Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts

Cancer Cell International

Laurence Le Moyec¹  Geneviève Leray²  Jacques D de Certaines²  Paiboon Reungpatthanaphong¹  Aurélie Mannechez² 

[1] Laboratoire LPBC-CSSB, UMR CNRS 7033, Université Paris 13, 74 avenue, Marcel Cachin, 93017 Bobigny Cedex. France;Laboratoire de Résonance Magnétique en Biologie et Médecine, Université Rennes 1, avenue du Professeur Léon Bernard, 35043 RENNES Cedex. France

关键词: Rafts;    Mobile lipids;    Multidrug resistance;    Proton Magnetic Resonance Spectroscopy;   

Others  :  796040
DOI  :  10.1186/1475-2867-5-2

 received in 2004-01-08, accepted in 2005-02-09,  发布年份 2005

【 摘 要 】

Background

Most cancer cells are characterized by mobile lipids visible on proton NMR (¹H-NMR), these being comprised mainly of methyl and methylene signals from lipid acyl chains. Erythroleukemia K562 cells show narrow signals at 1.3 and 0.9 ppm, corresponding to mobile lipids (methylene and methyl, respectively), which are reduced when K562 cells are multidrug resistant (MDR). While the significance of the mobile lipids is unknown, their subcellular localization is still a matter of debate and may lie in the membrane or the cytoplasm. In this study, we investigate the role of cholesterol in the generation of mobile lipid signals.

Results

The proportion of esterified cholesterol was found to be higher in K562-sensitive cells than in resistant cells, while the total cholesterol content was identical in both cell lines. Cholesterol extraction in the K562 wild type (K562wt) cell line and its MDR counterpart (K562adr), using methyl-β-cyclodextrin, was accompanied by a rise of mobile lipids in K562wt cells only. The absence of caveolae was checked by searching for the caveolin-1 protein in K562wt and K562adr cells. However, cholesterol was enriched in another membrane microdomain designated as "detergent-insoluble glycosphingomyelin complexes" or rafts. These microdomains were studied after extraction with triton X-100, a mild non-ionic detergent, revealing mobile lipid signals preserved only in the K562wt spectra. Moreover, following perturbation/disruption of these microdomains using sphingomyelinase, mobile lipids increased only in K562wt cells.

Conclusion

These results suggest that cholesterol and sphingomyelin are involved in mobile lipid generation via microdomains of detergent-insoluble glycosphingomyelin complexes such as rafts. Increasing our knowledge of membrane microdomains in sensitive and resistant cell lines may open up new possibilities in resistance reversion.

【 授权许可】

   
2005 Mannechez et al; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Hakumaki JM, Kauppinen RA: 1H NMR visible lipids in the life and death of cells. Trends Biochem Sci 2000, 25:357-62.
• [2]Mountford CE, Wright LC: Organization of lipids in the plasma membranes of malignant and stimulated cells: a new model. Trends Biochem Sci 1988, 13:172-7.
• [3]Iorio E, Di Vito M, Spadaro F, Ramoni C, Lococo E, Carnevale R, Lenti L, Strom R, Podo F: Triacsin C inhibits the formation of 1H NMR-visible mobile lipids and lipid bodies in HuT 78 apoptotic cells. Biochim Biophys Acta 2003, 1634:1-14.
• [4]Barba I, Cabanas ME, Arus C: The relationship between nuclear magnetic resonance-visible lipids, lipid droplets, and cell proliferation in cultured C6 cells. Cancer Res 1999, 59:1861-8.
• [5]Zoula S, Herigault G, Ziegler A, Farion R, Decorps M, Remy C: Correlation between the occurrence of 1H-MRS lipid signal, necrosis and lipid droplets during C6 rat glioma development. NMR Biomed 2003, 16:199-212.
• [6]Le Moyec L, Millot G, Tatoud R, Calvo F, Eugene M: Lipid signals detected by NMR proton spectroscopy of whole cells are not correlated to lipid droplets evidenced by the Nile red staining. Cell Mol Biol (Noisy-le-grand) 1997, 43:703-9.
• [7]Le Moyec L, Tatoud R, Degeorges A, Calabresse C, Bauza G, Eugene M, Calvo F: Proton nuclear magnetic resonance spectroscopy reveals cellular lipids involved in resistance to adriamycin and taxol by the K562 leukemia cell line. Cancer Res 1996, 56:3461-7.
• [8]Le Moyec L, Legrand O, Larue V, Kawakami M, Marie JP, Calvo F, Hantz E, Taillandier E: Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion. NMR Biomed 2000, 13:92-101.
• [9]Mannechez A, Collet B, Payen L, Lecureur V, Fardel O, Le Moyec L, de Certaines JD, Leray G: Differentiation of the P-gp and MRP1 multidrug resistance systems by mobile lipid 1H-NMR spectroscopy and phosphatidylserine externalization. Anticancer Res 2001, 21:3915-9.
• [10]Finegold L, Singer MA: Cholesterol-multilipid interactions in bilayers. Chem Phys Lipids 1991, 58:169-73.
• [11]Lange Y, Strebel F, Steck TL: Role of the plasma membrane in cholesterol esterification in rat hepatoma cells. J Biol Chem 1993, 268:13838-43.
• [12]Rooney MW, Lange Y, Kauffman JW: Acyl chain organization and protein secondary structure in cholesterol-modified erythrocyte membranes. J Biol Chem 1984, 259:8281-5.
• [13]Rintoul DA, Chou SM, Silbert DF: Physical characterization of sterol-depleted LM-cell plasma membranes. J Biol Chem 1979, 254:10070-7.
• [14]Sinensky M: Defective regulation of cholesterol biosynthesis and plasma membrane fluidity in a Chinese hamster ovary cell mutant. Proc Natl Acad Sci U S A 1978, 75:1247-9.
• [15]Sankaram MB, Thompson TE: Modulation of phospholipid acyl chain order by cholesterol. A solid-state 2H nuclear magnetic resonance study. Biochemistry 1990, 29:10676-84.
• [16]Kilsdonk EP, Yancey PG, Stoudt GW, Bangerter FW, Johnson WJ, Phillips MC, Rothblat GH: Cellular cholesterol efflux mediated by cyclodextrins. J Biol Chem 1995, 270:17250-6.
• [17]Ilangumaran S, Hoessli DC: Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem J 1998, 335:433-40.
• [18]Brown DA, London E: Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 2000, 275:17221-4.
• [19]Kurzchalia TV, Dupree P, Parton RG, Kellner R, Virta H, Lehnert M, Simons K: VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J Cell Biol 1992, 118:1003-14.
• [20]Hatanaka M, Maeda T, Ikemoto T, Mori H, Seya T, Shimizu A: Expression of caveolin-1 in human T cell leukemia cell lines. Biochem Biophys Res Commun 1998, 253:382-7.
• [21]Adam G, Alpes H, Blaser K, Neubert B: Cholesterol and phospholipid content of 3T3 cells and transformed derivatives. Z Naturforsch [C] 1975, 30:638-42.
• [22]Mackinnon WB, May GL, Mountford CE: Esterified cholesterol and triglyceride are present in plasma membranes of Chinese hamster ovary cells. Eur J Biochem 1992, 205:827-39.
• [23]Romsicki Y, Sharom FJ: The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter. Biochemistry 1999, 38:6887-96.
• [24]Grosse PY, Bressolle F, Pinguet F: Methyl-beta-cyclodextrin in HL-60 parental and multidrug-resistant cancer cell lines: effect on the cytotoxic activity and intracellular accumulation of doxorubicin. Cancer Chemother Pharmacol 1997, 40:489-94.
• [25]Parolini I, Sargiacomo M, Lisanti MP, Peschle C: Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G proteins, and CD55) selectively localize in Triton-insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. Blood 1996, 87:3783-94.
• [26]London E, Brown DA: Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts). Biochim Biophys Acta 2000, 1508:182-95.
• [27]Melkonian KA, Chu T, Tortorella LB, Brown DA: Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry 1995, 34:16161-70.
• [28]Lavie Y, Fiucci G, Liscovitch M: Upregulation of caveolin in multidrug resistant cancer cells: functional implications. Adv Drug Deliv Rev 2001, 49:317-23.
• [29]Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959, 37:911-7.