DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11-RAD50-NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.

• Publikační typ
• časopisecké články MeSH

Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX.

• MeSH
• fosforylace účinky záření   MeSH
• G1 fáze účinky záření   MeSH
• G2 fáze účinky záření   MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• záření gama škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

• MeSH
• biologické markery analýza   MeSH
• HeLa buňky MeSH
• lidé MeSH
• mikroskopie * MeSH
• pilotní projekty MeSH
• poškození DNA * MeSH
• reprodukovatelnost výsledků MeSH
• valinomycin toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF.

• MeSH
• 53BP1 metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• histony metabolismus   MeSH
• lidé MeSH
• lymfocyty metabolismus   účinky záření   MeSH
• poškození DNA * MeSH
• průtoková cytometrie metody   MeSH
• software MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama * MeSH
• zobrazování trojrozměrné metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND INFORMATION: The DNA damage response is a fundamental, well-regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non-irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins. RESULTS: We observed that spontaneous and irradiation-induced foci (IRIF) demonstrated a high abundance of phosphorylated H2AX, which was consistent with 53BP1 and BMI1 protein accumulation. However, NBS1 and MDC1 proteins were recruited to nuclear bodies (NBs) to a lesser extent. Irradiation by γ-rays significantly increased the number of 53BP1- and γH2AX-positive IRIF, but cell cycle-dependent differences were only observed for γH2AX-positive foci in both non-irradiated and γ-irradiated cells. In non-irradiated cells, the G2 phase was characterised by an increased number of spontaneous γH2AX-foci; this increase was more pronounced after γ-irradiation. Cells in G2 phase had the highest number of γH2AX-positive foci. Similarly, γ-irradiation increased the number of NBS1-positive NBs only in G2 phase. Moreover, NBS1 accumulated in nucleoli after γ-irradiation showed the slowest recovery after photobleaching. Analysis of protein accumulation kinetics at locally induced DNA lesions showed that in HeLa cells, BMI1, PCNA and coilin were rapidly recruited to the lesions, 10-15 s after UVA-irradiation, whereas among the other proteins studied, BRCA1 demonstrated the slowest recruitment: BRCA1 appeared at the lesion 20 min after local micro-irradiation by UVA laser. CONCLUSION: We show that the kinetics of the accumulation of selected DNA repair-related proteins is protein specific at locally induced DNA lesions, and that the formation of γH2AX- and NBS1-positive foci, but not 53BP1-positive NBs, is cell cycle dependent in HeLa cells. Moreover, γH2AX is the most striking protein present not only at DNA lesions, but also spreading out in their vicinity. SIGNIFICANCE: Our conclusions highlight the significant role of the spatiotemporal dynamics of DNA repair-related proteins and their specific assembly/disassembly at DNA lesions, which can be cell type- and cell cycle dependent.

• MeSH
• buněčný cyklus genetika   účinky záření   MeSH
• DNA genetika   metabolismus   MeSH
• fosforylace účinky záření   MeSH
• HeLa buňky MeSH
• histony genetika   metabolismus   MeSH
• intracelulární signální peptidy a proteiny genetika   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• oprava DNA genetika   účinky záření   MeSH
• poškození DNA genetika   účinky záření   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference 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

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

• MeSH
• buněčné dělení účinky léků   MeSH
• buněčné jadérko chemie   účinky léků   ultrastruktura   MeSH
• chromozomální proteiny, nehistonové analýza   MeSH
• daktinomycin farmakologie   MeSH
• epigeneze genetická účinky léků   MeSH
• G2 fáze účinky léků   MeSH
• guanylátcyklasa analýza   antagonisté a inhibitory   MeSH
• HeLa buňky chemie   účinky léků   MeSH
• histony analýza   metabolismus   MeSH
• intracelulární signální peptidy a proteiny analýza   MeSH
• kyselina ellagová farmakologie   MeSH
• lidé MeSH
• metylace MeSH
• nádorové proteiny analýza   MeSH
• organizátor jadérka chemie   účinky léků   ultrastruktura   MeSH
• poškození DNA MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• promotorové oblasti (genetika) MeSH
• ribozomální DNA účinky léků   genetika   MeSH
• RNA-polymerasa I analýza   MeSH
• signální adaptorové proteiny CARD analýza   antagonisté a inhibitory   MeSH
• transkripční iniciační komplex Pol1 - proteiny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The aim of our study was to determine whether phosphorylation of histone H2AX can be used as an indicator of received dose of gamma radiation after whole-body irradiation of rats. Wistar rats were irradiated by 1-10 Gy of gamma radiation by 60Co source. Value LD50/60 was 7.37 (4.68-8.05) Gy. Histone H2AX is phosphorylated by ATM kinase on serine 139 (γH2AX) quickly after the irradiation. It forms microscopically visible foci in the site of double strand breaks of DNA. Flow-cytometric method was used for quantitative detection. This study is the first one that evaluated dose-dependency of H2AX phosphorylation in peripheral lymphocytes of rats irradiated by whole-body dose 1-10 Gy. Our data show a dose-dependent increase in γH2AX in rat peripheral blood lymphocytes 1 h after whole-body irradiation by the dose of 1-10 Gy. We proved that phosphorylation of histone H2AX is a prompt and reliable indicator of the received radiation dose suitable for rapid measurement before the number of lymphocytes in peripheral blood starts to decrease. It can be used already 1 h after the irradiation for an estimation of the received dose of radiation. Blood samples can be stored in 4 °C for 23 h without significantly affecting the result.

• Klíčová slova
• histon H2AX,
• MeSH
• bioindikátory MeSH
• biologické markery MeSH
• celotělové ozáření MeSH
• dávka záření * MeSH
• fosforylace * MeSH
• histony * MeSH
• lymfocyty MeSH
• monitorování radiace * MeSH
• potkani Wistar MeSH
• průtoková cytometrie MeSH
• radiační expozice MeSH
• radiační účinky MeSH
• záření gama MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

PURPOSE: This study evaluates early changes in human mesenchymal stem cells (MSC) isolated from dental pulp and periodontal ligament after γ-irradiation and the effect of ataxia-telangiectasia mutated (ATM) inhibition. METHODS: MSC were irradiated with 2 and 20 Gy by (60)Co. For ATM inhibition, specific inhibitor KU55933 was used. DNA damage was measured by Comet assay and γH2AX detection. Cell cycle distribution and proteins responding to DNA damage were analyzed 2-72 h after the irradiation. RESULTS: The irradiation of MSC causes an increase in γH2AX; the phosphorylation was ATM-dependent. Irradiation activates ATM kinase, and the level of p53 protein is increased due to its phosphorylation on serine15. While this phosphorylation of p53 is ATM-dependent in MSC, the increase in p53 was not prevented by ATM inhibition. A similar trend was observed for Chk1 and Chk2. The increase in p21 is greater without ATM inhibition. ATM inhibition also does not fully abrogate the accumulation of irradiated MSC in the G2-phase of the cell-cycle. CONCLUSIONS: In irradiated MSC, double-strand breaks are tagged quickly by γH2AX in an ATM-dependent manner. Although phosphorylations of p53(ser15), Chk1(ser345) and Chk2(thr68) are ATM-dependent, the overall amount of these proteins increases when ATM is inhibited. In both types of MSC, ATM-independent mechanisms for cell-cycle arrest in the G2-phase are triggered.

• MeSH
• dávka záření MeSH
• DNA vazebné proteiny antagonisté a inhibitory   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mezenchymální kmenové buňky cytologie   fyziologie   účinky záření   MeSH
• nádorové supresorové proteiny antagonisté a inhibitory   MeSH
• periodontální vaz cytologie   fyziologie   účinky záření   MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   MeSH
• proteiny buněčného cyklu antagonisté a inhibitory   MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama MeSH
• zubní dřeň cytologie   fyziologie   účinky záření   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH