We applied a novel variant of GSE selection technique to identification of new regulators of FAS apoptosis. Library of randomly fragmented chicken cDNAs cloned in replication-competent avian retroviral vector RCASBP(A) was introduced into chicken DF-1 cells that ectopically expressed human FAS receptor, what made them susceptible to FAS apoptosis, were transduced with the GSE library followed by several rounds of selection of viruses conferring resistance of cells to FAS agonistic antibodies. A set of GSEs inhibiting FAS-dependent apoptosis were isolated from these viruses and individually confirmed in chicken and human cells. Surprisingly, several of isolated GSEs encoded short fragments of different components of mitochondrial electron-transport chain encoded by mitochondrial genome. We focused on characterizing cytochrome b (Cyt b) from complex III and showed that this protein undergoes cleavage after cell treatment with FAS and its C-terminal half became detectable in the cytoplasm within 4 hours after induction of apoptosis. Ectopic expression of the full-length and C-terminal fragment of Cyt b in the cytoplasm resulted in activation of caspases 3 and 9 and rapid apoptosis in a variety of human cell lines. This apoptosis can be suppressed by the isolated GSE but not with Bcl2 or shRNA against Apaf-1 that were effective in inhibiting staurosporin-mediated cell death. These results indicate that cytoplasmic release of mitochondrial Cyt b can serve as a trigger and a mediator of FAS-induced apoptosis.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA