Free radical and oxidants have been implicated as causative agents in a wide variety of pathological conditions, including cancer. Antioxidants, produced by the body, provide a safeguard against the damaging effects of oxidants. Thus, the ability to manipulate the expression of antioxidants could provide a useful strategy against diseases associated with oxidant-induced cell damage. We hypothesize that the Nrfl transcription factor plays a direct role in the coordinate regulation of genes in the anti-oxidative pathway, as it has been implicated in the regulation of antioxidant genes through the cis-acting antioxidant response element (ARE). The overall aim of this proposal is to define the role of Nrfl in knockout mice. Three types of projects are proposed. We will examine the response of cells from Nrfl mutants to oxidative stress. We hypothesize that cells lacking Nrfl will be defective in antioxidant defense, and will result in the accumulation of reactive oxygen species and display and display increased sensitivity to their cytotoxic effects. We will examine the role of Nrfl protein in the regulation of a number of antioxidant genes which we have identified to be down-regulated in nrfl mutant animals. We hypothesize that the activation of these genes should occur in an ARE-site-dependent and an Nrfl-dependent manner. Third, we will examine the molecular mechanisms by which oxidative stress is relayed to Nrfl. We hypothesize that induction through the ARE sequence by Nrfl occurs via a post- translational mechanism. We will measure changes in Nrfl's ability to activate transcription in response to oxidative stress. Finally, we will determine the nature by which Nrfl protein is modified in response to oxidative stress. We hope to determine the genetic and molecular basis of the oxidative stress response, and to develop strategies to manipulate this mechanism in disease states. Since these same pathways are affected in cancer cells, it is hoped that these studies will also contribute to a better understanding of malignancy and provide insights into novel therapeutics.
