From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 and § Wadsworth Center, Albany, New York 12201
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ABSTRACT |
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To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred ~4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.
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INTRODUCTION |
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The glutathione S-transferases (GSTs)1 catalyze the conjugation with glutathione of a number of electrophilic xenobiotics, including several carcinogens, mutagens, and anticancer drugs (1-6). Usually, but not invariably, these electrophiles are made less reactive by conjugation with glutathione, and the conjugates are thought to be less toxic to the cell. Consequently, GSTs are believed to play an important role in the defense of cells against these xenobiotic toxins.
Several antineoplastic drugs, particularly the reactive electrophilic alkylating agents, can form conjugates with glutathione both spontaneously and in GST-catalyzed reactions (7-14). Despite these catalytic activities, the role of GSTs in the protection of cells from the cytotoxicities of these cancer drugs remains equivocal due to the inconsistent results obtained by different laboratories (5, 15-25). Although some investigators have found associations between cellular resistance to some anticancer drugs and expression of particular isozymes of GST, other investigators have found no such associations in other cell lines. In MCF7 breast carcinoma cells, increased expression of Alpha, Mu, or Pi class GSTs failed to confer any consistent, significant resistance to a variety of anticancer drugs, including drugs known to be substrates of GSTs (19, 20, 24). We recently proposed that conjugation of some of these drugs and toxins with glutathione may represent only partial detoxification. In this view, export of the glutathione conjugate is required to fully potentiate the GST-mediated protection. The identification of MRP1 as an important glutathione-conjugate efflux pump (26-30) raises the possibility that MRP1 and GST may act in synergy to confer cellular resistance to some of these compounds (31). This hypothesis was recently validated for the model carcinogen, 4-nitroquinoline-1-oxide (31). These studies showed that GST P1-1-associated protection from 4-nitroquinoline-1-oxide cytotoxicity was dependent upon concomitant expression of MRP1. Additionally, GST P1-1-associated protection from 4-nitroquinoline-1-oxide-mediated DNA adduct formation was greatly enhanced by co-expression of MRP1.
Previous studies failed to demonstrate an association between increased MRP1 expression and resistance to alkylating agents in MCF7/VP cells (32). In another study examining paired cell lines that differed in the levels of MRP1 expressed, increased MRP1 was associated with chlorambucil resistance in one but not the other two paired cell lines (33). These inconsistent relationships between increased MRP1 and alkylating agent sensitivity indicate that other factors, such as GST or glutathione levels, may be important in determining whether or not MRP1 will mediate protection from the cytotoxicities of some alkylating cancer drugs. Additionally, properties of some of the bifunctional alkylating agents and their metabolites make them particularly interesting candidates for GST/MRP1-mediated detoxification. Even after formation of monoglutathionyl derivatives, some of these bifunctional alkylating agents retain significant reactivity at the unmodified alkylating group and may therefore remain cytotoxic. Moreover, glutathione conjugates of some of these compounds, the nitrogen mustards melphalan and chlorambucil, are known to be transported by MRP1-containing membrane vesicles in vitro (27, 33).
We have developed cellular models to examine the role of GST/MRP1 synergy in the emergence of anticancer drug resistance. Although cell-free, in vitro analyses of toxin conjugation and transport are informative, cellular models of MRP1 and GST function are essential to determine the cytoprotective consequences of coordinated MRP1 and GST expression and to determine the precise mechanisms of cellular detoxifications. Accordingly, from MCF7 cells that express extremely low levels of MRP1 and GST, we have developed derivative sublines that express MRP1 alone or in combination with representatives of three major classes of human cytosolic GSTs, GST A1-1, M1-1, and P1-1. Results show that GST A1-1 operates in synergy with MRP1 to confer resistance to the antineoplastic nitrogen mustard chlorambucil. The studies indicate the mechanism of synergy involves GST catalytic activity as well as MRP1-mediated efflux of glutathione conjugates.
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EXPERIMENTAL PROCEDURES |
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Drugs and Chemicals--
Mitoxantrone,
1,3-bis(2-chloroethyl)-1-nitrosourea, thiotepa, and hepsulfan were
provided by the Drug Synthesis and Chemistry Branch, Developmental
Therapeutics Program of the NCI, National Institutes of Health
(Bethesda, MD). Geneticin was from Life Technologies, Inc., and
hygromycin was from Calbiochem. All other drugs were from Sigma. Stock
solutions stored at 80 °C were VP-16 (5 mM in 50%
ethanol), doxorubicin (5 mM in H2O),
vincristine (1 mM in H2O), mitoxantrone (5 mM in dimethyl sulfoxide), dicumarol (50 mM in
0.1 N NaOH), and sulfinpyrazone (200 mM in dimethyl sulfoxide). The following were prepared fresh: chlorambucil (100 mM in ethanol), melphalan (16.4 mM in acidified
ethanol (~0.4 N HCl)),
1,3-bis(2-chloroethyl)-1-nitrosourea (250 mM in 71%
ethanol), and thiotepa (100 mM in H2O).
Cell Lines and Tissue Culture--
Cells were grown at 37 °C,
5% CO2 in DMEM supplemented with 10% fetal calf serum.
All cell lines were derived from cloned parental MCF7 breast carcinoma
cell lines, MCF7/WT (GST/MRP1
), and the MDR derivative MCF7/VP
(GST-/MRP1+) (32). Cells expressing human isozymes of GST, GST A1-1,
GST M1-1, or GST P1-1 were established by stable transfection of
MCF7/VP cells with: 1) the
pCEP4 vector (34) containing cDNA
inserts (35-37) encoding the human GSTA1 gene (MCF7/VP
)
or the GSTM1 gene (MCF7/VPµ), or 2) the pcDNA3.1 vector (Invitrogen, Carlsbad, CA) containing the cDNA insert
encoding the human GSTP1*A allele (MCF7/VP
) as described
previously (31). Control cell lines were produced by stable
transfection of MCF7/WT and MCF7/VP cells with pcDNA3.1 or
pCEP4
lacking GST cDNA inserts (MCF7/WT-neo, MCF7/WT-hyg, MCF7/VP-neo,
and MCF7/VP-hyg). Cells transfected with pcDNA3- or
pCEP4-based
vectors were selected in 1 mg/ml geneticin or 0.4 mg/ml hygromycin,
respectively. Transfected cells were routinely grown in the selecting
drug (geneticin or hygromycin) until a few days before experiments,
when they were transferred to drug-free medium.
Cytotoxicity Determinations-- Drug cytotoxicities were determined with the sulforhodamine B assay using 96-well microtiter plates (38) as described previously (31). For VP-16, doxorubicin, vincristine, and mitoxantrone, cells were exposed to drug or equivalent vehicle continuously in DMEM supplemented with 10% fetal calf serum. For chlorambucil, melphalan, 1,3-bis(2-chloroethyl)-1-nitrosourea, thiotepa, and hepsulfan, cells were exposed to drugs or vehicle for 1 h in either serum-free DMEM or DMEM supplemented with 1% fetal calf serum.
In some experiments, cells were preincubated for 15 min in the presence of vehicle or inhibitor (0.1 mM dicumarol (in HBSS) or 2 mM sulfinpyrazone (in DMEM plus 1% fetal bovine serum)) followed by co-incubation of chlorambucil with vehicle or inhibitor (0.1 mM dicumarol (in HBSS) or 2 mM sulfinpyrazone (DMEM/1% fetal calf serum)) for 1 h.Preparation of Affinity-purified, Recombinant Human GST
A1-1--
The human GSTA1 cDNA (36) was ligated into the
EcoRI site of the pPROK-1 plasmid expression vector
(CLONTECH). The protein was expressed following
transformation into E. coli DH5a. The culture was grown
overnight and then chilled, pelleted, and resuspended in 0.1 volume of
10 mM KPO4 (pH 8) plus 5 mM EDTA. The
suspension was warmed to 20 °C, incubated in lysozyme (0.2 mg/ml)
for 10 min, chilled on ice, sonicated, and then centrifuged at
10,000 × g for 20 min. The supernatant was adjusted to
0.2 M NaCl and loaded onto a glutathione affinity column
(Sigma G-4510) equilibrated with 50 mM Tris buffer (pH 7.4)
plus 0.2 M NaCl. The column was washed with 10 volumes of
equilibration buffer, and then GSTA1-1 was eluted with buffer
containing 50 mM Tris (pH 9.6), 0.2 M NaCl, and
5 mM glutathione. The eluate was neutralized with dilute
HCl, dialyzed against 10 mM KPO4 (pH 7.4) plus
50% glycerol, and stored at 20 °C.
Biochemical and in Vitro GST Enzyme Analyses-- Glutathione levels were measured by the enzymatic recycling method (39). GST activities were determined using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (40). Enzyme kinetic and in vitro inhibition analyses were accomplished using affinity-purified, recombinant human GST A1-1. For these assays, CDNB served as the varied substrate, and glutathione concentration was fixed at 2 mM. Activity was monitored spectrophotometrically at 25 °C and pH 6.5 as described by Habig et al. (40).
Northern blots of total cellular RNA were done as described (41) using probes derived from the entire human GST A1, GST M1, or GST P1 cDNAs (36, 37, 42) or the 5' 2629 base pairs of the MRP1 cDNA (43). The cDNA probes were labeled with [In Vivo (Intact Cells) Analyses of the Effects of Inhibitors on
MRP1-mediated DNP-SG Efflux and GST Activity--
For the analysis of
DNP-SG efflux, MCF7/VP cells were plated to a density of 0.6 × 106 cells/well in 6-well plates. 24 h later, cells
were preincubated for 2 min in HBSS plus vehicle or 2 mM
sulfinpyrazone. Following preincubations, cells were co-incubated at
25 °C with 1 µM CDNB plus vehicle or 2 mM
sulfinpyrazone. At the indicated times, medium was removed and
acidified to 10% perchloric acid. Cells were lysed in 10% perchloric
acid. Medium and lysate samples were prepared for reverse phase high
performance liquid chromatography analysis as described (31). Details
of DNP-SG chromatography will be described
elsewhere.2 Briefly,
acid-soluble samples were eluted isocratically from a C18 reverse phase
column in 20% methanol + 0.1% trifluoroacetic acid. Chromatographs
were monitored spectrophotometrically at 340 nm, and DNP-SG levels were
determined from the areas of peaks eluting at the position of authentic
DNP-SG by comparison with DNP-SG standards. Concentrations of DNP-SG
reference standards were determined using
340 = 10.2 cm
1 mM
1.
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RESULTS |
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Characterization of Cell Lines-- MCF7 breast carcinoma cells were chosen to study the effects of expression of MRP1 alone or in combination with isozymes of GST. These cell lines constitute a good model system for such studies because parental MCF7/WT cells express extremely low levels of cytosolic GSTs and MRP1. Additionally, a MDR derivative of MCF7/WT, MCF7/VP, is available that has as its primary genetic and phenotypic change the overexpression of high levels of MRP1 (32). Like MCF7/WT, MCF7/VP cells have very low endogenous cytosolic GST levels. Neither MCF7/WT nor MCF7/VP contain detectable MDR1 (P-glycoprotein) or the MRP2 (cMOAT or cMRP) isoform (32).3
Parental MCF7/VP cells were stably transfected with expression vectors containing cDNAs encoding the human GST A1-1 (MCF7/VP
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Effect of GSTs on the Sensitivities to Drug Cytotoxicities of
MRP1-expressing MCF7 Cells--
The effect of MRP1 expression, alone
or in combination with three different isozymes of cytosolic GST, on
cellular sensitivities to various cytotoxic anticancer drugs was
tested. Many of these data are summarized in Table
II. A comparison of the relative resistance of MCF7/WT versus MCF7/VP cells to four drugs of
the MDR phenotype, VP-16, doxorubicin, vincristine, and mitoxantrone, confirmed previous findings (32) that increased expression of MRP1
alone confers resistance to these drugs. Although VP-16, doxorubicin,
and vincristine are not known to form conjugates with glutathione, we
wondered whether these drugs or their metabolic derivatives might be
unrecognized, toxic substrates of any of the three cytosolic GST
isozymes tested and, if so, whether the GST isozymes would augment
MRP1-mediated resistance to these drugs. Although mitoxantrone can form
glutathione conjugates, it is not known whether any of the cytosolic
GSTs tested can catalyze these reactions. However, as shown in Table
II, expression of relatively high levels of GST A1-1 (MCF7/VP
cells), GST M1-1 (MCF7/VPµ cells), or GST P1-1 (MCF7/VP
cells) had
no significant effect on the level of MRP1-associated resistance to
these MDR-related drugs.
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Expression of GST A1-1 Protein and Catalytic Activity Are Required
for MCF7/VP Resistance to Chlorambucil--
Inspection of
cytotoxicity profiles of MCF7/WT versus MCF7/VP reveals that
MRP1 alone does not confer resistance to chlorambucil (Fig. 3). Indeed,
as shown here and reported previously (32), expression of MRP1 alone is
associated with modest sensitization to chlorambucil cytotoxicity. This
indicates that the resistance to chlorambucil in MRP1-positive cells is
absolutely dependent upon GST A1-1 protein expression. To confirm this
relationship and to eliminate the possibility that MCF7/VP
is an
exceptional MCF7/VP clone, in which the resistance to chlorambucil is
independent of GST A1-1, we tested whether inhibition of GST catalytic
activity in vivo would reverse resistance in MCF7/VP
cells.
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MRP1-dependent Glutathione Conjugate Efflux Activity Is
Required for Chlorambucil Resistance in MCF7/VP Cells--
To
evaluate the requirement for MRP1 in the observed chlorambucil
resistant phenotype, we used sulfinpyrazone to inhibit MRP1-mediated efflux of glutathione conjugates in vivo. The concentration
of sulfinpyrazone added (2 mM) was without significant
cytotoxicity. Moreover, addition of 2 mM sulfinpyrazone had
very little effect on in vivo GST A1-1 activity over the
entire 1-h incubation period (Fig.
6A). However, under these same
conditions, sulfinpyrazone effectively inhibited
MRP1-dependent efflux of the glutathione conjugate, DNP-SG,
(Fig. 6B, top panel) to efflux rates comparable to those
seen when MRP1-dependent efflux is inhibited by ATP
depletion (31).4
Additionally, inhibition of efflux by sulfinpyrazone resulted in a
profound increase in intracellular accumulation of DNP-SG (Fig.
6B, bottom panel). The effect of similar treatments with sulfinpyrazone on chlorambucil cytotoxicity is reported in Fig. 7. Sulfinpyrazone completely reverses
MCF7/VP
resistance to chlorambucil but has little effect on MCF7/WT
or MCF7/VP cells (Fig. 7, A and B). Thus,
sulfinpyrazone sensitization to chlorambucil toxicity is selective for
GST A1-1-expressing cells. We conclude that MRP1-dependent glutathione conjugate efflux activity is absolutely required to potentiate GST A1-1-mediated resistance to chlorambucil in MCF7 cells
under the conditions used.
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DISCUSSION |
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Our results establish that MRP1 and GST A1-1 act in synergy to
confer resistance to chlorambucil in MCF7 cells. The co-dependence of
resistance upon both GST A1-1 and MRP1 is remarkable. First, that GST
A1-1 activity alone does not confer protection indicates that the
glutathione conjugate(s), or other metabolites, of chlorambucil, the
formation of which is favored in the presence of GST A1-1, may be toxic
to the cells. Because the conjugate(s) and other derivatives of
chlorambucil formed are more water-soluble than the parent compound
and, therefore, less permeable to the plasma membrane, they may
accumulate to high intracellular levels in the absence of MRP1 or
another suitable efflux mechanism. This may explain why GST A1-1, at
the levels achieved, is insufficient to confer protection from
chlorambucil cytotoxicity in the absence of MRP1-dependent
efflux activity. It is perhaps even more remarkable that MRP1 alone
fails to confer protection from chlorambucil cytotoxicity. This finding
is particularly significant because glutathione conjugation of
chlorambucil can occur non-enzymatically (10, 49) and because glutathione conjugates of chlorambucil, especially the monoglutathionyl derivative, are reportedly substrates of MRP1-dependent
transport in isolated membrane vesicles (33). Thus, it is not
immediately obvious why MRP1 alone does not afford some protection from
chlorambucil toxicity. These issues have important implications for
understanding the dynamics of drug detoxification and the relative
importance of the components of drug detoxification studiedcomponents
that include phase II drug conjugation (GST/glutathione) and phase III
drug/conjugate efflux (MRP1).
GST A1-1 is known to catalyze the formation of the monoglutathionyl, but not the diglutathionyl, derivative of chlorambucil from glutathione and chlorambucil (49). However, the catalytic rate enhancement is relatively modest (10, 11, 49). Indeed, Meyer et al. (49) suggest that, rather than increasing the overall extent of conjugation with glutathione, the major effect of the human GST alpha class isoforms, A1-1, A1-2, and A2-2 on chlorambucil metabolism may be to increase the proportion of monoglutathionyl chlorambucil relative to diglutathionyl chlorambucil and other derivatives (including hydroxy- and phosphate-substituted metabolites) (49). This altered profile is believed to be the combined consequence of 1) GST Alpha class catalysis of monoglutathionyl chlorambucil formation, and 2) the ability of these GSTs to sequester monoglutathionyl chlorambucil at the enzyme active site with high affinity (49).
Because the monoglutathionyl chlorambucil retains one of the reactive chloroethyl groups, this metabolite of chlorambucil is only partially detoxified. Although GST A1-1 may sequester the monoglutathionyl derivative and thereby mitigate some toxicity, with continued exposure to chlorambucil, this GST:monoglutathionyl chlorambucil interaction will become saturated and will inhibit further catalysis (49, 50). Thus, in the absence of MRP1-dependent efflux of the monoglutathionyl derivative, the detoxification capacity of GST A1-1 may be quickly exceeded. It is possible that the other derivatives may also have some toxicities at high intracellular levels.
Our transfection data show that GST A1-1 protein is required for MRP1-associated resistance to chlorambucil in MCF7 cells. Moreover, results from in vivo inhibition of GST A1-1 with dicumarol indicate that GST catalytic activity is also an important requirement for maximum resistance. We have recently confirmed that purified GST A1-1 does bind radiolabeled monoglutathionyl chlorambucil with considerable avidity.3 Hence, both catalytic activity and chlorambucil conjugate binding could be important mechanisms of GST A1-1-associated cytoprotection. In our view, GST A1-1 may serve to catalyze the substitution of glutathione to one of the chloroethyl groups of chlorambucil. These monoglutathionyl derivatives, formed both enzymatically and non-enzymatically, can then be sequestered as relatively benign complexes with GST A1-1 until they can be delivered to MRP1 for export. In the absence of MRP1, as monoglutathionyl chlorambucil accumulates intracellularly, the catalytic activity GST A1-1 is compromised by product inhibition and the binding capacity of the enzyme for monoglutathionyl chlorambucil is exceeded. Consequently, the levels of chlorambucil remain high, and the levels of reactive, free monoglutathionyl chlorambucil and its other derivatives accumulate intracellularly resulting in increased cytotoxicity. In the absence of GST A1-1, not only is the monoglutathionyl derivative free to react with cellular macromolecules, but the distribution of chlorambucil metabolites is shifted away from the monoglutathionyl forms to other derivatives that are significantly poorer substrates for MRP1-mediated efflux. The potential importance of such GST A1-1-dependent changes in the profile of chlorambucil glutathione conjugates and derivatives is underscored by the recent findings of Barnouin et al. (33). These investigators show that the monoglutathionyl chlorambucil is by far the best chlorambucil derivative for MRP1-dependent transport in isolated membrane vesicles in vitro.
The three isozymes of GST tested confer no protection to MRP1-expressing MCF7/VP cells against the cytotoxicities of the four other alkylating agents examined. The reason for this is unknown. The GSTs examined may have little impact in vivo on the metabolism of thiotepa, hepsulfan, or 1,3-bis(2-chloroethyl)-1-nitrosourea. Additionally, it is not known whether MRP1 can support the efflux of glutathione conjugates of thiotepa or hepsulfan. Some GST isozymes are reported to catalyze the denitrosation of 1,3-bis(2-chloroethyl)-1-nitrosourea, but these studies have not identified stable glutathione conjugate intermediates (15, 22). Thus, there may be no stable glutathione conjugate of 1,3-bis(2-chloroethyl)-1-nitrosourea to serve as a potential MRP1 substrate. Particularly interesting is the failure of GST A1-1 and MRP1 to confer resistance to melphalan, a nitrogen mustard closely related to chlorambucil. The explanation for the difference in melphalan and chlorambucil resistance is unknown. Structural differences in these drugs may result in GST A1-1 having distinctly different effects on the metabolic profiles of melphalan and chlorambucil or their glutathione conjugates in vivo. Alternatively, glutathione conjugates of melphalan may be less efficiently exported by MRP1. Regardless of the explanation, our results show that MRP1/GST resistance synergy is both specific for a particular GST isozyme:drug pair and highly drug-selective, even among structurally related drugs.
The magnitude of MRP1-mediated resistance to four drugs of the MDR phenotype was not significantly augmented by co-expression of GST A1-1, GST M1-1, or GST P1-1. This result was not surprising for the drugs doxorubicin, VP-16, and vincristine, which are not known to form stable conjugates with glutathione. However, it was important to examine these drugs because it was possible that unrecognized metabolites or derivatives of these compounds might form glutathione conjugates that could influence their export by MRP1. In contrast, mitoxantrone reacts with glutathione to form conjugates (51). However, mitoxantone is not known to be a substrate of cytosolic GSTs, although it is reported to be a substrate of microsomal GST (52). The failure of the cytosolic GSTs tested to augment MRP1-mediated resistance to mitoxantrone is consistent with these data.
In summary, we demonstrate that GST A1-1 can act in synergy with MRP1 to confer resistance to the alkylating agent chlorambucil. The concept of coordinated action of phase II GST-dependent and phase III MRP1-dependent (and other membrane transport proteins) processes will very likely prove to be of general importance for detoxification of a variety of xenobiotic compounds, electrophiles that include genotoxic carcinogens (31) and anticancer drugs. The mechanism of GST A1-1/MRP1 resistance to chlorambucil depends upon both GST A1-1 catalytic activity and MRP1-dependent efflux activity. Moreover, because MRP1 is required to potentiate GST A1-1-associated resistance to chlorambucil, this suggests the possibility that some of the glutathione conjugates or other metabolites may themselves be important cellular toxins. Additionally, the removal of these conjugates by MRP1 may be required to relieve product inhibition and thereby maintain continued GST catalysis of drug conjugation. Finally, GST/MRP1 resistance synergy is highly specific for the particular xenobiotic: GST isozyme pair. There are multiple known and putative membrane-associated transport proteins related to MRP1 (53). It is possible that the complete detoxification of xenobiotic-glutathione conjugates will prove to be similarly dependent upon the specific membrane-associated transport protein expressed.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA70338 and ES06006.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Wake Forest University School of Medicine, Medical Center Blvd.,
Winston-Salem, NC 27157. Tel.: 336-710-9478; Fax: 336-710-7671; E-mail: cmorrow{at}wfubmc.edu.
The abbreviations used are: GST, glutathione S-transferaseCDNB, 1-chloro-2,4-dinitrobenzeneDMEM, Dulbecco's modified Eagle's mediumDNP-SG, glutathione conjugate of CDNB, S-(2,4-dinitrophenyl)-glutathioneHBSS, Hanks' balanced salt solutionMDR, multidrug resistanceMRP1, multidrug resistance protein 1VP-16, etoposide.
3 C. S. Morrow, P. K. Smitherman, S. K. Diah, E. Schneider, and A. J. Townsend, unpublished data.
4 S. Diah, unpublished data.
2 C. S. Morrow, P. K. Smitherman, S. K. Diah, E. Schneider, and A. Townsend, manuscript in preparation.
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REFERENCES |
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