Masters Degrees (Medical Physiology)

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Browsing Masters Degrees (Medical Physiology) by browse.metadata.advisor "Engelbrecht, Anna-Mart"

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    The effect of hypoxia on protein phosphatase 2A (PP2A) in MDA-MB-231 cells, and, a pilot study to establish a primary breast cancer cell model from Fine Needle Aspirations.
    (Stellenbosch : Stellenbosch University, 2018-03) Patten, Victoria Alexandra; Van Vuuren, Derick; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical Physiology.
    Background: Cancerous tumours are characterised by areas of insufficient blood supply, with resulting hypoxia and nutrient deprivation. The mechanisms by which cancer cells adapt to these conditions are of interest due to potential clinical relevance. Although immortalised cell lines have been used extensively in cancer research, a primary cancer cell model could potentially shed additional light on cancer biology. Protein phosphatase 2A (PP2A) is a Serine/Threonine protein phosphatase which has been implicated in hypoxia, although contradictory reports regarding its involvement has been published. Aims: 1. To establish a primary cell culture model using fine needle aspiration (FNA) for cell collection. 2. To investigate the effect of hypoxia on these primary cells. 3. To investigate the effect of hypoxia on PP2A expression, post-translational modification and activity in MDA-MB-231 cells. We hypothesized that hypoxia would induce a reduction in PP2A activity, thereby favouring the phosphorylation and activation of enzymes associated with survival and proliferation. Methods: Nine FNA samples were collected from patients at Tygerberg Hospital. Samples were subjected to processing and cultured at 37°C in suitable growth medium containing HEPES and epidermal growth factor. Cells were maintained in culture for 1.5–6 weeks and medium was refreshed initially after 3-4 days, and there after every second day. Although cells appeared to attach and survive initially, numbers dwindled until none were visible. We are therefore as of yet unable to establish a primary cancer cell model. Potential variables which could be of importance in future attempts to culture primary cells, include using a suitable attachment matrix, as well as optimizing the culture medium. Concerning PP2A in hypoxia in standard cell culture: MDA-MB-231 cells were cultured in a gas mixture of 5%CO2, 0.5%O2 for varying lengths of time, in growth medium containing only 1% FBS, where after cells were harvested and the following primary end-points investigated: (1.) The expression, phosphorylation and methylation of the catalytic subunit of PP2A; (2.) PP2A activity; and (3.) cell viability. Results: Following 72 hours hypoxia, Western blotting showed a decrease in total PP2A (control: 1.00±0.1426 arbitrary units (AU) vs hypoxia: 0.3513±0.07558 AU; n=3; p≤0.05), as well as unexpectedly a decrease in the relative phosphorylation of both PKB (phospho-tototal (p/t) ratio: control: 1.211±0.1820 AU vs hypoxia: 0.2088±0.03583 AU; n=3; p≤0.05) and ERK (control: 1.00±0.1519 AU vs hypoxia: 0.3493±0.05206 AU; n=3; p≤0.05). This led us to investigate shorter durations of hypoxia, specifically in the range between 2 and 8 hours. No significant changes in PP2A expression, post-translational modification or activity were observed, although there was a trend in increasing PP2A activity associated with hypoxia, which was highlighted by a significant increase in cells exposed to 6 hours of chemically stabilized HIF1ɑ (characteristic of hypoxia) (control: 134.7±33.74 AU vs positive control: 193.6±35.31 AU; n=2; p≤0.05). Four and six hours of hypoxia were consistently associated with a significant decrease in ATP. Discussion and Conclusion: Although we observed an interesting pattern of increased PP2A activity associated with hypoxia, our results failed to show a convincing link. Based on this, as well as literature, further research is needed: It would be interesting to investigate intermediate durations of less strenuous hypoxia (1% O2 instead of 0.5%). Modulating the activity of PP2A in hypoxia, followed by assessment of cell viability and pro-survival signalling would also contribute value.