Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to gamma-radiation but activation of this sequence is defective in radioresistant SQ20B cells.
OBJECTIVE
To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to 7-rays.


METHODS
Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (delta(psi)m), reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry.


RESULTS
In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in delta(psi)m and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, gamma-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway.


CONCLUSIONS
Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.
Alphonse
G
G
INSERM U189, Department of Biochemistry, Lyon-Sud Medical School, Oullins, France. galph@lyon-sud.univ-lyon1.fr
Aloy
M T
MT
Broquet
P
P
Gerard
J P
JP
Louisot
P
P
Rousson
R
R
Rodriguez-Lafrasse
C
C
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D001865:Bongkrekic Acid / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D005720:Gamma Rays; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008564:Membrane Potentials / Q000528:radiation effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects*; D009025:Morpholines / Q000494:pharmacology; D011836:Radiation Tolerance; D017382:Reactive Oxygen Species / Q000378:metabolism
D061945:Caspase Inhibitors; D002518:Ceramides; D009025:Morpholines; D017382:Reactive Oxygen Species; D001865:Bongkrekic Acid; C033110:RV 538; D020169:Caspases; D005978:Glutathione