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...

Background: Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional (“sub‐G1”) DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome. Materials and Methods: The model of activation‐induced apoptosis of human lymphocytes was chosen as it provides uniform cell population with identical DNA content (DI = 1.00) that undergo apoptosis. Their apoptosis was induced by multivalent mitogen phytohemagglutinin (PHA) in the absence and presence of geldanamycin (GA), the benzoquinone ansamycin antibiotic which binds to Hsp90 (Heat Shock Protein 90) and alters its function. The cells were stained with acridine orange, the metachromatic fluorochrome that differentially stains cellular DNA and RNA. Results: A sharp, discrete peak representing the subpopulation of “sub‐G1” cells with highly reproducible DI = 0.42 ± 0.02 (CV = 5.5 ± 1.2) was observed on DNA content histograms of lymphocytes whose apoptosis was induced by PHA alone. Two distinct peaks, one representing cell subpopulations with DI = 0.42 (as above) and another, with DI = 0.79 ± 0.04 (CV = 5.8 ± 0.4), respectively, were seen in apoptotic cells from cultures stimulated with PHA in the presence of GA. The frequency of cells represented by the sub‐G1 peaks varied depending on time of induction of apoptosis and GA concentration. Conclusions: Apoptosis‐induced DNA fragmentation is discontinuous; approximately 42% of DNA is relatively stable and remains within the cell. The data suggest that the stable DNA is associated with nuclear matrix while the degradable fraction represents DNA in loop domains. A transient DNA stabilization is apparent in the presence of GA as evidenced by the presence of cell subpopulations with 79% of DNA retained in the cell. The observed discontinuity of DNA fragmentation appears to reflect sequential involvement of different nucleases and may also be modulated by chromatin structure. © 2007 International Society for Analytical Cytology.

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 ...

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (γH2AX) can be revealed immunocytochemically; the intensity of γH2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of γH2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m 2 -3.45 kJ/m 2 ) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G 1 , G 2 and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced γH2AX in G 1 , G 2 and M was much higher in HeLa-than in HL-60-cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of γH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication by aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of γH2AX. Furthermore, aphidicolin itself induced γH2AX in early-S phase cells. The induction of γH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of γH2AX in G 1 , G 2 and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.

This review covers the topic of cytometric assessment of activation of Ataxia telangiectasia mutated (ATM) protein kinase and histone H2AX phosphorylation on Ser139 in response to DNA damage, particularly the damage that involves formation of DNA double-strand breaks. Briefly described are molecular mechanisms associated with activation of ATM and the downstream events that lead to recruitment of DNA repair machinery, engagement of cell cycle checkpoints, and activation of apoptotic pathway. Examples of multiparameter analysis of ATM activation and H2AX phosphorylation vis-a-vis cell cycle phase position and induction of apoptosis that employ flow-and laser scanning-cytometry are provided. They include cells treated with a variety of exogenous genotoxic agents, such as ionizing and UV radiation, DNA topoisomerase I (topotecan) and II (mitoxantrone, etoposide) inhibitors, nitric oxide-releasing aspirin, DNA replication inhibitors (aphidicolin, hydroxyurea, thymidine), and complex environmental carcinogens such as present in tobacco smoke. Also presented is an approach to identify DNA replicating (BrdU incorporating) cells based on selective photolysis of DNA that triggers H2AX phosphorylation. Listed are strategies to distinguish ATM activation and H2AX phosphorylation induced by primary DNA damage by genotoxic agents from those effects triggered by DNA fragmentation that takes place during apoptosis. While we review most published data, recent new findings also are included. Examples of multivariate analysis of ATM activation and H2AX phosphorylation presented in this review illustrate the advantages of cytometric flow-and image-analysis of these events in terms of offering a sensitive and valuable tool in studies of factors that induce DNA damage and/or affect DNA repair and allow one to explore the linkage between DNA damage, cell cycle checkpoints and initiation of apoptosis. ' International Society for Analytical CytologyKey terms ionizing radiation; DNA topoisomerase inhibitors; DNA double-strand breaks; carcinogens; tobacco smoke; replication stress; genotoxins; DNA photolysis ACTIVATION OF ATM AND PHOSPHORYLATION OF HISTONE H2AX TRIGGERED BY DNA DAMAGE Ataxia telangiectasia mutated (ATM) is a protein kinase that becomes activated in response to DNA damage, particularly when the damage involves formation of DNA double-strand breaks (DSBs) (1-9; Fig. 1). Interestingly, the initial activation of ATM does not takes place at the exact site of the DSB but at some distance from it, and appears to be triggered by a change in the higher order of chromatin structure caused by unwinding and relaxation of the topological stress of the DNA double helix upon induction of the DSB (4). Activation of ATM occurs through its autophosphorylation on Ser1981 and it requires prior ATM acetylation that is mediated by the Tip60 histone acetyltransferase (13). Ser1981 ATM phosphorylation causes dissociation of the inactive ATM dimer or multimer into single monomeric units