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Title: Measuring DNA damage and associated epigenetic changes genome-wide in cells following exposure to platinum analogue chemotherapeutic drugs
Author: Powell, James Rees
ISNI:       0000 0004 5351 0127
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2014
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Many chemotherapy drugs act by inducing DNA damage leading to cell death, and the platinum analogue class of anticancer drugs are the most commonly used DNA damaging chemotherapeutic drugs. Despite extensive analysis of platinum-DNA interactions, particularly characterising the individual adducts and their effects on DNA replication, transcription and cell survival, measurement of these adducts in cells with higher sensitivity and precision is necessary. Previous work studying platinum-DNA adduct formation has been performed using DNA damage assays such as immuno-slot-blots to detect whole genome DNA damage, or with combinations of chromatography and mass spectrometry to characterise each adduct individually. The ability to measure platinum-induced DNA damage genome-wide with high resolution in human cells could have profound implications for basic mechanistic research, as well as clinical translational research and treatment stratification, by providing a tool with the potential for predicting clinical response to these agents. The achievement of this PhD was developing an assay to measure platinuminduced DNA damage induction at high resolution, density and precision within the genome of human cells. This was achieved using DNA immunoprecipitation coupled with analysis using DNA microarrays, allowing measurements of platinum-induced DNA damage to be made at high resolution throughout the human genome for the first time. This assay was initially developed to measure cisplatin and oxaliplatin induced DNA damage in the genome of the yeast model organism Saccharomyces cerevisiae and experimental profiles of cisplatin and oxaliplatin-induced DNA damage were validated by demonstrating close correlation with mathematically generated predicted profiles for platinum-induced DNA damage. The assay was then applied and validated to measure cisplatin, oxaliplatin and ultraviolet-induced CPD formation in human fibroblast cells, and again, experimental profiles of cisplatin and oxaliplatin-induced DNA damage and UV-induced CPD formation were shown to correlate well with predicted profiles of DNA damage. Novel comparative analytical approaches for studying microarray data from these genome-wide DNA damage datasets are demonstrated and further validation of the assay is provided by demonstrating the contrast between platinum-induced DNA damage and UV-induced CPD formation genome-wide and in the context of repeat sequences of DNA. Finally, cisplatin and oxaliplatin-induced histone H3 acetylation changes are examined and histone H3 K14 acetylation is demonstrated to be a prominent histone modification following exposure to platinum analogues. Novel analysis is performed to investigate the influence of chromatin on platinum adduct formation, and greater platinum-induced DNA damage is demonstrated in DNA samples treated in vitro compared with DNA samples taken from cells treated in culture. Comparisons were also performed between histone H3 acetylation in yeast cells following exposure to cisplatin or UV irradiation and this comparison revealed very similar patterns of histone acetylation following exposure to these two different genotoxins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH426 Genetics ; RC0254 Neoplasms. Tumors. Oncology (including Cancer) ; RM Therapeutics. Pharmacology