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Title: Investigations into kinase associated genotoxicity
Author: Manville, Catriona Margaret
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2012
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This study used the GreenScreen HC assay to determine genotoxic damage in a panel of 68 commercially available kinase inhibitors encompassing 18 targets. The GreenScreen uses a reporter cell line where GFP is fused to the GADD45a promoter. In response to DNA damage, GADD45a is upregulated and GFP produced. Therefore, the genotoxic potential of a compound can be predicted by spectroscopic measurements of the cytotoxic and genotoxic responses of a population of cells when exposed to a compound. Positive results were not caused by the specific inhibition of any of the kinase targets tested but observed as a result of the ATP competitive nature and specificity of the inhibitor for the desired target. The GreenScreen data was complimented by in silico analysis using Derek for Windows. A subset of three PI3K inhibitors were also assessed in the bacterial Ames and mammalian in vitro micronucleus assays. The role of ERK1 in the cellular genotoxic response to the TOP2 poison, etoposide was assessed in a paired murine cell line (one knocked out for ERK1). There was a statistically significant increase in number of micronuclei in the ERK1 knockout compared to the wild type cell line. ERK1 sites of phosphorylation of the C-terminal domain of TOP2B were identified by site directed mutagenesis and mass spectrometry analysis. The micronucleus assay was also carried out on human and murine, wild type and TOP2B knockout, cells to determine the role of TOP2B in etoposide mediated genotoxicity. In the absence of TOP2B, significantly fewer micronuclei were induced and the genotoxic effect was removed. Therefore TOP2B appears to mediate the cell's genotoxic response to etoposide. This work also included a bioinformatics analysis of genome wide study of human TOP2B genomic occupancy sites revealed by ChIP-Seq. The location of TOP2B occupancy coincides with transcription factors including SP1 and REST. TOP2B is shown to bind preferentially to repetitive regions and CPG islands. Gene ontology analysis, using DAVID, was conducted to determine pathways and processes TOP2B is involved in, including neuron development and kinase activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available