Background: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted ␥H2AX. The intensity of immunofluorescence (IF) of ␥H2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the druginduced (DI) IF of ␥H2AX, and in particular to distinguish it from AA ␥H2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. Methods: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA crosslinking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of ␥H2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of ␥H2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. Results: In cells treated with TPT or MTX, the increase in DI-␥H2AX IF peaked at 1.5 or 2 h, and was maximal in Sor G 1 -phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the ␥H2AX IF was less intense, peaked later (3 h) and showed no cell cyclephase specificity. In the presence of phosphatase inhibitor

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 ...

Histone H2AX is phosphorylated on Ser-139 by ATM kinase in response to damage that induces dsDNA breaks. Immunocytochemical detection of phosphorylated H2AX (γH2AX), thus, reveals the presence of dsDNA breaks in chromatin. Multiparameter cytometry was presently used to correlate the appearance of γH2AX with: a. cell cycle phase; b. caspase-3 activation; and c. apoptosis-associated DNA fragmentation in individual human leukemic HL-60 cells treated with the DNA topoisomerase (topo1) inhibitors topotecan (TPT) and camptothecin (CPT) or with the topo2 inhibitor mitoxantrone (MTX).In response to TPT or CPT maximal increase of γH2AX immunofluorescence was seen in S-phase cells by 90 min. In contrast, following MTX treatment the maximal rise of γH2AX was detected at 2 h in G 1 cells and the cell cycle phase specificity was much less apparent. A linear relationship between the drug concentration and increase of γH2AX immunofluorescence was seen only up to 200 nM TPT; a decline in γH2AX was apparent at a concentration range between 0.4 and 1.6 γM TPT. Thus, the intensity of γH2AX immunofluorescence, as a marker of cell survival following TPT treatment, can be used only within a limited range of drug concentration. Following treatment with TPT, CPT or MTX the peak of H2AX phosphorylation preceded caspase-3 activation and the appearance of apoptosis-associated DNA fragmentation, both selective to S-phase cells. Progression of apoptosis was paralleled by a decrease in γH2AX immunofluorescence. The data also indicate that regardless whether treated with inhibitors of topo1 or topo2, at comparable levels of dsDNA breaks, the cells replicating DNA have a higher proclivity to undergo apoptosis compared to G 1 or G 2 /M cells.

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.