Project 3 will analyze the in vitro effects of radiomodulatory agents (RMAs) on human cells - from normal individuals (Aim 1), from patients with known DMA repair disorders (Aim 2), and from a panel of radiosensitive patients with undiagnosed disorders (Aim 3). The RMAs to be tested will have been identified by high throughput screening (Core D), followed by additional characterization in yeast and mouse models (Projects 2 and 3). We are uniquely poised to perform the studies outlined in Project 3 because pur laboratory has a large cell repository and long-standing experience with functional and diagnostic DNA repair assays, such as radiosensitivity testing by colony survival, radioresistant DNA synthesis, IRIF formation, serine kinase activity, and comet assays. Spectral karyotyping will identify characteristic chromosomal aberrations and help to quantify the effects of candidate RPAs (Core E). In the final stages of these analyses, intracellular tyrosine kinase signaling and global protein expression microarrays will profile the downstream effects of these compounds (Core D). Through the use of radiosensitive (RS) cells with known mutations in 1) ATM-related pathways (A-T, NBS, Mre11), 2) SMC1-independent radiosensitivity (Fanconi pathway), and 3) non-homologous (illegitimate) end joining repair (LIG4), candidate RMAs will be grouped into operational categories and common mechanisms of action. As new insights into the mechanisms of human radiosensitivity are discovered, a RS panel of >40 lymphoblastpid cell lines will allow these categories to be refined and applied towards the treatment of specific molecular defects and the underlying mechansims of action of the radiomodulatory compounds. The added value of these coordinated center studies will include the identification of a) new bipmarkers for evaluating human radiation exposure and b) novel radiomodulatory agents for mitigating and preventing the full effects of radiation damage.
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