Comparative genomics of species-specific tumor promotion

描述（由申请人提供）：本提案的总体目标是开发一种创新的毒物基因组学方法，用于定义细胞基因表达级联中的关键元素，这些元素在暴露于物理或化学试剂后会产生不同物种的差异效应。由于传统的毒物基因组学方法不能可靠地解决毒性的跨物种差异，并且主要依赖于将转录变化与病理终点联系起来的相关证据，因此需要一种新的方法。缺失的部分是对改变基因列表中包含的因果关系和由此产生的毒理学结果的机制理解。这项研究的首要假设是，定义基因表达变化的级联网络对于理解化学物质的毒性和预测它们的跨物种保护反应至关重要。这些变化包括初级、次级和三级基因表达反应之间的相互关系。这一假设将使用独特的基因组工具组合进行测试，这些工具可以解剖暴露于典型肝毒物苯巴比妥后的转录反应，苯巴比妥对啮齿动物和人类具有不同的致癌活性。本提案的具体目的是：(1)通过使用全基因组微阵列进行基因表达时序研究，验证苯巴比妥会引起小鼠和人原代肝细胞基因表达暂时可解决的变化的假设；(2)通过RNA干扰分别阻断早期诱导的关键调控基因，并测量其对下游基因表达变化的影响，验证苯巴比妥基因表达级联的假设；(3)验证了小鼠肝细胞中存在与细胞早期增殖事件相关的特异性次级基因表达应答的假设，而在人肝细胞中不存在。通过确定次级转录反应和主要调控基因之间的相互关系，我们将能够将增殖子网络与负责跨物种差异的主要事件或受体机械地联系起来。通过这项研究，我们相信一种新的毒理学方法将被开发出来，可以通过比较物种间的信号级联来预测毒性反应的保护，并为许多化学制剂的作用机制提供新的见解。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to develop an innovative toxicogenomics approach for defining critical elements in cellular gene expression cascades that confer differential effects across species following exposure to physical or chemical agents. A new approach is required since traditional toxicogenomic methods have not reliably addressed cross-species differences in toxicity and have primary relied on correlative evidence to associate transcriptional changes with a pathological end point. The missing component is a mechanistic understanding of the cause-and-effect relationships contained within the lists of altered genes and the resulting toxicological outcome. The overarching hypothesis for the proposed research is that defining the cascading network of gene expression changes is essential for understanding the toxicity of chemicals and predicting their cross-species conservation of response. These changes include the interrelationships between the primary, secondary, and tertiary gene expression responses. This hypothesis will be tested using unique combination of genomic tools that can dissect these transcriptional responses following exposure to a prototypical hepatotoxicant, phenobarbital that has different carcinogenic activity in rodents and humans. The specific aims of this proposal are: (1) test the hypothesis that phenobarbital will cause temporally resolvable changes in gene expression in primary mouse and human hepatocytes through time-course gene expression studies using whole-genome microarrays; (2) test the hypothesis that the gene expression cascade for phenobarbital can be delineated by individually blocking key regulatory genes induced at earlier time points using RNA interference and measuring the effect on down-stream changes in gene expression; (3) test the hypothesis that specific secondary gene expression responses related to early cell proliferation events exist in mouse hepatocytes, but not in human hepatocytes. By determining the interrelationships between the secondary transcriptional responses and the primary regulatory genes, we will be able to mechanistically link the proliferative subnetwork to the primary event or receptor responsible for cross-species differences. Through this study we believe a novel approach to toxicology will be developed that allows comparisons of the signaling cascades across species to predict the conservation of the toxic response as well as provide new insights into the mechanism of action for many chemical agents.