Factors to Consider for the Correct Use of γH2AX in the Evaluation of DNA Double-Strand Breaks Damage Caused by Ionizing Radiation

Abstract

People exposed to ionizing radiation (IR) both for diagnostic and therapeutic purposes is constantly increasing. Since the use of IR involves a risk of harmful effects, such as the DNA DSB induction, an accurate determination of this induced DNA damage and a correct evaluation of the risk-benefit ratio in the clinical field are of key relevance. γH2AX (the phosphorylated form of the histone variant H2AX) is a very early marker of DSBs that can be induced both in physiological conditions, such as in the absence of specific external agents, and by external factors such as smoking, heat, background environmental radiation, and drugs. All these internal and external conditions result in a basal level of γH2AX which must be considered for the correct assessment of the DSBs after IR exposure. In this review we analyze the most common conditions that induce H2AX phosphorylation, including specific exogenous stimuli, cellular states, basic environmental factors, and lifestyles. Moreover, we discuss the most widely used methods for γH2AX determination and describe the principal applications of γH2AX scoring, paying particular attention to clinical studies. This knowledge will help us optimize the use of available methods in order to discern the specific γH2AX following IR-induced DSBs from the basal level of γH2AX in the cells.

Keywords: DNA damage; DNA repair; basal level; double-strand breaks (DSBs); ionizing radiation; γH2AX scoring.

Figure 1

Double-strand break (DSB)- and H2AX-inducing factors. Exogenous, endogenous, individual factors, and lifestyle inducing DSBs and H2AX are described.

Figure 2

Phosphorylation of H2AX in the DSB response. Upon DSBs induced by IR, H2AX activity is regulated by Ser139 phosphorylation/dephosphorylation cycles. Phosphorylation is catalyzed by ATM, ATR, and DNA-PK. When H2AX is phosphorylated at the sites flanking DSBs, different repair proteins are recruited, including the MRN protein complex, 53BP1, and cohesins. After repair, γH2AX is removed by dephosphorylation catalyzed by the protein phosphatases PP2A, PP4C, PP6, and WIP1.

Figure 3

γH2AX foci repair kinetic. Immunofluorescence analysis of γH2AX in HF cells irradiated with 0.2 Gy. Following DNA damage, γH2AX is quickly induced and reaches the maximum peak in a very short time (about 30 min). Then, the γH2AX levels decrease more slowly until they reach the basal levels at about 24 h. Top panel: graph showing the amount of γH2AX foci/cells at the indicated time points after IR stimulus. Bottom panel: representative immunofluorescence images at the indicated time points after IR stimulus (scale bar is 10 µm).

Figure 4

Two different populations of γH2AX foci. Immunofluorescence analysis of γH2AX and 53BP1 in HF cells irradiated with 0.2 Gy. Two populations of γH2AX foci, distinguished by the size and colocalization with DNA DSB repair proteins, are shown. The small foci are more numerous and do not colocalize with 53BP1, while the large foci represent a small population and colocalize with 53BP1.