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Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death
M D Jacobsen1, M Weil, M C Raff
1MRC Laboratory for Molecular Cell Biology, University College London, United Kingdom.
Abstract:
In the accompanying paper by Weil et al. (1996) we show that staurosporine (STS), in the presence of cycloheximide (CHX) to inhibit protein synthesis, induces apoptotic cell death in a large variety of nucleated mammalian cell types, suggesting that all nucleated mammalian cells constitutively express all of the proteins required to undergo programmed cell death (PCD). The reliability of that conclusion depends on the evidence that STS-induced, and (STS + CHS)-induced, cell deaths are bona fide examples of PCD. There is rapidly accumulating evidence that some members of the Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are part of the basic machinery of PCD. Here we show that Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cell-permeable, irreversible, tripeptide inhibitor of some of these proteases, suppresses STS-induced and (STS + CHX)-induced cell death in a wide variety of mammalian cell types, including anucleate cytoplasts, providing strong evidence that these are all bona fide examples of PCD. We show that the Ced-3/ICE family member CPP32 becomes activated in STS-induced PCD, and that Bcl-2 inhibits this activation. Most important, we show that, in some cells at least, one or more CPP32-family members, but not ICE itself, is required for STS-induced PCD. Finally, we show that zVAD-fmk suppresses PCD in the interdigital webs in developing mouse paws and blocks the removal of web tissue during digit development, suggesting that this inhibition will be a useful tool for investigating the roles of PCD in various developmental processes.
Insights
Staurosporine (STS) induces programmed cell death (PCD) in mammalian cells. A protease inhibitor, zVAD-fmk, blocks STS-induced PCD, confirming it as a genuine cell death pathway and implicating CPP32-family proteases in the process.
Area of Science:
- Cell Biology
- Developmental Biology
- Biochemistry
Background:
- Staurosporine (STS) induces apoptotic cell death in mammalian cells, suggesting constitutive expression of programmed cell death (PCD) machinery.
- The Ced-3/Interleukin-1 beta converting enzyme (ICE) family of cysteine proteases are implicated in PCD.
- Reliable evidence is needed to confirm STS-induced cell death as bona fide PCD.
Purpose of the Study:
- To confirm that STS-induced cell death is a genuine example of PCD.
- To investigate the role of Ced-3/ICE family proteases in STS-induced PCD.
- To explore the utility of protease inhibitors in studying PCD during development.
Main Methods:
- Treatment of mammalian cells and anucleate cytoplasts with staurosporine (STS) and cycloheximide (CHX).
- Inhibition of STS-induced cell death using Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a cysteine protease inhibitor.
- Analysis of CPP32 activation and its inhibition by Bcl-2.
- Assessment of zVAD-fmk effects on PCD in developing mouse interdigital webs.
Main Results:
- zVAD-fmk suppressed STS-induced and (STS + CHX)-induced cell death in various mammalian cell types, including anucleate cytoplasts, validating them as PCD.
- CPP32, a Ced-3/ICE family member, was activated during STS-induced PCD, and Bcl-2 inhibited this activation.
- CPP32-family members, but not ICE, were required for STS-induced PCD in some cells.
- zVAD-fmk inhibited PCD in developing mouse paws, preventing web tissue removal during digit development.
Conclusions:
- STS-induced cell death in mammalian cells is a bona fide example of PCD.
- CPP32-family proteases play a crucial role in STS-induced PCD.
- Protease inhibitors like zVAD-fmk are valuable tools for studying PCD in developmental processes.