Cell-type-specific responses to DNA damage from acute and chronic radiation
It is well understood that radiation causes damage to DNA, which triggers a DNA-damage response often centred on the signalling pathways of ATM, with the subsequent involvement of proteins such as p53, p21 and H2AX. Many experiments have been performed with cell lines derived from different origins and donors which, while invaluable for studying specific effects, restrict direct assessment of response variability between different cells, tissues and individuals. This thesis investigates normal tissue responses to DNA damage from acute and chronic radiation, including exploration of cell-type and individual-specific differences in DNA-damage response. Due to the difficulty of obtaining different primary cell types of isogenic background (necessary to prevent confounding results by inter-individual variation), there are still unanswered questions regarding the extent and details of cell-type-specific responses to ionising radiation. I have developed a method for isolation of primary cells from human tissue and compared cellular outcomes and molecular signalling in response to an X-ray dose. Using these primary cells, I show that cell type causes far more variability in radiation response, with keratinocytes, which have highest p21 protein levels, being most susceptible to cell cycle arrest and senescence, while donor variability is limited. Another issue that has received insufficient attention is the biological effects of chronic radiation. Both experimental and epidemiological evidence highlight the damaging and disease-causing effects of high doses of radiation. As there are limited facilities worldwide to perform the experiments, understanding of the cellular response to chronic radiation is not well known. To address this, I focused on characterising the response of primary cells isolated from neonatal foreskin donors and exposed to chronic radiation. Not surprisingly, results show that chronic radiation elicited many classic DNA-damage response outcomes such as cell cycle arrest, apoptosis and senescence. However, unlike acute radiation, the chronically irradiated cells demonstrated a reduction in histone levels accompanied by significant senescence induction and increased global transcription, indicating deregulation of gene expression. These characteristics are associated with age-related pathologies. Therefore, I extensively tested epigenetic age in these cells, which uses consistent changes in DNA methylation with age to accurately predict age. This demonstrated that epigenetic age was not altered by chronic radiation, but since this only captures certain aspects of ageing, the results suggest that chronic radiation increases wear-and-tear aspects of ageing. Overall, the work described in this thesis contributes to understanding the extent of normal radiation response variability in donors and cell types. It also reveals histone reductions associated with cellular senescence as an effect of continuous exposure to low doses of ionising radiation that may have wider implications for ageing and the incidence of specific pathologies.
Cell type, DNA damage, Epigenetic, Histone, Inter-individual, Ionising radiation
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