Related Experiment Videos
Ceramide-induced apoptosis is mediated by caspase activation independently from retinoblastoma protein
A Spinedi1, A Amendola, S Di Bartolomeo
1Department of Biology, University of Rome Tor Vergata, Italy. spinedi@seneca.ccd.utovrm.it
Abstract:
Recent evidence suggests that untimely retinoblastoma protein (RB) dephosphorylation and/or proteolytic degradation might provide key events down-stream cysteine protease (caspase) activation in apoptosis induction. We have dealt with this issue by studying apoptosis induced by N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells, maintained in complete growth medium. We report that C6-Cer-induced apoptosis occurred predominantly in G1/S phases of the cycle and was associated with RB dephosphorylation, in the setting of negligible Bcl-2 expression. Apoptosis was also associated with poly(ADP-ribose) polymerase (PARP) cleavage, thus indicating activation of CPP32/Yama/apopain (caspase-3); however, while the tripeptide caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone was able to prevent both C6-Cer-induced PARP cleavage and apoptosis, it was ineffective in preventing RB dephosphorylation. Moreover proteolytic RB cleavage occurred only to a marginal extent after C6-Cer treatment. These results indicate that apoptosis induced by ceramide in CHP-100 cells is caspase-mediated, but RB post-translational modification does not provide a key step, downstream caspase activation, in apoptosis execution.
Insights
Ceramide-induced apoptosis in neuroepithelioma cells is caspase-mediated. However, retinoblastoma protein (RB) dephosphorylation is not a key step downstream of caspase activation during apoptosis execution.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Retinoblastoma protein (RB) dephosphorylation and degradation are implicated in apoptosis.
- Caspase activation is a critical event in programmed cell death.
Purpose of the Study:
- To investigate the role of RB post-translational modifications in ceramide-induced apoptosis.
- To determine if RB dephosphorylation is a key downstream event of caspase activation.
Main Methods:
- Induction of apoptosis using N-hexanoylsphingosine (C6-Cer) in CHP-100 human neuroepithelioma cells.
- Cell cycle analysis and assessment of RB dephosphorylation.
- Evaluation of poly(ADP-ribose) polymerase (PARP) cleavage as an indicator of caspase-3 activation.
- Inhibition of caspases using Z-Val-Ala-DL-Asp-fluoromethylketone.
Main Results:
- C6-Cer induced apoptosis predominantly in G1/S phases, associated with RB dephosphorylation and PARP cleavage.
- Caspase inhibition prevented apoptosis and PARP cleavage but not RB dephosphorylation.
- Proteolytic RB cleavage was minimal following C6-Cer treatment.
Conclusions:
- Ceramide-induced apoptosis in CHP-100 cells is mediated by caspases.
- RB post-translational modification, specifically dephosphorylation, is not a critical downstream event in caspase-mediated apoptosis execution.