Role of caspases in apoptotic execution

Apoptosis is a morphologically and biochemically distinct form of cell death that plays a major role in the deletion of cells under a variety of physiological and pathological conditions. Morphologically, this process is characterized by a variable latent phase, followed by a dramatic execution phase. Apoptotic execution is accompanied by the overall shrinkage of the cell, a dramatic upregulation of motility processes (plasma membrane blebbing), and condensation of chromatin, followed by fragmentation of the nucleus and cytoplasm into multiple membraneenclosed bodies. These changes are accompanied by cleavage of nuclear proteins by specialized cell death proteases termed caspases. We have developed a cell-free approach to the analysis of the events of apoptotic execution, using cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. So-called S/M extracts from morphologically normal “committed stage” cells induce apoptotic morphology and DNA cleavage in substrate nuclei (Lazebnik, Y.A., Cole, S., Cooke, C.A., Nelson, W.G. & Earnshaw, W.C. (1993). 1. Cell RioI. 123, 7-22). These apoptotic changes appear to involve the function of multiple caspases (cysteine aspartases specialized proteases that are critical for apoptosis) acting in parallel. Affinity labeling of active caspases with Z-EK(biotin)D-aomk reveals at least seventeen distinct active caspase species in the S/M extracts (Martins, L.M., Kottke, T.J., Kaufmann, S.H. & Earnshaw, W.C. (1998). Blood 92,3042-3049). Inhibition of these caspases using WAD-chloromethylketone abrogates all signs of apoptosis. Very early during nuclear disassembly in the extracts, one or more of the extract caspases cleaves poly (ADP-ribose)polymerase (PARP) at the sequence DEVD-G, the first cleavage site determined for any caspase substrate in apoptosis (Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G. & Earnshaw, W.C. (1994). Nature 371, 346-347). A second caspase subsequently catalyzes lamin cleavage, a reaction that is. required for disassembly of the nucleus into individual apoptotic bodies (Takahashi, A., Alnemri, E., Lazebnik, Y.A., Fernandes-Alnemri, T., Litwack, G., Moir, R.D., Goldman, R.D., Poirier, G.G., Kaufmann, S.H. & Earnshaw, W.C. (1996). PYOC. Nut. Acad. Sci. (USA) 93, 8395-8400). These experiments led to a view in which the events of apoptotic execution are largely due to the direct action of caspases on crucial cellular substrates. More recently, we have developed an alternative method for making extracts from cells in apoptotic execution. Surprisingly, these extracts induce apoptotic events in substrate nuclei in a caspase-independent manner, suggesting that nuclear disassembly is driven largely by factors activated downstream of the caspases at the onset of apoptotic execution (Samejima, K., Tone, S., Kottke, T.J., Enari, M., Sakahira, H., Cooke, C.A., Durrieu, F., Martins, L.M., Nagata, S., Kaufmann, S.H. & Earnshaw, W.C. (1998). 1. Cell Biol. 143, 225-239). We showed that one such factor, the caspase-activated DNase, CAD/CPAN/DFF40 can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors. However, CAD cannot be the sole morphogenic factor triggered by caspases, since inhibition of CAD by addition of the inhibitory chaperone ICAD/DFF to execution phase extracts has no effect on the ability of those extracts to induce apoptotic events in added nuclei. These observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves. They also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent