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Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein–Related Activities

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Purpose.

P-glycoprotein (P-gp), a membrane ATPase expelling many structurally unrelated compounds out of cells, is one of the major contributors to multidrug resistance. It is enriched in cold TritonX-100 insoluble membrane domains (i.e., rafts). The purpose of this work was to characterize the ATPase activities of raft preparations from P388 cells overexpressing P-gp (P388/ADR) or devoid of P-gp (P388) and to establish a P-gp–enriched screening system for P-gp–interfering compounds.

Methods.

Rafts were extracted with cold TritonX-100. The ATPase activity was characterized in 96-well plates using a fluorescence assay.

Results.

The ATPase activity per mg protein was about five times higher in P388/ADR rafts than in crude membranes. The anti–P-gp antibody C219 inhibited 20% of the activity in P388/ADR rafts but only about 10% of the activity in P388/ADR crude membranes and had no effect on the activity of P388 rafts. The known P-gp–activating compounds verapamil, progesterone, and valinomycin revealed the typical bell-shaped activity/concentration profiles in P388/ADR rafts, indicative for activation at low compound concentrations and inhibition at concentrations >10 to 100 μM. The inhibitory effect was also observed in P388 rafts.

Conclusions.

Extracted rafts are rich in functional ATPases. Rafts from P-gp–overexpressing cells display P-gp–typical ATPase activity and provide an easy, P-gp–enriched screening system.

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Abbreviations

ADP:

adenosine diphosphate

ATP:

adenosine triphosphate

BSA:

bovine serum albumin

DTT:

dithiothreitol

P-gp:

P-glycoprotein

SDS-PAGE:

sodium laurylsulfate-polyacrylamide gel electrophoresis

References

  1. 1. S. V. Ambudkar, S. Dey, C. A. Hrycyna, M. Ramachandra, I. Pastan, and M. M. Gottesman. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 39:361–398 (1999).

    Google Scholar 

  2. 2. T. Litman, T. Skovsgaard, and W. D. Stein. Pumping of drugs by P-glycoprotein: a two-step process? J. Pharmacol. Exp. Ther. 307:846–853 (2003).

    Google Scholar 

  3. 3. G. D. Eytan, R. Regev, G. Oren, and Y. G. Assaraf. The role of passive transbilayer drug movement in multidrug resistance and its modulation. J. Biol. Chem. 271:12897–12902 (1996).

    Google Scholar 

  4. 4. A. Seelig. A general pattern for substrate recognition by P-glycoprotein. Eur. J. Biochem. 251:252–261 (1998).

    Google Scholar 

  5. 5. J. E. Penzotti, M. L. Lamb, E. Evensen, and P. D. Grootenhuis. A computational ensemble pharmacophore model for identifying substrates of P-glycoprotein. J. Med. Chem. 45:1737–1740 (2002).

    Google Scholar 

  6. 6. R. Didziapetris, P. Japertas, A. Avdeef, and A. Petrauskas. Classification analysis of P-glycoprotein substrate specificity. J. Drug Target. 11:391–406 (2003).

    Google Scholar 

  7. 7. A. Seelig and E. Landwojtowicz. Structure-activity relationship of P-glycoprotein substrates and modifiers. Eur. J. Pharm. Sci. 12:31–40 (2000).

    Google Scholar 

  8. 8. T. Osterberg and U. Norinder. Theoretical calculation and prediction of P-glycoprotein-interacting drugs using MolSurf parametrization and PLS statistics. Eur. J. Pharm. Sci. 10:295–303 (2000).

    Google Scholar 

  9. 9. T. Langer, M. Eder, R. D. Hoffmann, P. Chiba, and G. F. Ecker. Lead identification for modulators of multidrug resistance based on in silico screening with a pharmacophoric feature model. Arch. Pharm. Pharm. Med. Chem. 337:317–327 (2004).

    Google Scholar 

  10. 10. D. Schwab, H. Fischer, A. Tabatabaei, S. Poli, and J. Huwyler. Comparison of in vitro P-glycoprotein screening assays: recommendations for their use in drug discovery. J. Med. Chem. 46:1716–1725 (2003).

    Google Scholar 

  11. 11. A. Garrigues, J. Nugier, S. Orlowski, and E. Ezan. A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein. Anal. Biochem. 305:106–114 (2002).

    Google Scholar 

  12. 12. F. J. Sharom, R. Liu, Q. Qu, and Y. Romsicki. Exploring the structure and function of the P-glycoprotein multidrug transporter using fluorescence spectroscopic tools. Sem. Cell Dev. Biol. 12:257–265 (2001).

    Google Scholar 

  13. 13. E. Landwojtowicz, P. Nervi, and A. Seelig. Real-time monitoring of P-glycoprotein activation in living cells. Biochemistry 41:8050–8057 (2002).

    Google Scholar 

  14. 14. S. P. Hammerle, B. Rothen-Rutishauser, S. D. Kramer, M. Gunthert, and H. Wunderli-Allenspach. P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope. Eur. J. Pharm. Sci. 12:69–77 (2000).

    Google Scholar 

  15. 15. N. Kokubu, D. Cohen, and T. Watanabe. Functional modulation of ATPase of P-glycoprotein by C219, a monoclonal antibody against P-glycoprotein. Biochem. Biophys. Res. Commun. 230:398–401 (1997).

    Google Scholar 

  16. 16. T. Litman, T. Zeuthen, T. Skovsgaard, and W. D. Stein. Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity. Biochim. Biophys. Acta 1361:159–168 (1997).

    Google Scholar 

  17. 17. S. Ambudkar, I. Lelong, J. Zhang, C. Cardarelli, M. Gottesman, and I. Pastan. Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc. Natl. Acad. Sci. USA 89:8472–8476 (1992).

    Google Scholar 

  18. 18. P. Lu, R. Liu, and F. J. Sharom. Drug transport by reconstituted P-glycoprotein in proteoliposomes. Effect of substrates and modulators, and dependence on bilayer phase state. Eur. J. Biochem. 268:1687–1697 (2001).

    Google Scholar 

  19. 19. G. D. Luker, C. M. Pica, S. Kumar, D. F. Covey, and D. Piwnica-Worms. Effects of cholesterol and enantiomeric cholesterol on P-glycoprotein localization and function in low-density membrane domains. Biochemistry 39:7645–7650.

    Google Scholar 

  20. 20. Y. Lavie, G. Fiucci, and M. Liscovitch. Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells. J. Biol. Chem. 273:32380–32383 (1998).

    Google Scholar 

  21. 21. M. Demeule, J. Jodoin, D. Gingras, and R. Beliveau. P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries. FEBS Lett. 466:219–224 (2000).

    Google Scholar 

  22. 22. J. W. J. Hinrichs, K. Klappe, I. Hummel, and J. W. Kok. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279:5734–5738 (2004).

    Google Scholar 

  23. 23. M.-A. Ghetie, R. Marches, S. Kufert, and E. S. Vitetta. An anti-CD19 antibody inhibits the interaction between P-glycoprotein (P-gp) and CD19, causes P-gp to translocate out of lipid rafts, and chemosensitizes a multidrug-resistant (MDR) lymphoma cell line. Blood 104:178–183 (2004).

    Google Scholar 

  24. 24. L. J. Pike. Lipid rafts: bringing order to chaos. J. Lipid Res. 44:655–667 (2003).

    Google Scholar 

  25. 25. D. A. Brown and J. K. Rose. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544 (1992).

    Article  CAS  PubMed  Google Scholar 

  26. 26. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275 (1951).

    CAS  PubMed  Google Scholar 

  27. 27. P. Gonzalo, B. Sontag, D. Guillot, and J. P. Reboud. Fluorometric assay of GTPase activity: application to the couple elongation factor eEF-2-ribosome. Anal. Biochem. 225:178–180 (1995).

    Google Scholar 

  28. 28. A. Cornish-Bowden. Fundamentals of Enzyme Kinetics, Portland Press Ltd, London, 1995.

    Google Scholar 

  29. 29. A. M. Brown. A step-by-step guide to non-linear regression analysis of experimental data using a Microsoft Excel spreadsheet. Comp. Meth. Prog. Biomed. 65:191–200 (2001).

    Google Scholar 

  30. 30. F. J. Sharom, R. Liu, Y. Romsicki, and P. Lu. Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies. Biochim. Biophys. Acta 1461:327–345 (1999).

    Google Scholar 

  31. 31. I. R. Gibbons, M. P. Cosson, J. A. Evans, B. H. Gibbons, B. Houck, K. H. Martinson, W. S. Sale, and W. J. Y. Tang. Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate. Proc. Natl. Acad. Sci. USA 75:2220–2224 (1978).

    Google Scholar 

  32. 32. E. Sabbioni, G. Pozzi, A. Pintar, L. Casella, and S. Garattini. Cellular retention, cytotoxicity and morphological transformation by vanadium(IV) and vanadium(V) in BALB/3T3 cell lines. Carcinogenesis 12:47–52 (1991).

    Google Scholar 

  33. 33. J. Robert and C. Jarry. Multidrug resistance reversal agents. J. Med. Chem. 46:4805–4817 (2003).

    Google Scholar 

  34. 34. E. Bakos, R. Evers, E. Sinko, A. Varadi, P. Borst, and B. Sarkadi. Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol. Pharmacol. 57:760–768 (2000).

    Google Scholar 

  35. 35. A. Rosati, L. Candussio, E. Crivellato, F. Klugmann, T. Giraldi, D. Damiani, A. Michelutti, and G. Decorti. Bodipy-FL-verapamil: a fluorescent probe for the study of multidrug resistance proteins. Cell. Oncol. 26:3–11 (2004).

    Google Scholar 

  36. 36. R. Liu and F. J. Sharom. Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. Biochemistry 35:11865–11873 (1996).

    Google Scholar 

  37. 37. T. Litman, T. Zeuthen, T. Skovsgaard, and W. D. Stein. Competitive, non-competitive and cooperative interactions between substrates of P-glycoprotein as measured by its ATPase activity. Biochim. Biophys. Acta 1361:169–176 (1997).

    Google Scholar 

  38. 38. E. Howard and P. Roepe. Purified human MDR 1 modulates membrane potential in reconstituted proteoliposomes. Biochemistry 42:3544–3555 (2003).

    Google Scholar 

  39. 39. K. M. Kerr, Z. E. Sauna, and S. V. Ambudkar. Correlation between steady-state ATP hydrolysis and vanadate-induced ADP trapping in human P-glycoprotein. J. Biol. Chem. 276:8657–8664 (2001).

    Google Scholar 

  40. 40. E. Georges, J. T. Zhang, and V. Ling. Modulation of ATP and drug binding by monoclonal antibodies against P-glycoprotein. J. Cell. Physiol. 148:479–484 (1991).

    Google Scholar 

  41. 41. K. Malinska, J. Malinsky, M. Opekarova, and W. Tanner. Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol. Biol. Cell 14:4427–4436 (2003).

    Google Scholar 

  42. 42. Q. Qu and F. J. Sharom. FRET analysis indicates that the two ATPase active sites of the P-glycoprotein multidrug transporter are closely associated. Biochemistry 40:1413–1422 (2001).

    Google Scholar 

  43. 43. M. Younes-Ibrahim, M. Barnese, P. Burth, and M. V. Castro-Faria. Inhibition of purified human kidney Na+,K+-ATPase by cyclosporine A: a possible mechanism for drug human nephrotoxicity. Ann. N. Y. Acad. Sci. 986:633–635 (2003).

    Google Scholar 

  44. 44. V. Calderaro, M. Boccellino, G. Cirillo, L. Quagliuolo, D. Cirillo, and A. Giovane. Cyclosporine A amplifies Ca2+ signaling pathway in LLC-PK1 cells through the inhibition of plasma membrane Ca2+ pump. J. Am. Soc. Nephrol. 14:1435–1442 (2003).

    Google Scholar 

  45. 45. I. G. Bilmen, L. L. Wootton, and F. Michelangeli. The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A. Biochem. J. 366:255–263 (2002).

    Google Scholar 

  46. 46. Y. Romsicki and F. J. Sharom. The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter. Biochemistry 38:6887–6896 (1999).

    Google Scholar 

  47. 47. A. Garrigues, A. E. Escargueil, and S. Orlowski. The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc. Natl. Acad. Sci. USA 99:10347–10352 (2002).

    Google Scholar 

  48. 48. W. A. Ritschel and G. L. Kearns. Handbook of Basic Pharmacokinetics, American Pharmaceutical Association, Washington, DC, 1999.

    Google Scholar 

  49. 49. S. Munro. Lipid rafts: elusive or illusive? Cell 115:377–388 (2003).

    Google Scholar 

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Authors and Affiliations

  1. Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH, Federal Institute of Technology, Zürich, Switzerland

    Karsten Bucher, Camille A. Besse, Sarah W. Kamau, Heidi Wunderli-Allenspach & Stefanie D. Krämer

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  1. Karsten Bucher
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  2. Camille A. Besse
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Correspondence to Stefanie D. Krämer.

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Bucher, K., Besse, C., Kamau, S. et al. Isolated Rafts from Adriamycin-Resistant P388 Cells Contain Functional ATPases and Provide an Easy Test System for P-glycoprotein–Related Activities. Pharm Res 22, 449–457 (2005). https://doi.org/10.1007/s11095-004-1883-x

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