Analysis of apoptosis in cell-free systems

Cell-free systems have been instrumental in the identification of several important components of the cell death machinery such as cytochrome c, APAF-1, ICAD/CAD (DFF45/DFF40) and Smac/Diablo. Such systems have also proved invaluable for the detailed analysis of caspase activation mechanisms, caspase activation cascades, proteolysis of caspase substrates, apoptosis-associated chromatin condensation and internucleosomal DNA fragmentation. Here, we describe a cell-free system that we have used routinely in our laboratory for the analysis of caspase activation and associated events. Caspase activation in this system can be triggered either through assembly of the APAF-1 apoptosome by addition of cytochrome c/dATP, or alternatively, by addition of the cytotoxic lymphocyte protease, granzyme B. In both cases, the order of caspase activation events has been established and the relative importance of individual caspases to apoptosis-associated nuclear events, as well as substrate proteolysis, is known. Cell-free systems are therefore very useful for screening potential caspase-inhibitory compounds or other agents that may positively or negatively affect caspase-dependent events in apoptosis.