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 Keapl, 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 Keapl 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 Keapl, as well as the spatio-temporal dynamics of Keapl-Nrf2 interactions in vitro and in living cells by fluorescence resonance energy transfer spectroscopy. Subequent aims will investigate the downstream consequences of Keapl-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, Keapl-disrupted and double knockout cells and mice.
Aim 3 will use these genetic models to probe the pharmacodynamic action of a 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.
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