Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

Radiotherapy is widely used for the management of cancer and relies on ionizing radiation (IR)-induced DNA damage to kill malignant cells. DNA double-strand breaks (DSBs), which are exceptionally lethal lesions can be formed either by direct energy deposition or indirectly through the radiolysis of water molecules, which generate clusters of reactive oxygen species that attack DNA molecules [1-4]. DSBs are essentially two single-stranded nicks in opposing DNA strands that occur in close proximity, severely compromising genomic stability [2,5-7]. Therefore, it is critical that DSBs are repaired quickly and efficiently to prevent cellular death, chromosomal aberrations and mutations [6,8]. A series of complex pathways collectively known as the DNA damage response (DDR) is responsible for the recognition, signalling and repair of DSBs in cells, ultimately resulting in either cell survival or cell death [9,10]. DSBs are repaired by two major pathways, homologous recombination (HR) or non-homologous end joining (NHEJ), each with distinct and overlapping roles in maintaining genomic integrity. NHEJ, the more error-prone pathway, is commonly employed following IR-induced damage [11]. IR-induced DSBs cause rapid phosphorylation of the histone H2A variant H2AX to form γH2AX. This phosphorylation event takes place at the highly conserved SQ motif, which is a common substrate for the phosphatidyl-inosito 3-kinase (PI3K) family of proteins including ataxia telangiectasia mutated (ATM) [12-16]. Discrete nuclear foci that form as a result of H2AX phosphorylation are now widely used as a sensitive and reliable marker of DSBs [17,18]. Following a discussion of the biology of γH2AX formation, this review will focus on the utility of γH2AX as a molecular marker for monitoring the efficacy of radiation-modifying compounds.Recent years have witnessed a remarkable proliferation in immunofluorescence-based assays dedicated to the visualization of γH2AX foci. This has emerged as the prefe