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

描述(申请人提供)：六价铬(六价铬)或铬酸盐是一个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。拟议的研究集中在铬(VI)诱导的致癌机制上，目前尚不清楚。最近的研究表明，微粒铬(VI)会导致染色体不稳定，这在人类肺肿瘤中很常见。然而，关于铬(VI)诱导染色体不稳定的机制还没有研究。我们的初步数据显示，慢性暴露于颗粒铬(VI)会导致中心体放大和纺锤体组装检查点绕过，因此，本研究的目的是了解这些事件导致铬(VI)诱导的染色体不稳定和致癌的机制。我们将通过四个相互关联的特定目标来验证这一假设，即颗粒铬(VI)诱导的G2/M期停滞延长，导致中心体放大和纺锤体组装检查点绕过，从而导致染色体不稳定和癌变：1)证实颗粒铬(VI)诱导的DNA双链断裂导致长时间的G2/M停滞导致中心体放大；2)确定铬(VI)诱导的G2/M停滞、中心体解偶联和细胞分裂导致中心体放大的分子机制；3)确定Nek2A、MAD1和MAD2相互作用在颗粒铬(VI)诱导的纺锤体组装检查点旁路中的作用；以及4)鉴定在特定目标1-3中发现的细胞和分子表型变化，这些变化发生在逃脱铬(VI)诱导的死亡和生长停滞的细胞以及被铬(VI)转化为肿瘤的细胞中。这四个目标将使用现有的和最先进的毒理学、细胞遗传学和分子生物学技术相结合的方法：1)将使用免疫荧光和细胞遗传学分析来测量铬(VI)诱导的中心体扩增和纺锤体组装检查点绕过；2)将用于基因表达、免疫定位和RNAi研究，以确定中心体扩增和检查点绕过的机制；以及3)将用于细胞生长和肿瘤转化的分析以及对人类肿瘤的研究，以确定具有微粒铬(VI)诱导的染色体不稳定的细胞的命运。这些结果将导致关于铬(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的首次报道，以及首次描述暴露于铬(VI)工人的肿瘤中染色体不稳定性的特征。这项研究具有重要意义，因为它将提供：1)对微粒铬(VI)S致癌机制的了解；2)更好地评估接触微粒风险的基本信息；以及3)进一步研究铬(VI)、其他金属和一般肺癌的机制方法。

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.