This is the FIRST award application to study oxidative stress during glucocorticoid-mediated lymphocyte apoptosis. The hypothesis is that oxidative stress is required for glucocorticoid-mediated lymphocyte apoptosis. This application will test whether the observed down-regulation of the antioxidant defense enzymes is essential for apoptosis. The applicant will establish the mechanism by which antioxidant defense transcripts are down-regulated. Finally the applicant proposes to develop sensitive bioassays for representative oxidative damages to macromolecules in cells and to define targets representing oxidative damages during apoptosis. Specifically, Aim 1 is to test whether a reduction in the antioxidant defense is essential for glucocorticoid-mediated lymphocyte apoptosis by transfecting the WEHI 7.2 lymphocyte cell line with sense and antisense antioxidant defense expression vectors and determining whether resistance to apoptosis depends on the strength of the cellular antioxidant defense.
Specific Aim 2 is to determine the mechanism by which glucocorticoids down-regulate the antioxidant defense by measuring RNA stability and gene transcription rates and using structural/functional analysis of the regulated transcripts or gene sequences to identify sites that are regulated during apoptosis, and Specific Aim 3 is to identify targets of oxidative stress during apoptosis by using gas chromatography with electron capture detection to measure lipid peroxidation. Immunoassay will be used to detect oxidized proteins, gel shifts, and reporter constructs to monitor activity of redox-sensitive transcript or transcription factors, and a PCR-based method will assay oxidative damages to mitochondrial DNA. The information gained from the proposed study will be significant to the understanding of mechanisms by which aberrant control of the cellular redox state promotes disease, specifically through an effect on apoptosis. The future goal is to identify new targets for treatments aimed at restoring control of apoptosis in diseased tissues.
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