Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

Scherthan, Harry; Lee, Jinho; Maus, Emanuel; Schumann, Sarah; Muhtadi, Razan; Chojowski, Robert; Port, Matthias; Laßmann, Michael; Bestvater, Felix; Hausmann, Michael

doi:10.3390/cancers11121877

Cited by 30 publications

(51 citation statements)

References 81 publications

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“…These structures are best described as "closely interspaced DSBs", leading to increased biological effectiveness when not properly repaired [23,40]. Indeed, super-resolution techniques previously uncovered "nanoclusters" in previously thought single DSB foci [37]. Closely interspaced DSBs would explain why we observed a relatively low number of 53BP1 and RPA foci, which have in turn high intensity.…”

Section: Discussionmentioning

confidence: 68%

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Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

Roobol

Bent

Cappellen

et al. 2020

IJMS

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High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.

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Section: Discussionmentioning

confidence: 68%

“…Clustered or complex DNA damage is suggested to be an important cause of the increased biological effectiveness of high-LET radiation, and both terms are used to describe multiple types of DNA damage in close vicinity [17,[37][38][39]. We observed multiple independent resection events, marked by RPA, localizing to single 53BP1 foci.…”

Section: Discussionmentioning

confidence: 79%

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

Roobol

Bent

Cappellen

et al. 2020

IJMS

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“…Single molecule microscopy reveals the structure of fluorescent DSB foci, as the accumulation of hundreds of nano-foci [104]. Some authors use the term super-foci in order to distinguish conventional microscopy foci from nano-foci [131]. At the nano-foci level, algorithms can distinguish different groups, such as (a) cluster core points (having many neighbors), (b) outliers (reachable from core points but with few neighbors), and (c) noise.…”

Section: Clustering Analysismentioning

confidence: 99%

“…An example of such algorithm is DBSCAN (density based spatial clustering of applications with noise) [132], also implemented in ImageJ as part of the BioVoxxel Toolbox (http://www.biovoxxel.de). Super resolution imaging of DSB or complex DNA lesions utilizing DBSCAN algorithm for (γH2AX) cluster recognition/cluster classification can be found in [111,112,131].…”

Section: Clustering Analysismentioning

confidence: 99%

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

Nikitaki

Pariset

Sudar

et al. 2020

Cancers

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Complexity of DNA damage is considered currently one if not the primary instigator of biological responses and determinant of short and long-term effects in organisms and their offspring. In this review, we focus on the detection of complex (clustered) DNA damage (CDD) induced for example by ionizing radiation (IR) and in some cases by high oxidative stress. We perform a short historical perspective in the field, emphasizing the microscopy-based techniques and methodologies for the detection of CDD at the cellular level. We extend this analysis on the pertaining methodology of surrogate protein markers of CDD (foci) colocalization and provide a unique synthesis of imaging parameters, software, and different types of microscopy used. Last but not least, we critically discuss the main advances and necessary future direction for the better detection of CDD, with important outcomes in biological and clinical setups.

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“…While flow cytometry offers fast automated quantification of integrated values of these repair signals in high cell numbers [44], microscopy allows detection of individual IRIFs in the context of their natural chromatin environment in individual cells and analysis of their properties development in time [34,45,46]. Characterization of morphological and behavioral parameters of IRIFs formed by repair proteins participating in different DSB repair pathways [47]-such as 53BP1 and γH2AX, which were 4 used in the present manuscript for illustration-opens the doors to the exploration of spatiotemporal interactions between repair proteins at individual DSB sites, deepening our insights into mechanisms of DSB induction and (mis)repair [13][14][15]36,[48][49][50][51][52][53][54][55].…”

Section: Introductionmentioning

confidence: 99%

DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci

Vičar

Gumulec

Kolář

et al. 2020

Preprint

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DNA double-strand breaks, marked by Ionizing Radiation-Induced (Repair) Foci (IRIF), are the most serious DNA lesions, dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold standard method in radiation biodosimetry and allows research of DSB induction and repair at the molecular and a single cell level. In this study, we introduce DeepFoci - a deep learning-based fully-automatic method for IRIF counting and its morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for the nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation.The proposed method was trained and tested on challenging datasets consisting of mixtures of non-irradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDF, U-87) and cell primary cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 1-4 Gy gamma-rays and fixed at multiple (0-24 h) post-irradiation times. Upon all circumstances, DeepFoci was able to quantify the number of IRIF foci with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software kept its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This allowed multiparameter IRIF categorization, thereby refining the analysis of DSB repair processes and classification of patient tumors with a potential to identify specific cell subclones.The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to an advanced DSB focus analysis and, in turn, a better understanding of (radiation) DNA damaging and repair.HighlightsNew method for DSB repair focus (IRIF) detection and multi-parameter analysisTrainable deep learning-based methodFully automated analysis of multichannel 3D datasetsTrained and tested on extremely challenging datasets (tumor primary cultures)Comparable to an expert analysis and superb to available methodsGraphical Abstract

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Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

Cited by 30 publications

References 81 publications

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells

In Situ Detection of Complex DNA Damage Using Microscopy: A Rough Road Ahead

DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci

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