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AAPS PharmSci. Mar 2004; 6(1): 77–85.
Published online Mar 9, 2004. doi:  10.1208/ps060108
PMCID: PMC2750943
Development and characterization of a recombinant madin-darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1)
Ziping Yang, Micha Horn, Joanne Wang, Danny D Shen, and Rodney JY Hocorresponding author
Departments of Pharmaceutics and Pharmacy, University of Washington, 98195-7610 Seattle, WA
Rodney JY Ho, Phone: (206) 543-9434, Fax: (206) 543-3204, rodneyho/at/u.washington.edu.
corresponding authorCorresponding author.
Received October 3, 2003; Accepted December 22, 2003.
Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs.
Keywords: rat, MRP1, drug resistance, chemotherapeutic agents, cytotoxicity, transport, ATP-binding cassette, transwell
1. Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. doi: 10.1146/annurev.cb.08.110192.000435. [PubMed] [Cross Ref]
2. Wijnholds J. Drug resistance caused by multidrug resistance-associated proteins. Novartis Found Symp. 2002;243:69–79. doi: 10.1002/0470846356.ch6. [PubMed] [Cross Ref]
3. Goldstein LJ, Pastan I, Gottesman MM. Multidrug resistance in human cancer. Crit Rev Oncol Hematol. 1992;12:243–253. doi: 10.1016/1040-8428(92)90057-W. [PubMed] [Cross Ref]
4. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385–427. doi: 10.1146/annurev.bi.62.070193.002125. [PubMed] [Cross Ref]
5. Cole SP, Bhardwaj G, Gerlach JH, et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science. 1992;258:1650–1654. doi: 10.1126/science.1360704. [PubMed] [Cross Ref]
6. Kruh GD, Zeng H, Rea PA, et al. MRP subfamily transporters and resistance to anticancer agents. J Bioenerg Biomembr. 2001;33:493–501. doi: 10.1023/A:1012827221844. [PubMed] [Cross Ref]
7. Shen H, Paul S, Breuninger LM, et al. Cellular and in vitro transport of glutathione conjugates by MRP. Biochemistry. 1996;35:5719–5725. doi: 10.1021/bi960098n. [PubMed] [Cross Ref]
8. Rappa G, Lorico A, Flavell RA, Sartorelli AC. Evidence that the multidrug resistance protein (MRP) functions as a co-transporter of glutathione and natural product toxins. Cancer Res. 1997;57:5232–5237. [PubMed]
9. Loe DW, Deeley RG, Cole SP. Characterization of vincristine transport by the M(r) 190,000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res. 1998;58:5130–5136. [PubMed]
10. Paul S, Belinsky MG, Shen H, Kruh GD. Structure and in vitro substrate specificity of the murine multidrug resistance-associated protein. Biochemistry. 1996;35:13647–13655. doi: 10.1021/bi9616615. [PubMed] [Cross Ref]
11. Stride BD, Grant CE, Loe DW, Hipfner DR, Cole SP, Deeley RG. Pharmacological characterization of the murine and human orthologs of multidrug-resistance protein in transfected human embryonic kidney cells. Mol Pharmacol. 1997;52:344–353. [PubMed]
12. Zhang DW, Cole SP, Deeley RG. Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines. J Biol Chem. 2001;276:13231–13239. doi: 10.1074/jbc.M010008200. [PubMed] [Cross Ref]
13. Lin ZP, Johnson DR, Finch RA, Belinsky MG, Kruh GD, Sartorelli AC. Comparative study of the importance of multidrug resistance-associated protein 1 and P-glycoprotein to drug sensitivity in immortalized mouse embryonic fibroblasts. Mol Cancer Ther. 2002;1:1105–1114. [PubMed]
14. Cherrington NJ, Hartley DP, Li N, Johnson DR, Klaassen CD. Organ distribution of multidrug resistance proteins 1, 2, and 3 (Mrpl, 2, and 3) mRNA and hepatic induction of Mrp3 by constitutive androstane receptor activators in rats. J Pharmacol Exp Ther. 2002;300:97–104. doi: 10.1124/jpet.300.1.97. [PubMed] [Cross Ref]
15. Regina A, Koman A, Piciotti M, Hafny B, Center MS, Bergmann R, Couraud PO, Roux F. Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells. J Neurochem. 1998;71:705–715. [PubMed]
16. Decleves X, Regina A, Laplanche JL, et al. Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes. J Neurosci Res. 2000;60:594–601. doi: 10.1002/(SICI)1097-4547(20000601)60:5<594::AID-JNR4>3.0.CO;2-6. [PubMed] [Cross Ref]
17. Hirrlinger J, Konig J, Dringen R. Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones. J Neurochem. 2002;82:716–719. doi: 10.1046/j.1471-4159.2002.01082.x. [PubMed] [Cross Ref]
18. Leazer TM, Klaassen CD. The presence of xenobiotic transporters in rat placenta. Drug Metab Dispos. 2003;31:153–167. doi: 10.1124/dmd.31.2.153. [PubMed] [Cross Ref]
19. Soroka CJ, Lee JM, Azzaroli F, Boyer JL. Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver. Hepatology. 2001;33:783–791. doi: 10.1053/jhep.2001.23501. [PubMed] [Cross Ref]
20. Yang Z, Li CS, Shen DD, Ho RJ. Cloning and characterization of the rat multidrug resistance-associated protein 1. AAPS Pharm Sci. 2002;4(3):15–15. doi: 10.1208/ps040315. [PMC free article] [PubMed] [Cross Ref]
21. Nunoya K, Grant CE, Zhang D, Cole SP, Deeley RG. Molecular cloning and pharmacological characterization of rat multidrug resistance protein 1 (mrp1) Drug Metab Dispos. 2003;31:1016–1026. doi: 10.1124/dmd.31.8.1016. [PubMed] [Cross Ref]
22. Makhey VD, Guo A, Norris DA, Hu P, Yan J, Sinko PJ. Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells. Pharm Res. 1998;15:1160–1167. doi: 10.1023/A:1011971303880. [PubMed] [Cross Ref]
23. Cole SP, Sparks KE, Fraser K, et al. Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res. 1994;54:5902–5910. [PubMed]
24. Grant CE, Valdimarsson G, Hipfner DR, Almquist KC, Cole SP, Deeley RG. Overexpression of multidrug resistance-associated protein (MRP) increases resistance to natural product drugs. Cancer Res. 1994;54:357–361. [PubMed]
25. Stride BD, Cole SP, Deeley RG. Localization of a substrate specificity domain in the multidrug resistance protein. J Biol Chem. 1999;274:22877–22883. doi: 10.1074/jbc.274.32.22877. [PubMed] [Cross Ref]
26. Hipfner DR, Gao M, Scheffer G, Scheper RJ, Deeley RG, Cole SP. Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP) Br J Cancer. 1998;78:1134–1140. [PMC free article] [PubMed]
27. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977;36:59–74. doi: 10.1099/0022-1317-36-1-59. [PubMed] [Cross Ref]
28. Goh LB, Spears KJ, Yao D, et al. Endogenous drug transporters in vitro and in vivo models for the prediction of drug disposition in man. Biochem Pharmacol. 2002;64:1569–1578. doi: 10.1016/S0006-2952(02)01355-2. [PubMed] [Cross Ref]
29. Lin JH. Drug-drug interaction mediated by inhibition and induction of P-glycoprotein. Adv Drug Deliv Rev. 2003;55:53–81. doi: 10.1016/S0169-409X(02)00171-0. [PubMed] [Cross Ref]
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