Optimisation of Cell Based Apoptosis Assay to Investigate Gastrin- Induced Circumvention of Apoptosis in AR42J Cells
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This Master’s thesis is part of a research project investigating the anti-apoptotic action of gastrin. Gastrin is a peptide hormone with many characteristics. First of all, it is known for its role in regulation of gastric acid. However, gastrin has also a malignant potential. It has found to affect proliferation, migration, promotion of angiogenesis, as well as counteraction of apoptosis. Through microarray studies several genes have previously been found induced by gastrin in the adenocarcinoma derived cell line AR42J, some of them known to be involved in apoptosis. One of these genes was clusterin, a pleiotrophic protein associated with protection against stress-induced apoptosis. In Situ Cell Death Detection Kit (TUNEL assay) in combination with a high-throughput automatic fluorescent microscope analysis (ScanR) was optimised for AR42J cells to meet the request of an apoptosis assay to study the anti-apoptotic effect of gastrin at a single cell level. The AR42J cell line was used due to its endogenous expression of gastrin receptors. However, since this cell lines grow in clusters, we first needed to optimise growth-conditions, coating of wells and cell density, for microscopic single cell analysis. By using the optimised apoptosis assay, we demonstrated that gastrin circumvents stress-induced apoptosis in AR42J cells. Results obtained by Western blot analysis suggested that gastrin up-regulated secretory clusterin in serum-starved cells. Intracellular localisation of clusterin was then studied using immunocytochemistry and confocal microscope, and clusterin protein was found in neuronlike protrusions in the cytoplasm in both unstimulated and gastrin-stimulated AR42J cells. The localisation of the clusterin protein supported the presence of the pro-survival factor secretory clusterin (sCLU) in AR42J cells. To investigate whether clusterin mediates the anti-apoptotic effect of gastrin, we reverse transfected short hairpin RNA targeting clusterin to knock down the clusterin expression. However, this approach did not seem to down-regulate the clusterin expression in AR42J cells. We therefore investigated the effect of gastrin-induced apoptosis in the absence and presence of an anti-clusterin antibody by using a caspase assay (Caspase Glo 3/7). Neutralisation of secretory clusterin by a specific antibody abolished the anti-apoptotic effects of gastrin on serum starvation-induced apoptosis, suggesting that clusterin plays a role in the anti-apoptotic action of gastrin.