Cytoplasmic Damage and Genotoxicity

描述(申请人提供)：细胞质损伤的生物学后果在很大程度上是未知的。普遍的教条认为，环境致癌物质，如多环芳烃和氡阿尔法粒子的遗传毒性效应主要是由于对核的直接损害。使用精确的带电粒子微束和双荧光染料分别定位细胞核和细胞胞浆，从而避免无意中穿越细胞核，申请人先前已经证明，细胞质照射实际上是人-仓鼠杂交(AL)细胞CD59基因的突变，同时造成的细胞毒性最小。此外，初步证据表明，活性氧物种参与了这一过程。这引发了以下问题：涉及哪些类型的氧自由基？它们的来源是什么？这种激进的生成过程是否涉及线粒体损伤？靶向细胞质照射诱导的突变是否也发生在人的支气管上皮细胞(环境氡的靶组织)中？最后，细胞质照射能否在哺乳动物细胞中诱导旁观者突变效应，就像申请人最近在AL细胞的核穿越中所展示的那样。为了解决这些问题，提出了一系列5个具体目标来解决4个可检验的假设。AL细胞的CD59基因座和原代人支气管上皮细胞的HPRT基因座将发生突变。拟议的研究将有助于解决细胞质辐射如何导致核内遗传事件的机制。与旁观者诱变效应一起，这项研究将解决一些关于核外靶标和细胞质损伤是如何在哺乳动物细胞中处理的基本问题。

DESCRIPTION (provided by applicant): The biological consequences of cytoplasmic damage are largely unknown. The prevailing dogma considered the genotoxic effects of environmental carcinogens such as polycyclic aromatic hydrocarbons and radon alpha particles as being due mostly to direct damage to the nucleus. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. Furthermore, preliminary evidence suggests that reactive oxygen species mediate this process. This raised the following questions: What types of oxyradicals are involved and what are their origins? Does this radical generating process involve mitochondrial damage? Do the mutations induced by targeted cytoplasmic irradiation occur in human bronchial epithelial cells (target tissues of environmental radon) as well? And finally, can cytoplasmic irradiation induce bystander mutagenic effect in mammalian cells in a manner similar to what the applicant has recently demonstrated with nuclear traversal of the AL cells. To address these issues, a series of 5 specific aims are proposed to address the 4 testable hypotheses. Mutations will be scored at the CD59 locus of the AL cells and at the HPRT locus in primary human bronchial epithelial cells. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.