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

描述（由申请人提供）：六价铬（Cr（VI））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，是公认的人类肺部致癌物。拟议的研究重点是Cr（VI）诱导的致癌机制，这是目前未知的。最近的研究表明，颗粒铬（VI）诱导染色体不稳定，这是常见的人类肺肿瘤。然而，铬（VI）诱导的染色体不稳定性的机制的研究还没有完成。我们的初步数据表明，慢性暴露于颗粒铬（VI）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的机制，导致铬（VI）诱导的染色体不稳定和致癌。我们将通过以下四个相互关联的具体目标来验证颗粒Cr（VI）诱导的G2/M期阻滞延长导致中心体扩增和纺锤体组装检查点绕过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期阻滞延长导致中心体扩增; 2）确定Cr（VI）诱导的G2/M期阻滞如何使中心体和细胞分裂解偶联，引起中心体扩增的分子机制，重点关注Mps 1和Nek 2A; 3）确定Nek 2A、Mad 1和Mad 2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;和4）鉴定特异性目的1-3中揭示的发生在逃避Cr（VI）诱导的死亡和生长停滞的细胞中以及发生在被Cr（VI）肿瘤转化的细胞中的那些细胞和分子表型变化。这四个目标将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的组合，并采用以下方法：1）用免疫荧光和细胞遗传学测定来测量Cr（VI）诱导的中心体扩增和纺锤体组装检查点旁路; 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绕过的机制;和3）人类肿瘤中的细胞生长和肿瘤转化分析和研究将用于确定具有颗粒Cr（VI）诱导的染色体不稳定性的细胞的命运。结果将导致第一次报告的详细信息的相互作用的Cr（VI）与中心体复制机制，纺锤体组装检查点和第一个特征的染色体不稳定性的肿瘤中的Cr（VI）暴露工人。这项研究是重要的，因为它将提供：1）颗粒铬（VI）的致癌机制的理解; 2）基本信息，以更好地评估暴露于颗粒的风险;和3）的机制方法，为进一步研究铬（VI），其他金属，和肺癌的一般。公共卫生相关性：六价铬（Cr（VI））是一种人类肺癌致癌物。这些研究将推进我们对细胞如何保护免受Cr（VI）诱导的基因组不稳定性的细胞和分子机制的基本理解。我们的研究结果将帮助我们设计新的治疗方法和方法，以减少或预防铬（VI）诱导的肺癌和可能的其他金属，这仍然是主要的公共卫生问题。最后，我们将在人肺细胞系统中建立一个机制模型，这将使我们能够对预防人类肺癌的其他重要因素进行研究。

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.