Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis.
To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.
Rodriguez-Lafrasse
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INSERM U189, Department of Biochemistry, Lyon-Sud Medical School, BP12, 69921 Oullins Cedex, France. rodriguez@lyon-sud.univ-lyon1.fr
Alphonse
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Broquet
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P
Aloy
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MT
Louisot
P
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Rousson
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R
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002294:Carcinoma, Squamous Cell; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002448:Cell Adhesion; D002518:Ceramides / Q000096:biosynthesis*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D006258:Head and Neck Neoplasms; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D019169:Jurkat Cells; D007700:Kinetics; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D061945:Caspase Inhibitors; D002518:Ceramides; D015853:Cysteine Proteinase Inhibitors; D019014:fas Receptor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D005978:Glutathione