Project 2. Our working hypothesis in this Program is that chemical species generated by phagocytes at sites of inflammation represent a causative link to human disease. Within the themes of colitis, neutrophils and NO resistance shared across the Program, Project 2 focuses on reactions of the chemical mediators of inflammation with DNA, lipids, carbohydrates and proteins in epithelial cells to generate toxic products that lead to altered physiology, cell death and mutations associated with cancer. The objectives are to explore the mechanisms of this damage, to develop surrogate markers of the labile inflammatory mediators, and to develop candidate biomarkers of inflammation. In the last grant period, we developed analytical methods for DNA, RNA and protein damage products (with Core 1) and applied these methods to cultured cells (with Project 3) and to animal models of inflammation (with Projects 4, Core 2), with the goal of linking the chemical models developed in Project 1 with Projects 3 and 4. In the proposed studies, we will expand biomarker development to include DNA, RNA and protein damage products derived from lipid peroxidation and neutrophils, and quantify these markers in inflamed and cancerous human tissues.
The Specific Aims are as follow:
Aim 1. Define the spectrum of DNA, RNA and protein lesions produced by NO, N{2}O{3}, ONOO* and HOCI in isolated nucleic acids and cultured cells (with Projects 1, 3,4). We will develop analytical methods for DNA, RNA and protein damage products: chlorotyrosine, nitrotyrosine;halogenated nucleobases, guanine oxidation products;and RNA versions of all DNA lesions. These methods will then be applied to model cell systems with Projects 1, 3 and 4.
Aim 2. Apply methods and define lesion spectra in mouse models of inflammation (with Projects 1, 3, 4). We will establish the utility of biomarkers in animal models of inflammation and colitis with Project 4 and quantify DNA, RNA and protein lesions in tissues in coordination with other projects to develop predictive models for production of reactive nitrogen and halogen species.
Aim 3. Apply biomarker methods to inflamed and cancerous human tissues (with Projects 1,3, 4). The biomarkers will be translated to samples of normal human colon tissue and from patients with Crohn's disease and ulcerative colitis, with the goal of comparing the profiles from human and mouse models. We will also develop methods to analyze markers of NO and HOCI chemistry in archived paraffin blocks of melanoma tumors, with the goal of distinguishing the effects of NO derived from the melanoma cells per se, from those of the innate immune cells attacking the tumor.
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