DNA Damage and Response in the Bladder Microenvironment.

膀胱微环境中的 DNA 损伤和反应。

基本信息

批准号：
8713975
负责人：
BRADLEY P DIXON
金额：
$ 14.79万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2015-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8713975
关键词：
AVPR2 gene Academic Medical Centers Alkylating Agents Animal Experimentation Animal Model Animals Apoptosis Apoptotic Area Award Binding Biochemistry Biological Assay Biological Process Biomedical Engineering Bladder Bladder Tissue Cancer Biology Carcinogens Caspase Cell Culture Techniques Cell Cycle Checkpoint Cell Cycle Regulation Cell Line Cell physiology Cells Cellular biology Child Childhood Clinical Clinical Trials Colon Comet Assay Complex Core Facility DNA Damage DNA Repair DNA repair protein Detection Development Plans Dysplasia Environment Epithelial Cells Epithelium Experimental Hematology Faculty Flow Cytometry Fostering Funding Future Gastrointestinal tract structure Genetic Code Genitourinary system Goals Grant Histologic Histology Immunoblotting Immunofluorescence Immunologic Immunohistochemistry In Situ Nick-End Labeling In Vitro Institution Instruction Investigation Kinetics Label Laboratories Lead Lesion Lower urinary tract Malignant - descriptor Malignant Neoplasms Malignant neoplasm of gastrointestinal tract Malignant neoplasm of urinary bladder Manuscripts Mass Spectrum Analysis Measurement Measures Medical center Medicine Mentors Metaplasia Microbiology Mitochondria Mitotic Activity Molecular Genetics Mus Mutagenesis Operative Surgical Procedures Osmolar Concentration Outcome Pathway interactions Patient Care Patients Pediatric Hospitals Positioning Attribute Preparation Process Proliferating Proteins Rattus Research Research Personnel Resources Risk Series Signal Transduction Small Intestines Sodium Chloride Sprague-Dawley Rats Stomach Stress Surface Techniques Time Tissues Training Universities Urea Urinary tract Urine Urologist Urothelial Cell Waste Products Water consumption Western Blotting Work Writing annexin A5 base carcinogenesis career career development college cytochrome c experience gastrointestinal in vivo in vivo Model interest member migration novel therapeutic intervention operation oxidative DNA damage pressure prevent public health relevance reconstruction repaired research study response scaffold skills tolvaptan tool urinary

项目摘要

DESCRIPTION (provided by applicant): Bladder augmentations using gastrointestinal tisue are frequently performed in children with complex urogenital anomalies. The gastrointestinal tissues in an augmented bladder are significantly more likely to develop cancer than the native bladder tissue, although the mechanisms leading to this risk are not known. High urinary concentrations of urea and other substances contribute to a hyperosmolal bladder microenvironment, and we have recently shown that this hyperosmolal stress disrupts gastrointestinal cell DNA damage signaling and repair. Our central hypothesis is that the hyperosmolal bladder microenvironment impedes the detection and repair of DNA damage in susceptible tissues such as the gastrointestinal portion of an augmented bladder, leading to mutagenesis and ultimately carcinogenesis. The Candidate's immediate goals are to investigate these biological processes in a bladder microenvironment through both in vitro and in vivo models of bladder augmentation, and to further his formal scientific training through didactic instruction and mentored laboratory experience. The Candidate's long-term goals are to explore the translational aspect of this work in clinical trials aimed at the reduction of bladder cancer in patients with bladder augmentations and to become an independent investigator in the broad field of DNA damage and repair as it pertains to the lower urinary tract. The Candidate and his co-mentors, Drs. Peter Stambrook and John Bissler, have developed a comprehensive career development plan with four key components: laboratory experimentation; mentored oversight; didactic instruction through seminars, additional graduate coursework, and training in grant-writing and manuscript preparation; and clinical participation with pediatric urologists in the care of patients with bladder augmentations at Cincinnati Children's Hospital Medical Center (CCHMC). This career development plan will provide the Candidate with the skills and tools necessary to launch an independent research career, with specific translational impact on children with bladder augmentations. The environment at CCHMC and the University of Cincinnati (UC) College of Medicine is highly supportive of the Candidate in the early portion of his research career. Both institutions offer a wealth of resources, both physical and intellectual, that will help foster the Candidate's research career development. These include a number of core facilities directly relevant to this project such as the flow cytometry core, mass spectrometry core, and vivarium at CCHMC as well as numerous faculty members in the Division of Experimental Hematology and Cancer Biology at CCHMC and the Departments of Cancer and Cell Biology and Molecular Genetics, Biochemistry & Microbiology at UC with specifically relevant research interests to the Candidate's project and area of research focus. The research plan described in this application has three Specific Aims: to measure the effects of a hyperosmolal bladder microenvironment on the DNA damage response pathway, to assess the efficacy of DNA repair under a hyperosmolal bladder microenvironment, and to evaluate the activation of cell cycle checkpoints and apoptosis following DNA damage within a hyperosmolal bladder microenvironment. Each of these aims will be investigated in both in vitro and in vivo models of bladder augmentation. In the in vitro portion of these experiments, gastric, colon, small intestine, and bladder epithelial cell lines from the H-2Kb- tsA58 mouse will be gradually adapted to hyperosmolal conditions with sodium chloride or urea, or maintained in isoosmolal conditions, then exposed to different forms of DNA damage. DNA damage will be measured by techniques dependent on the type of DNA damage lesion induced. Activation of the DNA damage response pathway will be evaluated by western blot. The kinetics of DNA repair will be assessed by serial comet assays, and the activity of different DNA repair mechanisms will be assessed by coimmunoprecipitation and immunofluorescence. Cell cycle checkpoint activation will be determined by flow cytometry and by western blot for activation of cell cycle checkpoint proteins. Activation of apoptosis will be quantified by western blot, as well as annexin V binding through flow cytometry. In the in vivo portion of these experiments, we will perform bladder augmentations using stomach, small intestine, or colon tissue as well as sham operations in Sprague Dawley rats. At sequential time points following surgery, the animals will be sacrificed and the gastrointestinal and native bladder tissues will be examined histologically for metaplasia and dysplasia. The tissues will be analyzed for DNA damage using the TUNEL and PANT assays; for activation of the DNA damage response, cell cycle checkpoints, and apoptosis through immunohistochemistry; and activation of DNA repair through EdU labeling.

描述（由申请人提供）：使用胃肠组织进行膀胱扩张术经常在患有复杂泌尿生殖系统异常的儿童中进行。增强膀胱中的胃肠组织比天然膀胱组织更容易患癌症，尽管导致这种风险的机制尚不清楚。尿液中高浓度的尿素和其他物质会导致高渗性膀胱微环境，我们最近发现这种高渗性应激会破坏胃肠道细胞 DNA 损伤信号传导和修复。我们的中心假设是，高渗膀胱微环境阻碍了易感组织（例如增强膀胱的胃肠部分）DNA损伤的检测和修复，导致突变并最终致癌。候选人的近期目标是通过膀胱增强的体外和体内模型研究膀胱微环境中的这些生物过程，并通过教学指导和指导的实验室经验进一步加强他的正式科学培训。候选人的长期目标是探索这项工作在临床试验中的转化方面，旨在减少膀胱增大患者的膀胱癌，并成为 DNA 损伤和修复广泛领域的独立研究者，因为它涉及下尿路。候选人和他的共同导师，博士。 Peter Stambrook 和 John Bissler 制定了一项全面的职业发展计划，其中包括四个关键部分：实验室实验；指导监督；通过研讨会、额外的研究生课程以及拨款写作和手稿准备方面的培训进行教学指导；并与儿科泌尿科医生一起临床参与辛辛那提儿童医院医疗中心 (CCHMC) 膀胱增大患者的护理。该职业发展计划将为候选人提供开展独立研究职业所需的技能和工具，对膀胱增强儿童产生特定的转化影响。 CCHMC 和辛辛那提大学 (UC) 医学院的环境对候选人研究生涯的早期阶段给予了大力支持。两个机构都提供丰富的物质和智力资源，这将有助于促进候选人的研究职业发展。其中包括与该项目直接相关的许多核心设施，例如 CCHMC 的流式细胞术核心、质谱核心和动物饲养室，以及 CCHMC 实验血液学和癌症生物学部门以及癌症和癌症部门的众多教职人员。加州大学细胞生物学和分子遗传学、生物化学和微生物学，研究兴趣与候选人的项目和研究重点领域特别相关。本申请中描述的研究计划有三个具体目标：测量高渗膀胱微环境对DNA损伤反应途径的影响，评估高渗膀胱微环境下DNA修复的功效，以及评估细胞周期检查点的激活以及高渗性膀胱微环境中 DNA 损伤后的细胞凋亡。这些目标中的每一个都将在膀胱增强的体外和体内模型中进行研究。在这些实验的体外部分中，来自 H-2Kb-tsA58 小鼠的胃、结肠、小肠和膀胱上皮细胞系将逐渐适应氯化钠或尿素的高渗条件，或维持在等渗条件，然后暴露于不同形式的 DNA 损伤。 DNA 损伤将通过取决于引起的 DNA 损伤病变类型的技术来测量。 DNA 损伤反应途径的激活将通过蛋白质印迹进行评估。 DNA 修复动力学将通过连续彗星测定进行评估，不同 DNA 修复机制的活性将通过免疫共沉淀和免疫荧光进行评估。细胞周期检查点激活将通过流式细胞术和细胞周期检查点蛋白激活的蛋白质印迹来确定。细胞凋亡的激活将通过蛋白质印迹以及膜联蛋白 V 结合通过流式细胞术进行定量。在这些实验的体内部分，我们将使用胃、小肠或结肠组织以及斯普拉格道利大鼠的假手术进行膀胱扩张。在手术后的连续时间点，将处死动物并对胃肠道和天然膀胱组织进行组织学检查以了解化生和发育不良。将使用 TUNEL 和 PANT 检测对组织进行 DNA 损伤分析；通过免疫组织化学激活 DNA 损伤反应、细胞周期检查点和细胞凋亡；以及通过 EdU 标记激活 DNA 修复。