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• W2075616327 abstract "Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C4 and the cholestatic estrogen, 17β-estradiol 17(β-d-glucuronide) (E217βG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E217βG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E217βG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959–1187 or 1188–1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C4 with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E217βG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E217βG. Multidrug resistance protein (MRP) confers resistance to a number of natural product chemotherapeutic agents. It is also a high affinity transporter of some physiological conjugated organic anions such as cysteinyl leukotriene C4 and the cholestatic estrogen, 17β-estradiol 17(β-d-glucuronide) (E217βG). We have shown that the murine orthologue of MRP (mrp), unlike the human protein, does not confer resistance to common anthracyclines and is a relatively poor transporter of E217βG. We have taken advantage of these functional differences to identify region(s) of MRP involved in mediating anthracycline resistance and E217βG transport by generating mrp/MRP hybrid proteins. All hybrid proteins conferred resistance to the Vinca alkaloid, vincristine, when transfected into human embryonic kidney cells. However, only those in which the COOH-terminal third of mrp had been replaced with the corresponding region of MRP-conferred resistance to the anthracyclines, doxorubicin, and epirubicin. Exchange of smaller segments of the COOH-terminal third of the mouse protein by replacement of either amino acids 959–1187 or 1188–1531 with those of MRP produced proteins capable of conferring some level of resistance to the anthracyclines tested. All hybrid proteins transported cysteinyl leukotriene C4 with similar efficiencies. In contrast, only those containing the COOH-terminal third of MRP transported E217βG with an efficiency comparable with that of the intact human protein. The results demonstrate that differences in primary structure of the highly conserved COOH-terminal third of mrp and MRP are important determinants of the inability of the murine protein to confer anthracycline resistance and its relatively poor ability to transport E217βG. Multidrug resistance protein (MRP) 1The abbreviations used are: MRP, multidrug resistance protein; Pgp, P-glycoprotein; MSD, membrane-spanning domain; NBD, nucleotide binding domain; E217βG, 17β-estradiol 17-(β-d-glucuronide); LTC4, leukotriene C4; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; BCECF, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; PCR, polymerase chain reaction; kb, kilobase; HEK, human embryonic kidney cells and P-glycoprotein (Pgp) are very distantly related members of the superfamily of ATP binding cassette transmembrane transporters (1Deeley R.G. Cole S.P.C. Hayes J.D. Wolf C.R. Molecular Genetics of Drug Resistance. 3. Harwood Academic Press, Modern Genetics, Langhorne, PA1997: 247-298Google Scholar, 2Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3386) Google Scholar, 3Cole S.P.C. Deeley R.G. Bioessays. 1998; 20: 931-940Crossref PubMed Scopus (333) Google Scholar). Primary structure similarity between the two proteins is confined mainly to their nucleotide binding domains, regions that are generally conserved among ATP binding cassette superfamily members, and phylogenetic analyses suggest that MRP and Pgp evolved from different ancestral proteins (4Cole S.P.C. Bhardwaj G. Gerlach J.H. Mackie J.E. Grant C.E. Almquist K.C. Stewart A.J. Kurz E.U. Duncan A.M.V. Deeley R.G. Science. 1992; 258: 1650-1654Crossref PubMed Scopus (3022) Google Scholar,5Grant C.E. Kurz E.U. Cole S.P.C. Deeley R.G. Genomics. 1997; 45: 368-378Crossref PubMed Scopus (82) Google Scholar). Despite the lack of structural similarity, both proteins confer resistance to a similar but not identical spectrum of natural product chemotherapeutic agents, which includes the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins (6Grant C.E. Valdimarsson G. Hipfner D.R. Almquist K.C. Cole S.P.C. Deeley R.G. Cancer Res. 1994; 54: 357-361PubMed Google Scholar, 7Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar, 8Zaman G.J.R. Flens M.J. Van Leusden M.R. de Haas M. Mulder H.S. Lankelma J. Pinedo H.M. Scheper R.J. Baas F. Broxterman H.J. Borst P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8822-8826Crossref PubMed Scopus (703) Google Scholar, 9Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Biol. 1996; 6: 610-617Google Scholar). However, several lines of evidence suggest that MRP and Pgp confer resistance to these drugs by different mechanisms. Using plasma membrane vesicles enriched in Pgp, it has been possible to demonstrate direct transport of a number of chemotherapeutic agents and to label the protein with photoaffinity analogs of some drugs to which it confers resistance (10Cornwell M.M. Gottesman M.M. Pastan I.H. J. Biol. Chem. 1986; 261: 7921-7928Abstract Full Text PDF PubMed Google Scholar, 11Schlemmer S.R. Sirotnak F.M. J. Biol. Chem. 1994; 269: 31059-31066Abstract Full Text PDF PubMed Google Scholar, 12Safa A.R. Cancer Invest. 1992; 10: 295-305Crossref Scopus (46) Google Scholar). More recently, purified Pgp reconstituted into a lipid environment has been shown to transport a number of chemotherapeutic agents when provided with a suitable energy source (13Shapiro A.B. Ling V. J. Bioenerg. Biomembr. 1995; 27: 7-13Crossref PubMed Scopus (61) Google Scholar, 14Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (417) Google Scholar). In contrast, it has not been possible to demonstrate direct active transport of unmodified drugs by MRP-enriched membrane vesicles under similar conditions (15Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 16Heijn M. Hooijberg J.H. Scheffer G.L. Szabo G. Westerhoff H.V. Lankelma J. Biochim. Biophys. Acta. 1997; 1326: 12-22Crossref PubMed Scopus (66) Google Scholar, 17Muller M. Meijer C. Zaman G.J.R. Borst P. Scheper R.J. Mulder N.H. de Vries E.G.E. Jansen P.L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar, 18Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5138PubMed Google Scholar, 19Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar), and reports to the contrary have been retracted (20Kruh G.D. Biochemistry. 1997; 36: 13972Google Scholar, 21Paul S. Breuninger L.M. Tew K.D. Shen H. Kruh G.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14976PubMed Google Scholar). However, we have shown that MRP can actively transport the Vinca alkaloid, vincristine, and the potent mutagen, aflatoxin B1, in such a membrane vesicle system but only in the presence of physiological concentrations of glutathione (15Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 18Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5138PubMed Google Scholar, 22Loe D.W. Stewart R.K. Massey T.E. Deeley R.G. Cole S.P.C. Mol. Pharmacol. 1997; 51: 1034-1041Crossref PubMed Scopus (190) Google Scholar). It has also been possible to demonstrate that MRP-dependent transport of unmodified vincristine is accompanied by co-transport of reduced glutathione (18Loe D.W. Deeley R.G. Cole S.P.C. Cancer Res. 1998; 58: 5130-5138PubMed Google Scholar). In contrast to studies with unmodified chemotherapeutic drugs, direct active transport of several glutathione, glucuronide, and sulfate conjugates by MRP-enriched vesicles has been described by a number of laboratories. Some of these compounds are potential physiological substrates. These include LTC4, GSSG, E217βG, the mono- and bis-glucuronosyl conjugates of bilirubin, 6α-glucuronosylhyodeoxycholate, 3α-sulfatolithocholyltaurine, and the glutathione conjugate of prostaglandin A2 (15Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 17Muller M. Meijer C. Zaman G.J.R. Borst P. Scheper R.J. Mulder N.H. de Vries E.G.E. Jansen P.L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar,19Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar, 22Loe D.W. Stewart R.K. Massey T.E. Deeley R.G. Cole S.P.C. Mol. Pharmacol. 1997; 51: 1034-1041Crossref PubMed Scopus (190) Google Scholar, 23Jedlitschky G. Leier I. Buchholz U. Center M. Keppler D. Cancer Res. 1994; 54: 4833-4836PubMed Google Scholar, 24Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 25Leier I. Jedlitschky G. Buchholz U. Center M. Cole S.P.C. Deeley R.G. Keppler D. Biochem. J. 1996; 314: 433-437Crossref PubMed Scopus (289) Google Scholar, 26Jedlitschky G. Leier I. Buchholz U. Hummel-Eisenbeiss J. Burchell B. Keppler D. Biochem. J. 1997; 327: 305-310Crossref PubMed Scopus (259) Google Scholar, 27Evers R. Cnubben N.H.P. Wijnholds J. van Deemter L. van Bladeren P.J. Borst P. FEBS Lett. 1997; 419: 112-116Crossref PubMed Scopus (130) Google Scholar). LTC4 was the first high affinity substrate identified for MRP (K m 70–100 nm) (15Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar,17Muller M. Meijer C. Zaman G.J.R. Borst P. Scheper R.J. Mulder N.H. de Vries E.G.E. Jansen P.L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13033-13037Crossref PubMed Scopus (640) Google Scholar, 23Jedlitschky G. Leier I. Buchholz U. Center M. Keppler D. Cancer Res. 1994; 54: 4833-4836PubMed Google Scholar, 28Leier I. Jedlitschky G. Buchholz U. Cole S.P.C. Deeley R.G. Keppler D. J. Biol. Chem. 1994; 269: 27807-27810Abstract Full Text PDF PubMed Google Scholar). Consistent with the premise that it is a physiologically relevant substrate, knock-out mice lacking mrp have an impaired response to a leukotriene-mediated inflammatory stimulus (29Wijnholds J. Evers R. Van Leusden M.R. Mol C.A.A.M. Zaman G.J.R. Mayer U. Beijnen J.H. van der Valk M. Krimpenfort P. Borst P. Nat. Med. 1997; 3: 1275-1279Crossref PubMed Scopus (403) Google Scholar). Whether E217βG is a physiological substrate is not yet known, butin vitro, MRP transports this cholestatic estrogen conjugate with a K m of 1–3 μm (19Jedlitschky G. Leier I. Buchholz U. Barnouin K. Kurz G. Keppler D. Cancer Res. 1996; 56: 988-994PubMed Google Scholar, 24Loe D.W. Almquist K.C. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1996; 271: 9683-9689Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). We previously reported the cloning and in vitropharmacological characterization of the highly conserved murine orthologue of MRP, mrp (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). These studies revealed that the amino acid sequences of MRP and mrp are 88% identical. Both proteins confer resistance to Vinca alkaloids and the epipodophyllotoxin VP-16, and both transport LTC4 with similar kinetic parameters. However, despite the high degree of primary structure identity, mrp did not confer resistance to any of several anthracyclines tested (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 32Slapak C.A. Fracasso P.M. Martell R.L. Toppmeyer D.L. Lecerf J.-M. Levy S.B. Cancer Res. 1994; 54: 5607-5613PubMed Google Scholar,33Slapak C.A. Martell R.L. Terashima M. Levy S.B. Biochem. Pharmacol. 1996; 52: 1569-1576Crossref PubMed Scopus (10) Google Scholar). In addition, the ability of the murine protein to transport E217βG was relatively poor when compared with MRP (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). In the present study, we have taken advantage of functional differences between the two proteins to search for a region(s) of MRP involved in mediating anthracycline resistance and/or transport of substrates such as E217βG. We have stably expressed several mrp/MRP hybrid proteins in human embryonic kidney (HEK 293) cells and shown that they all confer resistance to vincristine and transport LTC4 with similar efficiency. However, only those proteins containing the COOH-terminal third of MRP conferred resistance to two anthracyclines tested, and only these proteins transported E217βG with an efficiency comparable with that of wild-type MRP. By exchanging segments within the COOH-terminal third of the protein, we identified amino acids 959–1187 of MRP as a region particularly critical for mediating anthracycline resistance. Doxorubicin HCl, vincristine sulfate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Sigma. Epirubicin HCl was purchased from Amersham Pharmacia Biotech. [3H]LTC4 (165 Ci mmol−1) and [3H]E217βG (55 Ci mmol−1) were purchased from NEN Life Science Products. The mrp/MRP1–857 vector was generated by PCR amplification of nucleotides 2575–3630 of mrp using a 5′ hybrid primer complementary to nucleotides 2565–2574 of MRP, which included an XhoI site followed by nucleotides 2575–2592 of mrp and a reverse primer complementary to nucleotides 3610–3630 of mrp (nucleotides numbered relative to beginning of the coding region; the EMBL/GenBank™ accession numbers are L05628, AF017145, andAF022824–AF022853 for MRP and AF022908 for mrp). The PCR product was digested with XhoI and SacI, and the fragment containing nucleotides 2570–3554 was isolated. cDNA clone 16Spe, which contains nucleotides 1612–4910 of mrp, was digested with XhoI and SacI, leaving nucleotides 3554–4910 of mrp attached to the vector pBluescript II (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar). The digested vector and attached insert was then ligated to theXhoI-SacI PCR product. The resulting construct was digested with KpnI in the polylinker region of the vector 5′ to the insert and with XhoI at the site introduced by PCR. The pCEBV7-MRP1 construct was digested with KpnI andXhoI to yield a fragment comprised of nucleotides 1–2560 of MRP with some of the vector polylinker at its 5′ end (7Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). This 2.6-kbKpnI-XhoI MRP fragment was ligated to theKpnI-XhoI-digested construct. The insert was excised using KpnI and NotI and transferred intoKpnI/NotI-digested pCEBV7 expression vector to give construct pCEBV7-mrp/MRP1–857. The cDNA specifying mrp/MRP959–1531 was generated by PCR amplification using a 5′ primer corresponding to nucleotides 1861–1880 of mrp and a 3′ hybrid primer containing nucleotides 2875–2885 of MRP, which included a HindIII site followed by nucleotides 2847–2862 of mrp. The product was cloned into theEcoRV site of pBluescript II KS+. Digestion of this construct with XhoI yielded a 4-kb BsmI-XhoI fragment comprised of nucleotides 1947 to 2885 of mrp attached to the vector. This fragment was ligated to a 1.9-kb XhoI-BsmI fragment containing nucleotides 1–1946 of mrp from pCEBV7-mrp (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar). The resulting insert was excised using HindIII, which cut in the polylinker region 5′ to the insert and at the 3′ end of the insert at the HindIII site introduced by PCR. This fragment was then ligated to an 11.5-kbHindIII fragment containing nucleotides 2875–4823 of MRP attached to the pCEBV7 expression vector to generate construct pCEBV7-mrp/MRP959–1531 (7Cole S.P.C. Sparks K.E. Fraser K. Loe D.W. Grant C.E. Wilson G.M. Deeley R.G. Cancer Res. 1994; 54: 5902-5910PubMed Google Scholar). The vector encoding mrp/MRP959–1187 was generated by ligating a HindIII-EcoRI fragment encompassing nucleotides 2875–3880 of MRP into HindIII-EcoRI digested pBluescript II KS+. This construct was digested withStuI at nucleotide 3551 of the insert and withSpeI at a site in the polylinker region 3′ to the insert to generate a 3.7-kb StuI-SpeI fragment containing the vector attached to nucleotides 2875–3554 of MRP. The 3.7-kbStuI-SpeI fragment was isolated and ligated to aStuI-SpeI fragment containing nucleotides 3554–4910 of mrp. This construct was linearized by HindIII digestion, treated with calf intestinal phosphatase, and ligated to aHindIII fragment containing nucleotides 1–2875 of mrp isolated from pCEBV7-mrp/MRP959–1531. The resulting insert was excised using EcoRV and NotI and ligated into pCEBV7 digested with PvuII and NotI to give construct pCEBV7-mrp/MRP959–1187. The vector encoding mrp/MRP1188–1531 was constructed by digesting mrp cDNA clone 41 (containing nucleotides 2809–5881 of mrp) with StuI at nucleotide 3575 of the insert and withBamHI in the polylinker region 3′ to the insert to generate a fragment containing nucleotides 2809–3575 of mrp attached to pBluescript II SK+ (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar). The 3.8-kb StuI-BamHI fragment was then ligated to a StuI-BamH fragment containing nucleotides 3562–4823 of MRP isolated from pCEBV7-MRP1 (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). The resulting construct was then digested with DraIII. The fragment encompassing nucleotides 3218–4823 of the insert attached to nucleotides 668–230 of pBluescript II SK+ was isolated and ligated to a DraIII fragment containing nucleotides 231–667 of pBluescript SK+ attached to nucleotides 1–3218 of mrp obtained by digestion of full-length mrp in pBluescript II SK+ (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar). The hybrid insert was excised by digestion with HindIII andXhoI and then re-ligated intoHindIII-XhoI-digested pCEBV7 to generate the pCEBV7-mrp/MRP1188–1531 construct. Integrity of the hybrid constructs was confirmed by restriction analysis and by sequencing across cloning junctions and those portions of the constructs contributed by PCR products. Stable transfection of HEK 293 cells with the pCEBV7-MRP1 and pCEBV7-mrp constructs has been described previously (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). pCEBV7 vectors containing mrp/MRP hybrid cDNAs were used to stably transfect HEK 293 cells in an identical fashion (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). Subpopulations of cells expressing high levels of wild-type mrp or mrp/MRP hybrid proteins were obtained by limiting cell dilution. The levels of wild-type mrp or MRP as well as the hybrid proteins were determined by immunoblot and/or dot blot analysis of membrane protein fractions from transfected cells, as described previously (34Hipfner D.R. Gauldie S.D. Deeley R.G. Cole S.P.C. Cancer Res. 1994; 54: 5788-5792PubMed Google Scholar, 35Almquist K.C. Loe D.W. Hipfner D.R. Mackie J.E. Cole S.P.C. Deeley R.G. Cancer Res. 1995; 55: 102-110PubMed Google Scholar, 36Hipfner D.R. Gao M. Scheffer G. Scheper R. Deeley R.G. Cole S.P.C. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar).Wild-type or hybrid proteins were detected with the monoclonal antibody, MRPr1, which recognizes a linear epitope of 10 amino acids (238–247), 9 of which are identical in mrp (36Hipfner D.R. Gao M. Scheffer G. Scheper R. Deeley R.G. Cole S.P.C. Br. J. Cancer. 1998; 78: 1134-1140Crossref PubMed Scopus (71) Google Scholar). Antibody binding was detected with goat anti-rat IgG (Pierce) followed by enhanced chemiluminescence detection (NEN Life Science Products). Drug resistance was determined using the microtiter plate MTT assay (30Stride B.D. Valdimarsson G. Gerlach J.H. Wilson G.M. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1996; 49: 962-971PubMed Google Scholar, 31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar, 37Cole S.P.C. Cancer Chemother. Pharmacol. 1990; 26: 250-256Crossref PubMed Scopus (51) Google Scholar). Cells were seeded in 96-well plates (1 × 104 cells/well), incubated at 37 °C for 24 h before the addition of drug, and then incubated for a further 72 h before the addition of MTT (2 mg/ml). IC50 values and standard deviations were obtained from the best fit of the data to a sigmoidal curve using GraphPad software. The significance of the difference between IC50 values of control and mrp/MRP transfectants was determined using an unpaired Student's t test. Relative resistance was obtained by dividing the IC50 of cells transfected with vectors encoding either wild-type or mrp/MRP hybrid proteins by the IC50 of cells transfected with the pCEBV7 vector (HEKPC7) alone. The kinetic parameters of [3H]LTC4 transport by inside-out membrane vesicles were determined as described previously (15Loe D.W. Almquist K.C. Deeley R.G. Cole S.P.C. J. Biol. Chem. 1996; 271: 9675-9682Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). Vesicles (2.5 μg of membrane protein) were incubated at 23 °C in transport buffer (50 mm Tris-HCl, 250 mm sucrose, 0.02% sodium azide, pH 7.4) containing AMP or ATP (4 mm), MgCl2 (10 mm), and [3H]LTC4 (15–1000 nm) in a final volume of 25 μl. Uptake was terminated after 30 s by rapid dilution of 20-μl aliquots into 1 ml of ice-cold transport buffer and filtration under vacuum through glass fiber filters. Filters were washed and dried before determination of the filter bound radioactivity. All data were corrected for the amount of [3H]LTC4, which remained bound to the filter in the absence of vesicle protein (usually less then 5% of the total radioactivity). Data were plotted as V o versus [S] to confirm that the concentration range selected was appropriate to observe both zero-order and first-order kinetics. Kinetic parameters (K m andV max) for the transport of [3H]LTC4 were determined from regression analysis of the Lineweaver-Burk transformation of the net uptake data (ATP-dependent minus AMP-dependent uptake). ATP-dependent uptake of [3H]E217βG was measured in membrane vesicles prepared from the transfectants as described for LTC4 with the following modifications. Reactions were carried out at 37 °C in a volume of 90 μl at a single concentration of [3H]E217βG (400 nm; 120 nCi) and 20 μg of membrane protein. Uptake was terminated at various times by removing aliquots (20 μl) and samples processed as described above. Previously, we demonstrated that stable transfection of HEK 293 cells with either mrp or MRP expression vectors conferred similar drug resistance profiles, with the notable exception that only the human protein increased resistance to several anthracyclines tested (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar). However, the levels of vector-encoded protein were severalfold lower in the mrp transfectant populations used for the original study than in the MRP transfectants used for comparison. To eliminate the possibility that the lower levels of mrp were responsible for the inability to detect anthracycline resistance, we isolated higher-expressing subpopulations from the original HEKmrptransfectants by limiting cell dilution. The level of mrp in the HEKmrp1 subpopulation is approximately equivalent to that of MRP in the HEKMRP transfectants (Fig.1 A). Both populations of cells showed a similar increase in resistance to vincristine relative to control transfectants (27- and 23-fold resistance for the HEKmrp1 and HEKMRP transfectants, respectively). The HEKMRP population also displayed 8- and 11-fold resistance to the anthracyclines, doxorubicin and epirubicin, respectively (Table I). In contrast, despite the higher levels of mrp in the HEKmrp1transfectants, no significant increase in resistance to either anthracycline could be detected as reported previously (Table I) (31Stride B.D. Grant C.E. Loe D.W. Hipfner D.R. Cole S.P.C. Deeley R.G. Mol. Pharmacol. 1997; 52: 344-353Crossref PubMed Scopus (137) Google Scholar).Table IRelative drug resistance of HEK 293 cells transfected with wild-type and hybrid murine and human MRPsConstructDrug (relative resistance)VincristineDoxorubicinEpirubicinMRP22.5 ± 7.2 (22.5)8.4 ± 3.3 (8.4)11.1 ± 4.1 (11.1)n = 3n = 3n = 3mrp26.7 ± 3.8 (26.7)1.0 ± 0.3 (1.0)1.2 ± 0.4 (1.2)n = 3n = 3n = 3mrp/MRP (1–857)16.4 ± 0.9 (16.4)1.2 ± 0.1 (1.2)1.4 ± 0.25 (1.4)n = 6n = 4n = 5mrp/MRP (959–1531A)10.9 ± 4.0 (21)2.2 ± 0.8 (3.4)2.6 ± 0.5 (4.2)n = 3n = 4n = 6mrp/MRP (959–1531B)56 ± 16 (28)4.2 ± 1.1 (2.6)4.0 ± 1.0 (2.5)n = 4n = 4n = 8mrp/MRP (959–1187)5.6 ± 0.5 (24)2.0 ± 0.4 (5)2.3 ± 0.6 (6.2)n = 4n = 5n = 4mrp/MRP (1188–1531)14.9 ± 0.7 (29)2.3 ± 0.7 (3.6)1.7 ± 0.6 (2.4)n = 7n = 8n = 7The resistance of HEK cells transfected with expression vectors encoding wild-type and hybrid murine and human proteins relative to that of cells transfected with empty vector were determined using the tetrazolium salt-based microtiter plate assay. In each experiment, cell viability was determined in quadruplicate at each drug concentration. Data were then analyzed as described under “Experimental Procedures.” The relative resistance was obtained by dividing the IC50 values for mrp/MRP-transfected cells by the IC50value obtained for control transfectants. The values shown represent the mean ± S.D. of relative resistance values determined from ≥3 independent experiments. Resistance factors normalized for differences in the levels of mrp/MRP expression in the transfectant populations used are shown in parentheses. Open table in a new tab The resistance of HEK cells transfected with expression vectors encoding wild-type and hybrid murine and human proteins relative to that of cells transfected with empty vector were determined using the tetrazolium salt-based microtiter plate assay. In each experiment, cell viability was determined in quadruplicate at each drug concentration. Data were then analyzed as described under “Experimental Procedures.” The relative resistance was obtained by dividing the IC50 values for mrp/MRP-transfected cells by the IC50value obtained for control transfectants. The values shown represent the mean ± S.D. of relative resistance values determined from ≥3 independent experiments. Resistance factors normalized for differences in the levels of mrp/MRP expression in the transfectant populations used are shown in parentheses. To identify regions of the mouse and human proteins responsible for the differences in anthracycline resistance, we generated a series of hybrid mrp/MRP molecules (Fig. 2). Initially, we replaced either the NH2-terminal 857 amino acids (mrp/MRP1–857) or COOH-terminal 574 amino acids of mrp (mrp/MRP959–1531) with the corresponding human sequence. In both cases, locations used to connect between the segments of the hybrid proteins were in the poorly conserved cytoplasmic region linking the NH2-proximal NBD to the COOH-proximal membrane-spanning domain (Fig. 2 A). The locations were chosen because we have shown previously that they are in a part of the linker region that is not required for the LTC4 transport activity of MRP (38Gao M. Yamazaki M. Loe D.W. Westlake C.J. Grant C.E. Cole S.P.C. Deeley R.G. J. Biol. Chem. 1998; 273: 10733-10740Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Populations of transfectants expressing mrp/MRP hybrids were subjected to limiting cell dilution to isolate subpopulations with" @default.
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• W2075616327 title "Localization of a Substrate Specificity Domain in the Multidrug Resistance Protein" @default.
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