) The overall research objectives of this program project are directed toward understanding the mechanistic bases for the recognition, processing and consequences of radiation induced lesions, with a particular emphasis on radon progeny alpha particles and their impact on human health. Nuclear DNA changes significantly impact human health with clear relationships to cell death, mutation and oncogenic changes. There is uncertainty however as to the potential contributions of stresses induced in non-nuclear targets to long term deleterious effects. It is clear that molecular interplay between membrane, cytoplasm and nucleus contributes to damage recognition, signaling and response pathways. An understanding of the role of cellular components in radiation responses can efficiently be obtained by use of a microbeam. In this project we will used defined influences (down to 1) or 100 keV/um alpha-particles to irradiate cell nucleus, cytoplasm or known cells in a population. Non-hit cells (bystanders) may be stressed by reactive oxygen species and/or affected by induced mobile cytokines. Mouse embryo fibroblasts derived from knockout mice, wild-type, p53-, and atm- will be irradiated both as single and as pair wise populations. Responses will be separately assessed in hit versus non-hit cells. We will ask the questions: Does particle spacing influence nucleus based responses? Does cytoplasmic irradiation initiate a biological response? Does cell specific irradiation initiate responses in non-hit cells on a genotype dependent basis? We will examine incidence of micronuclei; apoptosis, cell growth and development; cell cycle delay; and gene expression both by protein expression and localization, and mRNA by RT-PCR. In concert with the other projects of this program project, the combination of endpoints and precise site specific microbeam irradiation to address definitive question will establish mechanistic parameters and will define alpha-particle responsiveness at low doses with emphasis on the contribution of the bystander effect.
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