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如何控制细胞 压力反应。这项提案的重点是确定动态地 转录因子P53和MYC的表达协同调节细胞凋亡和衰老 对基因毒性应激的反应。对P53和MYC的适当调控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容易患上癌症。而P53和P53的调控和功能 MYC已经被广泛研究，确切地说它们是如何在个体中产生不同的细胞命运结果的 对同一压力做出反应的人群中的细胞仍然知之甚少。我们最近的研究表明 表明P53的动态，P53积累和降解的时间模式，服务于 控制MYC水平和细胞对DNA损伤的命运反应的完整功能。我们已经展示了P53 个体细胞之间的动力学是高度不同的，但这种差异如何仍有待确定 对DNA损伤剂的异质性反应，这对理解 肿瘤细胞的异质性和治疗的逃避。为了回答这个问题，我们将结合延时 荧光显微镜定量分析Key的P53和MYC动力学 在单细胞水平上的转录靶点来确定触发的时间调节 DNA损伤引起的细胞凋亡和衰老。我们将把这一分析应用于三种情况：1) 未转化的细胞表达正常的P53和MYC，2)MYC表达的转化细胞 在一定浓度范围内升高，以及3.)表达P53功能增益的转化细胞 热点突变。这项工作将展示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损伤化合物的异质性反应。这些结果将为我们提供对 人类细胞中最重要的应激反应途径之一的基本功能，并可能 告知基于改进的治疗交付时间的创新治疗战略。更多 总的来说，这项研究可能会提供对其他重要信号不断增加的清单的总体洞察 使用动态调节的路径。

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.