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Vol. 2, 307-316, March 2003     Molecular Cancer Therapeutics
© 2003 American Association for Cancer Research

Cloning and Functional Characterization of the Multidrug Resistance-associated Protein (MRP1/ABCC1) from the Cynomolgus Monkey

Nathalie Godinot, Philip W. Iversen, Linda Tabas, Xiaoling Xia, Daniel C. Williams, Anne H. Dantzig and William L. Perry, III1

Division of Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285

1 To whom requests for reprints should be addressed, at Division of Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. Phone: (317) 276-1083; Fax: (317) 276-6510; E-mail: bperry{at}Lilly.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The multidrug resistance-associated protein 1 (ABCC1) gene from human (hMRP1), dog (canMRP1), and mouse (muMRP1) all encode proteins that efficiently transport the endogenous MRP1 substrate glutathione-S-leukotriene C4 and confer resistance to anticancer agents, including vincristine and etoposide. hMRP1 also confers resistance to anthracyclines, whereas this is not true of canMRP1 or muMRP1. To determine whether MRP1 from another animal species used in toxicological studies would be more functionally similar to hMRP1, we cloned and characterized two alleles of the MRP1 homologue from the cynomolgus monkey Macaca fascicularis (monMRP1). The monMRP1 cDNAs encode proteins of 1531 residues that are 98, 90, and 88% identical to hMRP1, canMRP1, and muMRP1, respectively. Stable overexpression of both monMRP1 alleles and hMRP1 in transformed human embryonic kidney cells was achieved using an episomal expression vector. Transporters encoded by both monMRP1 alleles were functionally very similar to hMRP1. monMRP1 conferred an increased resistance to vincristine and etoposide and transported glutathione-S-leukotriene C4 into membrane vesicles. In addition, MRP1-mediated drug resistance was effectively reversed in monMRP1 and hMRP1 transfectants by LY402913, a new MRP1-selective inhibitor in the class of tricyclic isoxazoles. However, monMRP1 transporters conferred a reduced level of resistance to the anthracyclines doxorubicin, daunorubicin, and epirubicin relative to hMRP1, although resistance levels were significant relative to vector control cells. These functional differences between human and monkey MRP1 transporters will need to be considered when designing pharmacokinetic and toxicological studies for the preclinical evaluation of MRP1 modulators.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A major obstacle to the successful treatment of cancer is the development of resistance to anticancer agents. When resistance develops, often the cancer cells are cross-resistant to other structurally and mechanistically unrelated drugs, and this is known as multidrug resistance. Several members of the ABC2 transporter superfamily have been implicated in the development of multidrug resistance by their ability to reduce intracellular drug concentrations of structurally diverse anticancer agents and by their increased expression in multidrug resistant tumor cells (1).

The MRP1 (MRP1/ABCC1) gene was originally cloned from a multidrug-resistant human small cell lung cancer cell line and encodes a Mr 190,000 protein of 1531 amino acids (2). hMRP1 has a wide tissue distribution, including the basolateral membrane of epithelial cells in most tissues, and hMRP1 is expressed at relatively high levels in lung, testes, and kidney (3). hMRP1 is a primary transporter of GSH, glutathione S-conjugates, and glucuronate- and sulfate-conjugated organic anions, including LTC4 (4). Overexpression of hMRP1 results in multidrug resistance through an ATP-dependent efflux of several important classes of anticancer agents, including anthracyclines, epipodophyllotoxins, and the Vinca alkaloids (5). Transport of some substrates is also GSH dependent. hMRP1 overexpression has been detected in drug-selected cell lines derived from lung, leukemia, breast, bladder, prostate, and cervical cancer (6), supporting a role for hMRP1 in multidrug resistance acquired by tumor cells during chemotherapy. hMRP1 may also play a role in the intrinsic resistance of certain tumor types to chemotherapy because substantial levels of hMRP1 mRNA has been detected in nonselected cell lines derived from thyroid, non-small cell lung carcinomas, gliomas, and neuroblastomas (6).

One approach to overcoming resistance to anticancer agents caused by overexpression of an ABC transporter in human tumors is to develop noncytotoxic inhibitors that can block its transport activity (7). Decreased efflux of an anticancer agent when coadministered with an inhibitor would make the cells more drug sensitive and responsive to therapy. However, blocking the activity of hMRP1 in normal cells could hypersensitize some tissues to the anticancer agent, resulting in dose-limiting toxicities. Such toxicities might be anticipated through the preclinical testing of modulators in an animal model if the substrate specificity of its MRP1 homologue is similar to that of hMRP1.

Despite the high level of sequence identity between human, bovine, canine, and murine MRP1 homologues, only hMRP1 confers resistance to a major class of anticancer agents, the anthracyclines (5, 8– 11). To determine whether MRP1 from another animal species used in toxicological studies would be more functionally similar to hMRP1, we cloned and functionally characterized two alleles of the MRP1 homologue from the cynomolgus monkey, Macaca fascicularis (monMRP1). Stable overexpression of monMRP1 cDNAs and hMRP1 in human embryonic kidney (PEAKSTABLE) cells was achieved using an episomal expression vector. These cell lines were characterized with respect to the subcellular localization of MRP1, resistance to anticancer drugs in cytotoxicity assays, and the effects of the hMRP1 inhibitor LY402913 on monMRP1 function. monMRP1 transporters were also functionally evaluated in LTC4 transport assays using membrane vesicles prepared from these cell lines. Our results indicate that monkey and hMRP1 transporters are functionally very similar. However, whereas monMRP1 is the first MRP1 homologue to confer significant levels of anthracycline resistance to transfected cells relative to vector control cells, anthracycline resistance levels were reduced relative to hMRP1 transfected cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of monMRP1.
Captive-bred cynomolgus monkeys (Macaca fascicularis) of Mauritius origin were obtained from Charles River-BRF (Houston, TX). Animal care was conducted in accordance with the "Guide for the Care and Use of Laboratory Animals." Total RNA was purified from snap-frozen kidney tissue from a single individual using the Trizol method (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. One µg total RNA was reverse transcribed using a polyT primer and RNase H digested using reagents and protocols in the Superscript preamplification system (Invitrogen). To amplify a portion of the monkey MRP1 cDNA, PCR primers F10: 5'-CACCGGCATGGCGCTCCG-3' and R2: 5'-AAAAGACCTCTCTGCTGC-3' were designed based on the hMRP1 mRNA sequence (GenBank accession no. L05628, positions 190–207 and complementary bases 4741–4758, respectively). RT-PCR was performed using TaqPlus Long DNA Polymerase (Stratagene, La Jolla, CA). A specific 4.5-kb band was purified and sequenced directly using the ABI Prism Big Dye Terminator version 1 cycle sequencing kit (Perkin-Elmer Applied Biosystems Incorporated, Foster City, CA) on an ABI 377XL. The 5' end of the message was obtained using the 5' random amplification of cDNA ends system (Invitrogen) using the primer GSP1: 5'-GCAACTTTAAGATCTCCG-3' for reverse transcription of monkey kidney RNA, GSP2: 5'-GGAAGTAGGGCCCAAAGGTC-3' for the first-round amplification, and RR2: 5'-GGTGAAGTCAGGGTTGCTGG-3' for reamplification. PCR products were sequenced directly as above. The 3' end of the message was amplified using the 3' random amplification of cDNA end system (Invitrogen) using primer F28: 5'-GCTTGGTGGGCCTCTCAGTGTCTTACTCGT-3' for the first round PCR and the nested primer F29:5'-CTCAAGGAGTATTCAGAGACCGAGAAGGAG-3' for reamplification. PCR products were sequenced directly as described above. The complete open reading frame was amplified by RT-PCR using primers FLF1: 5'-GAGAGTCGACGATATCTGCCCGCCGCCGCCCTC-3' and FLR1: 5'-GAGAGCGGCCGCCCTGCCTGGGCGCGGGCA-3' using the Expand High Fidelity Kit (Roche Molecular Biochemicals, Indianapolis, IN). The PCR product was digested with NotI and SalI and cloned into the same sites of pBC (Stratagene). Sequencing of several clones revealed the presence of two alleles in the RNA isolated from this individual monkey that we refer to as alleles A (monMRP1A) and B (monMRP1B). Mutations introduced by PCR (changes that did not match the sequence obtained by direct sequencing and only present in single clones) were corrected using the Quickchange site-directed mutagenesis kit (Stratagene) and conventional cloning techniques. monMRP1A and monMRP1B have been deposited in GenBank (accession nos. AY146672 and AY146673, respectively).

Episomal Expression Vector.
The EBNA-1-based episomal expression vector pPEAK10 (Edge Biosystems, Gaithersburg, MD) was modified as follows to generate the vector EW1969 used in these studies. This vector encodes a shortened EBNA-1 gene that was modified by the supplier to reduce plasmid size. A 4.9-kb SpeI/NotI fragment of pPEAK10 was ligated to a 2.8-kb MluI/XbaI fragment of pCMV-SEAP (Tropix, Inc., Bedford, MA) after both fragments were blunted by treatment with T4 DNA polymerase (Roche Molecular Biochemicals). A 5.4-kb BamHI fragment of this plasmid was treated with Klenow fragment to create blunt ends and was subsequently digested with NcoI. A 4.9-kb fragment from these digests was ligated to a 0.6-kb NcoI/PvuII fragment of pcDNA3.1-Hygro(+) vector (Invitrogen) containing a portion of the CMV promoter, cloning sites, and polyadenylation signals. The cloning region between the AseI site at the T7 promoter, and the NotI site was replaced by ligating synthetic linkers between these restriction sites. The resulting vector (EW1969) features a shortened EBNA-1 gene/Ori P cassette for episomal expression in mammalian cells (12), an SV40 replication origin for replication in T-antigen-expressing cells, a puromycin resistance gene for selection of stable pools of transfected cells, and a CMV-promoter-polyA signal cassette for high-level expression of cloned genes in mammalian cells. The multicloning region also contains a short segment of the Xenopus laevis ß-globin gene 5' untranslated region that has been reported to increase the translation of some genes (13, 14) in addition to sequences for generation of FLAG and 6-His-tagged proteins (15).

MRP Expression in PEAKSTABLE Cells.
SalI (made blunt with treatment with Klenow fragment)/Not I fragments containing the complete open reading frames of monMRP1A, monMRP1B, and hMRP1 (obtained from Drs. Susan Cole and Roger Deeley, Queens University, Kingston, Canada) were cloned into the EcoRV and NotI sites of EW1969. PEAKSTABLE cells (transformed human embryonic kidney cells that stably express the EBNA-1 gene; Edge Biosystems) were grown in 5% CO2 at 37°C in a humidified atmosphere in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 50 µg/ml gentamicin (Invitrogen). The day before transfection, 9 x 105 cells were seeded into a 25-cm2 flask. Cells were transfected with MRP1 constructs in addition to the parental vector using Fugene 6 transfection reagent (Roche Molecular Biochemicals) according to the provided protocol. The following day, cells were trypsinized and expanded to a 150-cm2 flask, and puromycin (Edge Biosystems) was added to a final concentration of 0.5 µg/ml. Puromycin selection was considered complete when untransfected cells grown in parallel were completely killed (6–10 days).

Western Analysis.
Puromycin-selected PEAKSTABLE cells transfected with monMRP1A, monMRP1B, hMRP1, or the EW1969 parental vector were trypsinized, resuspended in growth medium, and centrifuged. The cell pellets were washed once in 5 ml of PBS containing protease inhibitors (Complete EDTA-free protease inhibitor mixture; Roche Molecular Biochemicals), centrifuged again, and placed on dry ice. Whole cell lysates were prepared by resuspending the cell pellets at 1 mg/ml in M-PER reagent (Pierce/Endogen, Rockford, IL), centrifuging at 20,000 x g for 15 min and collecting the supernatant. Protein concentrations were determined using the bicinchonic acid protein assay reagent (Pierce/Endogen) as directed by the manufacturer. Samples were loaded at 10 µg/lane onto 8% Novex Tris-Glycine SDS polyacrylamide gels (Invitrogen). After transfer onto Optitran supported nitrocellulose membranes (Schleicher & Schuell, Keene, NH), both monMRP and hMRP were detected using MRPr1, MRPm6, and QCRL-1 monoclonal antibodies (Alexis Biochemicals, San Diego, CA) as the primary and peroxidase-conjugated antirat IgG (Sigma-Aldrich, St. Louis, MO) as the secondary with the Renaissance Western Blot Chemiluminescence Reagent (Perkin-Elmer Life Sciences-NEN, Boston, MA).

Immunostaining.
Cells were grown on 4-well chambered coverglasses (Nalgene, Rochester, NY) as above. Cells were fixed for 10 min in 1% formaldehyde at room temperature, followed by permeabilization for 15 min in PBS containing 1% BSA and 0.025% NP40 (Sigma-Aldrich). Background staining was blocked with serum-free protein blocking reagent (Dako, Carpinteria, CA) for 30 min. The samples were incubated with anti-MRP1 antibody QCRL-3 (Signet, Dedham, MA) at a 1:10 dilution for 1 h at room temperature. Alexa 488-conjugated goat antimouse (Molecular Probes, Eugene, OR) diluted 1:500 was used as secondary reagent. The samples were counterstained using propidium iodide (Molecular Probes) at a 1:20 dilution in PBS for 1 min at room temperature. Cells were examined with a MRC-1024 confocal microscope (Bio-Rad, Hercules, CA).

Membrane Vesicle Preparation.
Puromycin-selected PEAKSTABLE cells transfected with hMRP1, monMRP1A, monMRP1B, or parental vector were grown in monolayers as described above. Membrane vesicles were prepared as previously described for HeLa-T5 cells (7, 16).

LTC4 Transport Assay.
ATP-dependent transport of LTC4 into inside-out membrane vesicles was measured for 45 s, which was within the linear range of uptake as described previously (7, 16). ATP-dependent LTC4 uptake was calculated by subtracting the uptake measured in the presence of adenylyl-(ß,{gamma}-methylene)-diphosphonate from the uptake measured in the presence of ATP. The uptake rate was calculated based on the protein content of the membrane vesicles. Protein was determined by the bicinchonic acid protein assay (Pierce/Endogen).

Chemosensitivity Testing.
The tetrazolium salt-based CellTiter 96 cell proliferation assay (Promega Corporation, Madison, WI) was used to determine resistance of the transfected cells to various chemotherapeutic agents in 96-well plates. The cells were plated at 7.5 x 103 cells/well in RPMI medium supplemented with 5% bovine calf serum (Invitrogen). They were exposed to drug 24 h after seeding and incubated for an additional 72 h before assaying for cell activity. Modulation of drug resistance was measured in assays containing 5 µM of the MRP1-selective inhibitor LY402913 (Eli Lilly and Company, Indianapolis, IN; Ref. 17). Resistance was determined in three to five independent experiments, and within each experiment, assays were carried out in quadruplicate.

Statistical Analysis.
All calculations for analysis of drug resistance data were performed on a log scale (18) and then converted back to the original scale (µg/ml). IC50s were calculated using a 4-parameter logistic fit, and data from multiple experiments were used to calculate mean IC50s ± SE. The fold resistance data in the presence or absence of the MRP1 inhibitor LY402913 were analyzed separately using a two-way ANOVA model with effects for cell line, drug, and their interaction. An experimental effect was included in the model to adjust for between-experiment differences. The final SEs were obtained using the delta method:

where SE(x) is the SE of the log IC50s, and is the average of the log (IC50) values. LTC4 uptake rates were analyzed by one-way ANOVA on the original scale. The Tukey-Kramer Honestly Significant Difference test was used to adjust for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of Monkey MRP1.
Using primers based on the hMRP1 sequence, we amplified a portion of the MRP1 gene from the cynomolgus monkey (monMRP1) by RT-PCR from RNA isolated from the kidney of a single cynomolgus monkey. Direct sequencing of PCR products and sequencing of cloned products identified two alleles of this gene (monMRP1A and monMRP1B) that encode 1531 amino acid proteins. monMRP1A and monMRP1B contain 18 single nucleotide polymorphisms within the coding region, three of which result in amino acid differences (residues 173, 704, and 1047; Fig. 1).



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  		Fig. 1. Alignment of the predicted amino acid sequences of human and monkey MRP1. hMRP1 (GenBank accession no. 2828206) differs from monkey MRP1 alleles A and B by 37 and 38 residues, respectively. monMRP1A and monMRP1B differ from each other at three positions (residues 173, 704, and 1047). The Walker A and B motifs and the active transport family signature sequences (C) are conserved elements characteristic of the NBDs of ABC transporters and are indicated with single lines. monMRP1A and monMRP1B have been deposited in GenBank (accession nos. AY146672 and AY146673, respectively).

 
Monkey MRP1 Shares High Sequence Identity to MRP1 Transporter from Human and Other Species.
Both alleles are 96.6% identical to hMRP1 at the nucleotide level. They also share a high percentage of identity with homologous proteins: 98% to hMPR1 (2); 92% to canMRP1 (11); 90% to bMRP1 (8); 88% to muMRP1 (19); and 87% to rMRP1 (20). monMRP1 alleles A and B differ from the hMRP1 protein by 37 and 38 residues, respectively, and these differences are fairly dispersed across the molecule (Fig. 1). The longest stretch of identity between human and monkey MRP1 proteins is a 238 amino acid region between residues 1048 and 1285. The Walker A and B motifs and the active transport family signature sequence, elements characteristic of the NBDs of ABC transporters (21), are completely conserved between hMRP1 and monMRP1 proteins (Fig. 1).

Overexpression of Monkey and hMRP1 in PEAKSTABLE Cells.
monMRP1 and hMRP1 proteins were expressed in PEAKSTABLE cells using episomally replicating plasmids introduced by transfection and selected with puromycin. Puromycin-resistant cells were analyzed subsequently as a population. Increased MRP1 expression of all three PEAKSTABLE cell lines was shown by Western analysis of whole cell lysates using the two MRP1-specific monoclonal antibodies MRPr1 and MRPm6 (Fig. 2). A Mr 190,000 band was easily detected in lysates containing monMRP1A, monMRP1B, and hMRP1 but was only weakly detectable in control cells upon long exposures to film (Fig. 2 and data not shown). Epitope-mapping studies previously demonstrated that MRPm6 binds to amino acids 238GSDLWSLNKE247 and MRPr1 binds to amino acids 1511PSDLLQQRGL1520 of hMRP1 (22). These epitopes are completely conserved in monMRP1 proteins. Analysis by Western blotting demonstrated that hMRP1, monMRP1A, and monMRP1B were consistently expressed at comparable levels in PEAKSTABLE cells over several months (data not shown). To exclude the possibility that PEAKSTABLE-monMRP1 cell lines expressed significant levels of hMRP1, Western analysis was also performed using a MRP1 monoclonal antibody, the epitope of which is not completely conserved in monMRP1 proteins. QCRL-1 binds to amino acids 918SSYSGDI924 (23), but monMRP1 proteins contain a valine at position 924. Strong staining of a Mr 190,000 protein was observed in hMRP1-transfected cells, but only low-level staining of the endogenous hMRP1 gene was detected in cells transfected with monMRP1A or monMRP1B (Fig. 2). These data are consistent with the overexpression of monkey MRP1 transporters in these cell lines without significant changes in endogenous hMRP1 levels.



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  		Fig. 2. MRP1 expression analyzed by Western blotting. Western blots of PeakSTABLE cells expressing MRP1 proteins and vector control cells were probed with three antibodies shown at the right. The position of size markers run in an adjacent lane are shown at the left. The epitopes recognized by antibodies MRPr1 and MRPm6 are completely conserved in monMRP1 proteins, whereas the region of hMRP1 recognized by QCRL-1 differs from monMRP1 by 1 residue (see text for details).

 
MRP1 Transporters Are Localized to the Plasma Membrane in Stable Transfectants.
To examine the subcellular localization of MRP1 transporters, PEAKSTABLE transfectants grown in monolayer cultures were fixed and stained with anti-MRP1 monoclonal antibody QCRL-3 that recognizes a conformation-dependent internal epitope of MRP1. Nuclei were counterstained with propidium iodide, and staining was visualized by confocal microscopy (Fig. 3). Cells expressing monMRP1 and hMRP1 proteins showed strong plasma membrane staining at comparable levels, whereas vector-transfected cells showed negligible staining under the detection conditions used.



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  		Fig. 3. Immunostaining of cell monolayers. Localization of MRP1 in puromycin-selected PEAKSTABLE cells transfected with monMRP1A (top left), monMRP1B (bottom right), hMRP1 (bottom left), and vector EW1969 (bottom right) by staining with antibody QCRL-3 and imaged by confocal microscopy is shown in green. Counterstaining of cell nuclei with propidium iodide is shown in red.

 
monMRP1 Transports LTC4 into Membrane Vesicles.
To confirm that the MRP1 proteins in PEAKSTABLE transfectants were functionally active, uptake studies were conducted with a natural MRP1 substrate, LTC4. Inside-out membrane vesicles were prepared, and ATP-dependent uptake of 90 nM LTC4 was measured. monMRP1A, monMRP1B, and hMRP1 proteins transported LTC4 into membrane vesicles at comparable rates (10.3 ± 1.3, 17.1 ± 2.7, and 11.7 ± 1.0 pmol/min/mg protein, respectively), whereas that of the vector was at least 90% lower (0.9 ± 0.4 pmol/min/mg protein; Fig. 4). These results indicate that that the transfected cDNAs encode functionally active transporters of LTC4.



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  		Fig. 4. ATP-dependent transport of 90 nM LTC4 into membrane vesicles prepared from puromycin-selected PEAKSTABLE cells transfected with hMRP1, monMRP1A, monMRP1B, or the vector EW1969. Data are the average ± range of duplicate data points.

 
monMRP1 Confers Resistance to Anticancer Drugs.
To examine the drug-resistance profiles conferred by hMRP1 and monMRP1, cytotoxicity assays were performed using PEAKSTABLE transfectants with a 72-h exposure to drugs. When compared with the vector-transfected PEAKSTABLE cells, hMRP1 and monMRP1 conferred resistance to all of the drugs tested, including the Vinca alkaloid vincristine, the epidophyllotoxin etoposide, as well as the anthracyclines doxorubicin, daunorubicin, and epirubicin. As shown in Table 1, the IC50s for these drugs were statistically different from vector control cells (P < 0.05). Furthermore, hMRP1 and monMRP1 transfectants were quite similar in their level of resistance to each other with the exception of etoposide resistance for monMRP1A (P < 0.05). However, species-specific effects were observed with respect to the anthracyclines. Both monMRP1 alleles conferred a significantly lower level of resistance (1.6–2.9-fold) to doxorubicin, daunorubicin, and epirubicin than hMRP1 (5.8–6.3-fold) to PEAKSTABLE cells (P < 0.013).


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  		Table 1 Resistance of MRP1 transfectants to chemotherapeutic drugs

 
Modulation of Drug Resistance with MRP1-selective Inhibitor.
A novel class of MRP1 inhibitors is the tricyclic isoxazoles, with LY402913 possessing an EC50 of 0.9 µM in cytotoxicity assays (17). It was unknown, however, if this class of inhibitors would also be active against monMRP1 given the sequence divergence between monMRP1 and hMRP1. To answer this question, cytotoxicity assays were performed in the presence and absence of 5 µM LY402913 using MRP1 and vector transfectants. LY402913 modulated the drug sensitivity of MRP1 and vector transfectants; however, it had a more pronounced effect on the MRP1 transfectants (Fig. 5). Consequently, the change in drug resistance was compared with both the untreated vector control cells and the LY402913-treated vector control cells as noted in Table 2. LY402913 completely reversed the resistance of PEAKSTABLE cells expressing hMRP1 and monMRP1 to all chemotherapeutic drugs tested except vincristine (Table 2). However, the resistance for vincristine was partially modulated from 10–15-fold to <=2.5-fold. This confirms that the major mechanism of drug resistance in these transfected cells is MRP1-mediated and can be modulated by the LY402913 inhibitor that was selected for activity against hMRP1.



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  		Fig. 5. LY402913 modulates monMRP1-mediated multidrug resistance. Cytotoxicity assays were performed using PEAKSTABLE-monMRP1B in the absence ({blacksquare}) or presence ({square}) of 5 µM LY402913 and using PEAKSTABLE vector control cells in the absence (•) or presence ({circ}) of 5 µM LY402913. Data are the means ± SE of quadruplicate determinations and were fitted to sigmoidal curves using a 4-parameter logistic fit. Data are representative of at least three independent experiments.

 

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  		Table 2 Modulation of multidrug resistance in MRP1-PEAKSTABLE transfectants by LY402913

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Before clinical evaluation of new drugs can begin in humans, toxicological studies in rodents and a nonrodent species such as dogs or monkeys must be conducted to evaluate safety. Interspecies differences, however, may affect the function of a protein of interest or its interaction with drugs. Such interspecies differences in function are evident in the case of the MRP1 from human, canine, and mouse. Although all three proteins transport endogenous substrates such as LTC4 effectively and confer resistance to the anticancer agents vincristine and etoposide, only hMRP1 confers resistance to anthracyclines (9, 10). Recent characterization of bMRP1 found that this protein also lacks the ability to confer anthracycline resistance (8). It has been hypothesized that the ubiquitously expressed hMRP1 may play an important role in protecting normal tissues from anticancer agents (3). This is supported by studies of mrp1 knockout mice that were found to have increased sensitivity to etoposide, resulting in increased bone marrow toxicity, and increased damage to the seminiferous tubules of the testis and the oropharyngeal cavity (24–26). Potential toxicities associated with sensitizing human tissues to anthracyclines by treatment with a MRP1 inhibitor during chemotherapy may not be adequately predicted using murine or canine models.

To explore the possibility that MRP1 from monkeys would be more functionally similar to hMRP1, we cloned cDNAs encoding MRP1 from the cynomolgus monkey. Two monMRP1 alleles were identified that encode proteins 98% identical to hMRP1 and differ from each other by three residues. It is unknown which allele represents the true wild-type allele because cDNAs representing each allele were derived from a single heterozygote, and the frequency of these alleles in a larger population was not determined. However, both alleles encoded functional transporters with similar activities in LTC4 and cytotoxicity assays. A 114 amino acid region of complete identity between muMRP1 and hMRP1 (residues 1126–1239) suggested an important role for this region of the protein (19). This region is also completely conserved in monMRP1 transporters. Furthermore, sequence alignment of hMRP1, monMRP1, canMRP1, bMRP1, muMRP1, and rMRP1 revealed only three divergent positions within this region (hMRP1 residues 1176, 1215, and 1218) in support of the earlier prediction (19). To allow functional comparisons between hMRP1, monMRP1A, and monMRP1B proteins, they were independently expressed in human embryonic kidney-derived PEAKSTABLE cells. All three proteins were expressed at comparable levels as assessed by Western blotting. No differences in endogenous hMRP1 expression levels were detected in monMRP1 transfectants when blots were probed with a hMRP1-specific antibody.

Of three residues found to be glycosylated in hMRP1 (N19, N23, and N1006; Ref. 26), two are present in monMRP1 (N19 and N1006), and only N1006 is present in MRP1 from all six species listed above. The glycosylation state of the protein is not expected to directly affect function because underglycosylated MRP1 expressed in insect cells and deglycosylated MRP1 in human cells treated with tunicamycin have essentially normal function (27, 28). However, because the stability and subcellular localization of some proteins is affected by their glycosylation status (29, 30), the subcellular localization of PEAKSTABLE cells expressing hMRP1 and monMRP1 proteins were examined by immunostaining and confocal microscopy. High levels of plasma membrane staining were observed in MRP1 transfectants compared with vector control cells with no apparent difference in the subcellular localization of hMRP1 and monMRP1 proteins.

Functionally, hMRP1 and monMRP1 transporters are very similar. monMRP1 transported the hMRP1 substrate LTC4 into membrane vesicles in an ATP-dependent manner and conferred a significant level of resistance to vincristine and etoposide when compared with vector control cells. Although both alleles of monMRP1 conferred the same relative levels of resistance to vincristine as hMRP1, monMRP1A conferred a lower level of resistance to etoposide when compared with monMRP1B and hMRP1. In contrast to the negligible levels of resistance to the anthracyclines conferred by MRP1 from mice (9, 10), dogs (11), and cows (8), monMRP1 conferred significant levels of resistance to anthracyclines, albeit statistically lower and estimated to be ~45% of that conferred by hMRP1. No statistically significant differences between monMRP1A and monMRP1B transfectants were observed for any of the anticancer agents tested.

Through the characterization of mouse/human hybrid proteins, regions of MRP1 important for conferring anthracycline resistance were identified (31). Only hybrid proteins that contained residues 959-1187 or 1188–1531 of hMRP1 conferred significant resistance to anthracyclines. Site-directed mutagenesis later identified a glutamate at position 1089 in hMRP1 transmembrane domain 14 to be critical for conferring resistance to anthracyclines (32). Substitution of a glutamine with glutamate at the corresponding position in muMRP1 (Q1086E) resulted in a protein that conferred 60% of the anthracycline resistance of hMRP1. Furthermore, bMRP1 and canMRP1 transporters do not contain this glutamine and do not confer resistance to anthracyclines (8, 11). Our results are consistent with those studies because monMRP1 transporters contain the essential glutamine at this position and do confer ~45% of the anthracycline resistance of hMRP1. Two additional residues of hMRP1 (R433 and W1246) have also been shown to be important for anthracycline resistance (33, 34) and are conserved in MRP1 from all six species listed above.

Although the COOH-terminal region of muMRP1 that is important for anthracycline resistance (amino acids 955-1528) differs from hMRP1 (amino acids 959-1531) at 47 positions, the corresponding region of monMRP1 differs from hMRP1 at only 8 positions (Fig. 1). Three of these positions in hMRP1 and monMRP1 [983, 1047 (allele A only), and 1286] were previously evaluated in studies of muMRP1. Independent substitution of these residues of hMRP1 into the corresponding positions of muMRP1 (residues 979, 1044, and 1283) had no effect on the ability of the resulting proteins to confer anthracycline resistance (32, 35). At positions 988 and 1410, monMRP1 matches the residue found in MRP1 proteins from two other species that do not confer anthracycline resistance (canMRP1 plus bMRP1 and muMRP1 plus bMRP1, respectively). monMRP1 also differs at three positions (1360, 1523, and 1526) from residues that are identical in hMRP1, canMRP1, bMRP1, muMRP1, and rMRP1, suggesting these changes may affect anthracycline specificity. It is also notable that at 8 positions (117, 147, 182, 351, 368, 546, 581, and 938), hMRP1 differs from MRP1 homologues of the other five species that are all identical at these positions.

Several groups have reported that charged residues associated within or adjacent to transmembrane segments [E1089, K332, H335, D336, R433, and R1249 of hMRP1 (32, 33, 36, 37), K324, K483, R1210, and R1257 of human MRP2 (38), K325, R586, R1206, and E1208 of rat MRP2 (39)] and in NBDs [D579 of cystic fibrosis transmembrane resistance regulator (40)] are important to the transport activity and/or substrate specificity of ABC transporters. In this light, it is interesting that many of the differences between monMRP1 and hMRP1 involve charged residues (positions 287, 344, 351, 460, 573, 640, 723, 880, 927, 937, 938, 1360, 1410, and 1526). Of particular interest are residues L460, R723, K1360, and D1409 in hMRP1 and R460, Q723, R1360, and G1410 in monMRP1 that affect transmembrane region 8, NBD1, NBD2, and NBD2, respectively. Additional studies of monMRP1 should aid in the identification of other residues(s) important for anthracycline resistance and/or differences in the transport of other substrates.

Recently, a novel class of tricyclic isoxazoles was identified in a screen for hMRP1 inhibitors (17). LY402913 is noncytotoxic, reverses hMRP1-mediated drug resistance in HeLa-T5 cells (EC50 of 0.90 µM), and inhibits ATP-dependent LTC4 uptake into membrane vesicles prepared from these cells. This compound also shows selectivity (~22-fold) for hMRP1 versus a related transporter, P-glycoprotein, in HL60/Adr and HL60/Vinc cells (17). To test whether LY402913 inhibits monMRP1, cytotoxicity assays were performed in the presence of 5 µM of this MRP1 inhibitor. LY402913 was equally effective at reversing the MRP1-mediated drug resistance in both monMRP1 and hMRP1 PEAKSTABLE transfectants. GSH-dependent photoaffinity labeling of hMRP1 by another inhibitor in this series, LY475776, was recently demonstrated (41, 42). Those studies identified T1242 and W1246 in hMRP1 as important for LY475776 photoaffinity labeling, and these residues are conserved in monMRP1A and monMRP1B. The data presented here predict that the binding site for this class of inhibitors is conserved in monMRP1. Photoaffinity labeling studies using membranes prepared from monMRP1 transfectants should help confirm this prediction.

In summary, we have cloned MRP1 cDNAs from the cynomolgus monkey and functionally characterized monMRP1- and hMRP1-transfected cells with respect to subcellular localization, drug resistance conferred, transport of LTC4 into membrane vesicles, and modulation of resistance by LY402913. In most respects, monkey MRP1 function is very similar to that of hMRP1. The cynomolgus monkey is the first animal model and the only toxicology model identified with an MRP1 homologue capable of conferring significant resistance to the anthracycline class of anticancer agents. monMRP1 is also inhibited by a MRP1-selective modulator selected for activity against hMRP1. It is surprising, however, that monMRP1 transporters confer significantly less resistance to anthracyclines than hMRP1 despite 98% identity at the protein level. These functional differences between human and monkey MRP1 transporters will need to be consid-ered when designing pharmacokinetic and toxicological studies for the preclinical evaluation of MRP1 modulators.


   Acknowledgments
 
We thank Tracy Caudill for cell culture and molecular biology support. We also thank Don McClure, Steve Kovacevic, Raj Haldankar, Joe Berry, and Brigette Schoner for contributions to the development and use of the episomal expression vector EW1969.


   Footnotes
 
2 The abbreviations used are: ABC, ATP-binding cassette; MRP1, multidrug resistance-associated protein; bMRP1, bovine MRP1; canMRP1, canine MRP1; hMRP1, human MRP1; monMRP1A, cynomolgus monkey MRP1 allele A; monMRP1B, cynomolgus monkey MRP1 allele B; muMRP1, murine (mouse)MRP1; rMRP1, rat RMP1; CMV, cytomegalovirus; EBNA-1, Epstein-Barr virus nuclear antigen 1; GSH, reduced glutathione; LTC4, leukotriene C4; RT-PCR, reverse transcription-PCR; NBD, nucleotide-binding domain. Back

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

Received 11/19/02; revised 12/16/02; accepted 1/ 3/03.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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