QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.017988v1 69/4/1499    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morrow, C. S. Articles by Townsend, A. J. Search for Related Content PubMed PubMed Citation Articles by Morrow, C. S. Articles by Townsend, A. J.

Multidrug Resistance Protein 1 (MRP1, ABCC1) Mediates Resistance to Mitoxantrone via Glutathione-Dependent Drug Efflux

Departments of Biochemistry (C.S.M., C.P.-S., B.B., P.K.S., A.J.T.) and Pathology (T.E.K.), Wake Forest University School of Medicine, Winston-Salem, North Carolina

Received August 11, 2005; accepted January 24, 2006

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Based upon several previous reports, no consistent relationship between multidrug resistance protein 1 (MRP1, ABCC1) expression and cellular sensitivity to mitoxantrone (MX) toxicity can be ascertained; thus, the role of MRP1 in MX resistance remains controversial. The present study, using paired parental, MRP1-poor, and transduced MRP1-overexpressing MCF7 cells, unequivocally demonstrates that MRP1 confers resistance to MX cytotoxicity and that resistance is associated with reduced cellular accumulation of MX. This MRP1-associated reduced accumulation of MX was partially reversed by treatment of cells with 50 µM MK571 [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid]—an MRP inhibitor that increased MX accumulation in MRP1-expressing MCF7 cells but had no effect on MRP-poor MCF7 cells. Moreover, in vitro experiments using inside-out membrane vesicles show that MRP1 supports ATP-dependent, osmotically sensitive uptake of MX. Unlike ABCG2 (breast cancer resistance protein, mitoxantrone-resistant protein), MRP1-mediated MX transport is dependent upon the presence of glutathione or its S-methyl analog. In addition, MX stimulates transport of [³H]glutathione. Together, these data are consistent with the interpretation that MX efflux by MRP1 involves cotransport of MX and glutathione. The results suggest that MRP1—like the alternative MX transporters ABCG2 and ABCB1 (MDR1, P-glycoprotein)—can significantly influence tumor cell sensitivity to and pharmacological disposition of MX.

Previous work in our laboratory examined an etoposide-selected cell line, MCF7/VP, that is multidrug-resistant on the basis of MRP1 amplification and overexpression (Schneider et al., 1994; Diah et al., 2001). MCF7/VP cells are cross-resistant to MX (Schneider et al., 1994) and have reduced MX accumulation because of an ATP-dependent efflux mechanism (Diah et al., 2001). However, because other genetic and phenotypic changes may have occurred during drug selection, including altered expression of other ABC transporters, we could not unequivocally attribute MX resistance to MRP1. Moreover, in these studies, it was apparent that the behavior of MX in MCF7/VP cells was in some ways atypical for an MRP1 substrate. First, unlike many classic MRP1 substrates, MX failed to form detectable conjugates of glutathione (GSH) or glucuronide. Second, in contrast to resistance associated with other weakly basic lipophilic MRP1 substrates, cellular accumulation of and resistance to MX was insensitive to depletion of intracellular glutathione (Diah et al., 2001).

Lacking in all of these studies was direct evidence that demonstrated whether or not MRP1 could mediate ATP-dependent MX efflux. Hence, the current studies, using materials derived from parental MCF7 cells stably transduced with MRP1, were undertaken to determine whether selective overexpression of MRP1 can confer resistance to MX by mediating its efflux and, if so, to characterize MX transport and its glutathione dependence. The results—indeed showing MRP1-dependent MX resistance and transport—are discussed in terms of the role of MRP1 in tumor cell sensitivity to MX and the roles of MRP family proteins in the pharmacological disposition of MX.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. [Glycine-2-³H]GSH (50 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA), and [³H]MX (3.2 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Unlabeled MX and herring sperm DNA were obtained from Sigma-Aldrich (St. Louis, MO). Mouse monoclonal antibodies against ABCG2 (BXP-21), MRP1 (QCRL-1), and MK571 were from Alexis Biochemicals (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated anti-MRP1 (QCRL-3) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and horseradish peroxidase-conjugated secondary antibody (goat anti-mouse IgG) was obtained from BD Transduction Laboratories (Lexington, KY).

Cell Lines and Culture. MRP1-overexpressing MCF7 cells, MCF7/MRP1-10, were derived from MRP-poor parental MCF7 cells (MCF7/WT) by stable transduction with an MRP1-expression vector as described previously (Smitherman et al., 2004). Additional transgenic MRP1-expressing clones (MCF7/MRP1-M6 and MCF7/MRP1-M24) were similarly obtained by independent retroviral transductions. The levels of MRP1 expression in the three transgenic cell lines were similar by Western blot analysis (not shown). Drug-selected MCF7 derivatives expressing MRP1 (MCF7/VP) or ABCG2 (MCF7/MX) have been reported elsewhere (Nakagawa et al., 1992; Schneider et al., 1994; Ross et al., 1999). Cell culture, cytotoxicity assays, and [³H]MX accumulation studies were accomplished exactly as described previously (Diah et al., 2001). For some [³H]MX accumulation experiments, cells were coincubated with 50 µM MK571, an MRP inhibitor. For some cytotoxicity experiments, intracellular GSH was depleted by 48-h treatment with 50 µM BSO before a 1-h exposure to MX (Diah et al., 2001). Total intracellular GSH concentrations were determined and calculated as described previously (Diah et al., 1999).

Transport Studies. ATP-dependent MRP1- or ABCG2-mediated transport was done using inside-out plasma membrane vesicles derived from MCF7/WT (MRP1-negative/ABCG2-negative controls), MCF7/MRP1-10 (MRP1-positive), and MCF7/MX (ABCG2-positive) cells as described previously (Paumi et al., 2001; Smitherman et al., 2004) with the inclusion of the creatine kinase/creatine phosphate ATP-regenerating system (Peklak-Scott et al., 2005). Transport reactions contained radiolabeled substrate (5 µM [³H]MX or 100 µM [³H]GSH) and were supplemented with reduced GSH, S-methyl glutathione, and/or L-glutamate as indicated. Reactions were initiated at 37°C by the addition of vesicles (15-30 µg of protein/50 µl of reaction). Reactions (10-µl aliquots) were terminated at indicated times in 1 ml of ice-cold Tris (10 mM), pH 7.5, plus 250 mM sucrose containing 10 mg/ml herring sperm DNA and processed by the rapid filtration method using hydrophilic membrane filters (GVWP; Millipore, Billerica, MA) (Paumi et al., 2001). ATP-dependent radiolabeled substrate uptake was calculated by subtracting uptake observed in ATP-negative controls containing a 4 mM concentration of the nonhydrolyzable analog ,-methylene ATP from uptake observed in the presence of 4 mM ATP.

Immunochemical Analyses. Western blotting was done as described previously using mouse monoclonal antibodies directed against MRP1 (QCRL-1) or ABCG2 (BXP-21) (Morrow et al., 1998). For flow cytometry, trypsinized cells were washed and fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA) solution. Cells were permeabilized with 0.1% saponin in PBS-BSA solution. Permeabilized cells were incubated with FITC-conjugated QCRL-3 (20 µg/ml) for 2 h at room temperature, washed, and postfixed in 3.7% formaldehyde/PBS-BSA solution before analysis on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Sequence Analysis of MRP1 Expressed in MCF7/VP Cells. The entire coding region of MRP1 expressed in the drug-selected MCF7/VP cells was amplified by reverse transcription-polymerase chain reaction and sequenced by the DNA sequencing core laboratory of Wake Forest University School of Medicine.

	Results

Top Abstract Materials and Methods Results Discussion References

 
MX Resistance in MCF7/VP Cells Is Not Attributable to ABCG2. It was previously shown that etoposide-selected, MRP1-overexpressing MCF7/VP cells are cross-resistant to MX cytotoxicity and accumulate less intracellular MX than parental MCF7/WT cells (Schneider et al., 1994; Diah et al., 2001). Western analysis showed that ABCG2 expression was quite low in both MCF7/WT and MCF7/VP compared with MX-selected MCF7/MX cells (Fig. 1). Moreover, as suggested earlier (Ross et al., 1999; Yang et al., 2000), the levels of ABCG2 expressed in MCF7/WT and MCF7/VP cells were similar, indicating that ABCG2 cannot account for MX cross-resistance in MCF7/VP cells.

View larger version (63K): [in this window] [in a new window]   Fig. 1. Expression of ABCG2 in derivatives of MCF7 cells. Shown is a Western blot analyzed for ABCG2 expression as described under Materials and Methods. Each lane contained 30 µg of membrane-associated protein derived from parental (MCF7/WT), etoposide-selected MRP1-overexpressing (MCF7/VP), and MX-selected ABCG2-overexpressing (MCF7/MX) cells. Positions of molecular mass markers (kDa) are shown on the left.

MRP1 Confers Resistance to MX by Reducing Intracellular Drug Accumulation. MCF7 cells stably transduced with MRP1 were used to determine whether selective overexpression of MRP1 could confer resistance to MX. Flow cytometry and Western blot analyses (Fig. 2, A and B) demonstrate that the level of MRP1 expression in the transgenic MCF7/MRP1-10 cell line was similar to that of the drug-selected MCF7/VP cell line. In addition, DNA sequence analysis revealed that the mRNA expressed in MCF7/VP cells encoded the common MRP1 variant (Cole et al., 1992), which is also identical to that encoded by the cDNA used to generate the transduced MCF7 cell lines (Zaman et al., 1994). Overexpression of MRP1 in three independently derived stably transduced MCF7 clones conferred 2.4-fold (MCF7/MRP1-M6 and MCF7/MRP1-M24) and 3.1-fold (MCF7/MRP1-10) greater resistance compared with control cells stably transduced with empty vector (Fig. 3). Moreover, MRP1 expression was associated with reduced MX accumulation in both transduced MCF7/MRP1-10 (45%) and drug-selected MCF7/VP (60%) cells (Fig. 4A). This MRP1-dependent reduction in MX accumulation was partially reversed by the MRP inhibitor MK571; as shown in Fig. 4B, 50 µM MK571 increased MX accumulation in MCF7/MRP1-10 but not MRP-poor MCF7/WT cells.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Expression of MRP1 (ABCC1) in MCF7 cell derivatives. Flow cytometry (A) and Western blot (B) analyses demonstrate comparable high levels of MRP1 overexpression in stably transduced (MRP1-10) and etoposide-selected (VP) but not parental (WT) MCF7 cells. A, cell number counted (counts) and relative fluorescence (QCRL3-FITC) are shown. B, positions of molecular mass markers (kDa) are shown on the left.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Selective expression of MRP1 in stably transduced MCF7 cells confers resistance to MX cytotoxicity. Cells were treated with the indicated concentrations of MX or vehicle by continuous exposure. Cell lines used were control MRP1-negative MCF7 cells stably transduced with empty vector () and MRP1-positive MCF7 cells stably transduced with an MRP1 retroviral expression vector (, MCF7/MRP1-10; , MCF7/MRP1-M6; , MCF7/MRP1-M24). Points represent mean values ± 1 S.D. from eight replicate determinations. IC50 values for the four cell lines were as follows: MCF7/WT, 0.62 nM; MCF7/MRP1-10, 1.9 nM; MCF7/MRP1-M6, 1.5 nM; and MCF7/MRP1-M24, 1.5 nM.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Overexpression of MRP1 reduces MX accumulation in both transgenic (MCF7/MRP1-10) and drug-selected (MCF7/VP) cell lines. A, accumulation of [3H]MX (1 µM) examined in parental, MRP1-poor (MCF7/WT), and MRP1-overexpressing (MCF7/MRP1-10 and MCF7/VP) cells as described previously (Diah et al., 2001). Points are the averages ± the range of duplicate determinations. B, accumulation of [3H]MX (1 µM) over 4 h examined in MCF7/WT and MCF7/MRP1-10 cells in the presence or absence (-) of the MRP inhibitor MK571 (50 µM). Exposure to MK571 or vehicle control began 15 min before the addition of [3H]MX and continued throughout the subsequent 4-h incubation. Bars represent the mean values ± 1 S.D. from three to six independent determinations. N.S., not significant; **, P < 0.0001 (two-tailed unpaired t test).

MRP1 Mediates ATP- and GSH-Dependent Transport of MX. Drug accumulation studies suggested MRP1 supports MX efflux. To verify the ability of MRP1 to mediate MX transport and to characterize its GSH dependence, inside-out membrane vesicles were used for in vitro drug transport studies. As shown in Fig. 5A, MRP1-mediated ATP-dependent transport of MX, but only in the presence of GSH (5 mM). In contrast, vesicles derived from MRP1-negative parental cells did not support ATP-dependent MX transport, even in the presence of GSH. MX uptake into inside-out vesicles derived from MCF7/MRP1-10 cells was osmotically sensitive [transport was progressively reduced with increasing sucrose concentrations (Fig. 5B)], indicating that the retained [³H]MX measured represented true uptake into the intravesicular space. In contrast to MRP1-mediated uptake, transport of MX by ABCG2 was independent of GSH (Fig. 6).

View larger version (10K): [in this window] [in a new window]   Fig. 5. MRP1 mediates osmotically sensitive ATP- and GSH-dependent transport of MX. A, ATP-dependent uptake of [3H]MX (5 µM) into insideout membrane vesicles derived from control, MRP1-negative (MCF7/WT), and MRP1-expressing (MCF7/MRP1-10) cells examined as described under Materials and Methods. Uptake was determined in MCF7/MRP1-10-derived vesicles in the presence () or absence () of 5 mM GSH. In the latter (GSH-negative; ), 5 mM L-glutamate was substituted for GSH. Also shown are results from studies using control, MCF7/WT vesicles done in the presence of 5 mM GSH (). Points represent the mean ATP-dependent uptake ± 1 S.E.M. of triplicate determinations. B, ATP-dependent uptake of [3H]MX (5 µM) by vesicles derived from MCF7/MRP1-10 cells determined over 15 min in reactions containing 5 mM GSH and varying concentrations (0.25, 0.33, 0.5, and 1 M) of sucrose as indicated. Data were normalized to uptake at standard sucrose concentration (0.25 M) to obtain relative MX transport. Values represent the means ± 1 S.E.M. of six independent determinations.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Transport of MX by ABCG2 is independent of GSH. ATP-dependent uptake of [3H]MX (5 µM) into ABCG2-containing membrane vesicles derived from MCF7/MX cells was examined in the presence () or absence () of 5 mM GSH. For the latter (GSH-negative; ), 5 mM L-glutamate was substituted for GSH. Shown are the mean values ± 1 S.E.M. from triplicate determinations.

View larger version (11K): [in this window] [in a new window]   Fig. 7. Role of GSH in MRP1 transport of MX. A, unlabeled MX (5 µM) stimulates the uptake of [3H]GSH (100 µM). Shown are the relative ATP-dependent uptakes of [3H]GSH by MRP1-containing MCF7/MRP1-10-derived vesicles in the presence or absence of 5 µM MX. All reactions were for 15 min. Data were normalized to zero mitoxantrone, and the bars represent mean values ± 1 S.E.M. from 13 independent determinations (P < 0.024; two-tailed unpaired t test). B, S-methyl analog of glutathione, but not L-glutamate (L-glu), substitutes for glutathione in supporting MRP1-mediated transport of MX. ATP-dependent uptake of [3H]MX (5 µM) into MRP1-containing vesicles (derived from MCF7/MRP1-10 cells) was determined over 15 min in the presence of 5 mM L-Glu, GSH, or S-methyl glutathione. Bars indicate the mean values ± 1 S.E.M. of eight (L-glu and GSH) or three (me-SG) independent determinations. C, ATP-dependent uptake of [3H]MX (5 µM) by MRP1-containing vesicles derived from MCF7/MRP1-10 cells determined over 15 min in the presence of 0 to 5 mM GSH as indicated. L-glutamate was added to reactions to achieve a total GSH + L-glutamate concentration equal to 5 mM. Bars represent mean values ± 1 S.E.M. from three independent determinations.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The work described herein demonstrates that MRP1 mediates MX resistance via supporting ATP-dependent drug efflux, thus resolving issues raised by conflicting data on the role of MRP1 in MX resistance as well as uncertainties raised by our own studies using a drug-selected cell line (Mirski et al., 1987; Cole et al., 1994; Schneider et al., 1994; Breuninger et al., 1995; Borst et al., 2000; Diah et al., 2001). Moreover, transport of MX by MRP1—like transport of other neutral or cationic MRP1-substrates (Loe et al., 1996, 1997, 1998; Renes et al., 1999)—is GSH-dependent, whereas transport of MX by the alternative efflux protein ABCG2 is not. In addition, MX reciprocally stimulates GSH transport by MRP1. These data are consistent with the interpretation that for MX, as for some other MRP1 substrates such as vincristine (Loe et al., 1996, 1998; Rappa et al., 1997), the requirement for GSH involves drug-GSH cotransport. However, this cotransport mechanism does not exclude the possibility that GSH may also serve as a transport regulator by interacting at a functionally distinct, nontransporting site on MRP1 (Zelcer et al., 2003; Chu et al., 2004; Peklak-Scott et al., 2005). Indeed, for some other substrates, GSH does not seem to be cotransported, yet it still serves to modulate transport activity of MRP1 (Leslie et al., 2001; Qian et al., 2001; Peklak-Scott et al., 2005).

It is remarkable that a consistent relationship between MRP1 overexpression and MX resistance has not previously been forthcoming. This may be because the level of MX resistance conferred by MRP1, although significant, is considerably less than that observed with other MRP1 substrates such as anthracyclines and etoposide (Schneider et al., 1994; Borst et al., 2000). When drug-selected or poorly matched cell lines are compared, other mechanisms of MX resistance may confound analysis. Indeed, the transgenic MCF7/MRP1-10 cells are 3-fold resistant to MX (compared with NCF7/WT), whereas drug-selected MCF7/VP cells are 6- to 10-fold resistant—a difference observed even though the levels of MRP1 expressed (Fig. 2) and intracellular MX accumulated are comparable (Fig. 4). Here, the difference in drug sensitivity is probably due to a second mechanism of MX resistance, decreased topoisomerase II, that emerged during etoposide selection of MCF7/VP cells (Schneider et al., 1994). Thus, the use of carefully matched parental and transgenic cell lines was required to reliably establish the role of MRP1 in conferring these relatively more modest levels of MX resistance. Other explanations for the failure of some other groups to detect MRP1-mediated MX resistance, even using matched transgenic cell lines, may include the following. The cell lines may differ with respect to GSH levels or other modulators of MRP1 transport activity. On the other hand, some cell lines may coexpress high levels of other MX transporters (e.g., P-glycoprotein or ABCG2) that may have masked the contribution of MRP1 to overall MX efflux and cellular resistance. Last, some cell lines may have expressed other allelic variants, or mutations, of MRP1 with altered activities toward MX. With regard to this last possibility, it is notable that both groups of MRP1-overexpressing cell lines used in our studies, the drug-selected MCF7/VP and the transduced MCF7/MRP1-series cells, express the most common, or wildtype variant of MRP1.

Another significant finding was the absolute dependence upon GSH (or its S-methyl analog) of MRP1-mediated MX transport in vitro (Figs. 5 and 7). In contrast, our previous study showed no effect of intracellular GSH depletion by BSO on either MX resistance or accumulation in MCF7/VP cells (Diah et al., 2001). The reason for this apparent discrepancy regarding the role of GSH in cell-free transport versus intact cells may be explained as follows. In our previous studies, treatment of MCF7 cells with BSO resulted in 90 to 95% depletion of intracellular GSH (Morrow et al., 1998; Diah et al., 2001). Thus, in MCF7 cells, which contain approximately 6 to 12 mM GSH (Diah et al., 1999; present study), such BSO treatment would leave as much as 0.35 to 1.2 mM residual intracellular GSH. At these GSH concentrations, the level of MRP1-mediated MX transport estimated in vesicle studies (Fig. 7C), although reduced, would remain significant. Thus, cellular depletion of GSH, even at 90 to 95%, may have been insufficient to achieve discernible differences in MX sensitivity. In the present study, we found that MCF7/MRP1-10 cells are particularly sensitive to BSO-mediated GSH depletion. Indeed, 48-h BSO treatment reduced intracellular GSH to <20 µM and resulted in significant, selective sensitization of MCF7/MRP1-10 cells to MX cytotoxicity (Table 1). Thus, we conclude that MRP1-mediated MX resistance is at least partially GSH-dependent and that the failure to show this dependence in the earlier study was most likely to be due to insufficient depletion of intracellular GSH in the MCF7/VP cells.

In conclusion, like the other known MX transporters P-glycoprotein and ABCG2, MRP1 may have a significant impact on tumor cell sensitivity to MX cytotoxicity, with MRP1-overexpressing tumors being relatively refractory. Moreover, MRP1, which is ubiquitously expressed but at variable levels in normal cells and tissues, also probably plays a significant role in the pharmacological disposition of MX. Other MRP family proteins, with more restricted tissue distributions, may also be important (Leslie et al., 2005). In particular, MRP2, which has significant substrate overlap with MRP1, is strategically located at the apical, excretory surfaces of cells within the liver, gut, and kidney as well as at the blood-brain barrier; thus, MRP2, along with P-glycoprotein and ABCG2, may have a significant impact on the clearance and pharmacodynamics of MX.

	Acknowledgements

 
We thank Erasmus Schneider for helpful suggestions in the design of MX transport studies.

	Footnotes

 
This work was supported by National Institutes of Health grant CA70338.

ABBREVIATIONS: MX, mitoxantrone; MRP, multidrug resistance (or resistance-associated) protein; GSH, glutathione; MK571, (E)-3-((3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl) (3-dimethylamino)-3-oxopropylthio)propanoic acid; FITC, fluorescein isothiocyanate; BSO, L-buthionine-(S,R)-sulfoximine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; me-SG, S-methyl analog of glutathione.

Address correspondence to: Dr. Charles S. Morrow, Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail: cmorrow{at}wfubmc.edu

	References

Top Abstract Materials and Methods Results Discussion References

 
Allen JD, Brinkhuis RF, Wijnholds J, and Schinkel AH (1999) The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res 59: 4237-4241.[Abstract/Free Full Text]

Borst P, Evers R, Kool M, and Wijnholds J (2000) A family of drug transporters: the multidrug resistance-associated proteins. J Natl Cancer Inst 92: 1295-1302.[Abstract/Free Full Text]

Brangi M, Litman T, Ciotti M, Nishiyama K, Kohlhagen G, Takimoto C, Robey R, Pommier Y, Fojo T, and Bates SE (1999) Camptothecin resistance: role of the ATP-binding cassette (ABC), mitoxantrone-resistance half-transporter (MXR) and potential for glucuronidation in MXR-expressing cells. Cancer Res 59: 5938-5946.[Abstract/Free Full Text]

Breuninger LM, Paul S, Gaughan K, Miki T, Chan A, Aaronson SA, and Kruh GD (1995) Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution. Cancer Res 55: 5342-5347.[Abstract/Free Full Text]

Chu X-Y, Huskey S-EW, Braun MP, Sarkadi B, Evans DC, and Evers R (2004) Transport of ethinylestradiol glucuronide and ethinylestradiol sulfate by the multidrug resistance proteins MRP1, MRP2, and MRP3. J Pharmacol Exp Ther 309: 156-164.[Abstract/Free Full Text]

Cole SP, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AM, and Deeley RG (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science (Wash DC) 258: 1650-1654.[Abstract/Free Full Text]

Cole SPC, Sparks KE, Fraser K, Loe DW, Grant CE, Wilson GM, and Deeley RG (1994) Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res 54: 5902-5910.[Abstract/Free Full Text]

Consoli U, Van NT, Neamati N, Mahadevia R, Beran M, Zhao S, and Andreeff M (1997) Cellular pharmacology of mitoxantrone in p-glycoprotein-positive and -negative human myeloid leukemic cell lines. Leukemia 11: 2066-2074.[CrossRef][Medline]

Dalton WS, Durie BG, Alberts DS, Gerlach JH, and Cress AE (1986) Characterization of a new drug-resistant human myeloma cell line that expresses P-glycoprotein. Cancer Res 46: 5125-5130.[Abstract/Free Full Text]

Diah SK, Smitherman PK, Aldridge J, Volk EL, Schneider E, Townsend AJ, and Morrow CS (2001) Resistance to mitoxantrone in MRP1-expressing MCF7 breast cancer cells: characterization of mitoxantrone transport. Cancer Res 61: 5461-5467.[Abstract/Free Full Text]

Diah SK, Smitherman PK, Townsend AJ, and Morrow CS (1999) Detoxification of 1-chloro-2,4-dinitrobenzene in MCF7 breast cancer cells expressing glutathione S-transferase P1-1 and/or multidrug resistance protein 1. Toxicol Appl Pharmacol 157: 85-93.[CrossRef][Medline]

DiPaola RS, Chenven ES, Shih WJ, Lin Y, Amenta P, Goodin S, Shumate A, Capanna T, Cardiella M, Cummings KB, et al. (2001) Mitoxantrone in patients with prostate specific antigen progression after local therapy for prostate carcinoma. Cancer 92: 2065-2071.[CrossRef][Medline]

Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, and Ross DD (1998) A multidrug resistance transporter from human MCF-7 breast cancer cells [published erratum appears in Proc Natl Acad Sci USA 96:2569]. Proc Natl Acad Sci USA 95: 15665-15670.[Abstract/Free Full Text]

Errington F, Willmore E, Tilby MJ, Li L, Li G, Li W, Baguley BC, and Austin CA (1999) Murine transgenic cells lacking DNA topoisomerase II are resistant to acridines and mitoxantrone: analysis of cytotoxicity and cleavable complex formation. Mol Pharmacol 56: 1309-1316.[Abstract/Free Full Text]

Harker WG, Slade DL, Drake FH, and Parr RL (1991) Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform. Biochemistry 30: 9953-9961.[CrossRef][Medline]

Harker WG, Slade DL, Parr RL, Feldhoff PW, Sullivan DM, and Holguin MH (1995a) Alterations in the topoisomerase II alpha gene, messenger RNA and subcellular protein distribution as well as reduced expression of the DNA topoisomerase II beta enzyme in a mitoxantrone-resistant HL-60 human leukemia cell line. Cancer Res 55: 1707-1716.[Abstract/Free Full Text]

Harker WG, Slade DL, Parr RL, and Holguin MH (1995b) Selective use of an alternative stop codon and polyadenylation signal within intron sequences leads to a truncated topoisomerase II alpha messenger RNA and protein in human HL-60 leukemia cells selected for resistance to mitoxantrone. Cancer Res 55: 4962-4971.[Abstract/Free Full Text]

Kufe DW, Pollock RE, Weichselbaum RR, Bast RC, Gansler TS, Holland JF, and Frei E (2003) Cancer Medicine 6. BC Decker, Inc., Hamilton, ON, Canada.

Leslie EM, Deeley RG, and Cole SP (2005) Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2 and BCRP (ABCG2) in tissue defense. Toxicol Appl Pharmacol 204: 216-237.[CrossRef][Medline]

Leslie EM, Ito K, Upadhyaya P, Hecht SS, Deeley RG, and Cole SPC (2001) Transport of the -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 276: 27846-27854.[Abstract/Free Full Text]

Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, and Bates SE (2000) The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113: 2011-2021.[Abstract]

Loe DW, Almquist KC, Deeley RG, and Cole SPC (1996) Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles: demonstration of glutathione-dependent vincristine transport. J Biol Chem 271: 9675-9682.[Abstract/Free Full Text]

Loe DW, Deeley RG, and Cole SPC (1998) Characterization of vincristine transport by the Mr 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res 58: 5130-5136.[Abstract/Free Full Text]

Loe DW, Stewart RK, Massey TE, Deeley RG, and Cole SPC (1997) ATP-dependent transport of aflatoxin B1 and its glutathione conjugates by the product of the multidrug resistance protein (MRP) gene. Mol Pharmacol 51: 1034-1041.[Abstract/Free Full Text]

Maliepaard M, van Gastelen MA, de Jong LA, Pluim D, van Waardenburg RC, Ruevekamp-Helmers MC, Floot BG, and Schellens JH (1999) Overexpression of the BCRP/MXR/ABCP gene in a topotecan-selected ovarian tumor cell line. Cancer Res 59: 4559-4563.[Abstract/Free Full Text]

Mirski SE, Gerlach JH, and Cole SP (1987) Multidrug resistance in a human small cell lung cancer cell line selected in adriamycin. Cancer Res 47: 2594-2598.[Abstract/Free Full Text]

Morrow CS, Diah S, Smitherman PK, Schneider E, and Townsend AJ (1998) Multidrug resistance protein and glutathione S-transferase P1-1 act in synergy to confer protection from 4-nitroquinoline 1-oxide toxicity. Carcinogenesis 19: 109-115.[Abstract/Free Full Text]

Nakagawa M, Schneider E, Dixon KH, Horton J, Kelley K, Morrow C, and Cowan KH (1992) Reduced intracellular drug accumulation in the absence of P-glycoprotein (mdr1) overexpression in mitoxantrone-resistant human MCF-7 breast cancer cells. Cancer Res 52: 6175-6181.[Abstract/Free Full Text]

Paumi CM, Ledford BG, Smitherman PK, Townsend AJ, and Morrow CS (2001) 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 276: 7952-7956.[Abstract/Free Full Text]

Peklak-Scott C, Townsend AJ, and Morrow CS (2005) Dynamics of glutathione conjugation and conjugate efflux in detoxification of the carcinogen, 4-nitroquinoline 1-oxide: contributions of glutathione, glutathione S-transferase and MRP1. Biochemistry 44: 4426-4433.[CrossRef][Medline]

Qian Y-M, Song W-C, Cui H, Cole SPC, and Deeley RG (2001) Glutathione stimulates sulfated estrogen transport by multidrug resistance protein 1. J Biol Chem 276: 6404-6411.[Abstract/Free Full Text]

Rappa G, Lorico A, Flavell RA, and Sartorelli AC (1997) Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins. Cancer Res 57: 5232-5237.[Abstract/Free Full Text]

Renes J, De Vries EGE, Nienhuis EF, Jansen PLM, and Müller M (1999) ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1. Br J Pharmacol 126: 681-688.[CrossRef][Medline]

Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Green-berger L, Cole SP, and Doyle LA (1999) Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 91: 429-433.[Abstract/Free Full Text]

Schneider E, Horton J, Yang CH, Nakagawa M, and Cowan KH (1994) Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF-7 cell line selected for etoposide resistance. Cancer Res 54: 152-158.[Abstract/Free Full Text]

Schurr E, Raymond M, Bell JC, and Gros P (1989) Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse mdr1 cDNA. Cancer Res 49: 2729-2733.[Abstract/Free Full Text]

Smitherman PK, Townsend AJ, Kute TE, and Morrow CS (2004) 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 308: 260-267.[Abstract/Free Full Text]

Yang CH, Schneider E, Kuo ML, Volk EL, Rocchi E, and Chen YC (2000) BCRP/MXR/ABCP expression in topotecan-resistant human breast carcinoma cells. Biochem Pharmacol 60: 831-837.[CrossRef][Medline]

Zaman GJR, Flens MJ, Van Leusden MR, De Haas M, Mulder HS, Lankelma J, Pinedo HM, Scheper RJ, Baas F, Broxterman HJ, et al. (1994) The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump. Proc Natl Acad Sci USA 91: 8822-8826.[Abstract/Free Full Text]

Zelcer N, Huisman MT, Reid G, Wielinga P, Breedveld P, Kuil A, Knipscheer P, Schellens JHM, Schinkel AH, and Borst P (2003) Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 278: 23538-23544.[Abstract/Free Full Text]

Zhou R, Wang Y, Gruber A, Larsson R, Castanos-Velez E, and Liliemark E (1999) Topoisomerase II-mediated alterations of K562 drug resistant sublines. Med Oncol 16: 191-198.[Medline]

This article has been cited by other articles:

				A. Rothnie, G. Conseil, A. Y. T. Lau, R. G. Deeley, and S. P. C. Cole Mechanistic Differences between GSH Transport by Multidrug Resistance Protein 1 (MRP1/ABCC1) and GSH Modulation of MRP1-Mediated Transport Mol. Pharmacol., December 1, 2008; 74(6): 1630 - 1640. [Abstract] [Full Text] [PDF]

				P. Acharya, M. P. O'Connor, J. W. Polli, A. Ayrton, H. Ellens, and J. Bentz Kinetic Identification of Membrane Transporters That Assist P-glycoprotein-Mediated Transport of Digoxin and Loperamide through a Confluent Monolayer of MDCKII-hMDR1 Cells Drug Metab. Dispos., February 1, 2008; 36(2): 452 - 460. [Abstract] [Full Text] [PDF]

				J. Weiss, J. Rose, C. H. Storch, N. Ketabi-Kiyanvash, A. Sauer, W. E. Haefeli, and T. Efferth Modulation of human BCRP (ABCG2) activity by anti-HIV drugs J. Antimicrob. Chemother., February 1, 2007; 59(2): 238 - 245. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: mol.105.017988v1 69/4/1499    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morrow, C. S. Articles by Townsend, A. J. Search for Related Content PubMed PubMed Citation Articles by Morrow, C. S. Articles by Townsend, A. J.