ROLE OF ENZYME INDUCTION IN CANCER CHEMOPREVENTION

描述(由申请人提供)：拟议研究的总体目标是评估癌症化学预防药物诱导2期反应的潜在机制和功能意义。种类繁多的化学制剂可以保护动物免受多种不同类型致癌物的致癌作用。其中许多化学保护剂通过选择性地诱导(通过增强转录)第二阶段、抗氧化和抗炎基因来发挥其抗癌作用，这些基因有助于解毒生物分子破坏形式的亲电体和氧化剂，从而提高细胞存活率。这些保护性基因中的大多数是通过一种常见的增强子--抗氧化反应元件(ARE)与转录因子Nrf2相互作用而诱导的。反过来，Nrf2又被抑制因子Keap1隔离，该抑制因子调节Nrf2在细胞中的命运。在这个项目中，我们试图使用分子、遗传和化学方法来验证Keap1是激活依赖于Nrf2的细胞保护基因的试剂的主要传感器的假设。目的1研究Keap1的结构性质和反应动力学，以及通过荧光共振能量转移光谱研究Keap1与Nrf2在体外和活细胞内相互作用的时空动力学。后续的AIMS将调查Keap1-Nrf2激活的下游后果。目的2将通过使用野生型、Nrf2干扰、Keap1干扰和双基因敲除的细胞和小鼠的模型，在体外和体内确定该途径在调节氧化应激中的作用。目的3将使用这些遗传模型来探索一类特别有效的化学预防药物--三萜类化合物的药效作用，并评估Nrf2在其作用中的核心作用。目的4将描述Nrf2信号与其他途径的相互作用，这些途径影响影响细胞命运的适应性反应。特别是，芳香烃受体和Notch信号通路的交叉调节将被探索，并评估反式激活的功能后果。这些研究将坚定地确立诱导第二阶段反应在化学预防中的作用。了解化学预防药物与Keap1作为哨兵传感器相互作用的机制，从而促进Nrf2信号诱导细胞生存基因的产生，将有助于识别和利用更具选择性的化合物，并提高其在人类中的有效性。

DESCRIPTION (provided by applicant): The overall goals of the proposed studies are to assess the underlying mechanisms and the functional significance of induction of the phase 2 response by cancer chemopreventive agents. An extraordinary variety of chemical agents protect animals against the neoplastic effects of many different types of carcinogens. Many of these chemoprotectors exert their anticarcinogenic effects by selectively inducing (by enhanced transcription) phase 2, antioxidative and anti-inflammatory genes that serve to detoxify the biomolecule damaging forms of electrophiles and oxidants, thereby enhancing cell survival. Most of these protective genes are induced through a common enhancer, the Antioxidant Response Element (ARE), by interactions with the transcription factor Nrf2. Nrf2, in turn, is sequestered by the represser Keap1, which regulates the fate of Nrf2 in cells. In this project, we seek to use molecular, genetic and chemical approaches to test the hypothesis that Keap1 is the major sensor for agents that activate the Nrf2-dependent cytoprotective genes. Aim 1 is designed to characterize the structural properties and reaction kinetics of Keap1, as well as the spatio-temporal dynamics of Keap1-Nrf2 interactions in vitro and in living cells by fluorescence resonance energy transfer spectroscopy. Subequent aims will investigate the downstream consequences of Keap1-Nrf2 activation. Aim 2 will define the role of this pathway in modulating oxidative stress in vitro and in vivo through the use of models employing wild-type, Nrf2-disrupted, Keap1-disrupted and double knockout cells and mice. Aim 3 will use these genetic models to probe the pharmacodynamic action of an exceptionally potent class of chemopreventive agents, triterpenoids, and to assess the central role of Nrf2 in their actions. Aim 4 will characterize the interactions of Nrf2 signaling with other pathways affecting adpative responses affecting cell fate. In particular, cross regulation of the aryl hydrocarbon receptor and the Notch signaling pathways will be probed and functional consequences of transactivation assessed. These studies will firmly establish the role of induction of the phase 2 response in chemoprevention. Knowledge of the mechanisms by which chemopreventive agents interact with Keapl as a sentinel sensor, thereby facilitating signaling by Nrf2 for induction of cell survival genes, will facilitate the identification and utilization of more selective compounds and enhance their effectiveness in humans.