Abstract
Caspase activation and proteolytic cleavage of specific target proteins represents an integral step in the pathway leading to the apoptotic death of cells. Analysis of caspase activity in intact cells, however, has been generally limited to the measurement of end-point biochemical and morphological markers of apoptosis. In an effort to develop a strategy with which to monitor caspase activity, early in the cell death cascade and in real-time, we have generated cell lines that overexpress recombinant GFP-based caspase substrates that display a quantifiable change in their spectral properties when cleaved by group II caspases. Specifically, tandem GFP substrates linked by a caspase-sensitive cleavage site show diminished fluorescence resonance energy transfer (FRET), as a consequence of cleavage, due to physical separation of the GFP moieties in apoptotic cells. We have evaluated the influence of different caspase-sensitive linkers on both FRET efficiency and cleavage by caspase-3. We also demonstrate that caspase activity as well as inhibition by pharmacological agents can be monitored, with minimal manipulation, in intact adherent cells seeded in a 96-well cell culture dish. Finally, we have adapted this technology to a high throughput screening platform to identify novel small molecule and cell permeable inhibitors of apoptosis. Based on a biochemical analysis of the compounds identified it is clear that this assay can be used to detect drugs which inhibit caspases directly as well as those which target upstream components of the caspase cascade.
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Abbreviations
- FRET:
-
fluorescence resonance energy transfer
- GFP:
-
green fluorescent protein
- PARP:
-
poly-(ADP-ribose)-polymerase
- DEVD-CHO:
-
tetrapeptide aldehyde
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Tawa, P., Tam, J., Cassady, R. et al. Quantitative analysis of fluorescent caspase substrate cleavage in intact cells and identification of novel inhibitors of apoptosis. Cell Death Differ 8, 30–37 (2001). https://doi.org/10.1038/sj.cdd.4400769
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DOI: https://doi.org/10.1038/sj.cdd.4400769
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