Defining and manipulating quiescence associated DNA damage resistance in single cells

定义和操纵单细胞中与静止相关的 DNA 损伤抵抗力

• 批准号: 9162369
• 负责人: Jacob Stewart-Ornstein
• 金额: $ 16.61万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9162369
• 关键词: Apoptosis; Behavior; Biological Assay; Biology; Biopsy; Cell Culture Techniques; Cell Proliferation; Cells; Chemicals; Chemotherapy-Oncologic Procedure; Cisplatin; Clinical Markers; Complement; Complex; Computational Technique; DNA Damage; Future; GMNN gene; Gene Expression; Goals; Grant; Human; Image; In Vitro; Label; Learning; Life; Link; MCM2 gene; Malignant Neoplasms; Maps; Measures; Mentors; Microscopy; Mitotic; Modeling; Molecular; Mus; Nature; Normal tissue morphology; Nude Mice; Outcome; Pathway interactions; Phase; Phenotype; Population; Positioning Attribute; Postdoctoral Fellow; Proliferating; Proliferation Marker; Protein p53; Radiation; Regulation; Reporter; Research; Resistance; Resolution; Role; Signal Transduction; Solid Neoplasm; Sorting - Cell Movement; Staining method; Stains; Stress; Supervision; System; Systems Biology; TP53 gene; Technical Expertise; Testing; Time; Tissues; Toxic effect; Tumor Tissue; Work; Yeasts; abstracting; base; career; cellular imaging; chemical genetics; chemotherapy; graduate student; immunohistochemical markers; in vivo; insight; interest; irradiation; live cell microscopy; medical schools; molecular marker; mouse model; neoplastic cell; novel; nucleotide analog; recombinase; repaired; response; screening; skills; small molecule; small molecule libraries; therapy resistant; three dimensional cell culture; tool; tumor; tumor microenvironment

Project Abstract Defining and manipulating quiescence associated DNA damage resistance in single cells The goal of this proposal is to study the phenomena of quiescence as it exists in tumors and relate this state to chemoresistance. All solid tumors are a heterogenous mix of proliferating and quiescent cells and many studies on different tumors have determined the quiescent population to be therapy resistant. Defining which quiescent states contribute to this resistance and identifying tool compounds to modulate the quiescent state is therefore of great interest. I will use a novel panel of live cell proliferation reporters derived from classical immunohistochemical markers of proliferation to study the nature of quiescent states and directly relate these quiescent states to DNA damage responses at the single cell level. This study will be carried out in 2- and 3D culture (Aim 1), where we have the genetic and chemical control to precisely relate DNA damage responses to quiescent states, and later taken in vivo (Aim 3) to mouse tumor models. Complementing these more observational aims, I will establish a novel screening tool to identify molecules that specifically modify the chemoresistance of quiescent cells using a fate tracing approach (Aim 2). This project will describe the diversity of quiescent states and their inter-relationships, directly relate quiescence to chemoresistance, and identify tool compounds to manipulate this phenotype. This proposal draws on my analytical skills developed as a graduate student working on yeast stress signaling and as a postdoc over the last two years studying determinates of p53 regulation in normal tissues and tumors. I will apply the single cell techniques and computational approaches I have learned to study the basic biology underlying quiescence and chemoresistance in tumors and to identify small molecules that perturb this resistance. In the mentored phase of this grant I will work under the supervision of my co-mentors Dr. Lahav and Dr. Weissleder, who are experts on the core focus of my proposal: single cell responses to genotoxic therapy. During this phase, I will gain the technical skills in cell culture, imaging, small molecule screening, and mouse work and the professional skills and profile to obtain an independent academic research position. For the initial phase, I will be embedded in the department of Systems Biology at Harvard Medical School, a world class research center for the study of complex quantitative phenotypes and surrounded by clinicians and academics working on cancer, among other problems. This proposal will complete my transition into a cancer biologist and provided me with a bridge to an independent research career studying cancer.

项目摘要 定义和操纵单细胞中静止相关的DNA损伤抗性 该提案的目标是研究肿瘤中存在的静止现象， 将这种状态与化疗耐药性联系起来。所有的实体瘤都是一种异质性的增殖混合物， 许多对不同肿瘤的研究已经确定了 人群对治疗耐药。定义哪些静态会产生这种电阻 因此，识别调节静止状态的工具化合物是非常有意义的。我 将使用一组新的活细胞增殖报告基因， 增殖的免疫组织化学标记物，以研究静止状态的性质， 直接将这些静止状态与单细胞水平的DNA损伤反应联系起来。这 研究将在二维和三维培养中进行（目标1），在那里我们有遗传和化学 控制，以精确地将DNA损伤反应与静止状态联系起来， (Aim 3）小鼠肿瘤模型。为了补充这些更具观察性的目标，我将 建立一个新的筛选工具，以确定分子，具体修改 使用命运追踪方法测定静止细胞的化学抗性（Aim 2）。该项目将 描述静止状态的多样性及其相互关系，直接关系到静止 化学抗性，并确定工具化合物来操纵这种表型。 这个建议借鉴了我的分析能力，作为一个研究生， 酵母应激信号，并作为博士后在过去两年中研究p53的决定因素 在正常组织和肿瘤中的调节。我将应用单细胞技术， 我学会了用计算方法来研究静止背后的基本生物学， 肿瘤中的化学抗性，并鉴定干扰这种抗性的小分子。在 在此资助的指导阶段，我将在我的共同导师Lahav博士的监督下工作， 博士Weissleder，他们是我的建议的核心焦点的专家： 遗传毒性治疗在这个阶段，我将获得细胞培养，成像， 小分子筛选，小鼠工作和专业技能和概况，以获得一个 独立的学术研究立场。在最初的阶段，我将被嵌入到 哈佛医学院系统生物学系，世界级的研究中心， 研究复杂的定量表型，周围有临床医生和学者， 在癌症和其他问题上。这次求婚会让我彻底变成癌症 他是一位生物学家，为我提供了一座通往独立研究癌症的桥梁。