Aberrant apoptosis is a hallmark of cancer and various autoimmune diseases. Caspases play essential roles in the apoptotic pathway. Due to the rapid activation of caspases during apoptosis, it has been difficult to delineate the signaling cascade of caspases simply by time difference in their activation. Moreover, different caspase pathways can be triggered simultaneously. The intrinsic apoptotic pathway involves the release of cytochrome c and other mediators from the mitochondria. Cytochrome c plays an essential role in the activation of caspase-9, the initiator caspase of the intrinsic apoptotic pathway. To delineate the signaling events downstream of caspase-9, we have established a cell line that expresses caspase-9 fused to FKBP (Fv-CASP9). We have also created cell lines expressing Fv-CASP9 and RNAi knockdowns of other caspases. We plan to use these cell lines to 1) determine the caspases that are downstream of caspase-9 activation, 2) delineate the order of caspase activation downstream of caspase-9, and 3) determine intrinsic apoptotic events that arise specifically after caspase-9 activation. This system frees us of the non-specificity that arises with the non-specific release of mediators after mitochondrial disruption.
