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(Received for publication, November 30,
1995; and in revised form, January 17, 1996) From the
The 190-kDa multidrug resistance protein (MRP) has recently been
associated with the transport of cysteinyl leukotrienes and several
glutathione (GSH) S-conjugates. In the present study, we have
examined the transport of leukotriene C
Multidrug resistance can be conferred by overexpression of
either the multidrug resistance protein, MRP( Some insight into the normal physiological
role of MRP has been obtained by the demonstration that membrane
vesicles from MRP-overexpressing drug-selected and transfected cells
support ATP-dependent transport of cysteinyl leukotrienes (e.g. LTC Although current evidence
strongly suggests that MRP mediates an active transport process, the
mechanistic relationship between the involvement of MRP in GSH S-conjugate transport and its role in multidrug resistance is
unclear. GSH conjugation is not known to be an important pathway for
the biotransformation of chemotherapeutic agents to which MRP confers
resistance and there is no evidence that this reaction occurs to any
significant extent in tumor cells or drug-resistant tumor cell
lines(17) . In the present study, the mechanism by which MRP
confers resistance to multiple drugs was investigated in a plasma
membrane vesicle model system. For these studies, we used membrane
vesicles from a population of HeLa cells, termed T14, which had been
transfected with a MRP expression vector(8) . By using
transfected cells as the source of membrane vesicles, those aspects of
LTC
The sidedness of the membrane vesicles was assessed
by determining the activities of two plasma membrane-associated
ectoenzymes (alkaline phosphatase and 5`-nucleotidase) in the presence
or absence of 0.2% Nonidet P-40 (18, 23) and it was
determined that 30-34% of T14 PM vesicles were inside-out. T14
vesicles were treated with concanavalin A (1:1 (w/w) ratio) overnight
at 4 °C to agglutinate outside-out vesicles(24) . Following
centrifugation to remove agglutinated material, the supernatant
displayed a 2-fold increase in the rate of
[
ATP-dependent uptake of
[
Figure 1:
Time course of
[
Figure 2:
Osmotic sensitivity and nucleotide and
cation specificity of [
Since the activity of many ATPases is
often maintained when other divalent cations are substituted for
Mg
Figure 3:
Effect of substrate and ATP concentration
on [
The apparent K
Figure 4:
Photolabeling of vesicle proteins and
inhibition of [
To examine
the specificity of the interaction between LTC
Figure 5:
Effect of alkylated GSH derivatives on
[
Figure 6:
Effect of chemotherapeutic agents and
LTB
Figure 7:
Vincristine uptake in membrane vesicles
from MRP-transfected HeLa cells. Panel A, membrane vesicles
from HeLa C6 (
Both P-glycoprotein and MRP are capable of causing resistance
to a similar spectrum of drugs when overexpressed in mammalian
cells(1, 8) . Transfection of MRP or P-glycoprotein
into drug-sensitive cells has been shown to result in reduced drug
accumulation(8, 30) . In the case of P-glycoprotein,
there is considerable experimental evidence the protein causes
resistance by binding and transporting drugs out of the cell or plasma
membrane in an ATP-dependent
fashion(2, 9, 31) . The mechanism by which
MRP mediates reduced drug accumulation in resistant cells is less well
understood and in contrast to P-glycoprotein, there is no evidence that
unmodified chemotherapeutic agents bind directly to, or are transported
by, the protein(7, 8) . In the present study, we
further characterized the transport properties of MRP using membrane
vesicles derived from both drug-selected MRP-overexpressing cells and a
population of transfected HeLa cells(8, 18) . We found
high-affinity rapid transport of LTC LTC In contrast to cysteinyl leukotrienes
and alkylated GSH derivatives, we and others have found that
chemotherapeutic drugs are poor inhibitors of LTC We also found that
the presence of GSH resulted in demonstrable ATP-dependent
[ The mechanism by which GSH
enables ATP-dependent VCR transport by MRP, and possibly other
chemotherapeutic agents as well, is unclear. GSH has also been reported
to potentiate binding of the hydrophobic ligand MK 801 to the integral
membrane NMDA receptor (35) but whether the mechanism involved
is similar to that which potentiates VCR transport is unknown. It is
possible that MRP contains a bipartite binding site for hydrophobic and
anionic moieties that would allow binding of non-covalent drug-GSH
complexes, or the sequential binding of GSH and drug. Occupation of
both elements of the site may be necessary before transport can occur.
At present, there is no convincing evidence that GSH is actively
co-transported with drug. GSH by itself is not a substrate for
transport by MRP(33) , and neither does it inhibit LTC In addition to the cysteinyl leukotrienes
and vincristine in the presence of GSH, MRP can transport
17 We and others have clearly shown that
MRP-overexpressing cells are not resistant to
cisplatin(8, 20, 30, 41, 42) .
The presence of ATP-dependent transport of a glutathione-platinum
complex in a platinum-resistant cell line has been demonstrated and it
was suggested that the transporter or ``GS-X pump'' in these
cells may be MRP(43) . Although this transport activity is
pharmacologically similar to MRP in some respects, in that it is
inhibitable by LTC To date, there is little known about
the mechanism of ATP-dependent MRP-mediated transport of cysteinyl
leukotrienes, steroid glucuronides, or drugs in association with GSH.
However, it is possible that substrate binding by MRP (in the presence
of GSH in the case of chemotherapeutic drugs) facilitates the binding
and subsequent hydrolysis of ATP, which in turn induces a
conformational change in MRP that allows for the release of the
transported molecule(s) into the extracellular space. Experiments aimed
at determining whether the ATPase activity of MRP is stimulated by
LTC
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9675-9682
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
and Chemotherapeutic Agents in Membrane Vesicles
DEMONSTRATION OF GLUTATHIONE-DEPENDENT VINCRISTINE TRANSPORT (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(LTC)
in membrane vesicles from MRP-transfected HeLa cells (T14), as well as
drug-selected H69AR lung cancer cells which express high levels of MRP. V
and K
values for
LTC transport by membrane vesicles from T14 cells were 529
± 176 pmol mg
min and 105
± 31 nM, respectively. At 50 nM LTC, the K (ATP) was 70
µM. Transport in T14 vesicles was osmotically-sensitive
and was supported by various nucleoside triphosphates but not by non-
or slowly-hydrolyzable ATP analogs. LTC transport rates in
membrane vesicles derived from H69AR cells and their parental and
revertant variants were consistent with their relative levels of MRP
expression. A 190-kDa protein in T14 membrane vesicles was photolabeled
by [
H]LTC and immunoprecipitation
with MRP-specific monoclonal antibodies (mAbs) confirmed that this
protein was MRP. LTC transport was inhibited by an
MRP-specific mAb (QCRL-3) directed against an intracellular
conformational epitope of MRP, but not by a mAb (QCRL-1) which
recognizes a linear epitope. Photolabeling with
[H]LTC was also inhibitable by mAb
QCRL-3 but not mAb QCRL-1. GSH did not inhibit LTC transport. However, the ability of alkylated GSH derivatives to
inhibit transport increased markedly with the length of the alkyl
group. S-Decylglutathione was a potent competitive inhibitor
of [H]LTC transport (K
116 nM),
suggesting that the two compounds bind to the same, or closely related,
site(s) on MRP. Chemotherapeutic agents including colchicine,
doxorubicin, and daunorubicin were poor inhibitors of
[H]LTC transport. Taxol, VP-16,
vincristine, and vinblastine were also poor inhibitors of LTC transport but inhibition by these compounds was enhanced by GSH.
Uptake of [H]vincristine into T14 membrane
vesicles in the absence of GSH was low and not dependent on ATP.
However, in the presence of GSH, ATP-dependent vincristine transport
was observed. Levels of transport increased with concentrations of GSH
up to 5 mM. The identification of an MRP-specific mAb that
inhibits LTC transport and prevents photolabeling of MRP by
LTC, provides conclusive evidence of the ability of MRP to
transport cysteinyl leukotrienes. Our studies also demonstrate that MRP
is capable of mediating ATP-dependent transport of vincristine and that
transport is GSH-dependent.
)(1) ,
or by P-glycoprotein(2) . Both MRP and P-glycoprotein belong to
the ATP-binding cassette transporter superfamily but share only
approximately 15% amino acid identity(3) . Considerable
evidence suggests that P-glycoprotein reduces cellular drug
accumulation by acting as an ATP-dependent drug efflux
pump(2, 4) , but the mechanism of action of MRP is
much less certain. The ability to transport drugs directly into plasma
membrane vesicles has been firmly established for
P-glycoprotein(5, 6) , but not for MRP. There is also
no evidence that xenobiotics bind directly to
MRP(7, 8) , as has been demonstrated for
P-glycoprotein by cross-linking studies with photoaffinity analogs of
chemotherapeutic agents(9) . These observations suggest that
these two ATP-binding cassette proteins may confer multidrug resistance
by different mechanisms.) and certain other GSH S-conjugates(10, 11, 12) . Further
evidence of a role for MRP in LTC transport was suggested
by the observation that photolabeling of a 190-kDa protein with
LTC in MRP-expressing cells is inhibited by MK571 (an
inhibitor of LTC transport)(10) . The cysteinyl
leukotrienes are potent mediators of inflammation that increase
vascular permeability and smooth muscle contraction(13) .
LTC is synthesized from GSH and LTA by
LTC synthase(14) . It is then exported from the
cell by an ATP-dependent transport mechanism(15) . LTC synthase and the LTC transporter protein are
expressed in eosinophils and mast cells(16) , as well as
endothelial cells(13) . LTC is also transported in
the liver by at least two routes: uptake from the blood circulation
into hepatocytes, and excretion from hepatocytes across canalicular
membranes into the bile(13) . and drug transport kinetics that are solely
attributable to MRP overexpression can be
identified(8, 18) . Our initial objective was to
characterize MRP-mediated LTC transport in membrane
vesicles from this new population of transfected HeLa cells so that
subsequent studies of drug, or drug conjugate, transport could be
evaluated within this context. We provide evidence that
[H]LTC binds specifically to MRP and
demonstrate that a MRP-specific mAb inhibits both
[H]LTC transport and binding. We have
also characterized the ability of alkylated GSH derivatives and
hydrophobic chemotherapeutic agents to inhibit
[H]LTC transport. Finally, we show
that GSH not only enhances the ability of vincristine to inhibit
LTC transport, but also results in ATP-dependent transport
of the drug itself.
Materials
[14,15,19,20-H]-LTC (128 Ci mmol) and
[
S]methionine (710 Ci mmol)
were purchased from DuPont NEN (Mississauga, Ontario, Canada), and
[H]vincristine (VCR) (6.9 Ci
mmol) was from Amersham (Oakville, Ontario, Canada).
LTB, LTC, VP-16, AMP, AMP-PNP, AMP-PCP,
ATP
S, GTP, CTP, UTP, colchicine, GSH, GSSG, and S-alkyl
GSH derivatives were from Sigma. ATP, daunorubicin, doxorubicin,
vinblastine, and VCR were purchased from ICN Biochemicals (St. Laurent,
PQ, Canada) and taxol was from Omicron BioChemicals (San Antonio, TX).
Creatine phosphate and creatine kinase were from Boehringer Mannheim
(Dorval, PQ, Canada). QCRL-3 is a IgG
murine mAb that
recognizes a conformation dependent epitope of MRP and has been
described previously(19) .Cell Culture
The small cell lung cancer cell line
H69, its doxorubicin-selected multidrug resistant H69AR, and the
drug-sensitive revertant cell line, H69PR, have been described
previously(3, 20) . The HeLa cells transfected with
the pCEBV7 vector (C6) or the vector containing the MRP coding sequence
pCEBV7-MRP1 (T14) have also been described(8) . The 8226/Dox40
cell line overexpresses P-glycoprotein (21) and was provided
by Dr. W. Dalton. All cell lines were cultured in RPMI 1640 medium with
5% defined bovine calf serum (HyClone Laboratories, Logan, UT) in the
absence of antibiotics, except for the transfected HeLa cells which
were maintained in 100 µg ml hygromycin B. In
some experiments, cells were metabolically labeled with
[S]methionine (300 µCi
ml) as described(18) .Membrane Vesicle Preparation
Plasma membrane
vesicles were prepared as described (5, 22) with
modifications(18) . Briefly, cells were homogenized in buffer
containing 10 mM Tris-HCl, 250 mM sucrose, 3 mM KCl, 0.25 mM MgCl
, pH 7.5, and protease
inhibitors(18) . Cell pellets were frozen at -70 °C
for at least 1 h, thawed, and then disrupted by N cavitation (10 min equilibration at 175 p.s.i.). EDTA was added
to 1 mM and after centrifugation at 500 
g for
15 min, the supernatant was layered over 35% (w/w) sucrose in 10 mM Tris-HCl, 1 mM EDTA (specific gravity, 
1.15 g
cm
) and centrifuged at 100,000 g for 2 h. The interface was collected and washed twice by
centrifugation. The membrane pellet was resuspended in transport buffer
(50 mM Tris-HCl, 250 mM sucrose, pH 7.5) and passed
20 times through a 27-gauge needle for vesicle formation. The resulting
vesicles were enriched 4-8-fold in plasma membranes relative to
the post-nuclear supernatant, as assessed by
Na
,K-ATPase, alkaline phosphatase,
and 5`-nucleotidase activities ( (18) and references cited
therein). Some contamination from endoplasmic reticulum was observed,
as indicated by 2.3-fold enrichment of NADPH cytochrome c reductase.H]LTC uptake (see below), consistent
with ATP-dependent transport being attributable to inside-out vesicles. Vesicle Transport of LTC
ATP-dependent transport of LTC and
VCR into membrane
vesicles was measured by rapid filtration as described(16) ,
with modifications. Thawed membrane vesicles were diluted in transport
buffer and passed 5 times through a 27-gauge needle. Standard transport
assays were carried out at 23 °C in a 120-µl volume containing
10 µg of vesicle protein, 4 mM ATP, 10 mM MgCl, an ATP regenerating system consisting of 10
mM creatine phosphate and 100 µg ml creatine kinase, and [H]LTC (50
nM; 100 nCi) in transport buffer. At the indicated times,
20-µl aliquots were removed and added to 1 ml of ice-cold transport
buffer, which was then filtered through nitrocellulose filters (0.22
µm) on a Hoeffer filtration manifold under vacuum. Filters were
immediately washed twice with 5 ml of cold transport buffer,
solubilized, and radioactivity determined.
[H]LTC uptake was expressed relative
to the protein concentration of the membrane vesicles(25) . All
data were corrected for the amount of
[H]LTC that remained bound to the
filter in the absence of vesicle protein, which was usually 5-10%
of the total radioactivity.H]VCR (200 nM; 70 nCi) was measured as
described for [H]LTC except that
100-120 µg of vesicle protein was used and the incubations
were carried out at 37 °C. In some experiments, GSH (1-5
mM) was added to the transport buffer. Uptake of
[H]VCR was stopped by rapid dilution in transport
buffer and immediate filtration through glass fiber (Type A/E) filters
(Gelman Sciences, Dorval, PQ), which had been presoaked overnight at 37
°C in 10% (w/v) bovine serum albumin(5) .Photolabeling of Membrane Proteins with
[
Membrane proteins were photolabeled with
[H]LTC and Immunoprecipitation of
MRPH]LTC essentially as
described(16) . Briefly, vesicles prepared from different cell
types (150 µg of membrane protein in 50 µl) were incubated with
[H]LTC (0.5 µCi, 78 nM)
at room temperature for 10 min and frozen in liquid N.
Samples were alternately irradiated for 30 s at 312 nm in a
Stratalinker, followed by snap-freezing in liquid N, for a
total of 10 min. A portion of the radiolabeled proteins was
immunoprecipitated with a mixture of MRP-specific mAbs QCRL-1, QCRL-2,
and QCRL-3(19) . Immunoprecipitates were solubilized in
Laemmli's buffer and radiolabeled proteins (100 µg) were
resolved by SDS-PAGE and gels were subjected to fluorography. To
determine whether unlabeled LTC, LTB, mAb
QCRL-3, or mouse IgG (isotype control) could inhibit
[H]LTC labeling, T14 membrane
vesicles were coincubated with these reagents and
[H]LTC for 10 min at room temperature
prior to irradiation and SDS-PAGE.
LTC
The
time course and ATP dependence of [ Uptake in Membrane VesiclesH]LTC accumulation by vesicles prepared from HeLa T14 cells is shown in Fig. 1A. Accumulation was measured at room temperature
with an initial concentration of 50 nM LTC in the
presence of 4 mM ATP or AMP. No cations (Na,
K, Ca
) were present and the membrane
potential was not experimentally manipulated. ATP-dependent uptake was
rapid, linear up to 30 s and approached steady-state after 90 s (not
shown). During the linear phase, the rate of ATP-dependent uptake was
approximately 150 pmol mg min.
Increasing the amount of vesicle protein resulted in rates of uptake
which were difficult to quantify because of the short duration of
linearity. Similarly, quantification was difficult if the experiments
were carried out at 37 °C rather than room temperature because of
high initial rates of transport and rapid depletion of extravesicular
LTC. The ATP-dependent rate of LTC uptake in
T14 vesicles was at least 100-fold higher than in vesicles from control
C6 cells (Fig. 1A). Furthermore, the low levels of
uptake in T14 vesicles in the presence of AMP were similar to those
observed for C6 membrane vesicles with or without ATP. The correlation
between LTC transport and levels of MRP expression was also
examined by measuring rates of LTC uptake in vesicles
isolated from drug-sensitive H69, drug-resistant H69AR, and revertant
H69PR cells. As shown in Fig. 1B, the rate of
ATP-dependent [H]LTC uptake in H69AR
vesicles was approximately 2-fold faster than for T14 vesicles and was
below the limits of detection in vesicles from H69 cells. Low levels of
ATP-dependent [H]LTC uptake could be
detected in H69PR vesicles (approximately 1.5 pmol mg min at room temperature) (Fig. 1C) where the levels of MRP are 3-5-fold
higher than in H69 cells(3, 19) . Vesicles from human
8226/Dox40 cells also exhibited levels of
[H]LTC transport comparable to those
of H69PR-derived vesicles (approximately 2 pmol mg min) (Fig. 1D). These cells
overexpress P-glycoprotein and contain low levels of MRP similar to
those found in H69PR cells (data not shown).
H]LTC uptake by membrane vesicles
from MRP and control transfected HeLa cells, parental and drug-selected
H69 cells, and 8226/Dox40 cells. Membrane vesicles were incubated with
50 nM [H]LTC in transport
buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.5) for
the times indicated. Closed symbols represent uptake in the
presence of 4 mM AMP; open symbols represent uptake
in the presence of 4 mM ATP. Vesicles were derived as
described under ``Experimental Procedures'' from the
following cell lines: Panel A, HeLa C6 (
, 
) and T14
(
, 
); Panel B, H69 (, ) and H69AR
(
, 
); Panel C, H69PR (
, 
); Panel
D, 8226/Dox40 (
, 
). The dotted curve in Panel A (T14) and Panel B (H69AR) indicate
ATP-dependent uptake, which was calculated by subtracting
ATP-independent transport from transport in the presence of ATP. Data
points are means of triplicate determinations (±S.E.) in a
typical experiment.
Osmotic Sensitivity and Nucleotide and Cation Specificity
of [
To confirm that LTCH]LTC Transport by T14 Membrane
Vesicles accumulation reflected
transport of substrate into the vesicle lumen, rather than surface or
intramembrane binding, the effect of extravesicular osmolarity on
[H]LTC uptake was measured. As shown
in Fig. 2A, the rate of
[H]LTC uptake in T14 vesicles was
clearly osmotically-sensitive. To establish the nucleotide dependence
of LTC transport and to determine whether ATP hydrolysis
was required, [H]LTC uptake was
measured in the presence of various nucleoside triphosphates and
non-hydrolyzable ATP analogs. As shown in Fig. 2B,
AMP-PNP, AMP-PCP, and ATPS (4 mM) were unable to support
LTC uptake, suggesting that ATP hydrolysis is necessary for
transport. Of the four nucleoside triphosphates tested, LTC uptake was most efficient in the presence of ATP (Fig. 2B).
H]LTC transport by T14 membrane vesicles. Panel A, T14
membrane vesicles were preincubated for 10 min in transport buffer
containing sucrose (0.25-1 M). Rates of
[H]LTC uptake were measured in the
presence of 4 mM ATP () or 4 mM AMP ()
as described under ``Experimental Procedures.'' Panel
B, rates of [H]LTC uptake were
measured in the presence of the indicated nucleotides (4 mM).
No regenerating system was included in these experiments and the rate
of transport in 4 mM ATP was not affected by its omission (solid bar). Results obtained with other nucleotides (hatched bars) are plotted as a % of values obtained with 4
mM ATP. The data shown are means of triplicate determinations
(±S.E.) in a single experiment. Panel C, rates of
[H]LTC uptake were measured in the
presence of 4 mM ATP and the indicated divalent cations (10
mM) (hatched bars). Results are plotted as a % of
control LTC uptake values obtained with 10 mM Mg at 30 s (solid bar). The results
shown are means of triplicate determinations (±S.E.) in a single
experiment and similar results were obtained in one additional
experiment.
, we investigated the level of ATP-dependent
[H]LTC transport in T14 vesicles in
the presence of Mn, Ca,
Co, Cd, Ba, and
Zn (10 mM). The relative ability of these
cations to support LTC transport correlated well with their
abilities to support ATP hydrolysis by known ATPases. Thus, when
Mn, Ca, or Co were substituted for Mg, the
[H]LTC transport rates were
measureable but reduced by 25, 50, and 65%, respectively, whereas
Cd, Ba, and Zn did not support transport (Fig. 2C).Inhibition of [
The inability of
non-hydrolyzable ATP analogs to support MRP-mediated LTCH]LTC Transport by ATPase Inhibitors uptake and the effect of substituting various cations for
Mg suggests that ATP hydrolysis, rather than just
ATP-binding, is necessary for transport. Consequently, transport
activity might be expected to be sensitive to inhibitors of certain
classes of ATPases. For this reason, we examined the effects of the
P-type ATPase inhibitor sodium vanadate, the
Na,K-ATPase inhibitor ouabain, and
the sulfhydryl reagent N-ethylmaleimide.
[H]LTC uptake was insensitive to
ouabain and vanadate at concentrations up to 1 mM.
Preincubation of membrane vesicles with 1 mMN-ethylmaleimide at 23 °C had no effect, but at 37
°C, [H]LTC uptake was inhibited
by 70%. This observation suggests that at least one cysteine residue
may be important for transport, as has been shown for other transport
ATPases (26) .Kinetic Parameters of [
Rates of uptake were measured at
several LTCH]LTC Transport in T14 Vesicles concentrations (12.5-1000 nM) to
determine K
and V for
[H]LTC transport in T14 membrane
vesicles. As shown in Fig. 3A (inset), a
Lineweaver-Burk double reciprocal plot yielded an apparent K of 77 nM for LTC and a V of 291 pmol mg min. The average values (±S.E.) for K and V for six experiments
were 105 ± 31 nM and 529 ± 176 pmol
min mg, respectively. This K value is in good agreement with that reported
previously for vesicles from human MRP-transfected HeLa T5 cells (97
nM)(10) , and for [H]LTC transport in murine mastocytoma membrane vesicles (70
nM) (16) . The V obtained with
T14 vesicles is approximately 5-fold higher than the value previously
reported for vesicles prepared from HeLa T5 cells transfected with a
different MRP expression vector (100 pmol min mg)(10) . The levels of MRP in T14
cells are 2-fold higher than in T5 cells (15) which may explain
the differences in V. However, it is also
possible that differences in procedures used to prepare vesicles may
contribute to the higher V values obtained with
T14 vesicles.
H]LTC uptake. Panel A,
[H]LTC uptake by T14 membrane
vesicles was measured at various LTC concentrations
(12.5-1000 nM) for 30 s at 23 °C. Data are plotted
as V
versus [S] to confirm that
the appropriate concentration range was selected to observe both
zero-order and first-order rate kinetics. Kinetic parameters were
determined from regression analysis of the Lineweaver-Burk
transformation of the data (inset). Panel B,
ATP-dependent uptake of [H]LTC was
measured at various concentrations of ATP (2 µM to 4
mM) in the presence of 50 nM [H]LTC. Uptake at ATP
concentrations up to 250 µM are plotted. Kinetic
parameters were determined from regression analysis of the
Lineweaver-Burk data transformation (inset).
for ATP (70
µM) was determined by measuring initial rates of
[H]LTC uptake at 30 s in the presence
of different concentrations of the nucleotide (2-4000
µM) (Fig. 3B, inset).Photolabeling of Membrane Proteins with
[
Previous studies have shown that
LTCH]LTC and Inhibition of Transport
with MRP-specific MAb QCRL-3 will bind to a 190-kDa protein enriched in membranes
from MRP transfected cells(10) . To demonstrate conclusively
that the labeled 190-kDa protein was MRP, T14 vesicles were incubated
with [H]LTC, uv irradiated to
cross-link bound LTC, and solubilized membranes were
immunoprecipitated with MRP-specific mAbs(19) . After SDS-PAGE
of the immunoprecipitate, a
[H]LTC-labeled 190-kDa protein (Fig. 4A, lane 3) was detected which
co-migrated with [S]methionine-labeled MRP
immunoprecipitated with the same mAbs (Fig. 4A, lane 1). In addition, the MRP-specific mAb QCRL-3, which
recognizes a conformation-dependent epitope, inhibited
[H]LTC uptake with an IC
of 5 µg ml (50 ng of mAb µg
protein) (Fig. 4B). The MRP-specific
mAb QCRL-1, which recognizes a linear MRP epitope, had no effect on
LTC transport (not shown) nor did isotype control
immunoglobulins up to 30 µg ml .
H]LTC uptake and
photolabeling by MRP-specific mAb QCRL-3. Panel A, T14
membrane vesicles were photolabeled with
[H]LTC and membrane protein (50 or
120 µg) was analyzed by SDS-PAGE and fluorography. Alternatively,
membranes (100 µg of protein) were photolabeled and
immunoprecipitated with a mixture of three MRP-specific mAbs
([H]LTC, middle
lane)(19) . For comparison, immunoprecipitations were also
performed on membranes prepared from cells labeled in culture with
[S]methionine (S-Ippt, far left lane)(18) . Panel B,
[H]LTC uptake was measured in the
presence of MRP-specific mAb QCRL-3. Each point represents the mean of
triplicate determinations (±S.E.) in a single experiment. In the
experiment shown, the rate of uptake in control incubations was 174
pmol mg min. Panel C,
membrane vesicle protein (
150 µg) from 8226/Dox40, H69AR, C6,
or T14 cells was incubated with [H]LTC (0.5 µCi; 78 nM) in transport buffer, irradiated at
312 nm, and analyzed by SDS-PAGE and fluorography (see
``Experimental Procedures''). T14 membrane vesicles were also
incubated with [H]LTC in the presence
of excess unlabeled LTC, LTB, MRP-specific mAb
QCRL-3 (10 µg ml), and murine IgG (70 µg ml), prior to irradiation and
SDS-PAGE and fluorography. The autoradiograms shown represent a 14-day
exposure.
and MRP, the
ability of unlabeled LTC or the unconjugated leukotriene,
LTB, to compete for [H]LTC labeling of membrane proteins was determined (Fig. 4C). In the absence of competitor, photolabeling
of MRP was readily detectable in H69AR (Fig. 4C, lane 2) and T14 (Fig. 4C, lane 4),
but not in C6 membranes (Fig. 4C, lane 3), as
expected. The relative degree of labeling was consistent with the
relative level of MRP in these cells. Photolabeling was inhibited by
excess unlabeled LTC (Fig. 4C, lane
5), but not LTB (Fig. 4C, lane
6). Labeling was also inhibited by mAb QCRL-3 (Fig. 4C, lane 7) at a concentration which
abolishes [H]LTC transport (100 ng
µg protein). MAb QCRL-1 did not inhibit
photolabeling (not shown), consistent with its inability to inhibit
LTC transport. Finally, to exclude the possibility that
LTC labeling of MRP was an artifact resulting from
nonspecific labeling of an abundant integral membrane protein, labeling
experiments were carried out with 8226/Dox40 vesicles known to contain
high levels of P-glycoprotein. As shown in Fig. 4C (lane 1), no labeling with
[H]LTC was observed.Inhibition of [
It has been reported
previously that the transporter(s) responsible for GSH S-conjugate in sarcolemmal (27) and hepatocanalicular (28, 29) membranes is inhibited by alkylated GSH
derivatives. Consequently, we investigated whether these compounds had
a similar effect on MRP-mediated [H]LTC Uptake by Alkylated GSH DerivativesH]LTC transport in T14 vesicles. Inhibition of uptake increased with
the length of the alkyl chain and hence with the hydrophobicity of the
GSH derivative (Fig. 5A). For example, S-octyl- and S-decyl-GSH inhibited LTC uptake by more than 60% at concentrations as low as 100
nM. GSH alone did not inhibit LTC transport even
at 5 mM. In contrast, GSSG inhibited transport with an
IC of 100 µM (data not shown)(12) .
Inhibition of LTC transport by S-decyl-GSH was
further characterized by determining the kinetic parameters of
inhibition. These experiments showed that S-decyl-GSH is a
competitive inhibitor with an apparent K
of 116
nM (Fig. 5B).
H]LTC uptake in MRP-enriched
membrane vesicles. Panel A, [H]LTC uptake into T14 membrane vesicles was measured in the presence of
the indicated concentrations of GSH derivatives. Results are plotted as
a % of ATP-dependent [H]LTC uptake in
the absence of GSH derivative and each bar represent the mean (±
S.E.) of triplicate determinations in a typical experiment. The control
uptake rate in this experiment was 190 pmol mg min. Panel B, uptake of
[H]LTC was measured in the presence
of various concentrations of S-decyl-GSH (control, ; 50
nM, ; 100 nM, ; 250 nM, ).
Double-reciprocal plots were generated and an apparent K of 116 nM was calculated from
the apparent K and V in the presence of S-decyl-GSH.
Inhibition of [
Several chemotherapeutic agents and the
non-cysteinyl leukotriene LTBH]LTC Transport in Membrane Vesicles by Chemotherapeutic Agents and
LTB were tested for their ability
to inhibit [H]LTC transport and the
results are shown in Fig. 6. Daunorubicin, doxorubicin,
colchicine, and VP-16 at 100 µM and taxol at 40 µM inhibited uptake by only 35-55% (Fig. 6A). A
modest enhancement of [H]LTC transport inhibition was observed when the vesicles were
coincubated with GSH (1 mM), and colchicine, taxol, or VP-16,
but not daunorubicin or doxorubicin (Fig. 6A). The
small effect seen with colchicine, taxol, and VP-16 may be attributable
to the weak inhibition (<15%) of transport exhibited by GSH itself
(not shown). In contrast, although VCR and vinblastine alone were also
poor inhibitors of [H]LTC transport
(30-50% inhibition at 100 µM), this inhibition was
markedly enhanced by coincubation with GSH (1 mM). In the
presence of GSH, 50-60 and 90-100% inhibition by
vinblastine and VCR was observed at drug concentrations of 10 and 100
µM, respectively (Fig. 6B).
2-Mercaptoethanol or dithiothreitol (1 mM) had no effect on
the inhibitory potency of vinblastine or VCR (data not shown),
indicating that the enhancing effect of GSH was not simply due to its
reducing capacity. As expected, LTB was a poor inhibitor of
LTC transport, showing little effect at 10 µM,
and requiring 100 µM (2000-fold molar excess compared to
LTC) to inhibit uptake by 80% (Fig. 6B).
The inhibition by this non-cysteinyl leukotriene was not augmented by
GSH.
on [H]LTC uptake by
T14 vesicles. The ability of drugs and LTB to inhibit
[H]LTC uptake was measured in the
absence or presence of GSH (1 mM) for 30 s at 23 °C.
Results were calculated as a % of control values obtained in the
absence of both drug and GSH. The bars represent the means
(±S.E.) of triplicate determinations in a single experiment and
similar results were obtained in at least one additional experiment. Panel A, the control uptake rate in this experiment was 185
pmol mg min. DOX,
doxorubicin; DNR, daunorubicin; CLC, colchicine; TXL, taxol. Panel B, the control uptake rate in this
experiment was 155 pmol mg min. VBL, vinblastine.
[
The ability of GSH to enhance inhibition of
LTCH]Vincristine Transport in
Membrane Vesicles transport by vinblastine and VCR suggested that GSH may
also play a role in the transport of these Vinca alkaloids. To
investigate this possibility directly, we examined the effect of GSH on
[H]VCR uptake. These experiments were performed
as described for LTC uptake, except that they were carried
out at 37 °C, the concentration of substrate (VCR) was 200
nM, 10-12-fold more vesicle protein was used, and
washing conditions were modified to minimize nonspecific binding of VCR
to the filters. No ATP-dependent uptake of [H]VCR
uptake could be detected in either C6 or T14 vesicles and steady-state
levels of vesicle-associated VCR were very low (approximately 3 and 6
pmol mg for C6 and T14 vesicles, respectively, at 20
min) (Fig. 7A). In contrast, ATP-dependent
[H]VCR uptake was demonstrable in T14 but not C6
vesicles when GSH was added and this effect was concentration dependent (Fig. 7B). Thus, in the presence of 1, 3, and 5 mM GSH, steady state levels of ATP-dependent VCR uptake in T14
vesicles were increased to approximately 15, 22, and 31 pmol
mg, respectively, at 20 min. 2-Mercaptoethanol,
dithiothreitol, or L-cysteine (up to 5 mM) did not
increase VCR uptake, indicating that it was not the reducing capacity
of GSH that was responsible for the increase in VCR uptake (Fig. 7B). [H]VCR uptake by T14
vesicles was osmotically-sensitive (not shown) and was inhibitable by
500 µM GSSG (23 ± 5% control), and 200 µMN-ethylmaleimide (45 ± 5% control), but not by 200
µM vanadate (85 ± 8% control), consistent with the
effect of these reagents on LTC transport.
, ) and T14 (, ) cells were
incubated with 200 nM [H]VCR in
transport buffer at 37 °C for the times indicated. Closed
symbols represent uptake in the presence of 4 mM AMP; open symbols represent uptake in the presence of 4 mM ATP. Panel B, T14 vesicles were incubated for 10 min in
200 nM [H]VCR in buffer containing 4
mM AMP, ATP, or ATP and an ATP-regenerating system (ATP/RS) as
described under ``Experimental Procedures.'' Transport was
measured in the absence and presence of the indicated concentrations of
GSH and 2-mercaptoethanol (2-ME). Bars represent the
means (±S.E.) of triplicate determinations in a typical
experiment and similar results were found in three additional
experiments.
in T14 vesicles that
was osmotically sensitive, required hydrolyzable nucleotides, and was
supported only by those divalent cations that support the activity of
other membrane ATPases, thus providing evidence that ATP hydrolysis as
well as binding is required for transport. Transport rates were
somewhat lower in T14 vesicles than in vesicles from the drug-selected
H69AR cells, in keeping with the relative levels of MRP expression in
these two cell types(8) . The low but detectable transport in
revertant H69PR cells was also consistent with the low levels of MRP in
these cells(3, 19) . The absence of significant
LTC transport in vesicles from
P-glycoprotein-overexpressing 8226/Dox40 cells confirmed the
MRP-specificity of this transport process. These observations confirm
and extend previous studies demonstrating that MRP-enriched vesicles
are capable of LTC transport(10, 11, 12) . Binding of
LTC to MRP was shown by immunoprecipitation of a 190-kDa
[H]LTC photoaffinity labeled T14
membrane protein with MRP-specific mAbs. MAb QCRL-3 which detects a
conformation-dependent epitope of MRP, also strongly inhibited
LTC transport and prevented
[H]LTC labeling of MRP. Taken
together, these observations provide strong evidence that LTC binds directly to MRP before being transported and further
suggest that LTC binds to MRP at a site within or near the
epitope detected by mAb QCRL-3. is the highest
affinity substrate identified to date for MRP and while some
modifications of the glutathione or arachidonate moieties of the
molecule are tolerated, levels of transport are usually diminished by
these changes(10) . Our present studies demonstrate that
LTC transport by MRP is effectively inhibited by alkylated
GSH derivatives. The inhibitory potency of alkylated GSH derivatives
with respect to LTC transport in hepatocanalicular (28, 29) and sarcolemmal (27) membranes has
been shown to increase proportionately with the length of the alkyl
chain. We also found this to be the case in vesicles from MRP
transfectants. Indeed, LTC transport by MRP was
significantly more sensitive to inhibition by these compounds than
reported for the transporters in rat muscle and
liver(27, 29) . Inhibition of MRP-mediated LTC transport by the most potent GSH derivative, S-decyl-GSH, was competitive, suggesting that it binds to a
site in MRP that is similar or possibly overlapping the site to which
LTC binds. The K (116 nM) for S-decyl-GSH was similar to the apparent K (105 nM) for LTC transport in T14 membrane
vesicles, indicating that this GSH derivative is potentially a high
affinity substrate for MRP. transport, exerting significant inhibition only at concentrations
200-2000-fold greater than the K of
LTC(12, 32) . However, we observed that
inhibition of LTC transport by certain drugs, most notably
the Vinca alkaloids VCR and vinblastine, could be
significantly enhanced by incubation with physiological concentrations
of GSH. Why this enhancement is more pronounced with these two drugs
than with others is presently unclear. It does not appear to be simply
related to the relative degree of MRP-mediated resistance to a
particular agent since T14 cells are considerably more resistant to
VCR, doxorubicin, and daunorubicin than they are to
vinblastine(8) . Nevertheless, the ability of both Vinca alkaloids to inhibit LTC transport in T14 membrane
vesicles is similarly and markedly enhanced by GSH while inhibition by
doxorubicin and daunorubicin are unaffected. A similar potentiating
effect of GSH is observed for inhibition of 17
-estradiol
17-(-D-glucuronide) transport by MRP (44) . This
effect appears specific for GSH since other thiols (2-mercaptoethanol, L-cysteine, and dithiothreitol) or other organic anions such
as glucuronic acid could not substitute for GSH.H]VCR transport by MRP-enriched vesicles.
ATP-dependent uptake of [H]VCR was approximately
31 pmol mg at steady state in the presence of 200
nM VCR and 5 mM GSH. This is substantially lower than
steady state levels of LTC uptake, GSSG
uptake(33) , and 17-estradiol
17-(-D-glucuronide) uptake (44) in MRP-enriched
vesicles but is comparable to that reported for vinblastine uptake in
vesicles from certain cell lines overexpressing
P-glycoprotein(6, 34) . transport, even at 5 mM, the highest intracellular
concentration likely to be encountered in vivo. In contrast,
GSSG caused 50% inhibition of LTC transport at
approximately 100 µM. Finally, it has been reported that
transport of daunorubicin by MRP does not increase GSH release by
intact cells(36, 37) . Thus it appears unlikely that
MRP transports drugs in association with reduced GSH. Alternatively,
interaction of GSH with MRP may cause a conformational change (37) or an alteration in the exposure of nonpolar residues,
that may favor binding of some hydrophobic compounds prior to
transport. Further studies are required to elucidate precisely how GSH
enhances VCR transport.-estradiol 17-(-D-glucuronide) and possibly
certain other cholestatic steroid glucuronides(44) . We have
determined that LTC can inhibit 17-estradiol
17-(-D-glucuronide) transport and vice versa.
Consequently, the presence of a cysteinyl residue is not absolutely
required for a compound to be a substrate for MRP-mediated transport. A
number of organic anions and cyclic peptides which are neither GSH nor
glucuronide conjugates such as MK 571 (K 0.6
µM) (16, 38) and cyclosporin A (K 5 µM) (10) can behave as
competitive inhibitors of LTC transport in membrane
vesicles from drug-selected cells known to overexpress MRP. Since
cyclosporin A is not an effective chemosensitizer in MRP-overexpressing
cells(30, 39, 40) , the ability of a compound
to inhibit LTC transport is clearly not always indicative
of its capacity to reverse MRP-associated resistance nor its ability to
act as a substrate., GSSG, and S-dinitrophenylglutathione, the overexpression of MRP mRNA and
protein in these cells has not been shown. Furthermore, we found that
cisplatin did not inhibit LTC transport in T14 vesicles
either by itself or in combination with GSH (results not shown).
Finally, Muller and co-workers (12) were unable to demonstrate
LTC transport in a platinum-resistant lung cancer cell line
which exhibits reduced drug accumulation. Thus current evidence
suggests that MRP is not involved in conferring resistance to
platinum-containing drugs although it remains possible that a
transporter related to MRP may be responsible, at least in some
platinum-resistant cell lines., 17-estradiol 17-(-D-glucuronide),
or chemotherapeutic agents in the absence or presence of GSH are
underway. Mapping of the mAb QCRL-3 epitope is also in progress and
together with proteolytic mapping studies of
[H]LTC-labeled MRP, should allow the
LTC binding site on the MRP molecule to be identified.
),-methyleneadenosine 5`-triphosphate; AMP-PNP, adenosine
5`-[,-imido]triphosphate; ATPS, adenosine
5`-O-(3-thiotriphosphate); LTC, leukotriene
C; LTB, leukotriene B; mAb,
monoclonal antibody; VCR, vincristine; PAGE, polyacrylamide gel
electrophoresis.
We thank D. R. Hipfner for mAbs and C. E. Grant for
transfected HeLa cells. We also thank Drs. D. Keppler and I. Leier for
helpful discussions. The technical assistance of K. Sparks and K.
Fraser is gratefully acknowledged.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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D. Trompier, X.-B. Chang, R. Barattin, A. d. M. d'Hardemare, A. Di Pietro, and H. Baubichon-Cortay Verapamil and Its Derivative Trigger Apoptosis through Glutathione Extrusion by Multidrug Resistance Protein MRP1 Cancer Res., July 15, 2004; 64(14): 4950 - 4956. [Abstract] [Full Text] [PDF]
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A. Haimeur, G. Conseil, R. G. Deeley, and S. P.C. Cole Mutations of Charged Amino Acids in or near the Transmembrane Helices of the Second Membrane Spanning Domain Differentially Affect the Substrate Specificity and Transport Activity of the Multidrug Resistance Protein MRP1 (ABCC1) Mol. Pharmacol., June 1, 2004; 65(6): 1375 - 1385. [Abstract] [Full Text] [PDF]
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S. Dallas, L. Schlichter, and R. Bendayan Multidrug Resistance Protein (MRP) 4- and MRP 5-Mediated Efflux of 9-(2-Phosphonylmethoxyethyl)adenine by Microglia J. Pharmacol. Exp. Ther., June 1, 2004; 309(3): 1221 - 1229. [Abstract] [Full Text] [PDF]
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K. Koike, G. Conseil, E. M. Leslie, R. G. Deeley, and S. P. C. Cole Identification of Proline Residues in the Core Cytoplasmic and Transmembrane Regions of Multidrug Resistance Protein 1 (MRP1/ABCC1) Important for Transport Function, Substrate Specificity, and Nucleotide Interactions J. Biol. Chem., March 26, 2004; 279(13): 12325 - 12336. [Abstract] [Full Text] [PDF]
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J. D. Campbell, K. Koike, C. Moreau, M. S. P. Sansom, R. G. Deeley, and S. P. C. Cole Molecular Modeling Correctly Predicts the Functional Importance of Phe594 in Transmembrane Helix 11 of the Multidrug Resistance Protein, MRP1 (ABCC1) J. Biol. Chem., January 2, 2004; 279(1): 463 - 468. [Abstract] [Full Text] [PDF]
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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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D.-W. Zhang, H.-M. Gu, D. Situ, A. Haimeur, S. P. C. Cole, and R. G. Deeley Functional Importance of Polar and Charged Amino Acid Residues in Transmembrane Helix 14 of Multidrug Resistance Protein 1 (MRP1/ABCC1): IDENTIFICATION OF AN ASPARTATE RESIDUE CRITICAL FOR CONVERSION FROM A HIGH TO LOW AFFINITY SUBSTRATE BINDING STATE J. Biol. Chem., November 14, 2003; 278(46): 46052 - 46063. [Abstract] [Full Text] [PDF]
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L. F. Payen, M. Gao, C. J. Westlake, S. P. C. Cole, and R. G. Deeley Role of Carboxylate Residues Adjacent to the Conserved Core Walker B Motifs in the Catalytic Cycle of Multidrug Resistance Protein 1 (ABCC1) J. Biol. Chem., October 3, 2003; 278(40): 38537 - 38547. [Abstract] [Full Text] [PDF]
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S. Dallas, X. Zhu, S. Baruchel, L. Schlichter, and R. Bendayan Functional Expression of the Multidrug Resistance Protein 1 in Microglia J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 282 - 290. [Abstract] [Full Text] [PDF]
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R. Yang, L. Cui, Y.-x. Hou, J. R. Riordan, and X.-b. Chang ATP Binding to the First Nucleotide Binding Domain of Multidrug Resistance-associated Protein Plays a Regulatory Role at Low Nucleotide Concentration, whereas ATP Hydrolysis at the Second Plays a Dominant Role in ATP-dependent Leukotriene C4 Transport J. Biol. Chem., August 15, 2003; 278(33): 30764 - 30771. [Abstract] [Full Text] [PDF]
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K. Nunoya, C. E. Grant, D. Zhang, S. P. C. Cole, and R. G. Deeley MOLECULAR CLONING AND PHARMACOLOGICAL CHARACTERIZATION OF RAT MULTIDRUG RESISTANCE PROTEIN 1 (MRP1) Drug Metab. Dispos., August 1, 2003; 31(8): 1016 - 1026. [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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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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M. J. Grzywacz, J.-M. Yang, and W. N. Hait Effect of the Multidrug Resistance Protein on the Transport of the Antiandrogen Flutamide Cancer Res., May 15, 2003; 63(10): 2492 - 2498. [Abstract] [Full Text] [PDF]
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E. M. Leslie, R. J. Bowers, R. G. Deeley, and S. P. C. Cole Structural Requirements for Functional Interaction of Glutathione Tripeptide Analogs with the Human Multidrug Resistance Protein 1 (MRP1) J. Pharmacol. Exp. Ther., February 1, 2003; 304(2): 643 - 653. [Abstract] [Full Text] [PDF]
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C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3 Am J Physiol Gastrointest Liver Physiol, February 1, 2003; 284(2): G280 - G289. [Abstract] [Full Text] [PDF]
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Y.-x. Hou, J. R. Riordan, and X.-b. Chang ATP Binding, Not Hydrolysis, at the First Nucleotide-binding Domain of Multidrug Resistance-associated Protein MRP1 Enhances ADP{middle dot}Vi Trapping at the Second Domain J. Biol. Chem., January 31, 2003; 278(6): 3599 - 3605. [Abstract] [Full Text] [PDF]
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L. Manciu, X.-B. Chang, F. Buyse, Y.-X. Hou, A. Gustot, J. R. Riordan, and J. M. Ruysschaert Intermediate Structural States Involved in MRP1-mediated Drug Transport. ROLE OF GLUTATHIONE J. Biol. Chem., January 24, 2003; 278(5): 3347 - 3356. [Abstract] [Full Text] [PDF]
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E. M. Leslie, R. G. Deeley, and S. P. C. Cole Bioflavonoid Stimulation of Glutathione Transport by the 190-kDa Multidrug Resistance Protein 1 (MRP1) Drug Metab. Dispos., January 1, 2003; 31(1): 11 - 15. [Abstract] [Full Text] [PDF]
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K. Koike, C. J. Oleschuk, A. Haimeur, S. L. Olsen, R. G. Deeley, and S. P. C. Cole Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1 J. Biol. Chem., December 13, 2002; 277(51): 49495 - 49503. [Abstract] [Full Text] [PDF]
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Y. Yang, Q. Chen, and J.-T. Zhang Structural and Functional Consequences of Mutating Cysteine Residues in the Amino Terminus of Human Multidrug Resistance-associated Protein 1 J. Biol. Chem., November 8, 2002; 277(46): 44268 - 44277. [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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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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Q. Mao, W. Qiu, K. E. Weigl, P. A. Lander, L. B. Tabas, R. L. Shepard, A. H. Dantzig, R. G. Deeley, and S. P. C. Cole GSH-dependent Photolabeling of Multidrug Resistance Protein MRP1 (ABCC1) by [125I]LY475776. EVIDENCE OF A MAJOR BINDING SITE IN THE COOH-PROXIMAL MEMBRANE SPANNING DOMAIN J. Biol. Chem., August 2, 2002; 277(32): 28690 - 28699. [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. Rajagopal, A. C. Pant, S. M. Simon, and Y. Chen In Vivo Analysis of Human Multidrug Resistance Protein 1 (MRP1) Activity Using Transient Expression of Fluorescently Tagged MRP1 Cancer Res., January 1, 2002; 62(2): 391 - 396. [Abstract] [Full Text] [PDF]
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G. Lee, S. Dallas, M. Hong, and R. Bendayan Drug Transporters in the Central Nervous System: Brain Barriers and Brain Parenchyma Considerations Pharmacol. Rev., December 1, 2001; 53(4): 569 - 596. [Abstract] [Full Text] [PDF]
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Y.-M. Qian, W. Qiu, M. Gao, C. J. Westlake, S. P. C. Cole, and R. G. Deeley Characterization of Binding of Leukotriene C4 by Human Multidrug Resistance Protein 1. EVIDENCE OF DIFFERENTIAL INTERACTIONS WITH NH2- AND COOH-PROXIMAL HALVES OF THE PROTEIN J. Biol. Chem., October 12, 2001; 276(42): 38636 - 38644. [Abstract] [Full Text] [PDF]
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H. H. DeCory, K. M. Piech-Dumas, S.-S. Sheu, H. J. Federoff, and M. W. Anders Efflux of Glutathione Conjugate of Monochlorobimane from Striatal and Cortical Neurons Drug Metab. Dispos., October 1, 2001; 29(10): 1256 - 1262. [Abstract] [Full Text] [PDF]
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K. Ito, H. Suzuki, and Y. Sugiyama Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G1034 - G1043. [Abstract] [Full Text] [PDF]
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S. K. Diah, P. K. Smitherman, J. Aldridge, E. L. Volk, E. Schneider, A. J. Townsend, and C. S. Morrow Resistance to Mitoxantrone in Multidrug-resistant MCF7 Breast Cancer Cells: Evaluation of Mitoxantrone Transport and the Role of Multidrug Resistance Protein Family Proteins Cancer Res., July 1, 2001; 61(14): 5461 - 5467. [Abstract] [Full Text] [PDF]
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E. M. Leslie, Q. Mao, C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole Modulation of Multidrug Resistance Protein 1 (MRP1/ABCC1) Transport and ATPase Activities by Interaction with Dietary Flavonoids Mol. Pharmacol., April 16, 2001; 59(5): 1171 - 1180. [Abstract] [Full Text]
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H. Komatani, H. Kotani, Y. Hara, R. Nakagawa, M. Matsumoto, H. Arakawa, and S. Nishimura Identification of Breast Cancer Resistant Protein/Mitoxantrone Resistance/Placenta-Specific, ATP-binding Cassette Transporter as a Transporter of NB-506 and J-107088, Topoisomerase I Inhibitors with an Indolocarbazole Structure Cancer Res., April 1, 2001; 61(7): 2827 - 2832. [Abstract] [Full Text]
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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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 D. M. van der Kolk, E. G. E. de Vries, W. L. J. van Putten, L. F. Verdonck, G. J. Ossenkoppele, G. E. G. Verhoef, and E. Vellenga P-glycoprotein and Multidrug Resistance Protein Activities in Relation to Treatment Outcome in Acute Myeloid Leukemia Clin. Cancer Res., August 1, 2000; 6(8): 3205 - 3214. [Abstract] [Full Text]
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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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D. W. Loe, R. G. Deeley, and S. P. C. Cole Verapamil Stimulates Glutathione Transport by the 190-kDa Multidrug Resistance Protein 1 (MRP1) J. Pharmacol. Exp. Ther., May 1, 2000; 293(2): 530 - 538. [Abstract] [Full Text]
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M. Gao, H.-R. Cui, D. W. Loe, C. E. Grant, K. C. Almquist, S. P. C. Cole, and R. G. Deeley Comparison of the Functional Characteristics of the Nucleotide Binding Domains of Multidrug Resistance Protein 1 J. Biol. Chem., April 21, 2000; 275(17): 13098 - 13108. [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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 M. Sjolinder, L. Stenke, B. Nasman-Glaser, S. Widell, J. Doucet, P.-J. Jakobsson, and J. A. Lindgren Aberrant expression of active leukotriene C4 synthase in CD16+ neutrophils from patients with chronic myeloid leukemia Blood, February 15, 2000; 95(4): 1456 - 1464. [Abstract] [Full Text] [PDF]
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S. Prechtl, M. Roellinghoff, R. Scheper, S. P. C. Cole, R. G. Deeley, and M. Lohoff The Multidrug Resistance Protein 1: A Functionally Important Activation Marker for Murine Th1 Cells J. Immunol., January 15, 2000; 164(2): 754 - 761. [Abstract] [Full Text] [PDF]
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 E Bakos, R Evers, G Calenda, G. Tusnady, G Szakacs, A Varadi, and B Sarkadi Characterization of the amino-terminal regions in the human multidrug resistance protein (MRP1) J. Cell Sci., January 12, 2000; 113(24): 4451 - 4461. [Abstract] [PDF]
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M. L. O'Brien, B. Vulevic, S. Freer, J. Boyd, H. Shen, and K. D. Tew Glutathione Peptidomimetic Drug Modulator of Multidrug Resistance-Associated Protein J. Pharmacol. Exp. Ther., December 1, 1999; 291(3): 1348 - 1355. [Abstract] [Full Text]
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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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Z.-S. Chen, T. Kawabe, M. Ono, S. Aoki, T. Sumizawa, T. Furukawa, T. Uchiumi, M. Wada, M. Kuwano, and S.-I. Akiyama Effect of Multidrug Resistance-Reversing Agents on Transporting Activity of Human Canalicular Multispecific Organic Anion Transporter Mol. Pharmacol., December 1, 1999; 56(6): 1219 - 1228. [Abstract] [Full Text]
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M. Kitazono, T. Sumizawa, Y. Takebayashi, Z.-S. Chen, T. Furukawa, S. Nagayama, A. Tani, S. Takao, T. Aikou, and S.-i. Akiyama Multidrug Resistance and the Lung Resistance-Related Protein in Human Colon Carcinoma SW-620 Cells J Natl Cancer Inst, October 6, 1999; 91(19): 1647 - 1653. [Abstract] [Full Text] [PDF]
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R. A. M. H. Van Aubel, J. B. Koenderink, J. G. P. Peters, C. H. Van Os, and F. G. M. Russel Mechanisms and Interaction of Vinblastine and Reduced Glutathione Transport in Membrane Vesicles by the Rabbit Multidrug Resistance Protein Mrp2 Expressed in Insect Cells Mol. Pharmacol., October 1, 1999; 56(4): 714 - 719. [Abstract] [Full Text]
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B. D. Stride, S. P. C. Cole, and R. G. Deeley Localization of a Substrate Specificity Domain in the Multidrug Resistance Protein J. Biol. Chem., August 6, 1999; 274(32): 22877 - 22883. [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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D. R. Hipfner, Q. Mao, W. Qiu, E. M. Leslie, M. Gao, R. G. Deeley, and S. P. C. Cole Monoclonal Antibodies That Inhibit the Transport Function of the 190-kDa Multidrug Resistance Protein, MRP. LOCALIZATION OF THEIR EPITOPES TO THE NUCLEOTIDE-BINDING DOMAINS OF THE PROTEIN J. Biol. Chem., May 28, 1999; 274(22): 15420 - 15426. [Abstract] [Full Text] [PDF]
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T. Hirohashi, H. Suzuki, and Y. Sugiyama Characterization of the Transport Properties of Cloned Rat Multidrug Resistance-associated Protein 3 (MRP3) J. Biol. Chem., May 21, 1999; 274(21): 15181 - 15185. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, T. Furukawa, T. Sumizawa, K. Ono, K. Ueda, K. Seto, and S.-I. Akiyama ATP-Dependent Efflux of CPT-11 and SN-38 by the Multidrug Resistance Protein (MRP) and Its Inhibition by PAK-104P Mol. Pharmacol., May 1, 1999; 55(5): 921 - 928. [Abstract] [Full Text]
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Y. Cui, J. König, U. Buchholz, H. Spring, I. Leier, and D. Keppler Drug Resistance and ATP-Dependent Conjugate Transport Mediated by the Apical Multidrug Resistance Protein, MRP2, Permanently Expressed in Human and Canine Cells Mol. Pharmacol., May 1, 1999; 55(5): 929 - 937. [Abstract] [Full Text]
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M. Sjölinder, S. Tornhamre, H.-E. Claesson, J. Hydman, and J. A. Lindgren Characterization of a leukotriene C4 export mechanism in human platelets: possible involvement of multidrug resistance-associated protein 1 J. Lipid Res., March 1, 1999; 40(3): 439 - 446. [Abstract] [Full Text]
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 J. M. Drazen, E. Israel, and P. M. O'Byrne Treatment of Asthma with Drugs Modifying the Leukotriene Pathway N. Engl. J. Med., January 21, 1999; 340(3): 197 - 206. [Full Text] [PDF]
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R. Raggers, A van Helvoort, R Evers, and G van Meer The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane J. Cell Sci., January 2, 1999; 112(3): 415 - 422. [Abstract] [PDF]
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M. Homma, H. Suzuki, H. Kusuhara, M. Naito, T. Tsuruo, and Y. Sugiyama High-Affinity Efflux Transport System for Glutathione Conjugates on the Luminal Membrane of a Mouse Brain Capillary Endothelial Cell Line (MBEC4) J. Pharmacol. Exp. Ther., January 1, 1999; 288(1): 198 - 203. [Abstract] [Full Text]
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J. F. Rebbeor, G. C. Connolly, M. E. Dumont, and N. Ballatori ATP-dependent Transport of Reduced Glutathione on YCF1, the Yeast Orthologue of Mammalian Multidrug Resistance Associated Proteins J. Biol. Chem., December 11, 1998; 273(50): 33449 - 33454. [Abstract] [Full Text] [PDF]
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