6 based on the direct comparison of DSBs induced in tumor versus normal cells. Timing of γH2AX measurements after drug treatment may also be critical for the appropriate data analysis and interpretation. Induction of H2AX phosphorylation is, at least for some agents, very rapid and this requires quick methods of patient sample acquisition and analysis. In some situations, the choice between of diﬀerent sample material may be crucial for the assay (PBMLs versus tumor cells— leukemia, normal tissue, and tumor samples for solid tumors etc.). Moreover, anticancer treatment may lead to increased phosphorylation of H2AX due to death-associated DNA fragmentation, especially after long time periods after drug treatment or in case of agents which induce rapid apoptosis. However, cell death-related H2AX phosphorylation can be discriminated from that induced by anticancer treatment based on focal and diﬀuse or soluble γH2AX signals [98]. This requires selection of appropriate diagnostic methods and application of biparametric ﬂow cytometry, scanning cytometry, or ﬂuorescence microscopy to estimate γH2AX levels. Acknowledgments This work was supported by a research Grant from Polish Ministry of Science and Higher Education (Grant no. N402 208535) and statutory funds from the Faculty of Chemistry, Gdansk University of Technology. References [1] S. P. Jackson, “Sensing and repairing DNA double-strand breaks,” Carcinogenesis, vol. 23, no. 5, pp. 687–696, 2002. [2] E. Sonoda, H. Hochegger, A. Saberi, Y. Taniguchi, and S. Takeda, “Diﬀerential usage of non-homologous end-joining and homologous recombination in double strand break repair,” DNA Repair, vol. 5, no. 9-10, pp. 1021–1029, 2006. [3] V. Smider and G. Chu, “The end-joining reaction in V(D)J recombination,” Seminars in Immunology, vol. 9, no. 3, pp. 189–197, 1997. [4] S. P. Jackson, “Detecting, signalling and repairing DNA double-strand breaks,” Biochemical Society Transactions, vol. 29, no. 6, pp. 655–661, 2001. [5] T. Tanaka, H. D. Halicka, X. Huang, F. Traganos, and Z. Darzynkiewicz, “Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants,” Cell Cycle, vol. 5, no. 17, pp. 1940– 1945, 2006. [6] T. Tanaka, X. Huang, H. D. Halicka et al., “Cytometry of ATM activation and histone H2AX phosphorylation to esti- mate extent of DNA damage induced by exogenous agents,” Cytometry Part A, vol. 71, no. 9, pp. 648–661, 2007. [7] M. M. Vilenchik and A. G. Knudson, “Endogenous DNA double-strand breaks: production, ﬁdelity of repair, and induction of cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 22, pp. 12871–12876, 2003. [8] A. Takahashi and T. Ohnishi, “Does γH2AX foci formation depend on the presence of DNA double strand breaks?” Cancer Letters, vol. 229, no. 2, pp. 171–179, 2005. Journal of Nucleic Acids [9] T. Tanaka, M. Kajstura, H. D. Halicka, F. Traganos, and Z. Darzynkiewicz, “Constitutive histone H2AX phosphorylation and ATM activation are strongly ampliﬁed during mitogenic stimulation of lymphocytes,” Cell Proliferation, vol. 40, no. 1, pp. 1–13, 2007. [10] M. Cuadrado, B. Martinez-Pastor, and O. Fernandez- Capetillo, “ATR activation in response to ionizing radiation: still ATM territory,” Cell Division, vol. 1, article 7, pp. 1–4, 2006. [11] J. C. Wang, “DNA topoisomerases,” Annual Review of Bio- chemistry, vol. 65, pp. 635–692, 1996. [12] J. C. Wang, “Cellular roles of DNA topoisomerases: a molecu- lar perspective,” Nature Reviews Molecular Cell Biology, vol. 3, no. 6, pp. 430–440, 2002. [13] J. L. Nitiss, “DNA topoisomerase II and its growing repertoire of biological functions,” Nature Reviews Cancer, vol. 9, no. 5, pp. 327–337, 2009. [14] J. E. Deweese and N. Osheroﬀ, “The DNA cleavage reaction of topoisomerase II: wolf in sheep’s clothing,” Nucleic Acids Research, vol. 37, no. 3, pp. 738–748, 2009. [15] A. K. McClendon and N. Osheroﬀ, “DNA topoisomerase II, genotoxicity, and cancer,” Mutation Research, vol. 623, no. 1-2, pp. 83–97, 2007. [16] Y. Pommier, “DNA topoisomerase I Inhibitors: chemistry, biology, and interfacial inhibition,” Chemical Reviews, vol. 109, no. 7, pp. 2894–2902, 2009. [17] Y. Pommier, “Topoisomerase I inhibitors: camptothecins and beyond,” Nature Reviews Cancer, vol. 6, no. 10, pp. 789–802, 2006. [18] E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner, “DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139,” Journal of Biological Chemistry, vol. 273, no. 10, pp. 5858–5868, 1998. [19] C. E. Helt, W. A. Cliby, P. C. Keng, R. A. Bambara, and M. A. O’Reilly, “Ataxia telangiectasia mutated (ATM) and ATM and Rad3-related protein exhibit selective target speciﬁcities in response to diﬀerent forms of DNA damage,” Journal of Biological Chemistry, vol. 280, no. 2, pp. 1186–1192, 2005. [20] M. B. Kastan and D.-S. Lim, “The many substrates and functions of ATM,” Nature Reviews Molecular Cell Biology, vol. 1, no. 3, pp. 179–186, 2000. [21] C. J. Bakkenist and M. B. Kastan, “DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation,” Nature, vol. 421, no. 6922, pp. 499–506, 2003. [22] A. Dupre´, L. Boyer-Chatenet, and J. Gautier, “Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex,” Nature Structural and Molecular Biology, vol. 13, no. 5, pp. 451–457, 2006. [23] N. Srivastava, S. Gochhait, P. de Boer, and R. N. K. Bamezai, “Role of H2AX in DNA damage response and human cancers,” Mutation Research, vol. 681, no. 2-3, pp. 180–188, 2009. [24] Y. Sun, X. Jiang, S. Chen, N. Fernandes, and B. D. Price, “A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 37, pp. 13182–13187, 2005. [25] Y. Sun, Y. Xu, K. Roy, and B. D. Price, “DNA damage- induced acetylation of lysine 3016 of ATM activates ATM kinase activity,” Molecular and Cellular Biology, vol. 27, no. 24, pp. 8502–8509, 2007. [26] I. M. Ward and J. Chen, “Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress,” Journal of Biological Chemistry, vol. 276, no. 51, pp. 47759– 47762, 2001.