DNA Damage and Response in the Bladder Microenvironment.

描述（由申请人提供）：使用胃肠道组织的膀胱扩大术经常用于患有复杂泌尿生殖系统异常的儿童。增强膀胱中的胃肠道组织比天然膀胱组织更容易发生癌症，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有助于高渗透压膀胱微环境，我们最近发现这种高渗透压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，高渗透压膀胱微环境阻碍检测和修复敏感组织中的DNA损伤，如增强膀胱的胃肠道部分，导致诱变和最终致癌。候选人的直接目标是通过膀胱增大的体外和体内模型来研究膀胱微环境中的这些生物学过程，并通过教学指导和指导实验室经验来进一步进行正式的科学培训。候选人的长期目标是在临床试验中探索这项工作的转化方面，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Peter Stambrook博士和John Bissler博士制定了一个全面的职业发展计划，其中包括四个关键组成部分：实验室实验;指导监督;通过研讨会进行教学指导，额外的研究生课程以及赠款写作和手稿准备培训;和临床参与儿童泌尿科医生在辛辛那提儿童医院医疗中心（CCHMC）的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，并对膀胱增大儿童产生特定的转化影响。CCHMC和辛辛那提大学（UC）医学院的环境在候选人研究生涯的早期阶段给予了高度支持。这两个机构提供了丰富的资源，无论是身体和智力，这将有助于促进候选人的研究职业发展。其中包括一些与该项目直接相关的核心设施，如CCHMC的流式细胞术核心，质谱核心和动物园，以及CCHMC实验血液学和癌症生物学部门以及癌症和细胞生物学和分子遗传学部门的众多教职员工，生物化学和微生物学在UC与具体相关的研究兴趣，以候选人的项目和研究重点领域。本申请中描述的研究计划有三个特定目的：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，以及评价高渗膀胱微环境中DNA损伤后细胞周期检查点的激活和细胞凋亡。这些目标中的每一个都将在膀胱增大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-tsA 58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，或维持在等渗条件下，然后暴露于不同形式的DNA损伤. DNA损伤将通过依赖于诱导的DNA损伤损伤类型的技术来测量。将通过蛋白质印迹法评价DNA损伤反应途径的活化。DNA修复的动力学将通过系列彗星试验进行评估，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。将通过流式细胞术和蛋白质印迹法测定细胞周期检查点蛋白的活化，以确定细胞周期检查点活化。将通过蛋白质印迹以及通过流式细胞术的膜联蛋白V结合来定量细胞凋亡的活化。在这些实验的体内部分中，我们将在Sprague道利大鼠中使用胃、小肠或结肠组织以及假手术进行膀胱增大。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，以确定化生和异型增生。将使用TUNEL和PANT试验分析组织的DNA损伤;通过免疫组织化学分析DNA损伤反应、细胞周期检查点和细胞凋亡的激活;以及通过EdU标记分析DNA修复的激活。

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.