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Keywords:

  • P-glycoprotein;
  • multidrug resistance;
  • Hoechst 33342;
  • rhodamine 123;
  • quercetin

Abstract

  1. Top of page
  2. Abstract
  3. References

In this paper, we show that P-glycoprotein contains two distinct sites for drug binding and transport, and that, unexpectedly, these sites interact in a positively cooperative manner. The kinetics of transport of rhodamine 123 and Hoechst 33342 in isolated P-glycoprotein-rich plasma membrane vesicles from Chinese hamster ovary CHRB30 cells were followed by continuous fluorescence monitoring. Each substrate stimulated P-glycoprotein-mediated transport of the other. Colchicine and quercetin stimulated rhodamine 123 transport and inhibited Hoechst 33342 transport. In contrast, anthracyclines such as daunorubicin and doxorubicin stimulated Hoechst 33342 transport and inhibited rhodamine 123 transport. Vinblastine, actinomycin D, and etoposide inhibited transport of both dyes. The results are consistent with a functional model of P-glycoprotein containing at least two positively cooperative sites (H site and R site) for drug binding and transport. This model is consistent with earlier observations of competitive and non-competitive effects of P-glycoprotein substrates and chemosensitizers. Such a two-site model may be fundamental to multidrug transport by P-glycoprotein, and it may be a feature common to other ATP-dependent transporters belonging to the ATP-binding cassette superfamily.

Abbreviations
ABC

ATP-binding cassette

CHRB30

multidrug-resistant Chinese hamster ovary B30 cells overexpressing P-glycoprotein

pyranine

8-hydroxypyrene-1,3,6-trisulfonic acid

References

  1. Top of page
  2. Abstract
  3. References
  • Ayesh, S., Shao, Y.-M. & Stein, W. D. (1996) Co-operative, competitive and non-competitive interactions between modulators of P-glycoprotein, Biochim. Biophys. Acta 1316, 818.
  • Bruggemann, E. P., Currier, S. J., Gottesman, M. M. & Pastan, I. (1992) Characterization of the azidopine and vinblastine binding site of P-glycoprotein, J. Biol. Chem. 267, 2102021026.
  • Chen, A. Y., Yu, C., Bodley, A., Peng, L. F. & Liu, L. F. (1993) A new mammalian DNA topoisomerase I poison Hoechst 33342: cytotoxicity and drug resistance in human cell cultures, Cancer Res. 53, 13321337.
  • 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. & Deeley, R. G. (1992) Overexpression of a transporter gene in a multi-drug-resistant human lung cancer cell line, Science 258, 16501654.
  • Critchfield, J. W., Welsh, C. J., Phang, J. M. & Yeh, G. C. (1994) Modulation of Adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells; activation of P-glycoprotein as a possible mechanism, Biochem. Pharmacol. 48, 14371445.
  • Demmer, A., Dunn, T., Hoof, T., Kubesch, P. & Tümmler, B. (1996) Competitive inhibition of photoaffinity labeling of P-glycoprotein by anticancer drugs and modulators including S9788, Eur. J. Pharmacol. 315, 338343.
  • Ferry, D. R., Malkhandi, P. J., Russell, M. A. & Kerr, D. J. (1995) Allosteric regulation of [3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine, Biochem. Pharmacol. 49, 18511861.
  • Garrigos, M., Mir, L. M. & Orlowski, S. (1997) Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase: further experimental evidence for a multisite model, Eur. J. Biochem. 244, 664673.
  • Germann, U. A. (1996) P-glycoprotein – a mediator of multidrug resistance in tumour cells, Eur. J. Cancer 32A, 927944.
  • Goda, K., Krasznai, Z., Cáspár, R., Lankelma, J., Westerhoff, H. V., Damjanovich, S. & Szabó, G. Jr (1996) Reversal of multidrug resistance by valinomycin is overcome by CCCP, Biochem. Biophys. Res. Commun. 219, 306310.
  • Gottesman, M. M. & Pastan, I. (1993) Biochemistry of multidrug resistance mediated by the multidrug transporter, Annu. Rev. Biochem. 62, 385427.
  • Greenberger, L. M., Lisanti, C. J., Silva, J. T. & Horwitz, S. B. (1991) Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr 1b, J. Biol. Chem. 266, 2074420751.
  • Greenberger, L. M. (1993) Major photoaffinity drug labeling sites for iodoarylazidoprazosin in P-glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12, J. Biol. Chem. 268, 1141711425.
  • Guiral, M., Viratelle, O., Westerhoff, H. V. & Lankelma, J. (1994) Cooperative P-glycoprotein-mediated daunorubicin transport into DNA-loaded plasma membrane vesicles, FEBS Lett. 346, 141145.
  • Hunter, J., Jepson, M. A., Tsuruo, T., Simmons, N. L. & Hirst, B. H. (1993) Functional expression of P-glycoprotein in apical membranes of human Caco-2 cells: kinetics of vinblastine secretion and interaction with modulators, J. Biol. Chem. 268, 1499114997.
  • Kolaczkowski, M., van der Rest, M., Cybularz-Kolaczkowska, A., Soumillion, J.-P., Konings, W. N. & Goffeau, A. (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p, J. Biol. Chem. 271, 3154331548.
  • Loe, D. W., Almquist, K. C., Deeley, R. G. & Cole, S. P. C. (1996) Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles, J. Biol. Chem. 271, 96759682.
  • Malkhandi, J., Ferry, D. R., Boer, R., Gekeler, V., Ise, W. & Kerr, D. J. (1994) Dexniguldipine-HCl is a potent allosteric inhibitor of [3H]vinblastine binding to P-glycoprotein of CCRF ADR5000 cells, Eur. J. Pharmacol. 288, 105114.
  • Morris, D. I., Greenberger, L. M., Bruggemann, E. P., Cardarelli, C., Gottesman, M. M., Pastan, I. & Seamon, K. B. (1994) Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin, Mol. Pharmacol. 46, 329337.
  • Orlowski, S., Mir, L. M., Belehradek, J. Jr & Garrigos, M. (1996) Effects of steroids and verapamil on P-glycoprotein ATPase activity: progesterone, desoxycorticosterone, and verapamil are mutually non-exclusive modulators, Biochem. J. 317, 515522.
  • Pawagi, A. B., Wang, J., Silverman, M., Reithmeier, R. A. F. & Deber, C. M. (1994) Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity, J. Mol. Biol. 235, 554564.
  • Pereira, E., Borrel, M. N., Fiallo, M. & Garnier-Suillerot, A. (1994) Non-competitive inhibition of P-glycoprotein-associated efflux of THP-adriamycin by verapamil in living K562 leukemia cells, Biochim. Biophys. Acta 1225, 209216.
  • Phang, J. M., Poore, C. M., Lopaczynska, J. & Yeh, G. C. (1993) Flavonol-stimulated efflux of 7,12 dimethylbenz(a)anthracene in multi-drug-resistant breast cancer cells, Cancer Res. 53, 59775981.
  • Ruetz, S. & Gros, P. (1994) Phosphatidylcholine translocase: a physiological role for the mdr2 gene, Cell 77, 10711081.
  • Safa, A. R., Glover, C. J., Sewell, J. L., Meyers, M. B., Biedler, J. L. & Felsted, R. L. (1987) Identification of the multidrug resistance-related membrane glycoprotein as an acceptor of calcium channel blockers, J. Biol. Chem. 262, 78847888.
  • Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) N-(p-azido-3-[125I]iodophenethyl)spiperone binds to specific regions of P-glycoprotein and another multidrug binding protein, spiperophilin, in human neuroblastoma cells, Biochemistry 33, 256265.
  • Sarkadi, B., Müller, M. & Holló, Z. (1996) The multidrug transporters-proteins of an ancient immune system, Immunol. Lett. 54, 215219.
  • Scambia, G., Ranelletti, F. O., Panici, P. B., DeVincenzo, R., Bonanno, G., Ferrandina, G., Piantelli, M., Bussa, S., Rumi, C., Cianfriglia, M. & Mancuso, S. (1994) Quercetin potentiates the effect of Adriamycin in a multidrug-resistant MCF-7 human breast cancer cell line: P-glycoprotein as a possible target, Cancer Chemother. Pharmacol. 34, 459464.
  • Shao, Y., Ayesh, S. & Stein, W. D. (1997) Mutually co-operative interactions between modulators of P-glycoprotein, Biochim. Biophys. Acta 1360, 3038.
  • Shapiro, A. B. & Ling, V. (1994) ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells, J. Biol. Chem. 269, 37453754.
  • Shapiro, A. B. & Ling, V. (1995) Reconstitution of drug transport by purified P-glycoprotein, J. Biol. Chem. 270, 1616716175.
  • Shapiro, A. B. & Ling, V. (1997) Effect of quercetin on Hoechst 33342 transport by purified and reconstituted P-glycoprotein, Biochem. Pharmacol. 53, 587596.
  • Shapiro, A. B., Corder, A. B. & Ling, V. (1997) P-glycoprotein-mediated Hoechst 33342 transport out of the lipid bilayer, Eur. J. Biochem. 250, 115121.
  • Sharom, F. J., Yu, X. & Doige, C. A. (1993) Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein, J. Biol. Chem. 268, 2419724202.
  • Sharom, F. J., Yu, X., DiDiodato, G. & Chu, J. W. K. (1996) Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport, Biochem. J. 320, 421428.
  • Smit, J. J. M., Schinkel, A. H., Oude Elferink, R. P. J., Groen, A. K., Wagenaar, E., van Deemter, L., Moi, C. A. A. M., Ottenhofer, R., van der Lugt, N. M. T., van Roon, M. A., Offerhaus, G. J. A., Berns, A. J. M. & Borst, P. (1993) Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from the bile and to liver disease, Cell 75, 451462.
  • Spoelstra, E. C., Westerhoff, H. V., Pinedo, H. M., Dekker, H. & Lankelma, J. (1994) The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunorubicin as a non-competing substrate, Eur. J. Biochem. 221, 363373.
  • Tamai, I. & Safa, A. R. (1991) Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug-resistant cells, J. Biol. Chem. 266, 1679616800.
  • Urbatsch, I. L., Al-Shawi, M. K. & Senior, A. E. (1994) Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein, Biochemistry 33, 70697076.
  • van Veen, H. W., Venema, K., Bolhuis, H., Oussenko, I., Kok, J., Poolman, B., Driessen, A. J. M. & Konings, W. (1996) Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1, Proc. Natl Acad. Sci. USA 93, 1066810672.