Abstract
The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [3H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.
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Abraham, E. H., Prat, A. G., Gerweck, L., Seneveratne, T., Arceci, R. J., Kramer, R., Guidotti, G., and Cantiello, H. F. (1993).Proc. Natl. Acad. Sci. USA 90, 312–316.
Al-Shawi, M. K., and Senior, A. E. (1993).J. Biol. Chem. 268, 4197–4206.
Al-Shawi, M. K., Urbatsch, I. L., Senior, A. E. (1994).J. Biol. Chem. 269, 8986–8992.
Ambudkar, S. V., and Maloney, P. C. (1986a).J. Biol. Chem. 261, 10079–10086.
Ambudkar, S. V., and Maloney, P. C. (1986b).Methods Enzymol.125, 558–563.
Ambudkar, S. V., Lynn, A. R., Maloney, P. C. and Rosen, B. P. (1986).J. Biol. Chem. 261, 15596–15600.
Ambudkar, S. V., Anantharam, V., and Maloney, P. C. (1990).J. Biol. Chem. 265, 12287–12292.
Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1992).Proc. Natl. Acad. Sci. USA 89, 8472–8476.
Ambudkar, S. V., Pastan, I., and Gottesman, M. M. (1995). InDrug Transport in Antimicrobial and Anticancer Chemotherapy: Cellular and Biochemical Aspects of Multidrug Resistance (Georgapapadakou, N. H., ed.). Marcel Dekker, New York, 525–547.
Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen, T. J., Ramjeesing, M., and Riordan, J. R. (1992).Cell 68, 809–818.
Bishop, L., Agbayani, R., Ambudkar, S. V., Maloney, P. C., and Ames, G. F-L. (1989).Proc. Natl. Acad. Sci. USA 86, 6953–6957.
Chen, C-J., Chin, C. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986).Cell 47, 381–389.
Chen, C. J., Clark, D., Ueda, K., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1990).J. Biol. Chem. 265, 506–514.
Childs, S., and Ling, V. (1994). InImportant Advances in Oncology: The MDR Superfamily of Genes and its Biological Implications (DeVita, V. T., Hellman, S., and Rosenberg, S. A., eds.), Lippincott, Philadelphia, pp. 21–36.
Cole, 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., and Deeley, R. G. (1992).Science.258, 1650–1654.
Dano, K. (1973).Biochim. Biophys. Acta,323, 466–483.
Davidson, A. L., and Nikaido, H. (1990).J. Biol. Chem. 265, 4254–4260.
Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992).Proc. Natl. Acad. Sci. USA 89, 2360–2364.
Decottignies, A., Kolackowski, M., Balzi, E., and Goffeau, A. (1994).J. Biol. Chem. 269, 12797–12803.
Dhir, R., and Gros, P. (1992).Biochemistry 31, 6103–6110.
Doige, C. A., and Ames, G. F. L. (1993).Annu. Rev. Microbiol. 47, 291–319.
Doige, C. A., Yu, X. H., and Sharom, F. J. (1992).Biochim. Biophys. Acta 1109, 149–160.
D'Souza, P., Ambudkar, S. V., August, J. T., and Maloney, P. C. (1987).Proc. Natl. Acad. Sci. USA 84, 6980–6984.
Endicott, J. A., and Ling, V. (1989).Annu. Rev. Biochem. 58, 137–171.
Eytan, G. D., Borgnia, M. J., Regev, R., and Assaraf, Y. G. (1994).J. Biol. Chem. 269, 26058–26565.
Fojo, A. T., Akiyama, S., Gottesman, M. M., and Pastan, I. (1985).Cancer Res. 45, 3002–3007.
Foote, S. J., Thompson, J. K., Conman, A. F., and Kemp, D. J. (1989).Cell 57, 921–930.
Gartner, J., Moser, H., and Valle, D. (1992).Nature Genetics 1, 16–23.
Germann, U. A., Willingham, M. C., Pastan, I., and, Gottesman, M. M. (1990).Biochemistry 29, 2295–2303.
Gill, D. R., Hyde, S. C., Higgins, C. F., Valverde, M. A., Mintenig, G. M., and SepÚlveda, F. V. (1992).Cell 71, 23–32.
Glisson, B., Gupta, R., Smallwood-Kentro, S., and Ross, W. (1986).Cancer Res. 46, 1934–1938.
Gottesman, M. M., and Pastan, I. (1988).J. Biol. Chem. 263, 12163–12166.
Gottesman, M. M., and Pastan, I. (1993).Annu. Rev. Biochem. 62, 385–427.
Gros, P., Ben Neriah, Y., Croop, J., and Housman, D. E. (1986).Nature (London) 323, 728–731.
Gros, P., Croop, J., and Housman, D. E. (1986).Cell 47, 371–380.
Higgins, C. F. (1992).Annu. Rev. Cell Biol. 8, 67–113.
Higgins, C. F., and Gottesman, M. M. (1992).Trends Biochem. Sci. 17, 18–21.
Horio, M., Gottesman, M. M., and Pastan, I. (1988).Proc. Natl. Acad. Sci. USA 85, 3580–3584.
Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990).Nature (London) 346, 362–365.
Juliano, R. L., and Ling, V. (1976).Biochim. Biophys. Acta 455, 152–162.
Lockwich, T., Ambudkar, S. V., Chauthaiwale, J., and Ambudkar, I. S. (1995).Biophys. J.,68, A387.
Luz, J. G., Wei, L-Y., Basu, S., and Roepe, P. D. (1994).Biochemistry 33, 7239–7249.
Maloney, P. C. and Ambudkar, S. V. (1989).Arch. Biochem. Biophys. 269, 1–10.
McGrath, J. P., and Varshavsky, A. (1989).Nature (London) 340, 400–404.
Monaco, J. J., Cho, S., and Attaya, M. (1990).Science 250, 1723–1726.
Moscow, J. A., and Cowan, K. H. (1988).J. Natl. Cancer Inst. 80, 14–20.
Mosser, J., Douar, A-M., Sarde, C-O., Kioschis, P., Feil, R., Moser, H., Poustka, A-M., Mandel, J-L., Aubourg, P. (1993).Nature (London) 361, 726–730.
Newman, M. J., and Wilson, T. H. (1980).J. Biol. Chem. 255, 10583–10586.
Ng, W. F., Sarangi, F., Zastawny, R. L., Veinot-Drebot, L. and Ling, V. (1989).Mol. Cell. Biol. 9, 1224–1232.
Pedersen, P. L., and Amzel, L. M. (1993).J. Biol. Chem. 268, 9937–9940.
Racker, E., Violand, B., O'Neil, S., Alfonzo, M., and Telford, J. (1979).Arch. Biochem. Biophys. 198, 470–477.
Rasola, A., Galietta, L. J. V., Gruenert, D. C., Romeo, G. (1994).J. Biol. Chem. 269, 1432–1436.
Riordan, J. R. (1993).Annu. Rev. Physiol. 55, 609–630.
Riordan, J. R., Rommens, J. M., Kerem, B. S., Alon, N., Rozmahel, R., Grzelezak, A., Aielenski, J., Lok, S., Plavsic, N., Chou, J-I., Drumm, M. L., Iannuzzi, M. C., Collins, F. S., and Tsui, L-C. (1989).Science 245, 1066–1073.
Roepe, P. D., Wei, L. Y., Cruz, J., and Carlson, D. (1993).Biochemistry 32, 11042–11056.
Roninson, I. B. (1991).Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, New York.
Ruan, Z-S., Vellareddy, A., Crawford, I. T., Ambudkar, S. V., Rhee, S. Y., Allison, M. J., and Maloney, P. C. (1992).J. Biol. Chem. 267, 10537–10543.
Ruetz, S., and Gros, P. (1994).Cell 77, 1071–1081.
Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992).J. Biol. Chem. 267, 4854–4858.
Shapiro, A., and Ling, V. (1994).J. Biol. Chem. 269, 3745–3754.
Sharom, F. J., Yu, X., and Doige, C. A. (1993).J. Biol. Chem. 268, 24197–24202.
Shyamala, V., Baichwald, V., Gant, T. W., Beall, E., and Ames, G. F-L. (1991).J. Biol. Chem. 266, 18714–18719.
Smit, J. J. M., Schinkel, A. H., OudeElferink, R. P. J., Groen, A. K., Wagenaar, E., van Deemter, L., Mol, C. A. M., Ottenhoff, R., van der Lugt, N. M. T., van Roon, M. A., van der Valk, M. A., Offerhaus, G. J. A., Berns, A. J. M., and Borst, P. (1993).Cell 75, 451–462.
Trowsdale, J., Hanson, I., Mockridge, I., Beck, S., Townsend, A., and Kelly, A. (1990).Nature (London) 348, 741–743.
Ueda, K., Cornwell, M. M., Gottesman, M. M., Pastan, I., Roninson, I. B., Ling, V., and Riordan, J. R. (1986).Biochem. Biophys. Res. Commun. 141, 956–962.
Ueda, K., Cardarelli, C., Gottesman, M. M., and Pastan, I. (1987).Proc. Natl. Acad. Sci. USA 84, 3004–3008.
Urbatsch, I. L., Al-Shawi, M. K., and Senior, A. E. (1994).Biochemistry 33, 7069–7076.
Valverde, M. A., Diáz, M., SepÚlveda, F. V., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992).Nature (London) 355, 830–833.
Wang, X. Y., Wall, D.M., Parkin, J. D., Zalcberg, J. R., and Kemm, R. E. (1994).Clin. Exp. Pharmacol. Physiol. 21, 101–108.
Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M., and Pastan, I. (1986).Cancer Res..46, 5941–5946.
Zeidel, M. L., Ambudkar, S. V., Smith, B. L., and Argre, P. (1992).Biochemistry 31, 7436–7440.
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Ambudkar, S.V. Purification and reconstitution of functional human P-glycoprotein. J Bioenerg Biomembr 27, 23–29 (1995). https://doi.org/10.1007/BF02110327
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DOI: https://doi.org/10.1007/BF02110327