Dynamics of Signaling Pathways: Mechanism and Function

信号通路的动力学：机制和功能

• 批准号: 7371188
• 负责人: Galit Lahav
• 金额: $ 32.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7371188
• 关键词: ATM Signaling Pathway; Affect; Age; Alleles; Apoptosis; Behavior; Biological; Cell Count; Cell Cycle; Cell Cycle Arrest; Cell Cycle Stage; Cells; Commit; Count; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Depth; Development; Diagnostic; Dose; Estrogen Antagonists; Estrogens; Feedback; Female; Frequencies; Gene Targeting; Genes; Genetic Polymorphism; Goals; Gonadal Steroid Hormones; Height; Human; Image; Individual; Knowledge; Life; Malignant Neoplasms; Masks; Measurement; Measures; Methods; Modeling; Molecular; Numbers; Outcome; Pathway interactions; Pharmaceutical Preparations; Physiologic pulse; Population; Postdoctoral Fellow; Proteins; Pulse taking; RNA Interference; Radiation; Radiation Tolerance; Rate; Relative (related person); Reporting; Role; Series; Shapes; Signal Pathway; Signal Transduction; System; TP53 gene; Test Result; Testing; Thinking; Time; cell type; cellular imaging; chemical genetics; inhibitor/antagonist; insight; mathematical model; novel; p53 Signaling Pathway; predictive modeling; programs; promoter; prototype; repaired; response; sex

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the dynamic behavior of biological signals is controlled and how these dynamics affect cellular responses. This proposal focuses on fundamental cellular mechanisms of the p53 signaling pathway. We recently used long-term time-lapse imaging studies on single cells and discovered that p53 levels show a highly unexpected pulsatile response to specific types of DNA damage. These repeated pulses had been masked in previous studies that measured p53 levels in populations of cells. We will combine quantitative dynamic measurements in single living cells, mathematical modeling and manipulation of the p53 circuit to ask how, and why, the p53 signaling pathway generates this series of uniform pulses and why different cells show different numbers of pulses. Our first aim is to identify the molecular mechanism leading to p53 pulses and the feedbacks that control their amplitude and duration. We will perform quantitative population and single cell measurements of the dynamics of several proteins in the p53 pathway and integrate the information into a theoretical framework, with the goal of developing a credible predictive model for p53 dynamics. Next, we will determine how p53 pulsatile behavior is connected with specific cellular outcomes and with the activation of specific downstream programs such as apoptosis, cell cycle arrest and DNA repair. We will track p53 dynamics in parallel with marker proteins that report on downstream programs in single living cells, and identify the fate of each imaged cell. We will manipulate the control circuit to alter the frequency or amplitude of the pulses, or eliminate them altogether, and ask how these changes affect the outcome for the cell. As well as asking how p53 dynamics determine outcome, we will examine whether the amount of DNA damage affects the number of pulses. We have developed a novel system for quantitating DNA double- stranded breaks (DSBs) and cell cycle stage in live cells and we will use this system in parallel with tracking p53 pulses to ask whether the initial number of DSBs affects the number of p53 pulses, and whether the cell's sensitivity to radiation changes during the cell cycle. Finally, we will determine how estrogen influences p53 dynamics and cell fate following DNA damage in cells that carry a specific polymorphism in Mdm2 promoter (SNP309), which were shown to have high levels of Mdm2 and no p53 pulses. We will predict and test the effect of estrogen antagonists and p53/Mdm2 inhibitors on p53 dynamics in the background of SNP309 cells. Our results will provide new insights into the control and manipulation of the p53 pathway, perhaps the most important pathway protecting human cells against the development of cancer. These studies will give a deeper understanding of the biological mechanisms and function of p53, and will provide a prototype for the analysis, description, and understanding of the dynamics of other signaling pathways in single living human cells.

描述（由申请人提供）：我们的长期目标是了解如何控制生物信号的动态行为以及这些动态如何影响细胞反应。该提案重点关注 p53 信号通路的基本细胞机制。我们最近对单细胞进行了长期延时成像研究，发现 p53 水平对特定类型的 DNA 损伤表现出高度意想不到的脉冲反应。这些重复的脉冲在之前测量细胞群中 p53 水平的研究中被掩盖了。我们将结合单个活细胞的定量动态测量、数学建模和 p53 电路的操作来探究 p53 信号通路如何以及为何产生这一系列均匀脉冲，以及为什么不同的细胞显示不同数量的脉冲。我们的首要目标是确定导致 p53 脉冲的分子机制以及控制其幅度和持续时间的反馈。我们将对 p53 通路中几种蛋白质的动态进行定量群体和单细胞测量，并将信息整合到理论框架中，目标是开发 p53 动态的可靠预测模型。接下来，我们将确定 p53 脉冲行为如何与特定细胞结果以及特定下游程序（例如细胞凋亡、细胞周期停滞和 DNA 修复）的激活相关。我们将与报告单个活细胞下游程序的标记蛋白并行跟踪 p53 动态，并确定每个成像细胞的命运。我们将操纵控制电路来改变脉冲的频率或幅度，或者完全消除它们，并询问这些变化如何影响细胞的结果。除了了解 p53 动态如何决定结果外，我们还将检查 DNA 损伤的数量是否会影响脉冲数量。我们开发了一种新颖的系统，用于定量活细胞中的 DNA 双链断裂 (DSB) 和细胞周期阶段，我们将使用该系统与跟踪 p53 脉冲并行，以了解 DSB 的初始数量是否影响 p53 脉冲的数量，以及细胞对辐射的敏感性是否在细胞周期中发生变化。最后，我们将确定在 Mdm2 启动子 (SNP309) 中携带特定多态性的细胞中 DNA 损伤后，雌激素如何影响 p53 动态和细胞命运，这些细胞具有高水平的 Mdm2 而没有 p53 脉冲。我们将在 SNP309 细胞背景下预测和测试雌激素拮抗剂和 p53/Mdm2 抑制剂对 p53 动态的影响。我们的研究结果将为 p53 通路的控制和操纵提供新的见解，p53 通路可能是保护人类细胞免受癌症发展的最重要通路。这些研究将更深入地了解p53的生物学机制和功能，并将为分析、描述和理解单个活人类细胞中其他信号通路的动态提供原型。