DNA Damage and Response in the Bladder Microenvironment.

描述(申请人提供)：在患有复杂的泌尿生殖系统异常的儿童中，经常使用胃肠道组织进行膀胱扩大术。尽管导致这种风险的机制尚不清楚，但增大的膀胱中的胃肠道组织比天然的膀胱组织更有可能发生癌症。尿中高浓度的尿素和其他物质导致了高渗的膀胱微环境，我们最近的研究表明，这种高渗应激破坏了胃肠道细胞DNA损伤信号和修复。我们的中心假设是，高渗透压的膀胱微环境阻碍了敏感组织(如增大的膀胱的胃肠道部分)DNA损伤的检测和修复，从而导致突变和最终致癌。这位候选人的近期目标是通过体外和体内的膀胱增大模型来研究膀胱微环境中的这些生物学过程，并通过教学指导和有指导的实验室经验来深化他的正规科学训练。候选人的长期目标是在旨在减少膀胱增大患者膀胱癌的临床试验中探索这项工作的翻译方面，并成为与下尿路有关的DNA损伤和修复广泛领域的独立研究员。这位候选人和他的共同导师Peter Stambrook博士和John Bissler博士已经制定了一个全面的职业发展计划，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和手稿准备方面的培训进行教学指导；以及在辛辛那提儿童医院医疗中心(CCHMC)与儿科泌尿科医生一起临床参与膀胱扩张术患者的护理。这一职业发展计划将为应聘者提供必要的技能和工具，以开始独立的研究职业生涯，并对患有膀胱增大的儿童产生具体的翻译影响。CCHMC和辛辛那提大学(UC)医学院的环境高度支持候选人在其研究生涯的早期阶段。这两个机构都提供了丰富的物质和智力资源，这将有助于促进候选人的研究生涯发展。其中包括一些与该项目直接相关的核心设施，如CCHMC的流式细胞仪核心、质谱学核心和间隔膜，以及CCHMC实验血液学和癌症生物学分部以及加州大学癌症和细胞生物学系以及分子遗传学、生物化学和微生物学系的众多教职员工，他们与候选人的项目和研究重点领域有明确的相关研究兴趣。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，评估高渗透膀胱微环境下DNA修复的有效性，以及评估高渗透膀胱微环境中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体外和体内膀胱扩大术模型中进行研究。在这些实验的体外部分，来自H-2kb-tsA58小鼠的胃、结肠、小肠和膀胱上皮细胞株将逐渐适应含有氯化钠或尿素的高渗透条件，或保持在等渗条件下，然后暴露于不同形式的DNA损伤。DNA损伤将采用取决于所致DNA损伤类型的技术进行测量。DNA损伤反应通路的激活将通过蛋白质印迹进行评估。DNA修复的动力学将通过系列彗星分析来评估，不同DNA修复机制的活性将通过免疫共沉淀和免疫荧光来评估。细胞周期检查点的激活将通过流式细胞仪和蛋白质印迹来确定，以激活细胞周期检查点蛋白。免疫印迹法检测细胞凋亡的激活程度，流式细胞术检测膜联蛋白V结合情况。在这些实验的活体部分，我们将使用胃、小肠或结肠组织进行膀胱增大，并对SpragueDawley大鼠进行假手术。在手术后的连续时间点，动物将被处死，胃肠道和自然膀胱组织将被组织学检查化生和异型增生。组织将使用TUNEL和PANT分析进行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；通过EDU标记激活DNA修复。 与公共健康相关：膀胱内的细胞暴露在高浓度的废物产品中，如尿素，通常存在于尿液中，可能会干扰身体识别和修复其遗传密码受损的能力。这可能会导致膀胱癌，特别是在接受手术重建膀胱的儿童中。

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. PUBLIC HEALTH RELEVANCE: Exposure of cells inside the bladder to high concentrations of waste products like urea, which are normally present in the urine, can interfere with the ability of the body to recognize and repair damage to its genetic code. This may lead to cancer of the bladder, especially in children who have their bladders surgically reconstructed.