Proton NMR visible mobile lipid signals in sensitive and multidrug-resistant K562 cells are modulated by rafts.
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-beta-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.
Mannechez
Aurélie
A
Laboratoire LPBC-CSSB, UMR CNRS 7033, Université Paris 13, 74 avenue, Marcel Cachin, 93017 Bobigny Cedex, France. lemoyec@smbh.univ-paris13.fr.
Reungpatthanaphong
Paiboon
P
de Certaines
Jacques D
JD
Leray
Geneviève
G
Le Moyec
Laurence
L