A Model In Vitro System to Study Heat Shock Protein 60 (HSP60) Expression in Response to Mitochondrial Impairment in Human Cells
Hall, L. (2013). A Model In Vitro System to Study Heat Shock Protein 60 (HSP60) Expression in Response to Mitochondrial Impairment in Human Cells (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/7905
Permanent Research Commons link: https://hdl.handle.net/10289/7905
Heat Shock Proteins (HSPs) are a class of ubiquitously expressed and functionally related proteins found in all living organisms from humans to bacteria. Their expression is increased in response to various cellular stressors in what is referred to as the heat shock response. The induction of one particular HSP, HSP60, has been found to be correlated with mitochondria specific cell stress. Recently HSP60 has been found to be secreted and expressed extracellularly, after first being thought to be strictly intracellular. However, the mechanisms of these mechanisms of translocation and secretion have not been clearly identified. The aim of this study was to develop a model in vitro system for HSP60 expression in human cells so that treatments which resulted in mitochondrial impairment could be investigated. This study looked at several aspects of high glucose, hydrogen peroxide (H₂O₂), and sodium azide treatment on human HeLa cells. At low levels, each treatment had a hormetic effect on HeLa cell growth; however at high concentrations growth was significantly inhibited. Additionally, high treatment concentrations resulted in increased cell lysis as determined by LDH assays. Treatment with concentrations of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide were the only treatment concentrations that did not result in significantly different levels of cell lysis when compared to a control sample. Mitochondrial dehydrogenase activity was also found to be significantly decreased at high treatment concentrations as determined by MTT assays. Thus, 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were identified as optimal conditions for mitochondrial targeted cell stress, as each of these treatments impaired cell growth and inhibited mitochondrial activity while having no significant effect on the degree of cell lysis. The 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments were then investigated for the intracellular reactive oxygen species (ROS) activity they induced over treatment periods of 24 hours, 3 and 7 days. After 7 days of treatment, the 200μM H₂O₂ and 50μM sodium azide treatments resulted in an approximate three-fold increase in ROS activity, while the 100mM glucose treatment resulted in almost twice as much ROS compared to a control sample. Finally, the effects of 100mM glucose, 200μM H₂O₂, and 50μM sodium azide treatments on HSP60 and HSP70 expression was also examined over 3 and 7 day time periods. After the 7 day treatment period, the 100mM glucose treatment had induced a 2.43 fold increase in HSP60 expression and a 2.75 fold increase in HSP70 expression, the 200μM H₂O₂ induced a 3.48 fold increase in HSP60 expression and a 3.98 fold increase in HSP70 expression, and finally the 50μM sodium azide induced a 4.74 fold increase in HSP60 expression and a 5.08 fold increase in HSP70 expression. It can therefore be concluded that 100mM glucose, 200μM H₂O₂, and 50μM sodium azide results in the upregulation of HSP60 and HSP70 in human HeLa cells. Therefore this model may be used to investigate further aspects of HSP60 induction, such as its translocation and secretion.
University of Waikato
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