COORDINATED REGULATION OF ANTIOXIDANT ENZYMES

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This project will examine the possibility that a true bi-directional coordinated regulation exists between major anti-oxidant enzymes and determine the effect of disrupting cellular oxidant homeostasis on the life span of primary human normal diploid fibroblasts. Previous studies by others only demonstrate that exposure to pro-oxidant stimuli increases the activity of these enzymes. We recently demonstrated that genetically altering the expression of one anti-oxidant enzyme, copper-zinc superoxide dismutase, modifies expression of other anti-oxidant enzymes, as well as oxidant & non-oxidant associated proteins. Enzyme alterations are stable and occur without need of exogenous pro-oxidant stimuli (hyperoxia or paraquat). Also, some alterations appear to be coordinated in that both increases and decreases are produced. The regulation we have detected between these anti-oxidant enzymes may be uni-directional in that while copper-zinc superoxide dismutase expression influences expression of the other enzymes the reverse is not true. That is, if unidirectional then alterations in manganese superoxide or glutathione peroxidase would not influence copper-zinc superoxide dismutase expression. The regulation, however, could be bi-directional or truly coordinated in that alterations in manganese superoxide dismutase and/or glutathione peroxidase will alter expression of copperzinc superoxide dismutase and expression of each other. The first portion of this project will determine whether a true bi-directional coordinated regulation exists between anti-oxidant enzymes. Activity of a specific anti-oxidant enzyme will be altered by introduction of sense.or anti-sense expression vectors (using a viral promoter coupled to cDNA of an anti-oxidant enzyme). The regulation will be studied in both early passage and late passage (senescing) fibroblasts, as well as an immortalized daughter line to also determine if this regulation is disrupted during the aging process or upon immortalization. The relevance of determining whether a coordinated regulation exists amongst these anti-oxidant enzymes, and how the existence of this regulation could link a variety of seemingly unrelated diseases, is also discussed. The second portion of the project will examine the effect of modifying anti-oxidant enzymes, again through introduction of sense or anti-sense expression vectors, on the life span of these fibroblasts.