DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-β-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.
- MeSH
- DNA metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * účinky záření MeSH
- enzymy opravy DNA * metabolismus genetika MeSH
- exodeoxyribonukleasy * metabolismus genetika MeSH
- lidé MeSH
- oprava DNA * MeSH
- proliferační antigen buněčného jádra metabolismus genetika MeSH
- proteiny buněčného cyklu MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 μm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/μm) and 27 MeV carbon ions (LET = 500 keV/μm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 μm. Lithium ions produce (1.5 ± 0.1) IRIF/μm track length, for carbon ions (2.2 ± 0.2) IRIF/μm are counted. These values are enhanced by a factor of 2-3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/μm is possible.
- MeSH
- biologické markery MeSH
- dávka záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- fluorescenční mikroskopie metody MeSH
- fosforylace účinky záření MeSH
- HeLa buňky MeSH
- lidé MeSH
- lineární přenos energie MeSH
- lithium škodlivé účinky MeSH
- oprava DNA účinky záření MeSH
- proteinkinasy účinky záření MeSH
- radiační expozice MeSH
- radiometrie metody MeSH
- těžké ionty škodlivé účinky MeSH
- uhlík škodlivé účinky MeSH
- záření gama škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The complex relationship between linear energy transfer (LET) and cellular response to radiation is not yet fully elucidated. To better characterize DNA damage after irradiations with therapeutic protons, we monitored formation and disappearance of DNA double-strand breaks (DNA DSB) as a function of LET and time. Comparisons with conventional γ-rays and high LET carbon ions were also performed. MATERIALS AND METHODS: In the present work, we performed immunofluorescence-based assay to determine the amount of DNA DSB induced by different LET values along the 62 MeV therapeutic proton Spread out Bragg peak (SOBP) in three cancer cell lines, i.e. HTB140 melanoma, MCF-7 breast adenocarcinoma and HTB177 non-small lung cancer cells. Time dependence of foci formation was followed as well. To determine irradiation positions, corresponding to the desired LET values, numerical simulations were carried out using Geant4 toolkit. We compared γ-H2AX foci persistence after irradiations with protons to that of γ-rays and carbon ions. RESULTS: With the rise of LET values along the therapeutic proton SOBP, the increase of γ-H2AX foci number is detected in the three cell lines up to the distal end of the SOBP, while there is a decrease on its distal fall-off part. With the prolonged incubation time, the number of foci gradually drops tending to attain the residual level. For the maximum number of DNA DSB, irradiation with protons attain higher level than that of γ-rays. Carbon ions produce more DNA DSB than protons but not substantially. The number of residual foci produced by γ-rays is significantly lower than that of protons and particularly carbon ions. Carbon ions do not produce considerably higher number of foci than protons, as it could be expected due to their physical properties. CONCLUSIONS: In situ visualization of γ-H2AX foci reveal creation of more lesions in the three cell lines by clinically relevant proton SOBP than γ-rays. The lack of significant differences in the number of γ-H2AX foci between the proton and carbon ion-irradiated samples suggests an increased complexity of DNA lesions and slower repair kinetics after carbon ions compared to protons. For all three irradiation types, there is no major difference between the three cell lines shortly after irradiations, while later on, the formation of residual foci starts to express the inherent nature of tested cells, therefore increasing discrepancy between them.
- MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- lidé MeSH
- lineární přenos energie * MeSH
- nádorové buněčné linie MeSH
- oprava DNA účinky záření MeSH
- protony * MeSH
- relativní biologická účinnost MeSH
- viabilita buněk účinky záření MeSH
- vztah dávky záření a odpovědi MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In cancer therapy, the application of (fractionated) harsh radiation treatment is state of the art for many types of tumors. However, ionizing radiation is a "double-edged sword"-it can kill the tumor but can also promote the selection of radioresistant tumor cell clones or even initiate carcinogenesis in the normal irradiated tissue. Individualized radiotherapy would reduce these risks and boost the treatment, but its development requires a deep understanding of DNA damage and repair processes and the corresponding control mechanisms. DNA double strand breaks (DSBs) and their repair play a critical role in the cellular response to radiation. In previous years, it has become apparent that, beyond genetic and epigenetic determinants, the structural aspects of damaged chromatin (i.e., not only of DSBs themselves but also of the whole damage-surrounding chromatin domains) form another layer of complex DSB regulation. In the present article, we summarize the application of super-resolution single molecule localization microscopy (SMLM) for investigations of these structural aspects with emphasis on the relationship between the nano-architecture of radiation-induced repair foci (IRIFs), represented here by γH2AX foci, and their chromatin environment. Using irradiated HeLa cell cultures as an example, we show repair-dependent rearrangements of damaged chromatin and analyze the architecture of γH2AX repair clusters according to topological similarities. Although HeLa cells are known to have highly aberrant genomes, the topological similarity of γH2AX was high, indicating a functional, presumptively genome type-independent relevance of structural aspects in DSB repair. Remarkably, nano-scaled chromatin rearrangements during repair depended both on the chromatin domain type and the treatment. Based on these results, we demonstrate how the nano-architecture and topology of IRIFs and chromatin can be determined, point to the methodological relevance of SMLM, and discuss the consequences of the observed phenomena for the DSB repair network regulation or, for instance, radiation treatment outcomes.
- MeSH
- chromatin genetika ultrastruktura MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- HeLa buňky MeSH
- ionizující záření MeSH
- lidé MeSH
- mikroskopie metody MeSH
- nádorové buněčné linie MeSH
- nádory genetika MeSH
- oprava DNA genetika účinky záření MeSH
- poškození DNA genetika účinky záření MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Track structure based simulations valuably complement experimental research on biological effects of ionizing radiation. They provide information at the highest level of detail on initial DNA damage induced by diverse types of radiation. Simulations with the biophysical Monte Carlo code PARTRAC have been used for testing working hypotheses on radiation action mechanisms, for benchmarking other damage codes and as input for modelling subsequent biological processes. To facilitate such applications and in particular to enable extending the simulations to mixed radiation field conditions, we present analytical formulas that capture PARTRAC simulation results on DNA single- and double-strand breaks and their clusters induced in cells irradiated by ions ranging from hydrogen to neon at energies from 0.5 GeV/u down to their stopping. These functions offer a means by which radiation transport codes at the macroscopic scale could easily be extended to predict biological effects, exploiting a large database of results from micro-/nanoscale simulations, without having to deal with the coupling of spatial scales and running full track-structure calculations.
- MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- lidé MeSH
- lineární přenos energie MeSH
- metoda Monte Carlo * MeSH
- poškození DNA * MeSH
- protony * MeSH
- radioterapie * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The biophysical simulation tool PARTRAC has been primarily developed to model radiation physics, chemistry and biology on nanometre to micrometre scales. However, the tool can be applied in simulating radiation effects in an event-by-event manner over macroscopic volumes as well. Benchmark simulations are reported showing that PARTRAC does reproduce the macroscopic Bragg peaks of proton beams, although the penetration depths are underestimated by a few per cent for high-energy beams. PARTRAC also quantifies the increase in DNA damage and its complexity along the beam penetration depth. Enhanced biological effectiveness is predicted in particular within distal Bragg peak parts of therapeutic proton beams.
- MeSH
- algoritmy MeSH
- DNA účinky záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- lineární přenos energie MeSH
- metoda Monte Carlo MeSH
- počítačová simulace * MeSH
- poškození DNA MeSH
- protonová terapie MeSH
- protony MeSH
- relativní biologická účinnost MeSH
- software MeSH
- voda MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raška, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.
- MeSH
- DNA-ligasa ATP genetika metabolismus MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- homologní rekombinace MeSH
- kultivované buňky MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie fyziologie MeSH
- nestabilita genomu * MeSH
- oprava DNA spojením konců fyziologie účinky záření MeSH
- oprava DNA fyziologie účinky záření MeSH
- proteiny vázající poly-ADP-ribosu genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
From the very beginnings of radiotherapy, a crucial question persists with how to target the radiation effectiveness into the tumor while preserving surrounding tissues as undamaged as possible. One promising approach is to selectively pre-sensitize tumor cells by metallic nanoparticles. However, though the "physics" behind nanoparticle-mediated radio-interaction has been well elaborated, practical applications in medicine remain challenging and often disappointing because of limited knowledge on biological mechanisms leading to cell damage enhancement and eventually cell death. In the present study, we analyzed the influence of different nanoparticle materials (platinum (Pt), and gold (Au)), cancer cell types (HeLa, U87, and SKBr3), and doses (up to 4 Gy) of low-Linear Energy Transfer (LET) ionizing radiation (γ- and X-rays) on the extent, complexity and reparability of radiation-induced γH2AX + 53BP1 foci, the markers of double stand breaks (DSBs). Firstly, we sensitively compared the focus presence in nuclei during a long period of time post-irradiation (24 h) in spatially (three-dimensionally, 3D) fixed cells incubated and non-incubated with Pt nanoparticles by means of high-resolution immunofluorescence confocal microscopy. The data were compared with our preliminary results obtained for Au nanoparticles and recently published results for gadolinium (Gd) nanoparticles of approximately the same size (2⁻3 nm). Next, we introduced a novel super-resolution approach-single molecule localization microscopy (SMLM)-to study the internal structure of the repair foci. In these experiments, 10 nm Au nanoparticles were used that could be also visualized by SMLM. Altogether, the data show that different nanoparticles may or may not enhance radiation damage to DNA, so multi-parameter effects have to be considered to better interpret the radiosensitization. Based on these findings, we discussed on conclusions and contradictions related to the effectiveness and presumptive mechanisms of the cell radiosensitization by nanoparticles. We also demonstrate that SMLM offers new perspectives to study internal structures of repair foci with the goal to better evaluate potential differences in DNA damage patterns.
- MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- gadolinium chemie MeSH
- HeLa buňky MeSH
- konfokální mikroskopie MeSH
- kovové nanočástice chemie terapeutické užití MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- poškození DNA účinky záření MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs). METHODS: We measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. RESULTS: We demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages. CONCLUSIONS: Our observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.
- MeSH
- 53BP1 genetika metabolismus MeSH
- buněčné linie MeSH
- DNA genetika metabolismus MeSH
- dvouřetězcové zlomy DNA * účinky záření MeSH
- exprese genu MeSH
- fibroblasty cytologie metabolismus účinky záření MeSH
- fosforylace účinky záření MeSH
- histony genetika metabolismus MeSH
- indukované pluripotentní kmenové buňky cytologie metabolismus účinky záření MeSH
- kontrolní body fáze G1 buněčného cyklu genetika MeSH
- kontrolní body fáze G2 buněčného cyklu genetika MeSH
- lidé MeSH
- lidské embryonální kmenové buňky cytologie metabolismus účinky záření MeSH
- oprava DNA genetika MeSH
- přeprogramování buněk MeSH
- stárnutí buněk genetika účinky záření MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: Goeckerman therapy is based on combined exposure to UV radiation (UVA, UVB) and crude coal tar (PAHs). Some indicators suggest a genotoxic hazard, however, the level of genotoxic risk of the therapy has not yet been investigated sufficiently. This study aims to assesss the genotoxic risk. METHODS: The studied group consisted of patients with chronic stable plaque psoriasis treated by Goeckerman therapy (n = 29). Heparin-treated peripheral blood samples were collected one day before the first treatment and immediately after the last procedure. The lymphocytes were isolated from the blood. The level of genotoxicity was evaluated using an alkaline version of the Comet assay which detects DNA single strand breaks (DNA-SSBs), a neutral version of the Comet assay which detects DNA double strand breaks (DNA-DSBs), and using chromosomal aberrations. RESULTS: The level of DNA-SSBs increased insignificantly (median; Q1-Q3): 1.4 (0.4; 0.1-1.4) vs. 2.5 (0.6; 0.3-2.7) %tDNA (P = 0.11) and the level of DNA-DSBs increased significantly: 7.8 (6.5; 3.4-10.5) vs. 20.7 (19.3; 14.2-24.6) % DNA (P < 0.001). The total number of aberrated cells (P < 0.001) and structurally aberrated cells (P < 0.001) increased significantly. CONCLUSION: The elevated levels of the DNA-DSBs and the chromosomal aberrations in the peripheral lymphocytes indicated a genotoxic hazard. However, the elevated level of the chromosomal abnormalities was below the upper level of the reference range for healthy Czech adults. While, the genotoxic risk appears to be low, Goeckerman treatment represents a further contribution to the lifetime load of genotoxic factors.
- MeSH
- chromozomální aberace účinky léků účinky záření MeSH
- chronická nemoc MeSH
- dehet uhelný škodlivé účinky MeSH
- dospělí MeSH
- dvouřetězcové zlomy DNA účinky léků účinky záření MeSH
- keratolytika škodlivé účinky MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfocyty * MeSH
- psoriáza terapie MeSH
- senioři MeSH
- terapie ultrafialovými paprsky škodlivé účinky MeSH
- ultrafialové záření škodlivé účinky MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
