The long-term objective of this study is to provide a mechanistic basis for the development of new therapeutic approaches in radiation therapy. We have observed that low doses of radiation can induce several cellular processes that we believe are important in radiation therapy: apoptosis, differentiation, senescence and impaired endothelial cell wound response. Our working hypothesis is that all four of these processes are induced by a limited number of biochemical signals that are produced in irradiated cells. Our objective is to identify these signals and to elucidate the mechanism by which they bring about these cellular responses. The model systems we will use have been selected because they are relevant to radiation therapy and because they allow quantitative single- cell analysis. Apoptosis will be determined by in situ hybridization of PC-3 human prostate cancer cells for expression of the apoptosis marker TRPM-2. Differentiation will be determined by the expression of differentiation-=specific cytokeratins in F9 embryonal carcinoma cells, as a model for retinoic acid-mimetic effects and by laminin expression in retinoic acid treated F9 cells as a model for CAMP-mimetic effects. Senescence will be determined by expression of a senescence-specific fibronectin epitope in normal human fibroblasts (CSC-3-3). The endothelial cell wound healing response will be studied in monolayers of bovine microvascular endothelial cells using digital imaging to measure actin fiber alignment. Two strategies will be employed in our mechanistic studies. The first strategy is to determine the impact of modifiers of DNA damage, protein thiol oxidation and lipid metabolism on the effect of radiation on the regulatory endpoints described above. The second strategy is to use these regulatory endpoints as bioassays to detect bioactive radiation products, or to test potential radiomimetic agents. We will test our multi-signal hypothesis by determining the effect of radiation on the dose response relationship to other signaling agents.
