Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morrow, C. S.
Right arrow Articles by Townsend, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morrow, C. S.
Right arrow Articles by Townsend, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 32, 20114-20120, August 7, 1998


Coordinated Action of Glutathione S-Transferases (GSTs) and Multidrug Resistance Protein 1 (MRP1) in Antineoplastic Drug Detoxification
MECHANISM OF GST A1-1- AND MRP1-ASSOCIATED RESISTANCE TO CHLORAMBUCIL IN MCF7 BREAST CARCINOMA CELLS*

Charles S. MorrowDagger , Pamela K. Smitherman, Sri K. Diah, Erasmus Schneider§, and Alan J. Townsend

From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 and § Wadsworth Center, Albany, New York 12201

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 Delta pCEP4 vector (34) containing cDNA inserts (35-37) encoding the human GSTA1 gene (MCF7/VPalpha ) 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/VPpi ) as described previously (31). Control cell lines were produced by stable transfection of MCF7/WT and MCF7/VP cells with pcDNA3.1 or Delta pCEP4 lacking GST cDNA inserts (MCF7/WT-neo, MCF7/WT-hyg, MCF7/VP-neo, and MCF7/VP-hyg). Cells transfected with pcDNA3- or Delta 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 [alpha -32P]dCTP by random priming (44). Western blot analyses of GST isoforms were accomplished as described previously using affinity-purified rabbit polyclonal antibodies directed against human GST A1, GST M1, or GST P1 subunits (19). Western blotting of membrane protein preparations for MRP1 was done with the QCRL-1 antibody kindly provided by Dr. S. P. C. Cole (45).

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/VPalpha 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 epsilon 340 = 10.2 cm-1 mM-1.

The effect of sulfinpyrazone or dicumarol on GST activity in vivo was determined as follows. MCF7/VPalpha cells (0.5 × 106/well) were seeded in 6-well plates. 24 h later, cells were incubated in DMEM plus 1% fetal calf serum ± 2 mM sulfinpyrazone or in HBSS ± 0.1 mM dicumarol at 37 °C in 5% CO2. At indicated times, cells were washed and incubated in HBSS equilibrated to 25 °C containing inhibitor (2 mM sulfinpyrazone or 0.1 mM dicumarol) or vehicle plus 10 µM CDNB. Relative in vivo GST activities were determined as the rate of total DNP-SG formation (DNP-SG in medium plus cell lysates) for 2 and 5 min at 25 °C. DNP-SG levels were measured chromatographically as described above. The levels of DNP-SG formation increased linearly with time throughout the 5 min incubation periods.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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/VPalpha ), GST M1-1 (MCF7/VPµ), or GST P1-1 (MCF7/VPpi ). Additionally, control cell lines were generated by stable transfection of parental MCF7/WT and MCF7/VP cells with empty expression vectors encoding antibiotic resistance to hygromycin (hyg) or geneticin (neo) but not GST. Expression of GST isozyme-specific protein and mRNA was detected only in cell lines transfected with GST expression vectors (Fig. 1). In these cells (MCF7/VPalpha , MCF7/VPµ, and MCF7/VPpi ), relatively high levels of GST activity were confirmed by enzyme assays (Table I). GST activities remained uniformly low in the parental and control cell lines (Table I). High levels of MRP1 expression (membrane protein and mRNA) were seen only in parental MCF7/VP cells and their transfected derivatives. Co-expression of GST isozymes had no effect on the levels of MRP1 (Fig. 2). Finally, glutathione levels were comparable in all of the cell lines tested (Table I).


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 1.   Expression of GST isozymes in transfected MCF7 cells. Levels of GST isozyme protein (A) and mRNA (B) were determined by Western and Northern blot, respectively. Blots were examined using isozyme-specific probes as described under "Experimental Procedures." Examined are cytosolic protein (50 µg/lane) and whole cellular RNA (10 µg/lane) from parental MCF7/WT (WT) and MCF7/VP (VP) cells and MCF7VP cells transfected with expression vectors encoding GST A1-1 (MCF/VPalpha , VP/alpha ), GST M1-1 (MCF7/VPµ, VP/µ), and GST P1-1 (MCF7/VPpi , VP/pi ).

                              
View this table:
[in this window]
[in a new window]
 
Table I
GST activities and glutathione levels in model cell lines
Values reported are the means of >=  three determinations ± 1 S.D. ND, not determined.


View larger version (75K):
[in this window]
[in a new window]
  		Fig. 2.   Expression of MRP1 in parental and transfected MCF7 cells. MRP1 protein (A) and mRNA (B) were detected by Western and Northern blot, respectively, as described under "Experimental Procedures." Lanes included 50 µg of membrane protein (A) or 10 µg of total cellular RNA (B) derived from parental MCF7/WT (WT) and MCF7/VP (VP) cells or from MCF7/VP cells transfected with expression vectors encoding GST A1-1 (MCF/VPalpha , VP/alpha ), GST M1-1 (MCF7/VPµ, VP/µ), and GST P1-1 (MCF7/VPpi , VP/pi ).

Transfection of control vectors devoid of GST cDNA inserts into parental MCF7/WT and MCF7/VP cells had no effect on GST levels (MCF7/WT-neo and -hyg; MCF7/VP-neo and -hyg), glutathione levels (MCF7/WT-neo and MCF7/VP-neo), or MRP1 expression (Table I). Moreover, cytotoxicity profiles of parental cells MCF7/WT and MCF7/VP were similar to the corresponding cells transfected with control vectors (MCF7/WT-neo and -hyg; MCF7/VP-neo and -hyg). Thus, data reported in the text below will not differentiate between the parental cells and their corresponding derivatives transfected with control vectors.

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/VPalpha cells), GST M1-1 (MCF7/VPµ cells), or GST P1-1 (MCF7/VPpi cells) had no significant effect on the level of MRP1-associated resistance to these MDR-related drugs.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Effect of MRP and GST expression on cellular sensitivities to the cytotoxicities of antineoplastic drugs

In contrast to the MDR phenotype-associated drugs, a variety of alkylating anticancer drugs are known to form glutathione conjugates both spontaneously and in GST-catalyzed reactions (7-14). However, MRP1 has not consistently been associated with resistance to these drugs. Nevertheless, a number of glutathione conjugates, including monoglutathionyl derivatives of chlorambucil and melphalan, are known to be substrates of MRP1-dependent transport in isolated membrane vesicles (27, 33). Therefore, we investigated whether MRP1 could support resistance to some of these alkylating agents if co-expressed with the GST isozyme appropriate to accelerate conjugate formation. There are reports that implicate GST catalysis in detoxification reactions involving all of the alkylating agents listed in Table II (13, 15-18, 21-23, 25, 46). However, neither MRP1 nor the isozymes of GST tested had any significant effect on relative resistance to melphalan, 1,3-bis(2-chloroethyl)-1-nitrosourea, thiotepa, or hepsulfan. In contrast, co-expression of GST A1-1 and MRP1 conferred significant resistance to chlorambucil, a nitrogen mustard closely related to melphalan (Fig. 3). Indeed, cumulative results from 12 independent experiments showed that GST A1-1 expression is associated with ~4-fold (3.74 ± 0.19 S.E.) resistance to chlorambucil in MCF7/VPalpha cells compared with MCF7/VP cells (p < 0.0001). Resistance to chlorambucil was GST A1-1 isozyme-specific because co-expression of MRP1 with GST M1-1 or GST P1-1 had no effect on cellular sensitivity to chlorambucil (not shown).


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3.   GST A1-1 confers resistance to chlorambucil in MRP1-expressing MCF7 cells. Shown are the profiles for chlorambucil cytotoxicity (1-h drug exposure) in parental MCF7/WT (WT) and MCF7/VP (VP) cells and in MCF7/VP cells transfected with GST A1-1 (MCF7/VPalpha , VP/alpha ). Error bars represent ± 1 S.D. from the mean proportion of cells surviving (n = 8) at the indicated chlorambucil concentrations.

Expression of GST A1-1 Protein and Catalytic Activity Are Required for MCF7/VPalpha 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/VPalpha 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/VPalpha cells.

Dicumarol was chosen as a candidate inhibitor because it has been reported to inhibit a rodent Alpha class GST and was expected to inhibit the human ortholog, GST A1-1 (47). Additionally, dicumarol is lipophilic and therefore should freely traverse the cell membrane of intact cells. To determine whether the inhibitory properties and potency of dicumarol would likely be sufficient to inhibit cellular GST A1-1 at subcytotoxic levels, we first examined the kinetics of dicumarol inhibition of the conversion of CDNB to DNP-SG by purified, recombinant GST A1-1 in vitro. These results (Fig. 4A) show that dicumarol inhibition is of the mixed type and is quite potent for this GST A1-1-catalyzed reaction (Kic = 3.6 µM; Kiu = 4.4 µM) (48). The effect of exogenously added dicumarol on GST A1-1 activity in vivo is examined in Fig. 4B. These data show that 0.1 mM dicumarol inhibited GST activity in MCF7/VPalpha cells by at least 80%, that inhibition was maximal within 15 min, and that inhibition remained at this level throughout the 1-h incubation. Treatment of intact cells with 0.1 mM dicumarol had no effect on the kinetics of MRP1-dependent DNP-SG efflux (data not shown). Based upon these preliminary results, the effect of GST A1-1 inhibition by dicumarol on MCF7/VPalpha cell sensitivity to chlorambucil was examined.


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4.   Inhibition of GST A1-1 by dicumarol, in vitro and in vivo. A, kinetic analysis of dicumarol inhibition of GST A1-1 in vitro was accomplished using affinity-purified GST A1-1 and CDNB as the variable substrate (see under "Experimental Procedures"). Under these conditions, kinetic parameters estimated from the data shown in the Hane's plots are: V = 64.3 µmol/min/mg enzyme and Km = 0.36 mM for CDNB; and Kiu = 4.4 µM and Kic = 3.6 µM for dicumarol. B, inhibition of GST A1-1 in vivo was done as described (see under "Experimental Procedures"). Cells were incubated with 0.1 mM dicumarol (open circles, + dicumarol) or vehicle (closed circles, - dicumarol) at 37 °C, and at the indicated times, the rate of CDNB conversion to DNP-SG was measured in intact cells. Shown are mean values ± the range of duplicate determinations, expressed as the fraction of 0 time controls.

Treatment of MCF7/VPalpha cells with 0.1 mM dicumarol sufficient to inhibit GST activity by >=  80% resulted in nearly complete reversal of GST A1-1-associated resistance (Fig. 5A). Moreover, dicumarol-mediated sensitization was selective for GST A1-1-expressing MCF7/VPalpha cells because dicumarol had no significant effect on the sensitivities of MCF7/WT and MCF7/VP cells to chlorambucil cytotoxicity (Fig. 5, A and B). These results demonstrate that GST A1-1 catalytic activity is indeed required for chlorambucil resistance in MCF7/VPalpha cells. However, these data leave undefined the role of MRP1 in this resistance to chlorambucil.


View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5.   Selective reversal of GST A1-1-associated resistance to chlorambucil in MCF7/VPalpha cells by dicumarol. A, shown are representative cytotoxicity profiles of parental MCF7/WT (WT) and MCF7/VP (VP) cells and of GST A1-1-transfected MCF7/VP cells (MCF7/VPalpha , VP/alpha ) treated with chlorambucil for 1 h in the absence (-, closed symbols, vehicle control) or presence (+, open symbols) of 0.1 mM dicumarol. Error bars signify ± 1 S.D. from the mean fraction surviving. Note that because serum was eliminated from these drug incubations, the cytotoxicity profiles are shifted to the left compared with experiments shown in Figs. 3 and 7. However, omission of serum does not affect the relative sensitivities to chlorambucil among the cell lines. B, effect of dicumarol on chlorambucil cytotoxicity is expressed as fold sensitization (IC50 indicated cell line in the absence of dicumarol div  IC50 cell line in the presence of 0.1 mM dicumarol). Bars represent mean values (± 1 S.D.) of three independent experiments.

MRP1-dependent Glutathione Conjugate Efflux Activity Is Required for Chlorambucil Resistance in MCF7/VPalpha 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/VPalpha 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.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 6.   Sulfinpyrazone selectively inhibits MRP1-mediated DNP-SG efflux in vivo. A, MCF7/VPalpha cells were preincubated for 15 min at 37 °C in medium containing 2 mM sulfinpyrazone or vehicle. Medium containing sulfinpyrazone or vehicle was replaced, and at the indicated times, GST activity was determined in vivo (intact cells) as described under "Experimental Procedures." GST activities are expressed as the percent of vehicle treated (minus sulfinpyrazone) controls ± 1 S.D. (n = 4). B. MCF7/VPalpha cells were preincubated with 2 mM sulfinpyrazone or vehicle for 2 min. Medium + (open circles) or - (closed circles) 2 mM sulfinpyrazone and containing 1 µM CDNB was added at time 0 at 25 °C. The rates of DNP-SG efflux (appearance in medium, top panel) and intracellular accumulation (appearance in cell lysates, bottom panel) were determined at 25 °C as described (see under "Experimental Procedures").


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 7.   Inhibition of MRP1-dependent glutathione conjugate efflux with sulfinpyrazone completely reverses resistance to chlorambucil in MCF7/VPalpha cells. A, shown are representative cytotoxicity profiles of parental MCF7/WT (WT) and MCF7/VP (VP) cells and of GST A1-1-transfected MCF7/VP cells (MCF7/VPalpha , VP/alpha ) treated with chlorambucil for 1 h in the absence (-, closed symbols, vehicle control) or presence (+, open symbols) of 2 mM sulfinpyrazone as described under "Experimental Procedures." Error bars signify ± 1 S.D. from the mean fraction surviving. B, effect of sulfinpyrazone on chlorambucil cytotoxicity is expressed as fold sensitization (IC50 indicated cell line in the absence of sulfinpyrazone div  IC50 cell line in the presence of 2 mM sulfinpyrazone). Bars represent mean values (± 1 S.E.) of four independent experiments.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 studied---components 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.

    FOOTNOTES

* 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.

Dagger 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.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Black, S. M., and Wolf, C. R. (1991) Pharmacol. Ther. 51, 139-154[CrossRef][Medline] [Order article via Infotrieve]
  2. Coles, B., and Ketterer, B. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 47-70[Medline] [Order article via Infotrieve]
  3. Hayes, J. D., and Pulford, D. J. (1995) Crit. Rev. Biochem. Mol. Biol. 30, 445-600[Medline] [Order article via Infotrieve]
  4. Mannervik, B., and Danielson, U. H. (1988) Crit. Rev. Biochem. 23, 283-337[Medline] [Order article via Infotrieve]
  5. Morrow, C. S., and Cowan, K. H. (1990) Cancer Cells 2, 15-22[Medline] [Order article via Infotrieve]
  6. Tew, K. D. (1994) Cancer Res. 54, 4313-4320[Abstract/Free Full Text]
  7. Awasthi, S., Bajpai, K. K., Piper, J. T., Singhal, S. S., Ballaatore, A., Seifert, W. E., Awasthi, Y. C., and Ansari, G. A. S. (1996) Drug Metab. Dispos. 24, 371-374[Medline] [Order article via Infotrieve]
  8. Bolton, M. G., Colvin, O. M., and Hilton, J. (1991) Cancer Res. 51, 2410-2415[Abstract/Free Full Text]
  9. Bolton, M. G., Hilton, J., Robertson, K. D., Streeper, R. T., Colvin, O. M., and Noe, D. A. (1993) Drug Metab. Dispos. 21, 986-996[Abstract]
  10. Ciaccio, P. J., Tew, K. D., and LaCreta, F. P. (1990) Cancer Commun. 2, 279-285[Medline] [Order article via Infotrieve]
  11. Ciaccio, P. J., Tew, K. H., and LaCreta, F. P. (1991) Biochem. Pharmacol. 42, 1504-1507[CrossRef][Medline] [Order article via Infotrieve]
  12. Dirven, H. A. A. M., van Ommen, B., and van Bladeren, P. J. (1994) Cancer Res. 54, 6215-6220[Abstract/Free Full Text]
  13. Dirven, H. A. A. M., Dictus, E. L. J. T., Broeders, N. L. H. L., van Ommen, B., and van Bladeren, P. J. (1995) Cancer Res. 55, 1701-1706[Abstract/Free Full Text]
  14. Dulik, D. M., Fenselau, C., and Hilton, J. (1986) Biochem. Pharmacol. 35, 3405-3409[CrossRef][Medline] [Order article via Infotrieve]
  15. Berhane, K., Hao, X.-X., Egyhazi, S., Hansson, J., Ringborg, U., and Mannervik, B. (1993) Cancer Res. 53, 4257-4261[Abstract/Free Full Text]
  16. Black, S. M., Beggs, J. D., Hayes, J. D., Bartoszek, A., Muramatsu, M., Sakai, M., and Wolf, C. R. (1990) Biochem. J. 268, 309-315[Medline] [Order article via Infotrieve]
  17. Buller, A. L., Clapper, M. L., and Tew, K. D. (1987) Mol Pharmacol. 31, 575-578[Abstract]
  18. Lewis, A. D., Hickson, I. D., Robson, C. N., Harris, A. L., Hayes, J. D., Griffiths, S. A., Manson, M. M., Hall, A. E., Moss, J. E., and Wolf, C. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8511-8515[Abstract/Free Full Text]
  19. Leyland-Jones, B., Townsend, A. J., Tu, C. P., Cowan, K. H., and Goldsmith, M. E. (1991) Cancer Res. 51, 587-594[Abstract/Free Full Text]
  20. Moscow, J. A., Townsend, A. J., and Cowan, K. H. (1989) Mol. Pharmacol. 36, 22-28[Abstract]
  21. Puchalski, R. B., and Fahl, W. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2443-2447[Abstract/Free Full Text]
  22. Smith, M. T., Evans, C. G., Doane-Setzer, P., Castro, V. M., Tahir, M. K., and Mannervik, B. (1989) Cancer Res. 49, 2621-2625[Abstract/Free Full Text]
  23. Schecter, R. L., Alaoui-Jamali, M. A., Woo, A., Fahl, W. E., and Batist, G. (1993) Cancer Res. 53, 4900-4906[Abstract/Free Full Text]
  24. Townsend, A. J., Tu, C. P., and Cowan, K. H. (1992) Mol. Pharmacol. 41, 230-236[Abstract]
  25. Yang, W. Z., Asher, B., Johnston, J. B., Israels, L. G., and Mowat, R. A. (1992) Mol. Pharmacol. 41, 625-630[Abstract]
  26. Jedlitschky, G., Leier, I., Buchholz, U., Center, M., and Keppler, D. (1994) Cancer Res. 54, 4833-4836[Abstract/Free Full Text]
  27. Jedlitschky, G., Leier, I., Buchholz, U., Barnouin, K., Kurz, G., and Keppler, D. (1996) Cancer Res. 56, 988-994[Abstract/Free Full Text]
  28. Leier, I., Jedlitschky, G., Buchholz, U., Cole, S. P. C., Deeley, R. G., and Keppler, D. (1994) J. Biol. Chem. 269, 27807-27810[Abstract/Free Full Text]
  29. Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682[Abstract/Free Full Text]
  30. Muller, M., Meijer, C., Zaman, G. J. R., Borst, P., Scheper, R. J., Mulder, N. H., De Vries, E. G. E., and Jansen, P. L. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 13033-13037[Abstract/Free Full Text]
  31. Morrow, C. S., Diah, S., Smitherman, P. K., Schneider, E., and Townsend, A. J. (1998) Carcinogenesis 19, 109-115[Abstract/Free Full Text]
  32. Schneider, E., Horton, J., Yang, C.-h., Nakagawa, M., and Cowan, K. H. (1994) Cancer Res. 54, 152-158[Abstract/Free Full Text]
  33. Barnouin, K., Leier, I., Jedlitschky, G., Pourtier-Manzanedo, A., Konig, J., Lehmann, W.-D., and Keppler, D. (1998) Br. J. Cancer 77, 201-209[Medline] [Order article via Infotrieve]
  34. Bunting, K. D., and Townsend, A. J. (1996) J. Biol. Chem. 271, 11884-11890[Abstract/Free Full Text]
  35. DeJong, J. L., Chang, C.-M., Whang-Peng, J., Knudsen, T., and Tu, C.-P. D. (1988) Nucleic Acids Res. 16, 8541-8554[Abstract/Free Full Text]
  36. Tu, C.-P. D., and Qian, B. (1986) Biochem. Biophys. Res. Commun. 141, 229-237[CrossRef][Medline] [Order article via Infotrieve]
  37. Rhoads, D., Zarlengo, R. P., and Tu, C. D. (1987) Biochem. Biophys. Res. Commun. 145, 474-481[CrossRef][Medline] [Order article via Infotrieve]
  38. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R. (1990) J. Natl. Cancer Inst. 82, 1107-1112[Abstract/Free Full Text]
  39. Tietze, F. (1969) Anal. Biochem. 27, 502-522[CrossRef][Medline] [Order article via Infotrieve]
  40. Habig, W., Pabst, M., and Jakoby, W. (1974) J. Biol Chem. 249, 7130-7139[Abstract/Free Full Text]
  41. Davis, L., Kuehl, M., and Battey, J. (1994) Basic Methods in Molecular Biology, 2nd Ed., Appleton and Lange, Norwalk, CT
  42. Moscow, J. A., Townsend, A. J., Goldsmith, M. E., Whang-Peng, J., Vickers, P. J., Legault-Poisson, S., Myers, C. E., and Cowan, K. H. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6518-6522[Abstract/Free Full Text]
  43. Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M. V., and Deeley, R. G. (1992) Science 258, 1650-1654[Abstract/Free Full Text]
  44. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1997) Current Protocols in Molecular Biology, John Wiley & Sons, New York
  45. Hipfner, D. R., Gauldie, S. D., Deeley, R. G., and Cole, S. P. C. (1994) Cancer Res. 54, 5788-5792[Abstract/Free Full Text]
  46. Armstrong, D. K., Gordon, G. B., Hilton, J., Streeper, R. T., Colvin, O. M., and Davidson, N. E. (1992) Cancer Res. 52, 1416-1421[Abstract/Free Full Text]
  47. Mays, J. B., and Benson, A. M. (1992) Biochem. Pharmacol. 44, 921-925[CrossRef][Medline] [Order article via Infotrieve]
  48. Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics, Portland Press, London
  49. Meyer, D. J., Gilmore, K. S., Harris, J. M., Hartley, J. A., and Ketterer, B. (1992) Br. J. Cancer 66, 433-438[Medline] [Order article via Infotrieve]
  50. Meyer, D. J. (1993) Xenobiotica 23, 823-834[Medline] [Order article via Infotrieve]
  51. Mews, K., Blanz, J., Ehninger, G., Gebhardt, R., and Zeller, K.-P. (1993) Cancer Res. 53, 5135-5142[Abstract/Free Full Text]
  52. Wolf, C. R., MacPherson, J. S., and Smyth, J. F. (1986) Biochem. Pharmacol. 35, 1577-1581[CrossRef][Medline] [Order article via Infotrieve]
  53. Kool, M., de Haas, M., Scheffer, G. L., Scheper, R. J., van Eijk, M. J. T., Juijn, J. A., Baas, F., and Borst, P. (1997) Cancer Res. 57, 3537-3547[Abstract/Free Full Text]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Molecular Cancer TherapeuticsHome page
C. Peklak-Scott, P. K. Smitherman, A. J. Townsend, and C. S. Morrow
Role of glutathione S-transferase P1-1 in the cellular detoxification of cisplatin
Mol. Cancer Ther., October 1, 2008; 7(10): 3247 - 3255.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. B. Sibhatu, P. K. Smitherman, A. J. Townsend, and C. S. Morrow
Expression of MRP1 and GSTP1-1 modulate the acute cellular response to treatment with the chemopreventive isothiocyanate, sulforaphane
Carcinogenesis, April 1, 2008; 29(4): 807 - 815.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
M. E. Kushman, S. L. Kabler, S. Ahmad, J. Doehmer, C. S. Morrow, and A. J. Townsend
Protective Efficacy of hGSTM1-1 against B[a]P and (+)- or ( )-B[a]P-7,8-Dihydrodiol Cytotoxicity, Mutagenicity, and Macromolecular Adducts in V79 Cells Coexpressing hCYP1A1
Toxicol. Sci., September 1, 2007; 99(1): 51 - 57.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Yoshimoto, C. K. Augustine, J. S. Yoo, P. A. Zipfel, M. A. Selim, S. K. Pruitt, H. S. Friedman, F. Ali-Osman, and D. S. Tyler
Defining regional infusion treatment strategies for extremity melanoma: comparative analysis of melphalan and temozolomide as regional chemotherapeutic agents
Mol. Cancer Ther., May 1, 2007; 6(5): 1492 - 1500.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
D. S. Riddick, C. Lee, S. Ramji, E. C. Chinje, R. L. Cowen, K. J. Williams, A. V. Patterson, I. J. Stratford, C. S. Morrow, A. J. Townsend, et al.
CANCER CHEMOTHERAPY AND DRUG METABOLISM
Drug Metab. Dispos., August 1, 2005; 33(8): 1083 - 1096.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. A. Ralat and R. F. Colman
Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site
J. Biol. Chem., November 26, 2004; 279(48): 50204 - 50213.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Mauzeroll and A. J. Bard
Scanning electrochemical microscopy of menadione-glutathione conjugate export from yeast cells
PNAS, May 25, 2004; 101(21): 7862 - 7867.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
P. Depeille, P. Cuq, S. Mary, I. Passagne, A. Evrard, D. Cupissol, and L. Vian
Glutathione S-Transferase M1 and Multidrug Resistance Protein 1 Act in Synergy to Protect Melanoma Cells from Vincristine Effects
Mol. Pharmacol., April 1, 2004; 65(4): 897 - 905.
[Abstract] [Full Text]

Home page
 J. Pharmacol. Exp. Ther.Home page
P. K. Smitherman, A. J. Townsend, T. E. Kute, and C. S. Morrow
Role of Multidrug Resistance Protein 2 (MRP2, ABCC2) in Alkylating Agent Detoxification: MRP2 Potentiates Glutathione S-Transferase A1-1-Mediated Resistance to Chlorambucil Cytotoxicity
J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 260 - 267.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
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]

Home page
 Cancer Res.Home page
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]

Home page
 CarcinogenesisHome page
R. L. Divi, Y. P. Dragan, H. C. Pitot, and M. C. Poirier
Immunohistochemical localization and semi-quantitation of hepatic tamoxifen-DNA adducts in rats exposed orally to tamoxifen
Carcinogenesis, October 1, 2001; 22(10): 1693 - 1699.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
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]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. O'Brien, G. D. Kruh, and K. D. Tew
The Influence of Coordinate Overexpression of Glutathione Phase II Detoxification Gene Products on Drug Resistance
J. Pharmacol. Exp. Ther., August 1, 2000; 294(2): 480 - 487.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
J. Wang, S. Bauman, and R. F. Colman
Probing Subunit Interactions in Alpha Class Rat Liver Glutathione S-Transferase with the Photoaffinity Label Glutathionyl S-[4-(Succinimidyl)benzophenone]
J. Biol. Chem., February 25, 2000; 275(8): 5493 - 5503.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. Stanulla, M. Schrappe, A. M. Brechlin, M. Zimmermann, and K. Welte
Polymorphisms within glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) and risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: a case-control study
Blood, February 15, 2000; 95(4): 1222 - 1228.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
W. R. Fields, C. S. Morrow, J. Doehmer, and A. J. Townsend
Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1
Carcinogenesis, June 1, 1999; 20(6): 1121 - 1125.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. M. Paumi, B. G. Ledford, P. K. Smitherman, A. J. Townsend, and C. S. Morrow
Role of Multidrug Resistance Protein 1 (MRP1) and Glutathione S-Transferase A1-1 in Alkylating Agent Resistance. KINETICS OF GLUTATHIONE CONJUGATE FORMATION AND EFFLUX GOVERN DIFFERENTIAL CELLULAR SENSITIVITY TO CHLORAMBUCIL VERSUS MELPHALAN TOXICITY
J. Biol. Chem., March 9, 2001; 276(11): 7952 - 7956.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morrow, C. S.
Right arrow Articles by Townsend, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morrow, C. S.
Right arrow Articles by Townsend, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement