Does γH2AX foci formation depend on the presence of DNA double strand breaks?

2005 ◽  
Vol 229 (2) ◽  
pp. 171-179 ◽  
Author(s):  
Akihisa Takahashi ◽  
Takeo Ohnishi
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Γh2ax Foci

3H-Thymidine Influence on DNA Double Strand Breaks Induction in Cultured Human Mesenchymal Stem Cells

2018 ◽  
Vol 63 (1) ◽  
pp. 28-34 ◽  
Author(s):  
Н. Воробьева ◽  
N. Vorob'eva ◽  
В. Уйба ◽  
V. Uyba ◽  
О. Кочетков ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Medium ◽  
Specific Radioactivity ◽  
Human Mesenchymal Stem Cells ◽  
Living Cells ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Immunocytochemical Analysis ◽  
Γh2ax Foci

Purpose: To estimate the impact of 3H-thymidine on DNA double strand breaks (DSBs) induction in cultured human mesenchymal stem cells (MSC). Material and methods: Isolation and cultivation of human bone marrow MSC was carried out according to a standard procedure. A sterile solution of 3H-thymidine with different specific radioactivity was added to the cell culture and incubated under the conditions of the CO2 incubator for 24 hours. The specific radioactivity of 3H-thymidine in the incubation medium was 50–1600 kBq/ml. To evaluate quantitatively the DSBs, an immunocytochemical analysis of the DSB marker – γH2AX foci histone was used. Additionally, the proportion of dividing cells was estimated using an immunocytochemical analysis of the cell proliferation marker, the Ki67 protein. Results: It was shown that 24 h incubation of human MSC in a culture medium results in a dose-dependent increase in γH2AX foci. There is a linear increase in the foci γH2AX in the range of 50–400 kBq/ml, after which the relative quantitative yield of foci per unit of specific radioactivity begins to decrease. In general, the dose-effect relationship is approximated by the quadratic function y = 3.13 + 50.80x – 12.38x2 (R2 = 0.99), where y is the number of foci γH2AX in the cell nucleus, and x is the specific radioactivity in 1000 kBq/ml. It was found that incubation of human MSC in a culture medium containing 800 and 1600 kBq/ml of 3H-thymidine resulted in a statistically significant decrease in the cells proliferative activity compared to the control of ~1.25 and 1.41 respectively. The peculiar biological limitation of tritium accumulation in the cell nucleus explains well the nonlinear character of the dependence of the formation of DSBs on the specific radioactivity of 3H-thymidine in the culture medium observed in our study. Conclusion: Quantitative analysis of γH2AX foci has proved to be a highly reproducible and highly sensitive method for evaluating the induction of DSBs in living cells under the action of 3H-thymidine. An analysis of the foci of γH2AX will be useful for accurate estimating the quantitative yield of DBS in living cells per dose of 3H-thymidine β-radiation. To do this, it is necessary to make a correct calculation of the doses received by the cells taking into account the microdistribution of 3H-thymidine in the cell volume and its accumulation in the DNA of living cells.

scholarly journals Radiation-induced DNA double-strand breaks in peripheral leukocytes and therapeutic response of heel spur patients treated by orthovoltage X-rays or a linear accelerator

2020 ◽  
Vol 196 (12) ◽  
pp. 1116-1127
Author(s):  
Sebastian Zahnreich ◽  
Hans-Peter Rösler ◽  
Carina Schwanbeck ◽  
Heiko Karle ◽  
Heinz Schmidberger
Keyword(s):  
Linear Accelerator ◽  
Double Strand Breaks ◽  
X Rays ◽  
Dna Double Strand Breaks ◽  
Heel Spur ◽  
Strand Breaks ◽  
Γh2ax Foci ◽  
Radiation Induced ◽  
Peripheral Leukocytes ◽  
Painful Heel

Abstract Purpose Biodosimetric assessment and comparison of radiation-induced deoxyribonucleic acid (DNA) double-strand breaks (DSBs) by γH2AX immunostaining in peripheral leukocytes of patients with painful heel spur after radiation therapy (RT) with orthovoltage X‑rays or a 6-MV linear accelerator (linac). The treatment response for each RT technique was monitored as a secondary endpoint. Patients and methods 22 patients were treated either with 140-kV orthovoltage X‑rays (n = 11) or a 6-MV linac (n = 11) with two weekly fractions of 0.5 Gy for 3 weeks. In both scenarios, the dose was prescribed to the International Commission on Radiation Units and Measurements (ICRU) dose reference point. Blood samples were obtained before and 30 min after the first RT session. γH2AX foci were quantified by immunofluorescence microscopy to assess the yield of DSBs at the basal level and after radiation exposure ex vivo or in vivo. The treatment response was assessed before and 3 months after RT using a five-level functional calcaneodynia score. Results RT for painful heel spurs induced a very mild but significant increase of γH2AX foci in patients’ leukocytes. No difference between the RT techniques was observed. High and comparable therapeutic responses were documented for both treatment modalities. This trial was terminated preliminarily after an interim analysis (22 patients randomized). Conclusion Low-dose RT for painful heel spurs with orthovoltage X‑rays or a 6-MV linac is an effective treatment option associated with a very low and comparable radiation burden to the patient, as confirmed by biodosimetric measurements.

scholarly journals Radiation dose determines the method for quantification of DNA double strand breaks

2016 ◽  
Vol 88 (1) ◽  
pp. 127-136 ◽  
Author(s):  
TANJA BULAT ◽  
OTILIJA KETA ◽  
LELA KORIĆANAC ◽  
JELENA ŽAKULA ◽  
IVAN PETROVIĆ ◽  
...  
Keyword(s):  
Radiation Dose ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Microscopy ◽  
Double Strand Breaks ◽  
Immunofluorescent Staining ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Γh2ax Foci

ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

Induction and rejoining of DNA double-strand breaks in the lymphocytes of prostate cancer patients after radium-223 treatment as assessed by the γH2AX foci assay

2019 ◽  
Vol 58 (05) ◽  
pp. 387-394
Author(s):  
Roswitha Runge ◽  
Liane Oehme ◽  
Sabine Grosche-Schlee ◽  
Anja Braune ◽  
Robert Freudenberg ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Patients ◽  
Ex Vivo ◽  
Double Strand Breaks ◽  
Patient Specific ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Γh2ax Foci ◽  
Radium 223 ◽  
Radiation Induced

Abstract Aim The aim of this study is to assess if the number of radiation-induced double strand breaks (DSB) in lymphocytes of prostate cancer patients is affected after repeated Ra-223 therapies. In addition, we investigated the repair of ex vivo induced DSB to investigate the repair proficiency in patient’s lymphocytes over the therapy course. Methods Before each of six therapy cycles, blood samples were obtained from seventeen patients. After separation of lymphocytes, the cells were subjected to immunofluorescence staining for detection of DSB-marking γH2AX foci. The number of foci per cell per patient sample was determined for each cycle (X1-X6, baseline foci per cell). Additionally, appropriate samples were exposed ex vivo to an X-ray dose of 1 Gy. The number of γH2AX foci per cell were analyzed after 0.5 h, 2 h and 24 h of recovery. Results Patient-specific linear regression of the baseline foci per cell over the therapy cycles revealed no significant slopes in the regression lines. Likewise, the mean baseline foci per cell of all patients for cycles X2-X6 was not significantly elevated in comparison to the pre-therapeutic value (X1). The differences between the percentages of residual DSB and cycles were not significant, both at 2 h and 24 h repair time. Consideration of the X6/X1 ratios of both the number of lymphocytes and the amount of residual damage at 24 h indicated a significant correlation. Conclusion Our findings indicate that the number of γH2AX foci per cell was not changed in dependence on the Ra-223 therapy cycles. The ability of patient’s lymphocytes to repair ex vivo induced DSB remained unaffected throughout the entire therapy course.

scholarly journals In Vitro Evaluation of No-Carrier-Added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons

2021 ◽  
Vol 22 (9) ◽  
pp. 4622
Author(s):  
Honoka Obata ◽  
Atsushi B. Tsuji ◽  
Hitomi Sudo ◽  
Aya Sugyo ◽  
Katsuyuki Minegishi ◽  
...  
Keyword(s):  
Short Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Primary Target ◽  
Auger Electrons ◽  
Strand Breaks ◽  
Chemical Damage ◽  
Γh2ax Foci ◽  
No Carrier Added

Due to their short-range (2–500 nm), Auger electrons (Auger e−) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Auger e−, it remains challenging to maximize the interaction between Auger e− and DNA. To assess the DNA-damaging effect of Auger e− released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by immunofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incubation), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by the release of Auger e− very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e−.

scholarly journals Low Repair Capacity of DNA Double-Strand Breaks Induced by Laser-Driven Ultrashort Electron Beams in Cancer Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9488
Author(s):  
Nelly Babayan ◽  
Natalia Vorobyeva ◽  
Bagrat Grigoryan ◽  
Anna Grekhova ◽  
Margarita Pustovalova ◽  
...  
Keyword(s):  
Electron Energy ◽  
Ultrashort Pulse ◽  
Electron Beams ◽  
A549 Cells ◽  
Double Strand Breaks ◽  
Pulse Irradiation ◽  
Dna Double Strand Breaks ◽  
Continuous Irradiation ◽  
Strand Breaks ◽  
Γh2ax Foci

Laser-driven accelerators allow to generate ultrashort (from femto- to picoseconds) high peak dose-rate (up to tens of GGy/s) accelerated particle beams. However, the radiobiological effects of ultrashort pulsed irradiation are still poorly studied. The aim of this work was to compare the formation and elimination of γH2AX and 53BP1 foci (well known markers for DNA double-strand breaks (DSBs)) in Hela cells exposed to ultrashort pulsed electron beams generated by Advanced Research Electron Accelerator Laboratory (AREAL) accelerator (electron energy 3.6 MeV, pulse duration 450 fs, pulse repetition rates 2 or 20 Hz) and quasi-continuous radiation generated by Varian accelerator (electron energy 4 MeV) at doses of 250–1000 mGy. Additionally, a study on the dose–response relationships of changes in the number of residual γH2AX foci in HeLa and A549 cells 24 h after irradiation at doses of 500–10,000 mGy were performed. We found no statistically significant differences in γH2AX and 53BP1 foci yields at 1 h after exposure to 2 Hz ultrashort pulse vs. quasi-continuous radiations. In contrast, 20 Hz ultrashort pulse irradiation resulted in 1.27-fold higher foci yields as compared to the quasi-continuous one. After 24 h of pulse irradiation at doses of 500–10,000 mGy the number of residual γH2AX foci in Hela and A549 cells was 1.7–2.9 times higher compared to that of quasi-continuous irradiation. Overall, the obtained results suggest the slower repair rate for DSBs induced by ultrashort pulse irradiation in comparison to DSBs induced by quasi-continuous irradiation.

scholarly journals Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3833
Author(s):  
Anne-Sophie Wozny ◽  
Arnaud Gauthier ◽  
Gersende Alphonse ◽  
Céline Malésys ◽  
Virginie Varoclier ◽  
...  
Keyword(s):  
Ion Irradiation ◽  
Hypoxia Inducible Factor ◽  
Therapeutic Benefit ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Carbon Ion ◽  
Oxygen Homeostasis ◽  
Γh2ax Foci ◽  
Main Regulator

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

Ultra-High Dose Radiation on Cryopreserved Mesenchymal Stem Cells: DNA Double-Strand Breaks and Proliferative Activity

2019 ◽  
pp. 5-10
Author(s):  
А. Цишнатти ◽  
A. Tsishnatti ◽  
М. Пустовалова ◽  
M. Pustovalova ◽  
А. Грехова ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Proliferative Activity ◽  
Liquid Nitrogen Temperature ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Protein Marker ◽  
Strand Breaks ◽  
Γh2ax Foci

Purpose: To conduct a comparative assessment of the effect of irradiation of human mesenchymal stem cells (MSC)at ultrahigh doses at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of “residual” DNA double-strand breaks (DSB) and proliferative activity of thawed MSC. Material and methods: Isolation and cultivation of MSC was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, the accelerator UELR-10-100-T-100 (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of 22 °C and –196 °C. The yield of “residual” DNA DSB was assessed using an immunocytochemical analysis of the foci of the protein-marker DSB – γH2AX. To assess the proliferative activity, the number of Ki67 (protein marker of cell proliferation) of positive cells was analyzed. Results: The results of γH2AX foci assessment in MSCs after 48 hours irradiation at a dose of 50 Gy showed that the number of “residual” γH2AX foci in the MSC nuclei irradiated at 22 °C is about 3.2 times (p = 0.0002) higher than in the MSC nuclei irradiated at –196 °C. Analysis of the proliferative activity of cells using the molecular marker of cell proliferation of the Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of 22 °C, completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, is significantly reduced, but some of the cells (3.5 ± 1.1 %) still retain the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lose their ability to proliferate, but partially retain the ability to adhere. The integral fluorescence conjugated with the flurochrome of antibodies to γH2AX of MSC nuclei irradiated at a dose of 500 Gy at a temperature of –196 °C is 1.8 times lower than that of nuclei irradiated at a temperature of 22 °C. Conclusion: The results of the studies indicate that cryopreserved MSCs irradiated at liquid nitrogen temperature (–196 °C) in a preservation medium containing 10 % DMSO can tolerate the effects of ionizing radiation in large doses (up to 50 Gy). However, there is a rather high yield of “residual” DSB DNA and a very low proliferative activity, which makes them unsuitable in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.

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