Investigation and application of γH2AX as a potential biomarker of DNA double strand breaks in insect and human cells
Date
2016
Authors
Siddiqui, Mohammad Sabbir
Editors
Advisors
Leifert, Wayne
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Thesis
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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.
School/Discipline
School of Agriculture, Food and Wine
Dissertation Note
Thesis (Ph.D.)--University of Adelaide, School of Agriculture, Food and Wine, 2016
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