암억제 유전자 p53 타겟 유전자 발굴과 p53에 의한 세포사에서 이들 유전자의 역할

Abstract

Identification of p53 target genes and their roles in p53-mediated cell death

By: Sujan Piya Advisor: Prof. Kim, Tae-Hyoung Department of Biochemistry and Molecular biology School of Medicine Graduate School of Chosun University

Apoptosis, an essential altruistic behaviour of cell as a whole maintains the integrity of total organism in normal physiological condition. p53 is one of the key player in maintaining the integrity of organism by removing the harmful, unnecessary or damaged cells. A number of genes transactivated by p53 are involved in these phenomena through signaling or transcriptional regulation of apoptotic and pro-apoptotic or non-apoptotic molecules. In this study, I have identified several novel p53 target genes and characterized their functional roles in p53-induced cell death. Furthermore, I also characterized the role of Noxa, a known p53 target gene, in LPS-mediated tumor regression. Besides conventional cell killing BH3 domain, I proposed a novel killing domain, mitochondrial target domain (MTD) in Noxa that has been known as a responsible domain for mitochondrial translocation of Noxa. BH3 domain and newly explored mitochondrial targeting domain (MTD) of Noxa are essential for the release of Cytochrome C, pro-apoptotic and apoptotic molecules from mitochondria as well as mitochondrial fragmentation ultimately resulting cell death. p53 has been known to play critical roles in diverse cellular responses such as cell cycle arrest, senescence and apoptosis through transcriptional control of its target genes. Identification of the p53 upregulated genes by DNA chip analysis and characterization of the new p53 transactivated genes will advance our understanding of scene behind the p53 exerted multiple phenomena. Genotoxic stresses stabilize the p53 protein which, in turn, transactivates target genes to cause apoptosis. In this thesis I show that DUSP6, SSFA2 and PHLDA1 are the novel target genes in p53 mediated apoptosis. To explore the p53 transactivated genes and their role in cell death, microarray followed by introduction of short hairpin of RNA and functional experimental approach were adapted. The computational analysis of DUSP6 promoter region revealed the putative binding site for p53. Additionally, the Chromatin immunoprecipitation analysis revealed that p53 transactivates of DUSP6. The functional analysis of DUSP6 shows that it suppresses the ERK phosphorylation and elevates 5-FU induced cell death. Peptides derived from mitochondrial target domain (MTD) of Noxa are very effective to kill the tumor cells. So, the tumor homing synthetic peptide TU-1 MTD, TU-2 MTD and TU-3 MTD were evaluated for tumor suppression efficacy. Specifically, TU-2 MTD and TU-3 MTD dramatically cause the cell death of CT26.WT cells in vitro and in mice. These findings suggest that MTD peptide can be developed as to peptide-based cancer drug. In contrast to classical p53 transactivation of Noxa, I explored transactivation of Noxa gene in p53-independent manner and its pivotal role in LPS-induced rejection of the tumor in the syngeneic mice. Herein, architectural signaling of IRFs in Noxa promoter region was dissected to understand the underlying mechanism of LPS-mediated rejection of the tumor in the syngeneic mice. I demonstrate that LPS, a critical molecule signaling to the TLR4/TLR3, and IFNs regulate the Noxa expression by activating IRF1, IRF3, and IRF7. This induction of Noxa by LPS or IFNs in tumor cells is required for tumor regression mediated by LPS. Collectively, this thesis will contribute to the understanding of p53-transactivated genes and their role in cell death and physiological role of Noxa in spontaneous tumor regression.