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

Zhao, Hong; Dobrucki, Jurek; Rybak, Paulina; Traganos, Frank; Halicka, H. Dorota; Darzynkiewicz, Zbigniew

doi:10.1002/cyto.a.21137

Cited by 38 publications

(51 citation statements)

References 44 publications

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“…Thus, our present data are compatible with the view that while some DSBs induced by Mxt or Etp were caused by collisions of DNA replication forks with the stabilized complexes of Top2-DNA (7), other DSBs could have originated from collisions of the DNA transcription machinery with these complexes (9,10) and/or were caused by ROS induced by treatment with these inhibitors (22,25). As we have shown recently, the oxidative stress-induced γH2AX foci are predominant in S-phase cells and do co-localize with DNA replication sites (32). …”

Section: Discussionmentioning

confidence: 70%

“…Each experiment was performed with an IgG control in which cells were labeled only with the secondary AlexaFluor 647 Ab, without primary antibody incubation to estimate the extent of nonspecific binding of the secondary antibody to the cells. Other details of cell incubations with the primary and secondary Ab have been previously described (30,32). …”

Section: Methodsmentioning

confidence: 99%

“…Toward this end we have labeled DNA replicating cells with a short pulse of the DNA precursor 5-ethynyl-2′deoxyuridine (EdU) and subsequently exposed them to the inhibitors. The detection of the EdU incorporation using the “click chemistry” approach (26) makes it possible to concurrently detect intracellular epitopes (27), including their modification by phosphorylation, with the use of phospho-specific Abs (28–32). Using multiparameter LSC, we have been able, therefore, to directly correlate DNA replication with the induction of DNA damage signaling triggered by topoisomerase inhibitors as revealed by H2AX phosphorylation.…”

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confidence: 99%

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Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide

et al. 2011

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DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient (“cleavable”) complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2′deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as γH2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of γH2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of γH2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of γH2AX foci predominantly in DNA replicating cells and close association and co-localization of γH2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the γH2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of γH2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1–DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (γH2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.

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Section: Discussionmentioning

confidence: 70%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide

et al. 2011

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“…Therefore, we examined effect of TTB on DNA damage. γ-H2AX staining is a well-known marker of oxidative-related DNA damage [29,30]. TTB increased co-staining of DAPI and γH2AX foci in MDA-MB-231 cells (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling

et al. 2017

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Tuberatolide B (TTB, C27H34O4) is a diastereomeric meroterpenoid isolated from the Korean marine algae Sargassum macrocarpum. However, the anticancer effects of TTB remain unknown. In this study, we demonstrate that TTB inhibits tumor growth in breast, lung, colon, prostate, and cervical cancer cells. To examine the mechanism by which TTB suppresses cell growth, we determined the effect of TTB on apoptosis, ROS generation, DNA damage, and signal transduction. TTB induced ROS production in MDA-MB-231, A549, and HCT116 cells. Moreover, TTB enhanced DNA damage by inducing γH2AX foci formation and the phosphorylation of DNA damage-related proteins such as Chk2 and H2AX. Furthermore, TTB selectively inhibited STAT3 activation, which resulted in a reduction in cyclin D1, MMP-9, survivin, VEGF, and IL-6. In addition, TTB-induced ROS generation caused STAT3 inhibition, DNA damage, and apoptotic cell death. Therefore, TTB suppresses cancer progression by promoting ROS-mediated inhibition of STAT3 signaling, suggesting that TTB is useful for the treatment of cancer.

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“…During the past 5 years, LSC has been extensively used in studies of DDR (12, 40–45). In these studies, the DNA damage signaling was detected immunocytochemically using phospho-specific Abs reactive with activated members of the signaling pathways: histone H2AX phosphorylated on Ser139 (γH2AX), ATM phosphorylated on Ser1981, p53 phosphorylated on Ser15, and Chk2 phosphorylated on Thr68.…”

Section: The Micronucleus Assay and Dna Damage Signalingmentioning

confidence: 99%

Laser Scanning Cytometry: Principles and Applications—An Update

Pożarowski

Holden²,

Darżynkiewicz

2012

Methods in Molecular Biology

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Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

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

Cited by 38 publications

References 44 publications

Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide

Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide

Tuberatolide B Suppresses Cancer Progression by Promoting ROS-Mediated Inhibition of STAT3 Signaling

Laser Scanning Cytometry: Principles and Applications—An Update

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