3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Investigating telomere dynamics in oesophageal squamous carcinoma cells using standard and gold nanoparticle-based assays(2017) Bernert, MartinCancer is characterised by abnormal cell proliferation and is one of the leading causes of death in first world countries and the second leading cause in developing countries. In 2012 alone, over 14 million cases were reported and over 8 million deaths were attributed to cancer worldwide, with sub-Saharan Africa, especially South Africa having one of the highest oesophageal cancer rates in the world. An important aspect of cancer is the telomeres, which are 10-15kbp of TTAGGG DNA repeats in humans at the ends of chromosomes. These repeats are maintained by the enzyme telomerase. Up to 90% of all cancers show increased telomerase activity to overcome the "end-replication" problem in which the telomeres shorten after each cell division. This eventually leads to cellular senescence. Due to the high number of cancers relying on increased telomerase activity to bypass senescence, telomerase could be a viable target for anti-cancer therapies. The limiting factor of the multi-subunit telomerase enzyme is its telomerase reverse transcriptase component (hTERT). hTERT has also been shown to migrate to the mitochondria during times of high oxidative stress caused by reactive oxygen species (ROS). Here it confers protection to the mitochondria against ROS, potentially preventing the cell form undergoing apoptosis and reaching senescence. This can potentially be detrimental, as cells become damaged by the ROS and continue dividing. This could lead to further genetic damage. Metformin, a drug used for the treatment of type-2 diabetes, has been linked to lower incidences of cancer. The mode of action of metformin is not yet fully understood, however it is known that it affects the mitochondria. Since hTERT and metformin could co-localise, the drug may influence hTERT and potentially telomerase activity. This makes metformin an anticancer candidate to be used in conjunction with traditional anticancer therapies. To determine telomerase activity in metformin treated oesophageal carcinoma cells, qPCR based telomerase activity assays must be used. These assays can be very expensive and time consuming, so a faster and cheaper alternative would be beneficial. Therefore, the aim of this project was to alter and improve a nanoparticle based detection method for telomerase activity, by decreasing the time required to prepare the DNA functionalised nanoparticles as well as determining a more rapid method of data measurement, and compare it to conventional qPCR based techniques (TRAPeze RT Telomerase Activity Kit – Merck). Thereafter the effects of the metformin treatment on telomere dynamics, such as telomere length, telomerase activity and hTERT mRNA expression, in oesophageal squamous carcinoma cells were determined. Gold nanoparticles were synthesised and functionalised with thiolated-DNA (telomerase substrate). These functionalised particles were characterised using transmission electron microscopy. To assess telomerase activity the extracted protein was added to the functionalised nanoparticle solution and allowed to elongate the coupled DNA. A characteristic of gold nanoparticles is that the size of the particles as well as their proximity to one another determines the colour of the nanoparticle solution. Due to the steric hindrance caused by the now elongated DNA, a distinct colour change was observable. The change in absorption spectra of the nanoparticle solution was recorded after the enzyme elongated the substrate. This nanoparticle based assay was then compared to TRAPeze RT Telomerase detection kit (Merck-Millipore) as a positive control. Using the conventional qPCR based telomerase activity assay, it was found that metformin significantly decreased telomerase activity in oesophageal cancer cell lines, however this was not seen using the nanoparticle assay. A colour change was observed with the nanoparticle assay compared to the negative control reflecting detection of telomerase activity. However, no significant decrease in telomerase activity could be detected due to metformin treatment. More optimisation is required, however this technique has great potential, as nanoparticle based assays are also known for their high sensitivity. This technique is also far more rapid and significantly cheaper that the qPCR based method. The gold nanoparticle based telomerase activity assay could become an alternative to conventional qPCR based techniques.Item The effect of Src inhibition on ROS triggered signalling in human oesophageal squamous carcinoma cells(2017) Houston-McMillan, EmbethReactive oxygen species (ROS) have been specifically highlighted as instigators of aberrant pro-survival and proliferative signal transduction pathways in recent years. A possibility that ROS stimulates the Src-Protein Kinase B (PKB)-Glycogen Synthase Kinase (GSK)3-β pathway, a non-canonical pro-survival and anti-apoptotic pathway, was identified and investigated. Specifically, Human Oesophageal Squamous Cell Carcinoma (HOSCC) cells were exposed to oxidative conditions, Src inhibition, and a combination of the two to determine the role that Src plays in the phosphorylation of PKB and GSK3-β in terms of ROS stimulation. Oxidative conditions led to a significant increase in pSrc and pPKB in only one of the 5 HOSCC cell lines being studied; however the abundances of pGSK3-β increased significantly in two of these cells lines and decreased significantly in two of the others. This indicates that oxidative conditions lead to different downstream effects in the various HOSCC cell lines, which are most likely achieved via the activation of various pathways as a result of crosstalk. Src inhibition led to a decrease in pPKB levels across all HOSCC cell lines displaying detectable levels of pPKB, however the abundance of pGSK3-β increased in these cell lines, indicating again that other pathways are at play with respect to the activation of GSK3-β in HOSCC cells. This is due to the fact that GSK3-β is a downstream effector of PKB, and should thus decrease in abundance as pPKB does. Oxidative conditions coupled with Src inhibition resulted in an increase in the abundance of pPKB and pGSK3-β in four of the five HOSCC cell lines. These results indicate that Src inhibition under oxidative conditions may lead to cell survival and proliferation via the activation of pPKB, a pivotal pro-survival protein, and subsequent inactivation of pGSK3-β, a known pro-apoptotic protein. Thus, although more than one pathway is likely to be involved in the phosphorylation of PKB and GSK3-β in HOSCC in terms of ROS, it appears as though inactive Src in HOSCC cells undergoing oxidative stress could be used as a testable marker for HOSCC – a devastating disease affecting numerous South Africans.Item The influence of p90RSK on FAK-dependent signalling in human oesophageal squamous carcinoma cells(2017) Lachenicht, CandiceThe focal adhesion kinase or FAK plays an important role in detecting and transducing signals that are generated by cell-substrate attachment (Focal adhesions). When these pathways are activated under atypical conditions they may promote metastasis, uncontrolled proliferation and a chemo-resistant phenotype. However the mechanisms by which this protein is activated ectopically in human oesophageal squamous cell carcinomas cell lines (HOSCC) is unknown. In the current study it was hypothesised that the p90 ribosomal S6 kinase, a key member of multiple pro-survival pathways (activator of the Y-box binding protein-1), activates FAK. RSK may promote FAK activation directly, from its location at the plasma membrane, or it may modulate FAK activation indirectly via the regulation of one of its substrates. RSK inhibits the activation of the glycogen synthase kinase 3β (GSK3β) by phosphorylation at Ser9. GSK3β also localises at focal adhesions and may therefore play a role in mediating FAK activity. To ascertain the role RSK plays in FAK activation, 3 inhibition studies were performed. In the first assay, RSK was specifically inhibited within HOSCC and the levels of active FAK monitored (two different environmental conditions). FAK activation was monitored by detecting the auto-phosphorylation of FAK at Tyr397. A GSK3β inhibition assay was then performed in which GSK3β was specifically inhibited and the levels of active FAK monitored. Lastly, a dual inhibition assay was performed where both RSK and GSK3β were inhibited simultaneously and the levels of active FAK monitored. The overall net changes in the phospho-protein profile indicated that all of the HOSCC cells had distinct cellular responses to the three inhibitor combinations. However RSK did not appear to activate/inhibit FAK activity directly, in most of the HOSCC cells, but rather modulated FAK activation through the inhibition of GSK3β. The effects the RSK/GSK3β pathway had on FAK activation was partially dependent on the HOSCC cells containing active levels of PTEN. Interestingly, the inhibition of both GSK3β and RSK reduced the levels of active FAK in 3 of the 5 HOSCC cell lines, indicating that this might be a good anti-cancer therapeutic. RSK appeared to play a more context specific role in FAK activation within the HOSCC cells suggesting that the grading system for moderately differentiated carcinomas needs to be improved. This paper also highlights the importance of studying the effects the microenvironment has on neoplasmic transformation as varied environmental conditions, during the RSK inhibition studies, drastically impacted the effects the RSK inhibitor had on FAK activation.