- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference 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
Nowadays, the irradiation methodology in proton therapy is switching from the use of passively scattered beams to active pencil beams due to the possibility of more conformal dose distributions. The dose rates of active pencil beams are much higher than those of passive beams. The purpose of this study was to investigate whether there is any difference in the biological effectiveness of these passive and active irradiation modes. The beam qualities of double scattering and pencil beam scanning were measured dosimetrically and simulated using the Monte Carlo code. Using the medulloblastoma cell line DAOY, we performed an in vitro comparison of the two modes in two positions along the dose-deposition curve plateau and inside the Bragg peak. We followed the clonogenic cell survival, apoptosis, micronuclei, and γH2AX assays as biological endpoints. The Monte Carlo simulations did not reveal any difference between the beam qualities of the two modes. Furthermore, we did not observe any statistically significant difference between the two modes in the in vitro comparison of any of the examined biological endpoints. Our results do not show any biologically relevant differences related to the different dose rates of passive and active proton beams.
- MeSH
- apoptóza účinky záření MeSH
- histony metabolismus MeSH
- lidé MeSH
- lineární přenos energie MeSH
- metoda Monte Carlo MeSH
- mikrojaderné testy MeSH
- nádorové buněčné linie MeSH
- neutrony MeSH
- počítačová simulace MeSH
- protonová terapie * MeSH
- viabilita buněk účinky záření MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- MeSH
- biologie buňky * MeSH
- buněčná smrt fyziologie genetika MeSH
- buněčné dělení fyziologie genetika MeSH
- buněčné struktury fyziologie MeSH
- buněčný cyklus fyziologie genetika MeSH
- chromozomy fyziologie genetika MeSH
- Eukaryota fyziologie ultrastruktura MeSH
- genetické jevy fyziologie genetika MeSH
- hybridizace in situ fluorescenční MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- chromozomální aberace účinky záření MeSH
- lidé MeSH
- mutageneze účinky záření MeSH
- oprava DNA účinky záření MeSH
- poškození DNA účinky záření MeSH
- radiační poranění patologie MeSH
- radiační účinky * MeSH
- rizikové faktory MeSH
- stochastické procesy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
V léčbě nádorů hlavy a krku (NHK) zaznamenáváme odklon od chirurgie směrem k radioterapii, respektive chemoradioterapii, s důrazem na poléčebnou kvalitu života. Volba léčebné modality však vychází pouze z klinických zkušeností a preferencí pacienta, protože neznáme marker radiosenzitivity jednotlivých nádorů. Nové možnosti rozhodování o nejvhodnější primární terapii, směřující k personalizované medicíně založené na kvantifikovatelných biomarkerech, by mohlo nabídnout studium radiačního poškození a reparace DNA u jednotlivých nádorů. V předloženém článku diskutujeme, společně s významem radioterapie v léčbě NHK, své předběžné výsledky odhalující existenci několika skupin NHK s ohledem na stabilitu genomu a reparační schopnosti nádorových buněk po ozáření. Monitorování kinetiky tvorby a zániku reparačních ohnisek γH2AX/53BP1 v buněčných primokulturách nádorových tkání získaných od jednotlivých pacientů naznačuje, že specifické odchylky v reparaci DNA, charakteristické pro identifikované skupiny, korelují se zvýšenou nebo sníženou radiosenzitivitou nádorových buněk. Naše zjištění tak přispívají k lepšímu pochopení vzniku a progrese NHK. Souvislost reparačních skupin s odpovědí nádorů na radioterapii in vivo však zůstává předmětem výzkumu. Jelikož velká část NHK netrpí reparačními defekty, a přitom se jejich viabilita po ozáření vzájemně diametrálně liší, preterapeutické testy pokrývající celé spektrum příčin radiosenzitivity NHK budou muset využívat kombinaci více biomarkerů, jež však stále čekají na své odhalení.
In order to maximize post-therapeutic quality of life, radio(chemo) therapy becomes preferred over surgery in head-and-neck tumor (HNT) treatment. However, the therapy selection is only based on the clinical experience and patient's preferences as the radiosensitivity markers remain unknown. New possibilities of deciding on the best primary therapy, moving us towards personalized medicine based on quantifiable biomarkers, have been opened by studies on DNA radiation damage and repair in individual patients’ tumors. Together with the importance of radiotherapy in HNT oncology, we discuss here our preliminary results revealing the existence of several HNT groups with respect to genome stability and repair ability of tumor cells after irradiation. Monitoring of the formation and disappearance of γH2AX/53BP1 foci in tumor cell primo-cultures derived from individual patients suggests that DNA repair capacity of the identified groups correlates with the tumor cell radiosensitivity. Our findings thus improve understanding of HNT biology; nevertheless, the relationship between the repair groups and in vivo response of tumors to radiotherapy must be further studied. Since most HNTs do not suffer from repair defects, although their viability varies after irradiation, pre-therapeutic tests covering the full spectrum of HNT radiosensitivity causes will require the use of a combination of multiple, still undiscovered biomarkers.
- MeSH
- chemorezistence MeSH
- léčba šetřící orgány MeSH
- lidé MeSH
- nádorové biomarkery MeSH
- nádory hlavy a krku * chirurgie genetika radioterapie MeSH
- oprava DNA * MeSH
- poškození DNA účinky záření MeSH
- radioterapie * škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy 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
DNA double stranded breaks (DSBs) are the most serious type of lesions introduced into chromatin by ionizing radiation. During DSB repair, cells recruit different proteins to the damaged sites in a manner dependent on local chromatin structure, DSB location in the nucleus, and the repair pathway entered. 53BP1 is one of the important players participating in repair pathway decision of the cell. Although many molecular biology details have been investigated, the architecture of 53BP1 repair foci and its development during the post-irradiation time, especially the period of protein recruitment, remains to be elucidated. Super-resolution light microscopy is a powerful new tool to approach such studies in 3D-conserved cell nuclei. Recently, we demonstrated the applicability of single molecule localization microscopy (SMLM) as one of these highly resolving methods for analyses of dynamic repair protein distribution and repair focus internal nano-architecture in intact cell nuclei. In the present study, we focused our investigation on 53BP1 foci in differently radio-resistant cell types, moderately radio-resistant neonatal human dermal fibroblasts (NHDF) and highly radio-resistant U87 glioblastoma cells, exposed to high-LET 15N-ion radiation. At given time points up to 24 h post irradiation with doses of 1.3 Gy and 4.0 Gy, the coordinates and spatial distribution of fluorescently tagged 53BP1 molecules was quantitatively evaluated at the resolution of 10⁻20 nm. Clusters of these tags were determined as sub-units of repair foci according to SMLM parameters. The formation and relaxation of such clusters was studied. The higher dose generated sufficient numbers of DNA breaks to compare the post-irradiation dynamics of 53BP1 during DSB processing for the cell types studied. A perpendicular (90°) irradiation scheme was used with the 4.0 Gy dose to achieve better separation of a relatively high number of particle tracks typically crossing each nucleus. For analyses along ion-tracks, the dose was reduced to 1.3 Gy and applied in combination with a sharp angle irradiation (10° relative to the cell plane). The results reveal a higher ratio of 53BP1 proteins recruited into SMLM defined clusters in fibroblasts as compared to U87 cells. Moreover, the speed of foci and thus cluster formation and relaxation also differed for the cell types. In both NHDF and U87 cells, a certain number of the detected and functionally relevant clusters remained persistent even 24 h post irradiation; however, the number of these clusters again varied for the cell types. Altogether, our findings indicate that repair cluster formation as determined by SMLM and the relaxation (i.e., the remaining 53BP1 tags no longer fulfill the cluster definition) is cell type dependent and may be functionally explained and correlated to cell specific radio-sensitivity. The present study demonstrates that SMLM is a highly appropriate method for investigations of spatiotemporal protein organization in cell nuclei and how it influences the cell decision for a particular repair pathway at a given DSB site.
