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Chudy P, Kochan J, Wawro M, Nguyen P, Gorczyca M, Varanko A, Retka A, Ghadei SS, Napieralska E, Grochot-Przęczek A, Szade K, Berendes LS, Park J, Sokołowski G, Yu Q, Józkowicz A, Nowak WN, Krzeptowski W. Heme oxygenase-1 protects cells from replication stress. Redox Biol 2024; 75:103247. [PMID: 39047636 PMCID: PMC11321372 DOI: 10.1016/j.redox.2024.103247] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Accepted: 06/18/2024] [Indexed: 07/27/2024] Open
Abstract
Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.
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Affiliation(s)
- Patryk Chudy
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; Doctoral School of Exact and Natural Sciences, Jagiellonian University, Krakow, Poland
| | - Jakub Kochan
- Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Mateusz Wawro
- Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Phu Nguyen
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Monika Gorczyca
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Aliaksandra Varanko
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Aleksandra Retka
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Swati Sweta Ghadei
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Emilija Napieralska
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Anna Grochot-Przęczek
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Krzysztof Szade
- Laboratory of Stem Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Lea-Sophie Berendes
- Department of General Pediatrics, University Hospital Münster, Münster, Germany
| | - Julien Park
- Department of General Pediatrics, University Hospital Münster, Münster, Germany
| | - Grzegorz Sokołowski
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Qiuliyang Yu
- Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, China
| | - Alicja Józkowicz
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
| | - Witold N Nowak
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; August Chełkowski Institute of Physics, Faculty of Science and Technology, University of Silesia, Chorzów, Poland.
| | - Wojciech Krzeptowski
- Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
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Allen LH, Fenech M, LeVatte MA, West KP, Wishart DS. Multiomics: Functional Molecular Biomarkers of Micronutrients for Public Health Application. Annu Rev Nutr 2024; 44:125-153. [PMID: 39207879 DOI: 10.1146/annurev-nutr-062322-022751] [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] [Indexed: 09/04/2024]
Abstract
Adequate micronutrient intake and status are global public health goals. Vitamin and mineral deficiencies are widespread and known to impair health and survival across the life stages. However, knowledge of molecular effects, metabolic pathways, biological responses to variation in micronutrient nutriture, and abilities to assess populations for micronutrient deficiencies and their pathology remain lacking. Rapidly evolving methodological capabilities in genomics, epigenomics, proteomics, and metabolomics offer unparalleled opportunities for the nutrition research community to link micronutrient exposure to cellular health; discover new, arguably essential micronutrients of microbial origin; and integrate methods of molecular biology, epidemiology, and intervention trials to develop novel approaches to assess and prevent micronutrient deficiencies in populations. In this review article, we offer new terminology to specify nutritional application of multiomic approaches and encourage collaboration across the basic to public health sciences to advance micronutrient deficiency prevention.
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Affiliation(s)
- Lindsay H Allen
- Western Human Nutrition Research Center, United States Department of Agriculture, Agricultural Research Service, Davis, California, USA
- Department of Nutrition, University of California, Davis, California, USA
| | - Michael Fenech
- Clinical and Health Sciences, University of South Australia, Adelaide, South Australia, Australia
- Genome Health Foundation, North Brighton, South Australia, Australia
| | - Marcia A LeVatte
- Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
| | - Keith P West
- Center for Human Nutrition, Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA;
| | - David S Wishart
- Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
- Department of Computing Science, University of Alberta, Edmonton, Alberta, Canada
- Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada
- Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
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Steffens Reinhardt L, Moira Morás A, Gustavo Henn J, Ricardo Arantes P, Bernardes Ferro M, Braganhol E, Oliveira de Souza P, de Oliveira Merib J, Ramos Borges G, Silveira Dalanhol C, Cox Holanda de Barros Dias M, Nugent M, Jaqueline Moura D. Nek1-inhibitor and temozolomide-loaded microfibers as a co-therapy strategy for glioblastoma treatment. Int J Pharm 2022; 617:121584. [PMID: 35202726 DOI: 10.1016/j.ijpharm.2022.121584] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2021] [Revised: 01/29/2022] [Accepted: 02/11/2022] [Indexed: 11/16/2022]
Abstract
Malignant glioblastoma (GB) is the predominant primary brain tumour in adults, but despite the efforts towards novel therapies, the median survival of GB patients has not significantly improved in the last decades. Therefore, localised approaches that treat GB straight into the tumour site provide an alternative to enhance chemotherapy bioavailability and efficacy, reducing systemic toxicity. Likewise, the discovery of protein targets, such as the NIMA-related kinase 1 (Nek1), which was previously shown to be associated with temozolomide (TMZ) resistance in GB, has stimulated the clinical development of target therapy approaches to treat GB patients. In this study, we report an electrospun polyvinyl alcohol (PVA) microfiber (MF) brain-implant prepared for the controlled release of Nek1 protein inhibitor (iNek1) and TMZ or TMZ-loaded nanoparticles. The formulations revealed adequate stability and drug loading, which prolonged the drugs' release allowing a sustained exposure of the GB cells to the treatment and enhancing the drugs' therapeutic effects. TMZ-loaded MF provided the highest concentration of TMZ within the brain of tumour-bearing rats, and it was statistically significant when compared to TMZ via intraperitoneal (IP). All animals treated with either co-therapy formulation (TMZ + iNek1 MF or TMZ nanoparticles + iNek1 MF) survived until the endpoint (60 days), whereas the Blank MF (drug-unloaded), TMZ MF and TMZ IP-treated rats' median survival was found to be 16, 31 and 25 days, respectively. The tumour/brain area ratio of the rats implanted with either MF co-therapy was found to be reduced by 5-fold when compared to Blank MF-implanted rats. Taken together, our results strongly suggest that Nek1 is an important GB oncotarget and the inhibition of Nek1's activity significantly decreases GB cells' viability and tumour size when combined with TMZ treatment.
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Affiliation(s)
- Luiza Steffens Reinhardt
- Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil; Biosciences Graduation Course, UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil.
| | - Ana Moira Morás
- Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil; Biosciences Graduation Course, UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil.
| | - Jeferson Gustavo Henn
- Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil; Biosciences Graduation Course, UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil.
| | | | - Matheus Bernardes Ferro
- Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil.
| | - Elizandra Braganhol
- Biosciences Graduation Course, UFCSPA, Porto Alegre, Rio Grande do Sul, Brazil.
| | | | | | | | | | | | - Michael Nugent
- Materials Research Institute, TUS, Athlone, Co. Westmeath, Ireland.
| | - Dinara Jaqueline Moura
- Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil.
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Mirman Z, Sasi NK, King A, Chapman JR, de Lange T. 53BP1-shieldin-dependent DSB processing in BRCA1-deficient cells requires CST-Polα-primase fill-in synthesis. Nat Cell Biol 2022; 24:51-61. [PMID: 35027730 PMCID: PMC8849574 DOI: 10.1038/s41556-021-00812-9] [Citation(s) in RCA: 38] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2020] [Accepted: 11/09/2021] [Indexed: 12/19/2022]
Abstract
The efficacy of poly(ADP)-ribose polymerase 1 inhibition (PARPi) in BRCA1-deficient cells depends on 53BP1 and shieldin, which have been proposed to limit single-stranded DNA at double-strand breaks (DSBs) by blocking resection and/or through CST-Polα-primase-mediated fill-in. We show that primase (like 53BP1-shieldin and CST-Polα) promotes radial chromosome formation in PARPi-treated BRCA1-deficient cells and demonstrate shieldin-CST-Polα-primase-dependent incorporation of BrdU at DSBs. In the absence of 53BP1 or shieldin, radial formation in BRCA1-deficient cells was restored by the tethering of CST near DSBs, arguing that in this context, shieldin acts primarily by recruiting CST. Furthermore, a SHLD1 mutant defective in CST binding (SHLD1Δ) was non-functional in BRCA1-deficient cells and its function was restored after reconnecting SHLD1Δ to CST. Interestingly, at dysfunctional telomeres and at DNA breaks in class switch recombination where CST has been implicated, SHLD1Δ was fully functional, perhaps because these DNA ends carry CST recognition sites that afford SHLD1-independent binding of CST. These data establish that in BRCA1-deficient cells, CST-Polα-primase is the major effector of shieldin-dependent DSB processing.
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Affiliation(s)
- Zachary Mirman
- Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY, USA
| | - Nanda Kumar Sasi
- Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY, USA
| | - Ashleigh King
- Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - J Ross Chapman
- Medical Research Council (MRC) Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK
| | - Titia de Lange
- Laboratory for Cell Biology and Genetics, Rockefeller University, New York, NY, USA.
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5
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Rucinski A, Biernacka A, Schulte R. Applications of nanodosimetry in particle therapy planning and beyond. Phys Med Biol 2021; 66. [PMID: 34731854 DOI: 10.1088/1361-6560/ac35f1] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2021] [Accepted: 11/03/2021] [Indexed: 12/28/2022]
Abstract
This topical review summarizes underlying concepts of nanodosimetry. It describes the development and current status of nanodosimetric detector technology. It also gives an overview of Monte Carlo track structure simulations that can provide nanodosimetric parameters for treatment planning of proton and ion therapy. Classical and modern radiobiological assays that can be used to demonstrate the relationship between the frequency and complexity of DNA lesion clusters and nanodosimetric parameters are reviewed. At the end of the review, existing approaches of treatment planning based on relative biological effectiveness (RBE) models or dose-averaged linear energy transfer are contrasted with an RBE-independent approach based on nandosimetric parameters. Beyond treatment planning, nanodosimetry is also expected to have applications and give new insights into radiation protection dosimetry.
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Affiliation(s)
| | - Anna Biernacka
- University of Gdansk, Intercollegiate Faculty of Biotechnology of University of Gdańsk and Medical University of Gdansk, 80-307 Gdansk, Poland
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Ge J, Ngo LP, Kaushal S, Tay IJ, Thadhani E, Kay JE, Mazzucato P, Chow DN, Fessler JL, Weingeist DM, Sobol RW, Samson LD, Floyd SR, Engelward BP. CometChip enables parallel analysis of multiple DNA repair activities. DNA Repair (Amst) 2021; 106:103176. [PMID: 34365116 PMCID: PMC8439179 DOI: 10.1016/j.dnarep.2021.103176] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2021] [Revised: 06/09/2021] [Accepted: 07/08/2021] [Indexed: 12/28/2022]
Abstract
DNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.
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Affiliation(s)
- Jing Ge
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Le P Ngo
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Simran Kaushal
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, 02115, United States
| | - Ian J Tay
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Elina Thadhani
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Jennifer E Kay
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Patrizia Mazzucato
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Danielle N Chow
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Jessica L Fessler
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - David M Weingeist
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Robert W Sobol
- Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15232, United States; University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, PA 15213, United States
| | - Leona D Samson
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Scott R Floyd
- Department of Radiation Oncology, Duke University School of Medicine, Durham, NC 27514, United States
| | - Bevin P Engelward
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, United States.
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Nakamura AJ. Beyond visualization of DNA double-strand breaks after radiation exposure. Int J Radiat Biol 2021; 98:522-527. [PMID: 33989105 DOI: 10.1080/09553002.2021.1930268] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
PURPOSE Radiation science and radiation biology are fields where milestones have been set by numerous woman researchers, as represented by Marie Curie. This shows that it is a research field that is like a model of research diversity in modern society. In this review, I will describe what kind of research activities I have conducted as a Japanese woman researcher in the field of radiation science research. In addition, as a Japanese woman radiobiologist, I will describe the sense of mission I felt after the Fukushima Nuclear Power Plant accident and the research issues we must challenge in the future. CONCLUSION As a Japanese woman researcher, I have felt a bias in gender balance in the field of science in Japan. Also, after the Fukushima nuclear Power Plant accident, I sometimes felt that woman researchers would be more suitable when sharing research results and specialized knowledge with the general public. In recent years, the importance of STEAM (Science-Technology-Engineering-Art-Mathematics) education has been highlighted all over the world, and I believe that the field of radiation science falls exactly into the STEAM education category. STEAM education is for people of all gender. I hope that radiation science research will lead to various younger generations, and that the gender balance of Japanese scientific researchers will increase.
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Affiliation(s)
- Asako J Nakamura
- Department of Biological Science, College of Sciences, Ibaraki University, Mito, Japan
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Potential application of γ-H2AX as a biodosimetry tool for radiation triage. MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH 2020; 787:108350. [PMID: 34083048 DOI: 10.1016/j.mrrev.2020.108350] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/01/2020] [Revised: 11/11/2020] [Accepted: 11/13/2020] [Indexed: 01/01/2023]
Abstract
Radiation triage and biological dosimetry are two initial steps in the medical management of exposed individuals following radiological accidents. Well established biodosimetry methods such as the dicentric (DC) assay, micronucleus (MN) assay, and fluorescence in-situ hybridization (FISH) translocation assay (for residual damage) have been used for this purpose for several decades. Recent advances in scoring methodology and networking among established laboratories have increased triage capacity; however, these methods still have limitations in analysing large sample numbers, particularly because of the ∼ 48 h minimum culture time required prior to analysis. Hence, there is a need for simple, and high throughput markers to identify exposed individuals in case of radiological/nuclear emergencies. In recent years, a few markers were identified, one being phosphorylated histone 2AX (γ-H2AX), which measured a nuclear foci or nuclear staining intensity that was found to be suitable for triage. Measurement of γ-H2AX foci formed at and around the sites of DNA double-strand breaks is a rapid and sensitive biodosimetry method which does not require culturing and is thus promising for the analysis of a large number of samples. In this review, we have summarized the recent developments of γ-H2AX assay in radiation triage and biodosimetry, focusing chiefly on: i) the importance of baseline frequency and reported values among different laboratories, ii) the influence of known and unknown variables on dose estimation, iii) quality assurance such as inter-laboratory comparison between scorers and scoring methods, and iv) current limitations and potential for future development.
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9
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Steffens L, Morás AM, Arantes PR, Masterson K, Cao Z, Nugent M, Moura DJ. Electrospun PVA-Dacarbazine nanofibers as a novel nano brain-implant for treatment of glioblastoma: in silico and in vitro characterization. Eur J Pharm Sci 2020; 143:105183. [DOI: 10.1016/j.ejps.2019.105183] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2019] [Revised: 12/07/2019] [Accepted: 12/13/2019] [Indexed: 01/06/2023]
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Babayan N, Grigoryan B, Khondkaryan L, Tadevosyan G, Sarkisyan N, Grigoryan R, Apresyan L, Aroutiounian R, Vorobyeva N, Pustovalova M, Grekhova A, Osipov AN. Laser-Driven Ultrashort Pulsed Electron Beam Radiation at Doses of 0.5 and 1.0 Gy Induces Apoptosis in Human Fibroblasts. Int J Mol Sci 2019; 20:ijms20205140. [PMID: 31627284 PMCID: PMC6829387 DOI: 10.3390/ijms20205140] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2019] [Revised: 10/09/2019] [Accepted: 10/16/2019] [Indexed: 12/12/2022] Open
Abstract
Rapidly evolving laser technologies have led to the development of laser-generated particle accelerators as an alternative to conventional facilities. However, the radiobiological characteristics need to be determined to enhance their applications in biology and medicine. In this study, the radiobiological effects of ultrashort pulsed electron beam (UPEB) and X-ray radiation in human lung fibroblasts (MRC-5 cell line) exposed to doses of 0.1, 0.5, and 1 Gy are compared. The changes of γH2AX foci number as a marker of DNA double-strand breaks (DSBs) were analyzed. In addition, the micronuclei induction and cell death via apoptosis were studied. We found that the biological action of UPEB-radiation compared to X-rays was characterized by significantly slower γH2AX foci elimination (with a dose of 1 Gy) and strong apoptosis induction (with doses of 0.5 and 1.0 Gy), accompanied by a slight increase in micronuclei formation (dose of 1 Gy). Our data suggest that UPEB radiation produces more complex DNA damage than X-ray radiation, leading to cell death rather than cytogenetic disturbance.
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Affiliation(s)
- Nelly Babayan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
- Yerevan State University, 1 Manoogian, 0025 Yerevan, Armenia.
| | - Bagrat Grigoryan
- CANDLE Synchrotron Research Institute, 31 Acharyan, 0040 Yerevan, Armenia.
| | - Lusine Khondkaryan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
| | - Gohar Tadevosyan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
| | - Natalya Sarkisyan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
| | - Ruzanna Grigoryan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
| | - Lilit Apresyan
- Institute of Molecular Biology NAS RA, 7 Hasratyan, 0014 Yerevan, Armenia.
| | | | - Natalia Vorobyeva
- State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, 46 Zhivopisnaya, 123182 Moscow, Russia.
- Semenov Institute of Chemical Physics. Russian Academy of Sciences, 4 Kosygina, Moscow 119991, Russia.
| | - Margarita Pustovalova
- Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region 141700, Russia.
| | - Anna Grekhova
- Emanuel Institute for Biochemical Physics. Russian Academy of Sciences, Moscow 119991, Russia.
| | - Andreyan N Osipov
- State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, 46 Zhivopisnaya, 123182 Moscow, Russia.
- Semenov Institute of Chemical Physics. Russian Academy of Sciences, 4 Kosygina, Moscow 119991, Russia.
- Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region 141700, Russia.
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11
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Lopez Perez R, Nicolay NH, Wolf JC, Frister M, Schmezer P, Weber KJ, Huber PE. DNA damage response of clinical carbon ion versus photon radiation in human glioblastoma cells. Radiother Oncol 2019; 133:77-86. [PMID: 30935585 DOI: 10.1016/j.radonc.2018.12.028] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 12/14/2018] [Accepted: 12/30/2018] [Indexed: 02/06/2023]
Abstract
BACKGROUND AND PURPOSE Carbon ion radiotherapy is a promising therapeutic option for glioblastoma patients due to its high physical dose conformity and greater biological effectiveness than photons. However, the biological effects of carbon ion radiation are still incompletely understood. Here, we systematically compared the biological effects of clinically used carbon ion radiation to photon radiation with emphasis on DNA repair. MATERIALS AND METHODS Two human glioblastoma cell lines (U87 and LN229) were irradiated with carbon ions or photons and DNA damage response was systematically analyzed, including clonogenic survival, induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and apoptosis or autophagy. γH2AX foci were analyzed by flow cytometry, conventional light microscopy and 3D superresolution microscopy. RESULTS DSBs were repaired delayed and with slower kinetics after carbon ions versus photons. Carbon ions caused stronger and longer-lasting cell cycle delays, predominantly in G2 phase, and a higher rate of apoptosis. Compared to photons, the effectiveness of carbon ions was less cell cycle-dependent. Homologous recombination (HR) appeared to be more important for DSB repair after carbon ions versus photons in phosphatase and tensin homolog (PTEN)-deficient U87 cells, as opposed to PTEN-proficient LN229 cells. CONCLUSION Carbon ions induced more severe DSB damage than photons, which was repaired less efficiently in both cell lines. Thus, carbon ion radiotherapy may help to overcome resistance mechanisms of glioblastoma associated with DNA repair for example in combination with repair pathway-specific drugs in the context of personalized radiotherapy.
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Affiliation(s)
- Ramon Lopez Perez
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany.
| | - Nils H Nicolay
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany; Department of Radiation Oncology, Freiburg University Medical Center, Germany
| | - Jörg-Christian Wolf
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany
| | - Moritz Frister
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany
| | - Peter Schmezer
- Epigenomics and Cancer Risk Factors, German Cancer Research Center, Heidelberg, Germany
| | - Klaus-Josef Weber
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany
| | - Peter E Huber
- CCU Molecular and Radiation Oncology, German Cancer Research Center and Department of Radiation Oncology, Heidelberg University Hospital, Germany.
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12
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Repair characteristics and time-dependent effects in Saccharomyces cerevisiae cells after X-ray irradiation. World J Microbiol Biotechnol 2018; 35:1. [DOI: 10.1007/s11274-018-2566-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 11/23/2018] [Indexed: 11/26/2022]
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13
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Babayan N, Hovhannisyan G, Grigoryan B, Grigoryan R, Sarkisyan N, Tsakanova G, Haroutiunian S, Aroutiounian R. Dose-rate effect of ultrashort electron beam radiation on DNA damage and repair in vitro. JOURNAL OF RADIATION RESEARCH 2017; 58:894-897. [PMID: 28992052 PMCID: PMC5737585 DOI: 10.1093/jrr/rrx035] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/16/2017] [Indexed: 05/07/2023]
Abstract
Laser-generated electron beams are distinguished from conventional accelerated particles by ultrashort beam pulses in the femtoseconds to picoseconds duration range, and their application may elucidate primary radiobiological effects. The aim of the present study was to determine the dose-rate effect of laser-generated ultrashort pulses of 4 MeV electron beam radiation on DNA damage and repair in human cells. The dose rate was increased via changing the pulse repetition frequency, without increasing the electron energy. The human chronic myeloid leukemia K-562 cell line was used to estimate the DNA damage and repair after irradiation, via the comet assay. A distribution analysis of the DNA damage was performed. The same mean level of initial DNA damages was observed at low (3.6 Gy/min) and high (36 Gy/min) dose-rate irradiation. In the case of low-dose-rate irradiation, the detected DNA damages were completely repairable, whereas the high-dose-rate irradiation demonstrated a lower level of reparability. The distribution analysis of initial DNA damages after high-dose-rate irradiation revealed a shift towards higher amounts of damage and a broadening in distribution. Thus, increasing the dose rate via changing the pulse frequency of ultrafast electrons leads to an increase in the complexity of DNA damages, with a consequent decrease in their reparability. Since the application of an ultrashort pulsed electron beam permits us to describe the primary radiobiological effects, it can be assumed that the observed dose-rate effect on DNA damage/repair is mainly caused by primary lesions appearing at the moment of irradiation.
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Affiliation(s)
- Nelly Babayan
- Yerevan State University, Alex Manoogian 1, 0025 Yerevan, Armenia
- Institute of Molecular Biology of NAS, Hasratyan 7, 0014 Yerevan, Armenia
- Corresponding author. Institute of Molecular Biology of NAS, Hasratyan 7, 0014 Yerevan, Armenia. Tel: +37491-572573; Fax: +37410-282061;
| | | | - Bagrat Grigoryan
- CANDLE Synchrotron Research Institute, Acharyan 31, 0040, Yerevan, Armenia
| | - Ruzanna Grigoryan
- Institute of Molecular Biology of NAS, Hasratyan 7, 0014 Yerevan, Armenia
| | - Natalia Sarkisyan
- Institute of Molecular Biology of NAS, Hasratyan 7, 0014 Yerevan, Armenia
| | - Gohar Tsakanova
- Institute of Molecular Biology of NAS, Hasratyan 7, 0014 Yerevan, Armenia
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14
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Mavragani IV, Nikitaki Z, Souli MP, Aziz A, Nowsheen S, Aziz K, Rogakou E, Georgakilas AG. Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis. Cancers (Basel) 2017; 9:cancers9070091. [PMID: 28718816 PMCID: PMC5532627 DOI: 10.3390/cancers9070091] [Citation(s) in RCA: 110] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 07/06/2017] [Accepted: 07/14/2017] [Indexed: 12/26/2022] Open
Abstract
Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.
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Affiliation(s)
- Ifigeneia V Mavragani
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece.
| | - Zacharenia Nikitaki
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece.
| | - Maria P Souli
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece.
| | - Asef Aziz
- Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, MN 55905, USA.
| | - Somaira Nowsheen
- Mayo Medical Scientist Training Program, Mayo Medical School and Mayo Graduate School, Mayo Clinic, Rochester, MN 55905, USA.
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
| | - Khaled Aziz
- Mayo Medical Scientist Training Program, Mayo Medical School and Mayo Graduate School, Mayo Clinic, Rochester, MN 55905, USA.
| | - Emmy Rogakou
- First Department of Pediatrics, "Aghia Sophia" Children's Hospital, Medical School, University of Athens, 11527 Athens, Greece.
| | - Alexandros G Georgakilas
- DNA Damage Laboratory, Physics Department, School of Applied Mathematical and Physical Sciences, National Technical University of Athens, Zografou Campus, 15780 Athens, Greece.
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15
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Hasan Basri IK, Yusuf D, Rahardjo T, Nurhayati S, Tetriana D, Ramadhani D, Alatas Z, Purnami S, Kisnanto T, Lusiyanti Y, Syaifudin M. Study of γ-H2AX as DNA double strand break biomarker in resident living in high natural radiation area of Mamuju, West Sulawesi. JOURNAL OF ENVIRONMENTAL RADIOACTIVITY 2017; 171:212-216. [PMID: 28282532 DOI: 10.1016/j.jenvrad.2017.02.012] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/02/2016] [Revised: 12/16/2016] [Accepted: 02/15/2017] [Indexed: 06/06/2023]
Abstract
High expression of phospho histone γ-H2AX, a sensitive marker of double stranded DNA damage, is believed to be an indication of defective DNA repair pathway or genomic instability that may cause mutations and ultimately cancer. DNA damage can be caused by ionizing radiation exposure. Beside in medical treatment/diagnosis or industry, ionizing radiation exposure can also be found in naturally in regions of high natural back ground radiation. In this study we collect the blood from 45 volunteers living in Mamuju, a region with highest natural radiation in Indonesia (dose of ∼7 mSv/year). Subjects were grouped as high natural background area (HNBA) (n = 37) and control area (n = 8). The expression γ-H2AX foci were evaluated by one of researcher fluorescence microscope examination. Our results show that the average foci numbers per cell were in the normal range. While not statistical different, the average of γ-H2AX foci in exposed area higher in the exposed compared to the control area, 0.31 versus 0.13 (p > 0.05), respectively. Moreover, there was also no statistical difference of average γ-H2AX foci between man and woman, old and young people in exposed and control area (p > 0.05). In this preliminary study we find that γ-H2AX foci (and thus DNA double strand break) frequency in residents living in the HNBA of Mamuju, West Sulawesi, show a trend towards higher (albeit not significant) average values relative to the control area. More research is needed to further scrutinize these observations.
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Affiliation(s)
- Iin Kurnia Hasan Basri
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia.
| | - Darlina Yusuf
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Tur Rahardjo
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Siti Nurhayati
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Devita Tetriana
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Dwi Ramadhani
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Zubaidah Alatas
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Sofiati Purnami
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Teja Kisnanto
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Yanti Lusiyanti
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
| | - Mukh Syaifudin
- Center for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency, Jl. Lebakbulus Raya No. 49, Jakarta Indonesia
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16
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Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response. G3-GENES GENOMES GENETICS 2016; 6:3869-3881. [PMID: 27678521 PMCID: PMC5144958 DOI: 10.1534/g3.116.033910] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control.
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17
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Garty G, Turner HC, Salerno A, Bertucci A, Zhang J, Chen Y, Dutta A, Sharma P, Bian D, Taveras M, Wang H, Bhatla A, Balajee A, Bigelow AW, Repin M, Lyulko OV, Simaan N, Yao YL, Brenner DJ. THE DECADE OF THE RABiT (2005-15). RADIATION PROTECTION DOSIMETRY 2016; 172:201-206. [PMID: 27412510 PMCID: PMC5225976 DOI: 10.1093/rpd/ncw172] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
The RABiT (Rapid Automated Biodosimetry Tool) is a dedicated Robotic platform for the automation of cytogenetics-based biodosimetry assays. The RABiT was developed to fulfill the critical requirement for triage following a mass radiological or nuclear event. Starting from well-characterized and accepted assays we developed a custom robotic platform to automate them. We present here a brief historical overview of the RABiT program at Columbia University from its inception in 2005 until the RABiT was dismantled at the end of 2015. The main focus of this paper is to demonstrate how the biological assays drove development of the custom robotic systems and in turn new advances in commercial robotic platforms inspired small modifications in the assays to allow replacing customized robotics with 'off the shelf' systems. Currently, a second-generation, RABiT II, system at Columbia University, consisting of a PerkinElmer cell::explorer, was programmed to perform the RABiT assays and is undergoing testing and optimization studies.
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Affiliation(s)
- G Garty
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - H C Turner
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - A Salerno
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Pratt & Whitney Canada Corp., 1000 Marie-Victorin, Longueil, QC, Canada J4G 1A1
| | - A Bertucci
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - J Zhang
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Auris Surgical Robotics Inc., 125 Shoreway Rd, San Carlos, CA 94070, USA
| | - Y Chen
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - A Dutta
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
- Present address: BioReliance Corp., 9630 Medical Center Dr, Rockville, MD 20850, USA
| | - P Sharma
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - D Bian
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - M Taveras
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - H Wang
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: General Motors Co., 30500 Mound Road, Warren, MI 48090, USA
| | - A Bhatla
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Curiosity Lab Inc., 54 Mallard Pl. Secaucus, NJ, 07094, USA
| | - A Balajee
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
- Present address: Cytogenetic Biodosimetry Laboratory, Radiation Emergency Assistance Center and Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, Building SC-10, 1299, Bethel Valley Road, Oak Ridge, TN, 37830, USA
| | - A W Bigelow
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - M Repin
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - O V Lyulko
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
| | - N Simaan
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
- Present address: Department of Mechanical Engineering, Vanderbuilt University, PMB 351592, Nashville, TN, 37235, USA
| | - Y L Yao
- Department of Mechanical Engineering, Columbia University, 500 West 120th Street, New York, NY 10027, USA
| | - D J Brenner
- Center for Radiological Research, Columbia University, VC11-230, 630 West 168th Street, New York, NY 10032, USA
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18
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Vijayraghavan S, Tsai FL, Schwacha A. A Checkpoint-Related Function of the MCM Replicative Helicase Is Required to Avert Accumulation of RNA:DNA Hybrids during S-phase and Ensuing DSBs during G2/M. PLoS Genet 2016; 12:e1006277. [PMID: 27556397 PMCID: PMC4996524 DOI: 10.1371/journal.pgen.1006277] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Accepted: 08/04/2016] [Indexed: 01/10/2023] Open
Abstract
The Mcm2-7 complex is the catalytic core of the eukaryotic replicative helicase. Here, we identify a new role for this complex in maintaining genome integrity. Using both genetic and cytological approaches, we find that a specific mcm allele (mcm2DENQ) causes elevated genome instability that correlates with the appearance of numerous DNA-damage associated foci of γH2AX and Rad52. We further find that the triggering events for this genome instability are elevated levels of RNA:DNA hybrids and an altered DNA topological state, as over-expression of either RNaseH (an enzyme specific for degradation of RNA in RNA:DNA hybrids) or Topoisomerase 1 (an enzyme that relieves DNA supercoiling) can suppress the mcm2DENQ DNA-damage phenotype. Moreover, the observed DNA damage has several additional unusual properties, in that DNA damage foci appear only after S-phase, in G2/M, and are dependent upon progression into metaphase. In addition, we show that the resultant DNA damage is not due to spontaneous S-phase fork collapse. In total, these unusual mcm2DENQ phenotypes are markedly similar to those of a special previously-studied allele of the checkpoint sensor kinase ATR/MEC1, suggesting a possible regulatory interplay between Mcm2-7 and ATR during unchallenged growth. As RNA:DNA hybrids primarily result from transcription perturbations, we suggest that surveillance-mediated modulation of the Mcm2-7 activity plays an important role in preventing catastrophic conflicts between replication forks and transcription complexes. Possible relationships among these effects and the recently discovered role of Mcm2-7 in the DNA replication checkpoint induced by HU treatment are discussed.
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Affiliation(s)
- Sriram Vijayraghavan
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Feng-Ling Tsai
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Anthony Schwacha
- Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
- * E-mail:
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19
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Lapytsko A, Kollarovic G, Ivanova L, Studencka M, Schaber J. FoCo: a simple and robust quantification algorithm of nuclear foci. BMC Bioinformatics 2015; 16:392. [PMID: 26589438 PMCID: PMC4654864 DOI: 10.1186/s12859-015-0816-5] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Accepted: 11/07/2015] [Indexed: 12/11/2022] Open
Abstract
Background The number of γH2AX foci per nucleus is an accepted measure of the number of DNA double-strand breaks in single cells. One of the experimental techniques for γH2AX detection in cultured cells is immunofluorescent labelling of γH2AX and nuclei followed by microscopy imaging and analysis. Results In this study, we present the algorithm FoCo for reliable and robust automatic nuclear foci counting in single cell images. FoCo has the following advantages with respect to other software packages: i) the ability to reliably quantify even densely distributed foci, e.g., on images of cells subjected to radiation doses up to 10 Gy, ii) robustness of foci quantification in the sense of suppressing out-of-focus background signal, and iii) its simplicity. FoCo requires only 5 parameters that have to be adjusted by the user. Conclusions FoCo is an open-source user-friendly software with GUI for individual foci counting, which is able to produce reliable and robust foci quantifications even for low signal/noise ratios and densely distributed foci. Electronic supplementary material The online version of this article (doi:10.1186/s12859-015-0816-5) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Anastasiya Lapytsko
- Medical Faculty, Institute for Experimental Internal Medicine, Otto von Guericke University, Pfälzer Platz 2, 39106, Magdeburg, Germany.
| | - Gabriel Kollarovic
- Medical Faculty, Institute for Experimental Internal Medicine, Otto von Guericke University, Pfälzer Platz 2, 39106, Magdeburg, Germany. .,Cancer Research Institute, Slovak Academy of Sciences, Vlarska 7, 83391, Bratislava, Slovakia.
| | - Lyubomira Ivanova
- Medical Faculty, Institute for Experimental Internal Medicine, Otto von Guericke University, Pfälzer Platz 2, 39106, Magdeburg, Germany.
| | - Maja Studencka
- Medical Faculty, Institute for Experimental Internal Medicine, Otto von Guericke University, Pfälzer Platz 2, 39106, Magdeburg, Germany.
| | - Jörg Schaber
- Medical Faculty, Institute for Experimental Internal Medicine, Otto von Guericke University, Pfälzer Platz 2, 39106, Magdeburg, Germany.
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20
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Perumal V, Sekaran TSG, Raavi V, Basheerudeen SAS, Kanagaraj K, Chowdhury AR, Paul SFD. Radiation signature on exposed cells: Relevance in dose estimation. World J Radiol 2015; 7:266-278. [PMID: 26435777 PMCID: PMC4585950 DOI: 10.4329/wjr.v7.i9.266] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2015] [Revised: 07/03/2015] [Accepted: 08/03/2015] [Indexed: 02/06/2023] Open
Abstract
The radiation is considered as a double edged sword, as its beneficial and detrimental effects have been demonstrated. The potential benefits are being exploited to its maximum by adopting safe handling of radionuclide stipulated by the regulatory agencies. While the occupational workers are monitored by personnel monitoring devices, for general publics, it is not a regular practice. However, it can be achieved by using biomarkers with a potential for the radiation triage and medical management. An ideal biomarker to adopt in those situations should be rapid, specific, sensitive, reproducible, and able to categorize the nature of exposure and could provide a reliable dose estimation irrespective of the time of the exposures. Since cytogenetic markers shown to have many advantages relatively than other markers, the origins of various chromosomal abnormalities induced by ionizing radiations along with dose-response curves generated in the laboratory are presented. Current status of the gold standard dicentric chromosome assay, micronucleus assay, translocation measurement by fluorescence in-situ hybridization and an emerging protein marker the γ-H2AX assay are discussed with our laboratory data. With the wide choice of methods, an appropriate assay can be employed based on the net.
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21
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Siddiqui MS, François M, Fenech MF, Leifert WR. Persistent γH2AX: A promising molecular marker of DNA damage and aging. MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH 2015; 766:1-19. [PMID: 26596544 DOI: 10.1016/j.mrrev.2015.07.001] [Citation(s) in RCA: 167] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Revised: 07/13/2015] [Accepted: 07/14/2015] [Indexed: 12/12/2022]
Abstract
One of the earliest cellular responses to DNA double strand breaks (DSBs) is the phosphorylation of the core histone protein H2AX (termed γH2AX). Persistent γH2AX is the level of γH2AX above baseline, measured at a given time-point beyond which DNA DSBs are normally expected to be repaired (usually persist for days to months). This review summarizes the concept of persistent γH2AX in the context of exogenous source induced DNA DSBs (e.g. ionizing radiation (IR), chemotherapeutic drugs, genotoxic agents), and endogenous γH2AX levels in normal aging and accelerated aging disorders. Summary of the current literature demonstrates the following (i) γH2AX persistence is a common phenomenon that occurs in humans and animals; (ii) nuclei retain persistent γH2AX foci for up to several months after IR exposure, allowing for retrospective biodosimetry; (iii) the combination of various radiosensitizing drugs with ionizing radiation exposure leads to persistent γH2AX response, thus enabling the potential for monitoring cancer patients' response to chemotherapy and radiotherapy as well as tailoring cancer treatments; (iv) persistent γH2AX accumulates in telomeric DNA and in cells undergoing cellular senescence; and (v) increased endogenous γH2AX levels may be associated with diseases of accelerated aging. In summary, measurement of persistent γH2AX could potentially be used as a marker of radiation biodosimetry, evaluating sensitivity to therapeutic genotoxins and radiotherapy, and exploring the association of unrepaired DNA DSBs on telomeres with diseases of accelerated aging.
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Affiliation(s)
- Mohammad Sabbir Siddiqui
- CSIRO Food and Nutrition Flagship, Genome Health and Healthy Aging, Adelaide, South Australia 5000, Australia; University of Adelaide, School of Agriculture, Food & Wine, Urrbrae, South Australia 5064, Australia
| | - Maxime François
- CSIRO Food and Nutrition Flagship, Genome Health and Healthy Aging, Adelaide, South Australia 5000, Australia
| | - Michael F Fenech
- CSIRO Food and Nutrition Flagship, Genome Health and Healthy Aging, Adelaide, South Australia 5000, Australia
| | - Wayne R Leifert
- CSIRO Food and Nutrition Flagship, Genome Health and Healthy Aging, Adelaide, South Australia 5000, Australia.
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22
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Rothkamm K, Barnard S, Moquet J, Ellender M, Rana Z, Burdak-Rothkamm S. DNA damage foci: Meaning and significance. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2015; 56:491-504. [PMID: 25773265 DOI: 10.1002/em.21944] [Citation(s) in RCA: 248] [Impact Index Per Article: 24.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/28/2014] [Accepted: 02/13/2015] [Indexed: 06/04/2023]
Abstract
The discovery of DNA damage response proteins such as γH2AX, ATM, 53BP1, RAD51, and the MRE11/RAD50/NBS1 complex, that accumulate and/or are modified in the vicinity of a chromosomal DNA double-strand break to form microscopically visible, subnuclear foci, has revolutionized the detection of these lesions and has enabled studies of the cellular machinery that contributes to their repair. Double-strand breaks are induced directly by a number of physical and chemical agents, including ionizing radiation and radiomimetic drugs, but can also arise as secondary lesions during replication and DNA repair following exposure to a wide range of genotoxins. Here we aim to review the biological meaning and significance of DNA damage foci, looking specifically at a range of different settings in which such markers of DNA damage and repair are being studied and interpreted.
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Affiliation(s)
- Kai Rothkamm
- Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, United Kingdom
- Department of Radiotherapy, Laboratory of Radiation Biology and Experimental Radiation Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Stephen Barnard
- Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, United Kingdom
| | - Jayne Moquet
- Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, United Kingdom
| | - Michele Ellender
- Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, United Kingdom
| | - Zohaib Rana
- Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, United Kingdom
| | - Susanne Burdak-Rothkamm
- Department of Cellular Pathology, Oxford University Hospitals, Headley Way, Headington, Oxford, United Kingdom
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23
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Kovalchuk IP, Golubov A, Koturbash IV, Kutanzi K, Martin OA, Kovalchuk O. Age-dependent changes in DNA repair in radiation-exposed mice. Radiat Res 2015; 182:683-94. [PMID: 25409128 DOI: 10.1667/rr13697.1] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Ionizing radiation (IR) is a well-known human carcinogen. Young and adult individuals are known to respond to radiation in a different manner. In this study, we analyzed changes in the spleen of juvenile (two-week-old), adult (two-month-old) and old (18-month-old) C57BL/6 male mice subjected to a whole-body exposure to 1 Gy of X rays. We measured the number of γ-H2AX foci and ATM protein levels as a reflection of the level of DNA double-strand breaks (DSBs), and found that old animals had a high frequency of occurrence of noninduced DSBs. Exposure to X rays resulted in a rapid increase in the number of DSBs in juvenile and adult animals at 6 h postirradiation followed by a return to preirradiated DSB values at 96 h postirradiation. No changes were observed in old animals. The analysis of the levels of proteins involved in DNA damage base excision and mismatch repair pathways, including KU70, RAD51, POL β, POL δ, POL ε, APE1 and MSH2 showed substantial age-dependent radiation-induced differences. Finally, we demonstrated that old animals had a higher background level of cell apoptosis compared to younger animals, but in contrast to younger animals, old animals were not able to commit spleen cells to apoptosis after being irradiated. Thus, spleen cells of old mice have a high level of spontaneous DNA damage, but they are not able to deal with additional radiation-induced damage as efficiently as younger animals, substantiating age-depending differences in radiation-induced DNA damage and repair response and its outcomes.
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Affiliation(s)
- Igor P Kovalchuk
- a Department of Biological Sciences, University of Lethbridge, Lethbridge, Alberta, Canada
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Turner HC, Sharma P, Perrier JR, Bertucci A, Smilenov L, Johnson G, Taveras M, Brenner DJ, Garty G. The RABiT: high-throughput technology for assessing global DSB repair. RADIATION AND ENVIRONMENTAL BIOPHYSICS 2014; 53:265-72. [PMID: 24477408 PMCID: PMC3999265 DOI: 10.1007/s00411-014-0514-0] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/02/2013] [Accepted: 01/14/2014] [Indexed: 05/19/2023]
Abstract
At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a rapid automated biodosimetry tool (RABiT); this is a completely automated, ultra-high-throughput robotically based biodosimetry workstation designed for use following a large-scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high-speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time-dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7 and 24 h) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker.
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Affiliation(s)
- Helen C Turner
- Department of Radiation Oncology, Center for Radiological Research, Columbia University Medical Center, 630 W. 168th St. VC11-240, New York, NY, 10032, USA,
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Lyulko OV, Garty G, Randers-Pehrson G, Turner HC, Szolc B, Brenner DJ. Fast image analysis for the micronucleus assay in a fully automated high-throughput biodosimetry system. Radiat Res 2014; 181:146-61. [PMID: 24502354 PMCID: PMC4011502 DOI: 10.1667/rr13441.1] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The development of, and results from an image analysis system are presented for automated detection and scoring of micronuclei in human peripheral blood lymphocytes. The system is part of the Rapid Automated Biodosimetry Tool, which was developed at the Center for High-Throughput Minimally Invasive Radiation Biodosimetry for rapid radiation dose assessment of many individuals based on single fingerstick samples of blood. Blood lymphocytes were subjected to the cytokinesis-block micronucleus assay and the images of cell cytoplasm and nuclei are analyzed to estimate the frequency of micronuclei in binucleated cells. We describe an algorithm that is based on dual fluorescent labeling of lymphocytes with separate analysis of images of cytoplasm and nuclei. To evaluate the performance of the system, blood samples of seven healthy donors were irradiated in vitro with doses from 0-10 Gy and dose-response curves of micronuclei frequencies were generated. To establish the applicability of the system to the detection of high doses, the ratios of mononucleated cells to binucleated cells were determined for three of the donors. All of the dose-response curves generated automatically showed clear dose dependence and good correlation (R(2) from 0.914-0.998) with the results of manual scoring.
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Affiliation(s)
- Oleksandra V. Lyulko
- Center for Radiological Research, Columbia University Medical Center, New York, New York
| | - Guy Garty
- Center for Radiological Research, Columbia University Medical Center, New York, New York
| | | | - Helen C. Turner
- Center for Radiological Research, Columbia University Medical Center, New York, New York
| | - Barbara Szolc
- Center for Radiological Research, Columbia University Medical Center, New York, New York
- David B. Kriser Dental Center, New York University College of Dentistry, New York, New York
| | - David J. Brenner
- Center for Radiological Research, Columbia University Medical Center, New York, New York
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Evaluation of the gamma-H2AX assay for radiation biodosimetry in a swine model. Int J Mol Sci 2013; 14:14119-35. [PMID: 23880859 PMCID: PMC3742235 DOI: 10.3390/ijms140714119] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2013] [Revised: 06/18/2013] [Accepted: 06/25/2013] [Indexed: 02/07/2023] Open
Abstract
There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo γ-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans.
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Abstract
Most methods for examining telomere functionality have relied on measurements of telomeric DNA by hybridization or quantitative PCR. While these techniques yield measures of telomeric DNA length, they generate whole-population results. However, telomeric DNA lengths on different chromatids even in the same cell are usually heterogeneous. Also, these measurements do not reveal whether a particular telomere contains the critical minimum DNA length to be functional. Therefore, in order to gain a more complete knowledge of cellular health, an alternative method that reveals the functional status of each individual telomere is needed. Based on the fact that a dysfunctional telomere induces a DNA damage response, we developed a novel technique which combines a DNA damage marker with fluorescence in situ hybridization (FISH) of telomeric DNA on metaphase chromosomes to assess the functional status of individual telomeres. This technique reveals not only whether the telomeric DNA in each chromatid is significantly shortened, but also whether the telomere has induced a DNA damage response, i.e., has become dysfunctional. We describe here in detail the protocols for simultaneous assessment of telomere length and functionality.
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Affiliation(s)
- Asako J Nakamura
- Department of Anatomy and Cell Biology, Osaka Medical College, Takatsuki, Japan.
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Cornelissen B, Darbar S, Kersemans V, Allen D, Falzone N, Barbeau J, Smart S, Vallis KA. Amplification of DNA damage by a γH2AX-targeted radiopharmaceutical. Nucl Med Biol 2012; 39:1142-51. [PMID: 22819196 DOI: 10.1016/j.nucmedbio.2012.06.001] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2012] [Revised: 05/11/2012] [Accepted: 06/02/2012] [Indexed: 01/07/2023]
Abstract
UNLABELLED (111)In-DTPA-anti-γH2AX-Tat, which combines an anti-γH2AX antibody with a cell-penetrating peptide, Tat, and the Auger electron-emitting radioisotope, (111)In, targets the DNA damage signalling protein, γH2AX, and has potential as a probe for imaging DNA damage in vivo. The goal of this study was to investigate whether (111)In-DTPA-anti-γH2AX-Tat labelled to high specific activity (6MBq/μg) can amplify treatment-related DNA damage for therapeutic gain. METHODS MDA-MB-468 and MDA-MB-231/H2N (231-H2N) breast cancer cells were incubated with (111)In-DTPA-anti-γH2AX-Tat (3MBq, 6MBq/μg) or a control radioimmunoconjugate, (111)In-DTPA-mIgG-Tat, and exposed to IR or bleomycin. DNA damage was studied by counting γH2AX foci and by neutral comet assay. Cytotoxicity was evaluated using clonogenic assays. (111)In-DTPA-anti-γH2AX-Tat was administered intravenously to 231-H2N-xenograft-bearing Balb/c nu/nu mice in tumor growth inhibition studies. RESULTS The number of γH2AX foci was greater after exposure of cells to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat compared to IR alone (20.6±2.5 versus 10.4±2.3 foci/cell; P<.001).(111)In-DTPA-anti-γH2AX-Tat resulted in a reduced surviving fraction in cells co-treated with IR (4Gy) versus IR alone (5.2%±0.9% versus 47.8%±2.8%; P<.001). Similarly, bleomycin (25-200μg/mL) plus (111)In-DTPA-anti-γH2AX-Tat resulted in a lower SF compared to bleomycin alone. The combination of a single exposure to IR (10Gy) plus (111)In-DTPA-anti-γH2AX-Tat significantly decreased the growth rate of 231-H2N xenografts in vivo compared to either (111)In-DTPA-anti-γH2AX-Tat or IR alone (-0.002±0.004 versus 0.036±0.011 and 0.031±0.014mm(3)/day, respectively, P<.001). CONCLUSION (111)In-DTPA-anti-γH2AX-Tat amplifies anticancer treatment-related DNA damage in vitro and has a potent anti-tumor effect when combined with IR in vivo.
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Affiliation(s)
- Bart Cornelissen
- Department of Oncology, CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, OX3 7LJ Oxford, UK
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Garcia-Villa A, Balasubramanian P, Miller BL, Lustberg MB, Ramaswamy B, Chalmers JJ. Assessment of γ-H2AX levels in circulating tumor cells from patients receiving chemotherapy. Front Oncol 2012; 2:128. [PMID: 23112954 PMCID: PMC3480704 DOI: 10.3389/fonc.2012.00128] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2012] [Accepted: 09/12/2012] [Indexed: 11/13/2022] Open
Abstract
Circulating tumor cells (CTCs) are prognostic markers in a variety of solid tumor malignancies. The potential of CTCs to be used as a “liquid biopsy” to monitor a patient’s condition and predict drug response and resistance is currently under investigation. Using a negative depletion, enrichment methodology, CTCs isolated from the peripheral blood of breast cancer patients with stage IV breast cancer undergoing DNA damaging therapy with platinum-based therapy were enriched. The enriched cell suspensions were stained with an optimized labeling protocol targeting: nuclei, cytokeratins 8, 18, and 19, the surface marker CD45, and the presence of the protein γ-H2AX. As a direct or indirect result of platinum therapy, double-strand break of DNA initiates phosphorylation of the histone H2AX, at serine 139; this phosphorylated form is referred to as γ-H2AX. In addition to γ-H2AX staining in specific locations with the cell nuclei, consistent with previous reports and referred to as foci, more general staining in the cell cytoplasm was also observed in some cells suggesting the potential of cell apoptosis. Our study underscores the utility and the complexity of investigating CTCs as predictive markers of response to various therapies. Additional studies are ongoing to evaluate the diverse γ-H2AX staining patterns we report here which needs to be further correlated with patient outcomes.
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Affiliation(s)
- Alejandra Garcia-Villa
- William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University Columbus, OH, USA
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Gandhi M, Evdokimova VN, T.Cuenco K, Nikiforova MN, Kelly LM, Stringer JR, Bakkenist CJ, Nikiforov YE. Homologous chromosomes make contact at the sites of double-strand breaks in genes in somatic G0/G1-phase human cells. Proc Natl Acad Sci U S A 2012; 109:9454-9. [PMID: 22645362 PMCID: PMC3386068 DOI: 10.1073/pnas.1205759109] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.
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Affiliation(s)
- Manoj Gandhi
- Department of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Viktoria N. Evdokimova
- Department of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Karen T.Cuenco
- Department of Oral Biology, School of Dental Medicine and
- Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15219
| | - Marina N. Nikiforova
- Department of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - Lindsey M. Kelly
- Department of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - James R. Stringer
- Department of Molecular Genetics, University of Cincinnati, Cincinnati, OH 45267; and
| | - Christopher J. Bakkenist
- Department of Molecular Genetics, University of Cincinnati, Cincinnati, OH 45267; and
- Departments of Radiation Oncology and Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213
| | - Yuri E. Nikiforov
- Department of Pathology and Laboratory Medicine, University of Pittsburgh, Pittsburgh, PA 15213
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Cornelissen B, Waller A, Target C, Kersemans V, Smart S, Vallis KA. 111In-BnDTPA-F3: an Auger electron-emitting radiotherapeutic agent that targets nucleolin. EJNMMI Res 2012; 2:9. [PMID: 22348532 PMCID: PMC3298710 DOI: 10.1186/2191-219x-2-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2011] [Accepted: 02/20/2012] [Indexed: 11/10/2022] Open
Abstract
INTRODUCTION The F3 peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK), a fragment of the human high mobility group protein 2, binds nucleolin. Nucleolin is expressed in the nuclei of normal cells but is also expressed on the membrane of some cancer cells. The goal was to investigate the use of 111In-labeled F3 peptide for Auger electron-targeted radiotherapy. METHODS F3 was labeled with fluorescein isothiocyanate (FITC) for confocal microscopy and conjugated to p-SCN-benzyl-diethylenetriaminepentaacetic acid (BnDTPA) for labeling with 111In to form 111In-BnDTPA-F3. MDA-MB-231-H2N (231-H2N) human breast cancer cells were exposed to 111In-BnDTPA-F3 and used in cell fractionation, γH2AX immunostaining (a marker of DNA double-strand breaks), and clonogenic assays. In vivo, biodistribution studies of 111In-BnDTPA-F3 were performed in 231-H2N xenograft-bearing mice. In tumor growth delay studies, 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) was administered intravenously to 231-H2N xenograft-bearing mice once weekly for 3 weeks. RESULTS Membrane-binding of FITC-F3 was observed in 231-H2N cells, and there was co-localization of FITC-F3 with nucleolin in the nuclei. After exposure of 231-H2N cells to 111In-BnDTPA-F3 for 2 h, 1.7% of 111In added to the medium was membrane-bound. Of the bound 111In, 15% was internalized, and of this, 37% was localized in the nucleus. Exposure of 231-H2N cells to 111In-BnDTPA-F3 (1 μM, 6 MBq/μg) resulted in a dose-dependent increase in γH2AX foci and in a significant reduction of clonogenic survival compared to untreated cells or cells exposed to unlabeled BnDTPA-F3 (46 ± 4.1%, 100 ± 1.8%, and 132 ± 7.7%, respectively). In vivo, tumor uptake of 111In-BnDTPA-F3 (3 μg, 6 MBq/μg) at 3-h post-injection was 1% of the injected dose per gram (%ID/g), and muscle uptake was 0.5%ID/g. In tumor growth delay studies, tumor growth rate was reduced 19-fold compared to untreated or unlabeled BnDTPA-F3-treated mice (p = 0.023). CONCLUSION 111In-BnDTPA-F3 is internalized into 231-H2N cells and translocates to the nucleus. 111In-BnDTPA-F3 has a potent cytotoxic effect in vitro and an anti-tumor effect in mice bearing 231-H2N xenografts despite modest total tumor accumulation.
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Affiliation(s)
- Bart Cornelissen
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Andrew Waller
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Carol Target
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Veerle Kersemans
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Sean Smart
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
| | - Katherine A Vallis
- Department of Oncology, Cancer Research UK/Medical Research Council Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ, UK
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Jakob B, Durante M. Radiation dose detection by imaging response in biological targets. Radiat Res 2012; 177:524-32. [PMID: 22332929 DOI: 10.1667/rr2452.1] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
Imaging was one of the earliest techniques to quantify radiation dose. While films and active fluorescent detectors are still commonly used in physical dosimetry, biological imaging is emerging as a new method to visualize and quantify radiation dose in biological targets. Methods for biological imaging are normally based on molecular fluorescent probes, labeling chromatin-conjugated molecules or specific repair proteins. Examples are chromatin-binding coumarin compounds, which become fluorescent under irradiation, or the H2AX histone, which is rapidly phosphorylated at sites of DNA double-strand breaks and can be visualized by immunostaining. Many other DNA repair proteins can be expressed with fluorescent targets, such as green fluorescent protein, thus becoming visible for dose estimation in vivo. The possibility to visualize radiation damage in living biological targets is particularly important for repair kinetic studies, for estimating individual radiation response, and for remote control of living samples exposed to radiation, for instance in robotic space missions. In vivo dose monitoring in particle therapy exploits the production of positron emitters by nuclear interaction of the incident beam in the patient's body. Positron emission tomography (PET) can then be used to visualize and quantify the particle dose in the patient, and it can in principle also be used for radiotherapy with high-energy X rays. Alternatively, prompt γ rays or scattered secondary particles are under study for in vivo dosimetry of ion beams in therapy.
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Affiliation(s)
- B Jakob
- GSI Helmholtzzentrum für Schwerionenforschung, Biophysics Department, Planckstraße 1, 64291 Darmstadt, Germany
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Abstract
The DNA damage response can be initiated in response to a variety of stress signals that are encountered during physiological processes or in response to exogenous cues, such as ionizing radiation or DNA-damaging therapeutic agents. A number of methods have been developed to examine the morphological, biochemical, and molecular changes that take place during the DNA damage response. When cells are exposed to ionizing radiation or DNA-damaging chemotherapeutic agents, double-stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of histone H2A variant H2AX. Because phosphorylation of H2AX at Ser 139 (γ-H2AX) is abundant, fast, and correlates well with each DSB, it is the most sensitive marker that can be used to examine the DNA damage produced and the subsequent repair of the DNA lesion. γ-H2AX can be detected by immunoblotting and immunostaining using microscopic or flow cytometric detection. Since γ-H2AX can be also generated during DNA replication, as a consequence of apoptosis, or as it is found associated with residual DNA damage, it is important to determine the kinetics, number, size, and morphology of γ-H2AX-associated foci. This chapter describes a few standard protocols that we have successfully used in our laboratory for a number of experimental systems, primarily hematologic and epithelial cells grown in culture.
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Cornelissen B, Kersemans V, Darbar S, Thompson J, Shah K, Sleeth K, Hill MA, Vallis KA. Imaging DNA damage in vivo using gammaH2AX-targeted immunoconjugates. Cancer Res 2011; 71:4539-49. [PMID: 21586614 PMCID: PMC3130133 DOI: 10.1158/0008-5472.can-10-4587] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.
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Affiliation(s)
- Bart Cornelissen
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Veerle Kersemans
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Sonali Darbar
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - James Thompson
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Ketan Shah
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Kate Sleeth
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Mark A. Hill
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
| | - Katherine A. Vallis
- CR-UK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom
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Ivashkevich AN, Martin OA, Smith AJ, Redon CE, Bonner WM, Martin RF, Lobachevsky PN. γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis. Mutat Res 2011; 711:49-60. [PMID: 21216255 PMCID: PMC3101310 DOI: 10.1016/j.mrfmmm.2010.12.015] [Citation(s) in RCA: 101] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2010] [Revised: 12/17/2010] [Accepted: 12/31/2010] [Indexed: 11/26/2022]
Abstract
The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.
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Affiliation(s)
- Alesia N. Ivashkevich
- Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Melbourne, Victoria, 3002, Australia
| | - Olga A. Martin
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, D.H.H.S., Bethesda, MD 20892, USA
| | - Andrea J. Smith
- Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Melbourne, Victoria, 3002, Australia
| | - Christophe E. Redon
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, D.H.H.S., Bethesda, MD 20892, USA
| | - William M. Bonner
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, D.H.H.S., Bethesda, MD 20892, USA
| | - Roger F. Martin
- Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Melbourne, Victoria, 3002, Australia
| | - Pavel N. Lobachevsky
- Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Melbourne, Victoria, 3002, Australia
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Nishii M, Nakano S, Nakamura S, Wate R, Shinde A, Kaneko S, Kusaka H. Myonuclear breakdown in sporadic inclusion body myositis is accompanied by DNA double strand breaks. Neuromuscul Disord 2011; 21:345-52. [PMID: 21353553 DOI: 10.1016/j.nmd.2011.02.004] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2010] [Revised: 01/18/2011] [Accepted: 02/02/2011] [Indexed: 12/27/2022]
Abstract
Rimmed vacuoles in sporadic inclusion body myositis (s-IBM) contain nuclear remnants. We sought to determine if the nuclear degeneration seen in s-IBM is associated with DNA damage. In muscle biopsy specimens from ten patients with s-IBM and 50 controls, we immunolocalized 1) phosphorylated histone H2AX (γ-H2AX), which is a sensitive immunocytochemical marker of DNA double-strand breaks and 2) DNA-PK, which is an enzyme involved in double-strand break repair. In s-IBM, vacuolar peripheries often showed strong immunoreactivity to γ-H2AX and the three components of DNA-PK (DNA-PKcs, Ku70, and Ku80). A triple fluorescence study of Ku70, emerin, and DNA displayed nuclear breakdown and it suggested impaired nuclear incorporation of Ku70. The percentage of positive nuclei for γ-H2AX was significantly higher in vacuolated fibers than non-vacuolated fibers in s-IBM, or fibers in polymyosits. We hypothesize that a dysfunction of nuclear envelope may cause nuclear fragility, double-strand breaks and impaired nuclear transport in s-IBM.
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Affiliation(s)
- Makoto Nishii
- Department of Neurology and Brain Medical Research Center, Kansai Medical University, Japan
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37
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Paris L, Cordelli E, Eleuteri P, Grollino MG, Pasquali E, Ranaldi R, Meschini R, Pacchierotti F. Kinetics of γ-H2AX induction and removal in bone marrow and testicular cells of mice after X-ray irradiation. Mutagenesis 2011; 26:563-72. [DOI: 10.1093/mutage/ger017] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
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Löffler H, Fechter A, Matuszewska M, Saffrich R, Mistrik M, Marhold J, Hornung C, Westermann F, Bartek J, Krämer A. Cep63 Recruits Cdk1 to the Centrosome: Implications for Regulation of Mitotic Entry, Centrosome Amplification, and Genome Maintenance. Cancer Res 2011; 71:2129-39. [DOI: 10.1158/0008-5472.can-10-2684] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Turner HC, Brenner DJ, Chen Y, Bertucci A, Zhang J, Wang H, Lyulko OV, Xu Y, Shuryak I, Schaefer J, Simaan N, Randers-Pehrson G, Yao YL, Amundson SA, Garty G. Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects. Radiat Res 2011; 175:282-90. [PMID: 21388271 PMCID: PMC3121903 DOI: 10.1667/rr2125.1] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.
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Affiliation(s)
- Helen C Turner
- Center for Radiological Research, Columbia University Medical Center, New York, New York 10032, USA.
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Radioprotective effects of Bmi-1 involve epigenetic silencing of oxidase genes and enhanced DNA repair in normal human keratinocytes. J Invest Dermatol 2011; 131:1216-25. [PMID: 21307872 DOI: 10.1038/jid.2011.11] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Normal human keratinocytes (NHKs) undergo premature senescence following exposure to ionizing radiation (IR). This study investigates the effect of Bmi-1, a polycomb group protein, on radiation-induced senescence response. When exposed to IR, NHK transduced with Bmi-1 (NHK/Bmi-1) showed reduced senescent phenotype and enhanced proliferation compared with control cells (NHK/B0). To investigate the underlying mechanism, we determined the production of reactive oxygen species (ROS), expression of ROS-generating enzymes, and DNA repair activities in cells. ROS level was increased upon irradiation but notably reduced by Bmi-1 transduction. Irradiation led to strong induction of oxidase genes, e.g., Lpo (lactoperoxidase), p22-phox, p47-phox, and Gp91, in NHK/B0 but their expression was almost completely silenced in NHK/Bmi-1. Induction of oxidase genes upon irradiation was linked with loss of trimethylated histone 3 at lysine 27 (H3K27Me3), but NHK/Bmi-1 expressed a higher level of H3K27Me3 compared with NHK/B0. Bmi-1 transduction suppressed IR-associated induction of jumanji domain containing 3 while enhancing the expression of EZH2, thereby preventing the loss of H3K27Me3 in the irradiated cells. Furthermore, NHK/Bmi-1 demonstrated increased repair of IR-induced DNA damage compared with NHK/B0. These results indicate that Bmi-1 elicits radioprotective effects on NHK by mitigating the genotoxicity of IR through epigenetic mechanisms.
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γ-H2AX detection in peripheral blood lymphocytes, splenocytes, bone marrow, xenografts, and skin. Methods Mol Biol 2011; 682:249-70. [PMID: 21057933 DOI: 10.1007/978-1-60327-409-8_18] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Measurement of DNA double-strand break (DSB) levels in cells is useful in many research areas, including those related to DNA damage and repair, tumorigenesis, anti-cancer drug development, apoptosis, radiobiology, environmental effects, and aging, as well as in the clinic. DSBs can be detected in the nuclei of cultured cells and tissues with an antibody to H2AX phosphorylated on serine residue 139 (γ-H2AX). DSB levels can be obtained either by measuring overall γ-H2AX protein levels in a cell population or by counting γ-H2AX foci in individual nuclei. Total levels can be obtained in extracts of cell populations by immunoblot analysis, and in cell populations by flow cytometry. Furthermore, with flow cytometry, the cell cycle distribution of a population can be obtained in addition to DSB levels, which is an advantage when studying anti-cancer drugs targeting replicating tumor cells. These described methods are used in genotoxicity assays of compounds of interest or in analyzing DSB repair after exposure to drugs or radiation. Immunocyto/immunohistochemical analysis can detect γ-H2AX foci in individual cells and is very sensitive (a single DSB can be visualized), permitting the use of extremely small samples. Measurements of γ-H2AX focal numbers can reveal subtle changes found in the radiation-induced tissue bystander response, low dose radiation exposure, and in cells with mutations in genomic stability maintenance pathways. In addition, marking DNA DSBs in a nucleus with γ-H2AX is a powerful tool to identify novel DNA repair proteins by their abilities to co-localize with γ-H2AX foci at the DSB site. This chapter presents techniques for γ-H2AX detection in a variety of human and mouse samples.
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Protein phosphatase 5 is necessary for ATR-mediated DNA repair. Biochem Biophys Res Commun 2011; 404:476-81. [DOI: 10.1016/j.bbrc.2010.12.005] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2010] [Accepted: 12/02/2010] [Indexed: 11/30/2022]
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Kinders RJ, Hollingshead M, Lawrence S, Ji J, Tabb B, Bonner WM, Pommier Y, Rubinstein L, Evrard YA, Parchment RE, Tomaszewski J, Doroshow JH, National Cancer Institute Phase 0 Clinical Trials Team. Development of a validated immunofluorescence assay for γH2AX as a pharmacodynamic marker of topoisomerase I inhibitor activity. Clin Cancer Res 2010; 16:5447-57. [PMID: 20924131 PMCID: PMC2982895 DOI: 10.1158/1078-0432.ccr-09-3076] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Phosphorylated histone H2AX (γH2AX) serves as a biomarker for formation of DNA double-strand break repair complexes. A quantitative pharmacodynamic immunofluorescence assay for γH2AX was developed, validated, and tested in human tumor xenograft models with the use of clinically relevant procedures. EXPERIMENTAL DESIGN The γH2AX immunofluorescence assay uses a novel data quantitation and image processing algorithm to determine the extent of nuclear-specific γH2AX staining in tumor needle biopsies and hair follicles collected from mice bearing topotecan-responsive A375 xenografts. After method validation with the topoisomerase I (Top1) inhibitor topotecan, the assay was used to compare pharmacodynamic properties of three structurally related indenoisoquinoline Top1 inhibitors. RESULTS γH2AX response to topotecan was quantified over a 60-fold dose range (0.016-1.0 times the murine single-dose maximum tolerated dose), and significant pharmacodynamic response was measured at the mouse equivalent of the 1.5 mg/m(2) clinical dose as well as the lowest dose tested. Responses were within a time window amenable for biopsy collection in clinical trials. These studies enabled characterization of dose and time responses for three indenoisoquinolines, resulting in selection of two for clinical evaluation. γH2AX response to Top1 inhibitors in hair follicles was also observable above a minimal dose threshold. CONCLUSIONS Our γH2AX assay is sufficiently accurate and sensitive to quantify γH2AX in tumor samples and will be used in correlative studies of two indenoisoquinolines in a phase I clinical trial at the National Cancer Institute. Data suggest that hair follicles may potentially serve as a surrogate tissue to evaluate tumor γH2AX response to Top1 inhibitors.
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Affiliation(s)
- Robert J Kinders
- Laboratory of Human Toxicology and Pharmacology, Applied/Developmental Research Support Directorate, Science Applications International Corporation-Frederick, Inc., NationalCancer Institute-Frederick, Frederick, MD 21702, USA.
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Giunta S, Belotserkovskaya R, Jackson SP. DNA damage signaling in response to double-strand breaks during mitosis. J Cell Biol 2010; 190:197-207. [PMID: 20660628 PMCID: PMC2930281 DOI: 10.1083/jcb.200911156] [Citation(s) in RCA: 255] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2009] [Accepted: 06/22/2010] [Indexed: 01/11/2023] Open
Abstract
The signaling cascade initiated in response to DNA double-strand breaks (DSBs) has been extensively investigated in interphase cells. Here, we show that mitotic cells treated with DSB-inducing agents activate a "primary" DNA damage response (DDR) comprised of early signaling events, including activation of the protein kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), histone H2AX phosphorylation together with recruitment of mediator of DNA damage checkpoint 1 (MDC1), and the Mre11-Rad50-Nbs1 (MRN) complex to damage sites. However, mitotic cells display no detectable recruitment of the E3 ubiquitin ligases RNF8 and RNF168, or accumulation of 53BP1 and BRCA1, at DSB sites. Accordingly, we found that DNA-damage signaling is attenuated in mitotic cells, with full DDR activation only ensuing when a DSB-containing mitotic cell enters G1. Finally, we present data suggesting that induction of a primary DDR in mitosis is important because transient inactivation of ATM and DNA-PK renders mitotic cells hypersensitive to DSB-inducing agents.
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Affiliation(s)
- Simona Giunta
- Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Biochemistry, and Department of Zoology, University of Cambridge, CB2 1QN Cambridge, England, UK
| | - Rimma Belotserkovskaya
- Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Biochemistry, and Department of Zoology, University of Cambridge, CB2 1QN Cambridge, England, UK
| | - Stephen P. Jackson
- Wellcome Trust and Cancer Research UK Gurdon Institute, Department of Biochemistry, and Department of Zoology, University of Cambridge, CB2 1QN Cambridge, England, UK
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45
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Sedelnikova OA, Redon CE, Dickey JS, Nakamura AJ, Georgakilas AG, Bonner WM. Role of oxidatively induced DNA lesions in human pathogenesis. Mutat Res 2010; 704:152-9. [PMID: 20060490 PMCID: PMC3074954 DOI: 10.1016/j.mrrev.2009.12.005] [Citation(s) in RCA: 257] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2009] [Revised: 12/08/2009] [Accepted: 12/14/2009] [Indexed: 02/06/2023]
Abstract
Genome stability is essential for maintaining cellular and organismal homeostasis, but it is subject to many threats. One ubiquitous threat is from a class of compounds known as reactive oxygen species (ROS), which can indiscriminately react with many cellular biomolecules including proteins, lipids, and DNA to produce a variety of oxidative lesions. These DNA oxidation products are a direct risk to genome stability, and of particular importance are oxidative clustered DNA lesions (OCDLs), defined as two or more oxidative lesions present within 10 bp of each other. ROS can be produced by exposure of cells to exogenous environmental agents including ionizing radiation, light, chemicals, and metals. In addition, they are produced by cellular metabolism including mitochondrial ATP generation. However, ROS also serve a variety of critical cellular functions and optimal ROS levels are maintained by multiple cellular antioxidant defenses. Oxidative DNA lesions can be efficiently repaired by base excision repair or nucleotide excision repair. If ROS levels increase beyond the capacity of its antioxidant defenses, the cell's DNA repair capacity can become overwhelmed, leading to the accumulation of oxidative DNA damage products including OCDLs, which are more difficult to repair than individual isolated DNA damage products. Here we focus on the induction and repair of OCDLs and other oxidatively induced DNA lesions. If unrepaired, these lesions can lead to the formation of mutations, DNA DSBs, and chromosome abnormalities. We discuss the roles of these lesions in human pathologies including aging and cancer, and in bystander effects.
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Affiliation(s)
- Olga A Sedelnikova
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, MD 20892, USA.
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46
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Cogan N, Baird DM, Phillips R, Crompton LA, Caldwell MA, Rubio MA, Newson R, Lyng F, Case CP. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase. Mutat Res 2010; 683:1-8. [PMID: 19800897 DOI: 10.1016/j.mrfmmm.2009.09.012] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2009] [Revised: 09/23/2009] [Accepted: 09/24/2009] [Indexed: 01/29/2023]
Abstract
The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.
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Affiliation(s)
- Nicola Cogan
- Bristol Implant Research Centre, University of Bristol, Bristol, UK
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47
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Tsaousi A, Jones E, Case CP. The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2010; 697:1-9. [DOI: 10.1016/j.mrgentox.2010.01.012] [Citation(s) in RCA: 86] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/23/2009] [Revised: 10/28/2009] [Accepted: 12/22/2009] [Indexed: 12/28/2022]
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48
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Garty G, Chen Y, Salerno A, Turner H, Zhang J, Lyulko O, Bertucci A, Xu Y, Wang H, Simaan N, Randers-Pehrson G, Yao YL, Amundson SA, Brenner DJ. The RABIT: a rapid automated biodosimetry tool for radiological triage. HEALTH PHYSICS 2010; 98:209-17. [PMID: 20065685 PMCID: PMC2923588 DOI: 10.1097/hp.0b013e3181ab3cb6] [Citation(s) in RCA: 91] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
In response to the recognized need for high throughput biodosimetry methods for use after large-scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. The authors describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or gamma-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in situ analysis in multi-well plates, implementation of robotic-plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here the authors describe the development of each of the individual modules of the RABIT system and show preliminary data from key modules. System integration is ongoing, followed by calibration and validation.
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Affiliation(s)
- Guy Garty
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - Youhua Chen
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | - Alessio Salerno
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | - Helen Turner
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - Jian Zhang
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | - Oleksandra Lyulko
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - Antonella Bertucci
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - Yanping Xu
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - Hongliang Wang
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | - Nabil Simaan
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | | | - Y. Lawrence Yao
- Department of Mechanical Engineering, Columbia University, New York, NY 10027
| | - Sally A. Amundson
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
| | - David J. Brenner
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032
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Nakamura AJ, Rao VA, Pommier Y, Bonner WM. The complexity of phosphorylated H2AX foci formation and DNA repair assembly at DNA double-strand breaks. Cell Cycle 2010; 9:389-97. [PMID: 20046100 PMCID: PMC3086803 DOI: 10.4161/cc.9.2.10475] [Citation(s) in RCA: 111] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The maintenance of genome stability requires efficient DNA double-stranded break (DSB) repair mediated by the phosphorylation of multiple histone H2AX molecules near the break sites. The phosphorylated H2AX (gammaH2AX) molecules form foci covering many megabases of chromatin. The formation of gamma-H2AX foci is critical for efficient DNA damage response (DDR) and for the maintenance of genome stability, however, the mechanisms of protein organization in foci is largely unknown. To investigate the nature of gammaH2AX foci formation, we analyzed the distribution of gammaH2AX and other DDR proteins at DSB sites using a variety of techniques to visualize, expand and partially disrupt chromatin. We report here that gammaH2AX foci change composition during the cell cycle, with proteins 53BP1, NBS1 and MRE11 dissociating from foci in G(2) and mitosis to return at the beginning of the following G(1). In contrast, MDC1 remained colocalized with gamma-H2AX during mitosis. In addition, while gammaH2AX was found to span large domains flanking DSB sites, 53BP1 and NBS1 were more localized and MDC1 colocalized in doublets in foci. H2AX and MDC1 were found to be involved in chromatin relaxation after DSB formation. Our data demonstrates that the DSB repair focus is a heterogeneous and dynamic structure containing internal complexity.
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Affiliation(s)
- Asako J Nakamura
- Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
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50
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Janke R, Herzberg K, Rolfsmeier M, Mar J, Bashkirov VI, Haghnazari E, Cantin G, Yates JR, Heyer WD. A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae. Nucleic Acids Res 2010; 38:2302-13. [PMID: 20061370 PMCID: PMC2853130 DOI: 10.1093/nar/gkp1222] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55–S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.
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Affiliation(s)
- Ryan Janke
- Department of Microbiology, University of California, Davis, CA 95616-8665, USA
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