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. 2011 Nov;79(11):897-902.
doi: 10.1002/cyto.a.21137. Epub 2011 Sep 8.

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry"

Hong Zhao  1 , Jurek Dobrucki, Paulina Rybak, Frank Traganos, H Dorota Halicka, Zbigniew Darzynkiewicz
Affiliations

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry"

Hong Zhao et al. Cytometry A. 2011 Nov.

Abstract

Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .

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Figures

Figure 1
Figure 1
Correlation between DNA replication and induction of γH2AX in A549 cells treated with H2O2. Panel A: The cells were not exposed to EdU nor were they treated with H2O2. Panels B and D: cells were exposed in culture to 5 μM EdU for 120 min. The cells with variable level of EdU incorporation were entering (enS) or exiting (exS) S phase during the duration of the EdU pulse as their interval of exposure to the precursor at the time of DNA replication varied between 1 and 120 min. C, E, and F: The cultures were initially treated with 5 μM EdU for 60 min, then exposed to 200 μM of H2O2 (still in the presence of EdU) for an additional 60 min. All cultures (B—F) thus had a 120 min-pulse of EdU, while C,E,F cultures were also exposed to H2O2 for 60 min. Using the “paint-a-gate” gating analysis, the DNA replicating cells characterized by EdU incorporation (above the threshold of the of unexposed to EdU cells; A, dashed line) were colored red (B) and then replotted as γH2AX vs. DNA content (E) or γH2AX vs. EdU (F) bivariate distributions. It is quite evident that following exposure to H2O2, predominantly DNA replicating (red) cells had increased expression of γH2AX [above the level of the H2O2 untreated control cells (D, dashed line)]. A moderate correlation between EdU incorporation and the induction of H2AX estimated for the population of DNA replicating (red colored) cells is apparent, with the coefficient (Pearson) r = 0.36 (F). The insets in A and C show DNA frequency histograms from the respective cultures. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
Figure 2
Figure 2
Correlation between DNA replication and phosphorylation of ATM on Ser1981 in A549 cells treated with H2O2. Panels A and B: the cells were exposed in culture to 5 μM EdU for 120 min. Panels B, D, and F: The cultures were initially treated with 5 μM EdU for 60 min, then exposed to 200 μM of H2O2 (still in the presence of EdU) for an additional 60 min. As in Fig. 1, using the “paint-a-gate” gating analysis, the EdU incorporating cells were colored red (B) and then data were replotted as ATM-S1981P vs. DNA content (D) or ATM-S1981P vs. EdU (F) bivariate distributions. It is apparent that predominantly DNA replicating (red) cells exhibited an increased level of ATM-S1081P following exposure to H2O2 [above the upper threshold for 97% of cells in the H2O2 untreated control culture (C, dashed line)]. A modest correlation between EdU incorporation and the induction of ATM-S1981P estimated for the population of DNA replicating (red colored) cells is apparent, with the coefficient (Pearson) r = 0.32 (F). The insets in panels A and B show DNA frequency histograms from the respective cultures. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
Figure 3
Figure 3
Relationship between the induction of γH2AX or activation of ATM and the extent of EdU incorporation of cells exposed to H2O2. Panel A: The cells were exposed to EdU for 60 min and then to H2O2 for the next 60 min (as in Fig. 1C). The analysis gate was set in mid-S to exclude the enS and exS cells. Such analysis enabled one to correlate the extent of EdU incorporation of the cells that were exposed to EdU for the full 120 min at the time when they were replicating DNA compared to the expression of γH2AX (D) or ATM-S1981P(E). [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]
Figure 4
Figure 4
Relationship between the sites of EdU incorporation and the induction of γH2AX foci in A549 cells treated with H2O2 Confocal images of A549 nuclei (selected equatorial planes) that were exposed to EdU for 30 min and then to H2O2 for additional 30 min. The incorporation of EdU was detected using the “Click-iT® methodology utilizing AlexaFluor 488-tagged azide (green fluorescence), whereas γH2AX was detected immunocytochemically with the secondary Ab labeled with AlexaFluor 568 (red fluorescence). The bottom panels show the sites of EdU incorporation that are in close proximity or colocalize with γH2AX foci, selected from the respective cell images (enlarged). The size marker = 5 μm.
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