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Shen MZ, Zhang Y, Wu F, Shen MZ, Liang JL, Zhang XL, Liu XJ, Li XS, Wang RS. MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A. World J Gastrointest Oncol 2024; 16:1453-1464. [PMID: 38660649 PMCID: PMC11037043 DOI: 10.4251/wjgo.v16.i4.1453] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 12/31/2023] [Accepted: 02/02/2024] [Indexed: 04/10/2024] Open
Abstract
BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer (CRC); yet, the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission. AIM To elucidate the role played by microRNA-298 (miR-298) in CRC radio-resistance. METHODS To establish a radio-resistant CRC cell line, HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period. The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR, and protein expression determination was realized through Western blotting. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay. Radio-induced apoptosis was discerned through flow cytometry analysis. RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells. MiR-298 emerged as a key determinant of cell survival following radiation exposure, as its overexpression led to a notable reduction in radiation-induced apoptosis. Intriguingly, miR-298 expression exhibited a strong correlation with CRC cell viability. Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A (DYRK1A) as miR-298's direct target. CONCLUSION Taken together, our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation, thereby positioning miR-298 as a promising candidate for mitigating radio-resistance in CRC.
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Affiliation(s)
- Mei-Zhu Shen
- Department of Radiotherapy, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Yong Zhang
- Department of Radiotherapy, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Fang Wu
- Department of Radiotherapy, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Mei-Zhen Shen
- Department of Radiotherapy, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Jun-Lin Liang
- Department of Colorectal Anal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Xiao-Long Zhang
- Department of Colorectal Anal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Xiao-Jian Liu
- Department of Colorectal Anal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Xin-Shu Li
- Department of Clinical Medicine, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
| | - Ren-Sheng Wang
- Department of Radiotherapy, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China
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Shen M, Zhang Y, Wu F, Shen M, Zhang S, Guo Y, Gan J, Wang R. Knockdown of hCINAP sensitizes colorectal cancer cells to ionizing radiation. Cell Cycle 2024; 23:233-247. [PMID: 38551450 PMCID: PMC11057657 DOI: 10.1080/15384101.2024.2309015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 09/21/2023] [Accepted: 11/08/2023] [Indexed: 05/01/2024] Open
Abstract
Colorectal cancer (CRC) poses a significant challenge in terms of treatment due to the prevalence of radiotherapy resistance. However, the underlying mechanisms responsible for radio-resistance in CRC have not been thoroughly explored. This study aimed to shed light on the role of human coilin interacting nuclear ATPase protein (hCINAP) in radiation-resistant HT-29 and SW480 CRC cells (HT-29-IR and SW480-IR) and investigate its potential implications. Firstly, radiation-resistant CRC cell lines were established by subjecting HT-29 and SW480 cells to sequential radiation exposure. Subsequent analysis revealed a notable increase in hCINAP expression in radiation-resistant CRC cells. To elucidate the functional role of hCINAP in radio-resistance, knockdown experiments were conducted. Remarkably, knockdown of hCINAP resulted in an elevation of reactive oxygen species (ROS) generation upon radiation treatment and subsequent activation of apoptosis mediated by mitochondria. These observations indicate that hCINAP depletion enhances the radiosensitivity of CRC cells. Conversely, when hCINAP was overexpressed, it was found to enhance the radio-resistance of CRC cells. This suggests that elevated hCINAP expression contributes to the development of radio-resistance. Further investigation revealed an interaction between hCINAP and ATPase family AAA domain containing 3A (ATAD3A). Importantly, ATAD3A was identified as an essential factor in hCINAP-mediated radio-resistance. These findings establish the involvement of hCINAP and its interaction with ATAD3A in the regulation of radio-resistance in CRC cells. Overall, the results of this study demonstrate that upregulating hCINAP expression may improve the survival of radiation-exposed CRC cells. Understanding the intricate molecular mechanisms underlying hCINAP function holds promise for potential strategies in targeted radiation therapy for CRC. These findings emphasize the importance of further research to gain a comprehensive understanding of hCINAP's precise molecular mechanisms and explore its potential as a therapeutic target in overcoming radio-resistance in CRC. By unraveling the complexities of hCINAP and its interactions, novel therapeutic approaches may be developed to enhance the efficacy of radiation therapy and improve outcomes for CRC patients.
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Affiliation(s)
- Meizhu Shen
- Department of Radiotheraphy, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Yong Zhang
- Department of Radiotheraphy, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Fang Wu
- Department of Radiotheraphy, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Meizhen Shen
- Department of Radiotheraphy, People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China
| | - Sen Zhang
- Department of Colorectal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Yun Guo
- Department of Colorectal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Jialiang Gan
- Department of Colorectal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
| | - Rensheng Wang
- Department of Radiotheraphy, The First Affiliated Hospital of Guangxi Medical University, Nanning, China
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Oertel J, Sachs S, Flemming K, Obeid MH, Fahmy K. Distinct Effects of Chemical Toxicity and Radioactivity on Metabolic Heat of Cultured Cells Revealed by “Isotope-Editing”. Microorganisms 2023; 11:microorganisms11030584. [PMID: 36985158 PMCID: PMC10056173 DOI: 10.3390/microorganisms11030584] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 01/30/2023] [Accepted: 02/22/2023] [Indexed: 03/02/2023] Open
Abstract
Studying the toxicity of chemical compounds using isothermal microcalorimetry (IMC), which monitors the metabolic heat from living microorganisms, is a rapidly expanding field. The unprecedented sensitivity of IMC is particularly attractive for studies at low levels of stressors, where lethality-based data are inadequate. We have revealed via IMC the effect of low dose rates from radioactive β−-decay on bacterial metabolism. The low dose rate regime (<400 µGyh−1) is typical of radioactively contaminated environmental sites, where chemical toxicity and radioactivity-mediated effects coexist without a predominance or specific characteristic of either of them. We found that IMC allows distinguishing the two sources of metabolic interference on the basis of “isotope-editing” and advanced thermogram analyses. The stable and radioactive europium isotopes 153Eu and 152Eu, respectively, were employed in monitoring Lactococcus lactis cultures via IMC. β−-emission (electrons) was found to increase initial culture growth by increased nutrient uptake efficiency, which compensates for a reduced maximal cell division rate. Direct adsorption of the radionuclide to the biomass, revealed by mass spectrometry, is critical for both the initial stress response and the “dilution” of radioactivity-mediated damage at later culture stages, which are dominated by the chemical toxicity of Eu.
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Affiliation(s)
- Jana Oertel
- Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany
| | - Susanne Sachs
- Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany
| | - Katrin Flemming
- Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany
| | - Muhammad Hassan Obeid
- Protection and Safety Department, Atomic Energy Commission of Syria, Damascus P.O. Box 6091, Syria
| | - Karim Fahmy
- Helmholtz-Zentrum Dresden-Rossendorf, Bautzner Landstrasse 400, 01328 Dresden, Germany
- Cluster of Excellence Physics of Life, Technische Universität Dresden, 01062 Dresden, Germany
- Correspondence:
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4
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Lillhök J, Billnert-Maróti R, Anastasiadis A. MCNP 6.2 simulations of energy deposition in low-density volumes corresponding to unit-density volumes on the nanometre level. RADIAT MEAS 2022. [DOI: 10.1016/j.radmeas.2022.106831] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
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5
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Kyriakou I, Tremi I, Georgakilas AG, Emfietzoglou D. Microdosimetric investigation of the radiation quality of low-medium energy electrons using Geant4-DNA. Appl Radiat Isot 2021; 172:109654. [PMID: 33676082 DOI: 10.1016/j.apradiso.2021.109654] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Revised: 02/17/2021] [Accepted: 02/20/2021] [Indexed: 02/06/2023]
Abstract
The increasing clinical use of low-energy photon and electron sources (below few tens of keV) has raised concerns on the adequacy of the existing approximation of an energy-independent radiobiological effectiveness. In this work, the variation of the quality factor (Q) and relative biological effectiveness (RBE) of electrons over the low-medium energy range (0.1 keV-1 MeV) is examined using several microdosimetry-based Monte Carlo methodologies with input data obtained from Geant4-DNA track-structure simulations. The sensitivity of the results to the different methodologies, Geant4-DNA physics models, and target sizes is examined. Calculations of Q and RBE are based on the ICRU Report 40 recommendations, the Kellerer-Hahn approximation, the site version of the theory of dual radiation action (TDRA), the microdosimetric kinetic model (MKM) of cell survival, and the calculated yield of DNA double strand breaks (DSB). The stochastic energy deposition spectra needed as input in the above approaches have been calculated for nanometer spherical volumes using the different electron physics models of Geant4-DNA. Results are normalized at 100 keV electrons which is here considered the reference radiation. It is shown that in the energy range ~50 keV-1 MeV, the calculated Q and RBE are approximately unity (to within 1-2%) irrespective of the methodology, Geant4-DNA physics model, and target size. At lower energies, Q and RBE become energy-dependent reaching a maximum value of ~1.5-2.5 between ~200 and 700 eV. The detailed variation of Q and RBE at low energies depends mostly upon the adopted methodology and target size, and less so upon the Geant4-DNA physics model. Overall, the DSB yield predicts the highest RBE values (with RBEmax≈2.5) whereas the MKM the lowest RBE values (with RBEmax≈1.5). The ICRU Report 40, Kellerer-Hahn, and TDRA methods are in excellent agreement (to within 1-2%) over the whole energy range predicting a Qmax≈2. In conclusion, the approximation Q=RBE=1 was found to be valid only above ~50 keV whereas at lower energies both Q and RBE become strongly energy-dependent. It is envisioned that the present work will contribute towards establishing robust methodologies to determine theoretically the energy-dependence of radiation quality of individual electrons which may then be used in subsequent calculations involving practical electron and photon radiation sources.
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Affiliation(s)
- Ioanna Kyriakou
- Medical Physics Laboratory, University of Ioannina Medical School, 45110, Ioannina, Greece.
| | - Ioanna Tremi
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Zografou, Athens, Greece
| | - Alexandros G Georgakilas
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens (NTUA), Zografou, Athens, Greece
| | - Dimitris Emfietzoglou
- Medical Physics Laboratory, University of Ioannina Medical School, 45110, Ioannina, Greece
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6
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Margis S, Magouni M, Kyriakou I, Georgakilas AG, Incerti S, Emfietzoglou D. Microdosimetric calculations of the direct DNA damage induced by low energy electrons using the Geant4-DNA Monte Carlo code. Phys Med Biol 2020; 65:045007. [PMID: 31935692 DOI: 10.1088/1361-6560/ab6b47] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
To calculate the yield of direct DNA damage induced by low energy electrons using Monte Carlo generated microdosimetric spectra at the nanometer scale and examine the influence of various simulation inputs. The potential of classical microdosimetry to offer a viable and simpler alternative to more elaborate mechanistic approaches for practical applications is discussed. Track-structure simulations with the Geant4-DNA low-energy extension of the Geant4 Monte Carlo toolkit were used for calculating lineal energy spectra in spherical volumes with dimensions relevant to double-strand-break (DSB) induction. The microdosimetric spectra were then used to calculate the yield of simple and clustered DSB based on literature values of the threshold energy of DNA damage. The influence of the different implementations of the dielectric function of liquid water available in Geant4-DNA (Option 2 and Option 4 constructors), as well as the effect of particle tracking cutoff energy and target size are examined. Frequency- and dose-mean lineal energies in liquid-water spheres of 2, 2.3, 2.6, and 3.4 nm diameter, as well as, number of simple and clustered DSB/Gy/cell are presented for electrons over the 100 eV to 100 keV energy range. Results are presented for both the 'default' (Option 2) and 'Ioannina' (Option 4) physics models of Geant4-DNA applying several commonly used tracking cutoff energies (10, 20, 50, 100 eV). Overall, the choice of the physics model and target diameter has a moderate effect (up to ~10%-30%) on the DSB yield whereas the effect of the tracking cutoff energy may be significant (>100%). Importantly, the yield of both simple and clustered DSB was found to vary significantly (by a factor of 2 or more) with electron energy over the examined range. The yields of electron-induced simple and clustered DSB exhibit a strong energy dependence over the 100 eV-100 keV range with implications to radiation quality issues. It is shown that a classical microdosimetry approach for the calculation of DNA damage based on lineal energy spectra in nanometer-size targets predicts comparable results to computationally intensive mechanistic approaches which use detailed atomistic DNA geometries, thus, offering a relatively simple and robust alternative for some practical applications.
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Affiliation(s)
- Stefanos Margis
- Medical Physics Laboratory, University of Ioannina Medical School, 45110 Ioannina, Greece
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7
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Kumar A, Becker D, Adhikary A, Sevilla MD. Reaction of Electrons with DNA: Radiation Damage to Radiosensitization. Int J Mol Sci 2019; 20:E3998. [PMID: 31426385 PMCID: PMC6720166 DOI: 10.3390/ijms20163998] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2019] [Revised: 08/01/2019] [Accepted: 08/12/2019] [Indexed: 01/19/2023] Open
Abstract
This review article provides a concise overview of electron involvement in DNA radiation damage. The review begins with the various states of radiation-produced electrons: Secondary electrons (SE), low energy electrons (LEE), electrons at near zero kinetic energy in water (quasi-free electrons, (e-qf)) electrons in the process of solvation in water (presolvated electrons, e-pre), and fully solvated electrons (e-aq). A current summary of the structure of e-aq, and its reactions with DNA-model systems is presented. Theoretical works on reduction potentials of DNA-bases were found to be in agreement with experiments. This review points out the proposed role of LEE-induced frank DNA-strand breaks in ion-beam irradiated DNA. The final section presents radiation-produced electron-mediated site-specific formation of oxidative neutral aminyl radicals from azidonucleosides and the evidence of radiosensitization provided by these aminyl radicals in azidonucleoside-incorporated breast cancer cells.
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Affiliation(s)
- Anil Kumar
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA
| | - David Becker
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA
| | - Amitava Adhikary
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA
| | - Michael D Sevilla
- Department of Chemistry, Oakland University, Rochester, MI 48309, USA.
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8
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Yard BD, Gopal P, Bannik K, Siemeister G, Hagemann UB, Abazeed ME. Cellular and Genetic Determinants of the Sensitivity of Cancer to α-Particle Irradiation. Cancer Res 2019; 79:5640-5651. [PMID: 31387923 DOI: 10.1158/0008-5472.can-19-0859] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2019] [Revised: 06/12/2019] [Accepted: 07/29/2019] [Indexed: 12/20/2022]
Abstract
Targeted α-particle-emitting radionuclides have great potential for the treatment of a broad range of cancers at different stages of progression. A platform that accurately measures cancer cellular sensitivity to α-particle irradiation could guide and accelerate clinical translation. Here, we performed high-content profiling of cellular survival following exposure to α-particles emitted from radium-223 (223Ra) using 28 genetically diverse human tumor cell lines. Significant variation in cellular sensitivity across tumor cells was observed. 223Ra was significantly more potent than sparsely ionizing irradiation, with a median relative biological effectiveness of 10.4 (IQR: 8.4-14.3). Cells that are the most resistant to γ radiation, such as Nrf2 gain-of-function mutant cells, were sensitive to α-particles. Combining these profiling results with genetic features, we identified several somatic copy-number alterations, gene mutations, and the basal expression of gene sets that correlated with radiation survival. Activating mutations in PIK3CA, a frequent event in cancer, decreased sensitivity to 223Ra. The identification of cellular and genetic determinants of sensitivity to 223Ra may guide the clinical incorporation of targeted α-particle emitters in the treatment of several cancer types. SIGNIFICANCE: These findings address limitations in the preclinical guidance and prediction of radionuclide tumor sensitivity by identifying intrinsic cellular and genetic determinants of cancer cell survival following exposure to α-particle irradiation.See related commentary by Sgouros, p. 5479.
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Affiliation(s)
- Brian D Yard
- Department of Translational Hematology Oncology Research, Cleveland Clinic, Cleveland, Ohio
| | - Priyanka Gopal
- Department of Translational Hematology Oncology Research, Cleveland Clinic, Cleveland, Ohio
| | - Kristina Bannik
- Research and Development, Pharmaceuticals, Bayer AG, Berlin, Germany
| | | | - Urs B Hagemann
- Research and Development, Pharmaceuticals, Bayer AG, Berlin, Germany
| | - Mohamed E Abazeed
- Department of Translational Hematology Oncology Research, Cleveland Clinic, Cleveland, Ohio. .,Department of Radiation Oncology, Cleveland Clinic, Cleveland, Ohio
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9
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Erdmann E, Łabuda M, Aguirre NF, Díaz-Tendero S, Alcamí M. Furan Fragmentation in the Gas Phase: New Insights from Statistical and Molecular Dynamics Calculations. J Phys Chem A 2018. [PMID: 29543456 DOI: 10.1021/acs.jpca.8b00881] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
We present a complete exploration of the different fragmentation mechanisms of furan (C4H4O) operating at low and high energies. Three different theoretical approaches are combined to determine the structure of all possible reaction intermediates, many of them not described in previous studies, and a large number of pathways involving three types of fundamental elementary mechanisms: isomerization, fragmentation, and H/H2 loss processes (this last one was not yet explored). Our results are compared with the existing experimental and theoretical investigations for furan fragmentation. At low energies the first processes to appear are isomerization, which always implies the breaking of one C-O bond and one or several hydrogen transfers; at intermediate energies the fragmentation of the molecular skeleton becomes the most relevant mechanism; and H/H2 loss is the dominant processes at high energy. However, the three mechanisms are active in very wide energy ranges and, therefore, at most energies there is a competition among them.
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Affiliation(s)
- Ewa Erdmann
- Faculty of Applied Physics and Mathematics , Gdańsk University of Technology , Narutowicza 11/12 , 80-233 Gdańsk , Poland
| | - Marta Łabuda
- Faculty of Applied Physics and Mathematics , Gdańsk University of Technology , Narutowicza 11/12 , 80-233 Gdańsk , Poland
| | - Néstor F Aguirre
- Theoretical Division, Los Alamos National Laboratory , Los Alamos , New Mexico 87545 , United States
| | | | - Manuel Alcamí
- Instituto Madrileño de Estudios Avanzados en Nanociencias (IMDEA-Nanociencia) , 28049 Madrid , Spain
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10
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Villegas F, Bäckström G, Tilly N, Ahnesjö A. Energy deposition clustering as a functional radiation quality descriptor for modeling relative biological effectiveness. Med Phys 2016; 43:6322. [DOI: 10.1118/1.4966033] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
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11
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Isozaki T, Fujita M, Yamada S, Imadome K, Shoji Y, Yasuda T, Nakayama F, Imai T, Matsubara H. Effects of carbon ion irradiation and X-ray irradiation on the ubiquitylated protein accumulation. Int J Oncol 2016; 49:144-52. [PMID: 27175736 PMCID: PMC4902063 DOI: 10.3892/ijo.2016.3504] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2016] [Accepted: 03/26/2016] [Indexed: 02/07/2023] Open
Abstract
C-ion radiotherapy is associated with improved local control and survival in several types of tumors. Although C-ion irradiation is widely reported to effectively induce DNA damage in tumor cells, the effects of irradiation on proteins, such as protein stability or degradation in response to radiation stress, remain unknown. We aimed to compare the effects of C-ion and X-ray irradiation focusing on the cellular accumulation of ubiquitylated proteins. Cells from two human colorectal cancer cell lines, SW620 and SW480, were subjected to C-ion or X-ray irradiation and determination of ubiquitylated protein levels. High levels of ubiquitylated protein accumulation were observed in the C-ion-irradiated SW620 with a peak at 3 Gy; the accumulation was significantly lower in the X-ray-irradiated SW620 at all doses. Enhanced levels of ubiquitylated proteins were also detected in C-ion or X-ray-irradiated SW480, however, those levels were significantly lower than the peak detected in the C-ion-irradiated SW620. The levels of irradiation-induced ubiquitylated proteins decreased in a time-dependent manner, suggesting that the proteins were eliminated after irradiation. The treatment of C-ion-irradiated SW620 with a proteasome inhibitor (epoxomicin) enhanced the cell killing activity. The accumulated ubiquitylated proteins were co-localized with γ-H2AX, and with TP53BP1, in C-ion-irradiated SW620, indicating C-ion-induced ubiquitylated proteins may have some functions in the DNA repair system. Overall, we showed C-ion irradiation strongly induces the accumulation of ubiquitylated proteins in SW620. These characteristics may play a role in improving the therapeutic ratio of C-ion beams; blocking the clearance of ubiquitylated proteins may enhance sensitivity to C-ion radiation.
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Affiliation(s)
- Tetsuro Isozaki
- Department of Frontier Surgery, Chiba University Graduate School of Medicine, Chiba, Japan
| | - Mayumi Fujita
- Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba, Japan
| | - Shigeru Yamada
- Research Center Hospital, Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, Japan
| | - Kaori Imadome
- Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba, Japan
| | - Yoshimi Shoji
- Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba, Japan
| | - Takeshi Yasuda
- Radiation Emergency Medicine Research Program, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba, Japan
| | - Fumiaki Nakayama
- Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba, Japan
| | - Takashi Imai
- Advanced Radiation Biology Research Program, National Institute of Radiological Sciences, Chiba, Japan
| | - Hisahiro Matsubara
- Department of Frontier Surgery, Chiba University Graduate School of Medicine, Chiba, Japan
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12
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Emerson CH, Bertuch AA. Consider the workhorse: Nonhomologous end-joining in budding yeast. Biochem Cell Biol 2016; 94:396-406. [PMID: 27240172 DOI: 10.1139/bcb-2016-0001] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
DNA double strand breaks (DSBs) are dangerous sources of genome instability and must be repaired by the cell. Nonhomologous end-joining (NHEJ) is an evolutionarily conserved pathway to repair DSBs by direct ligation of the ends, with no requirement for a homologous template. While NHEJ is the primary DSB repair pathway in mammalian cells, conservation of the core NHEJ factors throughout eukaryotes makes the pathway attractive for study in model organisms. The budding yeast, Saccharomyces cerevisiae, has been used extensively to develop a functional picture of NHEJ. In this review, we will discuss the current understanding of NHEJ in S. cerevisiae. Topics include canonical end-joining, alternative end-joining, and pathway regulation. Particular attention will be paid to the NHEJ mechanism involving core factors, including Yku70/80, Dnl4, Lif1, and Nej1, as well as the various factors implicated in the processing of the broken ends. The relevance of chromatin dynamics to NHEJ will also be discussed. This review illustrates the use of S. cerevisiae as a powerful system to understand the principles of NHEJ, as well as in pioneering the direction of the field.
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Affiliation(s)
- Charlene H Emerson
- a Graduate Program in Genetics, Baylor College of Medicine, Houston, TX 77030, USA.,b Departments of Pediatrics and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
| | - Alison A Bertuch
- b Departments of Pediatrics and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA
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13
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Fukumoto M. Radiation pathology: From thorotrast to the future beyond radioresistance. Pathol Int 2014; 64:251-62. [DOI: 10.1111/pin.12170] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2014] [Accepted: 04/13/2014] [Indexed: 12/30/2022]
Affiliation(s)
- Manabu Fukumoto
- Department of Pathology; Institute of Development, Aging and Cancer; Tohoku University; Sendai Japan
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Kinashi Y, Okumura K, Kubota Y, Kitajima E, Okayasu R, Ono K, Takahashi S. Dose-rate effect was observed in T98G glioma cells following BNCT. Appl Radiat Isot 2013; 88:81-5. [PMID: 24360864 DOI: 10.1016/j.apradiso.2013.11.117] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2012] [Revised: 11/28/2013] [Accepted: 11/28/2013] [Indexed: 11/17/2022]
Abstract
BACKGROUND It is generally said that low LET radiation produce high dose-rate effect, on the other hand, no significant dose rate effect is observed in high LET radiation. Although high LET radiations are produced in BNCT, little is known about dose-rate effect of BNCT. MATERIALS AND METHODS T98G cells, which were tumor cells, were irradiated by neutron mixed beam with BPA. As normal tissue derived cells, Chinese hamster ovary (CHO-K1) cells and DNA double strand breaks (DNA-DSBs) repair deficient cells, xrs5 cells were irradiated by the neutrons (not including BPA). To DNA-DSBs analysis, T98G cells were stained immunochemically with 53BP1 antibody. The number of DNA-DSBs was determined by counting 53BP1 foci. RESULTS There was no dose-rate effect in xrs5 cells. D0 difference between 4cGy/min and 20cGy/min irradiation were 0.5 and 5.9 at the neutron and gamma-ray irradiation for CHO-K1, and 0.3 at the neutron for T98G cells. D0 difference between 20cGy/min and 80cGy/min irradiation for T98G cells were 1.2 and 0.6 at neutron irradiation plus BPA and gamma-ray. The differences between neutron irradiations at the dose rate in T98G cells were supported by not only the cell viability but also 53BP1 foci assay at 24h following irradiation to monitor DNA-DSBs. CONCLUSION Dose-rate effect of BNCT when T98G cells include 20ppm BPA was greater than that of gamma-ray irradiation. Moreover, Dose-rate effect of the neutron beam when CHO-K1 cells did not include BPA was less than that of gamma-ray irradiation These present results may suggest the importance of dose-rate effect for more efficient BNCT and the side effect reduction.
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Affiliation(s)
- Yuko Kinashi
- Kyoto University, Research Reactor Institute, Osaka 590-0494, Japan.
| | - Kakuji Okumura
- Kyoto University, Research Reactor Institute, Osaka 590-0494, Japan
| | | | | | | | - Koji Ono
- Kyoto University, Research Reactor Institute, Osaka 590-0494, Japan
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Autsavapromporn N, Suzuki M, Funayama T, Usami N, Plante I, Yokota Y, Mutou Y, Ikeda H, Kobayashi K, Kobayashi Y, Uchihori Y, Hei TK, Azzam EI, Murakami T. Gap junction communication and the propagation of bystander effects induced by microbeam irradiation in human fibroblast cultures: the impact of radiation quality. Radiat Res 2013; 180:367-75. [PMID: 23987132 PMCID: PMC4058832 DOI: 10.1667/rr3111.1] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Understanding the mechanisms underlying the bystander effects of low doses/low fluences of low- or high-linear energy transfer (LET) radiation is relevant to radiotherapy and radiation protection. Here, we investigated the role of gap-junction intercellular communication (GJIC) in the propagation of stressful effects in confluent normal human fibroblast cultures wherein only 0.036-0.144% of cells in the population were traversed by primary radiation tracks. Confluent cells were exposed to graded doses from monochromatic 5.35 keV X ray (LET ~6 keV/μm), 18.3 MeV/u carbon ion (LET ~103 keV/μm), 13 MeV/u neon ion (LET ~380 keV/μm) or 11.5 MeV/u argon ion (LET ~1,260 keV/μm) microbeams in the presence or absence of 18-α-glycyrrhetinic acid (AGA), an inhibitor of GJIC. After 4 h incubation at 37°C, the cells were subcultured and assayed for micronucleus (MN) formation. Micronuclei were induced in a greater fraction of cells than expected based on the fraction of cells targeted by primary radiation, and the effect occurred in a dose-dependent manner with any of the radiation sources. Interestingly, MN formation for the heavy-ion microbeam irradiation in the absence of AGA was higher than in its presence at high mean absorbed doses. In contrast, there were no significant differences in cell cultures exposed to X-ray microbeam irradiation in presence or absence of AGA. This showed that the inhibition of GJIC depressed the enhancement of MN formation in bystander cells from cultures exposed to high-LET radiation but not low-LET radiation. Bystander cells recipient of growth medium harvested from 5.35 keV X-irradiated cultures experienced stress manifested in the form of excess micronucleus formation. Together, the results support the involvement of both junctional communication and secreted factor(s) in the propagation of radiation-induced stress to bystander cells. They highlight the important role of radiation quality and dose in the observed effects.
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Affiliation(s)
- Narongchai Autsavapromporn
- Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, 263-8555, Japan
| | - Masao Suzuki
- Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, 263-8555, Japan
| | - Tomoo Funayama
- Microbeam Radiation Biology Group, Medical and Biotechnological Application Division, Quantum Beam Sciences Directorate, Japan Atomic Energy Agency, Takasaki, 370-1292, Japan
| | - Noriko Usami
- Photon Factory, High Energy Accelerator Research Organization, Tsukuba, 305-0801, Japan
| | - Ianik Plante
- University Space Research Association, NASA Johnson Space Center, Houston, Texas 77058
| | - Yuichiro Yokota
- Microbeam Radiation Biology Group, Medical and Biotechnological Application Division, Quantum Beam Sciences Directorate, Japan Atomic Energy Agency, Takasaki, 370-1292, Japan
| | - Yasuko Mutou
- Microbeam Radiation Biology Group, Medical and Biotechnological Application Division, Quantum Beam Sciences Directorate, Japan Atomic Energy Agency, Takasaki, 370-1292, Japan
| | - Hiroko Ikeda
- Microbeam Radiation Biology Group, Medical and Biotechnological Application Division, Quantum Beam Sciences Directorate, Japan Atomic Energy Agency, Takasaki, 370-1292, Japan
| | - Katsumi Kobayashi
- Photon Factory, High Energy Accelerator Research Organization, Tsukuba, 305-0801, Japan
| | - Yasuhiko Kobayashi
- Microbeam Radiation Biology Group, Medical and Biotechnological Application Division, Quantum Beam Sciences Directorate, Japan Atomic Energy Agency, Takasaki, 370-1292, Japan
| | - Yukio Uchihori
- Research, Development and Support Center, National Institute of Radiological Sciences, Chiba, 263-8555, Japan
| | - Tom K. Hei
- Center of Radiological Research, Columbia University Medical Center, New York, New York 10032
| | - Edouard I. Azzam
- Department of Radiology, Rutgers University, New Jersey Medical School, Cancer Center, Newark, New Jersey 07103
| | - Takeshi Murakami
- Research Center for Charged Particle Therapy, National Institute of Radiological Sciences, Chiba, 263-8555, Japan
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Loucas BD, Cornforth MN. The LET dependence of unrepaired chromosome damage in human cells: a break too far? Radiat Res 2013; 179:393-405. [PMID: 23578187 PMCID: PMC6058738 DOI: 10.1667/rr3159.2] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to "open breaks". This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with ¹³⁷Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from ²³⁸Pu and 1 GeV/amu ⁵⁶Fe ions. In agreement with previous studies, our results show that irrespective of radiation quality, the overall level of misrepaired damage exceeds that of unrepaired damage by a large margin. The unrepaired component of damage produced by γ rays and α particles was remarkably similar, about 5%. On that basis it is difficult to justify the popular notion that the strong LET-dependence for aberration formation is due to unrepaired DNA double-strand breaks (DSBs) that, by virtue of their complexity at the nanometer scale, are qualitatively different in nature. In marked contrast, this unrejoined component rose to about 14% after exposure to Fe ions. A closer look at the unrepaired component revealed that most of this roughly threefold difference was derived from incomplete exchanges. Despite vast differences in LET, unrejoined breaks from incomplete exchanges were far more likely to occur among exchanges that involved more than two breakpoints. We attempted to reconcile these observations in the form of a hypothesis that predicts that exchanges, irrespective of LET, should exhibit an increasing tendency for incompleteness as the number of initial breaks destined to take part in the exchange increases. This effect, we argue is not caused by the number of initial breaks per se, but instead reflects the maximum distance over which proximate breaks can interact. This adds a spatial aspect to multi-break interactions that we call "A Break Too Far".
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Affiliation(s)
- Bradford D Loucas
- Department of Radiation Oncology, The University of Texas Medical Branch, Galveston, Texas 77555, USA.
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Okumura K, Kinashi Y, Kubota Y, Kitajima E, Okayasu R, Ono K, Takahashi S. Relative biological effects of neutron mixed-beam irradiation for boron neutron capture therapy on cell survival and DNA double-strand breaks in cultured mammalian cells. JOURNAL OF RADIATION RESEARCH 2013; 54:70-75. [PMID: 22966174 PMCID: PMC3534280 DOI: 10.1093/jrr/rrs079] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/25/2012] [Revised: 07/27/2012] [Accepted: 08/03/2012] [Indexed: 06/01/2023]
Abstract
Understanding the biological effects of neutron mixed-beam irradiation used for boron neutron capture therapy (BNCT) is important in order to improve the efficacy of the therapy and to reduce side effects. In the present study, cell viability and DNA double-strand breaks (DNA-DSBs) were examined in Chinese hamster ovary cells (CHO-K1) and their radiosensitive mutant cells (xrs5, Ku80-deficient), following neutron mixed-beam irradiation for BNCT. Cell viability was significantly impaired in the neutron irradiation groups compared to the reference gamma-ray irradiation group. The relative biological effectiveness for 10% cell survival was 3.3 and 1.2 for CHO-K1 and xrs5 cells, respectively. There were a similar number of 53BP1 foci, indicators of DNA-DSBs, in the neutron mixed-beam and the gamma-ray groups. In addition, the size of the foci did not differ between groups. However, neutron mixed-beam irradiation resulted in foci with different spatial distributions. The foci were more proximal to each other in the neutron mixed-beam groups than the gamma-ray irradiation groups. These findings suggest that neutron beams may induce another type of DNA damage, such as clustered DNA-DSBs, as has been indicated for other high-LET irradiation.
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Affiliation(s)
- Kakuji Okumura
- Research Reactor Institute, Kyoto University, Kumatori-cho, Sennann-gun, Osaka 590-0494, Japan
| | - Yuko Kinashi
- Research Reactor Institute, Kyoto University, Kumatori-cho, Sennann-gun, Osaka 590-0494, Japan
| | | | | | | | - Koji Ono
- Research Reactor Institute, Kyoto University, Kumatori-cho, Sennann-gun, Osaka 590-0494, Japan
| | - Sentaro Takahashi
- Research Reactor Institute, Kyoto University, Kumatori-cho, Sennann-gun, Osaka 590-0494, Japan
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Arani LS, Mignon P, Abdoul-Carime H, Farizon B, Farizon M, Chermette H. DFT study of the fragmentation mechanism of uracil RNA base. Phys Chem Chem Phys 2012; 14:9855-70. [DOI: 10.1039/c2cp40384f] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Konopacka M, Rogoliński J, Slosarek K. Direct and bystander effects induced by scattered radiation generated during penetration of radiation inside a water-phantom. Mutat Res 2011; 721:6-14. [PMID: 21237284 DOI: 10.1016/j.mrgentox.2010.11.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2010] [Revised: 11/02/2010] [Accepted: 11/05/2010] [Indexed: 05/30/2023]
Abstract
In this study, the dose distribution of photon (6 MV) and electron (22 MeV) radiation in a water-phantom was compared with the frequency of apoptotic and micronucleated cells of two human cell lines (BEAS-2B normal bronchial epithelial cells and A549 lung cancer epithelial cells). Formation of micronuclei and apoptotic-like bodies was evaluated by the cytokinesis-block micronucleus test. Measurements were performed for five different phantom depths (3-20 cm). Irradiated cells were placed in a water-phantom in three variants: directly on the axis in the beam, under shielding (only in photon radiation) and outside the beam field. The results reveal a discrepancy between the distribution of physical dose at different depths of the water-phantom and biological effects. This discrepancy is of special significance in case of cells irradiated at a greater depth or placed outside the field and under shield during the exposure to radiation. The frequency of cytogenetic damage was higher than the expected value based on the physical dose received at different depths. Cells placed outside the beam axis were exposed to scattered radiation at very low doses, so we tested if bystander effects could have had a role in the observed discrepancy between physical radiation dose and biological response. We explored this question by use of a medium-transfer technique in which medium (ICM-irradiation conditioned medium) from irradiated cells was transferred to non-irradiated (bystander) cells. The results indicate that when cells were incubated in ICM transferred from cells irradiated at bigger depths or from cells exposed outside the radiation field, the number of apoptotic and micronucleated cells was similar to that after direct irradiation. This suggests that these damages are caused by factors released by irradiated cells into the medium rather than being induced directly in DNA by X-rays. Evaluation of biological effects of scattered radiation appears useful for clinical practice.
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Affiliation(s)
- M Konopacka
- Department of Experimental and Clinical Radiobiology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, Gliwice, Poland.
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20
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The radiosensitizing effect of Ku70/80 knockdown in MCF10A cells irradiated with X-rays and p(66)+Be(40) neutrons. Radiat Oncol 2010; 5:30. [PMID: 20423515 PMCID: PMC2877562 DOI: 10.1186/1748-717x-5-30] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2009] [Accepted: 04/27/2010] [Indexed: 11/17/2022] Open
Abstract
Background A better understanding of the underlying mechanisms of DNA repair after low- and high-LET radiations represents a research priority aimed at improving the outcome of clinical radiotherapy. To date however, our knowledge regarding the importance of DNA DSB repair proteins and mechanisms in the response of human cells to high-LET radiation, is far from being complete. Methods We investigated the radiosensitizing effect after interfering with the DNA repair capacity in a human mammary epithelial cell line (MCF10A) by lentiviral-mediated RNA interference (RNAi) of the Ku70 protein, a key-element of the nonhomologous end-joining (NHEJ) pathway. Following irradiation of control and Ku-deficient cell lines with either 6 MV X-rays or p(66)+Be(40) neutrons, cellular radiosensitivity testing was performed using a crystal violet cell proliferation assay. Chromosomal radiosensitivity was evaluated using the micronucleus (MN) assay. Results RNAi of Ku70 caused downregulation of both the Ku70 and the Ku80 proteins. This downregulation sensitized cells to both X-rays and neutrons. Comparable dose modifying factors (DMFs) for X-rays and neutrons of 1.62 and 1.52 respectively were obtained with the cell proliferation assay, which points to the similar involvement of the Ku heterodimer in the cellular response to both types of radiation beams. After using the MN assay to evaluate chromosomal radiosensitivity, the obtained DMFs for X-ray doses of 2 and 4 Gy were 2.95 and 2.66 respectively. After neutron irradiation, the DMFs for doses of 1 and 2 Gy were 3.36 and 2.82 respectively. The fact that DMFs are in the same range for X-rays and neutrons confirms a similar importance of the NHEJ pathway and the Ku heterodimer for repairing DNA damage induced by both X-rays and p(66)+Be(40) neutrons. Conclusions Interfering with the NHEJ pathway enhanced the radiosensitivity of human MCF10A cells to low-LET X-rays and high-LET neutrons, pointing to the importance of the Ku heterodimer for repairing damage induced by both types of radiation. Further research using other high-LET radiation sources is however needed to unravel the involvement of DNA double strand break repair pathways and proteins in the cellular response of human cells to high-LET radiation.
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21
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Davídková M, Juha L, Bittner M, Koptyaev S, Hájková V, Krása J, Pfeifer M, Stísová V, Bartnik A, Fiedorowicz H, Mikolajczyk J, Ryc L, Pína L, Horváth M, Babánková D, Cihelka J, Civis S. A High-Power Laser-Driven Source of Sub-nanosecond Soft X-Ray Pulses for Single-Shot Radiobiology Experiments. Radiat Res 2007; 168:382-7. [PMID: 17705629 DOI: 10.1667/rr0676.1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2006] [Accepted: 05/04/2007] [Indexed: 11/03/2022]
Abstract
A large-scale, double-stream gas puff target has been illuminated by sub-kJ, near-infrared (NIR) focused laser pulses at the PALS facility (Prague Asterix Laser System) to produce high-energy pulses of soft X rays from hot, dense plasma. The double-puff arrangement ensures high gas density and conversion efficiency from NIR to X rays approaching that typical for solid targets. In addition, its major advantage over solid targets is that it is free of debris and has substantially suppressed charged-particle emission. The X-ray emission characteristics of the source were determined for a range of gases that included krypton, xenon, N(2), CO and N(2)-CO. A demonstrated application of the xenon-based source is a single-shot damage induction to plasmid DNA. The yields of single-strand breaks (SSBs) and double-strand breaks (DSBs) were determined as a function of energy fluence adjusted by varying distance of sample from the source and thickness of aluminum filters.
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Affiliation(s)
- Marie Davídková
- Department of Radiation Dosimetry, Institute of Nuclear Physics, Academy of Sciences of the Czech Republic, 180 86, Prague 8, Czech Republic.
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22
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Li G, Zhou Z, Liu G, Zheng F, He C. Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Curr Genet 2007; 51:233-43. [PMID: 17372735 DOI: 10.1007/s00294-007-0122-5] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2006] [Revised: 01/27/2007] [Accepted: 01/29/2007] [Indexed: 11/29/2022]
Abstract
Agrobacterium tumefaciens-mediated transformation (ATMT) has been proven to be a powerful strategy for gene disruption in plants and fungi. Patterns associated with transferred DNA (T-DNA) integration in plants and yeast have been studied comprehensively, whereas no detailed analysis of T-DNA integration has been reported yet in filamentous fungi. Here, we reported the T-DNA insertion patterns in the genome of filamentous fungus Magnaporthe oryzae. Using ATMT, a T-DNA tagged population consisting of 6,179 transformants of M. oryzae was constructed. With thermal asymmetric interlaced-PCR (TAIL-PCR), 623 right border (RB) flanking sequences and 124 left border (LB) flanking sequences were generated. Analysis of these flanking sequences indicated a significant integration bias toward non-coding sequences, suggesting distribution of T-DNAs was not random. Comparing to T-DNA RB, LB was nicked inaccurately and truncated frequently during integration. Chromosomal rearrangements, such as deletion, inversion, and translocation, were associated with T-DNA integration in some transformants. Our data suggest that, comparing with plant cells, T-DNA integrates into this filamentous fungus with more precise and simpler patterns. Some phenotypic mutants were observed in our T-DNA tagged population, and these transformants will be very useful for functional genomics research of M. oryzae.
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Affiliation(s)
- Guihua Li
- State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, People's Republic of China
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Dunn J, Potter M, Rees A, Rünger TM. Activation of the Fanconi Anemia/BRCA Pathway and Recombination Repair in the Cellular Response to Solar Ultraviolet Light. Cancer Res 2006; 66:11140-7. [PMID: 17145857 DOI: 10.1158/0008-5472.can-06-0563] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Recombination repair plays an important role in the processing of DNA double-strand breaks (DSB) and DNA cross-links, and has been suggested to be mediated by the activation of the Fanconi anemia (FA)/BRCA pathway. Unlike DNA damage generated by ionizing radiation or DNA cross-linking, UV light-induced DNA damage is not commonly thought to require recombination for processing, as UV light does not directly induce DSBs or DNA cross-links. To elucidate the role of recombination repair in the cellular response to UV, we studied the FA/BRCA pathway in primary skin cells exposed to solar-simulated light. UV-induced monoubiquitination of the FANCD2 protein and formation of FANCD2 nuclear foci confirmed the activation of the pathway by UV light. This was only observed when cells were irradiated during S phase and was not caused by directly UV-induced DSBs. UV-exposed cells did not exhibit FANCD2 nuclear foci once they entered mitosis or when growth-arrested. In addition, UV-induced nuclear foci of the recombination proteins, RAD51 and BRCA1, colocalized with FANCD2 foci. We suggest that in response to UV light, when nucleotide excision repair failed to repair, or when translesional DNA synthesis failed to bypass UV-induced DNA photoproducts, the FA/BRCA pathway mediates the recombination repair of replication forks stalled at DNA photoproducts as a third line of defense.
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Affiliation(s)
- Jessica Dunn
- Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA
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Abstract
Nonhomologous end joining (NHEJ), the direct rejoining of DNA double-strand breaks, is closely associated with illegitimate recombination and chromosomal rearrangement. This has led to the concept that NHEJ is error prone. Studies with the yeast Saccharomyces cerevisiae have revealed that this model eukaryote has a classical NHEJ pathway dependent on Ku and DNA ligase IV, as well as alternative mechanisms for break rejoining. The evolutionary conservation of the Ku-dependent process includes several genes dedicated to this pathway, indicating that classical NHEJ at least is a strong contributor to fitness in the wild. Here we review how double-strand break structure, the yeast NHEJ proteins, and alternative rejoining mechanisms influence the accuracy of break repair. We also consider how the balance between NHEJ and homologous repair is regulated by cell state to promote genome preservation. The principles discussed are instructive to NHEJ in all organisms.
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Affiliation(s)
- James M Daley
- Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109-0602, USA.
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Mainardi E, Donahue RJ, Wilson WE, Blakely EA. Comparison of Microdosimetric Simulations Using PENELOPE and PITS for a 25 keV Electron Microbeam in Water. Radiat Res 2004; 162:326-31. [PMID: 15333002 DOI: 10.1667/rr3210] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The calculations presented compared the performances of two Monte Carlo codes used for the estimation of microdosimetric quantities: Positive Ion Track Structure code (PITS) and a main user code based on the PENetration and Energy Loss of Positrons and Electrons code (PENELOPE-2000). Event-by-event track structure codes like PITS are believed to be superior for microdosimetric applications, and they are written for this purpose. PITS tracks electrons in water down to 10 eV. PENELOPE is one of the few general-purpose codes that can simulate random electron-photon showers in any material for energies from 100 eV to 1 GeV. The model used in the comparison is a water cylinder with an internal scoring geometry made of spheres 1 microm in diameter where the scoring quantities are calculated. The source is a 25 keV electron pencil beam impinging normally on the sphere surface. This work shows only the lineal energy y and spectra graphical presentation as a function of y since for microdosimetry and biology applications, and for discussion of radiation quality in general, these results are more appropriate. The computed PENELOPE results are in agreement with those obtained with the PITS code and published previously in this journal. This paper demonstrates PENELOPE's usefulness at low energies and for small geometries. What is still needed are experimental results to confirm these analyses.
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Affiliation(s)
- Enrico Mainardi
- Dipartimento di Ingegneria Nucleare e Conversioni Energetiche, University of Rome La Sapienza, 00186 Roma, Italy.
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Mothersill C, Seymour C. Low-dose radiation effects: experimental hematology and the changing paradigm. Exp Hematol 2003; 31:437-45. [PMID: 12829018 DOI: 10.1016/s0301-472x(03)00078-x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
This review looks at the emerging field of nontargeted radiation effects and their impact on low-dose radiation risk assessment and radiotherapy. It identifies the major role of experimental hematologists and cytogeneticists in changing the old view of radiation action on living things. It also considers the history of radiobiology, seeking to explain why it is only now that we are considering indirect or nontargeted effects of low doses even though the evidence was there, though buried, in the old literature. Effects receiving major attention worldwide now include genomic instability and bystander effects. The impact of these effects, both on radiotherapy used to treat cancer and on radiation induction of cancer, still need to be clarified. Techniques developed by experimental hematologists are central to these efforts and have been instrumental in causing radiobiologists to consider that a paradigm shift is necessary. Throughout, we make a plea to think "outside the box" since the very construction of a framework necessarily limits our thinking and our experimental design.
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Affiliation(s)
- Carmel Mothersill
- Radiation and Environmental Science Centre, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland.
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