Cell-free reconstitution of Fas-, UV radiation- and ceramide-induced apoptosis

Cell-free systems are valuable tools for the dissection of complex cellular processes. Here we show that cytoplasmic extracts from cells exposed to anti-Fas antibody or UV radiation contain an activity capable of reproducing morphological changes typical of apoptosis in nuclei added to these extracts, as well as internucleosomal cleavage of DNA and proteolysis of a protein known to be cleaved during the apoptosis of intact cells. Extracts from control cell populations were inactive in this respect. These effects were partly blocked by the addition of purified Bcl-2 protein or a competitive inhibitor peptide of interleukin-1β-converting enzyme to the extracts. Furthermore, apoptotic activity was induced in cytoplasmic extracts from untreated cells by the addition of ceramide, a lipid second messenger implicated recently in apoptosis signaling. These extracts should prove highly useful in the dissection of molecular events that occur during apoptosis.