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Experimental Therapeutics |
Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [Z-S. C., K. L., S. W., R. B. R., H. Z., G. D. K.], and Department of Medicinal Biochemistry, Graduate School of Medical Sciences, Kyushu University School of Medicine, Fukuoka 812-8582, Japan [M. K.]
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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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MRP4 and MRP5 differ in their structures from MRPs 1–3 in that unlike the latter proteins, they do not possess a third (NH2-terminal) hydrophobic domain (15 , 31) , and this difference is reflected in their distinctive drug resistance profiles, substrate selectivities, and potential physiological functions. These two pumps do not confer resistance to natural product anticancer agents, but instead are capable of conferring resistance to nucleotide analogues such as 6-MP and 9-(2-phosphonylmethoxyethyl)adenine (32, 33, 34, 35, 36) . In addition, MRP4 and MRP5 have the facility for the MgATP-energized transport of cAMP and cGMP, a feature that suggests their involvement in the regulation of intracellular cyclic nucleotide levels (35 , 37) .
Although MRP4 and MRP5 confer resistance to nucleotide analogues and transport cyclic nucleotides, the substrate ranges and resistance profiles of these two pumps appear to be distinct in several regards. By contrast with MRP5, MRP4 has the facility for mediating the transport of glucuronides, such as estradiol E217ßG, and it is also a higher affinity transporter of cAMP than is MRP5 (35) . Another potential difference concerns the antimetabolite MTX. MRP4, similar to MRPs 1–3, and in contrast to MRP5, has been shown to confer resistance to this widely used antimetabolite (34) . Although the potency of MRP4 as assessed in transfected NIH3T3 cells is lower than that reported for MRPs 1–3 expressed in transduced 2008 cells (13 , 14) , this feature of MRP4 is of interest in that it raises the possibility that it is a component of the previously described energy-dependent efflux system for MTX (38) , a system for which MRP3 and MRP1 are now established components (39) . However, the notion that MRP4 is able to transport MTX has not been firmly established in that it is currently an inference based entirely upon a previous study in which we found a relatively low level of resistance to this agent and an associated cellular accumulation deficit in a single clone of transfected NIH3T3 cells (34) .
Here we analyze the potential involvement of MRP4 in the cellular pharmacology of MTX and related processes, by examining its ability to mediate the transport of this agent and folates in membrane vesicles prepared from insect cells infected with MRP4 baculovirus. In so doing, it is demonstrated that MRP4 is not only competent in the MgATP-energized transport of MTX and folates, but that its affinities for these substrates compare favorably with those we reported previously for MRP1 and MRP3 (39) . It is also shown that MRP4 can accommodate a critical property of previously described MTX efflux systems in that its capacity to transport this agent is abrogated by the addition of a single glutamyl residue, as is also the case for MRP1 and MRP3 (39) . In addition, it is shown that glutamylation similarly affects the transport activity of MRP2.
On the basis of these results, two conclusions are drawn: (a) MRP4 is a bona fide MTX resistance factor, and it along with MRP1, MRP2, and MRP3 are components of the energy-dependent efflux system for this agent and physiological folates; and (b) MRP4 is a common efflux pump for MTX and certain nucleotide analogues.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Membrane Vesicles and Transport Experiments.
Membrane vesicles were prepared by the nitrogen cavitation method as described previously (40)
. Transport experiments were performed using the rapid filtration method essentially as described (17)
and carried out in medium containing membrane vesicles (10 µg), 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 4 mM ATP, 10 mM phosphocreatine, 100 µg/ml creatine phosphokinase, and radiolabeled substrate ± unlabeled substrate in a total volume of 50 µl. Reactions were carried out at 37°C and stopped by the addition of 3 ml of ice-cold stop solution (0.25 M sucrose, 100 mM NaCl, and 10 mM Tris-HCl, pH 7.4) Samples were passed through 0.22 µm Durapore membrane filters (Millipore, Bedford, MA) under vacuum. The filters were washed three times with 3 ml of ice-cold stop solution and dried at room temperature for 30 min. Radioactivity was measured by the use of a liquid scintillation counter. Rates of net ATP-dependent transport were determined by subtracting the values obtained in the presence of 4 mM MgAMP from those obtained in the presence of 4 mM MgATP. Uptake rates were linear for >5 min, and rates for concentration dependence experiments were measured at 5 min.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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[3H]MTX was indeed subject to MRP4-mediated, MgATP-dependent transport (Fig. 1A)
. When measured at initial concentrations of 100 µM and at the 5-min time point of the assay, [3H]MTX was taken up by MRP4-enriched vesicles a rate of 103 pmol/mg/min from medium containing MgATP and a rate of 45 pmol/mg/min from medium containing MgAMP. By contrast, the rates of uptake of membranes prepared from uninfected insect cells were <34 pmol/mg/min, under either energized or nonenergized conditions. MgATP-stimulated uptake by MRP4-enriched membranes was linear over the first 10 min of the assay. Uptake by membranes prepared from insect cells infected with parental baculovirus was indistinguishable from that of uninfected insect cells, indicating that infection by itself did not influence MTX transport (data not shown).
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-linked glutamate residues, and a critical feature of previously described energy-dependent MTX efflux systems is the ability to transport the parent drug but not its polyglutamylated metabolites (38)
. To determine whether MRP4 can accommodate this criterion, its capacity to transport [3H]MTX-Glu2 was assessed. In sharp contrast to [3H]MTX and in strict agreement with the properties expected for a component of the MTX efflux system, 100 µM [3H]MTX-Glu2 was not transported to any extent by MRP4-enriched membrane vesicles. As shown in Fig. 1B
, the uptake rate of MRP4-enriched membranes in medium containing MgATP was indistinguishable from either the uptake rate of the same membranes in medium containing MgAMP or the uptake rates of membranes prepared from uninfected insect cells under either energized or nonenergized conditions. [3H]MTX-Glu3 was similarly not subject to MRP4-mediated transport (data not shown).
Osmotic Sensitivity of MTX Transport.
MgATP-dependent retention of MTX by MRP4-enriched membrane vesicles was predominately a consequence of transport into the intravesicular space as opposed to nonspecific binding to the filters or membranes. Uptake of 100 µM [3H]MTX by MRP4-enriched membrane vesicles in medium containing MgATP increased as a linear function of the reciprocal of sucrose concentration, indicating that the transported substrate was delivered into an osmotically active compartment (Fig. 2)
. By contrast, substrate retention measured in medium containing MgAMP was only moderately affected by the sucrose concentration. This suggested that under nonenergized conditions, the apparent uptake largely represented nonspecific binding.
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From these experiments, it was determined that folates are indeed susceptible to MRP4-mediated transport (Fig. 3)
. When measured at initial concentrations of 100 µM and over the first 5 min of the assay, [3H]folic acid and [3H]N5-formyl-THF were taken up from medium containing MgATP by MRP4-enriched vesicles at rates of 142 and 153 pmol/mg/min, respectively. By contrast, the corresponding rates for the same membranes under nonenergized conditions were 67 and 56 pmol/mg/min, respectively, for folic acid and N5-formyl-THF and <44 pmol/mg/min for control membranes under either energized or nonenergized conditions.
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The substrate concentration dependence of MgATP-energized [3H]MTX, [3H]folic acid, and [3H]N5-formyl-THF transport by MRP4-enriched membrane vesicles approximated Michaelis-Menten kinetics. The initial rates of MgATP-dependent uptake of all three compounds, enumerated as the difference between uptake in medium containing MgATP and uptake in medium containing MgAMP, and measured over a broad range of substrate concentrations, exhibited saturation kinetics (Fig. 4)
. Nonlinear least squares fitting of the data to the Michaelis-Menten equation (41)
yielded Km and Vmax values of 0.22 ± 0.01 mM and 0.24 ± 0.05 nmol/mg/min, 0.17 ± 0.02 mM and 0.68 ± 0.14 nmol/mg/min, and 0.64 ± 0.23 mM and 1.95 ± 0.18 nmol/mg/min for MTX, folic acid, and N5-formyl-THF, respectively (Table 1)
. The efficiencies of transport (Vmax/Km) fell in the rank order folic acid (4.0) > N5-formyl-THF (3.0) > MTX (1.1) (Table 1)
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. Of these compounds, E217ßG exerted the highest degree of inhibition (60.1% at 30 µM). By contrast, the inhibitions exerted by cGMP, cAMP, folic acid, and N5-formyl-THF were modest (53.5, 51.5, 55.0, and 28.8%, respectively, at 300 µM concentrations). The higher degree of inhibition exerted by E217ßG by comparison with MTX, folic acid, and N5-formyl-THF is commensurate with their respective Km values (Km E217ßG, 30.3 µM; Ref. 35
; Table 1
). However, cGMP (Km, 9.7 µM) and cAMP (Km, 44.5 µM), whose affinities are either higher than or roughly comparable with that of E217ßG (35)
, were by comparison unexpectedly weak inhibitors.
The inhibitory activities of three MRP1 reversing agents were examined to test their effects on MRP4 activity. These agents included MK571, a leukotriene D4 receptor antagonist, and two inhibitors of organic anion transporters, probenecid and sulfinpyrazone (17
, 42, 43, 44, 45)
. MK571 was not only the most potent inhibitor of the three MRP1 reversing agents, but it was also the single most efficacious inhibitor of all of the compounds tested (43.6% inhibition at 1 µM, 73.9% inhibition at 3 µM, and complete inhibition at 10 µM; Table 2
, and data not shown in Table 2
). By contrast, the inhibitions exerted by 300 µM concentrations of probenecid and sulfinpyrazone (50.6 and 76.7%, respectively) were modest.
The inhibitory activities of three P-glycoprotein modulating agents were next examined. These agents included verapamil, a calcium channel blocker, and two bulky lipophilic compounds, PSC833 and cyclosporin A. Of these agents, PSC833 was the most efficacious inhibitor (43.6% at 10 µM). The inhibitions exerted by cyclosporin A (14.5%) and verapamil (46.2%) at concentrations of 10 and 30 µM, respectively, were significantly lower by comparison.
Knowing that MRP4 is able to transport cyclic nucleotides and that inhibitors of phosphodiesterases have been reported to be potent inhibitors of MRP5 (37) , we next examined the inhibitory activity of this class of compounds. Three phosphodiesterase inhibitors were selected for analysis, trequinsin, zaprinast, and sildenafil. All three of these agents exhibited significant inhibitory activity (74.3, 65.9, and 54% inhibition at 10 µM concentrations, respectively). Trequinsin was the most potent inhibitor of this group of agents and the second most potent inhibitor overall.
Transport of MTX but not MTX-Glu2 by MRP2.
Having determined here that the properties of MRP4 are consistent with it being a component of the cellular MTX efflux system, and in a previous study that MRP1 and MRP3 are similarly able to transport MTX but not polyglutamylated derivatives (39)
, we next examined the MTX transport properties of MRP2 (cMOAT), the only remaining MRP family member whose expression has been associated with MTX resistance (14)
but whose in vitro transport properties with regard to MTX versus MTX polyglutamates has not been determined. For this purpose, membrane vesicles prepared from MRP2-transfected (LLC/cMOAT-1) and parental vector-transfected (LLC/CMV) LLC/PK1 cells were used (10)
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Membranes prepared from LLC/cMOAT-1 cells catalyzed the MgATP-dependent uptake of 100 µM [3H]MTX at an initial rate of 61.6 pmol/mg/min at the 5-min time point of the assay, whereas control LLC/CMV membranes did not mediate appreciable transport under energized conditions (Fig. 5A)
. However, in strict correspondence with the results obtained with MRP1, MRP3, and MRP4, glutamylation dramatically reduced the capacity of MRP2 to transport MTX, in that 100 µM [3H]MTX-Glu2 was not transported to any extent by either LLC/cMOAT-1 or LLC/CMV membrane vesicles (Fig. 5B)
. Similarly, [3H]MTX-Glu3 transport was not mediated by MRP2 (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As expected, based upon the structural resemblance of MTX to physiological folates and from our previous determination that MRP1 and MRP3 are able to transport folates, it is shown that MRP4 also has the facility for the low affinity, high capacity transport of folic acid and N5-formyl-THF. Although we did not examine this feature of MRP2, its involvement in folate transport has been inferred previously from transport studies using hepatocyte canalicular membrane vesicles prepared from a rat strain that is genetically deficient in this pump (47)
. In combination, these observations concerning folates, which are also susceptible to polyglutamylation by FPGS, suggest the possibility that their intracellular levels may be influenced by expression of MRPs 1–4.
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In addition to revealing differences between MRP4 and MRP5, analysis of MRP4 inhibitors also demonstrated an interesting similarity between the two pumps with regard to phosphodiesterase inhibitors. The activity of this class of agents, which resemble cyclic nucleotides, as inhibitors of cyclic nucleotide efflux pumps was initially indicated by their ability to attenuate cellular efflux of cAMP (48) . More recently, phosphodiesterase inhibitors have been reported to be extremely potent inhibitors of MRP5-mediated cGMP transport (37) . In the present study, it is shown that phosphodiesterase inhibitors are also good inhibitors of MRP4-mediated transport, although the degree of inhibition was not comparable with that described for MRP5. The weaker potency of these agents as inhibitors of MRP4 is commensurate with the lower affinity of the pump for cGMP (Km, 9.7 µM), by comparison with MRP5 (Km, 2.1 µM; Ref. 35 , 37 ). However, our measurements of inhibition were made in the context of MTX transport, and it is possible that phosphodiesterase inhibitors may be considerably more potent as inhibitors of MRP4-mediated cGMP transport. The susceptibility of MRP4 to inhibition by phosphodiesterase inhibitors may be of substantial pharmaceutical interest in that it suggests the possibility that these agents, which are currently used for the treatment of erectile dysfunction but which also have important potential applications in cardiovascular diseases, may enhance intracellular levels of cGMP not only by inhibiting phosphodiesterases and MRP5-mediated efflux, as suggested previously (37) , but by simultaneously inhibiting both of the currently known cGMP efflux pumps (MRP4 and MRP5). This knowledge may be of value in the development of agents that are specifically designed to be potent inhibitors of specific phosphodiesterases, MRP4 and MRP5.
The capability of MRP4 for conferring resistance to MTX in combination with the results of a previous study in which we found that MRP4-transfected NIH3T3 cells are resistant to 6-MP may have special significance for the treatment of childhood acute lymphoblastic leukemia, for which prolonged antimetabolite therapy with these two agents is an important component of remission maintenance therapy. Although our previous observation concerning MRP4 and 6-MP was based upon the analysis of a single transfected cell line and requires confirmation in other MRP4-transfected cell lines, it suggests that induction of MRP4 expression could simultaneously impair both arms of this treatment. Another potential consideration with regard to these two agents pertains to their synergistic cytotoxicity, which has been attributed in part to enhanced incorporation of thiopurine nucleotides into DNA consequent upon MTX-induced reductions in purine pools. Our results suggest that another potential source of synergistic activity might be the inhibition by MTX of MRP4-mediated thiopurine nucleotide efflux (Fig. 6)
.
In considering the potential for MRP4 to confer clinical resistance to MTX, as well as its capacity to influence folate homeostasis, certain considerations that we discussed in connection with MRP1 and MRP3 should be kept in mind (39)
. Although the affinities of MRP4 for MTX and folates (Table 1)
are higher than those we reported for the same compounds in the case of MRP3 (Km, 0.62, 1.96, and 1.74 mM, respectively, for MTX, folic acid, and N5-formyl-THF, respectively) and for MTX in the case of MRP1 (Km, 2.15 mM; Ref. 39
), the affinities of all of these pumps are quite high by comparison with serum MTX and folate levels (low micromolar concentrations). In addition, in transfected cell lines MTX resistance conferred by MRPs is time dependent in that high levels of resistance are observed only in growth assays in which MTX exposure is limited to the first 1–4 h of the assay (13
, 14
, 34)
, and the extent to which this feature conforms to the clinical pharmacokinetics of this agent in patients is unclear. For these reasons, the in vivo pharmacological significance of these pumps requires further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grant CA73728 and by an appropriation from the Commonwealth of Pennsylvania. Z-S. C. is the recipient of a W. J. Avery Fellowship from Fox Chase Cancer Center and a Japan Research Foundation Award for Clinical Pharmacology. K. L. is the recipient of National Institutes of Health Fellowship CA74518. 
2 Present address: Department of Pharmacology, Yale University, New Haven, CT 06520.
3 To whom requests for reprints should be addressed, at Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-5317; Fax: (215) 728-3603; E-mail: GD_Kruh{at}fccc.edu
4 The abbreviations used are: MRP, multidrug resistance protein; MOAT, multispecific organic anion transporter; 6-MP, 6-mercaptopurine; E217ßG, estradiol 17ß-D-glucuronide; N5-formyl-THF, N5-formyltetrahydrofolic acid; FPGS, folylpoly--glutamate synthetase; MTX, methotrexate.
Received 11/20/01. Accepted 4/ 1/02.
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S. H. Wright and W. H. Dantzler Molecular and Cellular Physiology of Renal Organic Cation and Anion Transport Physiol Rev, July 1, 2004; 84(3): 987 - 1049. [Abstract] [Full Text] [PDF]
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I. Ifergan, A. Shafran, G. Jansen, J. H. Hooijberg, G. L. Scheffer, and Y. G. Assaraf Folate Deprivation Results in the Loss of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression: A ROLE FOR BCRP IN CELLULAR FOLATE HOMEOSTASIS J. Biol. Chem., June 11, 2004; 279(24): 25527 - 25534. [Abstract] [Full Text] [PDF]
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 C. Kneuer, K. U. Honscha, and W. Honscha Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization Am J Physiol Renal Physiol, March 1, 2004; 286(3): F564 - F571. [Abstract] [Full Text]
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E. L. Volk and E. Schneider Wild-Type Breast Cancer Resistance Protein (BCRP/ABCG2) is a Methotrexate Polyglutamate Transporter Cancer Res., September 1, 2003; 63(17): 5538 - 5543. [Abstract] [Full Text] [PDF]
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Y. Guo, E. Kotova, Z.-S. Chen, K. Lee, E. Hopper-Borge, M. G. Belinsky, and G. D. Kruh MRP8, ATP-binding Cassette C11 (ABCC11), Is a Cyclic Nucleotide Efflux Pump and a Resistance Factor for Fluoropyrimidines 2',3'-Dideoxycytidine and 9'-(2'-Phosphonylmethoxyethyl)adenine J. Biol. Chem., August 8, 2003; 278(32): 29509 - 29514. [Abstract] [Full Text] [PDF]
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 G. Reid, P. Wielinga, N. Zelcer, I. van der Heijden, A. Kuil, M. de Haas, J. Wijnholds, and P. Borst The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs PNAS, August 5, 2003; 100(16): 9244 - 9249. [Abstract] [Full Text] [PDF]
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M. Stark, L. Rothem, G. Jansen, G. L. Scheffer, I. D. Goldman, and Y. G. Assaraf Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate Mol. Pharmacol., August 1, 2003; 64(2): 220 - 227. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, R. W. Robey, M. G. Belinsky, I. Shchaveleva, X.-Q. Ren, Y. Sugimoto, D. D. Ross, S. E. Bates, and G. D. Kruh Transport of Methotrexate, Methotrexate Polyglutamates, and 17{beta}-Estradiol 17-({beta}-D-glucuronide) by ABCG2: Effects of Acquired Mutations at R482 on Methotrexate Transport Cancer Res., July 15, 2003; 63(14): 4048 - 4054. [Abstract] [Full Text] [PDF]
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N. Zelcer, M. T. Huisman, G. Reid, P. Wielinga, P. Breedveld, A. Kuil, P. Knipscheer, J. H. M. Schellens, A. H. Schinkel, and P. Borst Evidence for Two Interacting Ligand Binding Sites in Human Multidrug Resistance Protein 2 (ATP Binding Cassette C2) J. Biol. Chem., June 20, 2003; 278(26): 23538 - 23544. [Abstract] [Full Text] [PDF]
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 S L Hider, C Morgan, E Bell, I N Bruce, P Ranganathan, and H L McLeod Will pharmacogenetics allow better prediction of methotrexate toxicity and efficacy in patients with RA? Ann Rheum Dis, June 1, 2003; 62(6): 591 - 591. [Full Text] [PDF]
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P. R. Wielinga, I. van der Heijden, G. Reid, J. H. Beijnen, J. Wijnholds, and P. Borst Characterization of the MRP4- and MRP5-mediated Transport of Cyclic Nucleotides from Intact Cells J. Biol. Chem., May 9, 2003; 278(20): 17664 - 17671. [Abstract] [Full Text] [PDF]
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G. Reid, P. Wielinga, N. Zelcer, M. de Haas, L. van Deemter, J. Wijnholds, J. Balzarini, and P. Borst Characterization of the Transport of Nucleoside Analog Drugs by the Human Multidrug Resistance Proteins MRP4 and MRP5 Mol. Pharmacol., May 1, 2003; 63(5): 1094 - 1103. [Abstract] [Full Text] [PDF]
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Y. G. Assaraf, L. Rothem, J. H. Hooijberg, M. Stark, I. Ifergan, I. Kathmann, B. A. C. Dijkmans, G. J. Peters, and G. Jansen Loss of Multidrug Resistance Protein 1 Expression and Folate Efflux Activity Results in a Highly Concentrative Folate Transport in Human Leukemia Cells J. Biol. Chem., February 21, 2003; 278(9): 6680 - 6686. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, E. Hopper-Borge, M. G. Belinsky, I. Shchaveleva, E. Kotova, and G. D. Kruh Characterization of the Transport Properties of Human Multidrug Resistance Protein 7 (MRP7, ABCC10) Mol. Pharmacol., February 1, 2003; 63(2): 351 - 358. [Abstract] [Full Text] [PDF]
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M. G. Belinsky, Z.-S. Chen, I. Shchaveleva, H. Zeng, and G. D. Kruh Characterization of the Drug Resistance and Transport Properties of Multidrug Resistance Protein 6 (MRP6, ABCC6) Cancer Res., November 1, 2002; 62(21): 6172 - 6177. [Abstract] [Full Text] [PDF]
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) or 4 mM MgAMP (
). Uptake was measured at 5 min. Values shown are means; bars, SE.
