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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

基本信息

批准号：
10084819
负责人：
Jacob Stewart-Ornstein
金额：
$ 24.5万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-15 至 2021-11-08
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10084819
关键词：
Apoptosis Behavior Biological Assay Biology Biopsy Cell Culture Techniques Cell Proliferation Cells Chemicals Chemoresistance Chemotherapy-Oncologic Procedure Cisplatin Clinical Clinical Markers Complement Complex DNA Damage Enterobacteria phage P1 Cre recombinase Future GMNN gene Gene Expression Genetic Goals Grant Human Image In Vitro Label Link MCM2 gene Malignant Neoplasms 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 Radiation Regulation Reporter Research Resistance Resolution Role Screening procedure Signal Transduction Solid Neoplasm Stains Stress Supervision System Systems Biology TP53 gene Technical Expertise Techniques Testing Time Tissues Toxic effect Work Yeasts base career cellular imaging chemotherapy gamma irradiation genotoxicity graduate student immunohistochemical markers in vivo insight interest live cell microscopy medical schools molecular marker neoplastic cell novel nucleotide analog repaired response screening skills small molecule small molecule libraries therapy resistant three dimensional cell culture tool tumor tumor microenvironment

项目摘要

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.

项目摘要单细胞静息相关DNA损伤抗性的确定和调控这项建议的目标是研究存在于肿瘤中的静止现象和将这种状态与化疗耐药性联系起来。所有实体瘤都是增殖的异质性混合体静止的细胞和对不同肿瘤的许多研究已经确定了静止的对治疗有抵抗力的人群。定义哪些静止状态对此阻力有贡献因此，识别工具化合物来调节静止状态是非常有意义的。我将使用一种新型的活细胞增殖记者小组，来自经典免疫组织化学标记物用于研究静止期和这些静止状态与单细胞水平上的DNA损伤反应直接相关。这研究将在2-和3D培养(目标1)中进行，在那里我们有遗传和化学对照以精确地将DNA损伤反应与静止状态相关联，并随后在体内进行目的3)建立小鼠肿瘤模型。为了补充这些更具观察性的目标，我将建立一种新的筛选工具来识别特定修改用命运追踪法研究静止细胞的化学抗性(目标2)。这个项目将描述静止状态的多样性及其相互关系，与静止直接相关化疗耐药，并确定工具化合物来操纵这种表型。这份提案利用了我作为研究生时培养出来的分析能力。酵母菌应激信号和作为博士后在过去两年中研究P53的决定在正常组织和肿瘤中的调节。我将应用单细胞技术和我学会了计算方法来研究静止和静止背后的基本生物学肿瘤中的化疗耐药性，并识别扰乱这种耐药性的小分子。在这笔赠款的指导阶段我将在我的共同导师拉哈夫博士和 Weissleder博士是我提案的核心焦点的专家：单细胞对基因毒性疗法。在这个阶段，我将获得细胞培养、成像、小分子筛选，鼠标工作和专业技能和个人资料，以获得独立的学术研究岗位。在初始阶段，我将嵌入到哈佛医学院系统生物学系，世界一流的研究中心复杂数量表型的研究和临床医生和学者的包围关于癌症，以及其他问题。这项提议将完成我向癌症的转变他是一位生物学家，并为我提供了一座通往研究癌症的独立研究生涯的桥梁。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Conservation and Divergence of p53 Oscillation Dynamics across Species.

DOI：
10.1016/j.cels.2017.09.012
发表时间：
2017-10-25
期刊：
Cell systems
影响因子：
9.3
作者：
Stewart-Ornstein J;Cheng HWJ;Lahav G
通讯作者：
Lahav G

Fluctuations in p53 Signaling Allow Escape from Cell-Cycle Arrest.

DOI：
10.1016/j.molcel.2018.06.031
发表时间：
2018-08-16
期刊：
Molecular cell
影响因子：
16
作者：
Reyes J;Chen JY;Stewart-Ornstein J;Karhohs KW;Mock CS;Lahav G
通讯作者：
Lahav G

p53 dynamics vary between tissues and are linked with radiation sensitivity.

DOI：
10.1038/s41467-021-21145-z
发表时间：
2021-02-09
期刊：
Nature communications
影响因子：
16.6
作者：
Stewart-Ornstein J;Iwamoto Y;Miller MA;Prytyskach MA;Ferretti S;Holzer P;Kallen J;Furet P;Jambhekar A;Forrester WC;Weissleder R;Lahav G
通讯作者：
Lahav G

p53 pulses lead to distinct patterns of gene expression albeit similar DNA-binding dynamics.