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Title: Functional characterisation of p300
Author: Iyer, N. G.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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p300, originally discovered as an adenoviral El A binding protein, is a putative tumour suppressor gene. Mutational analysis done in our laboratory found truncating mutations in a range of cell lines and primary tumours, in some cases associated with loss of the second allele. In order to study the possible pathways leading from p300 loss to cancer, I adopted a somatic cell line knock out strategy to study the gene function in a human epithelial system. Using homologous recombination mediated gene targeting I successfully disrupted p300 in HCT116, by targeting the single expressed, truncated allele. Resultant cells were null for p300 protein. Rescue clones were generated by re-introducing full-length p300 cDNA into the knock-out clones. p300 knock-out (KO) cells were defective in proliferation, with prolonged doubling times and increased S-phase. A fraction of cells were in senescence/quiescence, and included a flat cell phenotype. These changes were reversed in the rescue clones. KO cells also showed a Gl to S-phase transition defect with early S phase entry after serum depletion and nocodazole arrest and release experiments. Rb pathways were deranged, principally by Rb phosphorylation defects, which probably led to the activation of the E2F pathway and early S-phase entry. KO cells also demonstrated abnormalities in p21 response during the cell cycle, which could have contributed to the cell cycle phenotype. In addition, KO cells were found to have reduced cell-cell adhesion, due to a decrease in E-cadherin at tight junctions and total E-cadherin levels. DNA damage experiments suggest that p300 KO cells are especially sensitive to UV mediated DNA damage with increased apoptosis seen 24 h after irradiation. This was due to abnormalities in the p53 pathway- a combination of increased stability of p53, reduced acetylation at lysine 382 and abnormal transactivation of downstream factors. In contrast, long term growth of KO cells after UV and XR irradiation showed increased resistance to DNA damage.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available