The roles of p53 and MYC dynamics in regulating heterogeneous cell fate responses to genotoxic stress

项目摘要 我们的长期目标是了解信号转导系统的动态调节是如何控制细胞内 应激反应本建议的重点是确定动态的 转录因子p53和MYC的表达协同调节细胞凋亡和衰老， 遗传毒性应激反应。p53和MYC的适当调节在人类中具有不可否认的重要性。 健康，因为它们的突变使人类细胞易患癌症。而p53和p54的调节和功能， MYC已经被广泛研究，确切地说，它们是如何在个体中产生可变的细胞命运结果的。 对一个群体的细胞对同样的压力的反应仍然知之甚少。我们最近的研究 表明p53的动态，p53积累和降解的时间模式， 在控制MYC水平和细胞命运对DNA损伤的反应方面具有不可或缺的功能。我们发现p53 动力学在单个细胞之间高度可变，但仍有待确定这种可变性是如何产生的。 有助于对DNA损伤剂的异质性反应，这对于理解 肿瘤细胞异质性和逃避治疗。为了回答这个问题，我们将联合收割机 荧光显微镜定量p53和MYC动力学的关键定量分析 转录靶点，以确定触发的时间调节， 细胞凋亡和衰老响应DNA损伤。我们将把这种分析应用于三种情况：（1） 表达正常p53和MYC的非转化细胞，2.）转化细胞，其中MYC表达是 在一定浓度范围内升高，以及3.）表达p53功能获得性的转化细胞 热点突变这项工作将显示p53和MYC动力学如何控制终末细胞命运的启动 在生理和病理条件下，它将作为减少 对DNA损伤化合物的异质反应。这些结果将提供新的见解 人类细胞中最重要的应激反应途径之一的基本功能，并且很可能 为基于改进的治疗时机的创新治疗策略提供信息。更 总的来说，这项研究可能会为越来越多的其他重要信号提供一般性的见解 使用动态调节的途径。

Project Summary Our long-term goal is to understand how dynamic regulation of signal transduction systems control cellular stress responses. The focus of this proposal is on identifying the mechanisms by which dynamic expression of the transcription factors p53 and MYC coordinately regulate apoptosis and senescence in response to genotoxic stress. Proper regulation of p53 and MYC are of undeniable importance in human health, as their mutation predisposes human cells to cancer. While the regulation and functions of p53 and MYC have been extensively studied, exactly how they generate variable cell fate outcomes in individual cells of a population responding to the same stress remains poorly understood. Our recent studies have shown that the dynamics of p53, the temporal pattern of p53 accumulation and degradation, serves an integral function for controlling MYC levels and cell fate responses to DNA damage. We have shown p53 dynamics to be highly variable between individual cells, but it remains to be determined how such variability contributes to heterogeneous responses to DNA damaging agents, which is critical for understanding tumor cell heterogeneity and evasion of therapies. To answer this question, we will combine time-lapse fluorescence microscopy to quantify p53 and MYC dynamics with quantitative analysis of key transcriptional targets at the single cell level to determine the temporal regulation of the triggering of apoptosis and senescence in response to DNA damage. We will apply this analysis to three conditions: 1.) non-transformed cells expressing normal p53 and MYC, 2.) transformed cells in which MYC expression is elevated over a range of concentrations, and 3.) transformed cells expressing a p53 gain-of-function hotspot mutation. This work will show how p53 and MYC dynamics control initiation of terminal cell fates in physiological and pathological conditions, and it will serve as the basis for approaches to reduce heterogeneous responses to DNA damaging compounds. These results will provide novel insight into the basic functioning of one of the most important stress response pathways in human cells, and are likely to inform innovative therapeutic strategies based on improved timing of the delivery of therapies. More broadly, this study is likely to provide general insights into the growing list of other important signaling pathways that use dynamic regulation.