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Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, New York 12201
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence for the existence of an energy-dependent MTX transport or efflux mechanism has accumulated over several decades. Early reports described a rapid, energy-dependent, MTX efflux from Sarcoma-180 cells (11) , and a similar, azide-inhibitable, unidirectional export of MTX was also shown in leukemic L1210 cells (12) . Furthermore, efflux of both mono- and diglutamates of MTX was demonstrated in a cervical carcinoma cell line (13) . A series of reports throughout the last decade have further classified the cellular export of MTX as an ATP-dependent, outwardly directed mechanism that transports both MTX and its metabolites (14, 15, 16, 17, 18) .
Despite clear biochemical evidence, the mediator(s) of MTX efflux have only recently been described at the molecular level. Several members of the MRP or ABCC subfamily of the ABC transporters have been implicated in MTX export and resistance (19, 20, 21) . These ATP-dependent efflux pumps have been shown to transport MTX in vitro and to cause low levels of MTX resistance in both transfected and drug-selected cells, but only in short-term (1–3-h) exposure assays. This limited-resistance phenotype has been attributed to the inability of the MRPs to transport MTX in its polyglutamylated form; the addition of even a single extra glutamate residue to MTX caused an almost complete abrogation of drug transport (22 , 23) .
In contrast, we have described high-level MTX cross-resistance to continuous (7 days) drug exposure in the mitoxantrone-selected MCF7/MX breast carcinoma cell line, which expresses large amounts of the ABC transporter BCRP (24) , also known as mitoxantrone resistance protein (25) , placental-specific ABC transporter (26) , or ABCG2 (27) . Further characterization of these cells suggested that ATP-dependent, enhanced drug efflux caused the MTX resistance. MTX resistance in MCF7/MX cells was reversible by the BCRP inhibitors GF120918 and FTC, and levels of MTX resistance directly correlated with BCRP expression in this and multiple other cell lines of varied tissue origin (28) . Together, these data suggested that BCRP can function as a MTX transporter. However, resistance to MTX occurred only in the presence of the wild-type transporter, which contains an arginine at position 482, whereas cells that overexpress a mutated BCRP (R482T and R482G) remained sensitive to MTX. Here, we expand on the association between BCRP and MTX resistance by demonstrating that wild-type, but not mutated, BCRP is able to transport MTX and its polyglutamates in an in vitro membrane vesicle system.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vesicle Preparation
Vesicle preparation was adapted from Loe et al. (31)
. Cells were seeded onto 15-cm plates and were grown to 70% confluency, at which time they were washed twice with PBS and scraped into the residual PBS. Cells from 20 plates were combined and centrifuged at 500 x g at 4°C for 15 min; the pellet was frozen overnight at -80°C. Inside-out membrane vesicles were prepared by thawing the cell pellet at 4°C and resuspending the cells in 7 ml of homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 0.25 mM CaCl2, and one EDTA-free protease inhibitor mixture tablet (Roche, Indianapolis, IN). The cell suspension was then transferred to a 50-ml round-bottomed tube and placed on ice into the nitrogen cavitation chamber (Parr Instruments, Moline, IL). Under constant stirring, cells were saturated with nitrogen gas at a pressure of 1,250 p.s.i. for 20 min. After release of the pressure, membrane suspensions were centrifuged at 1,000 x g at 4°C for 15 min. The supernatant was overlayed on a sucrose cushion containing 35% sucrose, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and was centrifuged at 209,000 x g at 4°C for 120 min. The opaque interface was collected and diluted 
1:30 into TS buffer containing 10 mM Tris-HCl (pH 7.5) and 250 mM sucrose and was centrifuged at 123,000 x g at 4°C for 40 min. The vesicle-containing pellet was resuspended in 50 mM Tris-HCl (pH 7.5) and 250 mM sucrose, and dispersed 20 times through a 27-gauge needle. Vesicle preparations were assayed for protein concentration using the Bradford method (Bio-Rad, Hercules, CA), and membrane orientation was determined by measuring the activity of the endo-enzyme glyceraldehyde-3-phosphate dehydrogenase (32)
in the absence and presence of 0.1% Triton X-100.
Western Blot Analysis
Immunodetection of BCRP in the membrane vesicles was performed as described previously (28)
. Briefly, 50 µg of the protein of membrane vesicles were electrophoresed on a 10% polyacrylamide gel and were transferred to a PVDF membrane (Millipore, Bedford, MA). BCRP-specific signals were detected using a polyclonal rabbit anti-BCRP primary antibody (kindly provided by Dr. Susan Bates, National Cancer Institute, Bethesda, MD), horseradish peroxidase-conjugated goat antirabbit secondary antibody (Bio-Rad), and enhanced chemiluminescence (Pierce, Rockford, IL).
Vesicular Drug Accumulation Assays
Vesicular drug accumulation assays were adapted from Saxena and Henderson (33)
. All of the reaction mixtures were prepared as 20-µl samples containing the following: 0.1 mg/ml creatine kinase, 10 mM phosphocreatine (both from Sigma, St. Louis, MO), 10 mM Tris-HCl (pH 7.4), 10 mM magnesium chloride, 250 mM sucrose, 400 µM [3H]methotrexate (Moravek Biochemicals, Brea, CA), 4 mM ATP or AMP, and 5 µg of vesicles. Reaction mixtures were incubated at 37°C (0 time points were incubated at 4°C), and transport was terminated by dilution into 4 ml of ice-cold TS buffer. Vesicles were then separated from free drug by filtration of the reaction mixture through a glass microfiber filter (GF-F; Whatman, Fairfield, NJ) and rinsing five times with TS buffer. The amount of drug in the vesicles that remained on the filters was quantitated by liquid scintillation counting after 24 h in liquid-scintillation mixture. Unless otherwise indicated, all of the replicate assays were done with separate vesicle preparations.
Time Course Assays.
Reactions were incubated for 1, 2, 5, 10, 15, 30, 45, and 60 min at 37°C. Time 0 was incubated at 4°C.
Osmotic Sensitivity Assays.
MTX accumulation was measured in the presence of 0.25, 0.5, 0.67, 1.0, or 1.14 M sucrose and 200 µM [3H]methotrexate, in the presence or absence of ATP at 60 min.
Inhibitor Assays.
MTX accumulation was measured in the presence or absence of 5 µM FTC at 60 min.
Kinetic Studies.
Drug accumulation was measured after 2-min exposure to 0, 20, 50, 100, 200, 500, 1000, and 2000 µM [3H]methotrexate.
Competition Assays.
MTX accumulation was measured in the presence of 0, 10, 20, 50, and 100 µM mitoxantrone at 60 min.
Accumulation of MTX polyglutamates.
Transport of long-chain polyglutamates was assayed by incubation of vesicles in the presence of 400 µM [3H]methotrexate,4
[3H]methotrexate-Glu2, [3H]methotrexate-Glu3, or [3H]methotrexate-Glu4 (Moravek Biochemicals) for 60 min in either the presence or the absence of 5 µM FTC.
Calculation of Net Accumulation.
Unless otherwise indicated, all values shown correspond to net accumulation. Net MTX accumulation was determined by measurement of drug uptake in the presence of ATP or AMP at 4°C, and subtracting these background values (T0) from each respective measurement taken in the presence of ATP or AMP at 37°C, at time points ranging from 1 to 60 min. For example:
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RESULTS |
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To confirm the presence of BCRP in the inside-out vesicles, a Western blot analysis was performed. As expected, high levels of BCRP were detected in the vesicles from drug-selected MCF7/MX and S1-M1–80 cells, whereas moderate levels were observed in the vesicles derived from the BCRP-transfected cells MCF7/BCRP-Arg and MCF7/BCRP-Gly (Fig. 1)
. The apparent slight overexpression of BCRP in the MCF7/BCRP-Gly clone compared with the MCF7/BCRP-Arg clone is also seen in glyceraldehyde-3-phosphate dehydrogenase used as a loading control. It most likely can be attributed to slight differences in the amount of protein loaded and, therefore, does not represent a true difference in the level of BCRP in these vesicles. Using a serial dilution of membranes from MCF7/MX cells, we estimated that these cells expressed approximately 300–400-fold more BCRP than the transfectants (data not shown); this value corresponded well to that obtained by quantitative reverse transcription-PCR (28)
. Little or no BCRP was detected in vesicles from the parental cell lines MCF7/WT and S1, or from the control transfectants MCF7/pcDNA3. Together, these data indicated that the in vitro system was suitable for the intended studies; membrane vesicles were inverted and BCRP levels in vesicles were consistent with those observed in whole cells.
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, high levels of MTX accumulation were found in MCF7/MX and MCF7/BCRP-Arg vesicles, both of which express the arginine variant of BCRP, in the presence of ATP, but not in the presence of AMP. In contrast, vesicles generated from S1-M1–80 or MCF7/BCRP-Gly cells, which express the glycine variant of BCRP, displayed no significant increase in MTX accumulation in the presence of ATP compared with the increase in the presence of AMP. As expected, vesicles derived from MCF7/WT- and control-transfected cells exhibited little or no MTX transport in the presence of either ATP or AMP. Thus, it appeared that vesicles derived from cells containing the arginine variant of BCRP were able to accumulate MTX, and that accumulation was ATP dependent. In contrast, no accumulation was observed in vesicles containing BCRP with a glycine at position 482. These results were consistent with the previously observed energy-dependence of MTX efflux and the position-482 substrate specificity of BCRP in whole cells (24
, 28
, 34
, 35)
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8-fold lower in vesicles derived from the transfected cells, which is commensurate with the reduced levels of BCRP expression in the MCF7/BCRP-Arg cells relative to the MCF7/MX cells. In contrast, despite very high levels of BCRP in vesicles from S1-M1–80 cells, no accumulation of MTX was detected.
To further examine MTX transport via BCRP, we measured time-dependent drug accumulation in vesicles derived from both drug-selected and stably transfected cell lines. The MCF7/MX vesicles displayed an ATP-dependent accumulation of MTX that increased with time and was linear for at least two min (Fig. 2B)
. In contrast, no transport was detected in vesicles derived from either S1-M1–80 or parental cells, even after a 60-min drug exposure. The S1-M1–80 vesicles were, however, capable of ATP-dependent mitoxantrone accumulation (data not shown), indicating that the vesicles were functional. This provides further evidence that the amino acid at codon 482 is critical for BCRP substrate specificity. Similarly, MTX accumulation increased over time in the vesicles derived from the MCF7/BCRP-Arg cells, but no drug uptake was observed in either MCF7/BCRP-Gly vesicles or vesicles from control transfectants (Fig. 2C)
. Together, these data demonstrated that MTX transport was both energy and time dependent and appeared to occur only in the presence of the wild-type (Arg482) variant of BCRP.
Osmotic Sensitivity.
To assess whether the ATP-dependent MTX accumulation was caused by drug import into the lumen of the vesicles or was attributable to nonspecific binding to the membranes, we determined its osmotic sensitivity. ATP-dependent uptake of MTX into the MCF7/MX vesicles was measured under various osmotic conditions. Drug accumulation was linear, relative to the reciprocal of the sucrose concentration in the presence of ATP, whereas no drug accumulation was observed in the absence of ATP under otherwise identical reaction conditions (Fig. 3)
. These data demonstrated that the apparent intravesicular drug accumulation observed was, indeed, attributable to transport into the lumen of the membrane vesicles, rather than to nonspecific binding.
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, indicating that BCRP is the transporter of MTX in this system.
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, the velocity of substrate transport increased according to Michaelis-Menten kinetics in the presence of increasing levels of drug. MTX transport by BCRP was found to have a Km of 681 ± 280 µM and a Vmax of 2384 ± 445 pmol/mg/min. Thus, BCRP appeared to be a low-affinity, high-capacity transporter of MTX.
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. The inhibition of MTX transport ranged from 12%, in the presence of 10 µM mitoxantrone, to nearly 60%, in the presence of 100 µM mitoxantrone. These data suggested competition between mitoxantrone and MTX and provided further evidence that MTX was indeed a substrate for BCRP.
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and MCF7/BCRP-Arg (data not shown) cells. As expected, drug uptake into vesicles derived from both cell lines decreased as the MTX polyglutamate chain length increased; however, in contrast to the MRPs, significant amounts of MTX-Glu2 and MTX-Glu3 were still transported. Furthermore, MTX polyglutamate transport was sensitive to the BCRP inhibitor FTC, indicating that BCRP, indeed, was transporting MTX as well as its polyglutamylated metabolites.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In contrast, we present evidence that BCRP transports not only MTX but also MTX-polyglutamates. Using an in vitro inside-out vesicle system, we have demonstrated ATP-dependent transport of MTX into vesicles the membranes of which contain BCRP. MTX transport was osmotically sensitive and followed Michaelis-Menten kinetics. The Km and Vmax values of 680 µM and 2400 pmol/mg/min, respectively, suggest that BCRP is a low-affinity, high-capacity transporter. Furthermore, there was also substantial transport of polyglutamylated forms of MTX, MTX-Glu2, and MTX-Glu3. Accumulation of MTX-Glu2 and MTX-Glu3 was 60 and 40% of that observed with MTX. Finally, transport of all MTX species was sensitive to inhibition by the BCRP inhibitor FTC. Thus, the data presented clearly establish BCRP as a transporter of MTX and its polyglutamates.
Interestingly, MTX transport was only detected in vesicles with wild-type BCRP, which contains an arginine at amino acid position 482. In contrast, no uptake of MTX was observed into vesicles containing BCRP with an R482G mutation. This finding was consistent with the phenotype displayed by the cells from which the vesicles were derived, MCF7/MX (BCRP-Arg) and S1-M1–80 (BCRP-Gly), respectively. Whereas MCF7/MX cells are highly resistant to MTX, only minimal cross-resistance to MTX is observed in S1-M1–80 cells (28) . Thus, it appears that the R482G mutation resulted in a dramatic change in substrate specificity for BCRP. In agreement with this conclusion, Honjo et al. (34) have demonstrated that cells expressing the arginine variant were resistant to mitoxantrone and CPT-11 but remained largely sensitive to Adriamycin. In contrast, cells expressing the glycine variant exhibited enhanced levels of mitoxantrone resistance as well as high levels of Adriamycin resistance, both of which were accompanied by significantly reduced drug accumulation. Furthermore, cellular rhodamine 123 accumulation was drastically reduced in cells with BCRP-Gly but unaffected in cells with BCRP-Arg (34) . Similar effects were also observed with mouse bcrp when the corresponding arginine was mutated to either serine or methionine (35) . Thus, it seems clear that the identity of the amino acid at position 482 has a profound effect on the specificity of BCRP-mediated drug transport. However, additional studies are needed to elucidate the exact mechanism behind this observation.
Several members of the MRP family of ABC transporters have been shown to transport MTX, and their kinetic parameters have been determined. Reported Km values for MRP 1, 3, and 4 are 2150, 620, and 220 µM, respectively, and Vmax values are 2050, 2930, and 240 pmol/mg/min, respectively (22 , 23) . These values are comparable with those determined for BCRP in the present study. However, in contrast to what was observed for BCRP, the addition of even a single extra glutamate to MTX resulted in essentially complete abrogation of transport by the MRPs. Thus, although the affinities and capacities are in the same range, the ability to transport MTX-Glu2 and MTX-Glu3 makes BCRP clearly distinct from the MRPs, and unique among the ABC proteins reported to transport MTX.
It is tempting to speculate that the ability of BCRP to transport polyglutamylated MTX is responsible for the distinct MTX-resistance pattern of BCRP-overexpressing cells. Cells that overexpress BCRP are resistant against MTX in continuous exposure assays when polyglutamylation is likely to occur, suggesting that transport of polyglutamylated MTX may be an important requirement for resistance. In contrast, cells that overexpress MRPs are only resistant to short-term (<3 h) MTX exposure, when it is unlikely that MTX has undergone polyglutamylation (20 , 21 , 23) . Thus, despite the low affinity of BCRP for MTX, its capacity to also transport MTX-polyglutamates may account for the high levels of resistance in long-term assays. It further suggests that MTX-polyglutamates can be exported even as they are formed, as opposed to only after degradation, which is consistent with the observed lower steady-state levels of MTX-polyglutamates in MCF7/MX cells (24) .
The physiological significance of a Km as high as that observed for BCRP is unclear because it is doubtful whether the intracellular free MTX concentration will ever reach levels of that order of magnitude in vivo. For example, total MTX concentrations in the order of 500-3500 pmol/109 cells or 2–15 µM, have been found in pediatric leukemia cells (44, 45, 46) , which is two to three orders of magnitude below the Km for BCRP as well as for the MRPs. However, plasma MTX levels of 100 µM and greater have been measured 42 h after an initial high-dose MTX infusion (44) . Furthermore, it is conceivable that the local concentration on the cytoplasmic side of the plasma membrane may be higher than the average intracellular concentration. Nevertheless, despite the low affinity, when the overexpression of BCRP is sufficiently high, it can apparently cause enhanced MTX efflux and concomitant resistance (28) .
BCRP was originally identified and described as a mitoxantrone transporter. Therefore, we determined the effect of mitoxantrone on MTX transport. These studies have demonstrated that the presence of mitoxantrone reduced the transport of MTX. Increasing concentrations of mitoxantrone resulted in decreasing MTX accumulation into MCF7/MX vesicles, indicating that the presence of mitoxantrone interferes and possibly competes with MTX transport. These results further support the idea that BCRP is a MTX transporter. However, the data do not allow for further characterization of the type of competition between the two substrates.
Previously, MTX resistance in the MCF7/MX cells had been attributed to an ATP-dependent efflux mechanism, and it was shown that resistance was reversible with BCRP inhibitors (24 , 28) . Furthermore, MTX cross-resistance was not unique to the MCF7/MX cells; multiple BCRP-overexpressing cell lines exhibited similar cross-resistance, suggesting that it was caused by BCRP. Here, we extended this association and directly demonstrated that BCRP functions as a MTX efflux pump. BCRP appears to be unique among the MTX exporters described thus far in that it is also able to transport polyglutamylated forms of MTX. The efflux of MTX-polyglutamates may be a major contributor to MTX resistance under long-term exposure conditions.
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ACKNOWLEDGMENTS |
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Note Added in Proof
After this manuscript was accepted for publication, two reports appeared that independently came to essentially the same conclusions. (1) Z. S. Chen, et al. Transport of Methotrexate, Methotrexate Polyglutamates, and 17ß-Estradiol 17-(ß-D-glucuronide) by ABCG2: Effects of Acquired Mutations at R482 on Methotrexate Transport. Cancer Res., 63: 4048–4054, 2003. (2) H. Mitomo, et al. A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport. Biochem J, 373: 767–774, 2003.
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FOOTNOTES |
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1 Supported by NIH Grant CA72455 (to E. S.). 
2 To whom requests for reprints should be addressed, at Wadsworth Center, Empire State Plaza, Albany, NY 12201. Phone: (518) 474-2088; Fax: (518) 474-1850; E-mail: schneid{at}wadsworth.org
3 The abbreviations used are: MTX, methotrexate; ABC, ATP-binding cassette; BCRP, breast cancer resistance protein; FTC, fumitremorgin C; MRP, multidrug resistance-associated protein; PVDF, polyvinylidene difluoride.
4 MTX has an integral glutamate and, therefore, corresponds to MTX-Glu1.
Received 12/31/02. Revised 5/ 6/03. Accepted 6/30/03.
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REFERENCES |
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-glutamyl hydrolase activity. Cancer Res., 53: 2227-2230, 1993.This article has been cited by other articles:
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  I. Ifergan, G. Jansen, and Y. G. Assaraf The Reduced Folate Carrier (RFC) Is Cytotoxic to Cells under Conditions of Severe Folate Deprivation: RFC AS A DOUBLE EDGED SWORD IN FOLATE HOMEOSTASIS J. Biol. Chem., July 25, 2008; 283(30): 20687 - 20695. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Evans, J. P. Day, P. Cabrero, J. A. T. Dow, and S.-A. Davies A new role for a classical gene: White transports cyclic GMP J. Exp. Biol., March 15, 2008; 211(6): 890 - 899. [Abstract] [Full Text] [PDF]
|
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|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
 S. Chattopadhyay, R. G. Moran, and I. D. Goldman Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications Mol. Cancer Ther., February 1, 2007; 6(2): 404 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
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|
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|
|
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|
|
|
|
 |
|
 A. Shafran, I. Ifergan, E. Bram, G. Jansen, I. Kathmann, G. J. Peters, R. W. Robey, S. E. Bates, and Y. G. Assaraf ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates Cancer Res., September 15, 2005; 65(18): 8414 - 8422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
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|
|
|
|
|
|
I. Ifergan, G. Jansen, and Y. G. Assaraf Cytoplasmic Confinement of Breast Cancer Resistance Protein (BCRP/ABCG2) as a Novel Mechanism of Adaptation to Short-Term Folate Deprivation Mol. Pharmacol., April 1, 2005; 67(4): 1349 - 1359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-S. Chen, Y. Guo, M. G. Belinsky, E. Kotova, and G. D. Kruh Transport of Bile Acids, Sulfated Steroids, Estradiol 17-{beta}-D-Glucuronide, and Leukotriene C4 by Human Multidrug Resistance Protein 8 (ABCC11) Mol. Pharmacol., February 1, 2005; 67(2): 545 - 557. [Abstract] [Full Text] [PDF]
|
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|
 |
|
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P. Breedveld, N. Zelcer, D. Pluim, O. Sonmezer, M. M. Tibben, J. H. Beijnen, A. H. Schinkel, O. van Tellingen, P. Borst, and J. H. M. Schellens Mechanism of the Pharmacokinetic Interaction between Methotrexate and Benzimidazoles: Potential Role for Breast Cancer Resistance Protein in Clinical Drug-Drug Interactions Cancer Res., August 15, 2004; 64(16): 5804 - 5811. [Abstract] [Full Text] [PDF]
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), S1-M1–80 (
), and MCF7/WT (
), and stably transfected cell lines (C), MCF7/BCRP-Arg (
), MCF7/BCRP-Gly (
) and MCF7/pcDNA3 (*). The data represent the mean ± SE of three independent experiments performed using three separate vesicle preparations from MCF7/MX, MCF7/BCRP-Arg, and MCF7/BCRP-Gly cells, and the mean of two experiments for MCF7/WT and S1-M1–80 vesicles. (The scale of the Y axis is different between panels B and C.)
) of 4 mM ATP.
