Journal of Nucleic Acids observed, for example, in cancer cells. Thus, this form of homology-directed DNA repair of double-strand breaks helps to maintain a high level of genome stability, even though immediate cell survival may be only minimally aﬀected in its absence, because of other repair mechanisms (Figure 1 and reviewed in [75]). 4. Methods of γH2AX Detection The quantitative assessment of DSBs was initially based on methods, such as pulse-ﬁeld gel electrophoresis (PFGE), DNA elution tests, or the so-called “comet assay” (single cell gel electrophoresis—SCGE). Among these methods, the comet assay is a versatile, sensitive, and widely used one. In this method individual cells with damaged DNA embedded in agarose gels are subjected to an electric ﬁeld and generate a characteristic pattern of DNA distribution forming a tail that, after staining with ﬂuorescence dye, can be analyzed by ﬂuorescence microscopy. The extent and length of the comet’s tail correlates with the severity of DNA damage [76]. The sensitivity of the comet assay, however, depends on proper calibration and its speciﬁcity is not absolute [77, 78]. Development of an antibody, that is speciﬁc to γH2AX, made it possible to detect H2AX phosphorylation and thus detection of DNA damage and repair in situ in individual cells. The presence of γH2AX in chromatin can be detected shortly after induction of DSBs in the form of discrete nuclear foci. Since each focus represents a single DSB, their frequency reports the incidence of DSBs (reviewed in [79]). Compared with alternative methods of DNA damage assess- ment mentioned above, the immunocytochemical approach is less cumbersome and oﬀers much greater sensitivity [79, 80]. The presence of γH2AX-containing nuclear foci can be measured by microscopy, ﬂow cytometry, and possibly Western blotting of cell/tissue lysates, with normalization for the total H2AX levels. Measurement of γH2AX with the use of multiparameter ﬂow or laser scanning cytometry seems to be particular advantageous as H2AX phosphorylation can be determined in individual cells with high sensitivity and accuracy and γH2AX expression in cell populations can be correlated with DNA content or induction of apoptosis [79, 80]. 5. Applications of γH2AX Detection Assessment of H2AX phosphorylation as a reporter of DNA damage can be clinically useful. The most important clinical application of γH2AX measurements is to follow DSBs levels induced by radio- and chemotherapy as a marker of treatment eﬃcacy [81, 82] and in dose/scheduling estimation as well as to determine the eﬃciency of DNA repair to predict possible tumor sensitivity or resistance to DNA damaging anticancer agents. By assessing DSB levels induced by anticancer treatment in normal cells, one may also predict toxicity of anticancer treatment. Exposure to endogenous or environmental mutagens results in DNA damage which in turn activates the DDR cascade [83, 84]. Once activated, the DDR machinery 5 functions as an “anticancer barrier” and delays or suppresses tumor development by inducing cell death or replicative senescence [83, 85]. However, prolonged activation of DDR may sometimes lead to survival of malignant cells if only these cells are able to bypass cell growth barrier imposed by the DDR pathway [79]. Thus, activated components of the DDR can be used as cancer biomarkers, with γH2AX being the most sensitive one. In this way, γH2AX level measurements may help to detect precancerous lesions or cancer at its early stages [86–88]. In this case, increased background levels of γH2AX or the presence of DSBs on telomere ends may be indicative for replicative senescence, including premature senescence induced by anticancer drugs [89] or cancer progression. Finally, other possible practical applications of γH2AX include screening for eﬀects of environmental pollutants (air- or food-borne etc.) or follow- up of DNA lesions induced by mutagenic substances at work places, including nurses distributing anticancer drugs [90]. Similarly, γH2AX may help to evaluate eﬀects of chemopreventive agents. Endogenous γH2AX levels may also reﬂect genomic instability phenotype since optimal protection against cancer requires full functionality of both alleles of H2AX gene (H2AFX). Several diﬀerent tumor types have been reported to possess mutations, including single gene polymorphism, ampliﬁcation, or deletion in the region 11q23 that maps for H2AFX [62, 91]. Other groups reported alterations in H2AX gene copy number or changes in its promoter region to be associated with the risk of sporadic breast cancer and lymphomas [81, 92]. In this way, one may also relate H2AFX mutations or deletions with the susceptibility of individuals to tumors, such as lymphoma, acute myeloid leukemia, and head and neck cell carcinomas for tumor risk assessment studies [92–95]. 6. Conclusions As discussed above, determination of γH2AX levels is not only important from the fundamental perspective as a sen- sitive marker of the DNA damage response, including DNA repair processes. It has also many potential practical appli- cations in monitoring eﬀects induced by anticancer therapy and assessing tumor risk as well as in determination of DNA lesions resulting from exposure of animals and humans to environmental pollutants. However, it is important to discuss not only advantages but also potential limitations and problems of the application of γH2AX in clinical practice. An obvious advantage of γH2AX compared to other DNA damage markers is its extremely high sensitivity that enables one to detect very small changes in genome integrity on a single cell level. However, the lower limit of γH2AX detection depends on many factors, including background levels of nuclear foci observed in unstressed cells, which are associated with DNA replication and progression through mitosis [96, 97]. In addition, background γH2AX levels may diﬀer between various tumor cells and, more importantly, between tumor and normal cells due to changed functionality of H2AXF discussed above. This can make it more complicated to predict the toxicity of anticancer treatment in patients