Particulate Cr(VI) Toxicology in Human Lung Epithelial Cells and Fibroblasts

人肺上皮细胞和成纤维细胞中的颗粒 Cr(VI) 毒理学

基本信息

批准号：
7730926
负责人：
John Pierce Wise
金额：
$ 30.53万
依托单位：
UNIVERSITY OF SOUTHERN MAINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-06 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7730926
关键词：
Anaphase Arts Biological Biological Assay Bypass Cancer Etiology Carcinogen exposure Carcinogens Cell Cycle Cell Death Cell division Cells Centrosome Cessation of life Chromates Chromosomal Instability Chronic Cytogenetics DNA Double Strand Break Data Epithelial Cells Event Exposure to Failure Far-Western Blotting Fibroblasts G2/M Arrest Gene Expression Genomic Instability Goals Growth Human Immunofluorescence Immunologic Lead Link Lung Lung Neoplasms Malignant neoplasm of lung Measures Metals Modeling Molecular Molecular Cytogenetics Neoplastic Cell Transformation Particulate Phenotype Public Health RNA Interference Reporting Research Research Proposals Risk Role Spectral Karyotyping System Techniques Testing Toxicology Western Blotting carcinogenesis cell growth chromium hexavalent ion design interest prevent public health relevance tumor

项目摘要

DESCRIPTION (provided by applicant): Hexavalent chromium (Cr(VI)) or chromate is a major public health concern. Chromates, particularly the insoluble compounds, are well-established human lung carcinogens. The proposed research focuses on the mechanisms of Cr(VI)-induced carcinogenesis, which are currently unknown. Recent studies indicate that particulate Cr(VI) induces chromosome instability, which is often seen in human lung tumors. However, studies of the mechanisms of Cr(VI)-induced chromosome instability have not been done. Our preliminary data show that chronic exposure to particulate Cr(VI) induces centrosome amplification and spindle assembly checkpoint bypass, and thus, the goal of this research is to understand the mechanisms that cause these events leading to Cr(VI)-induced chromosome instability and carcinogenesis. We will test the hypothesis that particulate Cr(VI) induces prolonged G2/M arrest leading to centrosome amplification and spindle assembly checkpoint bypass resulting in chromosome instability and carcinogenesis through four interrelated specific aims: 1) Demonstrate that particulate Cr(VI)-induced DNA double strand breaks cause prolonged G2/M arrest leading to centrosome amplification; 2) Identify the molecular mechanisms of how Cr(VI)-induced G2/M arrest uncouples centrosome and cell division causing centrosome amplification focusing on Mps1 and Nek2A; 3) Determine the role of Nek2A, Mad1 and Mad2 interactions in particulate Cr(VI)-induced spindle assembly checkpoint bypass; and 4) Identify those cellular and molecular phenotypic changes revealed in Specific Aims 1-3 that occur in cells that escape Cr(VI)-induced death and growth arrest and in cells that are neoplastically transformed by Cr(VI). These four aims will use a combination of established and state-of-the-art toxicological, cytogenetic, and molecular biological techniques with the following approach: 1) Cr(VI)-induced centrosome amplification and spindle assembly checkpoint bypass will be measured with immunofluorescence and cytogenetic assays; 2) Gene expression, immunolocalization and RNAi studies will be used to determine the mechanisms of centrosome amplification and checkpoint bypass ; and 3) Cell growth and neoplastic transformation assays and studies in human tumors will be used to determine the fate of cells with particulate Cr(VI)-induced chromosome instability. Results will lead to the first reports of detailed information of the interactions of Cr(VI) with centrosome duplication machinery, spindle assembly checkpoint and the first characterizations of chromosome instability in tumors from Cr(VI)-exposed workers. This research is significant because it will provide: 1) An understanding of particulate Cr(VI)'s carcinogenic mechanism; 2) Essential information to better assess the risk of exposure to particulates; and 3) A mechanistic approach for further study of Cr(VI), other metals, and lung cancer in general. PUBLIC HEALTH RELEVANCE: Hexavalent chromium (Cr(VI)) is a human lung carcinogen. These studies will advance our basic understanding of the cellular and molecular mechanisms of how cells protect against Cr(VI)-induced genomic instability. Our findings will help us design new treatments and approaches to reduce or prevent Cr(VI)-induced lung cancer and possibly other metals, which continue to be major public health concerns. Finally, we will have established a mechanistic model in a human lung cell system that will allow us to conduct studies for other factors important for preventing human lung cancer both generally and by other agents.

描述（由申请人提供）：六价铬 (Cr(VI)) 或铬酸盐是主要的公共卫生问题。铬酸盐，特别是不溶性化合物，是公认的人类肺癌致癌物。拟议的研究重点是 Cr(VI) 诱导致癌的机制，目前尚不清楚。最近的研究表明，颗粒 Cr(VI) 会导致染色体不稳定，这在人类肺部肿瘤中很常见。然而，Cr(VI)引起的染色体不稳定机制的研究尚未完成。我们的初步数据表明，长期暴露于颗粒 Cr(VI) 会诱导中心体扩增和纺锤体组装检查点旁路，因此，本研究的目标是了解导致这些事件导致 Cr(VI) 诱导的染色体不稳定的机制，以及致癌作用。我们将通过四个相互关联的具体目标来检验颗粒 Cr(VI) 诱导延长的 G2/M 停滞，从而导致中心体扩增和纺锤体组装检查点旁路，从而导致染色体不稳定和致癌的假设：1) 证明颗粒 Cr(VI) 诱导的 DNA双链断裂导致 G2/M 期延长停滞，导致中心体扩增； 2) 确定 Cr(VI) 诱导的 G2/M 阻滞如何解偶联中心体和细胞分裂导致中心体扩增（重点关注 Mps1 和 Nek2A）的分子机制； 3) 确定 Nek2A、Mad1 和 Mad2 相互作用在颗粒 Cr(VI) 诱导的纺锤体组装检查点旁路中的作用； 4) 识别特定目标 1-3 中揭示的细胞和分子表型变化，这些变化发生在逃避 Cr(VI) 诱导的死亡和生长停滞的细胞中以及在被 Cr(VI) 肿瘤转化的细胞中。这四个目标将结合使用已建立的和最先进的毒理学、细胞遗传学和分子生物学技术，并采用以下方法：1) Cr(VI) 诱导的中心体扩增和纺锤体组装检查点旁路将通过免疫荧光进行测量和细胞遗传学检测； 2）基因表达、免疫定位和RNAi研究将用于确定中心体扩增和检查点旁路的机制； 3) 人类肿瘤中的细胞生长和肿瘤转化测定及研究将用于确定颗粒 Cr(VI) 诱导的染色体不稳定的细胞的命运。结果将导致首次报告 Cr(VI) 与中心体复制机制、纺锤体组装检查点相互作用的详细信息，以及接触 Cr(VI) 的工人肿瘤中染色体不稳定性的首次表征。这项研究意义重大，因为它将提供： 1) 了解颗粒 Cr(VI) 的致癌机制； 2) 更好地评估接触颗粒物风险的基本信息； 3) 进一步研究 Cr(VI)、其他金属和肺癌的机制方法。公共卫生相关性：六价铬 (Cr(VI)) 是一种人类肺癌致癌物。这些研究将增进我们对细胞如何防止 Cr(VI) 诱导的基因组不稳定性的细胞和分子机制的基本了解。我们的研究结果将帮助我们设计新的治疗方法和方法，以减少或预防六价铬诱发的肺癌以及可能的其他金属，这些金属仍然是主要的公共卫生问题。最后，我们将在人类肺细胞系统中建立一个机制模型，这将使我们能够研究对于一般预防人类肺癌和通过其他药物预防人类肺癌很重要的其他因素。