Characterization of additional targets of p53 in apoptosis

         p53 function as a transcriptional activator is important for its ability to induce apoptosis in response to certain cellular stresses, such as acute DNA damage signals.  Although some known p53 target genes, including Bax and Noxa, are required for a maximal apoptotic response, they are induced to similar levels during p53-mediated G1 cell cycle arrest and apoptosis, indicating that they are not responsible for determining whether cells undergo apoptosis.  To better define the pathway through which p53 specifies a cell death program, we used microarray analysis to identify genes selectively activated by p53 during apoptosis, which may dictate apoptotic cell fate.  By comparing the expression profiles of apoptotic, arresting, and p53-deficient mouse embryonic fibroblasts, we identified a number of genes that are selectively upregulated by p53 during apoptosis, including Perp and the pro-apoptotic gene Siva. Siva expression is broadly induced in multiple contexts of p53-dependent, but not p53-independent, cell death.  As might be expected of a protein involved in promoting a p53’s apoptotic response, we determined that expression of Siva is sufficient to induce apoptosis in MEFs, to a degree similar to p53.  To examine the requirement for endogenous Siva in the p53 apoptotic program, we turned to the nervous system, a context in which Siva is induced and where p53-dependent apoptosis has great physiological relevance in vivo.  Using RNA interference, we demonstrated that Siva is essential for DNA damage-induced, p53-dependent cell death in cerebellar granule neurons.  We characterized the mechanism of Siva-mediated apoptosis, by examined features of apoptosis induced by Siva expression in fibroblasts and neurons in comparison to cells undergoing p53-dependent apoptosis in response to DNA damage.  As with p53, Siva-induced apoptosis involves Bax translocation to the mitochondria, cytochrome c release, and caspase-3 activation, hallmarks of the intrinsic mitochondrial apoptosis pathway.  As a means of understanding to clarify Siva’s mode of action, we employed multiple fractionation techniques to identify Siva’s subcellular localization and determined that endogenous Siva is localized to the plasma membrane during neuronal cell death.  Furthermore, we established that components of the extrinsic apoptosis pathway, including caspase-8 and Bid, are activated during and are required for the complete p53 apoptotic response to DNA damage.  These studies highlight the participation of membrane signaling events in p53’s apoptotic program and have significant implications for understanding the mechanisms underlying pathogenesis after neuronal injury and in neurodegenerative diseases. We are continuing analysis of Siva and other apoptosis - specific p53 target genes such as Perp to gain further insight into pathways of p53-mediated apoptosis and tumor suppression.

Schematic outlining screen used to identify apoptosis-specific, p53-dependent target genes.
Northern blot analysis demonstrating high level expression of select novel p53 target genes, including Perp and Siva, during p53-dependent apoptosis compared to G1 arrest. These represent potential p53-dependent inducers of the apoptotic cell fate.


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