The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells

The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e,g,, many chemotherapeutic ag ents and radiation) require functional p53 and are inhibited by Bcl-2, Apop tin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptot ic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis . Although Apoptin activity was not affected by CrmA, p35 did inhibit Apopt in-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. Ho wever, in most of these cells, cytochrome c is still released and the mitoc hondria are not stained by CMX-Ros, indicating a drop in mitochondrial memb rane potential. These results imply that although the final apoptotic event s are blocked by p35, parts of the upstream apoptotic pathway that affect m itochondria are already activated by Apoptin, Taken together, these data sh ow that the viral protein Apoptin employs cellular apoptotic factors for in duction of apoptosis. Although activation of upstream caspases is not requi red, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.