γ-H2AX - A Novel Biomarker for DNA Double-strand Breaks

When DNA damage, whether it is endogenous or exogenous, forms double stranded breaks (DSBs), it is always followed by the phosphorylation of the histone, H2AX. H2AX is a variant of the H2A protein family, which is a component of the histone octomer in nucleosomes. It is phosphorylated by kinases such as ataxia telangiectasia mutated (ATM) and ATM-Rad3-related (ATR) in the PI3K pathway. This newly phosphorylated protein, γ- H2AX, is the first step in recruiting and localizing DNA repair proteins. DSBs can be induced by mechanisms such as ionizing radiation or cytotoxic agents and subsequently, γ- H2AX foci quickly form. These foci represent the DSBs in a 1:1 manner and can be used as a biomarker for damage. An antibody can be raised against γ- H2AX which can therefore be visualized by immunofluorescence through secondary antibodies. The detection and visualization of γ- H2AX by flow cytometry allow the assessment of DNA damage, related DNA damage proteins and DNA repair. γ- H2AX also has other applications in the detection of genomic damage caused by cytotoxic chemical agents and environmental and physical damage, especially in the context of cancer treatment and therapy. DNA damage occurs in all cells and the damage can be categorized into two types, endogenous and exogenous. Endogenous damage is caused by the cell itself and can arise from various pathways such as apoptosis, excision repair, oxidative damage or depurination. Exogenous damage occurs when cells are exposed to physical damage (radiation) or chemical agents (cytotoxic drugs). The DNA damage that ensues can be base damage, sugar damage, single stranded breaks (SSBs) or double stranded breaks (DSBs), the latter being lethal to cells. Measuring DSBs has been of interest in the research community because of its predictable nature. These lethal nicks in the DNA allow the prediction of toxicity in cells or the death of cells. There have been many methods for detecting DSBs, such as neutral elution, pulse field electrophoresis (2-D gel electrophoresis) and comet assays, but the γ- H2AX assay is particularly precise. This recently developed assay is not only simple but is also more sensitive to DSBs than the other techniques. Furthermore, it can detect DSBs in intact cells, which allows fluorescent visualization and the physical localization of the DSBs. The simplicity, ease and sensitivity of this assay have been the cause of its increasing use as a popular method for detecting DSBs.