Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair

Lorat, Yvonne; Brunner, Christina U.; Schanz, Stefanie; Jakob, Burkhard; Taucher-Scholz, G.; Rübe, Claudia E.

doi:10.1016/j.dnarep.2015.01.007

Cited by 104 publications

(116 citation statements)

References 43 publications

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“…Recent applications of electron-microscopy [25,26] and super-resolution light microscopy such as SMLM, STED or GSDIM [1,3,23,24,28,29,30,31,33,34] have demonstrated that it is feasible to study single molecular arrangements within a repair focus. With improving resolution of microscopy and data evaluation of structures on the meso- and nano-scale, the question for best suited analysis parameters and potentially useful classification criteria of repair foci and damaged chromatin sites has become important.…”

Section: Discussionmentioning

confidence: 99%

“…Nano-scaled analysis has reasoned several transmission electron and super-resolution light microscopic studies in order to elucidate the spatio-temporal internal organization of repair foci and their chromatin surroundings with molecular resolution [3,21,22,23,24,25,26,27,28,29]. Recently, it has been shown by super-resolution light microscopy, that γH2AX foci are built up by clusters that form nano-foci with different repair activities [23,28] and that inside these nano-foci repair proteins are well organized [23,24,29] whereas the chromatin environment is interacting in a characteristic arrangement [28,30,31].…”

Section: Introductionmentioning

confidence: 99%

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Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Hofmann

Krufczik

Heermann

et al. 2018

IJMS

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DNA double strand breaks (DSB) are the most severe damages in chromatin induced by ionizing radiation. In response to such environmentally determined stress situations, cells have developed repair mechanisms. Although many investigations have contributed to a detailed understanding of repair processes, e.g., homologous recombination repair or non-homologous end-joining, the question is not sufficiently answered, how a cell decides to apply a certain repair process at a certain damage site, since all different repair pathways could simultaneously occur in the same cell nucleus. One of the first processes after DSB induction is phosphorylation of the histone variant H2AX to γH2AX in the given surroundings of the damaged locus. Since the spatial organization of chromatin is not random, it may be conclusive that the spatial organization of γH2AX foci is also not random, and rather, contributes to accessibility of special repair proteins to the damaged site, and thus, to the following repair pathway at this given site. The aim of this article is to demonstrate a new approach to analyze repair foci by their topology in order to obtain a cell independent method of categorization. During the last decade, novel super-resolution fluorescence light microscopic techniques have enabled new insights into genome structure and spatial organization on the nano-scale in the order of 10 nm. One of these techniques is single molecule localization microscopy (SMLM) with which the spatial coordinates of single fluorescence molecules can precisely be determined and density and distance distributions can be calculated. This method is an appropriate tool to quantify complex changes of chromatin and to describe repair foci on the single molecule level. Based on the pointillist information obtained by SMLM from specifically labeled heterochromatin and γH2AX foci reflecting the chromatin morphology and repair foci topology, we have developed a new analytical methodology of foci or foci cluster characterization, respectively, by means of persistence homology. This method allows, for the first time, a cell independent comparison of two point distributions (here the point distributions of two γH2AX clusters) with each other of a selected ensample and to give a mathematical measure of their similarity. In order to demonstrate the feasibility of this approach, cells were irradiated by low LET (linear energy transfer) radiation with different doses and the heterochromatin and γH2AX foci were fluorescently labeled by antibodies for SMLM. By means of our new analysis method, we were able to show that the topology of clusters of γH2AX foci can be categorized depending on the distance to heterochromatin. This method opens up new possibilities to categorize spatial organization of point patterns by parameterization of topological similarity.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Hofmann

Krufczik

Heermann

et al. 2018

IJMS

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“…In previous TEM studies, we characterised the DNA-damage patterns caused in human fibroblasts by high-LET and low-LET irradiation, respectively [12]. Here, we report on the formation and subsequent repair of SSBs, DSBs, and clustered lesions directly after exposure to high-LET carbon ions.…”

mentioning

confidence: 85%

Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation

Lorat

Timm

Jakob

et al. 2016

Radiotherapy and Oncology

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“…The energy deposition by low-linear energy transfer (LET) radiation is distributed randomly throughout the cell, whereas the energy from high- linear energy transfer (LET) radiation is deposited as discrete tracks where the particle has passed through the cell [2]. As a result, the DNA damage induced by high-LET heavy ion radiation is more complex than that by X- or gamma rays and leads to more severe biological consequences [3].…”

Section: Introductionmentioning

confidence: 99%

pATM and γH2AX are effective radiation biomarkers in assessing the radiosensitivity of 12C6+ in human tumor cells

et al. 2017

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BackgroundTumour radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. The aim of the current study was to evaluate the potential value of phosphorylated H2AX (γH2AX) and ATM (pATM) in assessing 12C6+ radiosensitivity of tumour cells.MethodsHuman cervical carcinoma HeLa cells, hepatoma HepG2 cells, and mucoepidermoid carcinoma MEC-1 cells were irradiated with different doses of 12C6+. The survival fraction was assayed with clonogenic survival method and the foci of γH2AX and pATM was visualized using immunocytochemical methods. Flow cytometry was used to assay γH2AX, pATM and the cell cycle.ResultsThe survival fraction decreased immediately in dose-dependent manner, but in turn, significantly increased during 24 h after 12C6+ irradiation. Both γH2AX and pATM foci accumulated linearly with doses and with a maximum induction at 0.5 h for γH2AX and 0.5 or 4 h for pATM, respectively, and a fraction foci kept for 24 h. The expression of γH2AX and pATM was in relation to cell cycle. The G0/G1 phase cells had the highest expression of γH2AX after 0.5 h irradiation and then decreased to a lower level at 24 h after irradiation. An obvious increase of pATM in G2/M phase was shown after 24 h of 2 and 4 Gy irradiation. The significant G2/M phase arrest was shown. There was a close relationship between the clonogenic survival and γH2AX and pATM expression both in timing and dose in response to 12C6+.ConclusionsThe rate of γH2AX and pATM formation and loss may be an important factor in the response of cells to 12C6+. pATM and γH2AX are effective radiation biomarkers in assessing the radiosensitivity of 12C6+ in human tumor cells.

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Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair

Cited by 104 publications

References 43 publications

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Using Persistent Homology as a New Approach for Super-Resolution Localization Microscopy Data Analysis and Classification of γH2AX Foci/Clusters

Clustered double-strand breaks in heterochromatin perturb DNA repair after high linear energy transfer irradiation

pATM and γH2AX are effective radiation biomarkers in assessing the radiosensitivity of 12C6+ in human tumor cells

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