Role of Oxidative DNA Damage in Telomere Shortening and Cellular Aging
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The major aims of this research proposal were to develop assays for quantitating cellular aging and telomere shortening rates using yeast as a model system and to investigate factors that affect the kinetics of these processes. During the past year we have performed most of the experiments described in the Specific Aims and added additional experiments to clarify some of the results. For example, (a) we have created special yeast strains that have telomerase under the control of a new, regulatable GAL1 promoter and used these to develop assays for monitoring cell senescence, including measurement of changes in cell growth rate, killing and cell cycle checkpoint arrest, (b) we have tested effects of the pro-oxidant chemicals hydrogen peroxide, iron, and bleomycin, as well as the antioxidant chemicals N-acetylcysteine and resveratrol, on cell aging kinetics, and (c) we have constructed yeast strains with antioxidant genes GPX3 (glutathione peroxidase) and CTT1 (catalase) inactivated and evaluated the impact on cell aging. These early experiments have demonstrated that two of the pro-oxidants, bleomycin and iron, can act to accelerate cell senescence. The GPX3 and CTT1 mutations did not affect cell aging, but new mutant strains with multiple antioxidant genes inactivated (e.g. GPX3 + CTT1 as well as three other mutants) are currently being tested. In addition, during the past year we have worked to develop a new, simpler method for quantitating rates of telomeric DNA shortening that is based on the use of polymerase chain reaction (PCR) technique.