Characterisation of the UVA-induced DNA damage response in directly irradiated and bystander cells

Wilson, Kendal and Allinson, Sarah (2023) Characterisation of the UVA-induced DNA damage response in directly irradiated and bystander cells. Masters thesis, Lancaster University.

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Abstract

Ultraviolet (UV) radiation is a carcinogen responsible for causing skin cancer, one of the most common forms of cancer globally. UV irradiation leads to DNA damage including oxidised bases, cyclobutane pyrimidine dimer (CPD) formation and double strand breaks (DSB) activating the DNA damage response (DDR). Whilst UVB is directly more damaging to cellular DNA, it is becoming increasingly clear that UVA plays a large role in DNA damage induction and carcinogenesis. The DDR pathway is well understood, but the kinetics of protein activation need to be studied in more detail to develop our understanding of DNA damage induction, persistence and repair induced by UVA irradiation. Using dose dependent studies, human immortalised keratinocytes (HaCaTs) were exposed to UVA irradiation looking at DDR proteins, studying their activation and downregulation. A dose dependent change in activation of the DDR pathways was identified. Lower UVA doses demonstrated earlier activation of the ATR response whilst medium and higher doses indicated earlier activation of the ATM response. Furthermore, as the dose increased, peak activation of total γH2AX was observed later, as did possible repair of DNA damage. Finally, the late activation of sensor protein RPA could indicate the late generation of DNA damage resulting from oxidative stress. It is becoming increasingly apparent that direct irradiation is not the only cause of DNA damage and carcinogenesis. Indirect damage of neighbouring cells via factors released by the irradiated population is causing cellular stress and DNA damage. The resulting damage in bystander cells has been previously studied, but the DDR is less well understood. This research aimed to develop understanding of DDR protein activation and downregulation using a co-incubation technique. It was indicated there was a dose dependent change in DDR pathway activation where the ATM response occurred later at a higher dose whilst the reverse was observed for direct irradiations. The data implied that γH2AX was the result of ATM activation arguing against others which suggested it becomes activated by the ATR response.