Eastham, Matthew and Allinson, Sarah (2016) The effect of UVA irradiation on the DNA damage response in a human keratinocyte cell line. Masters thesis, Lancaster University.
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Abstract
The DNA damage response (DDR) recognises different types of DNA damage and initiates signalling pathways to bring about cell cycle arrest so the appropriate repair mechanisms can be activated, or to bring about cell death when the damage is unrepairable. DNA damage surveillance and repair pathways play an important role in protecting skin cells against the carcinogenic effects of ultraviolet radiation (UVR). In this research project, HaCaTs were used to investigate the direct and indirect effects of UVA irradiation on the DDR. Independent inhibition of ATM and ATR prior to irradiation with 50 kJ m-2 UVA caused cell survival to decrease in a dose-dependent manner over that seen where HaCaTs were irradiated in the absence of inhibitor pre-treatment, suggesting the involvement of ATM and ATR in processing UVA-induced lesions. The same observation was not seen when the dosage was increased to 100 kJ m-2. Western blot and immunofluorescence studies found that H2AX activation peaked between 1 and 4h following irradiation with 100 kJ m-2 UVA. Chk2 phosphorylation peaked immediately following UVA irradiation. Inhibition of ATM prior to UVA exposure resulted in a temporal decoupling of H2AX and Chk2 phosphorylation with peak H2AX activation occurring at a later timepoint whilst p-Chk2 activation was unchanged, implying that UVA-induced Chk2 phosphorylation may not be entirely ATM-dependent. The UVA-induced bystander effect was also investigated. H2AX activation in bystander cells increased at 48h over that seen at 24h when these cells were co-cultured with UVA-irradiated cells. A dose-dependent UVA-induced bystander response was observed following 1 week co-incubation as determined by clonogenic survival assays, which was not the case at 48h. These data suggest that the rate at which damage signals are released from irradiated cells decreased over time in a dose-dependent manner.