7-Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7-ketocholesterol-induced apoptosis: analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation.
BACKGROUND
Oxidized low-density lipoproteins play key roles in atherosclerosis. Their toxicity is at least in part due to 7-ketocholesterol (7KC), which is a potent inducer of apoptosis. In this study on human promonocytic U937 cells, we determined the effects and the interactions of 7KC with cellular lipids during 7KC-induced apoptosis.


METHODS
Morphologic and functional changes were investigated by microscopic and flow cytometric methods after staining with propidium iodide, 3,3'-dihexyloxacarbocyanine iodide, and Hoechst 33342. Cellular lipid content was identified by using filipin to quantify free cholesterol and Nile Red (NR), which emit a yellow or orange-red fluorescence in the presence of neutral and polar lipids, respectively. After staining with NR, interactions of 7KC with cellular lipids were identified by fluorescence resonance energy transfer biphoton spectral imaging confocal microscopy and by subcellular fractionation, gas chromatography, and mass spectrometry.


RESULTS
During 7KC-induced apoptosis the fluorescence from filipin and the ratio of measured (orange-red vs. yellow) fluorescence of NR were enhanced. Spectral analysis of images obtained in biphoton mode and resulting factor images demonstrated the occurrence of fluorescence resonance energy transfer between 7KC and NR and the subsequent colocalization of 7KC and NR. These data were in agreement with biochemical characterization and demonstrated that 7KC and neutral and polar lipids accumulate in NR-stained cytoplasmic structures.


CONCLUSIONS
During 7KC-induced apoptosis, 7KC modifies the cellular content of neutral and polar lipids, favors free cholesterol accumulation, and colocalizes with neutral and polar lipids that are inside NR-stained cytoplasmic structures.
Vejux
Anne
A
INSERM U498, Hôpital du Bocage, Dijon, France.
Kahn
Edmond
E
Dumas
Dominique
D
Bessède
Ginette
G
Ménétrier
Franck
F
Athias
Anne
A
Riedinger
Jean-Marc
JM
Frouin
Frédérique
F
Stoltz
Jean-François
JF
Ogier-Denis
Eric
E
Todd-Pokropek
Andrew
A
Lizard
Gérard
G
D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000187:drug effects; D002784:Cholesterol / Q000032:analysis / Q000378:metabolism; D022083:Cytoplasmic Structures / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D007653:Ketocholesterols / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D050356:Lipid Metabolism; D008564:Membrane Potentials / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D010713:Phosphatidylcholines / Q000032:analysis; D011419:Propidium / Q000737:chemistry / Q000378:metabolism; D013109:Sphingomyelins / Q000032:analysis; D013347:Subcellular Fractions / Q000737:chemistry; D020298:U937 Cells
D001562:Benzimidazoles; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007653:Ketocholesterols; D010078:Oxazines; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D011419:Propidium; D002784:Cholesterol; C003001:7-ketocholesterol; C044808:nile red; C017807:bisbenzimide ethoxide trihydrochloride