Investigation and application of γH2AX as a potential biomarker of DNA double strand breaks in insect and human cells

Abstract

Double strand breaks (DSBs) are one of the most biologically significant DNA damage lesions. Replication stress, endogenous reactive oxygen species, exogenous sources of DNA damage such as ionizing radiation (IR), and genotoxic compounds are key causes of DNA breaks in living systems. An early known response to DNA DSBs in the cell is the phosphorylation of the C-terminal of the core histone protein H2AX (termed γH2AX when phosphorylated). It is accepted that with the passage of time, the level of γH2AX declines as repair of DSBs is completed; however, DSBs can remain unrepaired and may result in persistent γH2AX signals and the knowledge of persistent γH2AX signals remain relatively unexplored. DNA damage has been associated with some agerelated diseases, including the neurodegenerative disorder, Alzheimer’s disease (AD). The aim of this PhD thesis was to (i) investigate IR-induced persistent γH2AX responses in Queensland fruit fly (Q-fly) (Bactrocera tryoni), and human buccal cell as a model system (ii) quantify endogenous γH2AX levels in buccal cells and lymphocytes of individuals with mild cognitive impairment (MCI) and AD relative to healthy controls in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL) and the South Australian Alzheimer’s Nutrition and DNA Damage (SAND) studies. Persistent and dose-dependent γH2AB (a homologue of γH2AX) signals were detected and quantified either by Western blot or laser scanning cytometry (LSC) for up to 17 days post-IR exposure in adult Q-flies (when irradiated as pupae). In human buccal cells irradiated (up to 4 Gy), LSC and visual scoring demonstrated a significant increase in γH2AX (n=6 individuals). Twenty-four hours after IR exposure the γH2AX levels remained significantly higher than baseline. The frequency of visually scored γH2AX in human buccal cell nuclei showed a strong positive correlation (up to r=0.999) with automated LSC scored γH2AX signals. In the SAND study, the endogenous γH2AX levels were significantly higher in lymphocytes from AD (n=20) compared to MCI (n=18) and controls (n=40). Plasma homocysteine, creatinine, and chitinase-3-like protein 1 (CHI3L1) were positively correlated with lymphocyte γH2AX signals, whilst glomerular filtration rate (GFR) was negatively correlated. In buccal cells, the endogenous γH2AX levels were significantly elevated in AD (n=16), compared to MCI (n=18) and controls (n=17), from the AIBL study. Nuclear circularity (irregular nuclear shapes) was significantly higher in buccal cell nuclei from AD compared to MCI and controls and there was a positive correlation between nuclear circularity and γH2AX signals. The elevated γH2AX levels in lymphocytes and buccal cells of AD patients may indicate defects in the efficiency of repairing the chronic endogenous DNA DSBs contributing to the accumulation of unrepaired or persistent DSBs. The measurement of persistent and endogenous γH2AX may have application in radiation biodosimetry as well as a potential biomarker in AD.