Dynamics of Signaling Pathways: Mechanism and Function

信号通路的动力学：机制和功能

• 批准号: 8214599
• 负责人: Galit Lahav
• 金额: $ 31.56万
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214599
• 关键词: ATM Signaling Pathway; Affect; Age; Alleles; Apoptosis; Behavior; Biological; Cell Count; Cell Cycle; Cell Cycle Arrest; Cell Cycle Stage; Cells; Commit; DNA Damage; DNA Double Strand Break; DNA Repair; Data; 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; Outcome; Pathway interactions; Pharmaceutical Preparations; Physiologic pulse; Population; Postdoctoral Fellow; Proteins; RNA Interference; Radiation; Radiation Tolerance; Relative (related person); Reporting; Role; Series; Shapes; Signal Pathway; Signal Transduction; System; Test Result; Testing; Time; cell type; cellular imaging; chemical genetics; imaging modality; 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。该提案的重点是p53信号通路的基本细胞机制。我们最近在单个细胞上使用了长期的延时成像研究，并发现p53水平对特定类型的DNA损伤表现出高度意外的脉冲反应。这些重复的脉冲已在先前的研究中掩盖了细胞种群中p53水平的研究。我们将结合单个活细胞中的定量动态测量，数学建模和对p53电路的操纵，以询问p53信号通路如何以及为什么会产生这一系列均匀的脉冲以及为什么不同的细胞显示出不同数量的脉冲。我们的第一个目的是确定导致p53脉冲的分子机制以及控制其振幅和持续时间的反馈。我们将对p53途径中几种蛋白质的动力学进行定量种群和单细胞测量值，并将信息整合到理论框架中，目的是为p53动力学开发可靠的预测模型。接下来，我们将确定p53搏动行为如何与特定的细胞结局以及特定下游程序（例如凋亡，细胞周期停滞和DNA修复）相关。我们将与标记蛋白并行跟踪P53动力学，这些蛋白会在单个活细胞中报告下游程序，并确定每个成像细胞的命运。我们将操纵控制电路以改变脉冲的频率或振幅，或完全消除它们，并询问这些变化如何影响细胞的结果。除了询问p53动力学如何确定结果外，我们还将检查DNA损伤的量是否影响脉冲的数量。我们已经开发了一种新型系统，用于定量活细胞中DNA双链断裂（DSB）和细胞周期阶段，我们将与跟踪p53脉冲并行使用该系统，以询问DSB的初始数量是否会影响p53脉冲的数量，以及细胞在细胞周期期间对辐射的敏感性是否影响。最后，我们将确定在MDM2启动子中携带特定多态性的细胞中，雌激素如何影响p53动力学和细胞命运（SNP309），这些细胞（SNP309）被证明具有高水平的MDM2和无p53脉冲。我们将预测并测试雌激素拮抗剂和p53/MDM2抑制剂对SNP309细胞背景中p53动力学的影响。我们的结果将为p53途径的控制和操纵提供新的见解，这可能是保护人类细胞免受癌症发展的最重要途径。这些研究将对p53的生物学机制和功能有更深入的了解，并为单个活着的人类细胞中其他信号通路的动力学提供了一个原型，以用于分析，描述和理解。