dietrich Keppler, Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany. E-mail: d.keppler{at}dkfz-heidelberg.de
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Abbreviations |
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MRP, human multidrug resistance protein (symbol ABCC1);
BSO, buthionine sulfoximine;
HEK-293, cell line derived from
human embryonic kidney;
LTC4, leukotriene C4;
LY335979, 4-(1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl)--[(5-quinolinyloxy)methyl]-1-piperazineethanol;
MDCK II, cell line derived from Madin-Darby canine kidney cells;
MRP2, human apical multidrug resistance protein (symbol ABCC2);
Mrp2, rat
apical multidrug resistance protein;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
PCR, polymerase chain reaction;
TMAP, transmembrane topology analysis
program.
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References |
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Top
Summary
Introduction
Experimental Procedures
Results
Discussion
References
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H. Lou, M. Ookhtens, A. Stolz, and N. Kaplowitz Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells Am J Physiol Gastrointest Liver Physiol, December 1, 2003; 285(6): G1335 - G1344. [Abstract] [Full Text] [PDF]
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J. B. Vaidyanathan and T. Walle Cellular Uptake and Efflux of the Tea Flavonoid (-)Epicatechin-3-gallate in the Human Intestinal Cell Line Caco-2 J. Pharmacol. Exp. Ther., November 1, 2003; 307(2): 745 - 752. [Abstract] [Full Text] [PDF]
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 G. Reid, P. Wielinga, N. Zelcer, I. van der Heijden, A. Kuil, M. de Haas, J. Wijnholds, and P. Borst The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs PNAS, August 5, 2003; 100(16): 9244 - 9249. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, R. W. Robey, M. G. Belinsky, I. Shchaveleva, X.-Q. Ren, Y. Sugimoto, D. D. Ross, S. E. Bates, and G. D. Kruh Transport of Methotrexate, Methotrexate Polyglutamates, and 17{beta}-Estradiol 17-({beta}-D-glucuronide) by ABCG2: Effects of Acquired Mutations at R482 on Methotrexate Transport Cancer Res., July 15, 2003; 63(14): 4048 - 4054. [Abstract] [Full Text] [PDF]
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A. C. Lockhart, R. G. Tirona, and R. B. Kim Pharmacogenetics of ATP-binding Cassette Transporters in Cancer and Chemotherapy Mol. Cancer Ther., July 1, 2003; 2(7): 685 - 698. [Abstract] [Full Text] [PDF]
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 A. Bodo, E. Bakos, F. Szeri, A. Varadi, and B. Sarkadi Differential Modulation of the Human Liver Conjugate Transporters MRP2 and MRP3 by Bile Acids and Organic Anions J. Biol. Chem., June 20, 2003; 278(26): 23529 - 23537. [Abstract] [Full Text] [PDF]
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N. Zelcer, M. T. Huisman, G. Reid, P. Wielinga, P. Breedveld, A. Kuil, P. Knipscheer, J. H. M. Schellens, A. H. Schinkel, and P. Borst Evidence for Two Interacting Ligand Binding Sites in Human Multidrug Resistance Protein 2 (ATP Binding Cassette C2) J. Biol. Chem., June 20, 2003; 278(26): 23538 - 23544. [Abstract] [Full Text] [PDF]
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T. Konno, T. Ebihara, K. Hisaeda, T. Uchiumi, T. Nakamura, T. Shirakusa, M. Kuwano, and M. Wada Identification of Domains Participating in the Substrate Specificity and Subcellular Localization of the Multidrug Resistance Proteins MRP1 and MRP2 J. Biol. Chem., June 13, 2003; 278(25): 22908 - 22917. [Abstract] [Full Text] [PDF]
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 K. Hidemura, Y. L. Zhao, K. Ito, A. Nakao, Y. Tatsumi, H. Kanazawa, K. Takagi, M. Ohta, and T. Hasegawa Shiga-Like Toxin II Impairs Hepatobiliary Transport of Doxorubicin in Rats by Down-Regulation of Hepatic P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2 Antimicrob. Agents Chemother., May 1, 2003; 47(5): 1636 - 1642. [Abstract] [Full Text] [PDF]
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M. Trauner and J. L. Boyer Bile Salt Transporters: Molecular Characterization, Function, and Regulation Physiol Rev, April 1, 2003; 83(2): 633 - 671. [Abstract] [Full Text] [PDF]
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O. Briz, R. I. R. Macias, M. Vallejo, A. Silva, M. A. Serrano, and J. J. G. Marin Usefulness of Liposomes Loaded with Cytostatic Bile Acid Derivatives to Circumvent Chemotherapy Resistance of Enterohepatic Tumors Mol. Pharmacol., March 1, 2003; 63(3): 742 - 750. [Abstract] [Full Text] [PDF]
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 K. Terasaka, N. Shitan, F. Sato, F. Maniwa, K. Ueda, and K. Yazaki Application of Vanadate-Induced Nucleotide Trapping to Plant Cells for Detection of ABC Proteins Plant Cell Physiol., February 15, 2003; 44(2): 198 - 200. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, E. Hopper-Borge, M. G. Belinsky, I. Shchaveleva, E. Kotova, and G. D. Kruh Characterization of the Transport Properties of Human Multidrug Resistance Protein 7 (MRP7, ABCC10) Mol. Pharmacol., February 1, 2003; 63(2): 351 - 358. [Abstract] [Full Text] [PDF]
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V. Keitel, A. T. Nies, M. Brom, J. Hummel-Eisenbeiss, H. Spring, and D. Keppler A common Dubin-Johnson syndrome mutation impairs protein maturation and transport activity of MRP2 (ABCC2) Am J Physiol Gastrointest Liver Physiol, January 1, 2003; 284(1): G165 - G174. [Abstract] [Full Text] [PDF]
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 P. T. Ronaldson and R. Bendayan Renal Drug Transport and Drug-Drug Interactions Journal of Pharmacy Practice, December 1, 2002; 15(6): 490 - 503. [Abstract] [PDF]
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 E. H. Rubin, D. P. de Alwis, I. Pouliquen, L. Green, P. Marder, Y. Lin, R. Musanti, S. L. Grospe, S. L. Smith, D. L. Toppmeyer, et al. A Phase I Trial of a Potent P-Glycoprotein Inhibitor, Zosuquidar.3HCl Trihydrochloride (LY335979), Administered Orally in Combination with Doxorubicin in Patients with Advanced Malignancies Clin. Cancer Res., December 1, 2002; 8(12): 3710 - 3717. [Abstract] [Full Text] [PDF]
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Y. Ohishi, Y. Oda, T. Uchiumi, H. Kobayashi, T. Hirakawa, S. Miyamoto, N. Kinukawa, H. Nakano, M. Kuwano, and M. Tsuneyoshi ATP-binding Cassette Superfamily Transporter Gene Expression in Human Primary Ovarian Carcinoma Clin. Cancer Res., December 1, 2002; 8(12): 3767 - 3775. [Abstract] [Full Text] [PDF]
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 C.M.F. Kruijtzer, J.H. Beijnen, and J.H.M. Schellens Improvement of Oral Drug Treatment by Temporary Inhibition of Drug Transporters and/or Cytochrome P450 in the Gastrointestinal Tract and Liver: An Overview Oncologist, December 1, 2002; 7(6): 516 - 530. [Abstract] [Full Text] [PDF]
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M. G. Belinsky, Z.-S. Chen, I. Shchaveleva, H. Zeng, and G. D. Kruh Characterization of the Drug Resistance and Transport Properties of Multidrug Resistance Protein 6 (MRP6, ABCC6) Cancer Res., November 1, 2002; 62(21): 6172 - 6177. [Abstract] [Full Text] [PDF]
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C. Michalski, Y. Cui, A. T. Nies, A. K. Nuessler, P. Neuhaus, U. M. Zanger, K. Klein, M. Eichelbaum, D. Keppler, and J. Konig A Naturally Occurring Mutation in the SLC21A6 Gene Causing Impaired Membrane Localization of the Hepatocyte Uptake Transporter J. Biol. Chem., November 1, 2002; 277(45): 43058 - 43063. [Abstract] [Full Text] [PDF]
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A. Haimeur, R. G. Deeley, and S. P. C. Cole Charged Amino Acids in the Sixth Transmembrane Helix of Multidrug Resistance Protein 1 (MRP1/ABCC1) Are Critical Determinants of Transport Activity J. Biol. Chem., October 25, 2002; 277(44): 41326 - 41333. [Abstract] [Full Text] [PDF]
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G. Hartmann, A. K. Y. Cheung, and M. Piquette-Miller Inflammatory Cytokines, but Not Bile Acids, Regulate Expression of Murine Hepatic Anion Transporters in Endotoxemia J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 273 - 281. [Abstract] [Full Text] [PDF]
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Z. P. Lin, D. R. Johnson, R. A. Finch, M. G. Belinsky, G. D. Kruh, and A. C. Sartorelli Comparative Study of the Importance of Multidrug Resistance-associated Protein 1 and P-Glycoprotein to Drug Sensitivity in Immortalized Mouse Embryonic Fibroblasts Mol. Cancer Ther., October 1, 2002; 1(12): 1105 - 1114. [Abstract] [Full Text] [PDF]
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Y.-M. Qian, C. E. Grant, C. J. Westlake, D.-W. Zhang, P. A. Lander, R. L. Shepard, A. H. Dantzig, S. P. C. Cole, and R. G. Deeley Photolabeling of Human and Murine Multidrug Resistance Protein 1 with the High Affinity Inhibitor [125I]LY475776 and Azidophenacyl-[35S]Glutathione J. Biol. Chem., September 13, 2002; 277(38): 35225 - 35231. [Abstract] [Full Text] [PDF]
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S. B. M. Fernandez, Z. Hollo, A. Kern, E. Bakos, P. A. Fischer, P. Borst, and R. Evers Role of the N-terminal Transmembrane Region of the Multidrug Resistance Protein MRP2 in Routing to the Apical Membrane in MDCKII Cells J. Biol. Chem., August 16, 2002; 277(34): 31048 - 31055. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, K. Lee, S. Walther, R. B. Raftogianis, M. Kuwano, H. Zeng, and G. D. Kruh Analysis of Methotrexate and Folate Transport by Multidrug Resistance Protein 4 (ABCC4): MRP4 Is a Component of the Methotrexate Efflux System Cancer Res., June 1, 2002; 62(11): 3144 - 3150. [Abstract] [Full Text] [PDF]
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D.-W. Zhang, S. P. C. Cole, and R. G. Deeley Determinants of the Substrate Specificity of Multidrug Resistance Protein 1. ROLE OF AMINO ACID RESIDUES WITH HYDROGEN BONDING POTENTIAL IN PREDICTED TRANSMEMBRANE HELIX 17 J. Biol. Chem., May 31, 2002; 277(23): 20934 - 20941. [Abstract] [Full Text] [PDF]
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A. Ilias, Z. Urban, T. L. Seidl, O. Le Saux, E. Sinko, C. D. Boyd, B. Sarkadi, and A. Varadi Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6) J. Biol. Chem., May 3, 2002; 277(19): 16860 - 16867. [Abstract] [Full Text] [PDF]
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A. Guo, W. Marinaro, P. Hu, and P. J. Sinko Delineating the Contribution of Secretory Transporters in the Efflux of Etoposide Using Madin-Darby Canine Kidney (MDCK) Cells Overexpressing P-Glycoprotein (Pgp), Multidrug Resistance-Associated Protein (MRP1), and Canalicular Multispecific Organic Anion Transporter (cMOAT) Drug Metab. Dispos., April 1, 2002; 30(4): 457 - 463. [Abstract] [Full Text] [PDF]
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M. Sasaki, H. Suzuki, K. Ito, T. Abe, and Y. Sugiyama Transcellular Transport of Organic Anions Across a Double-transfected Madin-Darby Canine Kidney II Cell Monolayer Expressing Both Human Organic Anion-transporting Polypeptide (OATP2/SLC21A6) and Multidrug Resistance-associated Protein 2 (MRP2/ABCC2) J. Biol. Chem., February 15, 2002; 277(8): 6497 - 6503. [Abstract] [Full Text] [PDF]
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N. J. Cherrington, D. P. Hartley, N. Li, D. R. Johnson, and C. D. Klaassen Organ Distribution of Multidrug Resistance Proteins 1, 2, and 3 (Mrp1, 2, and 3) mRNA and Hepatic Induction of Mrp3 by Constitutive Androstane Receptor Activators in Rats J. Pharmacol. Exp. Ther., January 1, 2002; 300(1): 97 - 104. [Abstract] [Full Text] [PDF]
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Y. Cui, J. Konig, and D. Keppler Vectorial Transport by Double-Transfected Cells Expressing the Human Uptake Transporter SLC21A8 and the Apical Export Pump ABCC2 Mol. Pharmacol., November 1, 2001; 60(5): 934 - 943. [Abstract] [Full Text] [PDF]
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P. Duesberg, R. Stindl, and R. Hehlmann Origin of multidrug resistance in cells with and without multidrug resistance genes: Chromosome reassortments catalyzed by aneuploidy PNAS, September 5, 2001; (2001) 201398998. [Abstract] [Full Text] [PDF]
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A. Yamada, K. Kawano, M. Koga, T. Matsumoto, and K. Itoh Multidrug Resistance-associated Protein 3 Is a Tumor Rejection Antigen Recognized by HLA-A2402-restricted Cytotoxic T Lymphocytes Cancer Res., September 1, 2001; 61(17): 6459 - 6466. [Abstract] [Full Text] [PDF]
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L. C. Young, B. G. Campling, S. P. C. Cole, R. G. Deeley, and J. H. Gerlach Multidrug Resistance Proteins MRP3, MRP1, and MRP2 in Lung Cancer: Correlation of Protein Levels with Drug Response and Messenger RNA Levels Clin. Cancer Res., June 1, 2001; 7(6): 1798 - 1804. [Abstract] [Full Text] [PDF]
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K. Ito, H. Suzuki, and Y. Sugiyama Charged Amino Acids in the Transmembrane Domains Are Involved in the Determination of the Substrate Specificity of Rat Mrp2 Mol. Pharmacol., April 16, 2001; 59(5): 1077 - 1085. [Abstract] [Full Text]
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L. Huang and M. Vore Multidrug Resistance P-Glycoprotein 2 Is Essential for the Biliary Excretion of Indocyanine Green Drug Metab. Dispos., April 13, 2001; 29(5): 634 - 637. [Abstract] [Full Text]
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K. Zhang, M. Chew, E. B. Yang, K. P. Wong, and P. Mack Modulation of Cisplatin Cytotoxicity and Cisplatin-Induced DNA Cross-Links in HepG2 Cells by Regulation of Glutathione-Related Mechanisms Mol. Pharmacol., April 1, 2001; 59(4): 837 - 843. [Abstract] [Full Text]
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D. Laouari, R. Yang, C. Veau, I. Blanke, and G. Friedlander Two apical multidrug transporters, P-gp and MRP2, are differently altered in chronic renal failure Am J Physiol Renal Physiol, April 1, 2001; 280(4): F636 - F645. [Abstract] [Full Text] [PDF]
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 K. Lee, A. J. P. Klein-Szanto, and G. D. Kruh Analysis of the MRP4 Drug Resistance Profile in Transfected NIH3T3 Cells J Natl Cancer Inst, December 6, 2000; 92(23): 1934 - 1940. [Abstract] [Full Text] [PDF]
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H. Xiong, K. C. Turner, E. S. Ward, P. L. M. Jansen, and K. L. R. Brouwer Altered Hepatobiliary Disposition of Acetaminophen Glucuronide in Isolated Perfused Livers from Multidrug Resistance-Associated Protein 2-Deficient TR- Rats J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 512 - 518. [Abstract] [Full Text]
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 M. V. St-Pierre, M. A. Serrano, R. I. R. Macias, U. Dubs, M. Hoechli, U. Lauper, P. J. Meier, and J. J. G. Marin Expression of members of the multidrug resistance protein family in human term placenta Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2000; 279(4): R1495 - R1503. [Abstract] [Full Text] [PDF]
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H. Zeng, G. Liu, P. A. Rea, and G. D. Kruh Transport of Amphipathic Anions by Human Multidrug Resistance Protein 3 Cancer Res., September 1, 2000; 60(17): 4779 - 4784. [Abstract] [Full Text]
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G. L. Scheffer, M. Kool, M. Heijn, Marcel de Haas, A. C. L. M. Pijnenborg, J. Wijnholds, A. van Helvoort, M. C. de Jong, J. H. Hooijberg, C. A. A. M. Mol, et al. Specific Detection of Multidrug Resistance Proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-Glycoprotein with a Panel of Monoclonal Antibodies Cancer Res., September 1, 2000; 60(18): 5269 - 5277. [Abstract] [Full Text]
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P. Borst, R. Evers, M. Kool, and J. Wijnholds A Family of Drug Transporters: the Multidrug Resistance-Associated Proteins J Natl Cancer Inst, August 16, 2000; 92(16): 1295 - 1302. [Abstract] [Full Text] [PDF]
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R. A. M. H. Van Aubel, R. Masereeuw, and F. G. M. Russel Molecular pharmacology of renal organic anion transporters Am J Physiol Renal Physiol, August 1, 2000; 279(2): F216 - F232. [Abstract] [Full Text] [PDF]
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J. Wijnholds, C. A. A. M. Mol, L. van Deemter, M. de Haas, G. L. Scheffer, F. Baas, J. H. Beijnen, R. J. Scheper, S. Hatse, E. De Clercq, et al. Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs PNAS, June 6, 2000; (2000) 120159197. [Abstract] [Full Text]
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E. Hinoshita, T. Uchiumi, K.-i. Taguchi, N. Kinukawa, M. Tsuneyoshi, Y. Maehara, K. Sugimachi, and M. Kuwano Increased Expression of an ATP-binding Cassette Superfamily Transporter, Multidrug Resistance Protein 2, in Human Colorectal Carcinomas Clin. Cancer Res., June 1, 2000; 6(6): 2401 - 2407. [Abstract] [Full Text]
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G. L. Scheffer, M. Maliepaard, A. C. L. M. Pijnenborg, M. A. van Gastelen, M. C. de Jong, A. B. Schroeijers, D. M. van der Kolk, J. D. Allen, D. D. Ross, P. van der Valk, et al. Breast Cancer Resistance Protein Is Localized at the Plasma Membrane in Mitoxantrone- and Topotecan-resistant Cell Lines Cancer Res., May 1, 2000; 60(10): 2589 - 2593. [Abstract] [Full Text]
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T. Cantz, A. T. Nies, M. Brom, A. F. Hofmann, and D. Keppler MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells Am J Physiol Gastrointest Liver Physiol, April 1, 2000; 278(4): G522 - G531. [Abstract] [Full Text] [PDF]
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E. Bakos, R. Evers, E. Sinkó, A. Váradi, P. Borst, and B. Sarkadi Interactions of the Human Multidrug Resistance Proteins MRP1 and MRP2 with Organic Anions Mol. Pharmacol., April 1, 2000; 57(4): 760 - 768. [Abstract] [Full Text]
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T. Hirohashi, H. Suzuki, H. Takikawa, and Y. Sugiyama ATP-dependent Transport of Bile Salts by Rat Multidrug Resistance-associated Protein 3 (Mrp3) J. Biol. Chem., January 28, 2000; 275(4): 2905 - 2910. [Abstract] [Full Text] [PDF]
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J. Konig, Y. Cui, A. T. Nies, and D. Keppler A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane Am J Physiol Gastrointest Liver Physiol, January 1, 2000; 278(1): G156 - G164. [Abstract] [Full Text] [PDF]
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H. Zeng, L. J. Bain, M. G. Belinsky, and G. D. Kruh Expression of Multidrug Resistance Protein-3 (Multispecific Organic Anion Transporter-D) in Human Embryonic Kidney 293 Cells Confers Resistance to Anticancer Agents Cancer Res., December 1, 1999; 59(23): 5964 - 5967. [Abstract] [Full Text] [PDF]
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A. H. Dantzig, R. L. Shepard, K. L. Law, L. Tabas, S. Pratt, J. S. Gillespie, S. N. Binkley, M. T. Kuhfeld, J. J. Starling, and S. A. Wrighton Selectivity of the Multidrug Resistance Modulator, LY335979, for P-Glycoprotein and Effect on Cytochrome P-450 Activities J. Pharmacol. Exp. Ther., August 1, 1999; 290(2): 854 - 862. [Abstract] [Full Text]
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 T. P. SCHAUB, J. KARTENBECK, J. KÖNIG, H. SPRING, J. DÖRSAM, G. STAEHLER, S. STÖRKEL, W. F. THON, and D. KEPPLER Expression of the MRP2 Gene-Encoded Conjugate Export Pump in Human Kidney Proximal Tubules and in Renal Cell Carcinoma J. Am. Soc. Nephrol., June 1, 1999; 10(6): 1159 - 1169. [Abstract] [Full Text]
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J. Konig, Y. Cui, A. T. Nies, and D. Keppler Localization and Genomic Organization of a New Hepatocellular Organic Anion Transporting Polypeptide J. Biol. Chem., July 21, 2000; 275(30): 23161 - 23168. [Abstract] [Full Text] [PDF]
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G. Jedlitschky, B. Burchell, and D. Keppler The Multidrug Resistance Protein 5 Functions as an ATP-dependent Export Pump for Cyclic Nucleotides J. Biol. Chem., September 22, 2000; 275(39): 30069 - 30074. [Abstract] [Full Text] [PDF]
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) or
4 mM 5'-AMP (
). Vesicle-associated radioactivity was determined by
rapid filtration (Keppler et al., 1998
) and from HEK-Co (
) was
calculated by subtracting values obtained in the presence of 5'-AMP
from those in the presence of ATP. Each point represents the mean ± S. D. (n = 4).
6). The significance of relative resistance
factors (RR) was calculated by Student's t test.