The effects of Phosphatase and Tensin Homolog (PTEN) overexpression on longevity of cultured Human Umbilical Vein Endothelial Cells (HUVEC)
Tait, Izak Schalk
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Ageing, the deterioration of organs and tissues and their ability to respond effectively to stressors, is a major cause of illnesses, disease and death. This is due largely to the shortening of telomeres resulting in cells undergoing senescence and apoptosis. This affects stem cells, without which organs cannot repair damage adequately. By removing oxidants that cause DNA and telomere damage the rate which telomeres shorten is reduced, resulting in a longer cellular lifespan. This can be accomplished by increasing the autophagic and antioxidant levels to remove oxidants and other toxic chemicals. Phosphatase and Tensin Homolog (PTEN) has been shown recently to be able to increase an organism’s lifespan by up-regulating pathways involved in DNA-damage repair and autophagy/antioxidants. Along with PTEN’s properties in metabolic increases, protection from insulin resistance and cancer prevention, PTEN is a good potential candidate to extend human longevity. The purpose of this thesis was to investigate whether these effects, having only been tested in animals, would have similar effects on humans. The goal was to explore the effects that overexpression of PTEN in Human Umbilical Vein Endothelial Cell (HUVEC) cultures would have on cell longevity via an increase in antioxidant potential. This was achieved by transfecting HUVEC cultures with PTEN plasmids using lipofection. Three doses of plasmids were tested: 0.01µg per ml, 0.1µg per ml and 1µg per ml. An Enzyme-Linked ImmunoSorbent Assay (ELISA) was performed to verify the success of the transfection and to quantify the levels of PTEN in each culture. The antioxidant potential of the cultures was assessed by a total antioxidant potential assay, expressing the total antioxidant potential in µM Copper Reducing Equivalents. The cultures were maintained until all cells achieved senescence and apoptosis in order to determine longevity. The results of each assay was then compared and correlated with each other and with the cells’ longevity in order to observe any patterns. The ELISA results showed a successful transfection. The transfected cultures showed a significant increase in PTEN levels compared with the non-transfected cultures (P < 0.001). The AntiOxidant Potential-450 assay (AOP-450) results showed significant increases in total antioxidant potential between the transfected and non-transfected cultures (P < 0.001). However, there was no significant difference between the 0.01 µg set and the Control set (P-value = 0.0957). This was due to a great degree of variance amongst the transfected cultures, ten times that of the non-transfected cultures. The cell culturing showed the transfected cultures had significantly longer lifespans than the non-transfected cultures (P < 0.001). Correlation between cell longevity and PTEN levels was 0.8727; and correlations between cell longevity and antioxidant potential was 0.6564. This thesis demonstrated that there is a potential for PTEN to be used to extend human longevity. The results show that the successful transfection of PTEN lead to an increase in PTEN levels, an increase in antioxidant potential and an increased cellular longevity. The higher correlation between cell longevity and PTEN than antioxidant shows there is a definitive link between increased levels of PTEN and longevity but there may be underlying factors which have influenced longevity independently of antioxidants.