Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Jul 1;365(Pt 1):259–268. doi: 10.1042/BJ20011880

Transport of phosphatidylserine via MDR1 (multidrug resistance 1)P-glycoprotein in a human gastric carcinoma cell line.

Antje Pohl 1, Hermann Lage 1, Peter Müller 1, Thomas Pomorski 1, Andreas Herrmann 1
PMCID: PMC1222671  PMID: 12071854

Abstract

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (264.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abulrob A. G., Gumbleton M. Transport of phosphatidylcholine in MDR3-negative epithelial cell lines via drug-induced MDR1 P-glycoprotein. Biochem Biophys Res Commun. 1999 Aug 19;262(1):121–126. doi: 10.1006/bbrc.1999.1120. [DOI] [PubMed] [Google Scholar]
  2. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  3. Bosch I., Dunussi-Joannopoulos K., Wu R. L., Furlong S. T., Croop J. Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. Biochemistry. 1997 May 13;36(19):5685–5694. doi: 10.1021/bi962728r. [DOI] [PubMed] [Google Scholar]
  4. Brüning A., Karrenbauer A., Schnabel E., Wieland F. T. Brefeldin A-induced increase of sphingomyelin synthesis. Assay for the action of the antibiotic in mammalian cells. J Biol Chem. 1992 Mar 15;267(8):5052–5055. [PubMed] [Google Scholar]
  5. Cardarelli C. O., Aksentijevich I., Pastan I., Gottesman M. M. Differential effects of P-glycoprotein inhibitors on NIH3T3 cells transfected with wild-type (G185) or mutant (V185) multidrug transporters. Cancer Res. 1995 Mar 1;55(5):1086–1091. [PubMed] [Google Scholar]
  6. Colleau M., Hervé P., Fellmann P., Devaux P. F. Transmembrane diffusion of fluorescent phospholipids in human erythrocytes. Chem Phys Lipids. 1991 Jan-Feb;57(1):29–37. doi: 10.1016/0009-3084(91)90046-e. [DOI] [PubMed] [Google Scholar]
  7. Cordon-Cardo C., O'Brien J. P., Boccia J., Casals D., Bertino J. R., Melamed M. R. Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem. 1990 Sep;38(9):1277–1287. doi: 10.1177/38.9.1974900. [DOI] [PubMed] [Google Scholar]
  8. Dachary-Prigent J., Freyssinet J. M., Pasquet J. M., Carron J. C., Nurden A. T. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood. 1993 May 15;81(10):2554–2565. [PubMed] [Google Scholar]
  9. Daleke D. L., Lyles J. V. Identification and purification of aminophospholipid flippases. Biochim Biophys Acta. 2000 Jun 26;1486(1):108–127. doi: 10.1016/s1388-1981(00)00052-4. [DOI] [PubMed] [Google Scholar]
  10. Dekkers D. W., Comfurius P., Schroit A. J., Bevers E. M., Zwaal R. F. Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1). Biochemistry. 1998 Oct 20;37(42):14833–14837. doi: 10.1021/bi981011w. [DOI] [PubMed] [Google Scholar]
  11. Devaux P. F. Is lipid translocation involved during endo- and exocytosis? Biochimie. 2000 May;82(5):497–509. doi: 10.1016/s0300-9084(00)00209-1. [DOI] [PubMed] [Google Scholar]
  12. Dietel M., Arps H., Lage H., Niendorf A. Membrane vesicle formation due to acquired mitoxantrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res. 1990 Sep 15;50(18):6100–6106. [PubMed] [Google Scholar]
  13. Ernest S., Bello-Reuss E. Secretion of platelet-activating factor is mediated by MDR1 P-glycoprotein in cultured human mesangial cells. J Am Soc Nephrol. 1999 Nov;10(11):2306–2313. doi: 10.1681/ASN.V10112306. [DOI] [PubMed] [Google Scholar]
  14. Hamon Y., Broccardo C., Chambenoit O., Luciani M. F., Toti F., Chaslin S., Freyssinet J. M., Devaux P. F., McNeish J., Marguet D. ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat Cell Biol. 2000 Jul;2(7):399–406. doi: 10.1038/35017029. [DOI] [PubMed] [Google Scholar]
  15. Hanson P. K., Nichols J. W. Energy-dependent flip of fluorescence-labeled phospholipids is regulated by nutrient starvation and transcription factors, PDR1 and PDR3. J Biol Chem. 2001 Jan 2;276(13):9861–9867. doi: 10.1074/jbc.M009065200. [DOI] [PubMed] [Google Scholar]
  16. Higgins C. F., Gottesman M. M. Is the multidrug transporter a flippase? Trends Biochem Sci. 1992 Jan;17(1):18–21. doi: 10.1016/0968-0004(92)90419-a. [DOI] [PubMed] [Google Scholar]
  17. Hu X. F., Slater A., Wall D. M., Kantharidis P., Parkin J. D., Cowman A., Zalcberg J. R. Rapid up-regulation of mdr1 expression by anthracyclines in a classical multidrug-resistant cell line. Br J Cancer. 1995 May;71(5):931–936. doi: 10.1038/bjc.1995.180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones T. R., Zamboni R., Belley M., Champion E., Charette L., Ford-Hutchinson A. W., Frenette R., Gauthier J. Y., Leger S., Masson P. Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist. Can J Physiol Pharmacol. 1989 Jan;67(1):17–28. doi: 10.1139/y89-004. [DOI] [PubMed] [Google Scholar]
  19. Kamp D., Haest C. W. Evidence for a role of the multidrug resistance protein (MRP) in the outward translocation of NBD-phospholipids in the erythrocyte membrane. Biochim Biophys Acta. 1998 Jun 24;1372(1):91–101. doi: 10.1016/s0005-2736(98)00049-2. [DOI] [PubMed] [Google Scholar]
  20. Kawamura T., Kusakabe T., Sugino T., Watanabe K., Fukuda T., Nashimoto A., Honma K., Suzuki T. Expression of glucose transporter-1 in human gastric carcinoma: association with tumor aggressiveness, metastasis, and patient survival. Cancer. 2001 Aug 1;92(3):634–641. doi: 10.1002/1097-0142(20010801)92:3<634::aid-cncr1364>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
  21. Kok J. W., Hoekstra K., Eskelinen S., Hoekstra D. Recycling pathways of glucosylceramide in BHK cells: distinct involvement of early and late endosomes. J Cell Sci. 1992 Dec;103(Pt 4):1139–1152. doi: 10.1242/jcs.103.4.1139. [DOI] [PubMed] [Google Scholar]
  22. Lage H., Jordan A., Scholz R., Dietel M. Thermosensitivity of multidrug-resistant human gastric and pancreatic carcinoma cells. Int J Hyperthermia. 2000 Jul-Aug;16(4):291–303. doi: 10.1080/02656730050074069. [DOI] [PubMed] [Google Scholar]
  23. Lage H., Perlitz C., Abele R., Tampé R., Dietel M., Schadendorf D., Sinha P. Enhanced expression of human ABC-transporter tap is associated with cellular resistance to mitoxantrone. FEBS Lett. 2001 Aug 17;503(2-3):179–184. doi: 10.1016/s0014-5793(01)02722-3. [DOI] [PubMed] [Google Scholar]
  24. Lehne G., Mørkrid L., den Boer M., Rugstad H. E. Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP). Int J Clin Pharmacol Ther. 2000 Apr;38(4):187–195. doi: 10.5414/cpp38187. [DOI] [PubMed] [Google Scholar]
  25. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell. 1990 Mar 9;60(5):821–836. doi: 10.1016/0092-8674(90)90096-w. [DOI] [PubMed] [Google Scholar]
  26. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  27. Marguet D., Luciani M. F., Moynault A., Williamson P., Chimini G. Engulfment of apoptotic cells involves the redistribution of membrane phosphatidylserine on phagocyte and prey. Nat Cell Biol. 1999 Nov;1(7):454–456. doi: 10.1038/15690. [DOI] [PubMed] [Google Scholar]
  28. Marx U., Lassmann G., Holzhütter H. G., Wüstner D., Müller P., Höhlig A., Kubelt J., Herrmann A. Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach. Biophys J. 2000 May;78(5):2628–2640. doi: 10.1016/S0006-3495(00)76807-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pagano R. E., Martin O. C., Schroit A. J., Struck D. K. Formation of asymmetric phospholipid membranes via spontaneous transfer of fluorescent lipid analogues between vesicle populations. Biochemistry. 1981 Aug 18;20(17):4920–4927. doi: 10.1021/bi00520a018. [DOI] [PubMed] [Google Scholar]
  30. Pomorski T., Muller P., Zimmermann B., Burger K., Devaux P. F., Herrmann A. Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach. J Cell Sci. 1996 Mar;109(Pt 3):687–698. doi: 10.1242/jcs.109.3.687. [DOI] [PubMed] [Google Scholar]
  31. Poot M., Zhang Y. Z., Krämer J. A., Wells K. S., Jones L. J., Hanzel D. K., Lugade A. G., Singer V. L., Haugland R. P. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J Histochem Cytochem. 1996 Dec;44(12):1363–1372. doi: 10.1177/44.12.8985128. [DOI] [PubMed] [Google Scholar]
  32. Raggers R. J., Pomorski T., Holthuis J. C., Kälin N., van Meer G. Lipid traffic: the ABC of transbilayer movement. Traffic. 2000 Mar;1(3):226–234. doi: 10.1034/j.1600-0854.2000.010305.x. [DOI] [PubMed] [Google Scholar]
  33. Raggers R. J., van Helvoort A., Evers R., van Meer G. The human multidrug resistance protein MRP1 translocates sphingolipid analogs across the plasma membrane. J Cell Sci. 1999 Feb;112(Pt 3):415–422. doi: 10.1242/jcs.112.3.415. [DOI] [PubMed] [Google Scholar]
  34. Roelofsen H., Vos T. A., Schippers I. J., Kuipers F., Koning H., Moshage H., Jansen P. L., Müller M. Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells. Gastroenterology. 1997 Feb;112(2):511–521. doi: 10.1053/gast.1997.v112.pm9024305. [DOI] [PubMed] [Google Scholar]
  35. Romsicki Y., Sharom F. J. Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter. Biochemistry. 2001 Jun 12;40(23):6937–6947. doi: 10.1021/bi0024456. [DOI] [PubMed] [Google Scholar]
  36. Rothnie A., Theron D., Soceneantu L., Martin C., Traikia M., Berridge G., Higgins C. F., Devaux P. F., Callaghan R. The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence. Eur Biophys J. 2001 Oct;30(6):430–442. doi: 10.1007/s002490100156. [DOI] [PubMed] [Google Scholar]
  37. Ruetz S., Gros P. Phosphatidylcholine translocase: a physiological role for the mdr2 gene. Cell. 1994 Jul 1;77(7):1071–1081. doi: 10.1016/0092-8674(94)90446-4. [DOI] [PubMed] [Google Scholar]
  38. Seigneuret M., Devaux P. F. ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3751–3755. doi: 10.1073/pnas.81.12.3751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smit J. J., Schinkel A. H., Oude Elferink R. P., Groen A. K., Wagenaar E., van Deemter L., Mol C. A., Ottenhoff R., van der Lugt N. M., van Roon M. A. Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease. Cell. 1993 Nov 5;75(3):451–462. doi: 10.1016/0092-8674(93)90380-9. [DOI] [PubMed] [Google Scholar]
  40. Smith A. J., Timmermans-Hereijgers J. L., Roelofsen B., Wirtz K. W., van Blitterswijk W. J., Smit J. J., Schinkel A. H., Borst P. The human MDR3 P-glycoprotein promotes translocation of phosphatidylcholine through the plasma membrane of fibroblasts from transgenic mice. FEBS Lett. 1994 Nov 14;354(3):263–266. doi: 10.1016/0014-5793(94)01135-4. [DOI] [PubMed] [Google Scholar]
  41. Stuart M. C., Bevers E. M., Comfurius P., Zwaal R. F., Reutelingsperger C. P., Frederik P. M. Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets. Thromb Haemost. 1995 Oct;74(4):1145–1151. [PubMed] [Google Scholar]
  42. Utsugi T., Schroit A. J., Connor J., Bucana C. D., Fidler I. J. Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 1991 Jun 1;51(11):3062–3066. [PubMed] [Google Scholar]
  43. Wüstner D., Pomorski T., Herrmann A., Müller P. Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone. Biochemistry. 1998 Dec 1;37(48):17093–17103. doi: 10.1021/bi981608b. [DOI] [PubMed] [Google Scholar]
  44. Zachowski A., Favre E., Cribier S., Hervé P., Devaux P. F. Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme. Biochemistry. 1986 May 6;25(9):2585–2590. doi: 10.1021/bi00357a046. [DOI] [PubMed] [Google Scholar]
  45. Zachowski A. Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem J. 1993 Aug 15;294(Pt 1):1–14. doi: 10.1042/bj2940001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zwaal R. F., Schroit A. J. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood. 1997 Feb 15;89(4):1121–1132. [PubMed] [Google Scholar]
  47. van Genderen I., van Meer G. Differential targeting of glucosylceramide and galactosylceramide analogues after synthesis but not during transcytosis in Madin-Darby canine kidney cells. J Cell Biol. 1995 Nov;131(3):645–654. doi: 10.1083/jcb.131.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. van Helvoort A., Giudici M. L., Thielemans M., van Meer G. Transport of sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-NBD-sphingomyelin is translocated across the plasma membrane by a multidrug transporter activity. J Cell Sci. 1997 Jan;110(Pt 1):75–83. doi: 10.1242/jcs.110.1.75. [DOI] [PubMed] [Google Scholar]
  49. van Helvoort A., Smith A. J., Sprong H., Fritzsche I., Schinkel A. H., Borst P., van Meer G. MDR1 P-glycoprotein is a lipid translocase of broad specificity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell. 1996 Nov 1;87(3):507–517. doi: 10.1016/s0092-8674(00)81370-7. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES