Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Nakajima, Naomasa; Brunton, Holly; Watanabe, Ritsuko; Shrikhande, Amruta; Hirayama, Ryoichi; Matsufuji, Naruhiro; Fujimori, A.; Murakami, Takeshi; Okayasu, Ryuichi; Jeggo, Penny A.; Shibata, Atsushi

doi:10.1371/journal.pone.0070107

Cited by 70 publications

(89 citation statements)

References 40 publications

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“…The existence of more than one DSB under a focus can be an issue for underestimation and the correct assignment of focus size can improve this situation but up to a point. The yields we found for low-LET (Figure 2) are compatible with previous findings of 10-20 DSB foci/Gy/cell [37,42,43] and the same stands also for a-particles [35,44,45] and Ar ions [46], although in the specific study 40 Ar ions of a much lower LET (125 keV/lm) were used. Previous studies using cold lysis PFGE protocols in order to minimize conversion of heat-labile sites to DSBs suggest relatively increased numbers of $25 DSBs/Gy/cell for normal human skin fibroblasts (GM5758) for low-LET radiations [47].…”

Section: Discussionsupporting

confidence: 92%

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Nikitaki

Nikolov

Mavragani

et al. 2016

Free Radical Research

102

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Detrimental effects of ionizing radiation (IR) are correlated to the varying efficiency of IR to induce complex DNA damage. A double strand break (DSB) can be considered the simpler form of complex DNA damage. These types of damage can consist of DSBs, single strand breaks (SSBs) and/or non-DSB lesions such as base damages and apurinic/apyrimidinic (AP; abasic) sites in different combinations. Enthralling theoretical (Monte Carlo simulations) and experimental evidence suggests an increase in the complexity of DNA damage and therefore repair resistance with linear energy transfer (LET). In this study, we have measured the induction and processing of DSB and non-DSB oxidative clusters using adaptations of immunofluorescence. Specifically, we applied foci colocalization approaches as the most current methodologies for the in situ detection of clustered DNA lesions in a variety of human normal (FEP18-11-T1) and cancerous cell lines of varying repair efficiency (MCF7, HepG2, A549, MO59K/J) and radiation qualities of increasing LET, that is c-, X-rays 0.3-1 keV/lm, a-particles 116 keV/lm and 36 Ar ions 270 keV/lm. Using c-H2AX or 53BP1 foci staining as DSB probes, we calculated a DSB apparent rate of 5-16 DSBs/cell/Gy decreasing with LET. A similar trend was measured for non-DSB oxidized base lesions detected using antibodies against the human repair enzymes 8-oxoguanine-DNA glycosylase (OGG1) or AP endonuclease (APE1), that is damage foci as probes for oxidized purines or abasic sites, respectively. In addition, using colocalization parameters previously introduced by our groups, we detected an increasing clustering of damage for DSBs and non-DSBs. We also make correlations of damage complexity with the repair efficiency of each cell line and we discuss the biological importance of these new findings with regard to the severity of IR due to the complex nature of its DNA damage. ARTICLE HISTORY

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Section: Discussionsupporting

confidence: 92%

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Nikitaki

Nikolov

Mavragani

et al. 2016

Free Radical Research

102

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“…Such DSB clustering was previously observed by others (40,(43)(44)(45)(46) and is also evident from our analysis of foci sizes and intensities showing that foci in the inner part of the particle track were on average larger and brighter than foci in the outer part of the track.…”

Section: Discussionsupporting

confidence: 88%

Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue

Mirsch

Tommasino

Frohns

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Charged particles are increasingly used in cancer radiotherapy and contribute significantly to the natural radiation risk. The difference in the biological effects of high-energy charged particles compared with X-rays or γ-rays is determined largely by the spatial distribution of their energy deposition events. Part of the energy is deposited in a densely ionizing manner in the inner part of the track, with the remainder spread out more sparsely over the outer track region. Our knowledge about the dose distribution is derived solely from modeling approaches and physical measurements in inorganic material. Here we exploited the exceptional sensitivity of γH2AX foci technology and quantified the spatial distribution of DNA lesions induced by charged particles in a mouse model tissue. We observed that charged particles damage tissue nonhomogenously, with single cells receiving high doses and many other cells exposed to isolated damage resulting from high-energy secondary electrons. Using calibration experiments, we transformed the 3D lesion distribution into a dose distribution and compared it with predictions from modeling approaches. We obtained a radial dose distribution with sub-micrometer resolution that decreased with increasing distance to the particle path following a 1/r 2 dependency. The analysis further revealed the existence of a background dose at larger distances from the particle path arising from overlapping dose deposition events from independent particles. Our study provides, to our knowledge, the first quantification of the spatial dose distribution of charged particles in biologically relevant material, and will serve as a benchmark for biophysical models that predict the biological effects of these particles.C harged particles, including protons, α-particles, and heavy ions, are increasingly used in cancer radiotherapy and represent a significant component of the natural background irradiation on earth and in space (1-3). Their biological effect is often very different from that of photons (X-or γ-rays), largely because charged particles deposit their energy along a track, whereas photons produce a fairly homogeneous dose distribution. Linear energy transfer (LET; typically given in units of keV/μm) has been introduced as a parameter to describe the amount of energy that charged particles deposit along their track. Particles with high LET are densely ionizing and typically biologically more effective than photons or low-LET particles. Energy deposition along a particle track is not restricted to the path itself (the so-called "track core"), but extends laterally into an area known as the penumbra of the particle, which can reach considerable distances for high-energy particles. Energy deposition in the penumbra arises from energetic secondary electrons, so-called δ-electrons, which are generated by ionization events of the charged particles and carry energy away from the immediate path into the penumbra. According to classical track structure theory, ∼50% of the total energy is deposited in the...

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“…Off-track gH2AX signals attributed to d electron-induced DSBs has been reported using conventional microscopy and image deconvolution (56). Consistent with our data, off-track gH2AX signals were smaller and showed less heterogeneity than foci within the ion track.…”

Section: Off-track Subfocisupporting

confidence: 91%

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

et al. 2016

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Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker gH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with

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Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Cited by 70 publications

References 40 publications

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue

Superresolution light microscopy shows nanostructure of carbon ion radiation‐induced DNA double‐strand break repair foci

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