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Experimental Therapeutics |
Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [H. Z., Z-S. C., M. G. B., G. D. K.], and Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 [P. A. R.]
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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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A crucial property of MTX is its susceptibility to polyglutamylation by the same enzymes that polyglutamylate physiological folates (5, 6, 7, 8, 9, 10)
. FPGS catalyzes the condensation of successive glutamate residues to the 
-carboxyl group of MTX, a monoglutamate, to yield MTX-Glu2–7 derivatives. By comparison with the parent molecule, which is subject to efflux via an energy-dependent process, polyglutamylated MTX is effluxed poorly and exhibits prolonged intracellular retention (11, 12, 13, 14, 15, 16, 17)
. As a consequence, polyglutamylation results in a massive enhancement of cytotoxicity. The precise mechanism of efflux of this agent is therefore fundamental to its potency as a chemotherapeutic and also has a bearing on the physiology of endogenous folates because their retention is similarly dependent on polyglutamylation (18)
. However, the long-standing issue of the identity of the cellular component(s) responsible for efflux of this agent remains unresolved (1)
.
Clues regarding the molecular identity of one or more components of the cellular efflux system have come recently from investigations of the transport properties of members of the MRP family of ATP-binding cassette transporters. Rat strains deficient in MRP2 are impaired in hepatobiliary excretion of MTX (19) , and canalicular membrane vesicles prepared from MRP2-deficient rat strains have a diminished capacity for the MgATP-dependent transport of physiological folates (20) . Cultured cells transfected with MRP1, MRP2, MRP3, or MRP4 are resistant to and accumulate lower levels of MTX, particularly in short-term drug exposure assays (21, 22, 23, 24) . Cloned human MRP3 is not only able to transport glutathione and glucuronate conjugates and monoanionic bile acids but is also able to transport MTX in the low micromolar range (25) . A similar substrate selectivity has been reported for rat MRP3 (26 , 27) . The kinetics have not been defined, but MRP1 and MRP2 have also been found to transport MTX into membrane vesicles (23) .
If these findings are indeed to be consistent with a role for at least some MRP family members in the energy-dependent cellular efflux system for MTX, then they must be able to accommodate a critical feature of the system: the capacity of the system to mediate the efflux of MTX but not that of its polyglutamylated metabolites. Here we examine the potential of MRP3 participation in these and related processes by determining the selectivity of MRP3 for MTX versus its metabolites and physiological folates. The results of these analyses show that the addition of only a single glutamate residue to MTX is sufficient to severely attenuate MRP3-mediated transport and that MRP3 is competent in the transport of FA and N5-formyltetrahydrofolic acid (leucovorin) as well as MTX. Parallel experiments on another MRP, MRP1, demonstrate similar properties.
Two conclusions derive from these findings: (a) MRP3 and MRP1 satisfy the requirements of components of the MTX efflux system; and (b) given that MRP3 and MRP1 are able to transport physiological folates at high capacity, they may not only indirectly influence antifolate cytotoxicity but may also contribute to cellular resistance in chemotherapeutic regimens in which leucovorin is a component.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MRP1-32), and parental vector-transfected NIH3T3 cell line (pSR) were as described previously (21
, 28)
.
Preparation of Membrane Vesicles and Transport Experiments.
Membrane vesicles were prepared as described previously (29)
, except that cell disruption was accomplished using a motor-driven Dounce homogenizer. The rates of MTX uptake were increased by approximately 25-fold over the values reported previously for this material (25)
as a result of this modification of the preparation protocol. Vesicular uptake of [3H]MTX and its derivatives into inside-out membrane vesicles was measured by the rapid filtration technique as described previously (25
, 29)
. For concentration dependence determinations, initial rates were measured at 5 min.
Data Analysis.
Kinetic parameters were computed by nonlinear least squares analysis (30)
using the Ultrafit computer software (BioSoft, Ferguson, MO).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies on membrane vesicles purified from HEK/MRP3-5 cells have established that MRP3 is competent in the high-capacity MgATP-energized transport of MTX (25)
. However, whereas these studies demonstrated transport of MTX, they did not assess transport of MTX polyglutamates (structures are shown in Fig. 1
). Thus, to explore the implications of polyglutamylation, the kinetics of uptake of MTX, MTX-Glu2, and MTX-Glu3 into MRP3-enriched membrane vesicles were compared. To assess the relative contribution of MRP3 to overall uptake, parallel experiments were also performed on membrane vesicles purified from HEK293 cells transfected with parental plasmid (HEK/pcDNA3 cells).
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. By comparison with the negligible rates of uptake of 1.0 µM [3H]MTX by HEK/MRP3-5 membrane vesicles in media containing MgAMP or HEK/pcDNA3 vesicles in media containing either MgATP or MgAMP, membrane vesicles purified from HEK/MRP3-5 cells mediated MgATP-dependent uptake at an initial rate of 5.7 pmol/mg/min (Fig. 2A)
. By contrast, uptake of MTX-Glu2, when measured by the same procedure on the same membrane preparations, was slow or undetectable. Whereas an increase in MgATP-dependent [3H]MTX-Glu2 uptake was evident in HEK/MRP3-5 vesicles versus the equivalent membrane fraction from HEK/pcDNA3 cells, the rate (0.2 pmol/mg/min) was <5% of that measured with [3H]MTX (Fig. 2B
; note that in Fig. 2
, the ordinates of B and C are 
10 times more sensitive than those of A). The degree to which the transport of MTX was attenuated by glutamylation increased with increase in the number of glutamyl repeats because MgATP-dependent uptake of 1.0 µM [3H]MTX-Glu3 was not detectable in any of the membrane vesicle preparations assayed (Fig. 2C)
.
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. By contrast, osmoticum exerted only a moderate effect on substrate retention measured in media containing MgAMP, suggesting that an appreciable fraction of the apparent uptake measured under nonenergized conditions represented binding to the membrane vesicles and/or membrane filters (Fig. 3)
.
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.
As shown in Fig. 4
, MRP3 is indeed able to transport FA and leucovorin. When measured at an initial concentration of 1.0 µM, [3H]FA and [3H]leucovorin were taken up by HEK/MRP3-5 vesicles at rates of 2.1 and 2.7 pmol/mg/min, respectively, from media containing MgATP and at rates of <0.6 pmol/mg/min from media containing MgAMP (Fig. 4)
. The corresponding values for HEK/pcDNA3 vesicles were consistently <0.4 pmol/mg/min.
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. Nonlinear least squares fitting of the data to the Michaelis-Menten equation (30)
yielded Km and Vmax values for the transport of MTX, FA, and leucovorin of 0.62 ± 0.23 mM and 2.93 ± 0.32 nmol/mg/min, 1.96 ± 0.13 mM and 1.71 ± 0.05 nmol/mg/min, and 1.74 ± 0.65 mM and 3.63 ± 1.20 nmol/mg/min, respectively (Table 1)
. The efficiencies of transport fell in the rank order MTX (Vmax/Km = 4.8) > leucovorin (Vmax/Km = 2.1) >> FA (Vmax/Km = 0.9; Table 1
). Because membrane vesicles purified from HEK/pcDNA3 cells catalyzed negligible MgATP-dependent uptake of all three folates at all of the concentrations examined (Fig. 5)
, the transport measured in the membranes from HEK/MRP3-5 cells was solely attributable to MRP3.
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With the exception of taurocholate, all of the compounds tested in this category inhibited MRP3-mediated MTX transport in a manner consistent with the results from previous resistance or transport studies. One hundred µM concentrations of the natural product anticancer agents inhibited MTX transport in the rank order etoposide > vincristine >> doxorubicin (89.4%, 28.3%, and 8.5% inhibition, respectively; Table 2
). One hundred µM concentrations of E217ßG and glycocholate inhibited MTX transport to degrees (91.7% and 73.2%; Table 2
) commensurate with their relative affinities for the transporter (Km(E217ßG) = 25.6 µM, Km(glycocholate) = 248 µM; Ref. 25
). The exception, taurocholate, inhibited MTX transport by 56.1% (Table 2)
, although this bile acid, unlike glycocholate, is not transported by human MRP3. In addition, although human MRP3 is able to transport glutathione conjugates, glutathione itself was a poor inhibitor (13% inhibition at 1 mM).
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. The inhibitions exerted by 111 µM concentrations of probenecid and AG-A (36.5% and 15.5%, respectively) were moderate by comparison (Table 2)
.
Neither MTX-Glu2 nor the quinazoline-based antifolate ZD1694, a monoglutamate that inhibits thymidylate synthase, was a potent inhibitor of MRP3-mediated MTX transport. Concentrations of 100 µM or more inhibited MTX uptake by only 12.7% and 26.6%, respectively (Table 2)
.
The inhibitory activities of three agents capable of modulating the drug resistance activity of Pgp, an ATP-binding cassette transporter that shares with MRP3 the facility for conferring resistance to natural product agents but has only a weak structural resemblance to MRP3, were examined to test for the effects of Pgp antagonists on MRP3-mediated MTX transport. Of these agents, CsA was a surprisingly potent inhibitor, whereas PSC833 exhibited moderate activity (73.7% and 38.6% inhibition at 10 µM, respectively). By comparison with the latter bulky lipophilic molecules, the calcium channel blocker verapamil was a weak inhibitor (13.1% inhibition at 111 µM). The effects of Pgp inhibitors on MRP3-mediated transport were therefore similar to their effects on MRP2, for which PSC833 is a more potent inhibitor than CsA (42) , and distinct from their effects on MRP1, for which these agents are weak inhibitors (43) .
In combination, these results showing that either amphipathic (glycocholate, E217ßG, PAK-104P, and MK571) or lipophilic (etoposide and CsA) substrates and antagonists are potent inhibitors of MRP3-mediated MTX transport add support to the possibility inferred previously from the finding that MRP3 transports glutathione and glucuronate conjugates and glycocholate (25) that the binding pocket of MRP3 has specific sites for both lipophilic and negatively charged ligands.
Transport of MTX and MTX Polyglutamates by MRP1.
Having established that the transport characteristics of MRP3 are consistent with those of the MTX efflux system, the potential participation of other MRPs was explored. Knowing that MTX efflux systems are widely distributed in tissues, the involvement of MRP1, the most ubiquitously expressed of the MRPs (44
, 45)
, was examined. For this purpose, uptake measurements were performed on membrane vesicles prepared from MRP1-transfected NIH3T3 cells (pSR-MRP1-32) and NIH3T3 cells transfected with parental vector (pSR; Ref. 28
). As judged by immunoblot analyses, membrane vesicles purified from pSR-MRP1-32 cells are a rich source of the Mr 190,000 MRP1 protein (data not shown).
Regardless of the property examined, MRP1-dependent MTX transport closely resembled that mediated by MRP3. Whereas pSR-MRP1-32 membrane vesicles catalyzed the MgATP-dependent uptake of 1.0 µM [3H]MTX at an initial rate of 1.4 pmol/mg/min, pSR membranes mediated little or no uptake (Fig. 6A)
. In strict correspondence with the results obtained with MRP3, glutamylation dramatically attenuated the amenability of MTX to transport because neither 1.0 µM [3H]MTX-Glu2 nor 1 µM [3H]MTX-Glu3 was transported by either pSR-MRP1-32 or pSR membranes (Fig. 6, B and C
; note that the ordinates in Fig. 6, B and C
, are >4-fold more sensitive than those in Fig. 6A
).
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-MRP1-32 cells approximated Michaelis-Menten kinetics (Fig. 7
and Table 1
) to yield Km and Vmax values of 2.15 ± 0.79 mM and 2.05 ± 0.91 nmol/mg/min, respectively. Membrane vesicles prepared from pSR cells catalyzed negligible MgATP-dependent uptake at all of the concentrations examined (Fig. 7)
, indicating that the transport measured was largely attributable to MRP1.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl hydrolase serves to cleave the poly--glutamate chain to regenerate the parental monoglutamate (46
, 47)
. Of these opposing reactions, the reaction catalyzed by -glutamyl hydrolase is considered to contribute least to intracellular MTX pool sizes. As a result, and by virtue of the inability of the energy-dependent efflux system to export polyglutamates, polyglutamylation likely plays a determinative role in modulating overall intracellular MTX levels.
In the experiments described here, we explored the possibility that certain members of the MRP family, which had previously been implicated in MTX transport, might satisfy the requirements predicted for components of the cellular MTX efflux system. In so doing, it has been determined that the two MRPs (MRP1 and MRP3) whose wide distributions (31
, 44
, 48, 49, 50)
coincide with that of the MTX efflux system do indeed satisfy the predicted requirements (Fig. 9)
. Both are high capacity, low affinity MTX transporters exhibiting little or no activity toward polyglutamates. We demonstrate that the addition of a single glutamyl residue is sufficient to abrogate transport by >95% in the case of MRP3 and to totally abrogate transport in the case of MRP1 and that the addition of further glutamyl residues augments this effect.
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-glutamyl hydrolase (2
, 51, 52, 53, 54, 55, 56, 57)
. Although enhanced efflux mediated by MRP1–MRP4 represents an additional MTX resistance mechanism, it remains to be determined whether this activity contributes to clinical resistance. The basic problem in understanding the relative contribution of cellular efflux to MTX resistance is the seeming strict time dependence of the phenomenon. The degree of resistance associated with the overexpression of MRPs depends on how the cells are exposed to MTX (21, 22, 23, 24)
. MRP1-MRP4 are highly efficacious resistance factors when drug exposure is limited to only 1–4 h of a 3-day growth assay, but they are poor resistance factors when a standard continuous exposure assay is used. The properties of MRP3 and MRP1 reported here are capable of explaining this unusual time dependence. If, as indicated from the results of measurements of cellular free and polyglutamylated MTX levels, this resistance phenotype is attributable to enhanced polyglutamylation consequent to continuous drug exposure (22 , 23) , then it might in fact be expected, given that MRP3 and MRP1 are severely limited in their capacity to transport MTX polyglutamates by comparison with the parent molecule, that the resistance conferred by these transporters will decrease with increase in time of exposure. If correct, a potential clinical implication follows from this interpretation; namely, that the capacity of MRP3 and MRP1 to confer resistance to MTX may be critically dependent on the schedule of drug administration in a way distinct from the resistance conferred by these pumps to natural product agents. Specifically, MRP-mediated MTX resistance may be less likely to be a factor when this agent is administered by prolonged continuous infusion rather than by short duration i.v. infusions. We speculate that these considerations may also apply to two other MRPs, MRP2 and MRP4, which have also been demonstrated to confer a time-dependent MTX resistance phenotype (23 , 24) .
A second issue pertaining to whether MRP3 and MRP1 are capable of influencing clinical resistance to MTX concerns their low affinity for this antimetabolite. The Km values we determined for MRP3 and MRP1 (0.62 and 2.15 mM, respectively) are in the range of plasma levels (0.1–1 mM) achieved after high-dose MTX infusions (e.g., 1.5 g/m2) but are significantly higher than the plasma levels (1–10 µM) associated with routinely used dosages of this agent (25–100 mg/m2; Ref. 58 ). Nevertheless, the possibility that the activities of MRP3 and MRP1 may be pharmacologically relevant is supported by the observation that cell lines transfected with these two transporters (as well as those transfected with MRP2 and MRP4) exhibit enhanced resistance at low micromolar MTX concentrations (22, 23, 24) .
Another property of MRP3 and MRP1, their facility for the transport of physiological folates, may also have clinical implications. Because sensitivity to antifolates is not determined solely by the intracellular level of drug but also by the size of the intracellular folate pool (physiological folates compete with antifolates for binding to DHFR as well as FPGS), any factor that influences physiological folate pool size could alter drug efficacy. This being the case, enhanced expression levels of MRPs could paradoxically diminish antifolate resistance by decreasing the size of the intracellular folate pool. Reciprocally, reduced expression levels of MRPs could enhance antifolate resistance. The observation that cell lines resistant to lipid-soluble antifolates have increased folate levels (59)
is at least consistent with such a scheme. However, similar to the case with MTX, the high Km value of MRP3 for FA (1.7 mM), in contrast with the low folate concentrations in plasma (
10–20 nM), is a consideration in understanding whether expression of the pump can influence intracellular folate homeostasis in vivo.
Analogous reasoning and kinetic considerations may apply to another MRP3 and MRP1 transport substrate, the reduced folate leucovorin (Km = 1.74 mM for MRP3). Leucovorin is used in cancer chemotherapy in two clinical situations. It is administered as a source of reduced folates after high-dose MTX treatment to reverse the MTX-induced metabolic block in normal tissues and thereby rescue bone marrow and mucosal surfaces from the massive cytotoxicity that would otherwise occur, and it is coadministered with 5-fluorouracil to increase binding of 5-fluoro-dUMP to its target, thymidylate synthetase, and thus increase its cytotoxicity (leucovorin serum concentrations are 5–100 µM as a rescue agent and 5–10 µM as an adjuvant). In the former case, increased MRP3 and MRP1 expression might diminish the rescue effect by decreasing the net uptake of reduced folate. In the latter case, increased expression of MRPs might diminish the cytotoxicity of the chemotherapeutic regimen by decreasing the intracellular concentration of adjuvant.
The susceptibility of MRP3-mediated MTX transport to inhibition by a number of natural product agents provides an explanation for the interesting observation made nearly three decades ago that vincristine and etoposide enhance intracellular MTX levels when these drugs are administered simultaneously (60
, 61)
. The demonstration that etoposide, which is presumed to be a substrate of MRP3 because resistance to this agent is conferred by the pump (21
, 22)
, is a potent inhibitor of MRP3-mediated MTX transport suggests that natural product agents, which are also known substrates for MRP1 and MRP2, may impair cellular efflux of MTX by acting as competitive inhibitors (Fig. 9)
. This finding is of potential clinical relevance in that MTX is sometimes combined with natural product agents in chemotherapy regimens. By the same token, the determination that several MRP1 and Pgp inhibitors are potent in vitro inhibitors of MRP3-mediated transport raises the possibility that these agents, when used in clinical trials with the intention of augmenting cellular levels of natural product agents, might concomitantly augment intracellular MTX levels. Additional studies should help to determine whether these and other speculative considerations concerning the potential involvement of MRPs in clinical MTX resistance are valid.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA73728 (to G. D. K.) and United States Department of Agriculture NRICGP Grant 99-35304-8094 (to P. A. R.). 
2 Present address: Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520.
3 Z-S. C. is the recipient of a Japan Research Foundation for Clinical Pharmacology Award.
4 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
5 The abbreviations used are: FA, folic acid; MRP, multidrug resistance protein; RFC1; reduced folate carrier 1; DHFR, dihydrofolate reductase; FPGS, folylpoly--glutamate synthetase; Pgp, P-glycoprotein; AG-A, agosterol A; E217ßG, 17ß-estradiol 17-(ß-D-glucuronide); CsA, cyclosporin A; MTX, methotrexate.
Received 4/ 6/01. Accepted 7/23/01.
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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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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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C. J. Oleschuk, R. G. Deeley, and S. P. C. Cole Substitution of Trp1242 of TM17 alters substrate specificity of human multidrug resistance protein 3 Am J Physiol Gastrointest Liver Physiol, February 1, 2003; 284(2): G280 - G289. [Abstract] [Full Text] [PDF]
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T.-F. Fu, J. N. Scarsdale, G. Kazanina, V. Schirch, and H. T. Wright Location of the Pteroylpolyglutamate-binding Site on Rabbit Cytosolic Serine Hydroxymethyltransferase J. Biol. Chem., January 17, 2003; 278(4): 2645 - 2653. [Abstract] [Full Text] [PDF]
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K. Koike, C. J. Oleschuk, A. Haimeur, S. L. Olsen, R. G. Deeley, and S. P. C. Cole Multiple Membrane-associated Tryptophan Residues Contribute to the Transport Activity and Substrate Specificity of the Human Multidrug Resistance Protein, MRP1 J. Biol. Chem., December 13, 2002; 277(51): 49495 - 49503. [Abstract] [Full Text] [PDF]
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A. Haimeur, R. G. Deeley, and S. P. C. Cole Charged Amino Acids in the Sixth Transmembrane Helix of Multidrug Resistance Protein 1 (MRP1/ABCC1) Are Critical Determinants of Transport Activity J. Biol. Chem., October 25, 2002; 277(44): 41326 - 41333. [Abstract] [Full Text] [PDF]
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E. L. Volk, K. M. Farley, Y. Wu, F. Li, R. W. Robey, and E. Schneider Overexpression of Wild-Type Breast Cancer Resistance Protein Mediates Methotrexate Resistance Cancer Res., September 1, 2002; 62(17): 5035 - 5040. [Abstract] [Full Text] [PDF]
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Z.-S. Chen, K. Lee, S. Walther, R. B. Raftogianis, M. Kuwano, H. Zeng, and G. D. Kruh Analysis of Methotrexate and Folate Transport by Multidrug Resistance Protein 4 (ABCC4): MRP4 Is a Component of the Methotrexate Efflux System Cancer Res., June 1, 2002; 62(11): 3144 - 3150. [Abstract] [Full Text] [PDF]
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). Values shown are the means ± SE (n = 3).
