3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Short hairpin RNA-directed knockdown of epidermal growth factor receptor in human oesophageal squamous carcinoma cell lines(2008-05-27T12:52:55Z) Killick, Mark AndrewEpidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase which activates, upon EGFR binding, a number of signaling pathways including the mitogenic protein kinase pathway (MAPK) and phosphatidylinositol 3-kinase cascade (PI3K). Over expression of EGFR is a common feature in variety of human cancers including lung, colorectal, breast, pancreatic and oesophageal cancers and results in autonomous cell growth, enhanced metastatic potential, tissue invasion and increased resistance to current cancer therapeutics. Thus EGFR has been identified as a potential target in cancer therapeutics. Using the RNA interference (RNAi) pathway, the aim was to specifically knockdown expression levels of endogeneous EGFR in human oesophageal squamous carcinoma cell (HOSCC) lines. The RNAi pathway was initiated through the transfection of three specifically designed short hairpin RNAs (shRNAs) against human EGFR. The shRNAs were specifically designed using bioinformatics tools and their individual knockdown efficacy determined through the introduction of an exogeneous based target reporter systems, psiCHECK and pcieGFP. Expression levels of EGFR were determined using Western blot analysis followed by densitometry. Knockdown of EGFR was achieved by all three EGFR shRNAs in the three HOSCC cell lines (WHCO1, WHCO5 and WHCO6) despite low transfection levels of ~10%. Greastest knockdown of EGFR (85%) was achieved by EGFR sh2 in WHCO5. EGFR sh2 and sh1 resulted in average knockdown of EGFR of ~ 65% in WHCO1 and WHCO5 respectively. Weakest knockdown of EGFR (~ 20%) was obtained by all three EGFR shRNAs following transfection of WHCO6. RNAi-based approaches therefore show substantial potential for the specific and efficient targeting of EGFR in human cancer cells.Item á-Catenin expression in oesophageal squamous cell carcinoma(2006-03-23) Schnugh, Desmond Joá-catenin plays a crucial role in cell adhesion. Expression levels of á-catenin have been shown to be decreased in almost all tumours studied. The levels of the epidermal growth factor receptor (EGFR) were shown to be increased in oesophageal squamous cell carcinoma (OSCC) cell lines. á-catenin therefore, may play a part in linking the EGF pathway or other signal transduction pathways, bringing about some of the changes in the OSCC cell lines. The á-catenin gene from five OSCC cell lines was sequenced. Three out of five OSCC cell lines studied were found to harbour mutations. One of the mutations resulted in a change in the amino acid sequence of á-catenin. It was concluded that this alteration may not have affected the functioning of á-catenin. á-catenin was largely expressed at the plasma membrane with some weaker cytoplasm/nuclear expression occurring in all of the OSCC cell lines. Treatment of the OSCC cells with EGF for a 12 hour period resulted in no noticeable change in the expression levels of á- catenin. The results obtained from this study indicated that á-catenin could play a role in signal transduction pathways in the OSCC cell lines.