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DNA in live cells undergoes continuous oxidative damage caused by metabolically generated endogenous as well as external oxidants and oxidant-inducers. The cumulative oxidative DNA damage is considered the key factor in aging and senescence while the effectiveness of anti-aging agents is often assessed by their ability to reduce such damage. Oxidative DNA damage also preconditions cells to neoplastic transformation. Sensitive reporters of DNA damage, particularly the induction of DNA double-strand breaks (DSBs), are activation of ATM, through its phosphorylation on Ser 1981, and phosphorylation of histone H2AX on Ser 139; the phosphorylated form of H2AX has been named γH2AX. We review the observations that constitutive ATM activation (CAA) and H2AX phosphorylation (CHP) take place in normal cells as well in the cells of tumor lines untreated by exogenous genotoxic agents. We postulate that CAA and CHP, which have been measured by multiparameter cytometry in relation to the cell cycle phase, are triggered by oxidative DNA damage. This review also presents the findings on differences in CAA and CHP in various cell lines as well as on the effects of several agents and growth conditions that modulate the extent of these histone and ATM modifications. Specifically, described are effects of the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC), and the glutathione synthetase inhibitor buthionine sulfoximine (BSO) as well as suppression of cell metabolism by growth at higher cell density or in the presence of the glucose antimetabolite 2-deoxy-D-glucose. Collectively, the reviewed data indicate that multiparameter cytometric measurement of the level of CHP and/or CAA allows one to estimate the extent of ongoing oxidative DNA damage and to measure the DNA protective-effects of antioxidants or agents that reduce or amplify generation of endogenous ROS. Keywordsaging; senescence; DNA replication; DNA double-strand breaks; cell cycle; free radicals; reactive oxygen species (ROS); reactive oxygen intermediates (ROIs); anti-oxidants; oxidative stress DNA DAMAGE BY ENDOGENOUS OXIDANTSBeing continuously exposed to oxidants produced during metabolic activity and to external oxidants or oxidant-inducers, DNA within cells undergoes oxidative damage. Estimates of the extent of endogenous DNA damage vary widely. [1][2][3][4][5] 5 Recombinatorial repair (also known as template-assisted repair or homologous recombination repair) and nonhomologous DNA-end joining (NHEJ) are two major pathways for repair of DSBs. The NHEJ pathway is error-prone, often resulting in deletion of a few base pairs. 6,7 This leads to accumulation of DNA damage with each sequential cell cycle, which is considered to be the primary cause of cell aging and senescence. 4,8,9 The cumulative DNA damage also promotes development of preneoplastic changes. Many strategies aimed at slowing down the aging process or preventing cancer are based on protection of DNA from oxidative damage, primarily by scavenging the endogenous oxidants. While appr...

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxy-nucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named γH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of γH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio-or chemotherapy may provide an early marker predictive of response to treatment. DNA FRAGMENTATION DURING APOPTOSIS Involvement of different nucleases in DNA fragmentationCondensation of chromatin and internucleosomal DNA fragmentation, together with cell shrinkage and shedding of apoptotic bodies ('blebbing'), are widely recognized hallmarks of apoptosis (Kerr et al. 1972;Arends et al. 1990;Nagata 2000;Nagata et al. 2003). Several nucleases have been identified as contributing towards DNA degradation; their activity is modulated by divalent cations. Depending on cation concentration, three distinct steps of DNA fragmentation, likely mediated by different enzymes, can be identified: (i) in the presence of Mg 2+ (2 mM, DNA is fragmented to about 0.05-1 megabase (Mb)-size sections (type-I, high molecular weight DNA fragmentation)); (ii) at low (nanomolar) Ca 2+ concentration, nuclear DNA is cleaved into intermediate (∼300 kb) fragments (type-II, intermediate DNA fragmentation); (iii) at micromolar levels of Ca 2+ , internucleosomal (type-III) DNA fragmentation takes place leading to formation of DNA sections of the size of mono and oligonucleosomes, which form a characteristic 'DNA-ladder' pattern during electrophoresis (Arends et al. 1990 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAmong the nucleases associated with DNA fragmentation during apoptosis, the best characterized is CAD (caspase-activated DNase) with its inhibitor ICAD (inhibitor of CAD) in mice, and its human homologue DFF40/DFF45 (DNA fragmentation factor) (Enari et al. 1998). CAD and ICAD (or ...