This grant application seeks to prove that the mechanism of acute esophageal injury in fractionated irradiation is through loss of critical esophageal stem cells (ESCs) by lipid peroxidation (LP), and that this process is modulated by expression of a hemagglutinin (HA) epitope-tagged human MnSOD transgene in these cells. We have developed a novel assay for murine Sca1-lin-Thy1+ ESCs which differentiate to cytokeratin-positive squamous or mucin-positive glandular cells in vitro and in vivo. We have shown that ESCs from green fluorescent protein-positive (GFP+) mice engraft in a direct cell dose-response fashion following intravenous injection and repopulate squamous and glandular tissue areas in irradiated esophagus and small intestine, respectively, of GFP-negative (GFP-) recipients. We have demonstrated in C3H/HeNsd and C57BL/6J mice that single or fractionated irradiation produces esophagitis and >25 percent weight loss as a premorbid marker of death by 30 days, and that while there is significant apoptosis, the toxicity is associated with lipid peroxidation (LP) and thiol depletion in esophagus in vivo. We have shown that ESC numbers decrease after irradiation, but are preserved if mice receive intraesophageal injection of hemagglutinin (HA)-MnSOD-plasmid/liposome (PL) complex prior to irradiation. The first specific aim tests the hypothesis that peroxidized lipids mediate cell death in fractionated irradiated esophagus in vivo and in explanted organ cell culture in vitro. The second specific aim tests the hypothesis that LP causes loss of ESCs after irradiation. The third specific aim tests the hypothesis that transplantation of bone marrow stem cells as another source of ESCs will improve tissue recovery after irradiation. In each aim, we will test the overall hypothesis that neutralization of irradiation-induced ROS production and LP in ESCs by administration of an epitope-tagged HA-MnSOD-PL decreases irradiation esophagitis and late stricture. Methods include: fractionated irradiation, measurement of ROS, LP, and DNA strand breaks by Comet assay, Apotag assay for apoptosis, immunohistochemical localization of p53 increase in ESCs and of HA-MnSOD delivered by PL in situ and in esophagus organ explant culture; FACS sorting and serial plating techniques for isolation of ESCs, and coculture of purified, unirradiated green fluorescent protein-positive (GFP+) ESCs with irradiated GFP-negative (GFP-) ESCs. These studies should identify the critical cells involved in irradiation esophagitis, define the mechanism of irradiation killing and potential for protection by antioxidant transgene therapy.
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