DNA Damage and Response in the Bladder Microenvironment.

膀胱微环境中的 DNA 损伤和反应。

• 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
• 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

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）：使用胃肠道tisue的膀胱增加在患有复杂的泌尿生殖器异常的儿童中经常进行。增强膀胱中的胃肠道组织比天然膀胱组织更有可能发生癌症，尽管导致这种风险的机制尚不清楚。尿素和其他物质的高尿液浓度有助于高温膀胱微环境，并且我们最近表明，这种高粘应力破坏了胃肠道细胞DNA DNA损伤信号传导和修复。我们的中心假设是，高温膀胱微环境阻碍了易感组织中DNA损伤的检测和修复，例如增强膀胱的胃肠道部分，导致诱变并最终导致致癌。候选人的直接目标是通过体外和体内膀胱增强模型在膀胱微环境中调查这些生物学过程，并通过教学教学和指导的实验室经验来进一步进一步进行他的正式科学培训。候选人的长期目标是在旨在减少膀胱增强患者膀胱癌的临床试验中探索这项工作的转化方面降低尿路。候选人及其联合给予人。彼得·斯坦布鲁克（Peter Stambrook）和约翰·比斯勒（John Bissler）制定了一项全面的职业发展计划，其中包含四个关键组成部分：实验室实验；指导的监督；通过研讨会，额外的研究生课程以及授予书写和手稿准备的教学教学；以及辛辛那提儿童医院医疗中心（CCHMC）的儿科泌尿科医生在儿科泌尿科医生的临床参与中。这项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，并对具有膀胱增强的儿童产生具体的翻译影响。 CCHMC和辛辛那提大学（UC）医学院的环境在他的研究生涯的早期中高度支持候选人。这两家机构都提供大量的物理和知识分子，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的流式细胞仪核心，质谱核心和Vivarium以及CCHMC实验血液学和癌症生物学的众多教师UC的细胞生物学和分子遗传学，生物化学和微生物学，具有针对候选人的项目和研究重点领域的特定相关研究兴趣。本应用中描述的研究计划具有三个具体目的：测量高玻璃体微环境对DNA损伤响应途径的影响，以评估在高温膀胱微环境下DNA修复的功效，并评估细胞周期检查点的激活在高玻璃微环境中DNA损伤后DNA损伤后的凋亡。这些目标都将在体外和体内膀胱增强模型中进行研究。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具有氯化钠或尿素的高温条件，或在等溶液条件下维持，然后暴露，然后暴露，然后暴露在外到不同形式的DNA损伤。 DNA损伤将通过取决于诱导的DNA损伤类型的技术来测量。 DNA损伤响应途径的激活将通过Western印迹评估。 DNA修复的动力学将通过串行彗星测定进行评估，不同DNA修复机制的活性将通过共免疫沉淀和免疫荧光评估。细胞周期检查点的激活将通过流式细胞仪和Western印迹确定，以激活细胞周期检查点蛋白。细胞凋亡的激活将通过蛋白质印迹以及通过流式细胞仪结合膜联蛋白V进行量化。在这些实验的体内部分中，我们将使用胃，小肠或结肠组织以及Sprague Dawley大鼠的假手术进行膀胱增大。在手术后的顺序时间点，将处死这些动物，并将在组织学上检查胃肠道和天然膀胱组织，以了解化生和发育不良。将使用TUNEL和PANT分析分析组织的DNA损伤。通过免疫组织化学激活DNA损伤反应，细胞周期检查点和凋亡；通过EDU标记激活DNA修复。