Our hypothesis is that localized energy deposition by single energetic particles introduces a significantly large insult into a very small cell volume, but leaves most cellular constituents initially unperturbed and able to respond to the damage. Using this localized damaging agent as a means to define a start point (in time and space) for cell signaling, we are beginning to address questions on the timing and localization of protein responses to cell damage. Three types of mammalian cells have been used: the HeLa human cervical carcinoma cell line, the NFF human neonatal foreskin fibroblast cell strain, and the 10T1/2 mouse embryo fibroblast-like cell line. Three fluences, plus the sham control, were used: 0, 1, 2, or 4 particles per cell nuclear area. Cobalt-60 gamma rays were used in parallel experiments for a low LET comparison. After irradiation, cells are incubated for varying lengths of time, then fixed and stored at 4oC. In subsequent in situ assays, the DNA is probed by enzymatic addition of labeled dNTP's to 3'-OH ends, or repair proteins are immunocytochemically labeled. The cells are then viewed by confocal laser scanning microscopy, to locate and quantify the fluoresceinated probes within the cell. Results so far have shown that irradiated HeLa cells show a distinct pattern of nuclear protein re-localization, that is a function of the LET/track structure of the radiation, the dose/fluence of the radiation, and the time after irradiation. In contrast, the human NFF fibroblasts show a more spread out concentration of repair proteins in the nucleus. The mouse 10T1/2 cell measurements have just begun, suggesting a novel method to visualize early events in the process of DNA repair in mammalian cells.
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