Characterizing Caspase-9 Mediated Signaling Events

描述（由申请人提供）：异常细胞凋亡是癌症和各种自身免疫性疾病的标志。半胱天冬酶在细胞凋亡途径中起重要作用。由于caspase在细胞凋亡过程中的快速激活，很难简单地通过其激活的时间差来描述caspase的信号级联。此外，不同的caspase途径可以同时被触发。内在凋亡途径涉及细胞色素c和其他介质从线粒体的释放。细胞色素c在caspase-9的激活中起重要作用，caspase-9是内源性凋亡途径的启动子。为了描述半胱天冬酶-9下游的信号传导事件，我们建立了表达与FKBP融合的半胱天冬酶-9（Fv-CASP 9）的细胞系。我们还创建了表达Fv-CASP 9和其他半胱天冬酶的RNAi敲除的细胞系。我们计划使用这些细胞系1）确定caspase-9激活下游的caspase，2）描绘caspase-9下游caspase激活的顺序，3）确定caspase-9激活后特异性出现的内在凋亡事件。该系统使我们摆脱了线粒体破坏后介质非特异性释放产生的非特异性。

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.