Imaging DNA double-strand breaks — are we there yet?

Atkinson, Jake; Bezak, Eva; Kempson, Ivan M.

doi:10.1038/s41580-022-00513-7

Cited by 8 publications

(4 citation statements)

References 9 publications

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“…We first confirmed the type of DNA damage and the time course produced by CSE in HUVECs. Since the initial cellular response upon double-strand break (DSBs) is the phosphorylation of histone H2AX, we performed immunofluorescent staining with antibody against phosphorylated H2AX (γH2AX), the most prominent marker of DSBs [ 23 ]. DSBs gradually increased until they became significant 72 h after CSE treatment ( Figure 1 A; Supplementary Figure S2A).…”

Section: Resultsmentioning

confidence: 99%

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway: application to a biomarker for atherosclerosis

et al. 2023

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Cigarette smoking is a major risk factor for atherosclerosis. We previously reported that DNA damage was accumulated in atherosclerotic plaque, and was increased in human mononuclear cells by smoking. As vascular endothelial cells are known to modulate inflammation, we investigated the mechanism by which smoking activates innate immunity in endothelial cells focusing on DNA damage. Further, we sought to characterize the plasma level of cell-free DNA (cfDNA), a result of mitochondrial and/or genomic DNA damage, as a biomarker for atherosclerosis. Cigarette smoke extract (CSE) increased DNA damage in the nucleus and mitochondria in human endothelial cells. Mitochondrial damage induced minority mitochondrial outer membrane permeabilization, which was insufficient for cell death but instead led to nuclear DNA damage. DNA fragments, derived from the nucleus and mitochondria, were accumulated in the cytosol, and caused a persistent increase in IL-6 mRNA expression via the cGAS-STING pathway. cfDNA, quantified with quantitative PCR in culture medium was increased by CSE. Consistent with in vitro results, plasma mitochondrial cfDNA (mt-cfDNA) and nuclear cfDNA (n-cfDNA) were increased in young healthy smokers compared to age-matched non-smokers. Additionally, both mt-cfDNA and n-cfDNA were significantly increased in patients with atherosclerosis compared to the normal controls. Our multivariate analysis revealed that only mt-cfDNA predicted the risk of atherosclerosis. In conclusion, accumulated cytosolic DNA caused by cigarette smoke and the resultant activation of the cGAS-STING pathway may be a mechanism of atherosclerosis development. The plasma level of mt-cfDNA, possibly as a result of DNA damage, may be a useful biomarker for atherosclerosis.

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Section: Resultsmentioning

confidence: 99%

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway: application to a biomarker for atherosclerosis

et al. 2023

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“…This promotes the recruitment of downstream DSB repair molecules, in addition to ensuring genomic stability [118]. However, there are common misconceptions when utilizing proteins within the DDR as markers for DSBs, as they are only an indication of whether DNA repair has been initiated, and not markers of where DSBs are located specifically [12]. Regarding γH2AX, it is generally true that the presence of DSBs induces the phosphorylation of H2AX.…”

Section: γH2axmentioning

confidence: 99%

“…However, fluorescence signals generated by these markers are not a direct indication of DSBs themselves. Rather, the presence of these markers signifies the initiation of DSB repair and not the presence of the actual breakage sites [12]. Tagging these proteins is an indirect method of DSB detection [13], but it is still extremely useful for identifying relationships among damage formation, repair efficacy, and repair kinetics.…”

Section: Introductionmentioning

confidence: 99%

DNA Double Strand Break and Response Fluorescent Assays: Choices and Interpretation

Atkinson,

Bezak,

et al. 2024

IJMS

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Accurately characterizing DNA double-stranded breaks (DSBs) and understanding the DNA damage response (DDR) is crucial for assessing cellular genotoxicity, maintaining genomic integrity, and advancing gene editing technologies. Immunofluorescence-based techniques have proven to be invaluable for quantifying and visualizing DSB repair, providing valuable insights into cellular repair processes. However, the selection of appropriate markers for analysis can be challenging due to the intricate nature of DSB repair mechanisms, often leading to ambiguous interpretations. This comprehensively summarizes the significance of immunofluorescence-based techniques, with their capacity for spatiotemporal visualization, in elucidating complex DDR processes. By evaluating the strengths and limitations of different markers, we identify where they are most relevant chronologically from DSB detection to repair, better contextualizing what each assay represents at a molecular level. This is valuable for identifying biases associated with each assay and facilitates accurate data interpretation. This review aims to improve the precision of DSB quantification, deepen the understanding of DDR processes, assay biases, and pathway choices, and provide practical guidance on marker selection. Each assay offers a unique perspective of the underlying processes, underscoring the need to select markers that are best suited to specific research objectives.

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“…These control and Tau knockdown cells were treated for 2 h with bleomycin (30 µg/mL), a drug known to induce double-strand breaks. We then quantified DSBs using a phosphorylated form of H2AX as an indicator of DNA damage [29]. Where we knocked down Tau, γ-H2AX fluorescence intensity increased 4-fold compared to just 2-fold in both MCF7 and MDA-MD-231 control cells (Figure 1).…”

Section: Tau Aids Clearance Of Double-strand Breaksmentioning

confidence: 99%

Cancer Cells Upregulate Tau to Gain Resistance to DNA Damaging Agents

Rico

Denechaud

Caillierez

et al. 2022

Cancers

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Recent reports suggested a role for microtubules in double-strand-DNA break repair. We herein investigated the role of the microtubule-associated protein Tau in radio- and chemotherapy. Noticeably, a lowered expression of Tau in breast cancer cell lines resulted in a significant decrease in mouse-xenograft breast tumor volume after doxorubicin or X-ray treatments. Furthermore, the knockdown of Tau impaired the classical nonhomologous end-joining pathway and led to an improved cellular response to both bleomycin and X-rays. Investigating the mechanism of Tau’s protective effect, we found that one of the main mediators of response to double-stranded breaks in DNA, the tumor suppressor p53-binding protein 1 (53BP1), is sequestered in the cytoplasm as a consequence of Tau downregulation. We demonstrated that Tau allows 53BP1 to translocate to the nucleus in response to DNA damage by chaperoning microtubule protein trafficking. Moreover, Tau knockdown chemo-sensitized cancer cells to drugs forming DNA adducts, such as cisplatin and oxaliplatin, and further suggested a general role of Tau in regulating the nuclear trafficking of DNA repair proteins. Altogether, these results suggest that Tau expression in cancer cells may be of interest as a molecular marker for response to DNA-damaging anti-cancer agents. Clinically targeting Tau could sensitize tumors to DNA-damaging treatments.

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Imaging DNA double-strand breaks — are we there yet?

Cited by 8 publications

References 9 publications

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway: application to a biomarker for atherosclerosis

Cigarette smoke induces mitochondrial DNA damage and activates cGAS-STING pathway: application to a biomarker for atherosclerosis

DNA Double Strand Break and Response Fluorescent Assays: Choices and Interpretation

Cancer Cells Upregulate Tau to Gain Resistance to DNA Damaging Agents

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