CHROMOSOME CHANGES, GENOME INSTABILITY, BYSTANDER EFFECT

描述（由申请人提供）：辐射诱发的旁观者效应是一种被广泛描述的现象，它挑战了公认的辐射作用范式，但在机制层面上仍然知之甚少。基因组不稳定性可能被认为是癌症发展的驱动力，而电离辐射已被证明是这种潜在反应的有效诱导剂。这两种非靶向效应之间的关系尚未确定。假设辐射诱导的旁观者反应和基因组不稳定反应是通过活性自由基介导的途径机制联系在一起的。此外，我们假设旁观者反应细胞将在与直接辐射损伤细胞相同的水平上表现出基因组不稳定性。这一发现可能会增加人们对低剂量/极低剂量辐射影响的关注。我们已经明确地展示了一个旁观者效应与精密带电粒子微束，其中核击中细胞与非击中的旁观者细胞被故意区分。我们将确定旁观者信号或非核[细胞质]照射是否会诱导哺乳动物细胞的基因组不稳定。在旁观者和不稳定反应途径中的反应性自由基需求将使用特定的化学抑制剂和基因特异性siRNA进行测试

DESCRIPTION (provided by applicant): The radiation-induced bystander effect is a widely described phenomenon which challenges the accepted paradigm of radiation action, but remains poorly understood at a mechanistic level. Genomic instability may be considered to be a driving force in the development of cancer, and ionizing radiations have been shown to be efficient inducers of this latent response. The relationship between these two nontargeted effects has not been established. It is hypothesized that the radiation-induced bystander response and the genomic instability response are mechanistically linked through reactive radical species mediated pathways. Furthermore, we hypothesize that bystander responding cells will show genomic instability at levels equal to that shown by directly radiation damaged cells. Such a finding could augment concerns about the effects of low/very low doses of radiation. We have definitively shown a bystander effect with a precision charged particle microbeam, where nuclear hit cells were knowingly discriminated from non-hit bystander cells. We will determine whether bystander signaling or non-nuclear [cytoplasmic] irradiation can induce genomic instability in mammalian cells. Reactive radical species requirement in bystander and instability response pathways will be tested using specific chemical inhibitors and gene specific siRNA approaches. Frequencies of chromosomal aberrations will be assessed as a function of time. Chromatid/chromosome aberration analysis plus/minus a telomere peptide nucleic acid probe and complete karyotype analyses by multiplex fluorescence in-situ hybridization [m-FISH] in human fibroblast cells, and human chromosome 11 m-BAND analysis in human-hamster hybrid AL cells will be undertaken. Further, we will compare 2D versus 3D responses by studying human bronchial epithelial cells both as 2D monolayers and as 3D airway like tissues, as more closely reflecting cellular behavior in vivo. Four Specific Aims and subsidiary hypotheses will aid in the definition of the mechanistic relationship between the bystander response and genomic instability. Interaction between the Aims of each project will ensure significant progress to fulfillment of the central hypothesis of this P01 in defining the mechanism/s of the radiation-induced bystander effect.