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

Atkinson, Jake; Bezak, Eva; Le, Hien; Kempson, Ivan

doi:10.3390/ijms25042227

Cited by 5 publications

(2 citation statements)

References 149 publications

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“…In response to a DSB, γH2AX occur to initiate repair, rapidly and meticulously. This promotes the recruitment of downstream DSB repair molecules, in addition to ensuring genomic stability ( 13 ). The location of labeled ARID1A and γ-H2AX was detected using the immunofluorescence technique to determine whether ARID1A was recruited to the DNA break site in the early stage of the DSBR.…”

Section: Resultsmentioning

confidence: 99%

ARID1A is involved in DNA double-strand break repair in gastric cancer

Zhang,

Qian,

et al. 2024

J Gastrointest Oncol

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Background Defects in DNA damage repair can cause genetic mutations, which in turn can cause different types of cancers. Chromatin remodeling complexes, which help repair damaged DNA, can cause the chromatin structure to change as a result of DNA damage. ARID1A may play a role in the process of DNA damage repair, and arid1a may be related to the occurrence and development of gastric cancer (GC). This study aimed to investigate the mechanism of ARID1A regulating the DNA damage repair of gastric adenocarcinoma cell lines AGS and SGC-7901 and its effect on migration, proliferation and apoptosis. Methods The expression of ARID1A plasmid was detected by Western blot and real-time polymerase chain reaction (PCR). The effect of etoposide (ETO) on the survival rate of AGS and SGC-7901 gastric adenocarcinoma cell lines was detected by MTT assay. The DNA double-strand break model was established by ETO and then passed through the comet assay and immunofluorescence co-localization to observe DNA damage; western blot method was used to detect the effect of ARID1A on the expression of related proteins in DNA damage repair pathway in gastric adenocarcinoma cells; scratch test and colony formation experiments were used to observe ARID1A migration and proliferation of gastric adenocarcinoma cells. The flow cytometry was used to detect the effect of ARID1A on apoptosis of gastric adenocarcinoma cells. Results The expression of mRNA and protein was increased after transfection of ARID1A plasmid. ETO was confirmed by MTT assay to inhibit cell survival in a dose-dependent manner. After the DNA double-strand break model was established by ETO, the expression levels of phospho-ataxia telangiectasia mutated (p-ATM) protein increased in the overexpressed ARID1A group. Meanwhile, the overexpressed ARID1A group had a shortened tail moment, and γ-H2AX and ARID1A co-localized in the DNA damage site of the nucleus. The over-expressed ARID1A group had weaker wound healing ability, reduced number of clone formation, and increased apoptosis rate. Conclusions ARID1A may repair DNA double-strand breaks caused by ETO by p-ATM pathway; ARID1A can inhibit the migration and proliferation of gastric adenocarcinoma cells and promote apoptosis. Our findings indicate that ARID1A could serve as a therapeutic target and biomarker for GC patients.

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Section: Resultsmentioning

confidence: 99%

ARID1A is involved in DNA double-strand break repair in gastric cancer

Zhang,

Qian,

et al. 2024

J Gastrointest Oncol

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“…Our rationale for focusing on γH2AX phosphorylation levels as a surrogate marker for DNA damage response was based on its established role in the early cellular response to DSBs and its utility as a sensitive indicator of DNA damage induction. Due to the intricate nature of DSB repair mechanisms and the consequent difficulty in identifying suitable markers for analysis, innovative fluorescence-based methods have been developed to advance the quantification of DSBs, although their interpretation can be questionable [ 40 ]. It is indeed recognized that the relationship between γH2AX levels and DSBs may exhibit variability under different experimental conditions and biological contexts, as highlighted in the literature [ 41 ].…”

Section: Discussionmentioning

confidence: 99%

Cosmic Whirl: Navigating the Comet Trail in DNA: H2AX Phosphorylation and the Enigma of Uncertain Significance Variants

Ustun Yilmaz,

Agaoglu,

Manto

et al. 2024

Genes

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Pathogenic variations in the BRCA2 gene have been detected with the development of next-generation sequencing (NGS)-based hereditary cancer panel testing technology. It also reveals an increasing number of variants of uncertain significance (VUSs). Well-established functional tests are crucial to accurately reclassifying VUSs for effective diagnosis and treatment. We retrospectively analyzed the multi-gene cancer panel results of 922 individuals and performed in silico analysis following ClinVar classification. Then, we selected five breast cancer-diagnosed patients’ missense BRCA2 VUSs (T1011R, T1104P/M1168K, R2027K, G2044A, and D2819) for reclassification. The effects of VUSs on BRCA2 function were analyzed using comet and H2AX phosphorylation (γH2AX) assays before and after the treatment of peripheral blood mononuclear cells (PBMCs) of subjects with the double-strand break (DSB) agent doxorubicin (Dox). Before and after Dox-induction, the amount of DNA in the comet tails was similar in VUS carriers; however, notable variations in γH2AX were observed, and according to combined computational and functional analyses, we reclassified T1001R as VUS-intermediate, T1104P/M1168K and D2819V as VUS (+), and R2027K and G2044A as likely benign. These findings highlight the importance of the variability of VUSs in response to DNA damage before and after Dox-induction and suggest that further investigation is needed to understand the underlying mechanisms.

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Gold Nanoparticles Cause Radiosensitization in 4T1 Cells by Inhibiting DNA Double Strand Break Repair: Single Cell Comparisons of DSB Formation and γH2AX Expression

Atkinson,

Chopin,

Bezak

et al. 2024

ChemPhysChem

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Metal nanoparticles sensitize cancers to radiotherapy however their mechanisms of action are complex. The conceptual inspiration arose from theories of physical dose deposition but various chemical and biological factors have also been identified. Interpretation of data has been limited by challenges in measuring true DNA damage compared to DNA damage repair factors. Here, we applied a new assay, STRIDE, for the first time to measure DNA double strand breaks (DSBs) in 4T1 cells as a model of triple negative breast cancer exposed to gold nanoparticles and radiation, and compared this to the common γH2AX assay for DSB repair. The STRIDE assay showed no increase in DSB detection 15 mins after irradiation for cells containing nanoparticles compared to cells without. Gold nanoparticles led to prolonged detection of DSBs after irradiation and delayed the DSB repair. The data show no evidence of increased radiation dose deposition with nanoparticles, but rather enhanced radiobiological effects resulting from nanoparticles which includes disruption of the recruitment of essential DDR machinery, thereby impairing DNA repair processes.

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

Cited by 5 publications

References 149 publications

ARID1A is involved in DNA double-strand break repair in gastric cancer

ARID1A is involved in DNA double-strand break repair in gastric cancer

Cosmic Whirl: Navigating the Comet Trail in DNA: H2AX Phosphorylation and the Enigma of Uncertain Significance Variants

Gold Nanoparticles Cause Radiosensitization in 4T1 Cells by Inhibiting DNA Double Strand Break Repair: Single Cell Comparisons of DSB Formation and γH2AX Expression

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