Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

Lee, Younghyun; Wang, Qi; Shuryak, Igor; Brenner, David J.; Turner, Helen

doi:10.1186/s13014-019-1344-7

Cited by 86 publications

(71 citation statements)

References 51 publications

(61 reference statements)

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“…The TDNA%, TM and OTM of the radiation+ANTP‐SmacN7 fusion peptide group were higher than those of the other groups, indicating more severe DNA damage. Measurement of γ‐H2AX focal levels in cells is a sensitive and reliable method for quantifying the radiation‐induced DNA damage response . In this study, the ANTP‐SmacN7 fusion peptide combined with irradiation significantly increased the radiation‐induced double‐stranded DNA breaks in A549 cells, and adding a caspase inhibitor reduced apoptosis but not the double‐stranded DNA breaks.…”

Section: Discussionmentioning

confidence: 72%

ANTP‐SmacN7 fusion peptide‐induced radiosensitization in A549 cells and its potential mechanisms

Zhang

Sun²,

Wang³

et al. 2020

Thoracic Cancer

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Background: Radioresistance in tumors limits the curative effect of the radiotherapy. Mimetic compounds of second mitochondria-derived activator of caspase (Smac) are potential new tumor radiation-sensitizing drugs because they can increase radiation-induced tumor cell apoptosis. Here, we observed the radiosensitization effect of a new Smac mimetic Antennapedia protein (ANTP)-SmacN7 fusion peptide in A549 cells and investigated the underlying mechanisms behind the effects of this protein on tumor cells. Methods: The ANTP-SmacN7 fusion peptide was synthesized and linked with fluorescein isothiocyanate to observe the protein's ability to penetrate cells. A549 cells were divided into the control, radiation-only, ANTP-SmacN7-only and ANTP-SmacN7 + radiation groups. The cells were exposed to 0, 2, 4 and 6 Gy, with 20 μmol/L of ANTP-SmacN7. The radiation-sensitizing effects of the ANTP-SmacN7 fusion proteins were observed via clonogenic assay. Apoptosis was detected using flow cytometry. A comet assay was used to assess DNA damage. The levels and degrees of cytochrome-c, PARP, H2AX, caspase-8, caspase-3, and caspase-9 activation were detected via western blot assay. The radiation sensitization of the fusion peptide, expression of γ-H2AX and C-PARP were compared after adding the caspase inhibitor, Z-VAD. Results: ANTP-SmacN7 fusion proteins entered the cells and promoted A549 cell radiosensitization. Treatment with ANTP-SmacN7 + radiation significantly reduced the A549 cell clone-forming rate, increased the cytochrome-c, cleaved caspase-8, cleaved caspase-3 and cleaved caspase-9 expression levels, promoted caspase activation, and increased the rate of radiation-induced apoptosis. The ANTP-SmacN7 fusion peptide significantly increased radiation-induced doublestranded DNA rupture in the A549 cells and increased DNA damage. Adding Z-VAD reduced the fusion peptide's proapoptotic effect but not the level of double-stranded DNA breakage. Conclusions: The ANTP-SmacN7 fusion peptide exerted a remarkable radiosensitization effect on A549 cells. This protein may reduce tumor cell radioresistance by inducing caspase activation and may be a potential new Smac mimetic that can be applied in radiosensitization therapy.

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Section: Discussionmentioning

confidence: 72%

ANTP‐SmacN7 fusion peptide‐induced radiosensitization in A549 cells and its potential mechanisms

Zhang

Sun²,

Wang³

et al. 2020

Thoracic Cancer

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“…However, non-specific signals raise detection limits above limits when using PMBCs. A technique using red blood cell lysis and leukocyte fixation in one step has been shown to be as effective as density gradient centrifugation for preparation of PMBCs for subsequent lymphocyte analysis [33,34]. Radiation sensitivity may affect various factors such as sex and age at exposure.…”

Section: Discussionmentioning

confidence: 99%

Induction of oxidative stress biomarkers following whole-body irradiation in mice

et al. 2020

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Dose assessment is an important issue for radiation emergency medicine to determine appropriate clinical treatment. Hematopoietic tissues are extremely vulnerable to radiation exposure. A decrease in blood cell count following radiation exposure is the first quantitative bio-indicator using hematological techniques. We further examined induction of oxidative stress biomarkers in residual lymphocytes to identify new biomarkers for dosimetry. In vivo whole-body radiation to mice exposed to 5 Gy significantly induces DNA double-strand breaks, which were visualized by γ-H2AX in mouse blood cells. Mouse blood smears and peripheral blood mononuclear cells (PBMC) isolated from irradiated mice were used for immunostaining for oxidative biomarkers, parkin or Nrf2. Parkin is the E3 ubiquitin ligase, which is normally localized in the cytoplasm, is relocated to abnormal mitochondria with low membrane potential (ΔΨm), where it promotes clearance via mitophagy. Nrf2 transcription factor controls the major cellular antioxidant responses. Both markers of oxidative stress were more sensitive and persistent over time than nuclear DNA damage. In conclusion, parkin and Nrf2 are potential biomarkers for use in radiation dosimetry. Identification of several biological markers which show different kinetics for radiation response is essential for radiation dosimetry that allows the assessment of radiation injury and efficacy of clinical treatment in emergency radiation incidents. Radiation-induced oxidative damage is useful not only for radiation dose assessment but also for evaluation of radiation risks on humans.

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“…It also allows to analyze cellular morphology and quantitate multi parametric fluorescent intensities (Vorobjev and Barteneva 2016). The application of multispectral imaging flow cytometry provides a novel and robust methodology for the estimation and quantitation of γ-H2AX intensity as well as foci in cells (Lee et al 2019).…”

Section: Discussionmentioning

confidence: 99%

“…We measured the following endpoints: 1) mouse leukocytes depletion, 2) apoptosis levels, 3) p53 expression and 4) γ-H2AX kinetics. We present here a γ-H2AX assay based on imaging flow cytometry (Lee et al 2019) to measure DNA damage in mouse blood leukocytes in vivo after progressively decreasing low dose rate external exposures.…”

Section: Introductionmentioning

confidence: 99%

DNA damage response in peripheral mouse blood leukocytesin vivoafter variable, low-dose rate exposure

Wang

Pujol

Taveras

et al. 2019

Preprint

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Environmental contamination and ingestion of the radionuclide Cesium-137 ( 137 Cs) is a large concern in fallout from a nuclear reactor accident and improvised nuclear device and highlights the need to develop biological assays for low dose rate, internal emitter radiation. To mimic low dose rates attributable to fallout, we have developed a VAriable Dose-rate External 137 Cs irradiatoR (VADER), which can provide arbitrarily varying and progressive low dose rate irradiations in the range of 1.2 to 0.1 Gy/day, while circumventing the complexities of dealing with radioactively-contaminated biomaterials. We investigated the kinetics of mouse peripheral leukocytes DNA damage response in vivo after variable, low-dose rate 137 Cs exposure. C57BL/6 mice were placed in the VADER over 7 days with total accumulated dose up to 2.7 Gy. Peripheral blood response including the leukocytes depletion, apoptosis signal protein p53 and DNA repair biomarker γ-H2AX were measured. The results illustrated that blood leukocyte count had significantly dropped by days 7. P53 levels peaked at day 2 (total dose=0.91Gy) and then declined whereas γ-H2AX yields generally increased with accumulated dose and peaked at day 5 (total dose=2.08Gy). ROC curve analysis for γ-H2AX provided a good discrimination of accumulated dose < 2 Gy and ≥ 2 Gy, highlighting the potential of γ-H2AX as a biomarker dosimetry in a protracted, environmental exposure scenario.

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Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

Cited by 86 publications

References 51 publications

ANTP‐SmacN7 fusion peptide‐induced radiosensitization in A549 cells and its potential mechanisms

ANTP‐SmacN7 fusion peptide‐induced radiosensitization in A549 cells and its potential mechanisms

Induction of oxidative stress biomarkers following whole-body irradiation in mice

DNA damage response in peripheral mouse blood leukocytesin vivoafter variable, low-dose rate exposure

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