|Author:||Yung, Ming-ho Mingo|
|Title:||The regulation of thioredoxin system in cells under oxidative stress|
|Subject:||Hong Kong Polytechnic University -- Dissertations|
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xvii, 171 leaves : ill. (some col.) ; 31 cm.|
|Abstract:||Aerobic organisms breathe fresh air instinctively. At the same time, they are exposed to the negative challenges and influences oxygen imposes on them. It can be expected that through selection forces, existing aerobic organisms should have developed effective anti-oxidative systems to help them survive under oxidative stress. Aerobic cells have a variety of means that protect them from the harmful effects of reactive oxygen species. One such protective mechanisms in diverse organisms and cells is the thioredoxin system. It is a simple but multifunctional antioxidant system that consists of a small ubiquitous dithiol protein thioredoxin (Trx), a pyridine nucleotide-disulphide oxidoreductase named thioredoxin reductase (TR) and a hydrogen donor NADPH. The thioredoxin system participates critically in combating against oxidative stress. Although the thioredoxin system in cells has been known and studied quite extensively, how it is regulated during oxidative stress conditions is not understood in most cases. To address this issue, the response of mammalian cells towards different levels of oxidative stress was studied to find out how the thioredoxin system was regulated. The levels and activities of the thioredoxin system in HeLa and HT-29 cells, were monitored upon challenged with various concentrations of hydrogen peroxide. Changes in cyctotoxicity, thioredoxin system proteins level, enzymatic activity and cell cycle were analysed. A decline in cell viabilities, both in HeLa cells and HT-29 cells, was observed upon a concentration-dependent hydrogen peroxide challenge. However, some increases of the thioredoxin reductase activity and thioredoxin activity were observed. Using immunoglobulin as substrate, HeLa cell was found with a 29% increase in the protein disulphide reduction activity upon treatment with 2mM hydrogen peroxide. Similarly, a 28% increase in the protein disulphide reduction activity was observed in HT-29 cell treated with 0.5 mM hydrogen peroxide. Western blotting and RT-PCR analyses revealed that the enhancements of activities in thioredoxin reductase and thioredoxin were probably due to the induction of protein synthesis and mRNA expression.|
In our previous investigations on erythrocytes, we found that a direct activation of the thioredoxin system could be involved in reaction to oxidative stress. In this study, we therefore looked into if regulations of the thioredoxin system do exist in cells generally without de novo protein synthesis. Cell cultures were pretreated with cycloheximide so as to inhibit de novo protein synthesis. The entire thioredoxin system activity, especially in cells treated with lower concentrations of hydrogen peroxide, was found functionally intact throughout the experiment. It was subsequently confirmed by the analyses of Western blot and RT-PCR which revealed insignificant changes in the cellular level of thioredoxin system proteins and mRNA respectively. Regulation of the thioredoxin system might therefore involve some endogenous factors instead of de novo protein synthesis. Multiple cellular processes such as proliferation, cell cycle and prosurvival signalling cascades are regulated by redox sensitive signalling factors. Thioredoxin and thioredoxin reductase, which are components of several redox regulated pathways, would exhibit their functional roles in the cellular defence against oxidative damage. To verify this notion, we looked into the relationship of the thioredoxin system and the changes in cell cycle stage in response to hydrogen peroxide treatment. HT-29 cells that had been exposed to hydrogen peroxide were subjected to fluorescence activated cell sorting analysis. A concomitant increase in thioredoxin system activity was observed in cells with an increase in the population of cells arrested at the G₂phase. Apoptosis was undetectable. We have provided some information on the protective effect of the thioredoxin system in cell viability and regulation of cell cycle in this study. A more in-depth understanding of the role of the thioredoxin system in fighting against oxidative stress will be further explored leading to potential applications of the system in the treatment of degenerated diseases and aging.
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