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.

scholarly journals Repair Kinetics of DNA Double Strand Breaks Induced by Simulated Space Radiation

Life ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 341
Author(s):  
Takashi Oizumi ◽  
Rieko Ohno ◽  
Souichiro Yamabe ◽  
Tomoo Funayama ◽  
Asako J. Nakamura
Keyword(s):  
Dna Damage ◽  
Ion Irradiation ◽  
Space Radiation ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Carbon Ion ◽  
Kinetics Of

Radiation is unavoidable in space. Energetic particles in space radiation are reported to induce cluster DNA damage that is difficult to repair. In this study, normal human fibroblasts were irradiated with components of space radiation such as proton, helium, or carbon ion beams. Immunostaining for γ-H2AX and 53BP1 was performed over time to evaluate the kinetics of DNA damage repair. Our data clearly show that the repair kinetics of DNA double strand breaks (DSBs) induced by carbon ion irradiation, which has a high linear energy transfer (LET), are significantly slower than those of proton and helium ion irradiation. Mixed irradiation with carbon ions, followed by helium ions, did not have an additive effect on the DSB repair kinetics. Interestingly, the mean γ-H2AX focus size was shown to increase with LET, suggesting that the delay in repair kinetics was due to damage that is more complex. Further, the 53BP1 focus size also increased in an LET-dependent manner. Repair of DSBs, characterized by large 53BP1 foci, was a slow process within the biphasic kinetics of DSB repair, suggesting non-homologous end joining with error-prone end resection. Our data suggest that the biological effects of space radiation may be significantly influenced by the dose as well as the type of radiation exposure.

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.

scholarly journals Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Takahiro Oike ◽  
Atsuko Niimi ◽  
Noriyuki Okonogi ◽  
Kazutoshi Murata ◽  
Akihiko Matsumura ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Carbon Ion Radiotherapy ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Carbon Ion

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 Hadron Therapy, Magnetic Nanoparticles and Hyperthermia: A Promising Combined Tool for Pancreatic Cancer Treatment

Nanomaterials ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1919 ◽  
Author(s):  
Francesca Brero ◽  
Martin Albino ◽  
Antonio Antoccia ◽  
Paolo Arosio ◽  
Matteo Avolio ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Magnetic Nanoparticles ◽  
Ion Irradiation ◽  
Clonogenic Survival ◽  
Double Strand Breaks ◽  
Carbon Ions ◽  
Dna Double Strand Breaks ◽  
Magnetic Fluid Hyperthermia ◽  
Strand Breaks

A combination of carbon ions/photons irradiation and hyperthermia as a novel therapeutic approach for the in-vitro treatment of pancreatic cancer BxPC3 cells is presented. The radiation doses used are 0–2 Gy for carbon ions and 0–7 Gy for 6 MV photons. Hyperthermia is realized via a standard heating bath, assisted by magnetic fluid hyperthermia (MFH) that utilizes magnetic nanoparticles (MNPs) exposed to an alternating magnetic field of amplitude 19.5 mTesla and frequency 109.8 kHz. Starting from 37 °C, the temperature is gradually increased and the sample is kept at 42 °C for 30 min. For MFH, MNPs with a mean diameter of 19 nm and specific absorption rate of 110 ± 30 W/gFe3o4 coated with a biocompatible ligand to ensure stability in physiological media are used. Irradiation diminishes the clonogenic survival at an extent that depends on the radiation type, and its decrease is amplified both by the MNPs cellular uptake and the hyperthermia protocol. Significant increases in DNA double-strand breaks at 6 h are observed in samples exposed to MNP uptake, treated with 0.75 Gy carbon-ion irradiation and hyperthermia. The proposed experimental protocol, based on the combination of hadron irradiation and hyperthermia, represents a first step towards an innovative clinical option for pancreatic cancer.

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