Mouse Models to Elucidate p53 Regulatory Mechanisms

描述（由申请人提供）：p53肿瘤抑制因子是一种转录因子，在DNA损伤剂、缺氧、癌基因、端粒异常和其他影响肿瘤生长和生存的条件激活的途径中起作用。在大多数人类肿瘤中，这一途径是失活的，使肿瘤细胞能够与受损的基因组一起循环，或在诱导基因组不稳定的条件下循环。细胞培养模型引出了p53调控的优雅机制和它们在体内的相关性的重大争议。本研究采用胚胎干细胞同源重组的方法，在假定的p53调控结构域中产生突变小鼠，研究其在p53正常表达条件下对不同组织中基因定义细胞中p53调控和功能的影响。具体来说，我们建议进行实验来继续分析我们最近产生的小鼠，其中缺失了一个n端假定的蛋白质相互作用结构域，据报道会影响细胞凋亡，但不会影响细胞周期阻滞。我们建议在c端区域产生影响p53稳定性和转录调控的突变小鼠。这些突变对p53稳定性、靶基因结合、细胞周期阻滞、细胞凋亡和衰老的影响将在对激活应激的反应中确定，如上述，因为它们与肿瘤的发生、进展和化疗反应最为相关。我们还将研究自发肿瘤发生，以及癌基因诱导的肿瘤发生的几种模型，以评估这些突变对p53依赖的肿瘤抑制的影响。所提出的模型将有助于进一步评估蛋白-蛋白相互作用在N端调控域中的作用，以及稳定和翻译后修饰对p53通路转录和生物学输出的重要性。这些研究对于在生理相关条件下不同细胞类型中p53调控的明确结论至关重要。获得的数据和开发的系统与p53靶向治疗相关，它们为转录因子如何将复杂的输入转化为不同的调控决策提供了范例。

DESCRIPTION (provided by applicant): The p53 tumor suppressor is a transcription factor functioning in a pathway activated by DNA damaging agents, hypoxia, oncogenes, telomere abnormalities and other conditions that impact on tumor growth and survival. This pathway is inactivated in most human neoplasms, enabling tumor cells to cycle with damaged genomes, or under conditions that induce genomic instability. Cell culture models elicited both elegant mechanisms of p53 regulation and significant controversy over their relevance in vivo. The proposed studies use homologous recombination in embryonic stem cells to generate mice with mutations in putative p53 regulatory domains to study their effects on p53 regulation and function in genetically defined ceils in different tissues under conditions of normal p53 expression. Specifically, experiments are proposed to continue analysis of mice we recently generated with a deletion of an N-terminal putative protein interaction domain reported to affect apoptosis but not cell cycle arrest. We propose to generate mice with mutations in a C-terminal domain that impacts on p53 stability and transcriptional regulation. The impact of these mutations on p53 stability, target gene binding, cell cycle arrest, apoptosis, and aging will be determined in response to activating stresses such as those described above as they are most relevant to tumor initiation, progression and chemotherapy response. We will also study spontaneous tumorigenesis, as well as several models of oncogene induced tumorigencity to assess the impact of these mutations on p53 dependent tumor suppression. The models proposed will enable in rive assessments of the contribution of protein-protein interactions in the N- terminal regulatory domain, and importance of stabilization and post-translational modifications on the transcriptional and biological output of the p53 pathway. These studies are essential for unambiguous conclusions about p53 regulation in different cell types under physiologically relevant conditions. The data obtained and systems developed have relevance for p53-targeted therapies, and they provide a paradigm for how transcription factors convert complex inputs into distinct regulatory decisions.