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
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
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
We evaluated the impact of ataxia-telangiectasia mutated kinase inhibitor KU55933, DNA-dependent protein kinase inhibitor NU7441 and ataxia telangiectasia and rad3-related kinase inhibitor VE821 in human peripheral lymphocytes in vitro. The lymphocytes were divided into 5 groups: non-irradiated control, irradiated group (2 Gy) and 3 groups pretreated with inhibitors 30 min before irradiation. We used flow cytometry to evaluate phosphorylated H2AX (γ-H2AX) and cytotoxicity (Apoptest). Micronucleus assay was used to assess genotoxicity. After irradiation, γ-H2AX, incidence of micronuclei (MN), nucleoplasmatic bridges (NPBs) and nuclear buds in binuclear cells, MN in mononuclear cells and apoptosis were increased. KU55933 decreased γ-H2AX and inhibited ionizing radiation-induced cytotoxicity. NU7441 showed no effect on γ-H2AX but it significantly increased MN and NPBs in binuclear cells and apoptosis. VE821 decreased γ-H2AX, whereas genotoxicity and cytotoxicity were not affected. In conclusion, KU55933 protected lymphocytes, which might be employed to preserve the immune system during anticancer therapy. NU7441 radiosensitized lymphocytes, thus, undesirable side effects toward immune system could be expected. VE821 showed decrease of γ-H2AX with no radiosensitizing effects in our model likely due to p53 positive status, which underlies the concept of its application in p53 negative environment.
- Klíčová slova
- Účinky ionizujícího záření způsobené inhibitory ATM (KU55933), DNA-PK (NU7441) a ATR (VE821) na lymfocyty periferní krve,
- MeSH
- ATM protein účinky záření MeSH
- biomedicínský výzkum MeSH
- financování organizované MeSH
- fosfatidylinositol-3-kinasy antagonisté a inhibitory účinky záření MeSH
- fosforylace imunologie účinky záření MeSH
- histony chemie účinky záření MeSH
- ionizující záření * MeSH
- krevní a imunitní systémy cytologie imunologie účinky záření MeSH
- lidé MeSH
- lymfocyty * cytologie imunologie účinky záření MeSH
- statistika jako téma MeSH
- techniky in vitro MeSH
- Check Tag
- lidé 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
DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)-triggered by radiation-induced double strand breaks-is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.
- MeSH
- ATM protein antagonisté a inhibitory MeSH
- fosforylace účinky léků účinky záření MeSH
- inhibitory proteinkinas farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- proteom metabolismus MeSH
- pyraziny farmakologie MeSH
- radiosenzibilizující látky farmakologie MeSH
- sulfony farmakologie MeSH
- záření gama * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 μM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.
- MeSH
- apoptóza účinky léků účinky záření MeSH
- časové faktory MeSH
- chromony farmakologie MeSH
- fosforylace účinky léků účinky záření MeSH
- histony metabolismus MeSH
- inhibitory proteinkinas farmakologie MeSH
- leukemie patologie MeSH
- lidé MeSH
- morfoliny farmakologie MeSH
- nádorové buněčné linie MeSH
- oprava DNA účinky léků účinky záření MeSH
- poškození DNA MeSH
- proliferace buněk účinky léků účinky záření MeSH
- proteinkinasa aktivovaná DNA antagonisté a inhibitory MeSH
- radiosenzibilizující látky farmakologie MeSH
- signální transdukce účinky léků účinky záření MeSH
- tolerance záření účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation. MATERIALS AND METHODS: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24-72 hours after irradiation in a dose range of 0-5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry. RESULTS: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation. CONCLUSION: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.
- MeSH
- antigeny CD45 metabolismus MeSH
- buňky NK imunologie metabolismus účinky záření MeSH
- fosfoproteiny chemie metabolismus MeSH
- fosforylace účinky záření MeSH
- histony chemie metabolismus MeSH
- imunofenotypizace MeSH
- krysa rodu rattus MeSH
- podskupiny B-lymfocytů imunologie metabolismus účinky záření MeSH
- podskupiny lymfocytů imunologie metabolismus účinky záření MeSH
- potkani Wistar MeSH
- T-lymfocyty - podskupiny imunologie metabolismus účinky záření MeSH
- tolerance záření MeSH
- vztah dávky záření a odpovědi MeSH
- záření gama MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.
- MeSH
- apoptóza účinky léků účinky záření MeSH
- fosforylace účinky léků účinky záření MeSH
- histony genetika metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- kyselina valproová farmakologie MeSH
- leukemie metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- oprava DNA účinky léků MeSH
- poškození DNA účinky léků účinky záření MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- radiosenzibilizující látky farmakologie MeSH
- regulace genové exprese účinky léků účinky záření MeSH
- rho proteiny vázající GTP genetika metabolismus MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: Mesenchymal stem cells isolated from bone marrow (BM-MSC) and periodontal ligament (PLSC) are cells with high proliferative potential and ability to self-renewal. Characterization of these cells under genotoxic stress conditions contributes to the assessment of their prospective usage. The aim of our study was to evaluate changes in BM-MSC and PLSC caused by ionizing radiation. METHODS: Human BM-MSC and PLSC were irradiated with the doses up to 20 Gy by Co(60) and observed 13 days; viability, proliferation, apoptosis and senescence induction, and changes in expression and phosphorylation status of related proteins were studied. RESULTS: Irradiation with the doses up to 20 Gy significantly reduces proliferation, but has no significant effect on cell viability. The activation of tumor suppressor protein 53 (p53) and its phosphorylations on serines 15 and 392 were detected from the first day after irradiation by 20 Gy and remained elevated to day 13. Expression of cyclin-dependent kinases inhibitor 1A (p21(Cip1/Waf1)) increased. The cell cycle was arrested in G2 phase. Instead of apoptosis we have detected hallmarks of stress-induced premature senescence: increase in cyclin-dependent kinases inhibitor 2A (p16(INK4a)) and increased activity of senescence-associated β-galactosidase. CONCLUSION: Mesenchymal stem cells isolated from bone marrow and periodontal ligament respond to ionizing radiation by induction of stress-induced premature senescence without apparent differences in their radiation response.
- MeSH
- aktivace enzymů účinky záření MeSH
- apoptóza účinky záření MeSH
- buňky kostní dřeně cytologie MeSH
- dospělé kmenové buňky cytologie metabolismus účinky záření MeSH
- dvouřetězcové zlomy DNA účinky záření MeSH
- fosforylace účinky záření MeSH
- histony metabolismus MeSH
- inhibitor p16 cyklin-dependentní kinasy metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy metabolismus MeSH
- kaspasy metabolismus MeSH
- kontrolní body buněčného cyklu účinky záření MeSH
- lidé MeSH
- mezenchymální kmenové buňky cytologie metabolismus účinky záření MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- periodontální vaz cytologie MeSH
- proliferace buněk účinky záření MeSH
- regulace genové exprese účinky záření MeSH
- stárnutí buněk účinky záření MeSH
- viabilita buněk účinky záření MeSH
- záření gama škodlivé účinky MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH