Characterizing Caspase-9 Mediated Signaling Events

描述(申请人提供)：异常的细胞凋亡是癌症和各种自身免疫性疾病的标志。半胱氨酸天冬氨酸氨基转移酶在细胞凋亡途径中起着重要作用。由于半胱氨酸天冬氨酸酶在细胞凋亡过程中的快速激活，单纯通过激活的时间差来描述半胱氨酸天冬氨酸氨基转移酶的信号级联已变得困难。此外，不同的caspase通路可以同时被触发。固有的细胞凋亡途径包括从线粒体释放细胞色素c和其他介质。细胞色素c在caspase-9的激活中起着至关重要的作用，caspase-9是固有的细胞凋亡途径的起始者。为了描述caspase-9下游的信号事件，我们建立了一个表达caspase-9与FKBP融合的细胞系(FV-CASP9)。我们还建立了表达FV-CASP9和RNAi敲除其他caspase的细胞系。我们计划使用这些细胞系来1)确定caspase-9激活下游的caspase，2)描绘caspase-9下游caspase激活的顺序，以及3)确定caspase-9激活后特定出现的固有凋亡事件。这个系统使我们摆脱了线粒体破坏后介质的非特异性释放所产生的非特异性。

DESCRIPTION (provided by applicant): 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.