Infrastructure Engineering - Theses
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ItemModelling of the Wnt signalling pathway and its role in colorectal cancer( 2011)Colorectal cancer is one of the most common forms of cancer and a leading cause of death in the western world (Parkin et al., 2005). Truncation of the tumour suppressor gene adenomatous polyposis coli (APC) (a key member of the intra-cellular canonical Wnt/β-catenin pathway) has been strongly linked with colorectal cancer (Kinzler et al., 1991b), being found in 85% of adenomatous carcinomas. During development, Wnt/β-Catenin signalling is known to regulate key cellular decisions, namely cell survival, proliferation and differentiation (Logan and Nusse, 2004, Cadigan and Peifer, 2009). The main function proposed for Wnt signalling is to regulate the cytosolic β-catenin concentration. However, recently other regulatory (Seo and Jho, 2007, Faux et al., 2008) and functional (Brembeck et al., 2006) roles have been proposed. Although many of the protein interactions in the Wnt/β-catenin pathway are now known, data on concentrations of the Wnt pathway components in mammalian cells are limited. This lack of quantitative data prevents a systems level approach via computational biology. This thesis presents a systems-level framework integrating experimental and computational techniques for the investigation of the Wnt/β-catenin pathway in cancerous and noncancerous mammalian epithelial cell lines. In order to acquire the experimental data necessary for system level modelling, new experimental techniques are developed in this thesis. These include a new fluorescence cytochemistry protocol employing 3D confocal microscopy and image processing techniques to determine cell volume and volume of cellular compartments in live cells, as well as a novel 3D confocal microscopy quantification technique to monitor spatial and temporal changes of specific target proteins in structurally intact cells. Using a previously published Xenopus cytosolic model of the Wnt/β-catenin pathway as a starting point (Lee et al., 2003), the Lee et al. 2003 β-catenin degradation experiment setup was repeated for mammalian epithelial kidney (HEK293T) cytosolic extracts, with β-catenin concentration quantified using Western blots. The experimental data acquired was integrated in a computational model to develop an initial cytosolic Wnt signalling model optimised for mammalian cells. To advance the Wnt model towards a whole cell system, cellular volumetric data was measured and used to calculate the total whole cell concentrations of five key Wnt pathway proteins: APC, Axin, GSK3β, β-Catenin and E-cadherin, for five mammalian cell lines (HEK293T, MDCK, Caco-2, SW480 and SW480APC) as quantified by Western blots. Applying the new 3D quantification technique, the dynamics of specific pathway proteins (i.e. β-catenin and N-cadherin) in intact HEK293T cells and L-cells were estimated under a range of Wnt perturbations (i.e. Wnt3A, partially purified Wnt3A (ppWnt3A), cycloheximide (CHX) and MG132). Computational models of Wnt pathway signalling are developed and results compared to the experimental data in order to formulate calibrated models of Wnt signalling. In the cytosolic experiment, the degradation dynamics of β-catenin is found to be significantly faster in the mammalian cells than that previously reported by Lee et al. (2003) for the Xenopus oocyte. Substantial differences in protein concentrations were also observed between mammalian cells and the Xenopus oocyte. In particular, it was found that the Axin level was much higher (>5000 times) in mammalian cells than the Xenopus oocyte, while the reverse was true for the APC level. It was also found that non-cancerous cells have a smaller APC:Axin ratio (APC:Axin<0.1) as compared to cancerous cells (APC:Axin>0.5), highlighting a potentially critical balance between these two scaffold proteins in Wnt signalling. 3D quantification of β-catenin dynamics for intact mammalian kidney epithelial cells (HEK293T) showed significant co-localisation at the cellular membrane. β-catenin degradation rates are consistent with the mammalian cytosolic extract experiment. Under Wnt 3A stimulation, Wnt activation dynamics are acquired, and 3D compartmental analysis reveals a sharp increase in nuclear β-catenin (compared to the increase in cytosol-membrane β-catenin) at the onset of Wnt stimulation. Spatial kinetics data is obtained for HEK293T and L-cells using a variety of perturbations (i.e. Wnt3A, ppWnt3A, MG132 and CHX). The sharp initial increase in nuclear:cytosol-membrane β-catenin ratio upon Wnt stimulation observed in HEK293T is only able to be reproduced in L-cells when stimulated with ppWnt3a. With L-cells having predominantly cytoplasmic β-catenin, this highlights the role of membrane localisation in β-catenin regulation. Furthermore, a potential synergistic effect was observed between Wnt stimulation and the inhibition of β-catenin degradation (with MG132). In both L-cells and HEK293T, only MG132+Wnt3A-stimulated cells were able to trigger a significant accumulation of β-catenin in the whole cell with no such accumulation observed in MG132+non-stimulated cells. These observations are contrary to reports indicating a significant β-catenin increase within an hour of Wnt3A incubation for L-cells (Hannoush, 2008) and requires further investigation. The approach adopted during this investigation is repeated iterations between computational modelling and experimental data collection to incrementally progress the model development and fundamental understanding of the Wnt signalling pathway. Future application of this methodology to colonic cells in crypts will provide a context for the interpretation of the cell model and cell data reported in this thesis, while establishing a platform for the interrogation of the role of Wnt signalling in colon cancer. It is hoped that a systems approach to signalling will eventually facilitate the prediction and development of optimal treatments for colon cancer.