Gamma‐H2AX immunofluorescence for the detection of tissue‐specific genotoxicity <i>in vivo</i>

Plappert‐Helbig, Ulla; Libertini, Silvana; Frieauff, Wilfried; Theil, Diethilde; Martus, Hans‐Jörg

doi:10.1002/em.22238

Cited by 28 publications

(14 citation statements)

References 37 publications

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“…This is supported by the proposal that in fibroblast cell lines UV-induced pan-nuclear γ-H2AX is a preapoptotic signaling event [49]. Whether high levels of pan-nuclear γ-H2AX are generated in patients [50] or in human tumor samples [1] following administration of cytotoxic doses of chemotherapeutics remains to be established. Thus, similarly to hyperphosphorylated RPA, which is now considered as a marker of ssDNA at the stalled forks [38], we propose that pan-nuclear γ-H2AX represents a surrogate marker of RS-induced cell death.…”

Section: Discussionmentioning

confidence: 98%

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Moeglin

Desplancq

Conic

et al. 2019

Cancers

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Phosphorylated histone H2AX (γ-H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of γ-H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear γ-H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear γ-H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear γ-H2AX pattern. Delivery of labeled anti-γ-H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear γ-H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of γ-H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy.

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Section: Discussionmentioning

confidence: 98%

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Moeglin

Desplancq

Conic

et al. 2019

Cancers

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“…Cat No. ab26350, Abcam, Cambridge, United Kingdom) against H2AX (histone 2 a family X), which is a standardized marker for DNA damage [ 43 ] and double stranded breaks at dilution of 1/500 (2 μ g/ml).…”

Section: Methodsmentioning

confidence: 99%

Aging Alters Olfactory Bulb Network Oscillations and Connectivity: Relevance for Aging-Related Neurodegeneration Studies

et al. 2020

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The aging process eventually cause a breakdown in critical synaptic plasticity and connectivity leading to deficits in memory function. The olfactory bulb (OB) and the hippocampus, both regions of the brain considered critical for the processing of odors and spatial memory, are commonly affected by aging. Using an aged wild-type C57B/6 mouse model, we sought to define the effects of aging on hippocampal plasticity and the integrity of cortical circuits. Specifically, we measured the long-term potentiation of high-frequency stimulation (HFS-LTP) at the Shaffer-Collateral CA1 pyramidal synapses. Next, local field potential (LFP) spectra, phase-amplitude theta-gamma coupling (PAC), and connectivity through coherence were assessed in the olfactory bulb, frontal and entorhinal cortices, CA1, and amygdala circuits. The OB of aged mice showed a significant increase in the number of histone H2AX-positive neurons, a marker of DNA damage. While the input-output relationship measure of basal synaptic activity was found not to differ between young and aged mice, a pronounced decline in the slope of field excitatory postsynaptic potential (fEPSP) and the population spike amplitude (PSA) were found in aged mice. Furthermore, aging was accompanied by deficits in gamma network oscillations, a shift to slow oscillations, reduced coherence and theta-gamma PAC in the OB circuit. Thus, while the basal synaptic activity was unaltered in older mice, impairment in hippocampal synaptic transmission was observed only in response to HFS. However, age-dependent alterations in neural network appeared spontaneously in the OB circuit, suggesting the neurophysiological basis of synaptic deficits underlying olfactory processing. Taken together, the results highlight the sensitivity and therefore potential use of LFP quantitative network oscillations and connectivity at the OB level as objective electrophysiological markers that will help reveal specific dysfunctional circuits in aging-related neurodegeneration studies.

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“…Immunocytochemical detection of the phosphorylated form of the histone protein component H2AX (γ-H2AX) is a well-known approach to estimate DNA DSB levels in both in vitro [47,53,66] and in vivo [67,68] studies. HCT 116 cells were grown on coverslips in 12-well plates at a density of 1.2 × 10 5 per well for 24 h (5% CO 2 ; 37 • C).…”

Section: Immunocytochemical Detection Of γ-H2axmentioning

confidence: 99%

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Cytotoxic and Genotoxic Activities In Vitro

et al. 2022

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In this paper, we present for the first time the evaluation of cytotoxicity and genotoxicity of de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides MM129, MM130, and MM131 in human tumor cell lines: HeLa, HCT 116, PC-3, and BxPC-3. Cytotoxic and genotoxic properties of the tested compounds were estimated using the MTT assay, comet assay (alkaline and neutral version), and γ-H2AX immuno-staining. Examined sulfonamides exhibited strong anticancer properties towards tested cells in a very low concentration range (IC50 = 0.17–1.15 μM) after 72 h exposure time. The results of the alkaline and neutral version of the comet assay following 24 h incubation of the cells with tested compounds demonstrated the capability of heterocycles to induce significant DNA damage in exposed cells. HCT 116 cells were the most sensitive to the genotoxic activity of novel tricyclic pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides in the neutral version of the comet assay. Immunocytochemical detection of γ-H2AX showed an increase in DNA DSBs level in the HCT 116 cell line, after 24 h incubation with all tested compounds, confirming the results obtained in the neutral comet assay. Among all investigated compounds, MM131 showed the strongest cytotoxic and genotoxic activity toward all tested cell types. In conclusion, our results suggest that MM129, MM130, and MM131 exhibit high cytotoxic and genotoxic potential in vitro, especially towards the colorectal cancer cell line HCT 116. However, further investigations and analyses are required for their future implementation in the field of medicine.

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Gamma‐H2AX immunofluorescence for the detection of tissue‐specific genotoxicity in vivo

Cited by 28 publications

References 37 publications

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Aging Alters Olfactory Bulb Network Oscillations and Connectivity: Relevance for Aging-Related Neurodegeneration Studies

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Cytotoxic and Genotoxic Activities In Vitro

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