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DNA Damage and Response in the Bladder Microenvironment.

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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.
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