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Vol. 1, 1335-1342, December 2002     Molecular Cancer Therapeutics
© 2002 American Association for Cancer Research

Identification and Characterization of the Canine Multidrug Resistance-associated Protein

Liandong Ma, Susan E. Pratt, Jin Cao1, Anne H. Dantzig, Robert E. Moore and Christopher A. Slapak2

Division of Cancer Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inherent or acquired resistance of tumor cells to a variety of structurally and functionally unrelated drugs, termed multidrug resistance, is a major obstacle to the successful use of chemotherapeutic agents. The acquisition of such a multidrug resistance phenotype in tumor cells is often associated with increased expression of either of two human MRPs,3 Pgp (1, 2) and/or MRP1. Human MRP1 (MRP1), like Pgp, is a member of the ATP-binding cassette transporter superfamily and functions as an ATP-dependent efflux transporter (3, 4). The ability of MRP1 to confer multidrug resistance has been demonstrated by transfection of the MRP1 gene into parental, drug-sensitive cell lines (5). MRP1 is capable of transporting anthracyclines (doxorubicin and daunorubicin), epipodophyllotoxins (etoposide and teniposide), and Vinca alkaloids (vincristine and vinblastine). The efflux activity of MRP1 is ATP dependent, and for certain anticancer drugs, glutathione dependent (4). In contrast to Pgp, MRP1 also transports organic anions such as glutathione S-conjugates, glucuronide conjugates, and sulfate conjugates (6, 7).

The expression of MRP1 has been detected in a variety of human tumors, including carcinomas of the lung, breast, bladder, stomach, prostate, and thyroid. It has also been detected in neuroblastoma, glioma, retinoblastoma, melanoma (8), and in blasts from patients with acute myeloid leukemia (9). These studies suggest a potential role of MRP1 in intrinsic or acquired resistance to anticancer drugs.

Significant efforts have been undertaken to evaluate the functional role of MRP1 as a mediator of clinical drug resistance. A number of compounds have been investigated for their ability to reverse MRP1-mediated multidrug resistance (10). Because MRP1 is also expressed at low, basal levels in epithelial, endocrine, and muscle tissues, as well as in peripheral blood cells, inhibition of MRP1 activity in humans may increase adverse effects of coadministered cytotoxic drugs (11). Understanding whether functional differences exist between MRP1 and the orthologs from species commonly used for preclinical models such as the mouse and dog is essential for developing and evaluating MRP1 modulators in these models.

The murine ortholog of MRP1 has been structurally and pharmacologically compared with MRP1 (12, 13). Although the proteins share 88% identity, phenotypic differences are present. Murine MRP1 (mrp1) is not capable of conferring resistance to anthracyclines, although it does confer resistance to vincristine and etoposide. These functional differences were further defined by constructing several mrp1/MRP1 hybrids and mapping of the region important for anthracycline resistance to amino acids 959-1187 in MRP1 (14). The amino acid glutamate at position 1089 (1086 in mrp1) has subsequently been demonstrated to be critical for conferring resistance to anthracyclines (15).

We used a similar approach to study canMRP1. We cloned and characterized the cDNA encoding the canine ortholog of MRP1. In the course of analyzing the canMRP1 cDNA, we identified five polymorphisms within the coding region, including one that leads to an amino acid change from alanine to serine at position 149. The ability of canMRP1 to confer multidrug resistance was demonstrated by transfection of canMRP1 cDNA into two recipient cell lines, HeLa and A2780 cells. The function of the canine protein was also examined by LTC4 transport and calcein-AM accumulation assays. Modulation of canMRP1 and MRP1 by the tricyclic isoxazole, LY402913, was compared in transfected HeLa cells by a growth inhibition assay. Although canMRP1 shares a high degree of identity with MRP1 (92%), canMRP1, like mrp1, did not confer resistance to doxorubicin in either HeLa or A2780 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture.
HeLa cells were cultured in MEM medium with 10% FCS, 0.1 mM nonessential amino acid, whereas A2780 cells were grown in RPMI 1640 with 10% FCS and 2 mM L-glutamine. The pMRP-fc-15 vector, HeLa C1 cells, and HeLa T5 cells were generously provided by Drs. Susan Cole and Roger Deeley (Queens University, Kingston, Ontario, Canada).

cDNA Amplification and Cloning.
Total RNA was extracted from canine (beagle dog) heart and kidney tissue with Trizol Reagent (Life Technologies, Inc.). Poly A + RNA was purified with the MessageMaker mRNA isolation System (Life Technologies, Inc.). First-strand cDNA was synthesized using the Superscript Preamplification System (Life Technologies, Inc.). cDNA fragments were amplified by Platinum Taq DNA polymerase (Life Technologies, Inc.). canMRP1 cDNAs in the regions of 345–990 and 3685–4021 were generated by PCR using primers based on MRP1 from the known MRP1 cDNA sequence. Subsequently, rapid amplification of cDNA end was carried out using primers derived from canine sequence using the Marathon cDNA Amplification Kit (Clontech Laboratories, Inc., Palo Alto, CA). On the basis of the untranslated cDNA sequence obtained from 5' and 3' rapid amplification of cDNA end, the forward and reverse primers, 5'-CCCGCCGCATGGCGCTCCGCGGGCTTCT-3' and 5'-CTGACGGGAACGCACCAGCTCC-3, were designed to amplify the full-length canMRP1 cDNA. To verify the fidelity of the canMRP1 cDNA sequence, the full-length canMRP1 cDNA was cloned from another tissue source, canine kidney. The cDNA was sequenced by an Applied Biosystems Automated DNA Sequencer (PE Applied Biosystems, Foster City, CA). Additional analyses were performed using SeqWeb version 1.2 (Genetics Computer Group, Madison, WI).

The cDNA corresponding to the complete coding region of the canMRP1 cDNA plus eight nucleotides of 5'- and 30 nucleotides of 3'-untranslated sequences was inserted into a pIRESneo eukaryotic expression vector (Clontech) to create a pIRESneo-canMRP1. A human/canMRP1 hybrid cDNA, encoding amino acids 1–900 of MRP1 and amino acids 901-1531 of canMRP1, was constructed: nucleotides 2701–4630 of MRP1 were excised and replaced by the same region of the canMRP. A second hybrid cDNA encoding amino acids 1–900 of canMRP1 and 901-1531 of MRP1 was generated using a similar approach. The two hybrid cDNAs were subcloned into a pIRESneo expression vector. A control vector (HC6) was generated by reversing the orientation of the hybrid cDNA.

Expression of canMRP1 in HeLa and A2780 Cells.
HeLa cells and A2780 cells were transfected using LipofectAmine Plus Reagent (Life Technologies, Inc.) and selected in 500 µg/ml G418. Individual G418-resistant colonies were expanded to determine canMRP1 levels by immunoblot and flow cytometric analysis.

Analysis of canMRP1 Protein Levels by Flow Cytometry.
Cells (5 x 105) cells in PBS were fixed and permeabilized by a Fixation Kit (Caltag Laboratories, Burlingame, CA). canMRP1 was detected by monoclonal antibody MRPm6 (1:200; Research Diagnostic, Inc., Flanders, NJ) that recognizes a linear epitope in MRP1 that is identical in canMRP1 (16). Antibody-binding was detected by fluorescein-conjugated goat antimouse immunoglobulin (1:200; BioSource, Camarillo, CA). Fluorescence was analyzed with Coulter Epics XL-M flow cytometer (Beckman/Coulter, Fullerton, CA).

Analysis of Calcein Accumulation by Flow Cytometry.
Assays were performed as reported previously (17). A total of 2.5 x 105 cells/ml was incubated with 0.25 µM calcein AM (Molecular Probes, Eugene, OR) in the presence or absence of 1.5 µM LY402913. Fluorescence was measured by flow cytometry.

Protein Blot Analysis.
Membrane protein (50 µg) was separated on 7.5% (w/v) polyacrylamide-SDS slab gels and then transferred to nitrocellulose filters. Filters were incubated with MRPm6 (1:500) for 1 h. Antibody binding was detected with a horseradish peroxidase-conjugated goat antirabbit IgG and a NEN Renaissance Kit (NEN, Boston, MA).

LTC4 Transport by Membrane Vesicles.
Assays were performed as described previously (18). Membrane vesicles (5–10 µg) were incubated at 37°C for 1 min in transport buffer [50 mM Tris, 250 mM sucrose, and 20 mM MgCl2, (pH 7.4)] containing 4 mM ATP or 4 mM Adenylylmethylenediphosphonate disodium magnesium salt (AMP-PCP; Roche Diagnostics Corporation, Indianapolis, IN), 100 µg/ml creatine phospho-kinase (Roche), 10 mM phospho-creatine and 50 nM [3H]LTC4 (110 µCi/nmol; NEN). To further compare canine protein with human MRP1, uptake was inhibited by various concentrations of MK 571, an established inhibitor of MRP1 (19). Uptake was terminated by aspirating membrane vesicles onto a glass fiber using a Packard 96-well harvester (Packard, Meriden, CT) and then washing three times with cold transport buffer. Assays were measured in triplicate.

Analysis of Drug Sensitivity.
Cells were seeded in triplicate at 10,000 cells/well in 96-well plates. The next day, drugs at various dilutions were added and incubated for 72 h in the presence or absence of 1.5 µM LY402913. Drug sensitivity was analyzed using the CellTiter 96 Cell Proliferation Assay (Promega Corporation, Madison, WI). The MTS Reagent was added after 72 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning, Sequencing, and Analysis of canMRP1 cDNA.
The full-length canMRP1 cDNA (nucleotides -8 to 4630, ATG referred to as +1) was amplified by RT-PCR from canine heart tissue. To confirm the fidelity of the cDNA sequence, the canMRP1 cDNA sequence was amplified, cloned, and sequenced in separate experiments using RNA isolated from canine kidney tissue.

Sequence analysis of canMRP1 cDNA clones revealed five polymorphisms located at nucleotides 69 (T/C), 445 (T/G), 523 (T/C), 738 (A/G), and 3150 (T/C), respectively. The T/G polymorphism at nucleotide 445 results in an amino acid change from alanine (A) to serine (S) at amino acid 149. Amino acid 149 is located in TM4. MRP1 has an alanine residue at this location. The two alleles were named canMRP1-A and canMRP1-B, respectively. The five polymorphisms were verified in canMRP1 cDNA isolated from kidney tissue (data not shown).

The lengths of the deduced amino acid sequences of canine and human MRP1 are identical (1531). Sequence alignment revealed that the canine protein is 92% identical to MRP1 (Fig. 1) and 88% identical to mrp1. Walker A, Walker B, and the ABC transporter signature motifs, characteristics of ABC superfamily proteins, are conserved in both the amino- and carboxyl-proximal nucleotide binding domains of the three proteins. The differences in the primary sequences result in few changes in potential sites for posttranslational modifications. canMRP1 possesses one potential glycosylation acceptor site, Asn23, compared with three sites, Asn19, Asn23, and Asn345 in the NH2-terminus of the MRP1. Limited proteolysis and site-directed mutagenesis in MRP1 have proven that Asn19 and Asn23 are actual glycosylation sites (20, 21). Moreover, the canine protein shares a common glutamine residue at position 1089 with murine protein (1086 in mrp1). This change, compared with MRP1, which carries a glutamate at this location, has been shown to result in a reduced anthracycline-resistance phenotype (15).



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  		Fig. 1. Alignment of the predicted amino acid sequences of canine, human, and murine MRP1. Light shaded, amino acid positions conserved in all three species; dark shaded, conserved residues in two of three species.

 
Transfection and Expression of canMRP1.
The ability of the canMRP1 or its hybrid proteins to confer drug resistance was confirmed by expressing canMRP1 or its hybrid cDNAs in two recipient cells, HeLa and A2780 cells. Three different pIRESneo expression vectors were constructed. pIRES/canMRP1 contains either canMRP1 allele A or allele B cDNA, pIRESneoMRP1/canMRP1 encodes a human/canine hybrid cDNA, which includes amino acids 1–900 of MRP1 and amino acids 901-1531 of canMRP1 (MRP1/canMRP1), and pIRESneocanMRP1/MRP1 contains amino acids 1–900 of canMRP1 and amino acids 901-1531 of MRP1.

HeLa or A2780 cells were transfected with the pIRESneo expression vectors or control vectors, including the pIRESneo vector and the pIRES-HC6 vector that contains a human/canine hybrid in the reverse orientation. After G418 selection, immunoblot and flow cytometric analyses were used to determine the levels of canMRP1 in the populations of MRP1-transfected cells. Increased canMRP1 expression in transfected HeLa cells relative to parental control vector-transfected cells was detected in several clones by immunoblot analysis of membrane-enriched fractions with monoclonal antibody MRPm6 (Fig. 2). An immunoreactive protein of Mr 190,000 was detected in membranes isolated from HeLa cells transfected with canMRP1-A (clone 14-10), canMRP-B (clones 15-3 and 15-27) or human MRP1 (HeLa T5). Under the same conditions, endogenous MRP1 was undetected in parental cells transfected with a vector control (pIRES). The levels of protein in canMRP1 transfectants were similar to the levels of MRP1 in HeLa T5. The levels of canMRP1 proteins in canMRP1-transfected HeLa and A2780 cells were additionally evaluated by flow cytometric analysis after staining with MRPm6. Representative flow cytometric histograms are shown in Fig. 3. canMRP1-transfected HeLa cell clones 14-10 (allele A), 15-3 (allele B) showed relatively strong immunofluorescent binding by MRPm6 compared with a control pIRES clone (Fig. 3A). The expression was similar to the cells transfected with MRP1 (HeLa-T5). A similar immunofluorescence intensity was detected in A2780 cells transfected with canMRP1 (clone 14-11) or MRP1/ canMRP1 hybrid (clone HC-7) cDNA, as compared with a control clone pIRES-HC6–3 (Fig. 3B). Unfortunately, the expression vector containing canine/human MRP1 hybrid cDNA (encompassing amino acids 1–900 of canine and 901-1531 of human) failed to yield an expressing subline in either HeLa or A2780 cells despite multiple attempts (data not shown).



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  		Fig. 2. Immumnoblot detection of MRP1 in canMRP1 cDNA-transfected HeLa cells. Membrane preparations (50 µg) were separated by SDS-PAGE and transferred to a nitrocellulose fiber. The blot was probed with monoclonal antibody MRPm6, which reacts with both the canine and human MRP1 proteins. The samples were obtained from HeLa cells transfected with MRP1 (HeLa T5), canMRP1-A (14-10), canMRP1-B cDNA (15-3 and 15-27), or pIRESneo.

 


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  		Fig. 3. Flow cytometry analysis of canMRP1, MRP1, or MRP1/canMRP1 hybrid proteins in HeLa and A2780 cells. Cells were fixed with Reagent A, permeabilized with Reagent B, and detected with MRPm6 (1:200). Antibody binding was stained with fluorescein-conjugated goat antimouse immunoglobulins (1:200). Shown are the histograms of relative fluorescence intensity for HeLa cell clones (A) or for A2780 cell clones (B), which were transfected by pIRES expression vectors containing MRP1 or hybrid MRP1 cDNA as indicated in the figure.

 
Functional Characterization of canMRP1.
To determine whether canMRP1 had transport activities, we examined the uptake of LTC4, a well-characterized substrate for MRP1, into membrane vesicles prepared from HeLa cells transfected with canMRP1 allele A, allele B, or with MRP1. As shown in Fig. 4, expression of canMRP1-A (clone 14-10) or canMRP1-B (clone 15-3) resulted in an ATP-dependent increase in vesicular accumulation of LTC4, which is similar to the uptake rate in HeLa T5 membrane vesicles (34 versus 44 pmol/min/mg protein, respectively). By contrast, the uptake rate into vesicles prepared from the vector control cells was minimal. To further characterize the canMRP1 transporter, inhibition of 50 nM LTC4 transport by MK571, which is a LTD4 receptor antagonist and a well-established inhibitor of MRP1 (19), was evaluated. The EC50s for MK 571 were 0.31, 0.15, and 0.46 µM for MRP1 (HeLa T5), canMRP1-A (14-10) or canMRP1-B, respectively (data not shown). These results indicate that the canine gene cloned encoded a functional transporter capable of transporting LTC4.



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  		Fig. 4. ATP-dependent [3H]LTC4 uptake in membrane vesicles prepared from canMRP1-transfected cells. ATP-dependent LTC4 transport by membrane vesicles prepared from human and canMRP1-transfected cells. Membrane vesicles were prepared from HeLa cells transfected by MRP1 (HeLa T5), canMRP1-A (14-10), canMRP1-B (15-3), or pIRES vector control (pIRES). Transport was performed for 1 min using 50 nM [3H]LTC4 and 5 µg of membrane protein. Previous experiments demonstrated that LTC4 transport was linear for at least 2 min under these conditions (data not shown). The uptake rate was corrected for the ATP-independent uptake by measuring uptake in the presence of Adenylylmethylenediphosphonate disodium magnesium salt (AMP-PCP) in place of ATP. Data are the mean ± SE from triplicate determinations with the exception that 15-3 is based upon duplicate points. The results are representative of three independent experiments.

 
Transfected cell populations were then tested for their sensitivity to a number of cytotoxic agents using MTS cell proliferation assays. HeLa cells expressing the canMRP1-A allele displayed an approximate 3-fold increased resistance to vincristine and a 2-fold resistance to etoposide (Table 1). Similar levels of resistance were observed with canMRP1-B-transfected HeLa cells, ~5-fold resistance to vincristine and 3-fold to etoposide. However, HeLa cells transfected by canMRP1 cDNA (allele A or B) did not display a doxorubicin resistance phenotype. Likewise, expression of canMRP1 in a second cell line, A2780, only conferred an increased resistance to vincristine (~7-fold), and etoposide (~4-fold) but not to doxorubicin (Table 2). The same spectrum of drug resistance was observed in cells expressing the human/canine hybrid MRP1 protein (HC-7). Human/canMRP1 hybrid conferred a 14-fold resistance to vincristine and a 5-fold resistance to etoposide but failed to confer detectable resistance to doxorubicin. In addition, we did not observe resistance to mitoxantrone in A2780 cell clones transfected by canMRP or the human/canMRP1 hybrid (data not shown).


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  		Table 1 Drug sensitivity of HeLa and A2780 cells transfected with canine and human MRP1 cDNA

 

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  		Table 2 Modulation of canine or human MRP1a

 
Modulation of canMRP1 by LY402913.
LY402913, a tricyclic isoxazole, inhibits MRP1 and reverses drug resistance to MRP1 substrates such as vincristine, doxorubicin, and etoposide in HeLa T5 cells. In addition, the combination of the MRP1 substrate vincristine with LY402913 delays the growth of MRP1-overexpressing tumors in mice (unpublished data). Understanding the reactivity of canMRP1 to LY402913 in vitro is essential for the pharmacokinetic and toxicokinetic studies of LY402913 in the canine models. The effects of LY402913 on vincristine sensitivity were examined in HeLa cells transfected with canMRP1. We found that LY402913 enhanced vincristine sensitivity in both canMRP1 transfected cells (15–3) and MRP1 transfected cells (HeLa-T5) by 6- and 3-fold, respectively (Table 2). These results indicate that canMRP1 shares the same reactivity to LY402913 with MRP1.

We further studied the effects of LY402913 on calcein AM accumulation in A2780 cells overexpressing canMRP1 (clone 14-11) or human/canine hybrid MRP1 (clone HC-7) as shown in Fig. 5. Calcein AM, a nonfluorescent hydrophobic molecule, rapidly penetrates cell membranes and becomes trapped upon conversion into fluorescent calcein (free calcein) by nonspecific cytoplasmic esterases. In the MRP1-expressing cells, calcein AM is extruded by MRP1 before its intracellular conversion (22). When extrusion is inhibited by an MRP1 modulator, free fluorescent calcein rapidly accumulates. Control cells (HC6–3) and cells expressing canMRP1 or MRP1/canMRP1 hybrid proteins were loaded with 0.25 µM calcein-AM in the presence (bottom panel) or absence (top panel) of 1.5 µM LY402913. The results showed that LY402913 restored calcein accumulation in both cell populations transfected by canine or human/canine hybrid MRP1 cDNA. LY402913 slightly increased calcein accumulation in control cells (HC6-3, bottom panel). Although by immunoblotting MRP1 is undetectable in A2789 cells, there may be a low-level of endogenous MRP1 that is modulated by LY402913.



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  		Fig. 5. Effects of LY402913 on calcein accumulation in A2780 cells transfected by canMRP1 cDNA (14-11) or MRP1/canMRP1 hybrid cDNA (HC-7). The cells, including the control HC6-3 line, were loaded with 0.25 µM calcein in the presence (bottom panel) or absence (top panel) of 1.5 µM LY402913. Cellular green fluorescence intensity was determined by flow cytometry. Representative data are shown as cell numbers plotted against log green fluorescence. Expression of canine or human canMRP1 in A2780 cells decreased calcein accumulation in the absence of LY402913 (top panel), whereas LY402913 restored dye accumulation close to the control level (bottom panel).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The development of new antitumor therapies has focused recently on molecularly defined, targeted agents. The preclinical development of such agents often uses murine and canine models for efficacy and/or safety analyses. Species differences between the targets in the human, murine, and canine orthologs are often not known or, at least, not fully understood. High degrees of preservation of amino acid identity may be interpreted as surrogates for identical function.

The identification of proteins capable of conferring multidrug resistance to clinically useful antitumor agents has led to the development of modulators to sensitize multidrug-resistant tumors. Whereas the human and murine genomes contain different numbers of Pgps (2 versus 3) with substantial sequence variation, few differences in the functional activity between species have been defined (23–25). As such, mice implanted with murine syngeneic and human xenograft tumors have been described using Pgp-modulating agents that have subsequently entered clinical trials (26).

Similarly, the development of reversal agents targeting MRP1 is an approach for overcoming MRP-related multidrug resistance in cancer patients. However, because of the broad expression of MRP1 in normal host tissues and its potential biological role in drug elimination, it is critical to understand fully the effects of MRP1 modulators in preclinical animal models. The identification and characterization of mrp1 have previously been described, but to date little is known about the structural or functional conservation between human and canMRP1 proteins. To provide a basis for studies of this transporter, we cloned and characterized the cDNA encoding the canMRP1 protein. Sequence alignments revealed that the canine protein is 92% identical to MRP1 and 88% to mrp1. The important conserved domains Walker A, Walker B, and the ABC transporter signature are identical among the proteins. In this study, we expressed and characterized canMRP1 functions in HeLa and A2780 cells.

Overall, our results showed that canMRP1 functioned in a manner similar to MRP1. canMRP1 conferred resistance to etopside and vincristine, as well as transported the MRP1 substrate LTC4. However, canMRP1 did not confer resistance to the anthracycline (doxorubicin) or the anthracenedione (mitoxantrone). Although canMRP1 shares a higher identity to MRP1 (92%) than to mrp1 (88%), this protein confers the same drug resistance profile against Vinca alkaloids, epipodophyllotoxins, and anthracyclines as does mrp1 (12, 13).

The region of the protein that mediates resistance to anthracyclines has been mapped to the COOH-terminal one-third of MRP1 by analyzing drug resistance profiles conferred by human/murine MRP1 hybrid protein (14). Site-directed mutagenesis studies of MRP1 have identified the glutamate at position 1089 of this region as critical for conferring drug resistance to anthracyclines (15). The resistance to anthracyclines was markedly reduced by the substitution of glutamate with glutamine, as is found in the corresponding position in murine mrp1. The canine protein shares the same glutamine residue as the murine protein at this position. These results indicate that primary sequence differences in COOH-terminus contribute to functional differences between the human and canine proteins.

We constructed human/canMRP1 hybrids to confirm the role of the COOH-terminus in mediating resistance to anthracyclines. A human/canMRP1 hybrid in which the COOH-terminus (amino acid 901-1531) of MRP1 was replaced by that of the canine protein, conferred resistance to vincristine and etoposide as effectively as the wild-type canine and human proteins. Thus, the regions critical for vincristine and etoposide binding are conserved between the two proteins. In contrast, the replacement of the COOH-terminal portion of MRP1 with that of the canine protein completely abolished the ability of the human MRP1 to confer resistance to doxorubicin. This result further suggested that glutamate at position 1089 is essential for mediating drug resistance to anthracyclines.

Analysis of deduced amino acid sequence showed differences between the human and canMRP1 in predicted glycosylation sites. canMRP1 does not have the first (of two) glycosylation sites, Asn19, at the NH2-terminus of the protein. This site has been shown to be a true glycosylation site in MRP1 by mutagenesis and peptide cleavage experiments (21). This change may alter the localization of the canMRP1 protein in cell membranes because glycosylation is an important step for trafficking of membrane proteins. However, strong plasma membrane staining of canMRP1, as observed with confocal microscopy in canMRP1-transfected cells (data not shown), demonstrated that canMRP1 does localize correctly to membranes in the cells. The failure of human/canine hybrid MRP1 to confer resistance to anthracyclines probably rules out the role of the glycosylation at Asn19 in mediating resistance to anthracyclines because the human portion of the MRP1 hybrid contains Asn19.

The data suggest that the vincristine and LY402913 binding domains are conserved between the human and canMRP1 proteins. LY402913 reversed drug resistance to vincristine in canMRP1 transfected cells as effectively as in MRP1 transfected cells. In a manner similar to MRP1, canMRP1 also effluxes calcein-a.m. Free calcein accumulation was decreased in A2780 cells transfected by canMRP1 cDNA. Treatment with LY402913 markedly increases the free calcein accumulation in A2780 cells expressing canMRP1 protein.

The alanine-serine heterozygous polymorphism identified in canMRP1 occurs at position 149, which is within TM4. Previous studies with Pgp have demonstrated that mutations in TM4 significantly changed the drug resistance profiles (27). We have examined the multidrug resistance profiles conferred by the canMRP1 A or B alleles and found that both alleles conferred a similar resistance profile. Therefore, the change that occurred in this TM region of canMRP1 appears to have no effect on its substrate specificity as tested thus far.

Taken together, these results indicate that canMRP1 confers a similar resistance profile as compared with MRP1, with respect to the Vinca alkaloids and epipodophyllotoxins. However, despite a high degree of primary sequence identity (92%), canMRP1 did not confer resistance to doxorubicin. These data will aid in the proper design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical development of MRP modulators.


   Acknowledgments
 
We thank Jennifer Davidson and Daniel C. Williams for critical advice on the manuscript and James Starling and William Perry for helpful discussion. We also thank Xiaoling Xia for assistance with the confocal laser scanning microscopy and Kevin L. Law and Robert L. Shepard for their important technical assistance. We thank Susan P. C. Cole and Roger G. Deeley for the MRP1 cDNA, and MRP1 cDNA transfectant. We also thank Michelle McNeil for the administrative assistance in the preparation of the manuscript.


   Footnotes
 
2 To whom requests for reprints should be addressed, at Division of Cancer Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285 Back

1 Present address: Department of Pharmacology, Merck Research Laboratories, Rahway, NH 07065. Back

3 The abbreviations used are: MRP, multidrug resistance protein; Pgp, P-glycoprotein; canMRP1, canine MRP1; TM4, transmembrane helix 4; LTC4, leukotriene C4. Back

Received 7/ 3/02; revised 10/ 4/02; accepted 10/23/02.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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