Pathology - Theses
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ItemAn assay to screen for mutant p53 gain of function using high content imaging( 2012)The p53 tumour suppressor gene is mutated in approximately half of all human cancers. These mutations not only inactivate the growth inhibitory functions of wild type p53 but certain mutations also confer additional oncogenic properties. These gained functions may contribute to the increased growth rate, resistance to apoptosis, reduced chemosensitivity and increased invasiveness of mutant p53 bearing tumours. For the purposes of discovering novel mediators of mutant p53 gain of function, a multi-parameter assay was developed for future use in a high throughput siRNA screen. Mutant p53 expressing cells were demonstrated to display an increased migratory phenotype in vitro compared to p53 null cells. A wound healing endpoint assay was adapted to a 96 well format using automated liquid handling and image capture. An image analysis algorithm was designed to accurately measure cell migration in a high throughput manner. Performing a low throughput pilot screen of a panel of siRNAs demonstrated that the general migration machinery was necessary for migration. However, knockdown of known components of the mutant p53 pathway failed to provide the robustness necessary for a high throughput screen. p53 protein stability is known to be regulated at multiple levels involving a complex network of feedback loops. Unlike the case with wtp53, the regulation of mutant p53 is only partially understood. A high content assay for mutant p53 stability was developed. The immunofluorescent staining protocol was adapted to a 384 well assay and an algorithm was designed to accurately measure pixel intensity in the nucleus and cytoplasm. A statistically significant and robust downregulation of mutant p53 was measured with siRNA against mutant p53. A pilot screen in endogenous mutant p53 cell lines demonstrated a sufficiently large assay window to identify siRNAs that reduce mutant p53 stability. A genome wide siRNA screen for genes that reduce mutant p53 could uncover novel therapeutic targets, which will enable the design of new molecularly targeted therapeutics. Drugs able to impede the action of mutant p53 will be relevant to a significant proportion of human cancers.