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(Received for publication, November 30,
1995; and in revised form, January 29, 1996) From the
In addition to its ability to confer resistance to a range of
natural product type chemotherapeutic agents, multidrug resistance
protein (MRP) has been shown to transport the cysteinyl leukotriene,
LTC
Increased expression of multidrug resistance protein (MRP) ( The ability of
MRP to function as an ATP-dependent transporter has been investigated
using plasma membrane vesicles prepared from drug-selected and MRP
transfected cells(13, 14, 15, 55) .
These studies have shown that vesicles enriched in MRP display elevated
levels of ATP-dependent, high affinity transport of cysteinyl
leukotrienes. Evidence has also been presented that the protein can
transport other organic glutathione
conjugates(13, 14) , as well as oxidized glutathione
itself(16) . In the accompanying article (55) , we
provide immunological confirmation that MRP is a primary active
transporter of LTC ATP-dependent transport systems for
organic GSH conjugates (17) , GSSG(18) , and other
organic anions (19, 20) have been characterized
functionally in a number of tissues. Studies with intact cells or
membrane vesicle preparations indicate that at least one of the
transporters involved, frequently referred to as the multispecific
organic anion transporter (MOAT), has a broad substrate specificity
that includes other organic anions in addition to glutathione
conjugates(21) . MOAT has been studied most extensively in bile
canalicular membranes where it is believed to contribute to the hepatic
clearance of a wide range of xenobiotic and endogenous organic anions,
notably the glucuronidated conjugates of bile acids and
bilirubin(22) . The transporter is functionally defective in
hepatocanalicular membranes of the TR Recent immunohistological data
indicate that subcellular localization of MRP, or an MRP-related
protein, may be abnormal in the livers of TR
Figure 1:
Time course of
[
Correlation
between ATP-dependent [
Figure 2:
Effect of
[
Figure 3:
Osmotic sensitivity and nucleotide
specificity of [
Nucleotide dependent
transport was not detectable when AMP-PNP, AMP-PCP, or ATP
Figure 4:
Effect of glucuronidated estradiols on
[
Lineweaver-Burk plots of
[
Figure 5:
Effect of LTC
Figure 6:
Photoaffinity labeling of T14 membrane
vesicles by [
Figure 7:
Effects of chemotherapeutic agents on
[
MRP is expressed in a wide range of normal tissues (1, 28, 29, 30) and is overexpressed
in many multidrug-resistant cell
lines(1, 3, 5, 6, 41) .
Elevated levels of MRP in membrane vesicles from drug-selected cell
lines and MRP transfectants have been correlated with increased
ATP-dependent transport of cysteinyl leukotrienes and other glutathione S-conjugates(13, 14, 15) . It has
been suggested on the basis of these data, that one of the
physiological roles of MRP is that of a GSH conjugate or GS-X
pump(42, 43) . However, the ability to transport
certain glutathione conjugates does not explain the drug
cross-resistance profile of MRP-transfected or selected
cells(1, 5, 12) . Current data indicate that
the major classes of drugs to which MRP confers resistance are not
metabolized via GSH conjugation(44) , although a GSH-dependent
transport mechanism may exist for some compounds(55) . Other
conjugation pathways, such as glucuronidation, appear more important
for the detoxification of natural product drugs such as
VP-16(45) , at least in the liver, where glucuronidation is a
major biotransformation pathway for steroid hormones and bile salts.
Our data demonstrate that some glucuronide conjugates are potential
substrates for MRP. The rates of ATP-dependent transport of
E Despite the ability of MRP to
transport anionic compounds with no apparent structural similarity,
studies with steroid conjugates reported here indicate that substrate
affinity can be markedly influenced by the site of anionic conjugation.
In human liver, E It has been suggested previously that ATP-dependent
hepatocanalicular transport of E Direct binding and transport of
chemotherapeutic drugs by MRP has not been demonstrated, but
30-60% decreases in MRP-dependent LTC Data presented here combined with those of
previous studies demonstrate that the substrate specificities of MRP
and MOAT overlap extensively (27, 21, 40, 54) . Our studies on
the transport of steroid glucuronides are also consistent with a
potential role for MRP in bile formation and in the development of
cholestasis. However, it remains to be established whether MOAT is a
single protein or whether functionally similar but structurally
distinct tissue-specific forms exist. With detailed knowledge of the
substrate specificities of MRP and the availability of mAbs capable of
specifically inhibiting its function, it should soon be possible to
determine whether MRP and MOAT are the same protein or different
functionally related transporters.
Volume 271,
Number 16,
Issue of April 19, 1996 pp. 9683-9689
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Estradiol 17-(-D
-Glucuronide) Transport by
Multidrug Resistance Protein (MRP)
INHIBITION BY CHOLESTATIC STEROIDS (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential
physiological substrates also includes cholestatic glucuronidated
steroids. ATP dependent, osmotically sensitive transport of the
naturally occurring conjugated estrogen, 17
-estradiol
17-(-D-glucuronide) (E
17G), was readily
demonstrable in plasma membrane vesicles from populations of
MRP-transfected HeLa cells (V
1.4 nmol
mg
min, K
2.5 µM). The involvement of MRP was confirmed
by demonstrating that transport was completely inhibited by a
monoclonal antibody specific for an intracellular conformational
epitope of the protein. MRP-mediated transport of LTC was
competitively inhibited by E17G (K
22 µM),
despite the lack of structural similarity between these two substrates.
Competitive inhibition of
[
H]E17G transport was also
observed with a number of other cholestatic conjugated steroids. All of
these compounds prevented photolabeling of MRP with
[H]LTC, demonstrating that the
cholestatic steroid and leukotriene conjugates compete either for the
same or possibly overlapping sites on the protein. Consistent with the
presence of overlapping but non-identical sites, studies using
chemotherapeutic drugs to inhibit MRP-mediated E17G
transport indicated that daunorubicin had the highest relative potency
of the drugs tested, whereas it was the least potent inhibitor of
LTC transport. Non-cholestatic steroids glucuronidated at
the 3 position of the steroid nucleus, such as 17-estradiol
3-(-D-glucuronide), did not compete for transport of
E17G by MRP, nor did they inhibit photolabeling of the
protein with [H]LTC. These data
identify MRP as a potential transporter of cholestatic conjugated
estrogens and demonstrate site-specific requirements for
glucuronidation of the steroid nucleus.
)or its cognate mRNA has been found in drug selected cell
lines from a wide range of tumor types (1, 2, 3, 4, 5, 6, 7, 8) .
These cell lines have a phenotype similar in many respects to that
conferred by P-glycoproteins. Transfection of MRP expression vectors
into drug-sensitive cells has confirmed that MRP confers resistance to
a spectrum of natural product type chemotherapeutic
agents(9, 10) . MRP, like the P-glycoproteins, is a
member of the ATP-binding cassette superfamily of transmembrane
transporters. However, unlike the P-glycoproteins, it has not been
possible to demonstrate that MRP binds and actively transports
unmodified forms of the drugs to which it confers
resistance(11, 12, 55) . and demonstrate for the first time that
in the presence of GSH, MRP can actively transport the Vinca alkaloid, vincristine.
mutant Wistar
rat, the phenotype of which is similar to that observed in the human
Dubin-Johnson syndrome (23, 24) and the mutant
Corriedale sheep(21, 25) . All of these congenital
conditions are associated with chronic conjugated hyperbilirubinemia.
In the TR rat model, although hepatocanalicular MOAT
activity is markedly reduced, organic anion transport in other tissues
appears to be normal(26) . Whether this indicates the existence
of structurally distinct hepatic and non-hepatic forms of the
transporter, or is attributable to a liver-specific defect in its
expression has not been established. rats(27) . This suggests that MRP could be the
transporter functionally described as MOAT. However, although MRP mRNA
can be detected in both rodent and human liver, its levels are low when
compared with other tissues such as muscle, testes, heart, and
lung(1, 28, 29, 30) . To further
define the substrate specificity of MRP, we have used plasma membrane
vesicles from drug-selected and MRP-transfected cells, to examine ATP
dependent transport of [H]17-estradiol
17-(-D-glucuronide) (E17G). This
estradiol metabolite is formed in the liver and subsequently excreted
into bile(31, 32) . Increases in the levels of
E17G and some other conjugated estrogens have been
implicated in the development of cholestasis during the later stages of
pregnancy, a condition which occurs with exceptional frequency in the
Dubin-Johnson syndrome(33) . Consequently, the potential for
other known cholestatic and non-cholestatic compounds to inhibit
MRP-mediated E17G transport has also been determined.
Finally, we have examined the relative abilities of several natural
product drugs to which MRP confers resistance to inhibit transport of
the conjugated steroid and determined the influence of GSH and
glucuronate on their potencies.
Materials
E17G,
E3G, E3G, E16
G,
E17G, E3SO17G,
glycolithocholate-3-sulfate, E17E, taurocholic acid,
glycocholic acid, LTC, AMP, AMP-PNP, AMP-PCP, ATP
S,
GTP, CTP, and UTP were purchased from Sigma. ATP was purchased from ICN
Biochemicals (St. Laurent, PQ, Canada). Chemotherapeutic agents were
obtained as described(55) . Creatine phosphate and creatine
kinase were purchased from Boehringer Mannheim (Dorval, PQ, Canada).
[14,15,19,20-H]LTC (128 Ci
mmol) and
[6,7-H]E17G (49 Ci
mmol) were purchased from DuPont NEN (Mississauga,
Ontario, Canada) and Amplify
was from Amersham
(Oakville, Ontario, Canada). All other reagents were from Sigma.
Isolation and purification of the murine MRP-specific mAbs (QCRL-1 and
-3) used in this study have been described(34) .Cell Culture
The human small cell lung cancer cell
line, H69, the doxorubicin-selected multidrug-resistant cell line,
H69AR, and the drug-sensitive revertant cell line, H69PR, have been
described(35, 36) . The production and maintenance of
MRP transfected (T14) or vector transfected (C6) HeLa cell populations
were as described(12) . The 8226/Dox40 myeloma cell line was
provided by Dr. W. Dalton (Arizona Cancer Center, Tucson,
AZ)(37) . All cell lines were cultured in RPMI 1640 medium
supplemented with 4 mML-glutamine and 5% defined
bovine calf serum (HyClone Laboratories, Logan, UT), in the absence of
antibiotics.Membrane Vesicle Preparation and Transport of
E
Plasma membrane vesicles were prepared as
described(38, 39, 55) . ATP-dependent
transport of E17G17G into membrane vesicles was measured
at 37 °C in a 120-µl reaction volume containing 20-40
µg of vesicle protein in the presence of
[H]E17G (50 nM; 80
nCi/reaction). At indicated times, 20-µl samples were removed,
diluted into 1 ml of ice-cold transport buffer (50 mM Tris-HCl, 250 mM sucrose, pH 7.5), and filtered under
vacuum through glass fiber filters (type A/E; Gelman Sciences,
Montreal, PQ) presoaked in transport buffer. Filters were washed twice
with 5 ml of transport buffer, dried, and subjected to liquid
scintillation counting. All data were corrected for the amount of
[H]E17G which remained bound to
the filter in the absence of vesicle protein (usually <5% of the
total radioactivity).Inhibition of Photoaffinity Labeling of MRP with
[
Vesicle membrane proteins (150 µg) were
photoaffinity labeled with [H]LTC by Conjugated
SteroidsH]LTC (0.5 µCi; 78 nM) as described (55) in the
presence of various concentrations of E17G, or other
steroid derivatives. Radiolabeled vesicles (100 µg of membrane
protein) were solubilized in Laemmli's buffer, analyzed on a 7%
gel by SDS-PAGE, and subjected to fluorography.
ATP-dependent Transport of
E
The kinetics and ATP dependence of
[17GH]E17G accumulation by vesicles
prepared from MRP-transfected HeLa T14 cells are shown in Fig. 1A. ATP-dependent uptake was linear up to 60 s and
approached steady-state after 180 s. During the linear phase, the rate
of uptake at 37 °C was approximately 22 pmol mg
protein min at an initial
concentration of 50 nM [H]E17G and the rate
increased linearly with the amount of membrane protein up to 40 µg
(data not shown). [H]E17G uptake
in the presence of AMP rather than ATP was approximately 1 pmol
mg min. At the same initial
substrate concentration, the rate of [H]LTC uptake by T14 vesicles was 150-200 pmol mg min at 23 °C. No ATP dependence of
[H]E17G uptake could be
demonstrated with vesicles from control C6 cells.
H]E17G uptake by membrane
vesicles from MRP-transfected HeLa cells (T14), control HeLa (C6)
cells, drug-sensitive H69 cells, multidrug-resistant H69AR cells,
revertant H69PR cells, and P-glycoprotein overexpressing 8226/Dox40
cells. Membrane vesicles were incubated at 37 °C in transport
buffer containing [H]E17G (50
nM, specific activity 13 Ci mmol) and ATP
(4 mM) (closed symbols) or AMP (4 mM) (open symbols) for the times indicated. Vesicles were derived
from the following cells, as described under ``Experimental
Procedures:'' Panel A, HeLa C6 (
, 
) and T14
(
, 
); Panel B, H69 (, ) and H69AR
(
,
); Panel C, H69PR (
, 
); Panel
D, 8226/Dox40 (
, 
). The broken curves in Panels A and B indicate ATP-dependent uptake for T14
and H69AR cells, respectively. Data points represent the means
(± S.E.) of triplicate determinations in a typical
experiment.
H]E17G
transport rates and levels of MRP expression was examined using
vesicles isolated from drug-sensitive H69, drug-resistant H69AR, and
revertant H69PR cells. The rate of ATP-dependent
[H]E17G uptake in H69AR vesicles
(60 pmol mg min) was
approximately 3-fold higher than for T14 vesicles (Fig. 1B), consistent with the 3-4-fold higher
levels of MRP in H69AR cells (12) and vesicle preparations
(data not shown). No ATP dependence of
[H]E17G uptake could be
demonstrated with H69 vesicles. The rate of ATP-dependent
[H]E17G uptake in H69PR membrane
vesicles was approximately 1% that of H69AR cells (Fig. 1C). This is consistent with the low, but
detectable levels of MRP mRNA (1) and protein (34) in
these cells. To examine the ability of P-glycoprotein to transport
E17G, we used vesicles from the multidrug-resistant
myeloma cell line, 8226/Dox40, which overexpresses P-glycoprotein (37) and has MRP levels comparable to those in H69PR cells
(data not shown). Vesicles from these cells exhibited levels of
[H]E17G transport no higher than
those of H69PR-derived vesicles (Fig. 1D). Thus the
data are consistent with E17G transport by 8226/Dox40
vesicles being attributable to their low levels of MRP. They also
indicate that there is little if any transport of E17G
by P-glycoprotein.Kinetic Parameters of
[
Rates of ATP-dependent uptake were determined at
several concentrations of EH]E17G Transport in HeLa T14
Vesicles17G (12.5 nM to 25
µM) and used to calculate K
and V for transport by T14 membrane vesicles. A
Lineweaver-Burk plot of the data yielded an apparent K of 2.5 µM for E17G and a V of 1.4 nmol mg min (Fig. 2A, inset).
An apparent K for ATP of 390 µM was
also determined by measuring initial rates of
[H]E17G uptake at 60 s in the
presence of different concentrations of nucleotide (1-4000
µM) (Fig. 2B, inset).
H]E17G and ATP concentration on
[H]E17G uptake by T14 vesicles. Panel A, the rate of ATP dependent
[H]E17G uptake by T14 membrane
vesicles was measured at various E17G concentrations
(12.5 nM to 25 µM) for up to 60 s at 37 °C in
the presence of a fixed concentration of nucleotide (4 mM), as
described under ``Experimental Procedures.'' Data were
plotted as V
versus [S] to
confirm that the concentration range selected was appropriate to
observe both zero-order and first-order rate kinetics. Kinetic
parameters (K 2.5 µM and V 1.4 nmol mg min) were determined from regression analysis
of the Lineweaver-Burk transformation of the data (inset). Panel B, ATP-dependent uptake of
[H]E17G was measured as
described for Panel A at various concentrations of nucleotide
(1 to 4000 µM) in the presence of a fixed concentration of
[H]E17G (50 nM). An
apparent K of 390 µM for ATP
was determined from regression analysis of the Lineweaver-Burk data
transformation (inset).
Osmotic Sensitivity and Nucleotide Specificity of
[
The rate of
[H]E17G Transport by T14
Membrane VesiclesH]E17G uptake in T14 vesicles
was osmotically-sensitive and decreased linearly with increasing
extravesicular sucrose concentration between 0.25 and 1.0 M (Fig. 3A). Steady-state levels of accumulation
showed similar osmotic sensitivity (data not shown) confirming that the
increase in vesicle associated
[H]E17G reflected transport into
the vesicle lumen rather than surface binding.
H]E17G transport
by T14 membrane vesicles. Panel A, T14 membrane vesicles were
preincubated for 10 min in transport buffer containing sucrose at
concentrations ranging from 250 to 1000 mM. Rates of
[H]E17G uptake at 37 °C
under various conditions of osmolarity were measured at a substrate
concentration of 50 nM in the presence of 4 mM AMP
() or ATP (), as described under ``Experimental
Procedures.'' Panel B, rates of
[H]E17G uptake were determined
at 37 °C, in the presence of various hydrolyzable and
non-hydrolyzable nucleotides (4 mM), as described under
``Experimental Procedures'' except that the nucleoside
triphosphate regenerating system was omitted. The rate observed in the
presence of 4 mM ATP was not affected by omission of the
regenerating system (data not shown). The rates of uptake supported by
various nucleotides have been expressed as a percentage of that
obtained with ATP. The results shown are the means (± S.E.) of
triplicate determinations in a single experiment. The rate of
ATP-dependent uptake in the control was 31.7 pmol mg min.
S (4
mM) were substituted for ATP, thus indicating a requirement
for ATP hydrolysis (Fig. 3B). As expected when compared
with other nucleotide triphosphates, ATP supported the highest rate of
[H]E17G transport. However, the
rate of transport in the presence of GTP was substantial and approached
approximately 70% of that achievable in the presence of ATP.Inhibition of Transport by MRP Specific mAbs
To
obtain additional evidence of a direct involvement of MRP in the
transport of E17G, we examined the ability of two MRP
specific mAbs to inhibit this process. mAb QCRL-3, which is specific
for an intracellular conformation-dependent epitope of
MRP(34) , completely inhibited
[H]E17G uptake at a
concentration of 20 µg ml. In contrast, the same
concentration of mAb QCRL-1, which recognizes a linear intracellular
epitope of MRP(34) , had no significant effect. Isotype control
immunoglobulins (mouse IgG, IgG
, and total
IgG) were also without effect at concentrations of 70 µg
ml.Inhibition of [
To determine whether
EH]LTC Transport by Estradiol Glucuronides17G and LTC interact with the same or
overlapping sites on the protein, we examined the ability of
E17G, and its structural isomer, E3G,
to inhibit ATP-dependent [H]LTC transport by T14 vesicles. E17G acted as a
competitive inhibitor, with an apparent K
of 22
µM (Fig. 4, upper panel). In contrast, the
non-cholestatic estrogen conjugate, E3G, did not
inhibit [H]E17G transport at
concentrations up to 100 µM (Fig. 4, lower
panel). These data suggest that LTC and
E17G interact with the same or overlapping sites on
MRP. They also demonstrate that interaction of the estrogen
glucuronides with MRP displays specificity with respect to the position
of the glucuronide moiety on the steroid nucleus.
H]LTC uptake by T14 membrane
vesicles. The rates of [H]LTC uptake
by T14 vesicles were determined at 23 °C, at various substrate
concentrations (25 to 1000 nM) in the absence () or
presence (, 20 µM; , 40 µM;
, 100 µM) of E17G (upper
panel) or E3G (lower panel).
Double-reciprocal plots were generated for each concentration of
potential inhibitor and used to determine K. The results shown are the means
(±S.E.) of triplicate determinations at each substrate and
inhibitor concentration.
Inhibition of
[
EH]E17G Transport by Various
Steroid Glucuronides and Bile Salt
Derivatives17G and other steroid D-ring
glucuronide conjugates have been shown to inhibit bile flow and to
cause a reversible type of
cholestasis(31, 32, 33) . In contrast,
steroid glucuronide conjugates of the A ring such as
E3G are not cholestatic (40, and references cited
therein). Consequently, we compared the cholestatic potency of various
estriol, estradiol, and bile salt derivatives with their ability to
inhibit [H]E17G transport by T14
membrane vesicles. Glucuronic acid itself was not inhibitory up to 5
mM and did not augment the modest inhibition (20%) of
[H]E17G transport by
17-estradiol (10 µM) (Table 1). Of the estrogen
conjugates tested, 17-(-D-glucuronides) of estradiol and
estriol and the 3-sulfate derivative of E17G were the
most potent inhibitors of transport (approximately 90% inhibition or
greater at 10 µM). In contrast, the
3-(-D-glucuronides) of estradiol and estriol were not
inhibitory and only modest inhibition was observed with
E16G. However, the cholestatic bile salt
glycolithocholate-3-sulfate was a potent inhibitor of transport (94%
inhibition at 10 µM) while glycocholate and taurocholate
(which are not derivatized at the 3-position of the A ring) were far
less inhibitory (30 and 35% at 10 µM, respectively). The
17-substituted synthetic estrogen,
17-ethinyl-17-estradiol, was not inhibitory.
H]E17G uptake by T14 membrane
vesicles indicated that LTC behaved as a competitive
inhibitor of [H]E17G transport,
with a K of 0.53 µM (Fig. 5). Inhibition by E17G (K 1.4 µM) and the
cholestatic bile salt glycolithocholate-3-sulfate (K 1.4 µM) (data not
shown) was also competitive. E16G, which is of
intermediate cholestatic potency (40) , was a less effective
competitive inhibitor of transport, with a K of 45 µM. Although
non-cholestatic in rodents, E3SO17G, was
also an effective competitive inhibitor of E17G
transport by MRP (K 1.7
µM).
, steroid
glucuronides, and bile salt derivatives on transport of
[H]E17G by T14 membranes. The
rates of uptake of [H]E17G by
T14 vesicles were determined at 37 °C, at various substrate
concentrations (0.05-25 µM) in the absence or
presence of the indicated concentration of LTC (1
µM) (upper panel), E17G (1
µM) or E16G (100 µM) (middle panel), and E3SO17G (1
µM) (lower panel). Double reciprocal plots were
generated for each inhibitor and used to determine a K. Results shown are the means
(±S.E.) of triplicates determinations at each substrate and
inhibitor concentration.
Photolabeling of Membrane Proteins with
[
To confirm the results of inhibition
studies, we determined the ability of various steroid derivatives to
compete for [H]LTC and Inhibition of Labeling by
Steroid GlucuronidesH]LTC binding to
MRP(55) . E17G inhibited photolabeling of MRP
by [H]LTC in a
concentration-dependent manner, with a IC
of approximately
20 µM (Fig. 6A). We also tested the
ability of a single concentration (100 µM) of cholestatic
and non-cholestatic steroid glucuronides and bile salts to inhibit
[H]LTC binding. Consistent with the
results of transport studies, photolabeling was not inhibited by
non-cholestatic E3G and E3G, but was
abolished by the cholestatic steroids, glycolithocholate-3-sulfate,
E17G, E3SO17G, and
E17G (Fig. 6B).
H]LTC and inhibition of
labeling by E17G and other steroid glucuronides. Panel A, T14 vesicles (150 µg of membrane protein) were
incubated with [H]LTC (50
nM) alone or in the presence of various concentrations of
E17G (5-100 µM) or 100 µM E3G, as indicated. Samples were irradiated at 312
nm prior to being subjected to SDS-PAGE and fluorography, as described (55) . Panel B, photoaffinity labeling of T14 membrane
vesicles with [H]LTC was carried out
as above in the presence of various glucuronides and bile salt
derivatives (100 µM), as indicated. Photolabeled membranes
were analyzed by SDS-PAGE and fluorography. DMSO, dimethyl
sulfoxide.
Inhibition of
[
Examples of each of the three major
classes of natural product type drugs to which MRP confers resistance
were compared for their ability to inhibit
[H]E17G Transport by
Chemotherapeutic AgentsH]E17G transport by T14
vesicles (Fig. 7, upper panel). VP-16 and vincristine
(100 µM) inhibited transport by 50-60%. Vinblastine
and the anthracyclines, daunorubicin and doxorubicin, inhibited
transport by 80-90% at the same concentration. Determination of
the concentration of drug (in the absence of GSH) required to inhibit
[H]E17G transport by 50%
indicated that daunorubicin (IC approximately 8
µM) was more potent than doxorubicin (IC approximately 50 µM), vinblastine (IC approximately 30 µM), and vincristine (IC approximately 70 µM) (Fig. 7, lower
panel). Physiological concentrations of GSH enhance the inhibitory
effect of vincristine and vinblastine (and to a lesser extent VP-16) on
MRP dependent transport of
[H]LTC(55) . Consequently, we
examined whether a similar effect was observed with
[H]E17G. GSH (1 mM)
increased inhibition from approximately 50 to 75% and from 60 to 90%,
for VP-16 and vincristine, respectively, but it did not enhance
transport inhibition by vinblastine or the anthracyclines (Fig. 7, upper panel). In contrast, glucuronic acid (5
mM) could not substitute for GSH and did not enhance
inhibition by any of the drugs tested (Fig. 7, upper
panel).
H]E17G uptake by T14 vesicles
in the absence or presence of GSH or glucuronic acid. Upper
panel, the rate of uptake of
[H]E17G (50 nM) by T14
vesicles was determined at 37 °C in the presence of various natural
product drugs (100 µM) alone (open bars) and in
combination with GSH (1 mM) (hatched bars) or
glucuronic acid (5 mM) (solid bars). Results are
expressed as a percentage of the control value obtained in the absence
of drug and anion. The drug vehicle (dimethyl sulfoxide) was present at
a concentration of 0.5% in control incubations lacking drug and had no
effect on uptake under the conditions of the assay. The results are the
means (±S.E.) of triplicate determinations in a single
experiment. The control uptake rate in this experiment was 19.2 pmol
mg min. DOX,
doxorubicin; DNR, daunorubicin; VBL, vinblastine; VCR, vincristine. Lower panel, inhibition of
[H]E17G uptake by T14 membrane
vesicles by various concentrations of DNR (), DOX (), VCR
(), or VBL (). Data points represent means of duplicate
determinations in a single experiment and are plotted as a percentage
of control values obtained in the absence of drug, but in the presence
of 0.5% (v/v) dimethyl sulfoxide. The control uptake rate in the
experiment shown was 25.8 pmol mg min.
17G by membrane vesicles from MRP-transfected HeLa
T14 cells were more than 20-fold higher than vesicles from cells
transfected with vector alone. In addition, the rates of
E17G transport obtained with vesicles from previously
characterized H69, H69AR, and H69PR small cell lung cancer cell lines
correlate well with their levels of MRP(1, 34) .
Several other lines of evidence confirm that MRP is the primary active
transporter involved. 1) A conformation dependent, MRP-specific mAb
that inhibits LTC transport by MRP(55) , also
inhibits E17G transport; 2) E17G and
LTC compete for ATP-dependent transport by MRP enriched
vesicles; and 3) E17G blocks the photolabeling of MRP
by [H]LTC in a
concentration-dependent manner. Thus LTC and
E17G appear to interact with similar or at least
mutually exclusive sites on MRP.17G and E3G are
formed in approximately equal amounts(46, 47) .
However, only the former is cholestatic. It has been suggested that one
mechanism by which some estrogen D-ring conjugates may diminish bile
flow is by competing for transport by one or more of the ATP-dependent
hepatocanalicular transport proteins. Whether or not MRP is the
transporter involved remains to be firmly established. However, the
substrate specificity of MRP correlates well with the cholestatic
potential of A-ring and D-ring steroid glucuronides. Non-cholestatic
E3G does not inhibit
[H]LTC transport by MRP nor does it
inhibit photolabeling of the protein. Similarly, E3G
and E3G do not compete for
[H]E17G transport. Thus A-ring
glucuronidation is insufficient to result in detectable interaction
with MRP. Alternative forms of anionic modification of the A-ring, such
as sulfation, also have little effect on the inhibitory potency of
17-(-D-glucuronides) (e.g. E3SO17G has a K of 1.7 µM with respect to E17G
transport). In contrast, sulfation of the A-ring of bile salts may
enhance inhibitory potency, since glycocholic acid itself is a
relatively weak inhibitor of E17G transport (30%
inhibition at 10 µM) compared with the cholestatic bile
salt glycolithocholate-3-sulfate (approximately 95% inhibition at 10
µM). More surprising, given the major structural
differences between some MRP substrates, is the finding that inhibitory
potency is sensitive to the position of glucuronidation within the
D-ring itself. The K of the moderately cholestatic
glucuronide, E16G, is more than 20-fold higher than
that of E17G. These studies demonstrate that the
protein can be highly selective with respect to the site of anionic
conjugation of some substrates and provide information of potential use
in designing agents capable of blocking MRP function. The behavior of
the synthetic estrogen, 17-ethinyl-17-estradiol is an
exception to the correlation between cholestatic potential and the
ability to inhibit MRP-dependent transport. This compound does not
inhibit transport of E17G in vitro but is
cholestatic. However, it is thought that the glucuronide of this
synthetic estrogen, produced in the liver, rather than the parent
compound is responsible for the cholestasis(33, 48) . 17G is attributable to
P-glycoprotein(32) . This suggestion stems from studies with
two drug-selected cell lines known to overexpress P-glycoprotein, which
displayed approximately 5-fold increased resistance to cytotoxic
concentrations of E17G and a 2-3-fold decrease
in accumulation of the compound. We have found that
E17G transport in vesicles from the P-glycoprotein
overexpressing myeloma cell line, 8226/Dox40, is approximately 20- and
100-fold lower than in vesicles from MRP transfected T14 and
drug-selected H69AR cells, respectively. Moreover, the drug-resistant
myeloma cells also express low but sufficient levels of MRP to account
for the low level of E17G transport observed. These
data combined with recent reports of multidrug-resistant cell lines
that express elevated levels of both P-glycoprotein and MRP (8, 49, 50) suggest that MRP rather than
P-glycoprotein is responsible for the previously observed transport of
E17G(32) . transport have
been observed in high concentrations (approximately 100
µM) of certain chemotherapeutic agents (15, 55) . With some drugs (e.g. vincristine
and vinblastine), we have shown that inhibition is potentiated by
physiological concentrations of GSH and have demonstrated
ATP/GSH-dependent transport of vincristine using T14
vesicles(55) . Data presented here demonstrate similar
inhibition of E17G transport with several
chemotherapeutic agents and potentiation of inhibition by vincristine
in the presence of GSH. A possible explanation of this behavior is that
inhibition and/or transport is enhanced as a result of independent
interactions of GSH and the Vinca alkaloids with distinct
regions of a composite binding site on MRP. Consequently, we determined
whether the anionic substituents of known MRP substrates, other than
GSH, might act in a similar fashion. However, no potentiation of
inhibition was observed in the presence of glucuronate with any of the
drugs tested and the anion also failed to enhance inhibition
E17G transport by 17-estradiol. Inorganic sulfate
was also without effect (data not shown). Thus the ability to enhance
inhibition and/or transport appears to be GSH-specific, rather than a
general property of the known anionic substituents of MRP substrates.
In addition, although all drugs tested inhibited E17G
and LTC transport, their relative potencies differ. For
example, daunorubicin is the most potent inhibitor of
E17G transport while it is the least potent inhibitor
of LTC transport(51, 55) . Similar
differences in the relative potency of various inhibitors have been
observed previously in the transport and/or binding of different
substrates by P-glycoprotein(52, 53) . One
interpretation is that interaction of potential substrates or
inhibitors with both proteins may occur via multiple, overlapping but
not identical sites.
),-methyleneadenosine 5`-triphosphate; AMP-PNP, adenosine
5`-[,-imido]triphosphate; ATPS, adenosine
5`-O-(3-thiotriphosphate); E17E,
17-ethinyl-17-estradiol;
E3SO17G, 17-estradiol
3-sulfato-17-(-D-glucuronide); E3G,
17-estradiol 3-(-D-glucuronide);
E3G, 16,17-estriol
3-(-D-glucuronide); E16G,
16,17-estriol 16-(-D-glucuronide);
E17G, 17-estradiol
17-(-D-glucuronide); E17G,
16,17-estriol 17-(-D-glucuronide);
glycolithocholate-3-sulfate, 3-hydroxy-5-cholan-24-oic acid N-[carboxymethyl]amide 3-sulfate; LTC,
leukotriene C; mAb, monoclonal antibody; MOAT,
multispecific organic anion transporter; PAGE, polyacrylamide gel
electrophoresis.
We thank D. R. Hipfner for providing MRP-specific
monoclonal antibodies and Dr. C. E. Grant for providing HeLa C6 and T14
transfected 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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E. Bakos, R. Evers, G. Szakacs, G. E. Tusnady, E. Welker, K. Szabo, M. de Haas, L. van Deemter, P. Borst, A. Varadi, et al. Functional Multidrug Resistance Protein (MRP1) Lacking the N-terminal Transmembrane Domain J. Biol. Chem., November 27, 1998; 273(48): 32167 - 32175. [Abstract] [Full Text] [PDF]
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Y. Yamane, M. Furuichi, R. Song, N. T. Van, R. T. Mulcahy, T. Ishikawa, and M. T. Kuo Expression of Multidrug Resistance Protein/GS-X Pump and gamma -Glutamylcysteine Synthetase Genes Is Regulated by Oxidative Stress J. Biol. Chem., November 20, 1998; 273(47): 31075 - 31085. [Abstract] [Full Text] [PDF]
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M. Alvarez, R. Robey, V. Sandor, K. Nishiyama, Y. Matsumoto, K. Paull, S. Bates, and T. Fojo Using the National Cancer Institute Anticancer Drug Screen to Assess the Effect of MRP Expression on Drug Sensitivity Profiles Mol. Pharmacol., November 1, 1998; 54(5): 802 - 814. [Abstract] [Full Text]
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X.-B. Chang, Y.-X. Hou, and J. R. Riordan Stimulation of ATPase Activity of Purified Multidrug Resistance-associated Protein by Nucleoside Diphosphates J. Biol. Chem., September 11, 1998; 273(37): 23844 - 23848. [Abstract] [Full Text] [PDF]
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M. Klein, E. Martinoia, and G. Weissenbock Directly Energized Uptake of beta -Estradiol 17-(beta -D-Glucuronide) in Plant Vacuoles Is Strongly Stimulated by Glutathione Conjugates J. Biol. Chem., January 2, 1998; 273(1): 262 - 270. [Abstract] [Full Text] [PDF]
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X.-B. Chang, Y.-X. Hou, and J. R. Riordan ATPase Activity of Purified Multidrug Resistance-associated Protein J. Biol. Chem., December 5, 1997; 272(49): 30962 - 30968. [Abstract] [Full Text] [PDF]
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C. Kast and P. Gros Topology Mapping of the Amino-terminal Half of Multidrug Resistance-associated Protein by Epitope Insertion and Immunofluorescence J. Biol. Chem., October 17, 1997; 272(42): 26479 - 26487. [Abstract] [Full Text] [PDF]
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B. D. Stride, C. E. Grant, D. W. Loe, D. R. Hipfner, S. P. C. Cole, and R. G. Deeley Pharmacological Characterization of the Murine and Human Orthologs of Multidrug-Resistance Protein in Transfected Human Embryonic Kidney Cells Mol. Pharmacol., September 1, 1997; 52(3): 344 - 353. [Abstract] [Full Text]
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D. W. Loe, R. K. Stewart, T. E. Massey, R. G. Deeley, and S. P. C. Cole ATP-Dependent Transport of Aflatoxin B1 and Its Glutathione Conjugates by the Product of the Multidrug Resistance Protein (MRP) Gene Mol. Pharmacol., June 1, 1997; 51(6): 1034 - 1041. [Abstract] [Full Text]
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M. Gao, D. W. Loe, C. E. Grant, S. P.C. Cole, and R. G. Deeley Reconstitution of ATP-dependent Leukotriene C4 Transport by Co-expression of Both Half-molecules of Human Multidrug Resistance Protein in Insect Cells J. Biol. Chem., November 1, 1996; 271(44): 27782 - 27787. [Abstract] [Full Text] [PDF]
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D. W. Loe, K. C. Almquist, R. G. Deeley, and S. P. C. Cole Multidrug Resistance Protein (MRP)-mediated Transport of Leukotriene C(4) and Chemotherapeutic Agents in Membrane Vesicles J. Biol. Chem., April 19, 1996; 271(16): 9675 - 9682. [Abstract] [Full Text] [PDF]
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K. Nagata, M. Nishitani, M. Matsuo, N. Kioka, T. Amachi, and K. Ueda Nonequivalent Nucleotide Trapping in the Two Nucleotide Binding Folds of the Human Multidrug Resistance Protein MRP1 J. Biol. Chem., June 2, 2000; 275(23): 17626 - 17630. [Abstract] [Full Text] [PDF]
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Y.-x. Hou, L. Cui, J. R. Riordan, and X.-b. Chang Allosteric Interactions between the Two Non-equivalent Nucleotide Binding Domains of Multidrug Resistance Protein MRP1 J. Biol. Chem., June 30, 2000; 275(27): 20280 - 20287. [Abstract] [Full Text] [PDF]
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Q. Mao, R. G. Deeley, and S. P. C. Cole Functional Reconstitution of Substrate Transport by Purified Multidrug Resistance Protein MRP1 (ABCC1) in Phospholipid Vesicles J. Biol. Chem., October 27, 2000; 275(44): 34166 - 34172. [Abstract] [Full Text] [PDF]
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S. Ryu, T. Kawabe, S. Nada, and A. Yamaguchi Identification of Basic Residues Involved in Drug Export Function of Human Multidrug Resistance-associated Protein 2 J. Biol. Chem., December 8, 2000; 275(50): 39617 - 39624. [Abstract] [Full Text] [PDF]
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Y.-M. Qian, W.-C. Song, H. Cui, S. P. C. Cole, and R. G. Deeley Glutathione Stimulates Sulfated Estrogen Transport by Multidrug Resistance Protein 1 J. Biol. Chem., February 23, 2001; 276(9): 6404 - 6411. [Abstract] [Full Text] [PDF]
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G. Liu, R. Sanchez-Fernandez, Z.-S. Li, and P. A. Rea Enhanced Multispecificity of Arabidopsis Vacuolar Multidrug Resistance-associated Protein-type ATP-binding Cassette Transporter, AtMRP2 J. Biol. Chem., March 16, 2001; 276(12): 8648 - 8656. [Abstract] [Full Text] [PDF]
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D.-W. Zhang, S. P. C. Cole, and R. G. Deeley Identification of an Amino Acid Residue in Multidrug Resistance Protein 1 Critical for Conferring Resistance to Anthracyclines J. Biol. Chem., April 13, 2001; 276(16): 13231 - 13239. [Abstract] [Full Text] [PDF]
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K.-i. Ito, S. L. Olsen, W. Qiu, R. G. Deeley, and S. P. C. Cole Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport J. Biol. Chem., May 4, 2001; 276(19): 15616 - 15624. [Abstract] [Full Text] [PDF]
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E. M. Leslie, K.-i. Ito, P. Upadhyaya, S. S. Hecht, R. G. Deeley, and S. P. C. Cole Transport of the beta -O-Glucuronide Conjugate of the Tobacco-specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by the Multidrug Resistance Protein 1 (MRP1). REQUIREMENT FOR GLUTATHIONE OR A NON-SULFUR-CONTAINING ANALOG J. Biol. Chem., July 20, 2001; 276(30): 27846 - 27854. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, K. Lee, and G. D. Kruh Transport of Cyclic Nucleotides and Estradiol 17-beta -D-Glucuronide by Multidrug Resistance Protein 4. RESISTANCE TO 6-MERCAPTOPURINE AND 6-THIOGUANINE J. Biol. Chem., August 31, 2001; 276(36): 33747 - 33754. [Abstract] [Full Text] [PDF]
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D.-W. Zhang, S. P. C. Cole, and R. G. Deeley Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity. EVIDENCE FOR FUNCTIONAL INTERACTION BETWEEN TRANSMEMBRANE HELICES 14 AND 17 J. Biol. Chem., September 7, 2001; 276(37): 34966 - 34974. [Abstract] [Full Text] [PDF]
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K.-i. Ito, C. J. Oleschuk, C. Westlake, M. Z. Vasa, R. G. Deeley, and S. P. C. Cole Mutation of Trp1254 in the Multispecific Organic Anion Transporter, Multidrug Resistance Protein 2 (MRP2) (ABCC2), Alters Substrate Specificity and Results in Loss of Methotrexate Transport Activity J. Biol. Chem., October 5, 2001; 276(41): 38108 - 38114. [Abstract] [Full Text] [PDF]
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