WorldWideScience

Sample records for h2ax phosphorylation dna

  1. H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication.

    Science.gov (United States)

    Botting, Carolyn; Lu, Xu; Triezenberg, Steven J

    2016-01-27

    Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.

  2. Mechanism of elimination of phosphorylated histone H2AX from chromatin after repair of DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Svetlova, M.P., E-mail: svetlma@mail.ru [Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., 194064 St. Petersburg (Russian Federation); Solovjeva, L.V.; Tomilin, N.V. [Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky ave., 194064 St. Petersburg (Russian Federation)

    2010-03-01

    Covalent modifications of histones in chromatin play an important role in regulation of eukaryotic gene expression and DNA repair. Formation of double-strand breaks (DSBs) in DNA is followed by the rapid local phosphorylation of the C-terminal serine in the replacement histone H2AX in megabase chromatin domains around DSBs and formation of discrete nuclear foci called {gamma}H2AX foci. This epigenetic modification of chromatin represents the 'histone code' for DNA damage signaling and repair and has been extensively studied during last decade. It is known that after DSB rejoining {gamma}H2AX foci are eliminated from the nucleus, but molecular mechanism of this elimination remains to be established. However, {gamma}H2AX elimination can serve as a useful marker of DSB repair in normal cells and tissues. In this paper the available data on kinetics and possible mechanisms of {gamma}H2AX elimination are reviewed.

  3. Phosphorylated fraction of H2AX as a measurement for DNA damage in cancer cells and potential applications of a novel assay.

    Directory of Open Access Journals (Sweden)

    Jiuping Ji

    Full Text Available Phosphorylated H2AX (γ-H2AX is a sensitive marker for DNA double-strand breaks (DSBs, but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.

  4. Histone H2AX phosphorylation is associated with most meiotic events in grasshopper.

    Science.gov (United States)

    Cabrero, J; Teruel, M; Carmona, F D; Camacho, J P M

    2007-01-01

    It is widely accepted that the H2AX histone in its phosphorylated form (gamma-H2AX) is related to the repair of DNA double-strand breaks (DSBs). In several organisms, gamma-H2AX presence has been demonstrated in meiotic processes such as recombination and sex chromosome inactivation during prophase I (from leptotene to pachytene). To test whether gamma-H2AX is present beyond pachytene, we have analysed the complete sequence of changes in H2AX phosphorylation during meiosis in grasshopper, a model organism for meiotic studies at the cytological level. We show the presence of phosphorylated H2AX during most of meiosis, with the exception only of diplotene and the end of each meiotic division. During the first meiotic division, gamma-H2AX is associated with i) recombination, as deduced from its presence in leptotene-zygotene over all chromosome length, ii) X chromosome inactivation, since at pachytene gamma-H2AX is present in the X chromosome only, and iii) chromosome segregation, as deduced from gamma-H2AX presence in centromere regions at first metaphase-anaphase. During second meiotic division, gamma-H2AX was very abundant at most chromosome lengths from metaphase to telophase, suggesting its possible association with the maintenance of chromosome condensation and segregation. Copyright 2007 S. Karger AG, Basel.

  5. Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

    Directory of Open Access Journals (Sweden)

    Serena Barral

    2014-01-01

    Full Text Available Phosphorylation of the histone H2AXH2AX form is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU, we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ and the granule cell precursors (cerebellum. Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS degenerative diseases.

  6. BAZ1B is dispensable for H2AX phosphorylation on Tyrosine 142 during spermatogenesis

    Directory of Open Access Journals (Sweden)

    Tyler J. Broering

    2015-07-01

    Full Text Available Meiosis is precisely regulated by the factors involved in DNA damage response in somatic cells. Among them, phosphorylation of H2AX on Serine 139 (γH2AX is an essential signal for the silencing of unsynapsed sex chromosomes during male meiosis. However, it remains unknown how adjacent H2AX phosphorylation on Tyrosine 142 (pTyr142 is regulated in meiosis. Here we investigate the meiotic functions of BAZ1B (WSTF, the only known Tyr142 kinase in somatic cells, using mice possessing a conditional deletion of BAZ1B. Although BAZ1B deletion causes ectopic γH2AX signals on synapsed autosomes during the early pachytene stage, BAZ1B is dispensable for fertility and critical events during spermatogenesis. BAZ1B deletion does not alter events on unsynapsed axes and pericentric heterochromatin formation. Furthermore, BAZ1B is dispensable for localization of the ATP-dependent chromatin remodeling protein SMARCA5 (SNF2h during spermatogenesis despite the complex formation between BAZ1B and SMARCA5, known as the WICH complex, in somatic cells. Notably, pTyr142 is regulated independently of BAZ1B and is dephosphorylated on the sex chromosomes during meiosis in contrast with the presence of adjacent γH2AX. Dephosphorylation of pTyr142 is regulated by MDC1, a binding partner of γH2AX. These results reveal the distinct regulation of two adjacent phosphorylation sites of H2AX during meiosis, and suggest that another kinase mediates Tyr142 phosphorylation.

  7. Characteristics of {gamma}-H2AX foci at DNA double-strand breaks sites

    Energy Technology Data Exchange (ETDEWEB)

    Pilch, D.R.; Sedelnikova, O.A.; Redon, C. [National Cancer Institute, National Institutes of Health, Lab. of Molecular Pharmacology, Bethesda, Maryland (United States); Celeste, A.; Nussenzweig, A. [National Cancer Institute, National Institutes of Health, Experimental Immunology Branch, Bethesda, Maryland (United States); Bonner, W.M. [National Cancer Institute, National Institutes of Health, Lab. of Molecular Pharmacology, Bethesda, Maryland (United States)

    2003-06-01

    Phosphorylated H2AX ({gamma}-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to {gamma}-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX{sup {delta}}{sup /{delta}} mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. {gamma}-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of {gamma}-H2AX formation. (author)

  8. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    Science.gov (United States)

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylatedH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

  9. PET imaging of DNA damage using {sup 89}Zr-labelled anti-γH2AX-TAT immunoconjugates

    Energy Technology Data Exchange (ETDEWEB)

    Knight, James C.; Topping, Caitriona; Mosley, Michael; Kersemans, Veerle; Cornelissen, Bart [University of Oxford, CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom); Falzone, Nadia [University of Oxford, CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom); Royal Marsden Hospital, Sutton, Surrey (United Kingdom); Fernandez-Varea, Jose M. [Universitat de Barcelona, Facultat de Fisica (ECM and ICC), Barcelona (Spain)

    2015-10-15

    The efficacy of most anticancer treatments, including radiotherapy, depends on an ability to cause DNA double-strand breaks (DSBs). Very early during the DNA damage signalling process, the histone isoform H2AX is phosphorylated to form γH2AX. With the aim of positron emission tomography (PET) imaging of DSBs, we synthesized a {sup 89}Zr-labelled anti-γH2AX antibody, modified with the cell-penetrating peptide, TAT, which includes a nuclear localization sequence. {sup 89}Zr-anti-γH2AX-TAT was synthesized using EDC/NHS chemistry for TAT peptide linkage. Desferrioxamine conjugation allowed labelling with {sup 89}Zr. Uptake and retention of {sup 89}Zr-anti-γH2AX-TAT was evaluated in the breast adenocarcinoma cell line MDA-MB-468 in vitro or as xenografts in athymic mice. External beam irradiation was used to induce DSBs and expression of γH2AX. Since {sup 89}Zr emits ionizing radiation, detailed radiobiological measurements were included to ensure {sup 89}Zr-anti-γH2AX-TAT itself does not cause any additional DSBs. Uptake of {sup 89}Zr-anti-γH2AX-TAT was similar to previous results using {sup 111}In-anti-γH2AX-TAT. Retention of {sup 89}Zr-anti-γH2AX-TAT was eightfold higher at 1 h post irradiation, in cells expressing γH2AX, compared to non-irradiated cells or to non-specific IgG control. PET imaging of mice showed higher uptake of {sup 89}Zr-anti-γH2AX-TAT in irradiated xenografts, compared to non-irradiated or non-specific controls (12.1 ± 1.6 vs 5.2 ± 1.9 and 5.1 ± 0.8 %ID/g, respectively; p < 0.0001). The mean absorbed dose to the nucleus of cells taking up {sup 89}Zr-anti-γH2AX-TAT was twofold lower compared to {sup 111}In-anti-γH2AX-TAT. Additional exposure of neither irradiated nor non-irradiated cells nor tissues to {sup 89}Zr-anti-γH2AX-TAT resulted in any significant changes in the number of observable DNA DSBs, γH2AX foci or clonogenic survival. {sup 89}Zr-anti-γH2AX-TAT allows PET imaging of DNA DSBs in a tumour xenograft mouse model

  10. Role of H2AX in DNA damage response and human cancers.

    NARCIS (Netherlands)

    Srivastava, N.; Gochhait, S.; Boer, P. de; Bamezai, R.N.

    2009-01-01

    H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and

  11. ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

    Directory of Open Access Journals (Sweden)

    Traganos Frank

    2007-06-01

    Full Text Available Abstract Background In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs such as ataxia telangiectasia mutated (ATM, ATM-and Rad-3 related (ATR kinase, or by DNA dependent protein kinase (DNA-PKcs. When DNA damage primarily involves formation of DNA double-strand breaks (DSBs, H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE to cigarette smoke (CS induced phosphorylation of H2AX. Results We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AXH2AX (R = 0.89. In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm. Conclusion These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences

  12. Use of the γ-H2AX assay to investigate DNA repair dynamics following multiple radiation exposures.

    Directory of Open Access Journals (Sweden)

    Luca G Mariotti

    Full Text Available Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX phosphorylation (γ-H2AX, a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

  13. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

    Directory of Open Access Journals (Sweden)

    Duensing Stefan

    2008-07-01

    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

  14. H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib.

    Science.gov (United States)

    Dong, Yaqiong; Xiong, Min; Duan, Lianning; Liu, Ze; Niu, Tianhui; Luo, Yuan; Wu, Xinpin; Xu, Chengshan; Lu, Chengrong

    2014-08-01

    Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

  15. Spatiotemporal kinetics of γ-H2AX protein on charged particles induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Niu, H., E-mail: hniu@mx.nthu.edu.tw [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Chang, H.C. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China); Cho, I.C. [Institute for Radiological Research, Chang Gung University and Chang Gung Memorial Hospital, Taoyuan, Taiwan (China); Chen, C.H. [Nuclear Science and Technology Development Center, National Tsing Hua University, Hsinchu, Taiwan (China); Liu, C.S. [Cancer Center of Taipei Veterans General Hospital, Taipei, Taiwan (China); Chou, W.T. [Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan (China)

    2014-08-15

    Highlights: • Charged particles can induce more complex DNA damages, and these complex damages have higher ability to cause the cell death or cell carcinogenesis. • In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in particle irradiated HeLa cells. • The HeLa cells were irradiated by 400 keV alpha-particles in four different dosages. • The result shows that a good linear relationship can be observed between foci number and radiation dose. • The data shows that the dissolution rate of γ-H2AX foci agree with the two components DNA repairing model, and it was decreasing as the radiation dose increased. • These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA repair. - Abstract: In several researches, it has been demonstrated that charged particles can induce more complex DNA damages. These complex damages have higher ability to cause the cell death or cell carcinogenesis. For this reason, clarifying the DNA repair mechanism after charged particle irradiation plays an important role in the development of charged particle therapy and space exploration. Unfortunately, the detail spatiotemporal kinetic of DNA damage repair is still unclear. In this study, we used γ-H2AX protein to investigate the spatiotemporal kinetics of DNA double strand breaks in alpha-particle irradiated HeLa cells. The result shows that the intensity of γ-H2AX foci increased gradually, and reached to its maximum at 30 min after irradiation. A good linear relationship can be observed between foci intensity and radiation dose. After 30 min, the γ-H2AX foci intensity was decreased with time passed, but remained a large portion (∼50%) at 48 h passed. The data show that the dissolution rate of γ-H2AX foci agreed with two components DNA repairing model. These results suggest that charged particles can induce more complex DNA damages and causing the retardation of DNA

  16. γ-H2AX as a biomarker for DNA double-strand breaks in ecotoxicology.

    Science.gov (United States)

    Gerić, Marko; Gajski, Goran; Garaj-Vrhovac, Vera

    2014-07-01

    The visualisation of DNA damage response proteins enables the indirect measurement of DNA damage. Soon after the occurrence of a DNA double-strand break (DSB), the formation of γ-H2AX histone variants is to be expected. This review is focused on the potential use of the γ-H2AX foci assay in assessing the genotoxicity of environmental contaminants including cytostatic pharmaceuticals, since standard methods may not be sensitive enough to detect the damaging effect of low environmental concentrations of such drugs. These compounds are constantly released into the environment, potentially representing a threat to water quality, aquatic organisms, and, ultimately, human health. Our review of the literature revealed that this method could be used in the biomonitoring and risk assessment of aquatic systems affected by wastewater from the production, usage, and disposal of cytostatic pharmaceuticals. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Deregulation of BRCA1 leads to impaired spatiotemporal dynamics of γ-H2AX and DNA damage responses in Huntington's disease.

    Science.gov (United States)

    Jeon, Gye Sun; Kim, Ki Yoon; Hwang, Yu Jin; Jung, Min-Kyung; An, Sungkwan; Ouchi, Mutsuko; Ouchi, Toru; Kowall, Neil; Lee, Junghee; Ryu, Hoon

    2012-06-01

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder of mid-life onset characterized by involuntary movements and progressive cognitive decline caused by a CAG repeat expansion in exon 1 of the Huntingtin (Htt) gene. Neuronal DNA damage is one of the major features of neurodegeneration in HD, but it is not known how it arises or relates to the triplet repeat expansion mutation in the Htt gene. Herein, we found that imbalanced levels of non-phosphorylated and phosphorylated BRCA1 contribute to the DNA damage response in HD. Notably, nuclear foci of γ-H2AX, the molecular component that recruits various DNA damage repair factors to damage sites including BRCA1, were deregulated when DNA was damaged in HD cell lines. BRCA1 specifically interacted with γ-H2AX via the BRCT domain, and this association was reduced in HD. BRCA1 overexpression restored γ-H2AX level in the nucleus of HD cells, while BRCA1 knockdown reduced the spatiotemporal propagation of γ-H2AX foci to the nucleoplasm. The deregulation of BRCA1 correlated with an abnormal nuclear distribution of γ-H2AX in striatal neurons of HD transgenic (R6/2) mice and BRCA1(+/-) mice. Our data indicate that BRCA1 is required for the efficient focal recruitment of γ-H2AX to the sites of neuronal DNA damage. Taken together, our results show that BRCA1 directly modulates the spatiotemporal dynamics of γ-H2AX upon genotoxic stress and serves as a molecular maker for neuronal DNA damage response in HD.

  18. Loss of H3K9me3 Correlates with ATM Activation and Histone H2AX Phosphorylation Deficiencies in Hutchinson-Gilford Progeria Syndrome.

    Directory of Open Access Journals (Sweden)

    Haoyue Zhang

    Full Text Available Compelling evidence suggests that defective DNA damage response (DDR plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS. Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3. In this study, we explore the potential connection(s between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox, double strand breaks (DSBs are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency.

  19. Calmodulin Mediates DNA Repair Pathways Involving H2AX in Response to Low-Dose Radiation Exposure of RAW 264.7 Macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Smallwood, Heather S.; Lopez Ferrer, Daniel; Eberlein, P. Elis; Watson, David J.; Squier, Thomas C.

    2009-02-05

    Understanding the molecular mechanisms that modulate macrophage radioresistance is necessary for the development of effective radiation therapies, as tumor-associated macrophages promote both angiogenesis and matrix remodeling that, in turn, enhance metastasis. In this respect, we have identified a dose-dependent increase in the abundance of the calcium regulatory protein calmodulin (CaM) in RAW 264.7 macrophages upon irradiation. CaM overexpression results in increased macrophage survival following radiation exposure, acting to diminish the sensitivity to low-dose exposures. Increases in CaM abundance also result in an increase in the number of phosphorylated histone H2AX protein complexes associated with DNA repair following macrophage irradiation, with no change in the extent of double-stranded DNA damage. In comparison, when NFκB-dependent pathways are inhibited, through the expression of a dominant-negative IκB construct, there is no significant increase in phosphorylated H2AX upon irradiation. These results indicate that the molecular basis for the up-regulation of histone H2AX mediated DNA-repair pathways is not the result of nonspecific NFκB-dependent pathways or a specific threshold of DNA damage. Rather, increases in CaM abundance act to minimize the low-dose hypersensitivity to radiation to enhance macrophage radioresistance through processes that include the upregulation of DNA repair pathways involving histone protein H2AX phosphorylation.

  20. Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study.

    Directory of Open Access Journals (Sweden)

    Michael Brand

    Full Text Available Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes.Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC and Q 10. Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX.For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%, selenium (14%, vitamin E (12%, vitamin C (25%, NAC (43% and Q 10 (18% led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect.Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes.

  1. Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs) Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study

    Science.gov (United States)

    Brand, Michael; Sommer, Matthias; Ellmann, Stephan; Wuest, Wolfgang; May, Matthias S.; Eller, Achim; Vogt, Sabine; Lell, Michael M.; Kuefner, Michael A.; Uder, Michael

    2015-01-01

    Background Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT) and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs) as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes. Materials and Methods Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC) and Q 10). Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX. Results For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%), selenium (14%), vitamin E (12%), vitamin C (25%), NAC (43%) and Q 10 (18%) led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect. Conclusion Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes. PMID:25996998

  2. Induction and Persistence of Large γH2AX Foci by High Linear Energy Transfer Radiation in DNA-Dependent protein kinase–Deficient Cells

    Energy Technology Data Exchange (ETDEWEB)

    Bracalente, Candelaria; Ibañez, Irene L. [Departamento de Micro y Nanotecnología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Molinari, Beatriz [Departamento de Radiobiología, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Palmieri, Mónica [Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (Argentina); Kreiner, Andrés [Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires (Argentina); Gerencia de Investigación y Aplicaciones, Comisión Nacional de Energía Atómica, San Martín, Buenos Aires (Argentina); Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); Valda, Alejandro [Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, San Martín, Buenos Aires (Argentina); and others

    2013-11-15

    Purpose: To evaluate the cell response to DNA double-strand breaks induced by low and high linear energy transfer (LET) radiations when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an essential protein of the nonhomologous end-joining repair pathway, lacks kinase activity. Methods and Materials: CHO10B2, a Chinese hamster ovary cell line, and its derived radiosensitive mutant cell line, irs-20, lacking DNA-PKcs activity, were evaluated after 0 to 3 Gy of γ-rays, plateau and Bragg peak protons, and lithium beams by clonogenic assay, and as a measurement of double-strand breaks, phosphorylated H2AXH2AX) foci number and size were quantified by immunocytofluorescence. Results: Irs-20 exhibited greater radiosensitivity and a higher amount of γH2AX foci than CHO10B2 at 6 hours after irradiation for all types of radiations. Remarkably, CHO10B2 and irs-20 maintained their difference in radiosensitivity after high-LET radiation. Six hours after low-LET radiations, irs-20 did not reach basal levels of γH2AX at high doses, whereas CHO10B2 recovered basal levels for all doses. After high-LET radiation, only CHO10B2 exhibited a reduction in γH2AX foci, but it never reached basal levels. Persistent foci in irs-20 confirmed a repair deficiency. Interestingly, after 30 minutes of high-LET radiation both cell lines exhibited large foci (size >0.9 μm{sup 2}) related to the damage nature, whereas at 6 hours irs-20 showed a higher amount of large foci than CHO10B2, with a 7-fold increase at 3 Gy, that could also be associated to radiosensitivity. Conclusions: We demonstrated, for the first time, an association between deficient DNA-PKcs activity and not only high levels of H2AX phosphorylation but also persistence and size increase of γH2AX foci after high-LET irradiation.

  3. Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homologue (H2AvB).

    Science.gov (United States)

    Siddiqui, Mohammad S; Filomeni, Erika; François, Maxime; Collins, Samuel R; Cooper, Tamara; Glatz, Richard V; Taylor, Phillip W; Fenech, Michael; Leifert, Wayne R

    2013-09-01

    The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or 'Q-fly') to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose-response of γH2AvB (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies.

  4. USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Aman Wang

    2017-05-01

    Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

  5. A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Bakkenist, Christopher J; Czambel, R Kenneth; Hershberger, Pamela A; Tawbi, Hussein; Beumer, Jan H; Schmitz, John C

    2015-01-01

    Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

  6. Phosphorylation of H2AX histones in response to double-strand breaks and induction of premature chromatin condensation in hydroxyurea-treated root meristem cells of Raphanus sativus, Vicia faba, and Allium porrum.

    Science.gov (United States)

    Rybaczek, Dorota; Maszewski, Janusz

    2007-01-01

    Histone H2A variant H2AX is rapidly phosphorylated on the induction of DNA double-strand breaks by ionizing radiation and hydroxyurea-mediated replication arrest, resulting in the formation of gamma-H2AX foci along megabase chromatin domains nearby the sites of incurred DNA damage. In an attempt to establish a relationship between species-specific nuclear architecture and H2AX phosphorylation in S/G(2) phase-arrested root meristem cells, immunocytochemical comparisons using an antibody raised against human gamma-H2AX were made among three plants differing with respect to DNA contents: Allium porrum, representing a reticulate type of DNA package, Vicia faba, having semireticulate cell nuclei, and Raphanus sativus, characterised by a chromocentric type of chromatin. Another approach was aimed at determining possible correlations between the extent of hydroxyurea-induced phosphorylation of H2AX histones and the quantities of root meristem cells induced by caffeine to enter aberrant mitotic division (premature chromosome condensation). It was concluded that the higher-order structure of chromatin may contribute to the accessibility of molecular factors engaged in the recognition and repair of genetic lesions. Consequently, in contrast to A. porrum and V. faba, a diffuse chromatin in chromocentric cell nuclei of R. sativus may become more vulnerable both to generate DNA double-strand breaks and to recruit molecular elements needed to arrange the cell cycle checkpoint functions, and thus, more resistant to factors which allow the cells to enter premature chromosome condensation spontaneously. On the other hand, however, caffeine-mediated overriding of the S-M checkpoint control system resulted in the typical appearance of premature chromosome condensation, irrespective of the genomic content of DNA.

  7. γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis.

    Science.gov (United States)

    Ivashkevich, Alesia N; Martin, Olga A; Smith, Andrea J; Redon, Christophe E; Bonner, William M; Martin, Roger F; Lobachevsky, Pavel N

    2011-06-03

    The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens. 2011 Elsevier B.V. All rights reserved.

  8. {gamma}H2AX foci as a measure of DNA damage: A computational approach to automatic analysis

    Energy Technology Data Exchange (ETDEWEB)

    Ivashkevich, Alesia N. [Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St. Andrew' s Place, East Melbourne, Victoria 3002 (Australia); Martin, Olga A. [Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, D.H.H.S., Bethesda, MD 20892 (United States); Smith, Andrea J. [Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St. Andrew' s Place, East Melbourne, Victoria 3002 (Australia); Redon, Christophe E.; Bonner, William M. [Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, D.H.H.S., Bethesda, MD 20892 (United States); Martin, Roger F. [Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St. Andrew' s Place, East Melbourne, Victoria 3002 (Australia); Lobachevsky, Pavel N., E-mail: pavel.lobachevsky@petermac.org [Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St. Andrew' s Place, East Melbourne, Victoria 3002 (Australia)

    2011-06-03

    The {gamma}H2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised {gamma}H2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of {gamma}H2AX focus induction for these biological specimens.

  9. Measurement of DNA damage in peripheral blood by the γ-H2AX assay as predictor of colorectal cancer risk.

    Science.gov (United States)

    Zhao, Lina; Chang, David W; Gong, Yilei; Eng, Cathy; Wu, Xifeng

    2017-05-01

    The detection of γ-H2AX focus is one of the most sensitive ways to monitor DNA double-strand breaks (DSBs). Although changes in γ-H2AX activity have been studied in tumor cells in colorectal cancer (CRC), changes in peripheral blood lymphocytes (PBLs) have not been examined previously. We hypothesize that higher levels of irradiation-induced γ-H2AX in PBLs may be associated with an elevated risk of colorectal cancer (CRC). In a case-control study, the baseline and ionizing radiation (IR)-induced γ-H2AX levels in PBLs from frequency-matched 320 untreated CRC patients and 320 controls were detected by a laser scanning cytometer-based immunocytochemical method. We used unconditional multivariable logistic regression to evaluate CRC risk by using the ratio of IR-induced γ-H2AX to the baseline levels with adjustment of age, sex and smoking status. We found CRC cases had significantly higher γ-H2AX ratio (1.5 vs. 1.41, Prisk of CRC (OR=6.72, 95% CI=4.54-9.94). Quartile analyses also showed significant dose-response relationship between higher γ-H2AX ratio and increased risk of CRC (P for trendrisk; however, no interactions with γ-H2AX ratio were observed. These results support the premise that DSBs in peripheral blood as measured by γ-H2AX level might represent an intermediate phenotype to assess the risk of CRC. Future prospective studies are necessary to confirm our findings in independent populations. Copyright © 2017. Published by Elsevier B.V.

  10. ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation

    Directory of Open Access Journals (Sweden)

    Yuko Atsumi

    2015-12-01

    Full Text Available In response to DNA double-strand breaks (DSBs, H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.

  11. Double strand break repair functions of histone H2AX

    Energy Technology Data Exchange (ETDEWEB)

    Scully, Ralph, E-mail: rscully@bidmc.harvard.edu; Xie, Anyong

    2013-10-15

    Chromosomal double strand breaks provoke an extensive reaction in neighboring chromatin, characterized by phosphorylation of histone H2AX on serine 139 of its C-terminal tail (to form “γH2AX”). The γH2AX response contributes to the repair of double strand breaks encountered in a variety of different contexts, including those induced by ionizing radiation, physiologically programmed breaks that characterize normal immune cell development and the pathological exposure of DNA ends triggered by telomere dysfunction. γH2AX also participates in the evolutionarily conserved process of sister chromatid recombination, a homologous recombination pathway involved in the suppression of genomic instability during DNA replication and directly implicated in tumor suppression. At a biochemical level, the γH2AX response provides a compelling example of how the “histone code” is adapted to the regulation of double strand break repair. Here, we review progress in research aimed at understanding how γH2AX contributes to double strand break repair in mammalian cells.

  12. Expression of DNA Damage Response Molecules PARP1, γH2AX, BRCA1, and BRCA2 Predicts Poor Survival of Breast Carcinoma Patients

    Directory of Open Access Journals (Sweden)

    See-Hyoung Park

    2015-08-01

    Full Text Available BACKGROUND: Poly(ADP-ribose polymerase 1 (PARP1, γH2AX, BRCA1, and BRCA2 are conventional molecular indicators of DNA damage in cells and are often overexpressed in various cancers. In this study, we aimed, using immunohistochemical detection, whether the co-expression of PARP1, γH2AX, BRCA1, and BRCA2 in breast carcinoma (BCA tissue can provide more reliable prediction of survival of BCA patients. MATERIALS AND METHODS: We investigated immunohistochemical expression and prognostic significance of the expression of PARP1, γH2AX, BRCA1, and BRCA2 in 192 cases of BCAs. RESULTS: The expression of these four molecules predicted earlier distant metastatic relapse, shorter overall survival (OS, and relapse-free survival (RFS by univariate analysis. Multivariate analysis revealed the expression of PARP1, γH2AX, and BRCA2 as independent poor prognostic indicators of OS and RFS. In addition, the combined expressional pattern of BRCA1, BRCA2, PARP1, and γH2AX (CSbbph was an additional independent prognostic predictor for OS (P < .001 and RFS (P < .001. The 10-year OS rate was 95% in the CSbbph-low (CSbbph scores 0 and 1 subgroup, but that was only 35% in the CSbbph-high (CSbbph score 4 subgroup. CONCLUSION: This study has demonstrated that the individual and combined expression patterns of PARP1, γH2AX, BRCA1, and BRCA2 could be helpful in determining an accurate prognosis for BCA patients and for the selection of BCA patients who could potentially benefit from anti-PARP1 therapy with a combination of genotoxic chemotherapeutic agents.

  13. Comparison of two methods for measuring γ-H2AX nuclear fluorescence as a marker of DNA damage in cultured human cells: applications for microbeam radiation therapy

    Science.gov (United States)

    Anderson, D.; Andrais, B.; Mirzayans, R.; Siegbahn, E. A.; Fallone, B. G.; Warkentin, B.

    2013-06-01

    Microbeam radiation therapy (MRT) delivers single fractions of very high doses of synchrotron x-rays using arrays of microbeams. In animal experiments, MRT has achieved higher tumour control and less normal tissue toxicity compared to single-fraction broad beam irradiations of much lower dose. The mechanism behind the normal tissue sparing of MRT has yet to be fully explained. An accurate method for evaluating DNA damage, such as the γ-H2AX immunofluorescence assay, will be important for understanding the role of cellular communication in the radiobiological response of normal and cancerous cell types to MRT. We compare two methods of quantifying γ-H2AX nuclear fluorescence for uniformly irradiated cell cultures: manual counting of γ-H2AX foci by eye, and an automated, MATLAB-based fluorescence intensity measurement. We also demonstrate the automated analysis of cell cultures irradiated with an array of microbeams. In addition to offering a relatively high dynamic range of γ-H2AX signal versus irradiation dose ( > 10 Gy), our automated method provides speed, robustness, and objectivity when examining a series of images. Our in-house analysis facilitates the automated extraction of the spatial distribution of the γ-H2AX intensity with respect to the microbeam array — for example, the intensities in the peak (high dose area) and valley (area between two microbeams) regions. The automated analysis is particularly beneficial when processing a large number of samples, as is needed to systematically study the relationship between the numerous dosimetric and geometric parameters involved with MRT (e.g., microbeam width, microbeam spacing, microbeam array dimensions, peak dose, valley dose, and geometric arrangement of multiple arrays) and the resulting DNA damage.

  14. Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci.

    Science.gov (United States)

    Jezkova, Lucie; Zadneprianetc, Mariia; Kulikova, Elena; Smirnova, Elena; Bulanova, Tatiana; Depes, Daniel; Falkova, Iva; Boreyko, Alla; Krasavin, Evgeny; Davidkova, Marie; Kozubek, Stanislav; Valentova, Olga; Falk, Martin

    2018-01-18

    Biological effects of high-LET (linear energy transfer) radiation have received increasing attention, particularly in the context of more efficient radiotherapy and space exploration. Efficient cell killing by high-LET radiation depends on the physical ability of accelerated particles to generate complex DNA damage, which is largely mediated by LET. However, the characteristics of DNA damage and repair upon exposure to different particles with similar LET parameters remain unexplored. We employed high-resolution confocal microscopy to examine phosphorylated histone H2AXH2AX)/p53-binding protein 1 (53BP1) focus streaks at the microscale level, focusing on the complexity, spatiotemporal behaviour and repair of DNA double-strand breaks generated by boron and neon ions accelerated at similar LET values (∼135 keV μm-1) and low energies (8 and 47 MeV per n, respectively). Cells were irradiated using sharp-angle geometry and were spatially (3D) fixed to maximize the resolution of these analyses. Both high-LET radiation types generated highly complex γH2AX/53BP1 focus clusters with a larger size, increased irregularity and slower elimination than low-LET γ-rays. Surprisingly, neon ions produced even more complex γH2AX/53BP1 focus clusters than boron ions, consistent with DSB repair kinetics. Although the exposure of cells to γ-rays and boron ions eliminated a vast majority of foci (94% and 74%, respectively) within 24 h, 45% of the foci persisted in cells irradiated with neon. Our calculations suggest that the complexity of DSB damage critically depends on (increases with) the particle track core diameter. Thus, different particles with similar LET and energy may generate different types of DNA damage, which should be considered in future research.

  15. gammaH2AX foci form preferentially in euchromatin after ionising-radiation.

    Directory of Open Access Journals (Sweden)

    Ian G Cowell

    Full Text Available BACKGROUND: The histone variant histone H2A.X comprises up to 25% of the H2A complement in mammalian cells. It is rapidly phosphorylated following exposure of cells to double-strand break (DSB inducing agents such as ionising radiation. Within minutes of DSB generation, H2AX molecules are phosphorylated in large chromatin domains flanking DNA double-strand breaks (DSBs; these domains can be observed by immunofluorescence microscopy and are termed gammaH2AX foci. H2AX phosphorylation is believed to have a role mounting an efficient cellular response to DNA damage. Theoretical considerations suggest an essentially random chromosomal distribution of X-ray induced DSBs, and experimental evidence does not consistently indicate otherwise. However, we observed an apparently uneven distribution of gammaH2AX foci following X-irradiation with regions of the nucleus devoid of foci. METHODOLOGY/PRINCIPLE FINDINGS: Using immunofluorescence microscopy, we show that focal phosphorylation of histone H2AX occurs preferentially in euchromatic regions of the genome following X-irradiation. H2AX phosphorylation has also been demonstrated previously to occur at stalled replication forks induced by UV radiation or exposure to agents such as hydroxyurea. In this study, treatment of S-phase cells with hydroxyurea lead to efficient H2AX phosphorylation in both euchromatin and heterochromatin at times when these chromatin compartments were undergoing replication. This suggests a block to H2AX phosphorylation in heterochromatin that is at least partially relieved by ongoing DNA replication. CONCLUSIONS/SIGNIFICANCE: We discuss a number of possible mechanisms that could account for the observed pattern of H2AX phosphorylation. Since gammaH2AX is regarded as forming a platform for the recruitment or retention of other DNA repair and signaling molecules, these findings imply that the processing of DSBs in heterochromatin differs from that in euchromatic regions. The

  16. Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines.

    Science.gov (United States)

    Bourton, Emma C; Plowman, Piers N; Zahir, Sheba Adam; Senguloglu, Gonul Ulus; Serrai, Hiba; Bottley, Graham; Parris, Christopher N

    2012-02-01

    The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types. Copyright © 2011 International Society for Advancement of Cytometry.

  17. The profiles of gamma-H2AX along with ATM/DNA-PKcs activation in the lymphocytes and granulocytes of rat and human blood exposed to gamma rays

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong [Fudan University, Department of Radiation Biology, Institute of Radiation Medicine, Shanghai (China)

    2016-08-15

    Establishing a rat model suitable for γ-H2AX biodosimeter studies has important implications for dose assessment of internal radionuclide contamination in humans. In this study, γ-H2AX, p-ATM and p-DNA-PKcs foci were enumerated using immunocytofluorescence method, and their protein levels were measured by Western blot in rat blood lymphocytes and granulocytes exposed to γ-rays compared with human blood lymphocytes and granulocytes. It was found that DNA double-strand break repair kinetics and linear dose responses in rat lymphocytes were similar to those observed in the human counterparts. Moreover, radiation induced clear p-ATM and p-DNA-PKcs foci formation and an increase in ratio of co-localization of p-ATM or p-DNA-PKcs with γ-H2AX foci in rat lymphocytes similar to those of human lymphocytes. The level of γ-H2AX protein in irradiated rat and human lymphocytes was significantly reduced by inhibitors of ATM and DNA-PKcs. Surprisingly, unlike human granulocytes, rat granulocytes with DNA-PKcs deficiency displayed a rapid accumulation, but delayed disappearance of γ-H2AX foci with essentially no change from 10 h to 48 h post-irradiation. Furthermore, inhibition of ATM activity in rat granulocytes also decreased radiation-induced γ-H2AX foci formation. In comparison, human granulocytes showed no response to irradiation regarding γ-H2AX, p-ATM or p-DNA-PKcs foci. Importantly, incidence of γ-H2AX foci in lymphocytes after total-body radiation of rats was consistent with that of in vitro irradiation of rat lymphocytes. These findings show that rats are a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. ATM and DNA-PKcs participate together in DSB repair in rat lymphocytes similar to that of human lymphocytes. Further, rat granulocytes, which have the characteristic of delayed disappearance of γ-H2AX foci in response to radiation, may be a useful experimental system for biodosimetry studies. (orig.)

  18. The profiles of gamma-H2AX along with ATM/DNA-PKcs activation in the lymphocytes and granulocytes of rat and human blood exposed to gamma rays.

    Science.gov (United States)

    Wang, Jing; Yin, Lina; Zhang, Junxiang; Zhang, Yaping; Zhang, Xuxia; Ding, Defang; Gao, Yun; Li, Qiang; Chen, Honghong

    2016-08-01

    Establishing a rat model suitable for γ-H2AX biodosimeter studies has important implications for dose assessment of internal radionuclide contamination in humans. In this study, γ-H2AX, p-ATM and p-DNA-PKcs foci were enumerated using immunocytofluorescence method, and their protein levels were measured by Western blot in rat blood lymphocytes and granulocytes exposed to γ-rays compared with human blood lymphocytes and granulocytes. It was found that DNA double-strand break repair kinetics and linear dose responses in rat lymphocytes were similar to those observed in the human counterparts. Moreover, radiation induced clear p-ATM and p-DNA-PKcs foci formation and an increase in ratio of co-localization of p-ATM or p-DNA-PKcs with γ-H2AX foci in rat lymphocytes similar to those of human lymphocytes. The level of γ-H2AX protein in irradiated rat and human lymphocytes was significantly reduced by inhibitors of ATM and DNA-PKcs. Surprisingly, unlike human granulocytes, rat granulocytes with DNA-PKcs deficiency displayed a rapid accumulation, but delayed disappearance of γ-H2AX foci with essentially no change from 10 h to 48 h post-irradiation. Furthermore, inhibition of ATM activity in rat granulocytes also decreased radiation-induced γ-H2AX foci formation. In comparison, human granulocytes showed no response to irradiation regarding γ-H2AX, p-ATM or p-DNA-PKcs foci. Importantly, incidence of γ-H2AX foci in lymphocytes after total-body radiation of rats was consistent with that of in vitro irradiation of rat lymphocytes. These findings show that rats are a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. ATM and DNA-PKcs participate together in DSB repair in rat lymphocytes similar to that of human lymphocytes. Further, rat granulocytes, which have the characteristic of delayed disappearance of γ-H2AX foci in response to radiation, may be a useful experimental system for biodosimetry studies.

  19. Nitric Oxide Induces Ataxia Telangiectasia Mutated (ATM) Protein-dependent γH2AX Protein Formation in Pancreatic β Cells*

    Science.gov (United States)

    Oleson, Bryndon J.; Broniowska, Katarzyna A.; Schreiber, Katherine H.; Tarakanova, Vera L.; Corbett, John A.

    2014-01-01

    In this study, the effects of cytokines on the activation of the DNA double strand break repair factors histone H2AX (H2AX) and ataxia telangiectasia mutated (ATM) were examined in pancreatic β cells. We show that cytokines stimulate H2AX phosphorylationH2AX formation) in rat islets and insulinoma cells in a nitric oxide- and ATM-dependent manner. In contrast to the well documented role of ATM in DNA repair, ATM does not appear to participate in the repair of nitric oxide-induced DNA damage. Instead, nitric oxide-induced γH2AX formation correlates temporally with the onset of irreversible DNA damage and the induction of apoptosis. Furthermore, inhibition of ATM attenuates cytokine-induced caspase activation. These findings show that the formation of DNA double strand breaks correlates with ATM activation, irreversible DNA damage, and ATM-dependent induction of apoptosis in cytokine-treated β cells. PMID:24610783

  20. Microwaves from UMTS/GSM mobile phones induce long-lasting inhibition of 53BP1/gamma-H2AX DNA repair foci in human lymphocytes.

    Science.gov (United States)

    Belyaev, Igor Y; Markovà, Eva; Hillert, Lena; Malmgren, Lars O G; Persson, Bertil R R

    2009-02-01

    We have recently described frequency-dependent effects of mobile phone microwaves (MWs) of global system for mobile communication (GSM) on human lymphocytes from persons reporting hypersensitivity to electromagnetic fields and healthy persons. Contrary to GSM, universal global telecommunications system (UMTS) mobile phones emit wide-band MW signals. Hypothetically, UMTS MWs may result in higher biological effects compared to GSM signal because of eventual "effective" frequencies within the wideband. Here, we report for the first time that UMTS MWs affect chromatin and inhibit formation of DNA double-strand breaks co-localizing 53BP1/gamma-H2AX DNA repair foci in human lymphocytes from hypersensitive and healthy persons and confirm that effects of GSM MWs depend on carrier frequency. Remarkably, the effects of MWs on 53BP1/gamma-H2AX foci persisted up to 72 h following exposure of cells, even longer than the stress response following heat shock. The data are in line with the hypothesis that the type of signal, UMTS MWs, may have higher biological efficiency and possibly larger health risk effects compared to GSM radiation emissions. No significant differences in effects between groups of healthy and hypersensitive subjects were observed, except for the effects of UMTS MWs and GSM-915 MHz MWs on the formation of the DNA repair foci, which were different for hypersensitive (P 0.05). The non-parametric statistics used here did not indicate specificity of the differences revealed between the effects of GSM and UMTS MWs on cells from hypersensitive subjects and more data are needed to study the nature of these differences. Copyright 2008 Wiley-Liss, Inc.

  1. Assessment of γ-H2AX levels in circulating tumor cells from patients receiving chemotherapy

    Directory of Open Access Journals (Sweden)

    Alejandra eGarcia-Villa

    2012-10-01

    Full Text Available Circulating tumor cells (CTCs are prognostic markers in a variety of solid tumor malignancies. The potential of CTCs to be used as a liquid biopsy to monitor a patient’s condition and predict drug response and resistance is currently under investigation. Using a negative depletion, enrichment methology, CTCs isolated from the peripheral blood of breast cancer patients with stage IV breast cancer undergoing DNA damaging therapy with platinum based therapy were enriched. The enriched cell suspensions, were stained with an optimized labeling protocol targeting: nuclei, cytokeratins 8, 18, and 19, the surface marker CD45, and the presence of the protein ɣ-H2AX. As a direct or indirect result of platinum therapy, double strand break of DNA initiates phosphorylation of the histone H2AX, at serine 139; this phosphorylated form is referred to as ɣ-H2AX. In addition to ɣ-H2AX staining in specific locations with the cell nuclei, consistent with previous reports and referred to as foci, more general staining in the cell cytoplamim was also observed in some cells suggesting the potential of cell apoptosis. Our study underscores the utility and the complexity of investigating CTCs as predictive markers of response to various therapies. Additional studies are ongoing to evaluate the diverse γ-H2AX staining patterns we report here which needs to be further correlated with patient outcomes

  2. Age-related disease association of endogenous γ-H2AX foci in mononuclear cells derived from leukapheresis.

    Directory of Open Access Journals (Sweden)

    Shepherd H Schurman

    Full Text Available The phosphorylated form of histone H2AX (γ-H2AX forms immunohistochemically detectable foci at DNA double strand breaks. In peripheral blood mononuclear cells (PBMCs derived from leukapheresis from patients enrolled in the Baltimore Longitudinal Study of Aging, γ-H2AX foci increased in a linear fashion with regards to age, peaking at ~57 years. The relationship between the frequency of γ-H2AX foci and age-related pathologies was assessed. We found a statistically significant (p = 0.023 50% increase in foci in PBMCs derived from patients with a known history of vitamin D deficiency. In addition, there were trends toward increased γ-H2AX foci in patients with cataracts (34% increase, p<0.10 and in sleep apnea patients (44%, p<0.10. Among patients ≥57 y/o, we found a significant (p = 0.037 36% increase in the number of γ-H2AX foci/cell for patients with hypertension compared to non-hypertensive patients. Our results support a role for increased DNA damage in the morbidity of age-related diseases. γ -H2AX may be a biomarker for human morbidity in age-related diseases.

  3. Immunofluorescence detection of clustered gamma-H2AX foci induced by HZE-particle radiation.

    Science.gov (United States)

    Desai, N; Davis, E; O'Neill, P; Durante, M; Cucinotta, F A; Wu, H

    2005-10-01

    We studied the spatial and temporal distributions of foci of the phosphorylated form of the histone protein H2AX (gamma-H2AX), which is known to be activated by double-strand breaks after irradiation of human fibroblast cells with high-energy silicon (54 keV/microm) and iron (176 keV/microm) ions. Here we present data obtained with the ion path parallel to a monolayer of human fibroblast cells that leads to gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane, thus enabling the analysis of the fluorescence distributions along the ion trajectories. Qualitative analyses of these distributions provide insights into DNA damage processing kinetics for high charge and energy (HZE) ions, including evidence of increased clustering of DNA damage and slower processing with increasing LET.

  4. Activation of H2AX and ATM in varicella-zoster virus (VZV)-infected cells is associated with expression of specific VZV genes.

    Science.gov (United States)

    Yamamoto, Takenobu; Ali, Mir A; Liu, XueQiao; Cohen, Jeffrey I

    2014-03-01

    Mammalian cells activate DNA damage response pathways in response to virus infections. Activation of these pathways can enhance replication of many viruses, including herpesviruses. Activation of cellular ATM results in phosphorylation of H2AX and recruits proteins to sites of DNA damage. We found that varicella-zoster (VZV) infected cells had elevated levels of phosphorylated H2AX and phosphorylated ATM and that these levels increased in cells infected with VZV deleted for ORF61 or ORF63, but not deleted for ORF67. Expression of VZV ORF61, ORF62, or ORF63 alone did not result in phosphorylation of H2AX. While BGLF4, the Epstein-Barr virus homolog of VZV ORF47 protein kinase, phosphorylates H2AX and ATM, neither VZV ORF47 nor ORF66 protein kinase phosphorylated H2AX or ATM. Cells lacking ATM had no reduction in VZV replication. Thus, VZV induces phosphorylation of H2AX and ATM and this effect is associated with the presence of specific VZV genes in virus-infected cells. Published by Elsevier Inc.

  5. γ-H2AX induced by linear alkylbenzene sulfonates is due to deoxyribonuclease-1 translocation to the nucleus via actin disruption

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Xiaoxu; Toyooka, Tatsushi; Kubota, Toru; Yang, Guang; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2015-07-15

    Graphical abstract: - Highlights: • Non-genotoxic linear alkylbenzene sulfonates (LAS) generated γ-H2AX. • The γ-H2AX was not induced through direct LAS-induced DNA damage. • LAS weakened interactions between actin and DNase I. • Released DNase I translocated to nucleus and broke DNA strands, generating γ-H2AX. • This is a novel pathway for chemically induced γ-H2AX. - Abstract: Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C{sub 10}–C{sub 14}LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl{sub 2}, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.

  6. Utility of γH2AX as a molecular marker of DNA double-strand breaks in nuclear medicine: applications to radionuclide therapy employing auger electron-emitting isotopes.

    Science.gov (United States)

    Mah, Li-Jeen; Orlowski, Christian; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-01-01

    There is an intense interest in the development of radiopharmaceuticals for cancer therapy. In particular, radiopharmaceuticals which involve targeting radionuclides specifically to cancer cells with the use of monoclonal antibodies (radioimmunotherapy) or peptides (targeted radiotherapy) are being widely investigated. For example, the ultra-short range Auger electron-emitting isotopes, which are discussed in this review, are being considered in the context of DNAtargeted radiotherapy. The efficient quantitative evaluation of the levels of damage caused by such potential radiopharmaceuticals is required for assessment of therapeutic efficacy and determination of relevant doses for successful treatment. The DNA double-strand break surrogate marker, γH2AX, has emerged as a useful biomonitor of damage and thus effectiveness of treatment, offering a highly specific and sensitive means of assessment. This review will cover the potential applications of γH2AX in nuclear medicine, in particular radionuclide therapy.

  7. Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells.

    Science.gov (United States)

    Pustovalova, Margarita; Grekhova, Anna; Astrelina, Тatiana; Nikitina, Viktoria; Dobrovolskaya, Ekaterina; Suchkova, Yulia; Kobzeva, Irina; Usupzhanova, Darya; Vorobyeva, Natalia; Samoylov, Aleksandr; Bushmanov, Andrey; Ozerov, Ivan V; Zhavoronkov, Alex; Leonov, Sergey; Klokov, Dmitry; Osipov, Andreyan N

    2016-12-11

    Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γaH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.

  8. Constitutive expression of gamma-H2AX has prognostic relevance in triple negative breast cancer

    NARCIS (Netherlands)

    Nagelkerke, A.P.; Kuijk, S.J. van; Sweep, F.C.; Nagtegaal, I.D.; Hoogerbrugge, N.; Martens, J.W.; Timmermans, M.A.Y.; Laarhoven, H.W.M. van; Bussink, J.; Span, P.N.

    2011-01-01

    BACKGROUND AND PURPOSE: Constitutive gamma-H2AX expression might indicate disruption of the DNA damage repair pathway, genomic instability, or shortened telomeric ends. Here, we quantified expression of endogenous gamma-H2AX and its downstream factor 53BP1 in a large number of breast cancer cell

  9. Evaluating γH2AX in spermatozoa from male infertility patients.

    Science.gov (United States)

    Zhong, Hui-zhi; Lv, Fu-tong; Deng, Xue-lian; Hu, Ying; Xie, Dan-ni; Lin, Bin; Mo, Zeng-nan; Lin, Fa-quan

    2015-09-01

    To investigate whether γH2AX levels were different in the spermatozoa of healthy men compared with infertility patients, and to assess the possible correlations between γH2AX and conventional semen parameters and double-stranded breaks (DSBs) identified with the use of comet assay. Prospective study. Clinical laboratory. Semen from 100 male infertile patients and 100 healthy sperm donors. Human sperm samples were analyzed in terms of World Health Organization parameters. The γH2AX levels were detected by means of flow cytometry. DSBs of sperm were detected by means of comet assay. Morphology slides were made and the sperm morphology assessed according to strict criteria. Conventional semen analyses, γH2AX levels in sperm, DNA DSBs in sperm, and correlations among γH2AX, conventional semen analyses, and DSBs. Concentration, viability, motility, and normal sperm morphology were significantly lower in male infertility patients compared with healthy men. Also, γH2AX levels and the number of DSBs were significantly higher in the sperm of infertile subjects compared with healthy men. γH2AX levels correlated negatively with conventional semen parameters and positively with DSBs. A threshold γH2AX level of 18.55% was identified as a cutoff value to discriminate infertile subjects from fertile control subjects with a specificity of 86.0% and a sensitivity of 83.0%. The positive and negative predictive values of the 18.55% γH2AX threshold were high: 87.7% and 85.5%, respectively. γH2AX levels were higher in the sperm of male infertility patients than in healthy men. γH2AX levels in sperm, as evaluated with the use of flow cytometry, might be a useful biomarker for evaluating DSBs in human spermatozoa. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  10. Phospho-histone H2AX is a diagnostic and prognostic marker for epithelial ovarian cancer.

    Science.gov (United States)

    Mei, Ling; Hu, Qian; Peng, Jing; Ruan, Jiaying; Zou, Juan; Huang, Qin; Liu, Shanling; Wang, He

    2015-01-01

    Histone H2AX phosphorylation is a sensitive marker for DSB which contributes to both genomic instability and cancer treatment. Monitoring its formation may be a sensitive means to monitor cancer progression and treatment effect. To define the role of phospho-H2AX (pH2AX) expression in development and prognosis of epithelial ovarian cancer (EOC). The expression of pH2AX in 87 EOC samples and 28 samples of normal ovarian tissues were examined by immunohistochemistry (IHC). The results were semi-quantitatively scored and analyzed by chi-square test. The overall survival time (OS) and disease free interval (DFI) were collected by follow-up and analyzed by Kaplan-Meier analysis. The expression level of pH2AX protein in EOC were higher than that in normal tissues (P<0.001). Among the sensitive cases, high expression of pH2AX was found in 53.2% cases while for resistant cases, high expression rate was 80% (P=0.025). However, pH2AX expression was not significantly correlated with age, histopathological type, tumor differentiation, lymph node metastasis or FIGO stages. Kaplan-Meier analysis found that DFI was negatively correlated with the pH2AX expression, where higher expression of pH2AX resulted in shorter DFI while no OS difference was detected in our study. pH2AX may be used to detect EOC at an early stage and identify women at higher risk for relapse.

  11. Targeting protein for xenopus kinesin-like protein 2 (TPX2) regulates γ-histone 2AX (γ-H2AX) levels upon ionizing radiation.

    Science.gov (United States)

    Neumayer, Gernot; Helfricht, Angela; Shim, Su Yeon; Le, Hoa Thi; Lundin, Cecilia; Belzil, Camille; Chansard, Mathieu; Yu, Yaping; Lees-Miller, Susan P; Gruss, Oliver J; van Attikum, Haico; Helleday, Thomas; Nguyen, Minh Dang

    2012-12-07

    The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

  12. Computational analysis of the number, area and density of gamma-H2AX foci in breast cancer cells exposed to (111)In-DTPA-hEGF or gamma-rays using Image-J software.

    Science.gov (United States)

    Cai, Zhongli; Vallis, Katherine A; Reilly, Raymond M

    2009-03-01

    To develop a simple method for the quantification of gamma-H2AX focus number, density and size. MDA-MB-468 human breast cancer cells were treated overnight with (111)In-diethylenetriaminepentaacetic acid human epidermal growth factor ((111)In-DTPA-hEGF, 0-142 kBq/pmol) or exposed to gamma-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (gamma-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of gamma-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify gamma-H2AX foci. The number of gamma-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R(2) = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R(2) values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of gamma-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R(2) values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the gamma-ray absorbed dose up to 3.0 Gy. The density, area and IntDen of individual foci, as well as the number of gamma-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.

  13. γ-H2AX expression detected by immunohistochemistry correlates with prognosis in early operable non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Matthaios D

    2012-10-01

    Full Text Available Dimitrios Matthaios,1 Periklis G Foukas,2 Maria Kefala,2 Panagiotis Hountis,3 Grigorios Trypsianis,4 Ioannis G Panayiotides,2 Ekaterini Chatzaki,5 Ekaterini Pantelidaki,6 Demosthenes Bouros,7 Petros Karakitsos,8 Stylianos Kakolyris11Department of Oncology, Democritus University of Thrace, Alexandroupolis, Greece; 2Department of Pathology, Attikon University Hospital, Athens, Greece; 3Cardiac Surgery Department, Athens Naval and Veterans Hospital, Athens, Greece; 4Laboratory of Statistics, Democritus University of Thrace, Alexandroupolis, Greece; 5Laboratory of Pharmacology, Democritus University of Thrace, Alexandroupolis, Greece; 6Department of Pathology, Evaggelismos Hospital, Athens, Greece; 7Department of Pneumonology, Democritus University of Thrace, Alexandroupolis, Greece; 8Department of Cytopathology, Attikon University Hospital, Athens, GreeceBackground: Phosphorylation of the H2AX histone is an early indicator of DNA double-strand breaks and of the resulting DNA damage response. In the present study, we assessed the expression and prognostic significance of γ-H2AX in a cohort of 96 patients with operable non-small cell lung carcinoma.Methods: Ninety-six paraffin-embedded specimens of non-small cell lung cancer patients were examined. All patients underwent radical thoracic surgery of primary tumor (lobectomy or pneumonectomy and regional lymph node dissection. γ-H2AX expression was assessed by standard immunohistochemistry. Follow-up was available for all patients; mean duration of follow-up was 27.50 ± 14.07 months (range 0.2–57 months, median 24 months.Results: Sixty-three patients (65.2% died during the follow-up period. The mean survival time was 32.2 ± 1.9 months (95% confidence interval [CI]: 28.5–35.8 months; median 30.0 months; 1-, 2- and 3-year survival rates were 86.5% ± 3.5%, 57.3% ± 5.1%, and 37.1% ± 5.4%, respectively. Low γ-H2AX expression was associated with a significantly better survival as compared with

  14. Effect of prolonging radiation delivery time on retention of gammaH2AX

    Directory of Open Access Journals (Sweden)

    Duzenli Cheryl

    2008-06-01

    Full Text Available Abstract Background and purpose Compared to conventional external beam radiotherapy, IMRT requires significantly more time to deliver the dose. Prolonging dose delivery potentially increases DNA repair which would reduce the biological effect. We questioned whether retention of γH2AX, a measure of lack of repair of DNA damage, would decrease when dose delivery was protracted. Materials and methods Exponentially growing SiHa cervical carinoma cells were irradiated with 6 MV photons in a water tank using a VarianEX linear accelerator. Cells held at 37°C received 2 Gy in 0.5 min and 4 Gy in 1 min. To evaluate effect of dose delivery prolongation, 2 and 4 Gy were delivered in 30 and 60 min. After 24 h recovery, cells were analyzed for clonogenic survival and for residual γH2AX as measured using flow cytometry. Results Increasing the dose delivery time from 0.5 or 1 min to 30 or 60 min produced a signficant increase in cell survival from 0.45 to 0.48 after 2 Gy, and from 0.17 to 0.20 after 4 Gy. Expression of residual γH2AX decreased from 1.27 to 1.22 relative to background after 2 Gy and 1.46 to 1.39 relative to background after 4 Gy, but differences were not statistically significant. The relative differences in the slopes of residual γH2AX versus dose for acute versus prolonged irradiation bordered on significant (p = 0.055, and the magnitude of the change was consistent with the observed increase in surviving fraction. Conclusion These results support the concept that DNA repair underlies the increase in survival observed when dose delivery is prolonged. They also help to establish the limits of sensitivity of residual γH2AX, as measured using flow cytometry, for detecting differences in response to irradiation.

  15. Effect of prolonging radiation delivery time on retention of gammaH2AX.

    Science.gov (United States)

    Moiseenko, Vitali; Banáth, Judit P; Duzenli, Cheryl; Olive, Peggy L

    2008-06-27

    Compared to conventional external beam radiotherapy, IMRT requires significantly more time to deliver the dose. Prolonging dose delivery potentially increases DNA repair which would reduce the biological effect. We questioned whether retention of gammaH2AX, a measure of lack of repair of DNA damage, would decrease when dose delivery was protracted. Exponentially growing SiHa cervical carcinoma cells were irradiated with 6 MV photons in a water tank using a VarianEX linear accelerator. Cells held at 37 degrees C received 2 Gy in 0.5 min and 4 Gy in 1 min. To evaluate effect of dose delivery prolongation, 2 and 4 Gy were delivered in 30 and 60 min. After 24 h recovery, cells were analyzed for clonogenic survival and for residual gammaH2AX as measured using flow cytometry. Increasing the dose delivery time from 0.5 or 1 min to 30 or 60 min produced a significant increase in cell survival from 0.45 to 0.48 after 2 Gy, and from 0.17 to 0.20 after 4 Gy. Expression of residual gammaH2AX decreased from 1.27 to 1.22 relative to background after 2 Gy and 1.46 to 1.39 relative to background after 4 Gy, but differences were not statistically significant. The relative differences in the slopes of residual gammaH2AX versus dose for acute versus prolonged irradiation bordered on significant (p = 0.055), and the magnitude of the change was consistent with the observed increase in surviving fraction. These results support the concept that DNA repair underlies the increase in survival observed when dose delivery is prolonged. They also help to establish the limits of sensitivity of residual gammaH2AX, as measured using flow cytometry, for detecting differences in response to irradiation.

  16. Suberoylanilide hydroxamic acid affects {gamma}H2AX expression in osteosarcoma, atypical teratoid rhabdoid tumor and normal tissue cell lines after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Blattmann, C.; Oertel, S.; Thiemann, M.; Weber, K.J.; Schmezer, P.; Zelezny, O.; Lopez Perez, R.; Kulozik, A.E.; Debus, J.; Ehemann, V. [Univ. Children' s Hospital, Heidelberg (Germany). Dept. of Pediatric Oncology, Hematology, Immunology and Pulmology

    2012-02-15

    Osteosarcoma and atypical teratoid rhabdoid tumors are tumor entities with varying response to common standard therapy protocols. Histone acetylation affects chromatin structure and gene expression which are considered to influence radiation sensitivity. The aim of this study was to investigate the effect of the combination therapy with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and irradiation on atypical teratoid rhabdoid tumors and osteosarcoma compared to normal tissue cell lines. Clonogenic assay was used to determine cell survival. DNA double-strand breaks (DSB) were examined by pulsed-field electrophoresis (PFGE) as well as by {gamma}H2AX immunostaining involving flow cytometry, fluorescence microscopy, and immunoblot analysis. SAHA lead to an increased radiosensitivity in tumor but not in normal tissue cell lines. {gamma}H2AX expression as an indicator for DSB was significantly increased when SAHA was applied 24 h before irradiation to the sarcoma cell cultures. In contrast, {gamma}H2AX expression in the normal tissue cell lines was significantly reduced when irradiation was combined with SAHA. Analysis of initial DNA fragmentation and fragment rejoining by PFGE, however, did not reveal differences in response to the SAHA pretreatment for either cell type. SAHA increases radiosensitivity in tumor but not normal tissue cell lines. The increased H2AX phosphorylation status of the SAHA-treated tumor cells post irradiation likely reflects its delayed dephosphorylation within the DNA damage signal decay rather than chromatin acetylation-dependent differences in the overall efficacy of DSB induction and rejoining. The results support the hypothesis that combining SAHA with irradiation may provide a promising strategy in the treatment of solid tumors. (orig.)

  17. Characteristics of changes in the number of yH2AX and Rad51 protein foci in human skin fibroblasts after prolonged exposure to low-dose rate X-ray radiation

    Directory of Open Access Journals (Sweden)

    Ozerov I.V.

    2014-12-01

    Full Text Available Aim: to compare the repair process of DNA double-strand breaks in mammalian cells after acute versus prolonged exposure to X-ray irradiation with different dose rates. Material and methods. Studies were performed on primary human fibroblasts isolated from skin biopsies of healthy volunteers (women, 29 and 30 years. Cells were irradiated using an X-ray machine RUB RUST-M1 (JSC "Ruselectronics", Moscow, Russia at 37°C temperature with a dose rate of 400 mGy/min (200 kV, 2*2.4 mA, a filter of 1.5mm AI or 4 mGy/min (50 kV, 2*0.4 mA, a filter of 1.5 mm AI. Immuno-cytochemical protein staining was utilized for yH2AX and Rad51 foci analysis. Results. Phosphorylated histone H2AX (yH2AX and the key protein of homologous recombination Rad51 foci formation and disappearance kinetics were investigated simultaneously in primary human dermal fibroblasts after acute and prolonged exposure to X-ray radiation at a same dose. It was shown that the relative yield of yH2AX foci per dose reduces with decrease in dose rate, while the relative yield of Rad51 foci conversely increases. Conclusion. Our findings suggest the fundamental differences in the ratio of non-homologous end joining and homologous recombination DNA repair in acute versus prolonged irradiated cells.

  18. High throughput measurement of γH2AX DSB repair kinetics in a healthy human population.

    Directory of Open Access Journals (Sweden)

    Preety M Sharma

    Full Text Available The Columbia University RABiT (Rapid Automated Biodosimetry Tool quantifies DNA damage using fingerstick volumes of blood. One RABiT protocol quantifies the total γ-H2AX fluorescence per nucleus, a measure of DNA double strand breaks (DSB by an immunofluorescent assay at a single time point. Using the recently extended RABiT system, that assays the γ-H2AX repair kinetics at multiple time points, the present small scale study followed its kinetics post irradiation at 0.5 h, 2 h, 4 h, 7 h and 24 h in lymphocytes from 94 healthy adults. The lymphocytes were irradiated ex vivo with 4 Gy γ rays using an external Cs-137 source. The effect of age, gender, race, ethnicity, alcohol use on the endogenous and post irradiation total γ-H2AX protein yields at various time points were statistically analyzed. The endogenous γ-H2AX levels were influenced by age, race and alcohol use within Hispanics. In response to radiation, induction of γ-H2AX yields at 0.5 h and peak formation at 2 h were independent of age, gender, ethnicity except for race and alcohol use that delayed the peak to 4 h time point. Despite the shift in the peak observed, the γ-H2AX yields reached close to baseline at 24 h for all groups. Age and race affected the rate of progression of the DSB repair soon after the yields reached maximum. Finally we show a positive correlation between endogenous γ-H2AX levels with radiation induced γ-H2AX yields (RIY (r=0.257, P=0.02 and a negative correlation with residuals (r=-0.521, P=<0.0001. A positive correlation was also observed between RIY and DNA repair rate (r=0.634, P<0.0001. Our findings suggest age, race, ethnicity and alcohol use influence DSB γ-H2AX repair kinetics as measured by RABiT immunofluorescent assay.

  19. High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Giovannini, Caterina [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy); Piaggi, Simona [Sezione di Patologia Sperimentale, Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Pisa University, Pisa (Italy); Federico, Giovanni [Unità di Endocrinologia Pediatrica e Diabete, Dipartimento di Medicina Clinica e Sperimentale Pisa University, Pisa (Italy); Scarpato, Roberto, E-mail: roberto.scarpato@unipi.it [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy)

    2014-12-15

    Highlights: • We aimed to detect signs of very early damage in peripheral cells of T1DM adolescents. • T1DM patients had high levels of oxidized cells and reduced expression of iNOS and NO. • Highly mutagenic lesions were markedly increased in the diabetic group, mainly in females. • The observed damage might increase the risk of cancer in the patients later in life. - Abstract: Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H{sub 2}O{sub 2}-induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O{sub 2}{sup −}; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H{sub 2}O{sub 2}-induced (47.13 ± 0.02) were significantly higher than in controls (35.90 ± 0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15 ± 1.08% of γ-H2AX{sup +} cells; 8.72 ± 2.14 γ-H2AXF/n; 9.26 ± 2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents

  20. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    Energy Technology Data Exchange (ETDEWEB)

    Yamagata, Kazutsune, E-mail: kyamagat@ncc.go.jp [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kitabayashi, Issay [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  1. Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

    Science.gov (United States)

    Nikolova, Teodora; Marini, Federico; Kaina, Bernd

    2017-10-01

    Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H2O2) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H2O2, less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier B

  2. Cell size matters in gamma-H2AX assay for low-dose alpha particle effect assessment

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ui seob; Kim, Eun Hee [Seoul National University, Daejeon (Korea, Republic of)

    2016-05-15

    Gamma-H2AX assay is an immuno-fluorescence experiment that enables detecting the location and number of DNA double strand breaks (DSBs) in cells. Under uniform radiation beam intensity, cells would respond with similar numbers of gamma-H2AX if they are similar in cross section. If cells are not represented by a common size, however, a larger cell has a greater chance of radiation exposure and has a better chance of counting a greater number of foci. In other words, the cell size distribution would be reflected in the FPC distribution. In the conventional gamma-H2AX assay, the mean FPC value solely indicates the level of cellular damage under a certain radiation exposure. The purpose of this study is to investigate the FPC distribution in connection with the cell size distribution. The high-LET alpha beam was employed for radiation exposure so that a single track of radiation leaves a meaningful amount of energy in the cell. Gamma-H2AX is a powerful tool for investigating the cellular response at low-dose exposure. If the gamma-H2AX assay is performed with cells of the same size, 'the average number of foci per cell' may accord with the overall response of sample cells to radiation exposure. With cells of non-uniform size, however, one should be cautious in taking the value as an index of the severity in cellular effect of radiation exposure. According to our experiments, a portion of sample cells carried DSBs of more than 5 times greater number than the mean FPC value and might play a critical role in radio-response.

  3. Identification of the elementary structural units of the DNA damage response

    OpenAIRE

    De Natale, F.; Rapp, A.; Yu, W.; Maiser, A.; Harz, H; Scholl, A.; Grulich, S.; Anton, T.; Hoerl, D.; Chen, W.; Durante, M; Taucher-Scholz, G.; Leonhardt, H.; Cardoso, M. C.

    2017-01-01

    Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heter...

  4. Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

    Directory of Open Access Journals (Sweden)

    Yuko Atsumi

    Full Text Available Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

  5. Effect of antioxidants on X-ray-induced γ-H2AX foci in human blood lymphocytes: preliminary observations.

    Science.gov (United States)

    Kuefner, Michael A; Brand, Michael; Ehrlich, James; Braga, Larissa; Uder, Michael; Semelka, Richard C

    2012-07-01

    To investigate the effect of a radioprotective oral agent containing a formulation of antioxidants and glutathione-elevating compounds on the extent of x-ray-induced γ-H2AX foci formation. The study was approved by local ethics committee and informed consent was obtained from each subject. In vitro experiments with blood lymphocytes of 25 healthy volunteers were performed without antioxidants and with antioxidants added either before or immediately after irradiation (10 mGy). For in vivo/in vitro tests, blood samples were obtained before, 15, 30, and 60 minutes (n=17) after, and 2, 3, and 5 hours (n=11) after oral ingestion of antioxidant pills and were irradiated (10 mGy). DNA double-strand breaks (DSBs) were quantified in isolated lymphocytes 5 minutes (in vitro and in vivo/in vitro) and 15 minutes (in vitro) after irradiation by enumerating γ-H2AX foci. To validate the data, additional in vitro experiments with use of 53BP1 as another independent marker for DSBs were performed. Nonirradiated samples served as controls. Statistical analyses were performed by using Wilcoxon rank-sum tests (in vitro), repeated-measures test, and Dunnett test (in vivo/in vitro). In the in vitro experiments, 15-minute preincubation with antioxidants significantly reduced mean γ-H2AX foci levels by 23% (Plead to a reduction of x-ray-induced foci (P=.6905). Mean 53BP1 foci were also reduced by preincubation with the radioprotectant. In the in vivo/in vitro tests, oral pretreatment with antioxidants also led to a significant reduction of γ-H2AX foci formation; administration 60 minutes before irradiation resulted in a mean foci reduction of 58% (Pleads to a significant reduction in foci. © RSNA, 2012.

  6. X-ray induced formation of γ-H2AX foci after full-field digital mammography and digital breast-tomosynthesis.

    Directory of Open Access Journals (Sweden)

    Siegfried A Schwab

    Full Text Available PURPOSE: To determine in-vivo formation of x-ray induced γ-H2AX foci in systemic blood lymphocytes of patients undergoing full-field digital mammography (FFDM and to estimate foci after FFDM and digital breast-tomosynthesis (DBT using a biological phantom model. MATERIALS AND METHODS: The study complies with the Declaration of Helsinki and was performed following approval by the ethic committee of the University of Erlangen-Nuremberg. Written informed consent was obtained from every patient. For in-vivo tests, systemic blood lymphocytes were obtained from 20 patients before and after FFDM. In order to compare in-vivo post-exposure with pre-exposure foci levels, the Wilcoxon matched pairs test was used. For in-vitro experiments, isolated blood lymphocytes from healthy volunteers were irradiated at skin and glandular level of a porcine breast using FFDM and DBT. Cells were stained against the phosphorylated histone variant γ-H2AX, and foci representing distinct DNA damages were quantified. RESULTS: Median in-vivo foci level/cell was 0.086 (range 0.067-0.116 before and 0.094 (0.076-0.126 after FFDM (p = 0.0004. In the in-vitro model, the median x-ray induced foci level/cell after FFDM was 0.120 (range 0.086-0.140 at skin level and 0.035 (range 0.030-0.050 at glandular level. After DBT, the median x-ray induced foci level/cell was 0.061 (range 0.040-0.081 at skin level and 0.015 (range 0.006-0.020 at glandular level. CONCLUSION: In patients, mammography induces a slight but significant increase of γ-H2AX foci in systemic blood lymphocytes. The introduced biological phantom model is suitable for the estimation of x-ray induced DNA damages in breast tissue in different breast imaging techniques.

  7. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamashita, Sachiko [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Tanaka, Masakazu [Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010 (Japan); Sato, Teruaki [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Ida, Chieri [Department of Applied Life Studies, College of Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610 (Japan); Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Moss, Joel [Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590 (United States); Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)

    2016-08-05

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

  8. γ-H2AX as a marker for dose deposition in the brain of wistar rats after synchrotron microbeam radiation.

    Directory of Open Access Journals (Sweden)

    Cristian Fernandez-Palomo

    Full Text Available Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT.Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.

  9. Identification of the elementary structural units of the DNA damage response.

    Science.gov (United States)

    Natale, Francesco; Rapp, Alexander; Yu, Wei; Maiser, Andreas; Harz, Hartmann; Scholl, Annina; Grulich, Stephan; Anton, Tobias; Hörl, David; Chen, Wei; Durante, Marco; Taucher-Scholz, Gisela; Leonhardt, Heinrich; Cardoso, M Cristina

    2017-06-12

    Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.

  10. Establishment of a γ-H2AX foci-based assay to determine biological dose of radon to red bone marrow in rats

    Science.gov (United States)

    Wang, Jing; He, Linfeng; Fan, Dunhuang; Ding, Defang; Wang, Xufei; Gao, Yun; Zhang, Xuxia; Li, Qiang; Chen, Honghong

    2016-07-01

    The biodosimetric information is critical for assessment of cancer risk in populations exposed to high radon. However, no tools are available for biological dose estimation following radon exposure. Here, we established a γ-H2AX foci-based assay to determine biological dose to red bone marrow (RBM) in radon-inhaled rats. After 1-3 h of in vitro radon exposure, a specific pattern of γ-H2AX foci, linear tracks with individual p-ATM and p-DNA-PKcs foci, was observed, and the yield of γ-H2AX foci and its linear tracks displayed a linear dose-response manner in both rat peripheral blood lymphocytes (PBLs) and bone-marrow lymphocytes (BMLs). When the cumulative doses of radon inhaled by rats reached 14, 30 and 60 working level months (WLM), the yields of three types of foci markedly increased in both PBLs and BMLs, and γ-H2AX foci-based dose estimates to RBM were 0.97, 2.06 and 3.94 mGy, respectively. Notably, BMLs displayed a more profound increase of three types of foci than PBLs, and the absorbed dose ratio between BMLs and PBLs was similar between rats exposed to 30 and 60 WLM of radon. Taken together, γ-H2AX foci quantitation in PBLs is able to estimate RBM-absorbed doses with the dose-response curve of γ-H2AX foci after in vitro radon exposure and the ratio of RBM- to PBL-absorbed doses in rats following radon exposure.

  11. Histone H2AX Is Involved in FoxO3a-Mediated Transcriptional Responses to Ionizing Radiation to Maintain Genome Stability

    Directory of Open Access Journals (Sweden)

    Stephane Tarrade

    2015-12-01

    Full Text Available Histone H2AX plays a crucial role in molecular and cellular responses to DNA damage and in the maintenance of genome stability. It is downstream of ataxia telangiectasia mutated (ATM damage signaling pathway and there is an emerging role of the transcription factor FoxO3a, a regulator of a variety of other pathways, in activating this signaling. We asked whether H2AX may feedback to FoxO3a to affect respective FoxO3a-dependent pathways. We used a genetically matched pair of mouse embryonic fibroblast H2AX+/+ and H2AX−/− cell lines to carry out comprehensive time-course and dose-response experiments and to show that the expression of several FoxO3a-regulated genes was altered in H2AX−/− compared to H2AX+/+ cells at both basal and irradiated conditions. Hspa1b and Gadd45a were down-regulated four- to five-fold and Ddit3, Cdkn1a and Sod2 were up-regulated 2–3-fold in H2AX−/− cells. Using the luciferase reporter assay, we directly demonstrated that transcriptional activity of FoxoO3a was reduced in H2AX−/− cells. FoxO3a localization within the nuclear phospho-ATM (Ser1981 foci in irradiated cells was affected by the H2AX status, as well as its posttranslational modification (phospho-Thr32. These differences were associated with genomic instability and radiosensitivity in H2AX−/− cells. Finally, knockdown of H2AX in H2AX+/+ cells resulted in FoxO3a-dependent gene expression patterns and increased radiosensitivity that partially mimicked those found in H2AX−/− cells. Taken together, our data suggest a role for FoxO3a in the maintenance of genome integrity in response to DNA damage that is mediated by H2AX via yet unknown mechanisms.

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

    Science.gov (United States)

    Nakajima, Nakako Izumi; Brunton, Holly; Watanabe, Ritsuko; Shrikhande, Amruta; Hirayama, Ryoichi; Matsufuji, Naruhiro; Fujimori, Akira; Murakami, Takeshi; Okayasu, Ryuichi; Jeggo, Penny; Shibata, Atsushi

    2013-01-01

    Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle. PMID:23967070

  13. Visualisation of γH2AX foci caused by heavy ion particle traversal; distinction between core track versus non-track damage.

    Directory of Open Access Journals (Sweden)

    Nakako Izumi Nakajima

    Full Text Available Heavy particle irradiation produces complex DNA double strand breaks (DSBs which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.

  14. Biochemical Kinetics Model of DSB Repair and GammaH2AX FOCI by Non-homologous End Joining

    Science.gov (United States)

    Cucinotta, Francis, A.; Pluth, Janice M.; Anderson, Jennifer A.; Harper, Jane V.; O'Neill, Peter

    2007-01-01

    We developed a biochemical kinetics approach to describe the repair of double strand breaks (DSB) produced by low LET radiation by modeling molecular events associated with the mechanisms of non-homologous end-joining (NHEJ). A system of coupled non-linear ordinary differential equations describes the induction of DSB and activation pathways for major NHEJ components including Ku(sub 70/80), DNA-PK(sub cs), and the Ligase IV-XRCC4 hetero-dimer. The autophosphorylation of DNA-PK(sub cs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of DNA-PK(sub cs) regulation of repair was developed with the initial step allowing access of other NHEJ components to breaks, and a second step limiting access to Ligase IV-XRCC4. Our model assumes that the transition from the first to second-step depends on DSB complexity, with a much slower-rate for complex DSB. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field electrophoresis (PFGE), quantification of the induction of gamma-H2AX foci, and live cell imaging of the induction of Ku(sub 70/80). Predictions are made for the behaviors of NHEJ components at low doses and dose-rates, where a steady-state is found at dose-rates of 0.1 Gy/hr or lower.

  15. Jumonji domain-containing protein 2B silencing induces DNA damage response via STAT3 pathway in colorectal cancer

    National Research Council Canada - National Science Library

    Chen, L; Fu, L; Kong, X; Xu, J; Wang, Z; Ma, X; Akiyama, Y; Chen, Y; Fang, J

    2014-01-01

    .... Immunofluorescence and western blotting detected phosphorylated histone H2AX, characteristic of double-strand breaks, and comet assay was used to investigate DNA damage, in CRC cells after JMJD2B small interfering RNA (siRNA) transfection...

  16. Relative biological efficiency of protons at low and therapeutic doses in induction of 53BP1/γH2AX foci in lymphocytes from umbilical cord blood.

    Science.gov (United States)

    Sorokina, Svetlana; Markova, Eva; Gursky, Jan; Dobrovodsky, Jozef; Belyaev, Igor

    2013-09-01

    In order to evaluate DNA damage induced by protons at low and radiotherapeutic doses at the therapeutic proton complex at Ružomberok, Slovak Republic, lymphocytes from umbilical cord blood (UCB) of the same four probands were irradiated in the dose range of 1-200 cGy with γ-rays and protons (200 MeV, irradiation in the Bragg peak). DNA repair γH2AX/53BP1 foci were analyzed by fluorescent microscopy and flow cytometry. Statistically significant effects of radiations were detected by fluorescent microscopy at all doses higher 1 cGy. Almost all distributions of foci in irradiated cells fitted to the Poisson distribution. In general, there was no difference in the levels of γH2AX and 53BP1 foci in irradiated cells. Flow cytometry was less sensitive and detected radiation induced effects at doses of 50 cGy and higher. Factorial analysis of variance in the whole studied dose range has shown no significant effect of radiation quality on number of γH2AX and 53BP1 foci. The ratio of proton-induced foci to γ-ray-induced foci was 0.86 ± 0.16 (53BP1) and 0.99 ± 0.34 (γH2AX) as measured by fluorescent microscopy and 0.99 ± 0.16 (γH2AX) as measured by flow cytometry at the radiotherapeutic dose of 2 Gy. Both flow cytometry and fluorescent microscopy indicated that the average value of relative biological efficiency (RBE) at radiation doses ≥ 20 cGy was about 1.0. Our data that RBE increased at low doses ≤ 20 cGy are relevant both to the development of treatment modalities and exposures that take place during space exploration and should be verified by further studies.

  17. Investigating γ H2AX as a Biomarker of Radiosensitivity Using Flow Cytometry Methods.

    Science.gov (United States)

    Beaton, Lindsay A; Marro, Leonora; Malone, Shawn; Samiee, Sara; Grimes, Scott; Malone, Kyle; Wilkins, Ruth C

    2013-01-01

    Background and Purpose. This project examined the in vitro   γ H2AX response in lymphocytes of prostate cancer patients who had a radiosensitive response after receiving radiotherapy. The goal of this project was to determine whether the γ H2AX response, as measured by flow cytometry, could be used as a marker of individual patient radiosensitivity. Materials and Methods. Patients were selected from a randomized clinical trial evaluating the optimal timing of Dose Escalated Radiation and short-course Androgen Deprivation Therapy. Of 438 patients, 3% developed Grade 3 late radiation proctitis and were considered to be radiosensitive. Blood was drawn from 10 of these patients along with 20 matched samples from patients with Grade 0 proctitis. Dose response curves up to 10 Gy along with time response curves after 2 Gy (0-24 h) were generated for each sample. The γ H2AX response in lymphocytes and lymphocyte subsets was analyzed by flow cytometry. Results. There were no significant differences between the radiosensitive and control samples for either the dose course or the time course. Conclusions. Although γ H2AX response has previously been demonstrated to be an indicator of individual patient radiosensitivity, flow cytometry lacks the sensitivity necessary to distinguish any differences between samples from control and radiosensitive patients.

  18. Survival Fraction at 2 Gy and γH2AX Expression Kinetics in Peripheral Blood Lymphocytes From Cancer Patients: Relationship With Acute Radiation-Induced Toxicities

    Energy Technology Data Exchange (ETDEWEB)

    Pouliliou, Stamatia E. [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Lialiaris, Theodoros S. [Department of Medical Genetics, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Dimitriou, Thespis [Department of Anatomy, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Giatromanolaki, Alexandra [Department of Pathology, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Papazoglou, Dimitrios [Department of Internal Medicine, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Pappa, Aglaia [Department of Molecular Biology and Genetics, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Pistevou, Kyriaki [Department of Radiotherapy/Oncology, Aristotle University of Thessalonica, Thessalonica (Greece); Kalamida, Dimitra [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece); Koukourakis, Michael I., E-mail: targ@her.forthnet.gr [Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, School of Health Sciences, Democritus University of Thrace, Alexandroupolis (Greece)

    2015-07-01

    Purpose: Predictive assays for acute radiation toxicities would be clinically relevant in radiation oncology. We prospectively examined the predictive role of the survival fraction at 2 Gy (SF2) and of γH2AX (double-strand break [DSB] DNA marker) expression kinetics in peripheral blood mononuclear cells (PBMCs) from cancer patients before radiation therapy. Methods and Materials: SF2 was measured with Trypan Blue assay in the PBMCs from 89 cancer patients undergoing radiation therapy at 4 hours (SF2{sub [4h]}) and 24 hours (SF2{sub [24h]}) after ex vivo irradiation. Using Western blot analysis and band densitometry, we further assessed the expression of γH2AX in PBMC DNA at 0 hours, 30 minutes, and 4 hours (33 patients) and 0 hour, 4 hours, and 24 hours (56 patients), following ex vivo irradiation with 2 Gy. Appropriate ratios were used to characterize each patient, and these were retrospectively correlated with early radiation therapy toxicity grade. Results: The SF2{sub (4h)} was inversely correlated with the toxicity grade (P=.006). The γH2AX-ratio{sub (30min)} (band density of irradiated/non-irradiated cells at 30 minutes) revealed, similarly, a significant inverse association (P=.0001). The DSB DNA repair rate from 30 minutes to 4 hours, calculated as the relative RγH2AX-ratio (γH2AX-ratio{sub (4h)}/γH2AX-ratio{sub (30min)}) showed a significant direct association with high toxicity grade (P=.01). Conclusions: Our results suggest that SF2 is a significant radiation sensitivity index for patients undergoing radiation therapy. γH2AX Western blot densitometry analysis provided 2 important markers of normal tissue radiation sensitivity. Low γH2AX expression at 30 minutes was linked with high toxicity grade, suggesting that poor γH2AX repair activity within a time frame of 30 minutes after irradiation predicts for poor radiation tolerance. On the other hand, rapid γH2AX content restoration at 4 hours after irradiation, compatible with

  19. Q{sub {gamma}-H2AX}, an analysis method for partial-body radiation exposure using {gamma}-H2AX in non-human primate lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Redon, Christophe E., E-mail: redonc@mail.nih.gov [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Nakamura, Asako J.; Gouliaeva, Ksenia [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Rahman, Arifur; Blakely, William F. [Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603 (United States); Bonner, William M. [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States)

    2011-09-15

    We previously used the {gamma}-H2AX assay as a biodosimeter for total-body irradiation (TBI) exposure ({gamma}-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant {gamma}-H2AX responses days after total-body exposure to 1-8.5 Gy ({sup 60}Co {gamma}-rays at 55 cGy min{sup -1}). Here, we introduce a partial-body exposure analysis method, Q{sub {gamma}-H2AX}, which is based on the number of {gamma}-H2AX foci per damaged cells as evident by having one or more {gamma}-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q{sub {gamma}-H2AX} dose-response calibration curves to assess acute partial-body exposures. {gamma}-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of {gamma}-H2AX may provide a robust biodosimeter for analyzing partial-body exposures to ionizing radiation in humans.

  20. Poor prognosis of constitutive gamma-H2AX expressing triple-negative breast cancers is associated with telomere length

    NARCIS (Netherlands)

    Nagelkerke, A.P.; Kuijk, S.J. van; Martens, J.W.; Sweep, F.C.; Hoogerbrugge, N.; Bussink, J.; Span, P.N.

    2015-01-01

    AIM: Here, we set out to establish whether endogenous gamma-H2AX is a biomarker in triple-negative breast cancer. METHODS: We explored the association of gamma-H2AX with mutation status and sensitivity to 139 different anticancer drugs in up to 41 breast cancer cell lines. Further, we correlated

  1. Geldanamycin, an inhibitor of Hsp90, increases paclitaxel-mediated toxicity in ovarian cancer cells through sustained activation of the p38/H2AX axis.

    Science.gov (United States)

    Mo, Qingqing; Zhang, Yu; Jin, Xin; Gao, Yue; Wu, Yuan; Hao, Xing; Gao, Qinglei; Chen, Pingbo

    2016-11-01

    Paclitaxel is a mitotic inhibitor used in ovarian cancer chemotherapy. Unfortunately, due to the rapid genetic and epigenetic changes in adaptation to stress induced by anticancer drugs, cancer cells are often able to become resistant to single or multiple anticancer agents. However, it remains largely unknown how paclitaxel resistance happens. In this study, we generated a cell line of acquired resistance to paclitaxel therapy, A2780T, which is cross-resistant to other antimitotic drugs, such as PLK1 inhibitor or AURKA inhibitor. Immunoblotting revealed significant alterations in cell-cycle-related and apoptotic-related proteins involved in key signaling pathways. In particular, phosphorylation of p38, which activates H2AX, was significantly decreased in A2780T cells compared to the parental A2780 cells. Geldanamycin (GA), an inhibitor of Hsp90, sustained activation of the p38/H2AX axis, and A2780T cells were shown to be more sensitive to GA compared to A2780 cells. Furthermore, treatment of A2780 and A2780T cells with GA significantly enhanced sensitivity to paclitaxel. Meanwhile, GA cooperated with paclitaxel to suppress tumor growth in a mouse ovarian cancer xenograft model. In conclusion, GA may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by inactivation of p38/H2AX axis.

  2. Gamma-H2AX-based dose estimation for whole and partial body radiation exposure.

    Directory of Open Access Journals (Sweden)

    Simon Horn

    Full Text Available Most human exposures to ionising radiation are partial body exposures. However, to date only limited tools are available for rapid and accurate estimation of the dose distribution and the extent of the body spared from the exposure. These parameters are of great importance for emergency triage and clinical management of exposed individuals. Here, measurements of γ-H2AX immunofluorescence by microscopy and flow cytometry were compared as rapid biodosimetric tools for whole and partial body exposures. Ex vivo uniformly X-irradiated blood lymphocytes from one donor were used to generate a universal biexponential calibration function for γ-H2AX foci/intensity yields per unit dose for time points up to 96 hours post exposure. Foci--but not intensity--levels remained significantly above background for 96 hours for doses of 0.5 Gy or more. Foci-based dose estimates for ex vivo X-irradiated blood samples from 13 volunteers were in excellent agreement with the actual dose delivered to the targeted samples. Flow cytometric dose estimates for X-irradiated blood samples from 8 volunteers were in excellent agreement with the actual dose delivered at 1 hour post exposure but less so at 24 hours post exposure. In partial body exposures, simulated by mixing ex vivo irradiated and unirradiated lymphocytes, foci/intensity distributions were significantly over-dispersed compared to uniformly irradiated lymphocytes. For both methods and in all cases the estimated fraction of irradiated lymphocytes and dose to that fraction, calculated using the zero contaminated Poisson test and γ-H2AX calibration function, were in good agreement with the actual mixing ratios and doses delivered to the samples. In conclusion, γ-H2AX analysis of irradiated lymphocytes enables rapid and accurate assessment of whole body doses while dispersion analysis of foci or intensity distributions helps determine partial body doses and the irradiated fraction size in cases of partial body

  3. Induction and disappearance of γH2AX foci and formation of micronuclei after exposure of human lymphocytes to ⁶⁰Co γ-rays and p(66)+ Be(40) neutrons.

    Science.gov (United States)

    Vandersickel, Veerle; Beukes, Philip; Van Bockstaele, Bram; Depuydt, Julie; Vral, Anne; Slabbert, Jacobus

    2014-02-01

    To investigate both the formation of micronuclei (MN) and the induction and subsequent loss of phosphorylated histone H2AX foci (γH2AX foci) after in vitro exposure of human lymphocytes to either (60)Co γ-rays or p(66)+ Be(40) neutrons. MN dose response (DR) curves were obtained by exposing isolated lymphocytes of 10 different donors to doses ranging from 0-4 Gy γ-rays or 0-2 Gy neutrons. Also, γH2AX foci DR curves were obtained following exposure to doses ranging from 0-0.5 Gy of either γ-rays or neutrons. Foci kinetics for lymphocytes for a single donor exposed to 0.5 Gy γ-rays or neutrons were studied up to 24 hours post-irradiation. Micronuclei yields following neutron exposure were consistently higher compared to that from (60)Co γ-rays. All MN yields were over-dispersed compared to a Poisson distribution. Over-dispersion was higher after neutron irradiation for all doses > 0.1 Gy. Up to 4 hours post-irradiation lower yields of neutron-induced γH2AX foci were observed. Between 4 and 24 hours the numbers of foci from neutrons were consistently higher than that from γ-rays. The half-live of foci disappearance is only marginally longer for neutrons compared to that from γ-rays. Foci formations were more likely to be over-dispersed for neutron irradiations. Although neutrons are more effective to induce MN, the absolute number of induced γH2AX foci are less at first compared to γ-rays. With time neutron-induced foci are more persistent. These findings are helpful for using γH2AX foci in biodosimetry and to understand the repair of neutron-induced cellular damage.

  4. Persistence of gamma-H2AX and 53BP1 foci in proliferating and nonproliferating human mammary epithelial cells after exposure to gamma-rays or iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Groesser, Torsten; Chang, Hang; Fontenay, Gerald; Chen, James; Costes, Sylvain V.; Barcellos-Hoff, Mary Helen; Parvin, Bahram; Rydberg, Bjorn

    2010-12-22

    To investigate {gamma}-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionizing radiation under different cell culture conditions. HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced {gamma}-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both {gamma}-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after {gamma}-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. Conclusions: The disappearance of radiation induced {gamma}-H2AX and 53BP1 foci in HMEC have different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent {gamma}-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodeling.

  5. A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells

    Directory of Open Access Journals (Sweden)

    Huber Peter E

    2011-02-01

    Full Text Available Abstract Background The protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2. Methods MRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB, or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay. Results TBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy or foci formation (1 Gy. While DNA fragment rejoining (PFGE was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination, but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival enhanced radiation-induced cell killing in the clonogenic assay. Conclusions The data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.

  6. Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation.

    Science.gov (United States)

    Liubavičiūtė, Aušra; Kraśko, Jan Aleksander; Mlynska, Agata; Lagzdina, Jelena; Sužiedėlis, Kęstutis; Pašukonienė, Vita

    2015-11-01

    The aim of this study was to evaluate the efficiency of proton beam irradiation in pancreatic cancer cell line MIA PaCa-2 and its role in the cell cycle, apoptosis, and formation of histone γH2AX in different reparation times (72-h follow-up). The MIA PaCa-2 pancreatic carcinoma cell line was irradiated with 1.6-Gy proton beam. After irradiation, cell viability was measured colorimetrically, and the cell cycle, apoptosis, and γH2AX expression were evaluated on a FACScan cytometer. Low-dose proton beam irradiation had an effect on the MIA PaCa-2 tumor cell line already 1h after exposure, but maximal lethality was reached after 72h postirradiation with a cell viability rate of 24%. The cell cycle went into partial G1/0 arrest, and was released after 72h. The expression of γH2AX was strong and its levels were significantly elevated as late as 48h post radiation. The apoptosis levels increased with post radiation incubation time to reach 79% after 72h. Our data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase. Copyright © 2015 Lithuanian University of Health Sciences. Production and hosting by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  7. γ-H2AX: A Novel Prognostic Marker in a Prognosis Prediction Model of Patients with Early Operable Non-Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    E. Chatzimichail

    2014-01-01

    Full Text Available Cancer is a leading cause of death worldwide and the prognostic evaluation of cancer patients is of great importance in medical care. The use of artificial neural networks in prediction problems is well established in human medical literature. The aim of the current study was to assess the prognostic value of a series of clinical and molecular variables with the addition of γ-H2AX—a new DNA damage response marker—for the prediction of prognosis in patients with early operable non-small cell lung cancer by comparing the γ-H2AX-based artificial network prediction model with the corresponding LR one. Two prognostic models of 96 patients with 27 input variables were constructed by using the parameter-increasing method in order to compare the predictive accuracy of neural network and logistic regression models. The quality of the models was evaluated by an independent validation data set of 11 patients. Neural networks outperformed logistic regression in predicting the patient’s outcome according to the experimental results. To assess the importance of the two factors p53 and γ-H2AX, models without these two variables were also constructed. JR and accuracy of these models were lower than those of the models using all input variables, suggesting that these biological markers are very important for optimal performance of the models. This study indicates that neural networks may represent a potentially more useful decision support tool than conventional statistical methods for predicting the outcome of patients with non-small cell lung cancer and that some molecular markers, such as γ-H2AX, enhance their predictive ability.

  8. DNA double-strand break repair: a theoretical framework and its application.

    Science.gov (United States)

    Murray, Philip J; Cornelissen, Bart; Vallis, Katherine A; Chapman, S Jon

    2016-01-01

    DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γH2AX. Many copies of γH2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti-γH2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo. Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, (111)In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti-γH2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti-γH2AX-TAT and γH2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti-γH2AX antibody is labelled with Auger electron-emitting (111)In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti-γH2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti-γH2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage

  9. Induction and Processing of the Radiation-Induced Gamma-H2AX Signal and Its Link to the Underlying Pattern of DSB: A Combined Experimental and Modelling Study.

    Directory of Open Access Journals (Sweden)

    Francesco Tommasino

    Full Text Available We present here an analysis of DSB induction and processing after irradiation with X-rays in an extended dose range based on the use of the γH2AX assay. The study was performed by quantitative flow cytometry measurements, since the use of foci counting would result in reasonable accuracy only in a limited dose range of a few Gy. The experimental data are complemented by a theoretical analysis based on the GLOBLE model. In fact, original aim of the study was to test GLOBLE predictions against new experimental data, in order to contribute to the validation of the model. Specifically, the γH2AX signal kinetics has been investigated up to 24 h after exposure to increasing photon doses between 2 and 500 Gy. The prolonged persistence of the signal at high doses strongly suggests dose dependence in DSB processing after low LET irradiation. Importantly, in the framework of our modelling analysis, this is related to a gradually increased fraction of DSB clustering at the micrometre scale. The parallel study of γH2AX dose response curves shows the onset of a pronounced saturation in two cell lines at a dose of about 20 Gy. This dose is much lower than expected according to model predictions based on the values usually adopted for the DSB induction yield (≈ 30 DSB/Gy and for the γH2AX foci extension of approximately 2 Mbp around the DSB. We show and discuss how theoretical predictions and experimental findings can be in principle reconciled by combining an increased DSB induction yield with the assumption of a larger genomic extension for the single phosphorylated regions. As an alternative approach, we also considered in our model the possibility of a 3D spreading-mechanism of the H2AX phosphorylation around the induced DSB, and applied it to the analysis of both the aspects considered. Our results are found to be supportive for the basic assumptions on which GLOBLE is built. Apart from giving new insights into the H2AX phosphorylation process

  10. DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies.

    Science.gov (United States)

    Viera, Alberto; Santos, Juan L; Page, Jesús; Parra, M Teresa; Calvente, Adela; Cifuentes, Marta; Gómez, Rocío; Lira, Renee; Suja, José A; Rufas, Julio S

    2004-04-01

    The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.

  11. Genome-wide transcriptional analysis of apoptosis-related genes and pathways regulated by H2AX in lung cancer A549 cells.

    Science.gov (United States)

    Lu, Chengrong; Xiong, Min; Luo, Yuan; Li, Jing; Zhang, Yanjun; Dong, Yaqiong; Zhu, Yanjun; Niu, Tianhui; Wang, Zhe; Duan, Lianning

    2013-09-01

    Histone H2AX is a novel tumor suppressor protein and plays an important role in apoptosis of cancer cells. However, the role of H2AX in lung cancer cells is unclear. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. We showed that H2AX was involved in apoptosis of lung cancer A549 cells as in other tumor cells. Knockdown of H2AX strongly suppressed apoptosis of A549 cells. We clarified the molecular mechanisms of apoptosis regulated by H2AX based on genome-wide transcriptional analysis. Microarray data analysis demonstrated that H2AX knockdown in A549 cells affected expression of 3,461 genes, including upregulation of 1,435 and downregulation of 2,026. These differentially expressed genes were subjected to bioinformatic analysis for exploring biological processes regulated by H2AX in lung cancer cells. Gene ontology analysis showed that H2AX affected expression of many genes, through which, many important functions including response to stimuli, gene expression, and apoptosis were involved in apoptotic regulation of lung cancer cells. Pathway analysis identified the mitogen-activated protein kinase signaling pathway and apoptosis as the most important pathways targeted by H2AX. Signal transduction pathway networks analysis and chromatin immunoprecipitation assay showed that two core genes, NFKB1 and JUN, were involved in apoptosis regulated by H2AX in lung cancer cells. Taken together, these data provide compelling clues for further exploration of H2AX function in cancer cells.

  12. Comparative potency approach based on H2AX assay for estimating the genotoxicity of polycyclic aromatic hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Audebert, M., E-mail: marc.audebert@toulouse.inra.fr [INRA UMR1331, TOXALIM (Research Center in Food Toxicology), 180 chemin de Tournefeuille, F-31027 Toulouse (France); Université de Toulouse, INP, ENVT, EIP, UPS, UMR1331, Toxalim, F-31076 Toulouse (France); Zeman, F.; Beaudoin, R.; Péry, A. [Unité “Modèles pour l' écotoxicologie et la toxicologie” (METO), INERIS, BP2, F-60550 Verneuil-en-Halatte (France); Cravedi, J.-P. [INRA UMR1331, TOXALIM (Research Center in Food Toxicology), 180 chemin de Tournefeuille, F-31027 Toulouse (France); Université de Toulouse, INP, ENVT, EIP, UPS, UMR1331, Toxalim, F-31076 Toulouse (France)

    2012-04-01

    Polycyclic Aromatic Hydrocarbons (PAHs) constitute a family of over one hundred compounds and can generally be found in complex mixtures. PAHs metabolites cause DNA damage which can lead to the development of carcinogenesis. Toxicity assessment of PAH complex mixtures is currently expressed in terms of toxic equivalents, based on Toxicity Equivalent Factors (TEFs). However, the definition of new TEFs for a large number of PAH could overcome some limitations of the current method and improve cancer risk assessment. The current investigation aimed at deriving the relative potency factors of PAHs, based on their genotoxic effect measured in vitro and analyzed with mathematical models. For this purpose, we used a new genotoxic assay (γH2AX) with two human cell lines (HepG2 and LS-174T) to analyze the genotoxic properties of 13 selected PAHs at low doses after 24 h treatment. The dose–response for genotoxic effects was modeled with a Hill model; equivalency between PAHs at low dose was assessed by applying constraints to the model parameters. In the two cell lines tested, we observed a clear dose–response for genotoxic effects for 11 tested compounds. LS-174T was on average ten times more sensitive than HepG2 towards PAHs regarding genotoxicity. We developed new TEFs, which we named Genotoxic Equivalent Factor (GEF). Calculated GEF for the tested PAHs were generally higher than the TEF usually used. Our study proposed a new in vitro based method for the establishment of relevant TEFs for PAHs to improve cancer risk assessment. -- Highlights: ► Examination of the genotoxic properties of 13 PAHs on two human cell lines. ► Modelization with a Hill model of the genotoxic dose–response. ► First investigation of the genotoxicity of benzo[c]fluorene on human cell lines. ► Establishment of relevant TEFs for PAHs to improve cancer risk assessment.

  13. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AXH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  14. Radiation dose determines the method for quantification of DNA double strand breaks

    Directory of Open Access Journals (Sweden)

    TANJA BULAT

    2016-03-01

    Full Text Available ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs that trigger phosphorylation of the histone protein H2AXH2AX. Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany. Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

  15. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Ikuma; Ibuki, Yuko, E-mail: ibuki@u-shizuoka-ken.ac.jp

    2014-12-15

    Graphical abstract: - Highlights: • Formaldehyde modified histones. • The phosphorylation of H3S10 was increased at the promoter regions of proto-oncogenes. • The phosphorylation of H2AXS139 was attributed to FA-induced DNA damage. • The FA-induced initiation and promotion of cancer could be judged by these modifications. - Abstract: Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.

  16. Quantitative image analysis of gamma-H2AX foci induced by ionizing radiation applying open source programs.

    Science.gov (United States)

    González, Jorge Ernesto; Lee, Manual; Barquinero, Joan Francesc; Valente, Marco; Roch-Lefèvre, Sandrine; García, Omar

    2012-04-01

    To test a CellProfiler pipeline for automated counting and characterization of gamma-H2AX foci in color images of human cultured cells. A431 cells were irradiated and stained for gamma-H2AX foci detection. Sets of color images were analyzed visually, and findings were compared with those using FociCounter and CellProfiler software. The CellProfiler pipeline includes some proprieties not present in FociCounter, such as the automatic detection of nuclei, the detection of touching nuclei and the rejection of nuclei that touch the border of the image. The time required for manual operation is associated with the number of images analyzed visually or by FociCounter but not for the CellProfiler program. CellProfiler reduced manual operation time and is about 4 times faster than semiautomatic detection using FociCounter and 10 times faster than visual counting. We conclude that CellProfiler and FociCounter are reliable tools for measuring gamma-H2AX foci. However, CellProfiler overcomes the limitations of the FociCounter program and is able to detect nuclei automatically, saving considerable manual operation.

  17. Ratio of γ-H2AX level in lymphocytes to that in granulocytes detected using flow cytometry as a potential biodosimeter for radiation exposure.

    Science.gov (United States)

    Wang, Zhidong; Hu, Hailiang; Hu, Ming; Zhang, Xueqing; Wang, Qi; Qiao, Yulei; Liu, Haixiang; Shen, Liping; Zhou, Pingkun; Chen, Ying

    2014-05-01

    This study aims to assess utilisation of the ratio of γ-H2AX in lymphocytes to that in granulocytes (RL/G of γ-H2AX) in blood as a rapid method for population triage and dose estimation during large-scale radiation emergencies. Blood samples from healthy volunteers exposed to 0-10 Gy of (60)Co irradiation were collected. The samples were cultured for 0-24 h and then analysed using flow cytometry to measure the levels of γ-H2AX in lymphocytes and granulocytes. The basal RL/G levels of γ-H2AX in healthy human blood, the response of RL/G of γ-H2AX to ionising radiation and its relationship with doses, time intervals after exposure and individual differences were also analysed. The level of γ-H2AX in lymphocytes increased in a dose-dependent manner after irradiation, whereas the level in granulocytes was not affected. A linear dose-effect relationship with low inter-experimental and inter-individual variations was observed. The RL/G of γ-H2AX may be used as a biomarker for population triage and dose estimation during large-scale radiation emergencies if blood samples can be collected within 24 h.

  18. gammaH2AX signalling during sperm chromatin remodelling in the mouse zygote.

    NARCIS (Netherlands)

    Derijck, A.H.A.; Heijden, G.W. van der; Giele, M.M.; Philippens, M.E.P.; Bavel, C.C.A.W. van; Boer, P. de

    2006-01-01

    In the mouse, the paternal post-meiotic chromatin is assumed to be devoid of DNA repair after nuclear elongation and protamine-induced compaction. Hence, DNA lesions induced thereafter will have to be restored upon gamete fusion in the zygote. Misrepair of such lesions often results in chromosome

  19. Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

    Directory of Open Access Journals (Sweden)

    Jean-Luc Perfettini

    Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

  20. γH2AX/53BP1 foci as a potential pre-treatment marker of HNSCC tumors radiosensitivity - preliminary methodological study and discussion

    Science.gov (United States)

    Falk, Martin; Horakova, Zuzana; Svobodova, Marketa; Masarik, Michal; Kopecna, Olga; Gumulec, Jaromir; Raudenska, Martina; Depes, Daniel; Bacikova, Alena; Falkova, Iva; Binkova, Hana

    2017-09-01

    In order to improve patients' post-treatment quality of life, a shift from surgery to non-surgical (chemo)radio-treatment is recognized in head and neck oncology. However, about half of HNSCC tumors are resistant to irradiation and an efficient marker of individual tumor radiosensitivity is still missing. We analyzed whether various parameters of DNA double strand break (DSB) repair determined in vitro can predict, prior to clinical treatment initiation, the radiosensitivity of tumors. We compared formation and decrease of γH2AX/53BP1 foci in 48 h after irradiating tumor cell primocultures with 2 Gy of γ-rays. To better understand complex tumor behavior, three different cell type primocultures - CD90-, CD90+, and a mixed culture of these cells - were isolated from 1 clinically radioresistant, 2 radiosensitive, and 4 undetermined HPV-HNSCC tumors and followed separately. While DSB repair was delayed and the number of persisting DSBs increased in the radiosensitive tumors, the results for the radioresistant tumor were similar to cultured normal human skin fibroblasts. Hence, DSB repair kinetics/efficiency may correlate with clinical response to radiotherapy for a subset of HNSCC tumors but the size (and therefore practical relevance) of this subset remains to be determined. The same is true for contribution of different cell type primocultures to tumor radioresistance.

  1. Micronucleus and H2AX phosphorylation assessment of silica-coated iron oxide nanoparticles in human neuronal cells

    OpenAIRE

    Sánchez-Flores, Maria; Valdiglesias, Vanessa; Kiliç, Gozde; Costa, Carla; Fernandez-Bertolez, Natalia; Costa, Solange; Teixeira, João Paulo; Pasaro, Eduardo; Laffon, Blanca

    2015-01-01

    As clinically approved metal oxide nanoparticles, iron oxide nanoparticles (ION) hold immense potential in a vast variety of applications in various fields of biomedicine and biotechnology. With the increase in ION usage, particularly in diagnostics and therapeutics, concerns regarding their interactions with cellular components and possible deleterious effects are also growing. This work was supported by Xunta de Galicia (EM 2012/079), the project NanoToxClass (ERA ERASllNN/00...

  2. DNA double-strand break repair is impaired in presenescent Syrian hamster fibroblasts.

    Science.gov (United States)

    Solovjeva, Ljudmila; Firsanov, Denis; Vasilishina, Anastasia; Chagin, Vadim; Pleskach, Nadezhda; Kropotov, Andrey; Svetlova, Maria

    2015-10-12

    Studies of DNA damage response are critical for the comprehensive understanding of age-related changes in cells, tissues and organisms. Syrian hamster cells halt proliferation and become presenescent after several passages in standard conditions of cultivation due to what is known as "culture stress". Using proliferating young and non-dividing presenescent cells in primary cultures of Syrian hamster fibroblasts, we defined their response to the action of radiomimetic drug bleomycin (BL) that induces DNA double-strand breaks (DSBs). The effect of the drug was estimated by immunoblotting and immunofluorescence microscopy using the antibody to phosphorylated histone H2AX (gH2AX), which is generally accepted as a DSB marker. At all stages of the cell cycle, both presenescent and young cells demonstrated variability of the number of gH2AX foci per nucleus. gH2AX focus induction was found to be independent from BL-hydrolase expression. Some differences in DSB repair process between BL-treated young and presenescent Syrian hamster cells were observed: (1) the kinetics of gH2AX focus loss in G0 fibroblasts of young culture was faster than in cells that prematurely stopped dividing; (2) presenescent cells were characterized by a slower recruitment of DSB repair proteins 53BP1, phospho-DNA-PK and phospho-ATM to gH2AX focal sites, while the rate of phosphorylated ATM/ATR substrate accumulation was the same as that in young cells. Our results demonstrate an impairment of DSB repair in prematurely aged Syrian hamster fibroblasts in comparison with young fibroblasts, suggesting age-related differences in response to BL therapy.

  3. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV

    Energy Technology Data Exchange (ETDEWEB)

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Ding, Nan [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Qi, Yongmei; Zhang, Yingmei [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China); Wang, Jufang [Gansu Key laboratory of Space Radiobiology, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Huang, Dejun, E-mail: huangdj@lzu.edu.cn [Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000 (China)

    2015-09-15

    Highlights: • Cadmium (Cd) exposure delayed the repair of DNA damage induced by X-ray. • Cd exposure altered the phosphorylation of DNA-PKcs on Thr-2609 and Ser-2056 sites. • Cd impaired the formation of XRCC4 and Ligase IV foci, and down-regulated their protein expression. • Zinc mitigated the effects of Cd on DDR by regulating pDNA-PKcs (Thr-2609), XRCC4 and Ligase IV. - Abstract: Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1–8 h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

  4. MORC2 Signaling Integrates Phosphorylation-Dependent, ATPase-Coupled Chromatin Remodeling during the DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Da-Qiang Li

    2012-12-01

    Full Text Available Chromatin dynamics play a central role in maintaining genome integrity, but how this is achieved remains largely unknown. Here, we report that microrchidia CW-type zinc finger 2 (MORC2, an uncharacterized protein with a derived PHD finger domain and a conserved GHKL-type ATPase module, is a physiological substrate of p21-activated kinase 1 (PAK1, an important integrator of extracellular signals and nuclear processes. Following DNA damage, MORC2 is phosphorylated on serine 739 in a PAK1-dependent manner, and phosphorylated MORC2 regulates its DNA-dependent ATPase activity to facilitate chromatin remodeling. Moreover, MORC2 associates with chromatin and promotes gamma-H2AX induction in a PAK1 phosphorylation-dependent manner. Consequently, cells expressing MORC2-S739A mutation displayed a reduction in DNA repair efficiency and were hypersensitive to DNA-damaging agent. These findings suggest that the PAK1-MORC2 axis is critical for orchestrating the interplay between chromatin dynamics and the maintenance of genomic integrity through sequentially integrating multiple essential enzymatic processes.

  5. The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements.

    Science.gov (United States)

    Thorne, David; Larard, Sophie; Baxter, Andrew; Meredith, Clive; Gaҫa, Marianna

    2017-01-04

    DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated. E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a "cigalike" and a closed "modular" system) and cigarette smoke. A Vitrocell® VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120min exposure (diluting airflow, 0.25-1L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% γH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform. The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM

  6. Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

    Science.gov (United States)

    Mitchell, Jonathan K.; Byers, Nathaniel M.

    2013-01-01

    The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7's N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein. PMID:24027328

  7. Formaldehyde-induced histone H3 phosphorylation via JNK and the expression of proto-oncogenes.

    Science.gov (United States)

    Yoshida, Ikuma; Ibuki, Yuko

    2014-12-01

    Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Application of translocation, γ-H2AX, and Sam68 as a biological indicators for the assessment of radiation exposure in nuclear power plant workers

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Kwang Hee; Park, Hyung Sun; Nam, Seon Young [Korea Hydro Nuclear Power Co., Seoul (Korea, Republic of)

    2014-05-15

    This study showed that confirmation of the initial dose estimated by dicentric analysis is provided by the subsequent FISH analysis for translocation frequency and provides further evidence for the valid use of FISH as a retrospective biological dosimeter. The IAEA manual on cytogenetic dosimetry recommends a halftime value of 3 y to correct for the decrease of dicentrics in case of delayed sampling based on the patient data of Buckton. Support for this comes from the cytogenetic follow up of an individual exposed to tritium, which also indicated a decline in dicentrics with a half-time of ∼3 y. Naturally, the RBE of tritium, as well as other kinds of ionizing radiation, depends on the dose, exposure conditions, and studied parameters. The information about the RBE of tritium that is most important from an applied standpoint is that associated with the range of low doses. In our study, the dose dependence of tritium RBE was not identified because of very low dose Tritium (< 1mSv). However, The strong smooth relationship between translocation yield and age is shown in Table 2. The translocation yields reported here are only slightly lower than already published. The implication is that the increase of yield with age could be due to environmental factors, to a natural aging process or both. In addition, we confirmed that γ-H2AX and Sam68 associated with DNA damage and apoptosis, can be new biological indicators for radiation exposure. Radiation workers are exposed to ionizing radiation from various sources. Ionizing radiation produces several types of DNA lesion, including DNA base alterations, DNA. DNA cross-links, and single- and double-strand breaks. As a protocol for biological dosimetry recommended by IAEA (2001), the analysis of solid stained dicentric chromosomes has been used since the mid 1960s. The intervening years have seen great improvements bringing the technique to a point where dicentric analysis has become a routine component of the radiological

  9. DNA damage precedes apoptosis during the regression of the interdigital tissue in vertebrate embryos

    Science.gov (United States)

    Montero, Juan A.; Sanchez-Fernandez, Cristina; Lorda-Diez, Carlos I.; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2016-01-01

    DNA damage independent of caspase activation accompanies programmed cell death in different vertebrate embryonic organs. We analyzed the significance of DNA damage during the regression of the interdigital tissue, which sculpts the digits in the embryonic limb. Interdigit remodeling involves oxidative stress, massive apoptosis and cell senescence. Phosphorylation of H2AX mediated by ATM precedes caspase dependent apoptosis and cell senescence during interdigit regression. The association of γH2AX with other downstream DNA repair factors, including MDC1, Rad50 and 53BP1 suggests a defensive response of cells against DNA damage. The relative distribution of cells γH2AX-only positive, TUNEL-only positive, and cells double positive for both markers is consistent with a sequence of degenerative events starting by damage of the DNA. In support of this interpretation, the relative number of γH2AX-only cells increases after caspase inhibition while the relative number of TUNEL-only cells increases after inhibition of ATM. Furthermore, cultured interdigits survived and maintained intense chondrogenic potential, even at advanced stages of degeneration, discarding a previous commitment to die. Our findings support a new biological paradigm considering embryonic cell death secondary to genotoxic stimuli, challenging the idea that considers physiological cell death a cell suicide regulated by an internal death clock that pre-programmes degeneration. PMID:27752097

  10. Roles of Kruppel-associated Box (KRAB)-associated Co-repressor KAP1 Ser-473 Phosphorylation in DNA Damage Response.

    Science.gov (United States)

    Hu, Chen; Zhang, Shengping; Gao, Xuan; Gao, Xiaojing; Xu, Xiaohong; Lv, Ya; Zhang, Yan; Zhu, Zhenhong; Zhang, Changqing; Li, Qiao; Wong, Jiemin; Cui, Yongping; Zhang, Wen; Ma, Lin; Wang, Chuangui

    2012-06-01

    The Kruppel-associated box (KRAB)-associated co-repressor KAP1 is an essential nuclear co-repressor for the KRAB zinc finger protein superfamily of transcriptional factors. Ataxia telangiectasia mutated (ATM)-Chk2 and ATM- and Rad3-related (ATR)-Chk1 are two primary kinase signaling cascades activated in response to DNA damage. A growing body of evidence suggests that ATM and ATR phosphorylate KAP1 at Ser-824 in response to DNA damage and regulate KAP1-dependent chromatin condensation, DNA repair, and gene expression. Here, we show that, depending on the type of DNA damage that occurs, KAP1 Ser-473 can be phosphorylated by ATM-Chk2 or ATR-Chk1 kinases. Phosphorylation of KAP1 at Ser-473 attenuated its binding to the heterochromatin protein 1 family proteins and inhibited its transcriptional repression of KRAB-zinc finger protein (KRAB-ZFP) target genes. Moreover, KAP1 Ser-473 phosphorylation induced by DNA damage stimulated KAP1-E2F1 binding. Overexpression of heterochromatin protein 1 significantly inhibited E2F1-KAP1 binding. Elimination of KAP1 Ser-473 phosphorylation increased E2F1-targeted proapoptotic gene expression and E2F1-induced apoptosis in response to DNA damage. Furthermore, loss of phosphorylation of KAP1 Ser-473 led to less BRCA1 focus formation and slower kinetics of loss of γH2AX foci after DNA damage. KAP1 Ser-473 phosphorylation was required for efficient DNA repair and cell survival in response to DNA damage. Our studies reveal novel functions of KAP1 Ser-473 phosphorylation under stress.

  11. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AXH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  12. Mean frequency and relative fluorescence intensity measurement of γ‐H2AX foci dose response in PBL exposed to γ‐irradiation: An inter‐ and intra‐laboratory comparison and its relevance for radiation triage

    National Research Council Canada - National Science Library

    Venkateswarlu, Raavi; Tamizh, Selvan G; Bhavani, Manivannan; Kumar, Arun; Alok, Amit; Karthik, Kanagaraj; Kalra, Namita; Vijayalakshmi, J; Paul, Solomon F. D; Chaudhury, N. K; Venkatachalam, Perumal

    2015-01-01

    Measurement of γ‐H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation...

  13. Modulation of LSD1 phosphorylation by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment in response to DNA damage.

    Science.gov (United States)

    Peng, Bin; Wang, Jing; Hu, Yuan; Zhao, Hongli; Hou, Wenya; Zhao, Hongchang; Wang, Hailong; Liao, Ji; Xu, Xingzhi

    2015-07-13

    Proper DNA damage response is essential for the maintenance of genome integrity. The E3 ligase RNF168 deficiency fully prevents both the initial recruitment and retention of 53BP1 at sites of DNA damage. In response to DNA damage, RNF168-dependent recruitment of the lysine-specific demethylase LSD1 to the site of DNA damage promotes local H3K4me2 demethylation and ubiquitination of H2A/H2AX, facilitating 53BP1 recruitment to sites of DNA damage. Alternatively, RNF168-mediated K63-linked ubiquitylation of 53BP1 is required for the initial recruitment of 53BP1 to sites of DNA damage and for its function in repair. We demonstrated here that phosphorylation and dephosphorylation of LSD1 at S131 and S137 was mediated by casein kinase 2 (CK2) and wild-type p53-induced phosphatase 1 (WIP1), respectively. LSD1, RNF168 and 53BP1 interacted with each other directly. CK2-mediated phosphorylation of LSD1 exhibited no impact on its interaction with 53BP1, but promoted its interaction with RNF168 and RNF168-dependent 53BP1 ubiquitination and subsequent recruitment to the DNA damage sites. Furthermore, overexpression of phosphorylation-defective mutants failed to restore LSD1 depletion-induced cellular sensitivity to DNA damage. Taken together, our results suggest that LSD1 phosphorylation modulated by CK2/WIP1 regulates RNF168-dependent 53BP1 recruitment directly in response to DNA damage and cellular sensitivity to DNA damaging agents. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Phosphorylation: The Molecular Switch of Double-Strand Break Repair

    Directory of Open Access Journals (Sweden)

    K. C. Summers

    2011-01-01

    Full Text Available Repair of double-stranded breaks (DSBs is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ, homologous recombination (HR, or the inclusive DNA damage response (DDR. These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

  15. Smoking cessation reverses DNA double-strand breaks in human mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Mari Ishida

    Full Text Available OBJECTIVE: Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs, the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs. APPROACH AND RESULTS: Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX. Young healthy active smokers (n = 15 showed increased γ-H2AX foci number when compared with non-smokers (n = 12 (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p<0.0001. Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36-5.24 to 0.28/cell; IQR, 0.12-0.53, p<0.05. A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01. CONCLUSIONS: Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.

  16. Assembly and function of DNA double-strand break repair foci in mammalian cells

    DEFF Research Database (Denmark)

    Bekker-Jensen, Simon; Mailand, Niels

    2010-01-01

    DNA double-strand breaks (DSBs) are among the most cytotoxic types of DNA damage, which if left unrepaired can lead to mutations or gross chromosomal aberrations, and promote the onset of diseases associated with genomic instability such as cancer. One of the most discernible hallmarks...... of the cellular response to DSBs is the accumulation and local concentration of a plethora of DNA damage signaling and repair proteins in the vicinity of the lesion, initiated by ATM-mediated phosphorylation of H2AX (¿-H2AX) and culminating in the generation of distinct nuclear compartments, so-called Ionizing...... of such DNA repair foci still remains limited. In this review, we focus on recent discoveries on the mechanisms that govern the formation of IRIF, and discuss the implications of such findings in light of our understanding of the physiological importance of these structures....

  17. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  18. Duodenal crypt health following exposure to Cr(VI): Micronucleus scoring, γ-H2AX immunostaining, and synchrotron X-ray fluorescence microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Chad M.; Wolf, Jeffrey C.; Elbekai, Reem H.; Paranjpe, Madhav G.; Seiter, Jennifer M.; Chappell, Mark A.; Tappero, Ryan V.; Suh, Mina; Proctor, Deborah M.; Bichteler, Anne; Haws, Laurie C.; Harris, Mark A.

    2015-08-01

    Lifetime exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal damage and an increase in duodenal tumors in B6C3F1 mice. To assess whether these tumors could be the result of a direct mutagenic or genotoxic mode of action, we conducted a GLP-compliant 7-day drinking water study to assess crypt health along the entire length of the duodenum. Mice were exposed to water (vehicle control), 1.4, 21, or 180 ppm Cr(VI) via drinking water for 7 consecutive days. Crypt enterocytes in Swiss roll sections were scored as normal, mitotic, apoptotic, karyorrhectic, or as having micronuclei. A single oral gavage of 50 mg/kg cyclophosphamide served as a positive control for micronucleus induction. Exposure to 21 and 180 ppm Cr(VI) significantly increased the number of crypt enterocytes. Micronuclei and γ-H2AX immunostaining were not elevated in the crypts of Cr(VI)-treated mice. In contrast, treatment with cyclophosphamide significantly increased numbers of crypt micronuclei and qualitatively increased γ-H2AX immunostaining. Synchrotron-based X-ray fluorescence (XRF) microscopy revealed the presence of strong Cr fluorescence in duodenal villi, but negligible Cr fluorescence in the crypt compartment. Together, these data indicate that Cr(VI) does not adversely effect the crypt compartment where intestinal stem cells reside, and provide additional evidence that the mode of action for Cr(VI)-induced intestinal cancer in B6C3F1 mice involves chronic villous wounding resulting in compensatory crypt enterocyte hyperplasia.

  19. Gamma-H2AX biodosimetry for use in large scale radiation incidents: comparison of a rapid ‘96 well lyse/fix’ protocol with a routine method

    Directory of Open Access Journals (Sweden)

    Jayne Moquet

    2014-03-01

    Full Text Available Following a radiation incident, preliminary dose estimates made by γ-H2AX foci analysis can supplement the early triage of casualties based on clinical symptoms. Sample processing time is important when many individuals need to be rapidly assessed. A protocol was therefore developed for high sample throughput that requires less than 0.1 ml blood, thus potentially enabling finger prick sampling. The technique combines red blood cell lysis and leukocyte fixation in one step on a 96 well plate, in contrast to the routine protocol, where lymphocytes in larger blood volumes are typically separated by Ficoll density gradient centrifugation with subsequent washing and fixation steps. The rapid ‘96 well lyse/fix’ method reduced the estimated sample processing time for 96 samples to about 4 h compared to 15 h using the routine protocol. However, scoring 20 cells in 96 samples prepared by the rapid protocol took longer than for the routine method (3.1 versus 1.5 h at zero dose; 7.0 versus 6.1 h for irradiated samples. Similar foci yields were scored for both protocols and consistent dose estimates were obtained for samples exposed to 0, 0.2, 0.6, 1.1, 1.2, 2.1 and 4.3 Gy of 250 kVp X-rays at 0.5 Gy/min and incubated for 2 h. Linear regression coefficients were 0.87 ± 0.06 (R2 = 97.6% and 0.85 ± 0.05 (R2 = 98.3% for estimated versus actual doses for the routine and lyse/fix method, respectively. The lyse/fix protocol can therefore facilitate high throughput processing for γ-H2AX biodosimetry for use in large scale radiation incidents, at the cost of somewhat longer foci scoring times.

  20. Mean frequency and relative fluorescence intensity measurement of γ-H2AX foci dose response in PBL exposed to γ-irradiation: An inter- and intra-laboratory comparison and its relevance for radiation triage.

    Science.gov (United States)

    Venkateswarlu, Raavi; Tamizh, Selvan G; Bhavani, Manivannan; Kumar, Arun; Alok, Amit; Karthik, Kanagaraj; Kalra, Namita; Vijayalakshmi, J; Paul, Solomon F D; Chaudhury, N K; Venkatachalam, Perumal

    2015-12-01

    Measurement of γ-H2AX protein changes in the peripheral blood lymphocytes (PBL) of individuals exposed to ionizing radiation is a simple, sensitive, and rapid assay for radiation triage and early marker of dose estimation. The qualitative and quantitative measurements of the protein changes were examined using flow cytometry and microscopy. Whole blood and isolated lymphocytes were exposed in vitro between 0.1 and 5 Gy doses of (60) Co γ-radiation at a dose rate of 1 Gy/min. Radiation induced γ-H2AX foci frequency (n = 3) and relative fluorescence intensity (n = 7) in PBL was measured at 0.5 and 2 hrs postexposure. The observed dose response for γ-H2AX foci frequency at both time points, for whole blood and isolated lymphocytes did not show any significant (P > 0.05) differences. However, when compared with γ-H2AX foci frequency scored manually (microscopy), the semiautomated analysis (captured images) showed a better correlation (r(2) = 0.918) than that obtained with automated (Metafer) scoring (r(2) = 0.690). It is noteworthy to mention that, the γ-H2AX foci frequency quantified using microscopy showed a dose dependent increase up to 2 Gy and the relative fluorescence intensity (RFI) measured with flow cytometry revealed an increase up to 5 Gy in the PBL exposed in vitro. Moreover, a better correlation was observed between the γ-H2AX foci frequency obtained by manual scoring and RFI (r(2) = 0.910). Kinetic studies showed that the γ-H2AX foci remain more or less unchanged up to 4 hrs and reduces gradually over 48 hrs of postexposure at 37°C. Further, inter and intra-laboratory comparisons showed consistency in the scoring of γ-H2AX foci frequency by manual and semiautomated scoring. The overall results suggest that measurement of γ-H2AX (microscopy and flow cytometry) should be employed within 4 to 6 hrs for a reliable dosimetry either by sharing the work load between the laboratories or investing more manpower; however, triage can be possible even up

  1. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    , initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis......Malignant gliomas, the deadliest of brain neoplasms, show rampant genetic instability and resistance to genotoxic therapies, implicating potentially aberrant DNA damage response (DDR) in glioma pathogenesis and treatment failure. Here, we report on gross, aberrant constitutive activation of DNA...... that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal...

  2. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

    Science.gov (United States)

    Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

    2015-01-01

    Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AXH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  3. DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV Lytic DNA Replication and in Response to Expression of ZEBRA.

    Directory of Open Access Journals (Sweden)

    Ruth Wang'ondu

    Full Text Available Epstein Barr virus (EBV, like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM, H2AXH2AX, or 53BP1 (p53BP1, were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E and Z(S186E, did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

  4. DNA damage response during mouse oocyte maturation.

    Science.gov (United States)

    Mayer, Alexandra; Baran, Vladimir; Sakakibara, Yogo; Brzakova, Adela; Ferencova, Ivana; Motlik, Jan; Kitajima, Tomoya S; Schultz, Richard M; Solc, Petr

    2016-01-01

    Because low levels of DNA double strand breaks (DSBs) appear not to activate the ATM-mediated prophase I checkpoint in full-grown oocytes, there may exist mechanisms to protect chromosome integrity during meiotic maturation. Using live imaging we demonstrate that low levels of DSBs induced by the radiomimetic drug Neocarzinostatin (NCS) increase the incidence of chromosome fragments and lagging chromosomes but do not lead to APC/C activation and anaphase onset delay. The number of DSBs, represented by γH2AX foci, significantly decreases between prophase I and metaphase II in both control and NCS-treated oocytes. Transient treatment with NCS increases >2-fold the number of DSBs in prophase I oocytes, but less than 30% of these oocytes enter anaphase with segregation errors. MRE11, but not ATM, is essential to detect DSBs in prophase I and is involved in H2AX phosphorylation during metaphase I. Inhibiting MRE11 by mirin during meiotic maturation results in anaphase bridges and also increases the number of γH2AX foci in metaphase II.  Compromised DNA integrity in mirin-treated oocytes indicates a role for MRE11 in chromosome integrity during meiotic maturation.

  5. Constitutive activation of the DNA damage response pathway as a novel therapeutic target in diffuse large B-cell lymphoma

    Science.gov (United States)

    Derenzini, Enrico; Agostinelli, Claudio; Imbrogno, Enrica; Iacobucci, Ilaria; Casadei, Beatrice; Brighenti, Elisa; Righi, Simona; Fuligni, Fabio; Di Rorà, Andrea Ghelli Luserna; Ferrari, Anna; Martinelli, Giovanni; Pileri, Stefano; Zinzani, Pier Luigi

    2015-01-01

    The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AXH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. PMID:25544753

  6. AZD1775 sensitizes T cell acute lymphoblastic leukemia cells to cytarabine by promoting apoptosis over DNA repair.

    Science.gov (United States)

    Ford, James B; Baturin, Dmitry; Burleson, Tamara M; Van Linden, Annemie A; Kim, Yong-Mi; Porter, Christopher C

    2015-09-29

    While some children with acute lymphoblastic leukemia (ALL) have excellent prognoses, the prognosis for adults and children with T cell ALL is more guarded. Treatment for T-ALL is heavily dependent upon antimetabolite chemotherapeutics, including cytarabine. Targeted inhibition of WEE1 with AZD1775 has emerged as a strategy to sensitize cancer cells to cytarabine and other chemotherapeutics. We sought to determine if this strategy would be effective for T-ALL with clinically relevant anti-leukemia agents. We found that AZD1775 sensitizes T-ALL cells to several traditional anti-leukemia agents, acting synergistically with cytarabine by enhancing DNA damage and apoptosis. In addition to increased phosphorylation of H2AX at serine 139 (γH2AX), AZD1775 led to increased phosphorylation of H2AX at tyrosine 142, a signaling event associated with promotion of apoptosis over DNA repair. In a xenograft model of T-ALL, the addition of AZD1775 to cytarabine slowed leukemia progression and prolonged survival. Inhibition of WEE1 with AZD1775 sensitizes T-ALL to several anti-leukemia agents, particularly cytarabine and that mechanistically, AZD1775 promotes apoptosis over DNA repair in cells treated with cytarabine. These data support the development of clinical trials including AZD1775 in combination with conventional chemotherapy for acute leukemia.

  7. Enhanced susceptibility of ovaries from obese mice to 7,12-dimethylbenz[a]anthracene-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Nteeba, Jackson, E-mail: nteeba@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2014-12-01

    7,12-Dimethylbenz[a]anthracene (DMBA) depletes ovarian follicles and induces DNA damage in extra-ovarian tissues, thus, we investigated ovarian DMBA-induced DNA damage. Additionally, since obesity is associated with increased offspring birth defect incidence, we hypothesized that a DMBA-induced DNA damage response (DDR) is compromised in ovaries from obese females. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice were dosed with sesame oil or DMBA (1 mg/kg; intraperitoneal injection) at 18 weeks of age, for 14 days. Total ovarian RNA and protein were isolated and abundance of Ataxia telangiectasia mutated (Atm), X-ray repair complementing defective repair in Chinese hamster cells 6 (Xrcc6), breast cancer type 1 (Brca1), Rad 51 homolog (Rad51), poly [ADP-ribose] polymerase 1 (Parp1) and protein kinase, DNA-activated, catalytic polypeptide (Prkdc) were quantified by RT-PCR or Western blot. Phosphorylated histone H2AXH2AX) level was determined by Western blotting. Obesity decreased (P < 0.05) basal protein abundance of PRKDC and BRCA1 proteins but increased (P < 0.05) γH2AX and PARP1 proteins. Ovarian ATM, XRCC6, PRKDC, RAD51 and PARP1 proteins were increased (P < 0.05) by DMBA exposure in lean mice. A blunted DMBA-induced increase (P < 0.05) in XRCC6, PRKDC, RAD51 and BRCA1 was observed in ovaries from obese mice, relative to lean counterparts. Taken together, DMBA exposure induced γH2AX as well as the ovarian DDR, supporting that DMBA causes ovarian DNA damage. Additionally, ovarian DDR was partially attenuated in obese females raising concern that obesity may be an additive factor during chemical-induced ovotoxicity. - Highlights: • DMBA induces markers of ovarian DNA damage. • Obesity induces low level ovarian DNA damage. • DMBA-induced DNA repair response is altered by obesity.

  8. DNA double strand breaks and Hsp70 expression in proton irradiated living cells

    Energy Technology Data Exchange (ETDEWEB)

    Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

    2007-07-15

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

  9. Replication stress and oxidative damage contribute to aberrant constitutive activation of DNA damage signalling in human gliomas

    DEFF Research Database (Denmark)

    Bartkova, J; Hamerlik, P; Stockhausen, Marie

    2010-01-01

    that the DDR machinery is constitutively activated in gliomas, as documented by phosphorylated histone H2AX (gammaH2AX), activation of the ATM-Chk2-p53 pathway, 53BP1 foci and other markers. Oxidative DNA damage (8-oxoguanine) was high in some GBM cell lines and many GBM tumors, while it was low in normal......-oxygen culture conditions and in clinical specimens of both low- and high-grade tumors. The observed global checkpoint signaling, in contrast to only focal areas of overabundant p53 (indicative of p53 mutation) in grade II astrocytomas, are consistent with DDR activation being an early event in gliomagenesis......, initially limiting cell proliferation (low Ki-67 index) and selecting for mutations of p53 and likely other genes that allow escape (higher Ki-67 index) from the checkpoint and facilitate tumor progression. Overall, these results support the potential role of the DDR machinery as a barrier to gliomagenesis...

  10. The DNA-repair Ku70 protein is located in the nucleus and tail of elongating spermatids in grasshoppers.

    Science.gov (United States)

    Cabrero, Josefa; Palomino-Morales, Rogelio J; Camacho, Juan Pedro M

    2007-01-01

    Fluorescence immunostaining for the phosphorylated H2AX histone (gammaH2AX) in the grasshopper Eyprepocnemis plorans has shown abundance of gammaH2AX in the nuclei of round and elongating spermatids, suggesting that DNA double-strand breaks (DSBs) occur regularly during spermiogenesis. Immunofluorescence patterns for Ku70, a DNA-repair protein participating in the non-homologous end-joining (NHEJ) pathway, showed that this protein is present in round and elongating spermatids, implying that the NHEJ DNA-repair pathway operates during chromatin compaction in spermiogenesis. In addition, during the final stages of spermiogenesis, the Ku70 protein concentrates on the region forming the sperm tail. Since Ku70 was also abundant in spermatid tails, it is reasonable to assume that Ku70 might play a novel function in sperm-tail formation. The analysis of Ku70 immunofluorescence patterns in 13 other grasshopper species also showed the presence of this protein in the nucleus and tail of elongating spermatids, indicating that this is a general characteristic in grasshoppers.

  11. Double-strand break induction and DNA damage response after {sup 12}C ion and photon radiation in U87 glioblastoma cells; Doppelstrangbruch-Induktion und DNA-Schadensantwort nach {sup 12}C-Ionen- und Photonenstrahlung in U87 Glioblastomzellen

    Energy Technology Data Exchange (ETDEWEB)

    Lopez Perez, Ramon

    2015-04-22

    Heavy ion radiation has greater biological effectiveness than the same physical dose of photon radiation. In this work the underlying reasons in the DNA damage response were analyzed in U87 glioblastoma cells. DNA double-strand breaks (DSBs) are the decicive lesions for the effectiveness of ionizing radiation. Their induction and repair was measured in the context of the cell cycle based on the DSB marker γH2AX (the phosphorylated form of the histone variant H2AX). Further, radiation-specific differences in choice of the DSB repair pathway was analyzed, as well as the consequences of repair failure. The results showed that in contrast to photons, {sup 12}C ion radiation produces more severe DSBs that are repaired delayed and with slower kinetics. Accordingly, stronger and longer lasting cell cycle delays, predominantly at the G2/M border, and a higher rate of apoptosis was detected for {sup 12}C ion radiation. Autophagy, an alternative mechanism of programmed cell death, was not relevant for neither of the two types of radiation. The effect of {sup 12}C ion radiation was less dependent on the cell cycle stage than for photon radiation. This became particularly evident in the DSB repair velocities during S- and G2-phase. After {sup 12}C ion radiation, cells were more dependent on homologous recombination repair (HRR) compared to photon radiation. The reason therefore that in contrast to photons, {sup 12}C ion radiation induced graver DSBs that were repaired slower and more dependent on HRR, was most probably enhanced clustering of DSBs due to the higher ionization density of {sup 12}C ion radiation. Microscopic inspection of immunofluorently stained γH2AX revealed that {sup 12}C ion radiation induced bigger DSB repair foci containing more γH2AX molecules (higher fluorescence intensity), although their initial number was smaller. Besides the foci, a weaker pan-nuclear γH2AX staining was observed that increased in a dose-dependent manner and was more pronounced

  12. Atrazine Triggers DNA Damage Response and Induces DNA Double-Strand Breaks in MCF-10A Cells

    Directory of Open Access Journals (Sweden)

    Peixin Huang

    2015-06-01

    Full Text Available Atrazine, a pre-emergent herbicide in the chloro-s-triazine family, has been widely used in crop lands and often detected in agriculture watersheds, which is considered as a potential threat to human health. Although atrazine and its metabolites showed an elevated incidence of mammary tumors in female Sprague–Dawley (SD rats, no molecular evidence was found relevant to its carcinogenesis in humans. This study aims to determine whether atrazine could induce the expression of DNA damage response-related proteins in normal human breast epithelial cells (MCF-10A and to examine the cytotoxicity of atrazine at a molecular level. Our results indicate that a short-term exposure of MCF-10A to an environmentally-detectable concentration of atrazine (0.1 µg/mL significantly increased the expression of tumor necrosis factor receptor-1 (TNFR1 and phosphorylated Rad17 in the cells. Atrazine treatment increased H2AX phosphorylationH2AX and the formation of γH2AX foci in the nuclei of MCF-10A cells. Atrazine also sequentially elevated DNA damage checkpoint proteins of ATM- and RAD3-related (ATR, ATRIP and phospho-Chk1, suggesting that atrazine could induce DNA double-strand breaks and trigger the DNA damage response ATR-Chk1 pathway in MCF-10A cells. Further investigations are needed to determine whether atrazine-triggered DNA double-strand breaks and DNA damage response ATR-Chk1 pathway occur in vivo.

  13. Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

    Energy Technology Data Exchange (ETDEWEB)

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

    2014-08-01

    DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

  14. Establishment of a semi-biological phantom model for the study of the effect of dose reducing measures on radiation-induced DNA double strand breaks in CT using the example of risk organ based tube current modulation; Etablierung eines semibiologischen Phantommodells zur Untersuchung des Effekts dosisreduzierender Massnahmen auf strahleninduzierte DNA-Doppelstrangbrueche in der CT am Beispiel der risikoorganbasierten Roehrenstrommodulation

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, Matthias

    2013-12-12

    The number of computed tomography (CT) examinations has been rising during the last decades. Therefore techniques for dose reduction receive increasing attention. Risk organ-based tube current modulation (RCM) in CT is a new approach and works by lowering the tube current, while the tube is in front of the patient's body. Therefore it should lead to a dose reduction for radiosensitive organs like the female breast, the eye lenses and the thyroid gland. Biological radiation effects cannot be estimated by physical-based dose measurements. γ-H2AX is a sensitive marker for the determination of x-ray induced DNA double-strand breaks (DSB). Hence the aim of this study was to establish a biological phantom model based on the γ-H2AX immunofluorescence microscopy method and to investigate the effect of RCM on radiation induced DNA damages. The γ-H2AX method is based on the phosphorylation of the histone variant H2AX. The phosphorylated histone γ-H2AX can be visualised using antibodies and is specific for radiation induced DSB. Blood lymphocytes from healthy volunteers, skin fibroblasts (LN) and mammary epithelial cells (HMEpC-p) were placed in different positions of an Alderson-phantom and exposed to x-rays using a 128-slice dual-source CT scanner. Standard head, neck and chest-CT scan protocols either with or without risk-organ based tube current modulation were used. RCM reduces the tube current to 20 percent at an angle of 130 degree anterior to the body, whereas tube current is increased at an angle of 230 degree posterior to the body. Afterwards cells were isolated, fixed on slides und stained with specific primary γ-H2AX antibodies and fluorescent secondary antibodies. Tiny green dots (named foci) can be detected and quantified with a fluorescence microscope and represent distinct DSB. Non-irradiated samples served as controls and CT-induced DSB were calculated by subtraction of pre- from post-exposure values. In this study a semibiological phantom model

  15. Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.

    Science.gov (United States)

    Mellinas-Gomez, Maria; Spanswick, Victoria J; Paredes-Moscosso, Solange R; Robson, Matthew; Pedley, R Barbara; Thurston, David E; Baines, Stephen J; Stell, Anneliese; Hartley, John A

    2015-08-19

    Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies. In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by

  16. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang, E-mail: songyangwenrong@hotmail.com

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

  17. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  18. Microvesicles Contribute to the Bystander Effect of DNA Damage.

    Science.gov (United States)

    Lin, Xiaozeng; Wei, Fengxiang; Major, Pierre; Al-Nedawi, Khalid; Al Saleh, Hassan A; Tang, Damu

    2017-04-07

    Genotoxic treatments elicit DNA damage response (DDR) not only in cells that are directly exposed but also in cells that are not in the field of treatment (bystander cells), a phenomenon that is commonly referred to as the bystander effect (BE). However, mechanisms underlying the BE remain elusive. We report here that etoposide and ultraviolet (UV) exposure stimulate the production of microvesicles (MVs) in DU145 prostate cancer cells. MVs isolated from UV-treated DU145 and A431 epidermoid carcinoma cells as well as etoposide-treated DU145 cells induced phosphorylation of ataxia-telangiectasia mutated (ATM) at serine 1981 (indicative of ATM activation) and phosphorylation of histone H2AX at serine 139 (γH2AX) in naïve DU145 cells. Importantly, neutralization of MVs derived from UV-treated cells with annexin V significantly reduced the MV-associated BE activities. Etoposide and UV are known to induce DDR primarily through the ATM and ATM- and Rad3-related (ATR) pathways, respectively. In this regard, MV is likely a common source for the DNA damage-induced bystander effect. However, pre-treatment of DU145 naïve cells with an ATM (KU55933) inhibitor does not affect the BE elicited by MVs isolated from etoposide-treated cells, indicating that the BE is induced upstream of ATM actions. Taken together, we provide evidence supporting that MVs are a source of the DNA damage-induced bystander effect.

  19. Unsuitability of lymphoblastoid cell lines as surrogate of cryopreserved isolated lymphocytes for the analysis of DNA double-strand break repair activity

    Energy Technology Data Exchange (ETDEWEB)

    Zijno, Andrea [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Porcedda, Paola [Department of Clinical and Biological Sciences, University of Turin (Italy); Saini, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Allione, Alessandra [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Garofalo, Bruno; Marcon, Francesca [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Guarrera, Simonetta [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Turinetto, Valentina; Minieri, Valentina [Department of Clinical and Biological Sciences, University of Turin (Italy); Funaro, Ada [Department of Genetics, Biology and Biochemistry, University of Turin (Italy); Crebelli, Riccardo [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Rome (Italy); Giachino, Claudia [Department of Clinical and Biological Sciences, University of Turin (Italy); Matullo, Giuseppe, E-mail: giuseppe.matullo@unito.it [Institute for Scientific Interchange (ISI) Foundation, Villa Gualino, Turin (Italy); Department of Genetics, Biology and Biochemistry, University of Turin (Italy)

    2010-02-03

    As first task of a comprehensive investigation on DNA repair genotype-phenotype correlations, the suitability of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) as surrogate of cryopreserved peripheral blood mononuclear cells (PBMCs) in DNA repair phenotypic assays was evaluated. To this aim the amount of DNA damage induced by {gamma}-rays and DNA repair capacity were evaluated in unstimulated (G{sub 0}) and mitogen-simulated (G{sub 2}) PBMC from 20 healthy subjects and in EBV-transformed LCL obtained from the same individuals. Phosphorylation of histone H2AX, micronuclei and chromosomal aberrations were the end-points investigated. The results obtained show higher basal frequencies of binucleated cells bearing micronuclei and nucleoplasmic bridge (NPB) in LCL with respect to PBMC, suggesting that EBV transformation may be associated with chromosomal instability. After irradiation, higher levels of micronuclei were induced in G{sub 0}-treated PBMC compared to cycling LCL; conversely, NPB were more frequent in LCL than in PBMC. Moreover, higher levels of chromosomal aberrations were observed in G{sub 2}-treated PBMC compared to LCL. Concerning {gamma}-H2AX measurements, phosphorylation levels 1 h after treatment and dephosphorylation kinetics were basically similar in LCL and in PBMC. However, while Spearman's test showed a strong correlation between the results obtained in replicated experiments with PBMC, high inter-experimental variability and poor reproducibility was observed in the experiments performed with LCL, possibly due to the intrinsic instability of LCL. In summary, both the analysis of {gamma}-H2AX and the evaluation of chromosome damage highlighted a larger inter-experimental variability in the results obtained with LCL compared to PBMC. Noteworthy, the two set of results proved to lack any significant correlation at the individual level. These results indicate that LCL may be unsuitable for investigating genotype

  20. Evaluation of low-dose proton beam radiation efficiency in MIA PaCa-2 pancreatic cancer cell line vitality and H2AX formation

    Directory of Open Access Journals (Sweden)

    Aušra Liubavičiūtė

    2015-11-01

    Conclusions: Our data demonstrate that low-doses proton beam irradiation had an effect on MIA PaCa-2 pancreatic carcinoma cell line. Full extent of irradiation had an impact only 24 h postirradiation, triggering DNA arrested cell cycle in G1/0 phase. Formed DNA DSBs were found to be repaired via the NHEJ pathway mechanism within 72 h. Unsuccessful repaired DSBs induced apoptotic cell death. After 72 h reparation processes were completed, and cell cycle was released from arrest in G1/0 phase.

  1. DNA damage response in male gametes of Cyrtanthus mackenii during pollen tube growth.

    Science.gov (United States)

    Hirano, Tomonari; Takagi, Keiichi; Hoshino, Yoichiro; Abe, Tomoko

    2013-01-01

    Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AXH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired.

  2. Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis.

    Science.gov (United States)

    Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-07-01

    Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Attenuation of the DNA Damage Response by Transforming Growth Factor-Beta Inhibitors Enhances Radiation Sensitivity of Non–Small-Cell Lung Cancer Cells In Vitro and In Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Du, Shisuo; Bouquet, Sophie; Lo, Chen-Hao; Pellicciotta, Ilenia; Bolourchi, Shiva [Department of Radiation Oncology, New York University School of Medicine, New York, New York (United States); Parry, Renate [Varian Medical Systems, Palo Alto, California (United States); Barcellos-Hoff, Mary Helen, E-mail: mhbarcellos-hoff@nyumc.org [Department of Radiation Oncology, New York University School of Medicine, New York, New York (United States)

    2015-01-01

    Purpose: To determine whether transforming growth factor (TGF)-β inhibition increases the response to radiation therapy in human and mouse non–small-cell lung carcinoma (NSCLC) cells in vitro and in vivo. Methods and Materials: TGF-β–mediated growth response and pathway activation were examined in human NSCLC NCI-H1299, NCI-H292, and A549 cell lines and murine Lewis lung cancer (LLC) cells. Cells were treated in vitro with LY364947, a small-molecule inhibitor of the TGF-β type 1 receptor kinase, or with the pan-isoform TGF-β neutralizing monoclonal antibody 1D11 before radiation exposure. The DNA damage response was assessed by ataxia telangiectasia mutated (ATM) or Trp53 protein phosphorylation, γH2AX foci formation, or comet assay in irradiated cells. Radiation sensitivity was determined by clonogenic assay. Mice bearing syngeneic subcutaneous LLC tumors were treated with 5 fractions of 6 Gy and/or neutralizing or control antibody. Results: The NCI-H1299, A549, and LLC NSCLC cell lines pretreated with LY364947 before radiation exposure exhibited compromised DNA damage response, indicated by decreased ATM and p53 phosphorylation, reduced γH2AX foci, and increased radiosensitivity. The NCI-H292 cells were unresponsive. Transforming growth factor-β signaling inhibition in irradiated LLC cells resulted in unresolved DNA damage. Subcutaneous LLC tumors in mice treated with TGF-β neutralizing antibody exhibited fewer γH2AX foci after irradiation and significantly greater tumor growth delay in combination with fractionated radiation. Conclusions: Inhibition of TGF-β before radiation attenuated DNA damage recognition and increased radiosensitivity in most NSCLC cells in vitro and promoted radiation-induced tumor control in vivo. These data support the rationale for concurrent TGF-β inhibition and RT to provide therapeutic benefit in NSCLC.

  4. ASPM influences DNA double-strand break repair and represents a potential target for radiotherapy.

    Science.gov (United States)

    Kato, Takamitsu A; Okayasu, Ryuichi; Jeggo, Penny A; Fujimori, Akira

    2011-12-01

    In a previous study using HiCEP (High coverage expression profiling), we demonstrated that ASPM (abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene was selectively down-regulated by IR (ionizing radiation). The roles of ASPM on radiosensitivity, however, have never been studied. Using glioblastoma cell lines and normal human fibroblasts, we investigated how IR sensitivity (survived fraction, DNA repair and chromosome aberration) was affected by the reduction of ASPM by specific siRNA (small interfering RNA). Down-regulation of ASPM by siRNA enhanced radiosensitivity in three human cell lines examined. Constant-field gel electrophoreses and γ-H2AX (phosphorylated form of Histone H2A variant H2AX) foci analysis showed that ASPM-specific siRNA impaired DNA double-strand breaks (DSB) in irradiated cells. Elevated levels of abnormal chromosomes were also observed following ASPM siRNA. In addition IR-sensitization by ASPM knockdown was not enhanced in DNA-PK (DNA-dependent protein kinase) deficient glioblastoma cells suggesting that ASPM impacts upon a DNA-PK-dependent pathway. Our results show for the first time that ASPM is required for efficient non-homologous end-joining in mammalian cells. In clinical applications, ASPM could be a novel target for combination therapy with radiation as well as a useful biomarker for tumor prognosis as ever described.

  5. Utilisation de l'essai comete et du biomarqueur gamma-H2AX pour detecter les dommages induits a l'ADN cellulaire par le 5-bromodeoxyuridine post-irradiation

    Science.gov (United States)

    La Madeleine, Carole

    Ce memoire est presente a la Faculte de medecine et des sciences de la sante de l'Universite de Sherbrooke en vue de l'obtention du grade de maitre es sciences (M.Sc.) en radiobiologie (2009). Un jury a revise les informations contenues dans ce memoire. Il etait compose de professeurs de la Faculte de medecine et des sciences de la sante soit : Darel Hunting PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Leon Sanche PhD, directeur de recherche (departement de medecine nucleaire et radiobiologie), Richard Wagner PhD, membre du programme (departement de medecine nucleaire et radiobiologie) et Guylain Boissonneault PhD, membre exterieur au programme (departement de biochimie). Le 5-bromodeoxyuridine (BrdU), un analogue halogene de la thymidine reconnu depuis les annees 60 comme etant un excellent radiosensibilisateur. L'hypothese la plus repandue au sujet de l'effet radio sensibilisant du BrdU est qu'il augmente le nombre de cassures simple et double brin lorsqu'il est incorpore dans l'ADN de la cellule et expose aux radiations ionisantes. Toutefois, de nouvelles recherches semblent remettre en question les observations precedentes. Ces dernieres etudes ont confirme que le BrdU est un bon radiosensibilisateur, car il augmente les dommages radio-induits dans l'ADN. Mais, c'est en etant incorpore dans une region simple brin que le BrdU radiosensibilise l'ADN. Ces recherches ont egalement revele pour la premiere fois un nouveau type de dommages produits lors de l'irradiation de l'ADN contenant du BrdU : les dimeres interbrins. Le but de ces travaux de recherche est de determiner si la presence de bromodeoxyuridine dans l'ADN augmente l'induction de bris simple et / ou double brin chez les cellules irradiees en utilisant de nouvelles techniques plus sensibles et specifiques que celles utilisees auparavant. Pour ce faire, les essais cometes et la detection des foci H2AX phosphorylee pourraient permettre d'etablir les effets engendres par

  6. DNA Damage and Inhibition of Akt Pathway in MCF-7 Cells and Ehrlich Tumor in Mice Treated with 1,4-Naphthoquinones in Combination with Ascorbate

    Directory of Open Access Journals (Sweden)

    Fabiana Ourique

    2015-01-01

    Full Text Available The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9 were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

  7. Replication independent DNA double-strand break retention may prevent genomic instability

    Directory of Open Access Journals (Sweden)

    Pornthanakasem Wichai

    2010-03-01

    Full Text Available Abstract Background Global hypomethylation and genomic instability are cardinal features of cancers. Recently, we established a method for the detection of DNA methylation levels at sites close to endogenous DNA double strand breaks (EDSBs, and found that those sites have a higher level of methylation than the rest of the genome. Interestingly, the most significant differences between EDSBs and genomes were observed when cells were cultured in the absence of serum. DNA methylation levels on each genomic location are different. Therefore, there are more replication-independent EDSBs (RIND-EDSBs located in methylated genomic regions. Moreover, methylated and unmethylated RIND-EDSBs are differentially processed. Euchromatins respond rapidly to DSBs induced by irradiation with the phosphorylation of H2AX, γ-H2AX, and these initiate the DSB repair process. During G0, most DSBs are repaired by non-homologous end-joining repair (NHEJ, mediated by at least two distinct pathways; the Ku-mediated and the ataxia telangiectasia-mutated (ATM-mediated. The ATM-mediated pathway is more precise. Here we explored how cells process methylated RIND-EDSBs and if RIND-EDSBs play a role in global hypomethylation-induced genomic instability. Results We observed a significant number of methylated RIND-EDSBs that are retained within deacetylated chromatin and free from an immediate cellular response to DSBs, the γ-H2AX. When cells were treated with tricostatin A (TSA and the histones became hyperacetylated, the amount of γ-H2AX-bound DNA increased and the retained RIND-EDSBs were rapidly repaired. When NHEJ was simultaneously inhibited in TSA-treated cells, more EDSBs were detected. Without TSA, a sporadic increase in unmethylated RIND-EDSBs could be observed when Ku-mediated NHEJ was inhibited. Finally, a remarkable increase in RIND-EDSB methylation levels was observed when cells were depleted of ATM, but not of Ku86 and RAD51. Conclusions Methylated RIND-EDSBs are

  8. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

    Science.gov (United States)

    Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

    2016-01-01

    DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

  9. DNA damages induced by Ar F laser

    Energy Technology Data Exchange (ETDEWEB)

    Chapel, C.; Rose, S.; Chevrier, L.; Cordier, E.; Courant, D. [CEA Fontenay-aux-Roses, 92 (France). Dept. de Radiobiologie et de Radiopathologie

    2006-07-01

    The photo ablation process used in corneal refractive surgery by the Argon Fluoride (Ar F) laser emitting in ultraviolet C at 193 nm, exposes viable cells round the irradiated zone to sub ablative doses (< 400 joules.m -2). Despite that DNA absorption is higher at 193 nm than 254 nm, cytotoxicity of 193 nm laser radiation is lower than radiation emitted by 254 nm UV-C lamps. In situ, DNA could be protected of laser radiation by cellular components. Consequently, some authors consider that this radiation does not induce genotoxic effect whereas others suspect it to be mutagenic. These lasers are used for fifteen years but many questions remain concerning the long term effects on adjacent cells to irradiated area. The purpose of this study is to describe the effect of 193 nm laser radiation on DNA of stromal keratocytes which are responsible of the corneal structure. The 193 nm laser irradiation induces directly DNA breakage in keratocytes as it has been shown by the comet assay under alkaline conditions. Two hours post irradiation, damages caused by the highest exposure (150 J.m-2) are not repaired as it has been measured with the Olive Tail Moment (product of tail length and tail DNA content). They give partly evidence of induction of an apoptotic process in cells where DNA could be too damaged. In order to characterize specifically double strand breaks, a comparative analysis by immunofluorescence of the H2 Ax histone phosphorylation (H2 Ax) has been performed on irradiated keratocytes and unirradiated keratocytes. Results show a dose dependent increase of the number of H2 Ax positive cells. Consequences of unrepaired DNA lesions could be observed by the generation of micronuclei in cells. Results show again an increase of micronuclei in laser irradiated cells. Chromosomal aberrations have been pointed out by cytogenetic methods 30 mn after irradiation. These aberrations are dose dependent (from 10 to 150 J.m-2). The number of breakage decreases in the long run

  10. Induction and reparation of double-strand DNA breaks in V79 cells continuously exposed to low dose-rate Y-radiation

    Directory of Open Access Journals (Sweden)

    Ozerov I.V.

    2013-12-01

    Full Text Available Aim: to study the patterns of changes in the number of DNA double-strand breaks (DSB in mammalian cells continuously exposed to low dose-rate y- radiation. Material and methods. Chinese hamster lung fibroblasts (V79 were used in this study. The y- irradiation of cells at a dose rate of 0.1 mGy/min was performed using the «Gamma-Panorama» unit (Cs-137. The fluorescence immunoassay of the phosphorylated H2AX-histone (y-H2AX foci was used to investigate the DNA DSBs formation. Frequency of apoptotic cells was evaluated using «DNA halo» assay. 5 (6 — chloromethyl-2,7-dichlorodihydrofluorescein diacetate was used to estimate the reactive oxygen species (ROS production. Results, it was showed that continuous low dose-rate irradiation of Chinese hamster V79 cells induces an increase of the y-H2AX foci number and ROS production rate at the early stages of exposure time (6-24 h, doses 3.6-14.4 cGy, while increasing exposition time and, therefore, the radiation dose (48-72 h, 28.8-43.2 cGy caused a decrease in these endpoints to almost the control level. There was observed no significant changes in the frequency of apoptotic cells. Conclusion. It is assumed that the processes causing the DSB amount changes in mammalian cells continuously exposed to low dose-rate y-radiation are associated with the development of oxidative stress and subsequent activation of cellular antioxidant defense systems.

  11. The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

    Science.gov (United States)

    Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

    2012-09-01

    We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

  12. Image-based modeling reveals dynamic redistribution of DNA damage into nuclear sub-domains.

    Directory of Open Access Journals (Sweden)

    Sylvain V Costes

    2007-08-01

    Full Text Available Several proteins involved in the response to DNA double strand breaks (DSB form microscopically visible nuclear domains, or foci, after exposure to ionizing radiation. Radiation-induced foci (RIF are believed to be located where DNA damage occurs. To test this assumption, we analyzed the spatial distribution of 53BP1, phosphorylated ATM, and gammaH2AX RIF in cells irradiated with high linear energy transfer (LET radiation and low LET. Since energy is randomly deposited along high-LET particle paths, RIF along these paths should also be randomly distributed. The probability to induce DSB can be derived from DNA fragment data measured experimentally by pulsed-field gel electrophoresis. We used this probability in Monte Carlo simulations to predict DSB locations in synthetic nuclei geometrically described by a complete set of human chromosomes, taking into account microscope optics from real experiments. As expected, simulations produced DNA-weighted random (Poisson distributions. In contrast, the distributions of RIF obtained as early as 5 min after exposure to high LET (1 GeV/amu Fe were non-random. This deviation from the expected DNA-weighted random pattern can be further characterized by "relative DNA image measurements." This novel imaging approach shows that RIF were located preferentially at the interface between high and low DNA density regions, and were more frequent than predicted in regions with lower DNA density. The same preferential nuclear location was also measured for RIF induced by 1 Gy of low-LET radiation. This deviation from random behavior was evident only 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AX and 53BP1 RIF showed pronounced deviations up to 30 min after exposure. These data suggest that DNA damage-induced foci are restricted to certain regions of the nucleus of human epithelial cells. It is possible that DNA lesions are collected in these nuclear sub-domains for more efficient repair.

  13. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  14. XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage

    Science.gov (United States)

    Lévy, Nicolas; Martz, Adeline; Bresson, Anne; Spenlehauer, Catherine; de Murcia, Gilbert; Ménissier-de Murcia, Josiane

    2006-01-01

    The two BRCT domains (BRCT1 and BRCT2) of XRCC1 mediate a network of protein–protein interactions with several key factors of the DNA single-strand breaks (SSBs) and base damage repair pathways. BRCT1 is required for the immediate poly(ADP–ribose)-dependent recruitment of XRCC1 to DNA breaks and is essential for survival after DNA damage. To better understand the biological role of XRCC1 in the processing of DNA ends, a search for the BRCT1 domain-associated proteins was performed by mass spectrometry of GST-BRCT1 pulled-down proteins from HeLa cell extracts. Here, we report that the double-strand break (DSB) repair heterotrimeric complex DNA-PK interacts with the BRCT1 domain of XRCC1 and phosphorylates this domain at serine 371 after ionizing irradiation. This caused XRCC1 dimer dissociation. The XRCC1 R399Q variant allele did not affect this phosphorylation. We also show that XRCC1 strongly stimulates the phosphorylation of p53-Ser15 by DNA-PK. The pseudo phosphorylated S371D mutant was a much weaker stimulator of DNA-PK activity whereas the non-phosphorylable mutant S371L endowed with a DNA-PK stimulating capacity failed to fully rescue the DSB repair defect of XRCC1-deficient EM9 rodent cells. The functional association between XRCC1 and DNA-PK in response to IR provides the first evidence for their involvement in a common DSB repair pathway. PMID:16397295

  15. Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance.

    Science.gov (United States)

    Mori, Eiichiro; Davis, Anthony J; Hasegawa, Masatoshi; Chen, David J

    2016-08-19

    DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that plays an essential role in the repair of DNA double-strand breaks (DSBs) in the non-homologous end-joining (NHEJ) pathway. The DNA-PK holoenzyme consists of a catalytic subunit (DNA-PKcs) and DNA-binding subunit (Ku70/80, Ku). Ku is a molecular sensor for double-stranded DNA and once bound to DSB ends it recruits DNA-PKcs to the DSB site. Subsequently, DNA-PKcs is activated and heavily phosphorylated, with these phosphorylations modulating DNA-PKcs. Although phosphorylation of DNA-PKcs is well studied, other post-translational modifications of DNA-PKcs are not. In this study, we aimed to determine if acetylation of DNA-PKcs regulates DNA-PKcs-dependent DSB repair. We report that DNA-PKcs is acetylated in vivo and identified two putative acetylation sites, lysine residues 3241 and 3260. Mutating these sites to block potential acetylation results in increased radiosensitive, a slight decrease in DSB repair capacity as assessed by γH2AX resolution, and increased chromosomal aberrations, especially quadriradial chromosomes. Together, our results provide evidence that acetylation potentially regulates DNA-PKcs. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  17. MOF phosphorylation by ATM regulates 53BP1-mediated DSB repair pathway choice

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R.; Hegdec, Muralidhar L.; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh1, Mayank; Ramnarain, Deepti B.; Hittelman, Walter N.; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K.; Ludwig, Thomas; Pandita, Raj K.; Tyler, Jessica K.; Pandita, Tej K.

    2014-01-01

    Cell cycle phase is a critical determinant of the choice between DNA damage repair by non-homologous end joining (NHEJ) or homologous recombination (HR). Here we report that DSBs induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF co-localizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S- and G2-phase but not G1-phase cells. Expression of MOF-T392A also reverses the reduction in DSB associated 53BP1 seen in wild type S/G2-phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair and decreased cell survival following irradiation. These data support a model whereby ATM mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2-phase. PMID:24953651

  18. MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice.

    Science.gov (United States)

    Gupta, Arun; Hunt, Clayton R; Hegde, Muralidhar L; Chakraborty, Sharmistha; Chakraborty, Sharmistha; Udayakumar, Durga; Horikoshi, Nobuo; Singh, Mayank; Ramnarain, Deepti B; Hittelman, Walter N; Namjoshi, Sarita; Asaithamby, Aroumougame; Hazra, Tapas K; Ludwig, Thomas; Pandita, Raj K; Tyler, Jessica K; Pandita, Tej K

    2014-07-10

    Cell-cycle phase is a critical determinant of the choice between DNA damage repair by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Here, we report that double-strand breaks (DSBs) induce ATM-dependent MOF (a histone H4 acetyl-transferase) phosphorylation (p-T392-MOF) and that phosphorylated MOF colocalizes with γ-H2AX, ATM, and 53BP1 foci. Mutation of the phosphorylation site (MOF-T392A) impedes DNA repair in S and G2 phase but not G1 phase cells. Expression of MOF-T392A also blocks the reduction in DSB-associated 53BP1 seen in wild-type S/G2 phase cells, resulting in enhanced 53BP1 and reduced BRCA1 association. Decreased BRCA1 levels at DSB sites correlates with defective repairosome formation, reduced HR repair, and decreased cell survival following irradiation. These data support a model whereby ATM-mediated MOF-T392 phosphorylation modulates 53BP1 function to facilitate the subsequent recruitment of HR repair proteins, uncovering a regulatory role for MOF in DSB repair pathway choice during S/G2 phase. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    Science.gov (United States)

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Ubiquitin Accumulation on Disease Associated Protein Aggregates Is Correlated with Nuclear Ubiquitin Depletion, Histone De-Ubiquitination and Impaired DNA Damage Response.

    Directory of Open Access Journals (Sweden)

    Adi Ben Yehuda

    Full Text Available Deposition of ubiquitin conjugates on inclusion bodies composed of protein aggregates is a definitive cytopathological hallmark of neurodegenerative diseases. We show that accumulation of ubiquitin on polyQ IB, associated with Huntington's disease, is correlated with extensive depletion of nuclear ubiquitin and histone de-ubiquitination. Histone ubiquitination plays major roles in chromatin regulation and DNA repair. Accordingly, we observe that cells expressing IB fail to respond to radiomimetic DNA damage, to induce gamma-H2AX phosphorylation and to recruit 53BP1 to damaged foci. Interestingly ubiquitin depletion, histone de-ubiquitination and impaired DNA damage response are not restricted to PolyQ aggregates and are associated with artificial aggregating luciferase mutants. The longevity of brain neurons depends on their capacity to respond to and repair extensive ongoing DNA damage. Impaired DNA damage response, even modest one, could thus lead to premature neuron aging and mortality.

  1. Quercetin-3-O-glucronide inhibits noradrenaline binding to α2-adrenergic receptor, thus suppressing DNA damage induced by treatment with 4-hydroxyestradiol and noradrenaline in MCF-10A cells.

    Science.gov (United States)

    Yamazaki, Shunsuke; Sakakibara, Hiroyuki; Takemura, Hitomi; Yasuda, Michiko; Shimoi, Kayoko

    2014-09-01

    Risk factors for breast cancer include estrogens such as 17β-estradiol (E2) and high stress levels. 4-Hydroxyestradiol (4-OHE2), a metabolite of E2 formed preferentially by cytochrome P450 1B1, is oxidized to E2-3,4-quinone, which reacts with DNA to form depurinating adducts that exert genotoxicity and carcinogenicity. Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. Here, we found that treatment with 4-OHE2 (3 μM) and NA (3 nM) significantly induced the phosphorylation of histone H2AX (γ-H2AX), one of the earliest indicators of DNA damage, and apurinic (AP) sites via the α2-adrenergic receptor (α2-AR) in human mammary epithelial MCF-10A cells. As an inverse association between a higher intake of flavonoids and breast cancer risk has previously been suggested from epidemiological studies, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin in the blood, on 4-OHE2- and NA-induced γ-H2AX and AP sites. Q3G (0.1 μM) suppressed their induction and inhibited the binding of [(3)H]-NA to α2-AR. These results suggest that Q3G acts as an α2-AR antagonist and that it could be used as a chemopreventive agent for stress-promoted breast cancer. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication

    DEFF Research Database (Denmark)

    Kamalyukova, Ilnaz M; Young, Clifford; Strømme, Caroline B

    2014-01-01

    During DNA replication, nucleosomes are rapidly assembled on newly synthesized DNA to restore chromatin organization. Asf1, a key histone H3-H4 chaperone required for this process, is phosphorylated by Tousled-like kinases (TLKs). Here, we identify TLK phosphorylation sites by mass spectrometry...

  3. ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

    Science.gov (United States)

    Ray, Alo; Blevins, Chessica; Wani, Gulzar; Wani, Altaf A

    2016-01-01

    Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

  4. Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency.

    NARCIS (Netherlands)

    Hinttala, R.; Smeets, R.; Moilanen, J.S.; Ugalde, C.; Uusimaa, J.; Smeitink, J.A.M.; Majamaa, K.

    2006-01-01

    BACKGROUND: Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. OBJECTIVE: To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying

  5. Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

    Directory of Open Access Journals (Sweden)

    Fernet Marie

    2003-07-01

    Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

  6. Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

    Science.gov (United States)

    Lu, Tao; Wu, Honglu; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Wong, Michael

    2016-07-01

    Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not

  7. Induction of in situ DNA double-strand breaks and apoptosis by 200 MeV protons and 10 MV X-rays in human tumour cell lines.

    Science.gov (United States)

    Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Suzuki, Kenshi; Terunuma, Toshiyuki; Yasuoka, Kiyoshi; Sakae, Takeji; Moritake, Takashi; Tsuboi, Koji

    2011-01-01

    To clarify the properties of clinical high-energy protons by comparing with clinical high-energy X-rays. Human tumor cell lines, ONS76 and MOLT4, were irradiated with 200 MeV protons or 10 MV X-rays. In situ DNA double-strand breaks (DDSB) induction was evaluated by immunocytochemical staining of phosphorylated histone H2AX (γ-H2AX). Apoptosis was measured by flow-cytometry after staining with Annexin V. The relative biological effectiveness (RBE) was obtained by clonogenic survival assay. DDSB induction was significantly higher for protons than X-rays with average ratios of 1.28 (ONS76) and 1.59 (MOLT4) at 30 min after irradiation. However the differences became insignificant at 6 h. Also, apoptosis induction in MOLT4 cells was significantly higher for protons than X-rays with an average ratio of 2.13 at 12 h. However, the difference became insignificant at 20 h. RBE values of protons to X-rays at 10% survival were 1.06 ± 0.04 and 1.02 ± 0.15 for ONS76 and MOLT4, respectively. Cell inactivation may differ according to different timings and/or endpoints. Proton beams demonstrated higher cell inactivation than X-rays in the early phases. These data may facilitate the understanding of the biological properties of clinical proton beams.

  8. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search ...... of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria....

  9. Hepatitis B virus X protein increases the Cdt1-to-geminin ratio inducing DNA re-replication and polyploidy.

    Science.gov (United States)

    Rakotomalala, Lova; Studach, Leo; Wang, Wen-Horng; Gregori, Gerald; Hullinger, Ronald L; Andrisani, Ourania

    2008-10-17

    Hepatitis B virus X protein (pX) is implicated in hepatocellular carcinoma pathogenesis by an unknown mechanism. Employing the tetracycline-regulated pX-expressing 4pX-1 cell line, derived from the murine AML12 hepatocyte cell line, we demonstrate that pX induces partial polyploidy (>4N DNA). Depletion of p53 in 4pX-1 cells increases by 5-fold the polyploid cells in response to pX expression, indicating that p53 antagonizes pX-induced polyploidy. Dual-parameter flow cytometric analyses show pX-dependent bromodeoxyuridine (BrdUrd) incorporation in 4pX-1 cells containing 4N and >4N DNA, suggesting pX induces DNA re-replication. Interestingly, pX increases expression of endogenous replication initiation factors Cdc6 and Cdtl while suppressing geminin expression, a negative regulator of rereplication. In comparison to a geminin knockdown 4pX-1 cell line used as DNA re-replication control, the Cdt1/geminin ratio is greater in 4pX-1 cells expressing pX, indicating that pX promotes DNA re-replication. In support of this conclusion, pX-expressing 4pX-1 cells, similar to the geminin knockdown 4pX-1 cells, continue to incorporate BrdUrd in the G2 phase and exhibit nuclear Cdc6 and MCM5 co-localization and the absence of geminin. In addition, pX expression activates the ATR kinase, the sensor of DNA re-replication, which in turn phosphorylates RAD17 and H2AX. Interestingly, phospho-H2AX-positive and BrdUrd -positive cells progress through mitosis, demonstrating a link between pX-induced DNA re-replication and polyploidy. Our studies high-light a novel function of pX that likely contributes to hepatocellular carcinoma pathogenesis.

  10. Rad50 zinc hook is important for the Mre11 complex to bind chromosomal DNA double-stranded breaks and initiate various DNA damage responses.

    Science.gov (United States)

    He, Jing; Shi, Linda Z; Truong, Lan N; Lu, Chi-Sheng; Razavian, Niema; Li, Yongjiang; Negrete, Alejandro; Shiloach, Joseph; Berns, Michael W; Wu, Xiaohua

    2012-09-14

    The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.

  11. Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses*

    Science.gov (United States)

    He, Jing; Shi, Linda Z.; Truong, Lan N.; Lu, Chi-Sheng; Razavian, Niema; Li, Yongjiang; Negrete, Alejandro; Shiloach, Joseph; Berns, Michael W.; Wu, Xiaohua

    2012-01-01

    The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn2+ promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation. PMID:22833675

  12. Spatiotemporal characterization of ionizing radiation induced DNA damage foci and their relation to chromatin organization

    Energy Technology Data Exchange (ETDEWEB)

    Costes, Sylvain V; Chiolo, Irene; Pluth, Janice M.; Barcellos-Hoff, Mary Helen; Jakob, Burkhard

    2009-09-15

    DNA damage sensing proteins have been shown to localize to the sites of DSB within seconds to minutes following ionizing radiation (IR) exposure, resulting in the formation of microscopically visible nuclear domains referred to as radiation-induced foci (RIF). This review characterizes the spatio-temporal properties of RIF at physiological doses, minutes to hours following exposure to ionizing radiation, and it proposes a model describing RIF formation and resolution as a function of radiation quality and nuclear densities. Discussion is limited to RIF formed by three interrelated proteins ATM (Ataxia telangiectasia mutated), 53BP1 (p53 binding protein 1) and ?H2AX (phosphorylated variant histone H2AX). Early post-IR, we propose that RIF mark chromatin reorganization, leading to a local nuclear scaffold rigid enough to keep broken DNA from diffusing away, but open enough to allow the repair machinery. We review data indicating clear kinetic and physical differences between RIF emerging from dense and uncondensed regions of the nucleus. At later time post-IR, we propose that persistent RIF observed days following exposure to ionizing radiation are nuclear ?scars? marking permanent disruption of the chromatin architecture. When DNA damage is resolved, such chromatin modifications should not necessarily lead to growth arrest and it has been shown that persistent RIF can replicate during mitosis. Thus, heritable persistent RIF spanning over tens of Mbp may affect the transcriptome of a large progeny of cells. This opens the door for a non DNA mutation-based mechanism of radiation-induced phenotypes.

  13. Boric Acid Reduces the Formation of DNA Double Strand Breaks and Accelerates Wound Healing Process.

    Science.gov (United States)

    Tepedelen, Burcu Erbaykent; Soya, Elif; Korkmaz, Mehmet

    2016-12-01

    Boron is absorbed by the digestive and respiratory system, and it was considered that it is converted to boric acid (BA), which was distributed to all tissues above 90 %. The biochemical essentiality of boron element is caused by boric acid because it affects the activity of several enzymes involved in the metabolism. DNA damage repair mechanisms and oxidative stress regulation is quite important in the transition stage from normal to cancerous cells; thus, this study was conducted to investigate the protective effect of boric acid on DNA damage and wound healing in human epithelial cell line. For this purpose, the amount of DNA damage occurred with irinotecan (CPT-11), etoposide (ETP), doxorubicin (Doxo), and H2O2 was determined by immunofluorescence through phosphorylation of H2AX((Ser139)) and pATM((Ser1981)) in the absence and presence of BA. Moreover, the effect of BA on wound healing has been investigated in epithelial cells treated with these agents. Our results demonstrated that H2AX((Ser139)) foci numbers were significantly decreased in the presence of BA while wound healing was accelerated by BA compared to that in the control and only drug-treated cells. Eventually, the results indicate that BA reduced the formation of DNA double strand breaks caused by agents as well as improving the wound healing process. Therefore, we suggest that boric acid has important therapeutical effectiveness and may be used in the treatment of inflammatory diseases where oxidative stress and wound healing process plays an important role.

  14. 7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2013-11-01

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4 days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and maintained in culture for 4 or 8 days. Alternately, PND4 F344 rat ovaries were exposed to 1 μM DMBA at the start of culture for 2 days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. γH2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and γH2AX protein. Additionally, DMBA (12.5 nM and 1 μM) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8 days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75 nM d4; 12.5 nM d8), while pATM was localized in large primary and secondary follicles and increased after 8 days of 75 nM DMBA exposure compared to both control and 12.5 nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss. - Highlights: • DMBA exposure increases ovarian caspase-3 protein expression. • DMBA exposure increases the γH2AX protein in oocytes. • DMBA exposure activates a DNA repair response in the ovary.

  15. Phosphorylation of human INO80 is involved in DNA damage tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Utsugi, Takahiko [Bio Matrix Research Inc., 105 Higashifukai, Nagareyama, Chiba 275-0101 (Japan); Ohtsu, Masaya [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Murakami, Yasufumi, E-mail: yasufumi@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Bio Matrix Research Inc., 105 Higashifukai, Nagareyama, Chiba 275-0101 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

  16. DNA Damage Caused by Metal Nanoparticles: the Involvement of Oxidative Stress and Activation of ATM

    Science.gov (United States)

    Wan, Rong; Mo, Yiqun; Feng, Lingfang; Chien, Sufan; Tollerud, David J.; Zhang, Qunwei

    2012-01-01

    Nanotechnology is a fast growing emerging field, the benefits of which are widely publicized. Our current knowledge of the health effects of metal nanoparticles such as nano-sized cobalt (Nano-Co) and titanium dioxide (Nano-TiO2) is limited but suggests that metal nanoparticles may exert more adverse pulmonary effects as compared with standard-sized particles. To investigate metal nanoparticle-induced genotoxic effects and the potential underlying mechanisms, human lung epithelial cell lines A549 cells were exposed to Nano-Co and Nano-TiO2. Our results showed that exposure of A549 cells to Nano-Co caused reactive oxygen species (ROS) generation that was abolished by pretreatment of cells with ROS inhibitors or scavengers, such as catalase and N-acetyl-L(+)-cysteine (NAC). However, exposure of A549 cells to Nano-TiO2 did not cause ROS generation. Nano-Co caused DNA damage in A549 cells which was reflected by an increase in length, width, and DNA content of the comet tail by Comet assay. Exposure of A549 cells to Nano-Co also caused a dose-and a time- response increased expression of phosphorylated histone H2AX (γ-H2AX), Rad51 and phosphorylated p53. These effects were significantly attenuated when A549 cells were pre-treated with catalase or NAC. Nano-TiO2 did not show these effects. These results suggest that oxidative stress may be involved in Nano-Co-induced DNA damage. To further investigate the pathways involved in the Nano-Co-induced DNA damage, we measured the phosphorylation of ataxia telangiectasia mutant (ATM). Our results showed that phosphorylation of ATM was increased when A549 cells were exposed to Nano-Co, and this effect was attenuated when cells were pretreated with catalase or NAC. Pre-treatment of A549 cells with an ATM specific inhibitor, KU55933, significantly abolished Nano-Co-induced DNA damage. Furthermore, pre-treatment of A549 cells with ROS scavengers, such as catalase and NAC, significantly abolished Nano-Co-induced increased expression

  17. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis.

    Science.gov (United States)

    Deniz, Miriam; Kaufmann, Julia; Stahl, Andreea; Gundelach, Theresa; Janni, Wolfgang; Hoffmann, Isabell; Keimling, Marlen; Hampp, Stephanie; Ihle, Michaela; Wiesmüller, Lisa

    2016-11-01

    Dysfunction of homologous recombination is a common denominator of changes associated with breast cancer-predisposing mutations. In our previous work, we identified a functional signature in peripheral blood lymphocytes from women who were predisposed that indicated a shift from homologous recombination to alternative, error-prone DNA double-strand break (DSB) repair pathways. To capture both hereditary and nonhereditary factors, we newly established a protocol for isolation and ex vivo analysis of epithelial cells, epithelial-mesenchymal transition cells (EMTs), and fibroblasts from breast cancer specimens (147 patients). By applying a fluorescence-based test system, we analyzed the error-prone DSB repair pathway microhomology-mediated end joining in these tumor-derived cell types and peripheral blood lymphocytes. In parallel, we investigated DNA lesion processing by quantitative immunofluorescence microscopy of histone H2AX phosphorylated on Ser139 focus after radiomimetic treatment. Our study reveals elevated histone H2AX phosphorylated on Ser139 damage removal in epithelial cells, not EMTs, and poly(ADP-ribose)polymerase inhibitor sensitivities, which suggested a DSB repair pathway shift with increasing patient age. Of interest, we found elevated microhomology-mediated end joining in EMTs, not epithelial cells, from patients who received a treatment recommendation of adjuvant chemotherapy, that is, those with high-risk tumors. Our discoveries of altered DSB repair activities in cells may serve as a method to further classify breast cancer to predict responsiveness to adjuvant chemotherapy and/or therapeutics that target DSB repair-dysfunctional tumors.-Deniz, M., Kaufmann, J., Stahl, A., Gundelach, T., Janni, W., Hoffmann, I., Keimling, M., Hampp, S., Ihle, M., Wiesmüller, L. In vitro model for DNA double-strand break repair analysis in breast cancer reveals cell type-specific associations with age and prognosis. © FASEB.

  18. Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing.

    Science.gov (United States)

    Jester, James V; Nien, Chyong Jy; Vasiliou, Vasilis; Brown, Donald J

    2012-01-01

    The purpose of this study was to determine the acute and long-term effects of mitomycin C (MMC) on quiescent rabbit corneal keratocytes regarding cell proliferation, myofibroblast differentiation and DNA repair. Quiescent keratocytes cultured in serum-free media were exposed to various concentrations of MMC and then treated with transforming growth factor-β (TGFβ). DNA damage was evaluated in both cultured keratocytes and live rabbit eyes following treatment with MMC. The long-term ability of quiescent keratocytes to repair MMC induced damage in vivo was evaluated in rabbits treated with MMC 2 months before 100 μm deep lamellar keratectomy (LK) injury. MMC significantly blocked TGFβ-induced cell proliferation and myofibroblast differentiation in cultured quiescent keratocytes and altered the transcriptional regulation of macrophage chemotactic protein-1 (MCP-1) and alpha smooth muscle actin (αSMA). MMC also induced phosphorylation of the nuclear histone marker of DNA damage, γH2AX (a member of the H2A histone family), without induction of cell cycle entry or immediate DNA repair measured by Comet assay. In live rabbits, 0.2 mg/ml MMC significantly induced γH2AX nuclear immunostaining (pMMC treatment 2 months before LK injury showed complete absence of any corneal scarring. MMC induces DNA damage to quiescent corneal keratocytes, which remains unrepaired, resulting in abnormal cell replication and gene transcription that leads to long-term effects on corneal repair. Overall these findings suggest that there may be long-term and perhaps permanent consequences to the application of MMC as an anti-fibrotic therapy.

  19. Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV.

    Science.gov (United States)

    Yang, Ying; Wu, Nandan; Tian, Sijia; Li, Fan; Hu, Huan; Chen, Pei; Cai, Xiaoxiao; Xu, Lijun; Zhang, Jing; Chen, Zhao; Ge, Jian; Yu, Keming; Zhuang, Jing

    2016-11-17

    Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia-reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair.

  20. Role of Protein Phosphorylation in the Regulation of Cell Cycle and DNA-Related Processes in Bacteria

    DEFF Research Database (Denmark)

    Garcia-Garcia, Transito; Poncet, Sandrine; Derouiche, Abderahmane

    2016-01-01

    In all living organisms, the phosphorylation of proteins modulates various aspects of their functionalities. In eukaryotes, protein phosphorylation plays a key role in cell signaling, gene expression, and differentiation. Protein phosphorylation is also involved in the global control of DNA repli...

  1. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Brandon J.; Dickey, Jennifer S.; Nakamura, Asako J.; Redon, Christophe E.; Parekh, Palak [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Griko, Yuri V. [Radiation and Space Biotechnology Branch, NASA Ames Research Center, Moffett Field, CA 94035 (United States); Aziz, Khaled; Georgakilas, Alexandros G. [Biology Department, East Carolina University, Greenville, NC 27858 (United States); Bonner, William M. [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Martin, Olga A., E-mail: sedelnio@mail.nih.gov [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States)

    2011-06-03

    Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0 {sup o}C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7 {sup o}C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13 {sup o}C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13 {sup o}C during and 12 h after irradiation. Mild hypothermia at 20 and 30 {sup o}C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13 {sup o}C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX ({gamma}-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13 {sup o}C compared to the rapid repair at 37 {sup o}C. For both {gamma}-H2AX foci and OCDLs, the return of lymphocytes to 37 {sup o}C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against

  2. DNA supercoiling depends on the phosphorylation potential in Escherichia coli

    DEFF Research Database (Denmark)

    Van Workum, M.; van Dooren, S.J.M; Oldenburg, N

    1996-01-01

    ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of protonophore (dinitrophenol) or by potassium cyanide addition, DNA...

  3. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2.

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V; Mason, Jennifer M; Memisoglu, Gonen; Waterman, David P; Long, Marcus J; Bishop, Douglas K; Haber, James E

    2015-08-18

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5' to 3' end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5' to 3' resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Caffeine impairs resection during DNA break repair by reducing the levels of nucleases Sae2 and Dna2

    Science.gov (United States)

    Tsabar, Michael; Eapen, Vinay V.; Mason, Jennifer M.; Memisoglu, Gonen; Waterman, David P.; Long, Marcus J.; Bishop, Douglas K.; Haber, James E.

    2015-01-01

    In response to chromosomal double-strand breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint, which is orchestrated by the PI3 kinase-like protein kinases ATR and ATM (Mec1 and Tel1 in budding yeast). Following DSB formation, Mec1 and Tel1 phosphorylate histone H2A on serine 129 (known as γ-H2AX). We used caffeine to inhibit the checkpoint kinases after DSB induction. We show that prolonged phosphorylation of H2A-S129 does not require continuous Mec1 and Tel1 activity. Unexpectedly, caffeine treatment impaired homologous recombination by inhibiting 5′ to 3′ end resection, independent of Mec1 and Tel1 inhibition. Caffeine treatment led to the rapid loss, by proteasomal degradation, of both Sae2, a nuclease that plays a role in early steps of resection, and Dna2, a nuclease that facilitates one of two extensive resection pathways. Sae2's instability is evident in the absence of DNA damage. A similar loss is seen when protein synthesis is inhibited by cycloheximide. Caffeine treatment had similar effects on irradiated HeLa cells, blocking the formation of RPA and Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends. Our findings provide insight toward the use of caffeine as a DNA damage-sensitizing agent in cancer cells. PMID:26019182

  5. The role of p53.S389 phosphorylation in DNA damage response pathways and tumorigenesis

    NARCIS (Netherlands)

    Bruins, Wendy

    2007-01-01

    The results presented in this thesis provide new information on the role of the p53.S389A point mutation in chemical-induced tumorigenesis. After DNA damage, p53 protein levels increase due to several post-translational activation processes. Phosphorylation of p53.S389 seems to be partly required

  6. Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

    Science.gov (United States)

    Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

    2017-05-11

    Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

  7. DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae.

    Science.gov (United States)

    Huang, Dongqing; Piening, Brian D; Kennedy, Jacob J; Lin, Chenwei; Jones-Weinert, Corey W; Yan, Ping; Paulovich, Amanda G

    2016-05-01

    In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMS-responsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance. Copyright © 2016 by the Genetics Society of America.

  8. Cytotoxicity and induction of DNA double-strand breaks by components leached from dental composites in primary human gingival fibroblasts.

    Science.gov (United States)

    Shehata, Mohamed; Durner, Jürgen; Eldenez, Ayce; Van Landuyt, Kirsten; Styllou, Panorea; Rothmund, Lena; Hickel, Reinhard; Scherthan, Harry; Geurtsen, Werner; Kaina, Bernd; Carell, Thomas; Reichl, Franz X

    2013-09-01

    The public interest steadily increases in the biological adverse effects caused by components released from resin-based dental restorations. In this study, the cytotoxicity and the genotoxicity were investigated of following released components from dental resin restorations in human gingival fibroblasts (HGF): tetraethyleneglycol dimethacrylate (TEEGDMA), neopentylglycol dimethacrylate (Neopen), diphenyliodoniumchloride (DPIC), triphenyl-stibane (TPSB) and triphenylphosphane (TPP). XTT based cell viability assay was used for cytotoxicity screening of substances. γ-H2AX assay was used for genotoxicity screening. In the γ-H2AX assay, HGFs were exposed to the substances for 6h. Induced foci represent double DNA strand breaks (DSBs), which can induce ATM-dependent phosphorylation of the histone H2AX. Cell death effects (apoptosis and necrosis), induced by the substances were visually tested by the same investigator using the fluorescent microscope. All tested substances induced a dose-dependent loss of viability in HGFs. Following toxicity ranking among the substances at EC50-concentration were found in the XTT assay (mM, mean±SEM; n=5): DPIC>Neopen>TPSB>TPP>TEEGDMA. DSB-foci per HGF-cell were obtained, when HGFs were exposed to the EC50-concentration of each substance in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA. Multi-foci cells (cells that contain more than 40 foci each) in 80 HGF-cells at EC50-concentration of each substance were found as follow (mean±SEM; n=3): DPIC>Neopen>TPP>TPSB>TEEGDMA. Cell apoptosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP >TEEGDMA. Cell necrosis contained in each substance at EC50-concentration in the following order (mean±SEM; n=3): DPIC>Neopen>TPSB>TPP>TEEGDMA. Leached components from dental resin restorations can induce DNA DSBs and cell death effects in HGFs. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All

  9. Influence of shieldings or antioxidants on DNA damage and early and delyed cell death induced in human fibroblasts by accelerated 595 MeV/u Fe ions

    Science.gov (United States)

    Antonelli, Francesca; Esposito, Giuseppe; Dini, Valentina; Belli, Mauro; Campa, Alessandro; Sorrentino, Eugenio; Antonella Tabocchini, Maria; Lobascio, Cesare; Berra, Bruno

    HZE particles from space radiation raise an important protection concern during long-term astronauts' travels. As high charge, high energy particles interact with a shield, both projec-tile and target fragmentation may occurs, so that the biological properties of the emerging radiation field depend on the nature and energy of the incident particles, and on the nature and thickness of the shield. We have studied the influence of PMMA and Kevlar shielding as well as the antioxidant compounds Rosmarinic acid or Resveratrol on DNA damage induction and processing (as evaluated by the g-H2AX phosphorylation assay) and on early and delayed cell death in AG01522 human fibroblasts irradiated with Fe ions of 595 MeV/u at the NASA Space Radiation Laboratory (NSRL), Brookhaven National Laboratory (BNL, Upton, USA). Insertion of PMMA or Kevlar shields (10 g/cm2 thick) gave no substantial change in the bio-logical effect per unit dose on the sample for all the end points studied. When irradiation was performed in the presence of 300 mM Rosmarinic acid or Resveratrol no difference were found for both early and delayed cell death, while a slight protective effect was observed for the initial and residual DNA damage. For both early and delayed cell death, Fe-ions are more effective than g-rays. The number of Fe-ion induced g-H2AX foci is instead lower than that induced by g-rays, due to the presence of multiple DSB within a single focus induced by Fe-ions. From a comparison of the g-H2AX data with the results on DNA fragmentation obtained with 414 MeV/u Fe ions at the Heavy Ions Medical Accelerator (HIMAC, Chiba, Japan) and with 1 GeV/u Fe ions at BNL, in the absence or in the presence of PMMA shields (Esposito et al, Advance in Space Research 2004) we speculate that the overall effect of the shield is a balance between the contributions due to the slowing down of the primary particles and that due to the nuclear fragmentation. Acknowledgment: Financial support from ASI project

  10. The WSTF-ISWI chromatin remodeling complex transiently associates with the human inactive X chromosome during late S-phase prior to BRCA1 and γ-H2AX.

    Directory of Open Access Journals (Sweden)

    Ashley E Culver-Cochran

    Full Text Available Replicating the genome prior to each somatic cell division not only requires precise duplication of the genetic information, but also accurately reestablishing the epigenetic signatures that instruct how the genetic material is to be interpreted in the daughter cells. The mammalian inactive X chromosome (Xi, which is faithfully inherited in a silent state in each daughter cell, provides an excellent model of epigenetic regulation. While much is known about the early stages of X chromosome inactivation, much less is understood with regards to retaining the Xi chromatin through somatic cell division. Here we report that the WSTF-ISWI chromatin remodeling complex (WICH associates with the Xi during late S-phase as the Xi DNA is replicated. Elevated levels of WICH at the Xi is restricted to late S-phase and appears before BRCA1 and γ-H2A.X. The sequential appearance of WICH and BRCA1/γ-H2A.X implicate each as performing important but distinct roles in the maturation and maintenance of heterochromatin at the Xi.

  11. Hsp90α regulates ATM and NBN functions in sensing and repair of DNA double-strand breaks.

    Science.gov (United States)

    Pennisi, Rosa; Antoccia, Antonio; Leone, Stefano; Ascenzi, Paolo; di Masi, Alessandra

    2017-08-01

    The molecular chaperone heat shock protein 90 (Hsp90α) regulates cell proteostasis and mitigates the harmful effects of endogenous and exogenous stressors on the proteome. Indeed, the inhibition of Hsp90α ATPase activity affects the cellular response to ionizing radiation (IR). Although the interplay between Hsp90α and several DNA damage response (DDR) proteins has been reported, its role in the DDR is still unclear. Here, we show that ataxia-telangiectasia-mutated kinase (ATM) and nibrin (NBN), but not 53BP1, RAD50, and MRE11, are Hsp90α clients as the Hsp90α inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) induces ATM and NBN polyubiquitination and proteosomal degradation in normal fibroblasts and lymphoblastoid cell lines. Hsp90α-ATM and Hsp90α-NBN complexes are present in unstressed and irradiated cells, allowing the maintenance of ATM and NBN stability that is required for the MRE11/RAD50/NBN complex-dependent ATM activation and the ATM-dependent phosphorylation of both NBN and Hsp90α in response to IR-induced DNA double-strand breaks (DSBs). Hsp90α forms a complex also with ph-Ser1981-ATM following IR. Upon phosphorylation, NBN dissociates from Hsp90α and translocates at the DSBs, while phThr5/7-Hsp90α is not recruited at the damaged sites. The inhibition of Hsp90α affects nuclear localization of MRE11 and RAD50, impairs DDR signaling (e.g., BRCA1 and CHK2 phosphorylation), and slows down DSBs repair. Hsp90α inhibition does not affect DNA-dependent protein kinase (DNA-PK) activity, which possibly phosphorylates Hsp90α and H2AX after IR. Notably, Hsp90α inhibition causes H2AX phosphorylation in proliferating cells, this possibly indicating replication stress events. Overall, present data shed light on the regulatory role of Hsp90α on the DDR, controlling ATM and NBN stability and influencing the DSBs signaling and repair. © 2017 Federation of European Biochemical Societies.

  12. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  13. A cytosolic activator of DNA replication is tyrosine phosphorylated in its active form.

    Science.gov (United States)

    Fresa, K L; Autieri, M V; Coffman, F D; Georgoff, I; Cohen, S

    1993-04-01

    Cytosolic extracts from actively dividing lymphoid cells have been shown to induce DNA synthesis in isolated, quiescent nuclei. An initiating factor in such extracts (activator of DNA replication; ADR) is a > 90-kDa aprotinin-binding protein whose activity is inhibitable not only by aprotinin, but also by several other protease inhibitors as well. Although cytosol from non-proliferating lymphocytes is devoid of ADR activity, we have shown that these preparations can be induced to express ADR activity by brief exposure to a membrane-enriched fraction of spontaneously proliferating MOLT-4 cells via a kinase-dependent mechanism. In the present study, we examine the role of tyrosine kinases in this process. Three inhibitors of tyrosine kinases (genistein, kaempferol, and quercetin) can inhibit the in vitro generation of ADR activity. In vitro generation of ADR activity is associated with the de novo phosphorylation of several proteins, many of which are detectable using anti-phosphotyrosine monoclonal antibodies. ADR itself may be tyrosine phosphorylated in active form as immunoprecipitation using such monoclonal antibodies leads to the depletion of its activity. Moreover, immunoprecipitation results in the removal of several de novo tyrosine-phosphorylated proteins, including species at approximately 122, 105, 93, 86, 79, and 65 kDa. A subset of de novo-phosphorylated proteins, migrating at approximately 105, 93, and 70 kDa, also bound to aprotinin, suggesting that at least one of these proteins may represent ADR itself.

  14. Differential Processing of Low and High LET Radiation Induced DNA Damage: Investigation of Switch from ATM to ATR Signaling

    Science.gov (United States)

    Saha, Janapriya; Wang, Minli; Hada, Megumi; Cucinotta, Francis A.

    2011-01-01

    majority of RPA-coated ssDNA is generally present only during DNA replication, ATR activation in G1 and G2-phase might still require formation of RPA-coated ssDNA, probably initiated by the MRN-CtIP complex and then extended by the Exo1- or BLM-dependent mechanisms at the sites of DSBs. Evidence accumulates that activation of ATM and ATR are oppositely regulated by the length of single stranded overhangs generated at the break sites by processes mentioned above and these stretches of single stranded overhangs hold the clue for ATM to ATR switch at broken DNA ends. We irradiated 82-6hTERT human fibroblast cells with low LET gamma-rays and high LET Fe and Si particles. Preliminary results with cells exposed to 1Gy gamma-rays show that the kinetics of pChk2-pT68 foci formation is comparable to that of gamma-H2AX although they appear to recede quicker. The number and intensity of observed foci reaches a maximum at 30 min and 60 min post IR for Chk2-pT68 and gamma-H2AX foci respectively and all Chk2-pT68 foci colocalize with gamma-H2AX foci. The kinetics of Chk1-pS345 and ATRIP are being determined. Results of Chk2-pT68 foci kinetics was also corroborated by western blot experiments, although phosphorylation was detected as early as 10 min and started receding 30 min post IR with 2Gy of gamma-rays. On the other hand, level of ATR-pS428 reached its maximum between 60 and 120 min and was maintained until the last measured time point of 4 hours post IR as determined by western blotting. Experiments performed with high LET Fe and Si particles will be reported.

  15. Aryl Hydrocarbon Receptor Suppresses the Prostate Cancer LNCaP Cell Growth and Invasion by Promoting DNA Damage Response Under Oxidative Stress.

    Science.gov (United States)

    Yu, Jing-Song; Leng, Peng-Fei; Li, Yi-Fu; Wang, Yong-Quan; Wang, Yan; An, Rui-Hua; Qi, Ji-Ping

    2017-11-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that interacts with multiple signaling pathways during prostate development. In the present study, LNCaP cells were knocked down of AhR by siRNA, or treated with the AhR agonist 3-methylcholanthrene (3MC). The effects of AhR on LNCaP cells and the associated mechanisms were studied both under normal condition and under hydrogen peroxide (H 2 O 2 ) - induced oxidative stress. MTT, transwell chamber assays and flow cytometry were employed to investigate cell proliferation, invasion, and apoptosis, respectively, whereas the DNA damage response (DDR) signaling (phosphorylation of ataxia-telangiectasia mutated [ATM], check-point kinase 2 [Chk2], histone H2AX, p53, and cleaved poly-ADP-ribose polymerase [PARP]) was detected by western blotting. Exposure of LNCaP cells to H 2 O 2 inhibited their viability and migration, and induced apoptosis, at a greater extent compared with the culture under normal conditions. In addition, the oxidative stress increased p-ATM, p-Chk2, p-p53, and p-H2AX expression levels significantly. Knockdown of AhR attenuated the aforementioned effects caused by H 2 O 2 -induced oxidative stress. Activation of AhR by 3MC treatment, further aggravated these changes of LNCaP cells on oxidative stress. The findings indicated that AhR suppresses the viability and migration of LNCaP cells notably under oxidative stress, and this process is associated with positive regulation of the responses to oxidative DNA damage.

  16. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA...... phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation...... assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38...

  17. Increased oxidative phosphorylation in response to acute and chronic DNA damage.

    Science.gov (United States)

    Brace, Lear E; Vose, Sarah C; Stanya, Kristopher; Gathungu, Rose M; Marur, Vasant R; Longchamp, Alban; Treviño-Villarreal, Humberto; Mejia, Pedro; Vargas, Dorathy; Inouye, Karen; Bronson, Roderick T; Lee, Chih-Hao; Neilan, Edward; Kristal, Bruce S; Mitchell, James R

    2016-01-01

    Accumulation of DNA damage is intricately linked to aging, aging-related diseases and progeroid syndromes such as Cockayne syndrome (CS). Free radicals from endogenous oxidative energy metabolism can damage DNA, however the potential of acute or chronic DNA damage to modulate cellular and/or organismal energy metabolism remains largely unexplored. We modeled chronic endogenous genotoxic stress using a DNA repair-deficient Csa -/- |Xpa -/- mouse model of CS. Exogenous genotoxic stress was modeled in mice in vivo and primary cells in vitro treated with different genotoxins giving rise to diverse spectrums of lesions, including ultraviolet radiation, intrastrand crosslinking agents and ionizing radiation. Both chronic endogenous and acute exogenous genotoxic stress increased mitochondrial fatty acid oxidation (FAO) on the organismal level, manifested by increased oxygen consumption, reduced respiratory exchange ratio, progressive adipose loss and increased FAO in tissues ex vivo . In multiple primary cell types, the metabolic response to different genotoxins manifested as a cell-autonomous increase in oxidative phosphorylation (OXPHOS) subsequent to a transient decline in steady-state NAD+ and ATP levels, and required the DNA damage sensor PARP-1 and energy-sensing kinase AMPK. We conclude that increased FAO/OXPHOS is a general, beneficial, adaptive response to DNA damage on cellular and organismal levels, illustrating a fundamental link between genotoxic stress and energy metabolism driven by the energetic cost of DNA damage. Our study points to therapeutic opportunities to mitigate detrimental effects of DNA damage on primary cells in the context of radio/chemotherapy or progeroid syndromes.

  18. Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line.

    Science.gov (United States)

    Li, X; Liu, W; Wang, H; Yang, L; Li, Y; Wen, H; Ning, H; Wang, J; Zhang, L; Li, J; Fan, D

    2015-03-30

    The telomeric protein TRF2, involving in telomeric and extratelomeric DNA damage response, has been previously reported to facilitate multidrug resistance (MDR) in gastric cancer cells by interfering ATM-dependent DNA damage response induced by anticancer drugs. Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Here we focus on the effects of Rap1 on TRF2 function in DNA damage response induced by anticancer drugs. Both Rap1 and TRF2 expression were upregulated in SGC7901 and its MDR variant SGC7901/VCR after etoposide treatment, which was more marked in SGC7901/VCR than in SGC7901. Rap1 silencing by siRNA in SGC7901/VCR partially reversed the etoposide resistance. And Rap1 silencing partially reversed the TRF2-mediated resistance to etoposide in SGC7901. Rap1 silencing did not affect the TRF2 upregulation induced by etoposide, but eliminated the inhibition effect of TRF2 on ATM expression and ATM phosphorylation at serine 1981 (ATM pS1981). Furthermore, phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were increased in Rap1 silencing cells in response to etoposide. Thus, we confirm that Rap1, interacting with TRF2 in the shelterin complex, also has an important role in TRF2-mediated DNA damage response in gastric cancer cells treated by etoposide.

  19. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Matthew L Hirsch

    Full Text Available Human embryonic stem cells (hESCs are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  20. Radiation induced DNA double-strand breaks in radiology; Strahleninduzierte DNA-Doppelstrangbrueche in der Radiologie

    Energy Technology Data Exchange (ETDEWEB)

    Kuefner, M.A. [Dornbirn Hospital (Austria). Dept. of Radiology; Brand, M.; Engert, C.; Uder, M. [Erlangen University Hospital (Germany). Dept. of Radiology; Schwab, S.A. [Radiologis, Oberasbach (Germany)

    2015-10-15

    Shortly after the discovery of X-rays, their damaging effect on biological tissues was observed. The determination of radiation exposure in diagnostic and interventional radiology is usually based on physical measurements or mathematical algorithms with standardized dose simulations. γ-H2AX immunofluorescence microscopy is a reliable and sensitive method for the quantification of radiation induced DNA double-strand breaks (DSB) in blood lymphocytes. The detectable amount of these DNA damages correlates well with the dose received. However, the biological radiation damage depends not only on dose but also on other individual factors like radiation sensitivity and DNA repair capacity. Iodinated contrast agents can enhance the x-ray induced DNA damage level. After their induction DSB are quickly repaired. A protective effect of antioxidants has been postulated in experimental studies. This review explains the principle of the γ-H2AX technique and provides an overview on studies evaluating DSB in radiologic examinations.

  1. JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

    Science.gov (United States)

    Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

    2017-08-01

    Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H2AXH2AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

    Science.gov (United States)

    Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

    2016-04-01

    To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  3. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel target of bisphenol A.

    Directory of Open Access Journals (Sweden)

    Yuki Ito

    Full Text Available Bisphenol A (BPA forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80, is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM, high doses of BPA were required before cellular effects were observed (100-300 μM. The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient. Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  4. Interplay between the DNA Damage Proteins MDC1 and ATM in the Regulation of the Spindle Assembly Checkpoint

    Science.gov (United States)

    Eliezer, Yifat; Argaman, Liron; Kornowski, Maya; Roniger, Maayan; Goldberg, Michal

    2014-01-01

    To avoid genomic instability, cells have developed surveillance mechanisms such as the spindle assembly checkpoint (SAC) and the DNA damage response. ATM and MDC1 are central players of the cellular response to DNA double-strand breaks. Here, we identify a new role for these proteins in the regulation of mitotic progression and in SAC activation. MDC1 localizes at mitotic kinetochores following SAC activation in an ATM-dependent manner. ATM phosphorylates histone H2AX at mitotic kinetochores, and this phosphorylation is required for MDC1 localization at kinetochores. ATM and MDC1 are needed for kinetochore localization of the inhibitory mitotic checkpoint complex components, Mad2 and Cdc20, and for the maintenance of the mitotic checkpoint complex integrity. This probably relies on the interaction of MDC1 with the MCC. In this work, we have established that ATM and MDC1 maintain genomic stability not only by controlling the DNA damage response, but also by regulating SAC activation, providing an important link between these two essential biological processes. PMID:24509855

  5. Purkinje Cell Degeneration in pcd Mice Reveals Large Scale Chromatin Reorganization and Gene Silencing Linked to Defective DNA Repair*

    Science.gov (United States)

    Baltanás, Fernando C.; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R.; Berciano, María T.; Lafarga, Miguel

    2011-01-01

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death. PMID:21700704

  6. Purkinje cell degeneration in pcd mice reveals large scale chromatin reorganization and gene silencing linked to defective DNA repair.

    Science.gov (United States)

    Baltanás, Fernando C; Casafont, Iñigo; Lafarga, Vanesa; Weruaga, Eduardo; Alonso, José R; Berciano, María T; Lafarga, Miguel

    2011-08-12

    DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death.

  7. DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-SIN1 association mediates ultraviolet B (UVB)-induced Akt Ser-473 phosphorylation and skin cell survival.

    Science.gov (United States)

    Tu, Ying; Ji, Chao; Yang, Bo; Yang, Zhi; Gu, Hua; Lu, Chun-Cheng; Wang, Rong; Su, Zhong-Lan; Chen, Bin; Sun, Wei-Ling; Xia, Ji-Ping; Bi, Zhi-Gang; He, Li

    2013-12-24

    The exposure of skin keratinocytes to Ultraviolet (UV) irradiation leads to Akt phosphorylation at Ser-473, which is important for the carcinogenic effects of excessive sun exposure. The present study investigated the underlying mechanism of Akt Ser-473 phosphorylation by UVB radiation. We found that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mammalian target of rapamycin (mTOR) complex 2 (mTORC2) were both required for UVB-induced Akt Ser-473 phosphorylation in keratinocytes. Inhibition of DNA-PKcs activity via its inhibitor NU7026, a dominant-negative kinase-dead mutation, RNA interference (RNAi) or gene depletion led to the attenuation of UVB-induced Akt Ser-473 phosphorylation. Meanwhile, siRNA silencing or gene depletion of SIN1, a key component of mTORC2, abolished Akt Ser-473 phosphorylation by UVB. Significantly, we discovered that DNA-PKcs was associated with SIN1 in cytosol upon UVB radiation, and this complexation appeared required for Akt Ser-473 phosphorylation. Meanwhile, this DNA-PKcs-SIN1 complexation by UVB was dependent on epidermal growth factor receptor (EGFR) activation, and was disrupted by an EGFR inhibitor (AG1478) or by EGFR depletion. UVB-induced complexation between DNA-PKcs and mTORC2 components was also abolished by NU7026 and DNA-PKcs mutation. Finally, we found that both DNA-PKcs and SIN1 were associated with apoptosis resistance of UVB radiation, and inhibition of them by NU7026 or genetic depletion significantly enhanced UVB-induced cell death and apoptosis. Taken together, these results strongly suggest that DNA-PKcs-mTORC2 association is required for UVB-induced Akt Ser-473 phosphorylation and cell survival, and might be important for tumor cell transformation.

  8. Relationship between internal dosimetry and DNA double strand breaks in lymphocytes after radionuclide therapy; Zusammenhang zwischen physikalischer Dosimetrie und DNA Doppelstrangbruechen in Lymphozyten nach Radionuklidtherapie

    Energy Technology Data Exchange (ETDEWEB)

    Eberlein, Uta

    2015-09-30

    In radionuclide therapy radiopharmaceuticals are administered mostly systemically. Primarily, beta-emitters are used because of their short range in tissue. As a result the radiopharmaceutical distributes within the human body and accumulates in organs and target structures. Thus, the body is irradiated internally, in contrast to external irradiation in radiotherapy. The pattern of the activity distribution within the human body is determined by the physical and chemical properties of the radiopharmaceutical. Furthermore, the amount of activity and its accumulation in organs or tissues is essential for the calculation of the absorbed dose which defines the energy deposited in the body by ionizing radiation. During internal or external irradiation, patients are exposed to ionizing radiation which does not only destroy the malignant cells but also damages healthy tissue and cells. This is mainly caused by direct and indirect interaction of the radiation with the DNA which damages the DNA structure. Most frequently, there are single strand breaks and base damages. DNA double strand breaks (DSBs) are rare; nevertheless, they are the most critical lesions for cells as repairing the damage is difficult. Unrepaired or misrepaired DNA could cause mutations, chromosomal aberrations or lead to cell death. The formation of a DNA DSB in nuclear chromatin results in the rapid phosphorylation of the histone H2 variant H2AX, then called gamma-H2AX. Furthermore, DSBs also recruit the damage sensor 53BP1 to the chromatin surrounding the DSBs, which leads to 53BP1 and gamma-H2AX co-localization in the chromatin surrounding a DSB. By immunofluorescence staining with gamma-H2AX and 53BP1 antibodies those biomarkers can be addressed by microscopically visible DNA damage protein foci, this is also known as the DNA damage focus assay. With progression of DSB repair, gamma-H2AX and 53BP1 foci disappear. It is assumed that one focus corresponds to one DSB. Therefore, the number of foci per

  9. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    Science.gov (United States)

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA.

    Science.gov (United States)

    Hayner, Jaclyn N; Douma, Lauren G; Bloom, Linda B

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. RNF8 Transduces the DNA-Damage Signal Via Histone Ubiquitylation And Checkpoint Protein Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Huen, M.S.Y.; Grant, R.; Manke, I.; Minn, K.; Yu, X.; Yaffe, M.B.; Chen, J.

    2009-06-01

    DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 {angstrom}. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

  12. ATM-mediated mitochondrial damage response triggered by nuclear DNA damage in normal human lung fibroblasts.

    Science.gov (United States)

    Shimura, Tsutomu; Sasatani, Megumi; Kawai, Hidehiko; Kamiya, Kenji; Kobayashi, Junya; Komatsu, Kenshi; Kunugita, Naoki

    2017-11-03

    Ionizing radiation (IR) elevates mitochondrial oxidative phosphorylation (OXPHOS) in response to the energy requirement for DNA damage responses. Reactive oxygen species (ROS) released during mitochondrial OXPHOS may cause oxidative damage to mitochondria in irradiated cells. In this paper, we investigated the association between nuclear DNA damage and mitochondrial damage following IR in normal human lung fibroblasts. In contrast to low-doses of acute single radiation, continuous exposure of chronic radiation or long-term exposure of fractionated radiation (FR) induced persistent Rad51 and γ-H2AX foci at least 24 hours after IR in irradiated cells. Additionally, long-term FR increased mitochondrial ROS accompanied with enhanced mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity. Mitochondrial ROS released from the respiratory chain complex I caused oxidative damage to mitochondria. Inhibition of ATM kinase or ATM loss eliminated nuclear DNA damage recognition and mitochondrial radiation responses. Consequently, nuclear DNA damage activates ATM which in turn increases ROS level and subsequently induces mitochondrial damage in irradiated cells. In conclusion, we demonstrated that ATM is essential in the mitochondrial radiation responses in irradiated cells. We further demonstrated that ATM is involved in signal transduction from nucleus to the mitochondria in response to IR.

  13. ERCC6L2 mutations link a distinct bone-marrow-failure syndrome to DNA repair and mitochondrial function.

    Science.gov (United States)

    Tummala, Hemanth; Kirwan, Michael; Walne, Amanda J; Hossain, Upal; Jackson, Nicholas; Pondarre, Corinne; Plagnol, Vincent; Vulliamy, Tom; Dokal, Inderjeet

    2014-02-06

    Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  14. Image-Based Modeling Reveals Dynamic Redistribution of DNA Damageinto Nuclear Sub-Domains

    Energy Technology Data Exchange (ETDEWEB)

    Costes Sylvain V., Ponomarev Artem, Chen James L.; Nguyen, David; Cucinotta, Francis A.; Barcellos-Hoff, Mary Helen

    2007-08-03

    Several proteins involved in the response to DNA doublestrand breaks (DSB) f orm microscopically visible nuclear domains, orfoci, after exposure to ionizing radiation. Radiation-induced foci (RIF)are believed to be located where DNA damage occurs. To test thisassumption, we analyzed the spatial distribution of 53BP1, phosphorylatedATM, and gammaH2AX RIF in cells irradiated with high linear energytransfer (LET) radiation and low LET. Since energy is randomly depositedalong high-LET particle paths, RIF along these paths should also berandomly distributed. The probability to induce DSB can be derived fromDNA fragment data measured experimentally by pulsed-field gelelectrophoresis. We used this probability in Monte Carlo simulations topredict DSB locations in synthetic nuclei geometrically described by acomplete set of human chromosomes, taking into account microscope opticsfrom real experiments. As expected, simulations produced DNA-weightedrandom (Poisson) distributions. In contrast, the distributions of RIFobtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) werenon-random. This deviation from the expected DNA-weighted random patterncan be further characterized by "relative DNA image measurements." Thisnovel imaging approach shows that RIF were located preferentially at theinterface between high and low DNA density regions, and were morefrequent than predicted in regions with lower DNA density. The samepreferential nuclear location was also measured for RIF induced by 1 Gyof low-LET radiation. This deviation from random behavior was evidentonly 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AXand 53BP1 RIF showed pronounced deviations up to 30 min after exposure.These data suggest that DNA damage induced foci are restricted to certainregions of the nucleus of human epithelial cells. It is possible that DNAlesions are collected in these nuclear sub-domains for more efficientrepair.

  15. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Landvik, N.E. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Arlt, V.M.; Nagy, E. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Solhaug, A. [Section for Toxicology, Department of Feed and Food Safety, National Veterinary Institute Pb 750 Sentrum, N-0106 Oslo (Norway); Tekpli, X. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Schmeiser, H.H. [Research Group Genetic Alteration in Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Refsnes, M. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Phillips, D.H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Lagadic-Gossmann, D. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Holme, J.A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway)

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ({sup 32}P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of I{kappa}B-{alpha} (suggesting activation of NF-{kappa}B) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-{kappa}B play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

  16. DNA damage in lymphocytes induced by cardiac CT and comparison with physical exposure parameters

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Wataru; Tatsugami, Fuminari; Awai, Kazuo [Department of Diagnostic Radiology, Institute of Biomedical Health Sciences, Hiroshima University, Hiroshima (Japan); Ishida, Mari; Sakai, Chiemi [Institute of Biomedical and Health Sciences, Department of Cardiovascular Physiology and Medicine, Hiroshima University, Hiroshima (Japan); Tashiro, Satoshi [Hiroshima University, Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima (Japan); Ishida, Takafumi [Institute of Clinical Research West Medical Center, Hiroshima (Japan); Nakano, Yukiko [Hiroshima University Hospital, Department of Cardiovascular Medicine, Hiroshima (Japan)

    2017-04-15

    To investigate whether physical exposure parameters such as the dose index (CTDI), dose length product (DLP), and size-specific dose estimate (SSDE) are predictive of DNA damage. In vitro, we scanned a phantom containing blood samples from five volunteers at CTDI 50, 100, and 150 mGy. One sample was not scanned. We also scanned samples in three different-size phantoms at CTDI 100 mGy. In vivo, we enrolled 45 patients and obtained blood samples before and after cardiac CT. The γ-H2AX foci were counted. In vitro, in the control and at CTDI 50, 100, and 150 mGy, the number of γ-H2AX was 0.94 ± 0.24 (standard error, SE), 1.28 ± 0.30, 1.91 ± 0.47, and 2.16 ± 0.20. At SSDE 180, 156, and 135 mGy, it was 2.41 ± 0.20, 1.91 ± 0.47, and 1.42 ± 0.20 foci/cell. The γ-H2AX foci were positively correlated with the radiation dose and negatively correlated with the body size. In vivo, the γ-H2AX foci were significantly increased after CT (from 1.21 ± 0.19 to 1.92 ± 0.22 foci/cell) and correlated with CTDI, DLP, and SSDE. DNA damage was induced by cardiac CT. There was a correlation between the physical exposure parameters and γ-H2AX. (orig.)

  17. Nitric Oxide Suppresses β-Cell Apoptosis by Inhibiting the DNA Damage Response

    Science.gov (United States)

    Oleson, Bryndon J.; Broniowska, Katarzyna A.; Naatz, Aaron; Hogg, Neil; Tarakanova, Vera L.

    2016-01-01

    Nitric oxide, produced in pancreatic β cells in response to proinflammatory cytokines, plays a dual role in the regulation of β-cell fate. While nitric oxide induces cellular damage and impairs β-cell function, it also promotes β-cell survival through activation of protective pathways that promote β-cell recovery. In this study, we identify a novel mechanism in which nitric oxide prevents β-cell apoptosis by attenuating the DNA damage response (DDR). Nitric oxide suppresses activation of the DDR (as measured by γH2AX formation and the phosphorylation of KAP1 and p53) in response to multiple genotoxic agents, including camptothecin, H2O2, and nitric oxide itself, despite the presence of DNA damage. While camptothecin and H2O2 both induce DDR activation, nitric oxide suppresses only camptothecin-induced apoptosis and not H2O2-induced necrosis. The ability of nitric oxide to suppress the DDR appears to be selective for pancreatic β cells, as nitric oxide fails to inhibit DDR signaling in macrophages, hepatocytes, and fibroblasts, three additional cell types examined. While originally described as the damaging agent responsible for cytokine-induced β-cell death, these studies identify a novel role for nitric oxide as a protective molecule that promotes β-cell survival by suppressing DDR signaling and attenuating DNA damage-induced apoptosis. PMID:27185882

  18. Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM).

    Science.gov (United States)

    Chen, Nai-Tzu; Wu, Chia-Yan; Chung, Chao-Yu; Hwu, Yeukuang; Cheng, Shih-Hsun; Mou, Chung-Yuan; Lo, Leu-Wei

    2012-01-01

    Doxorubicin is a potent anthracycline antibiotic, commonly used to treat a wide range of cancers. Although postulated to intercalate between DNA bases, many of the details of doxorubicin's mechanism of action remain unclear. In this work, we demonstrate the ability of fluorescence lifetime imaging microscopy (FLIM) to dynamically monitor doxorubicin-DNA intercalation during the earliest stages of apoptosis. The fluorescence lifetime of doxorubicin in nuclei is found to decrease rapidly during the first 2 hours following drug administration, suggesting significant changes in the doxorubicin-DNA binding site's microenvironment upon apoptosis initiation. Decreases in doxorubicin fluorescence lifetimes were found to be concurrent with increases in phosphorylation of H2AX (an immediate signal of DNA double-strand breakage), but preceded activation of caspase-3 (a late signature of apoptosis) by more than 150 minutes. Time-dependent doxorubicin FLIM analyses of the effects of pretreating cells with either Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)-hydrazine (a histone acetyltransferase inhibitor) or Trichostatin A (a histone deacetylase inhibitor) revealed significant correlation of fluorescence lifetime with the stage of chromatin decondensation. Taken together, our findings suggest that monitoring the dynamics of doxorubicin fluorescence lifetimes can provide valuable information during the earliest phases of doxorubicin-induced apoptosis; and implicate that FLIM can serve as a sensitive, high-resolution tool for the elucidation of intercellular mechanisms and kinetics of anti-cancer drugs that bear fluorescent moieties.

  19. Probing the dynamics of doxorubicin-DNA intercalation during the initial activation of apoptosis by fluorescence lifetime imaging microscopy (FLIM.

    Directory of Open Access Journals (Sweden)

    Nai-Tzu Chen

    Full Text Available Doxorubicin is a potent anthracycline antibiotic, commonly used to treat a wide range of cancers. Although postulated to intercalate between DNA bases, many of the details of doxorubicin's mechanism of action remain unclear. In this work, we demonstrate the ability of fluorescence lifetime imaging microscopy (FLIM to dynamically monitor doxorubicin-DNA intercalation during the earliest stages of apoptosis. The fluorescence lifetime of doxorubicin in nuclei is found to decrease rapidly during the first 2 hours following drug administration, suggesting significant changes in the doxorubicin-DNA binding site's microenvironment upon apoptosis initiation. Decreases in doxorubicin fluorescence lifetimes were found to be concurrent with increases in phosphorylation of H2AX (an immediate signal of DNA double-strand breakage, but preceded activation of caspase-3 (a late signature of apoptosis by more than 150 minutes. Time-dependent doxorubicin FLIM analyses of the effects of pretreating cells with either Cyclopentylidene-[4-(4-chlorophenylthiazol-2-yl-hydrazine (a histone acetyltransferase inhibitor or Trichostatin A (a histone deacetylase inhibitor revealed significant correlation of fluorescence lifetime with the stage of chromatin decondensation. Taken together, our findings suggest that monitoring the dynamics of doxorubicin fluorescence lifetimes can provide valuable information during the earliest phases of doxorubicin-induced apoptosis; and implicate that FLIM can serve as a sensitive, high-resolution tool for the elucidation of intercellular mechanisms and kinetics of anti-cancer drugs that bear fluorescent moieties.

  20. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    Science.gov (United States)

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  1. Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

    Directory of Open Access Journals (Sweden)

    Mehtap Kilic Eren

    Full Text Available The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD-dependent histone deacetylase family member sirtuin-1 (SIRT1 protein. In mammals seven members (SIRT1-7 of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging

  2. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA binding, transcriptional repression and transforming ability.

    Directory of Open Access Journals (Sweden)

    Daniel J White

    Full Text Available The EVI1 (ecotropic viral integration site 1 gene at 3q26 codes for a transcriptional regulator with an essential role in haematopoiesis. Overexpression of EVI1 in acute myeloid leukaemia (AML is frequently associated with 3q26 rearrangements and confers extremely poor prognosis. EVI1 mediates transcriptional regulation, signalling, and epigenetic modifications by interacting with DNA, proteins and protein complexes. To explore to what extent protein phosphorylation impacts on EVI1 functions, we analysed endogenous EVI1 protein from a high EVI1 expressing Fanconi anaemia (FA derived AML cell line. Mass spectrometric analysis of immunoprecipitated EVI1 revealed phosphorylation at serine 196 (S196 in the sixth zinc finger of the N-terminal zinc finger domain. Mutated EVI1 with an aspartate substitution at serine 196 (S196D, which mimics serine phosphorylation of this site, exhibited reduced DNA-binding and transcriptional repression from a gene promotor selectively targeted by the N-terminal zinc finger domain. Forced expression of the S196D mutant significantly reduced EVI1 mediated transformation of Rat1 fibroblasts. While EVI1-mediated serial replating of murine haematopoietic progenitors was maintained by EVI1-S196D, this was associated with significantly higher Evi1-trancript levels compared with WT-EVI1 or EVI1-S196A, mimicking S196 non-phosphorylated EVI1. These data suggest that EVI1 function is modulated by phosphorylation of the first zinc finger domain.

  3. MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in response to DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Khan, Shafqat A.; Sharda, Asmita; Reddy, Divya V; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-08-15

    Highlights: • Reversible reduction of H3S10 phosphorylation after DNA damage is G1 phase specific. • Dynamic balance between MAP kinases, MKP1 and MSK1 regulate H3S10P during DDR. • MKP1 associates with chromatin bearing γH2AX in response to DNA damage. • Inhibition of MKP1 activity with specific inhibitor promotes radiation-induced cell death. - Abstract: Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.

  4. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    Directory of Open Access Journals (Sweden)

    Natalia Bailon-Moscoso

    Full Text Available Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL, a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

  5. Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

    Science.gov (United States)

    Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

    2017-05-01

    Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

  6. Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

    Directory of Open Access Journals (Sweden)

    Serena Guidotti

    Full Text Available Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA, but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763 were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS production (2',7'-dichlorofluorescin diacetate staining. Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21, flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via

  7. Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

    Science.gov (United States)

    Guidotti, Serena; Minguzzi, Manuela; Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

    2015-01-01

    Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of

  8. Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

    Directory of Open Access Journals (Sweden)

    Federico González

    2013-03-01

    Full Text Available Induced pluripotent stem cells (iPSCs hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs. Additional mechanistic studies uncover a direct role of the homologous recombination (HR pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

  9. Phosphorylation of MCT-1 by p44/42 MAPK is required for its stabilization in response to DNA damage

    DEFF Research Database (Denmark)

    Nandi, S; Reinert, Line; Hachem, A

    2007-01-01

    that there were significantly increased levels of MCT-1 protein in a subset of primary diffuse large B-cell lymphomas. Levels of MCT-1 protein were shown to be increased after exposure to DNA damaging agents. This increase did not require new protein synthesis, suggesting that post-translational mechanisms were...... growth and proliferation through phosphorylation-dependent regulation of several substrates. The MCT-1 protein is predicted to have numerous putative phosphorylation sites. Using a combination of genetic and pharmacological approaches, we established that phosphorylation of MCT-1 protein by p44/p42...... mitogen-activated protein kinases is critical for stabilization of MCT-1 protein and for its ability to promote cell proliferation. Our data suggests that targeting the RAS/MEK/ERK signal transduction cascade may provide a potential therapeutic approach in lymphomas and related malignancies that exhibit...

  10. Human RECQ1 is a DNA damage responsive protein required for genotoxic stress resistance and suppression of sister chromatid exchanges.

    Directory of Open Access Journals (Sweden)

    Sudha Sharma

    2007-12-01

    Full Text Available DNA helicases are ubiquitous enzymes that unwind DNA in an ATP-dependent and directionally specific manner. Unwinding of double-stranded DNA is essential for the processes of DNA repair, recombination, transcription, and DNA replication. Five human DNA helicases sharing sequence similarity with the E. coli RecQ helicase have been identified. Three of the human RecQ helicases are implicated in hereditary diseases (Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome which display clinical symptoms of premature aging and cancer. RECQ1 helicase is the most highly expressed of the human RecQ helicases; however, a genetic disease has yet not been linked to mutations in the RECQ1 gene, and the biological functions of human RECQ1 in cellular DNA metabolism are not known.In this study, we report that RECQ1 becomes phosphorylated upon DNA damage and forms irradiation-induced nuclear foci that associate with chromatin in human cells. Depletion of RECQ1 renders human cells sensitive to DNA damage induced by ionizing radiation or the topoisomerase inhibitor camptothecin, and results in spontaneous gamma-H2AX foci and elevated sister chromatid exchanges, indicating aberrant repair of DNA breaks. Consistent with a role in homologous recombinational repair, endogenous RECQ1 is associated with the strand exchange protein Rad51 and the two proteins directly interact with high affinity.Collectively, these results provide the first evidence for a role of human RECQ1 in the response to DNA damage and chromosomal stability maintenance and point to the vital importance of RECQ1 in genome homeostasis.

  11. Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

    Science.gov (United States)

    Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

    2013-01-01

    Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

  12. Determination of radiation-induced DNA double-strand breaks for the biological dose monitoring in cardiac computerized tomography; Bestimmung von strahleninduzierten DNA-Doppelstrangbruechen zum Monitoring der biologischen Dosis in der Herz-Computertomographie

    Energy Technology Data Exchange (ETDEWEB)

    Wegener, Jasmin

    2013-11-12

    Background and aims: X-rays cause relevant DNA damage to cells. DNA double-strand breaks (DSBs) are considered to be the most biologically significant radiation induced DNA-lesions. Recently a sensitive immunofluorescence microscopic method was developed to quantify x-ray induced DSBs as nuclear foci, even after doses as used in computed tomography. The method is based on the phosphorylation of the histone variant H2AX after formation of DSBs and distinct foci representing DSBs can be visualised. The number of foci correlates well with the delivered radiation dose. The importance of cardiac CT has increased during the last years. The radiation exposure of cardiac CT is rather high compared to other radiologic diagnostic procedures and techniques for dose-reduction receive increasing attention. In this context the purpose of this study was to determine to what extent the γ-H2AX-based method is able to measure x-ray induced DSBs in patients undergoing cardiac CT. Furthermore the objective was to evaluate whether CT-induced DSBs correlate with exposure parameters (dose length product, DLP) and to assess the influence of the scan protocols on the biological radiation damage. Materials and methods: 32 patients undergoing coronary CT angiography either using a 64-slice (n = 5: SOMATOM Sensation 64 {sup registered}) or a dual-source CT scanner (n = 27: SOMATOM Definition {sup registered}) were included in the study. Venous blood samples were taken before and 0.5 h, 2.5 h, and 24 h after the CT scan. Additional venous blood samples obtained before CT were irradiated in-vitro at various radiation doses (10 mGy, 50 mGy, 100 mGy) to obtain reference values of foci. Lymphocytes were separated and incubated with a specific γ-H2AX primary and a fluorescent secondary antibody. The number of γ-H2AX-foci was quantified using a fluorescence microscope. Every distinct focus represents one DNA-DSB. The number of radiation-induced DSBs was calculated by subtracting the foci number

  13. HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration.

    Science.gov (United States)

    Cooper, Arik; García, Mayra; Petrovas, Constantinos; Yamamoto, Takuya; Koup, Richard A; Nabel, Gary J

    2013-06-20

    Human immunodeficiency virus-1 (HIV-1) has infected more than 60 million people and caused nearly 30 million deaths worldwide, ultimately the consequence of cytolytic infection of CD4(+) T cells. In humans and in macaque models, most of these cells contain viral DNA and are rapidly eliminated at the peak of viraemia, yet the mechanism by which HIV-1 induces helper T-cell death has not been defined. Here we show that virus-induced cell killing is triggered by viral integration. Infection by wild-type HIV-1, but not an integrase-deficient mutant, induced the death of activated primary CD4 lymphocytes. Similarly, raltegravir, a pharmacologic integrase inhibitor, abolished HIV-1-induced cell killing both in cell culture and in CD4(+) T cells from acutely infected subjects. The mechanism of killing during viral integration involved the activation of DNA-dependent protein kinase (DNA-PK), a central integrator of the DNA damage response, which caused phosphorylation of p53 and histone H2AX. Pharmacological inhibition of DNA-PK abolished cell death during HIV-1 infection in vitro, suggesting that processes which reduce DNA-PK activation in CD4 cells could facilitate the formation of latently infected cells that give rise to reservoirs in vivo. We propose that activation of DNA-PK during viral integration has a central role in CD4(+) T-cell depletion, raising the possibility that integrase inhibitors and interventions directed towards DNA-PK may improve T-cell survival and immune function in infected individuals.

  14. CHK1-driven histone H3.3 serine 31 phosphorylation is important for chromatin maintenance and cell survival in human ALT cancer cells.

    Science.gov (United States)

    Chang, Fiona T M; Chan, F Lyn; R McGhie, James D; Udugama, Maheshi; Mayne, Lynne; Collas, Philippe; Mann, Jeffrey R; Wong, Lee H

    2015-03-11

    Human ALT cancers show high mutation rates in ATRX and DAXX. Although it is well known that the absence of ATRX/DAXX disrupts H3.3 deposition at heterochromatin, its impact on H3.3 deposition and post-translational modification in the global genome remains unclear. Here, we explore the dynamics of phosphorylated H3.3 serine 31 (H3.3S31ph) in human ALT cancer cells. While H3.3S31ph is found only at pericentric satellite DNA repeats during mitosis in most somatic human cells, a high level of H3.3S31ph is detected on the entire chromosome in ALT cells, attributable to an elevated CHK1 activity in these cells. Drug inhibition of CHK1 activity during mitosis and expression of mutant H3.3S31A in these ALT cells result in a decrease in H3.3S31ph levels accompanied with increased levels of phosphorylated H2AX serine 139 on chromosome arms and at the telomeres. Furthermore, the inhibition of CHK1 activity in these cells also reduces cell viability. Our findings suggest a novel role of CHK1 as an H3.3S31 kinase, and that CHK1-mediated H3.3S31ph plays an important role in the maintenance of chromatin integrity and cell survival in ALT cancer cells. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Mutations in the ND5 subunit of complex I of the mitochondrial DNA are a frequent cause of oxidative phosphorylation disease.

    NARCIS (Netherlands)

    Blok, M.J.; Spruijt, L.; Coo, I.F.M. de; Schoonderwoerd, K.C.; Hendrickx, A.; Smeets, H.J.M.

    2007-01-01

    BACKGROUND: Detection of mutations in the mitochondrial DNA (mtDNA) is usually limited to common mutations and the transfer RNA genes. However, mutations in other mtDNA regions can be an important cause of oxidative phosphorylation (OXPHOS) disease as well. OBJECTIVE: To investigate whether regions

  16. Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

    2008-01-01

    Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...

  17. The sesquiterpene lactone dehydroleucodine triggers senescence and apoptosis in association with accumulation of DNA damage markers.

    Directory of Open Access Journals (Sweden)

    Valeria V Costantino

    Full Text Available Sesquiterpene lactones (SLs are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL, a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser, a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.

  18. The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers

    Science.gov (United States)

    Costantino, Valeria V.; Mansilla, Sabrina F.; Speroni, Juliana; Amaya, Celina; Cuello-Carrión, Darío; Ciocca, Daniel R.; Priestap, Horacio A.; Barbieri, Manuel A.; Gottifredi, Vanesa; Lopez, Luis A.

    2013-01-01

    Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models. PMID:23341930

  19. ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

    Science.gov (United States)

    Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

    2017-12-07

    Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, Ann-Sofie, E-mail: ann-sofie.gustafsson@bms.uu.se; Abramenkovs, Andris; Stenerlöw, Bo

    2014-11-15

    Highlights: • We reduced the level of DNA-PKcs with siRNA and examined cells after γ-irradiation. • Low DNA-PKcs levels lead to radiosensitivity but did not affect repair of DSB. • Low DNA-PKcs levels may block progression of mitosis. • DNA-PKcs role in mitotic progression is independent of its role in DSB repair. • We suggest different mechanisms by which loss of DNA-PKcs function sensitize cells. - Abstract: Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure

  1. Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins.

    Science.gov (United States)

    Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R

    2015-03-01

    Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.

  2. Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress.

    Science.gov (United States)

    Shi, Xinli; Wang, Li; Li, Xiaoming; Bai, Jing; Li, Jianchun; Li, Shenghao; Wang, Zeming; Zhou, Mingrui

    2017-07-11

    Dihydroartemisinin is an effective antimalarial agent with multiple biological activities. In the present investigation, we elucidated its therapeutic potential and working mechanism on human tongue squamous cell carcinoma (TSCC). It was demonstrated that dihydroartemisinin could significantly inhibit cell growth in a dose- and time-dependent manner by the Cell Counting Kit-8 and colony formation assay in vitro. Meanwhile, autophagy was promoted in the Cal-27 cells treated by dihydroartemisinin, evidenced by increased LC3B-II level, increased autophagosome formation, and increased Beclin-1 level compared to dihydroartemisinin-untreated cells. Importantly, dihydroartemisinin caused DNA double-strand break with simultaneously increased γH2AX foci and oxidative stress; this inhibited the nuclear localization of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), finally leading to autophagic cell death. Furthermore, the antitumor effect of dihydroartemisinin-monotherapy was confirmed with a mouse xenograft model, and no kidney injury associated with toxic effect was observed after intraperitoneal injection with dihydroartemisinin for 3 weeks in vivo. In the present study, it was revealed that dihydroartemisinin-induced DNA double-strand break promoted oxidative stress, which decreased p-STAT3 (Tyr705) nuclear localization, and successively increased autophagic cell death in the Cal-27 cells. Thus, dihydroartemisinin alone may represent an effective and safe therapeutic agent for human TSCC.

  3. Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Julie L Boerner

    Full Text Available Patients with metastatic triple-negative breast cancer (TNBC have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose polymerase (PARP, a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

  4. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R

    1997-01-01

    delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show...... that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF...

  5. CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Mariela C Marazita

    Full Text Available DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

  6. CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions.

    Directory of Open Access Journals (Sweden)

    Isabel Caetano de Abreu da Silva

    Full Text Available BACKGROUND: The helminth Schistosoma mansoni parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1, a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1 is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. PRINCIPAL FINDINGS: We showed in vitro and in vivo that CK2 phosphorylation was involved in the nucleocytoplasmic shuttling of SmHMGB1. By site-directed mutagenesis we mapped the two serine residues of SmHMGB1 that were phosphorylated by CK2. By DNA bending and supercoiling assays we showed that CK2 phosphorylation of SmHMGB1 had no effect in the DNA binding activities of the protein. We showed by electron microscopy, as well as by cell transfection and fluorescence microscopy that SmHMGB1 was present in the nucleus and cytoplasm of adult schistosomes and mammalian cells. In addition, we showed that treatments of the cells with either a phosphatase or a CK2 inhibitor were able to enhance or block, respectively, the cellular traffic of SmHMGB1. Importantly, we showed by confocal microscopy and biochemically that SmHMGB1 is significantly secreted by S. mansoni eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated. CONCLUSIONS: We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis.

  7. hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage

    Science.gov (United States)

    Duckett, Derek R.; Bronstein, S. Maynard; Taya, Yoichi; Modrich, Paul

    1999-01-01

    hMSH2⋅hMSH6 heterodimer (hMutSα) and hMLH1⋅hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O6-methylguanine (O6MeG) adducts. This study demonstrates that O6MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the SN1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N′-nitro-N-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O6MeG⋅T and O6MeG⋅C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage. PMID:10535931

  8. Individual repair of radiation-induced DNA double-strand breaks in lymphocytes. Implications for radiation-induced dermatitis in breast cancer; Die individuelle Reparatur von strahleninduzierten DNA-Doppelstrangbruechen in Lymphozyten. Implikationen fuer die radiogene Dermatitis beim Mammakarzinom

    Energy Technology Data Exchange (ETDEWEB)

    Melchior, Patrick Wilhelm

    2011-07-01

    Purpose: Adjuvant 'whole breast radiotherapy' (WBRT) is the standard of care after breast conserving surgery in women with breast cancer. Throughout different cancer stages the addition of WBRT leads to significantly improved rates of freedom from local failure and overall survival. WBRT is generally well tolerated. A 5-10%-rate of severe acute or long-term side effects is commonly observed. For both radiation-mediated tumor-cell-elimination and induction of side effects, DNA-double-strand-breaks (DSB) presumably play the decisive role. The intensity of normal tissue reactions in radiotherapy can, in part, be attributed to the intrinsic DSB repair-capacity. In this study in vivo and in vitro experiments are carried through in order to assess DSB repair-kinetics in blood lymphocytes of women with breast cancer. These findings are to be correlated with the degree of radiation-induced normal tissue toxicity. Patients and Methods: Eighteen patients with breast cancer, in whom WBRT was indicated, were examined. A total WBRT dose of 50 Gy (single dose 2 Gy) with an additional boost-radiotherapy to the initial tumor-region to a total dose of 60-66 Gy was administered. DSB repair was determined by means of counting γ-H2AX foci in blood lymphocytes at predefined points in time, i.e. before and 0.5 h; 2.5 h; 5 h and 24 h after in vivo irradiation (1st fraction of WBRT) and before and 0.5 h; 2.5 h and 5 h after in vitro irradiation with increasing radiation doses in the range of 10 - 500 mGy. Acute normal tissue toxicity was scored on the basis of a modified RTOG-classification (main aspects were erythema and dry or moist skin desquamation). Results: DSB repair-halflife-times did not differ between patients with a higher or lower than average incidence of acute side effects. In patients with 'above average' side effects larger irradiation volumes were treated (volume surrounded by the 50%-isodose). Adjusted for these, no single patients showed elevated

  9. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Directory of Open Access Journals (Sweden)

    Mehmet Karaca

    Full Text Available Reactivation of androgen receptor (AR may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  10. Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

    Science.gov (United States)

    Karaca, Mehmet; Liu, Yuanbo; Zhang, Zhentao; De Silva, Dinuka; Parker, Joel S; Earp, H Shelton; Whang, Young E

    2015-01-01

    Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

  11. Inactivation of nuclear GSK3β by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

    Science.gov (United States)

    Thornton, Tina M; Delgado, Pilar; Chen, Liang; Salas, Beatriz; Krementsov, Dimitry; Fernandez, Miriam; Vernia, Santiago; Davis, Roger J; Heimann, Ruth; Teuscher, Cory; Krangel, Michael S; Ramiro, Almudena R; Rincón, Mercedes

    2016-01-29

    Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3β (GSK3β) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3β on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3β and promote survival of cells undergoing DSBs. Inability to inactivate GSK3β through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrβ repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3β knockin mice. Thus, GSK3β emerges as an important modulator of the adaptive immune response.

  12. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    kinases and bacterial protein tyrosine kinases) are also much more promiscuous than the TCS kinases, i.e. each of them can phosphorylate several substrate proteins. As a consequence, the dynamics and topology of the signal transduction networks depending on these kinases differ significantly from the TCSs...... regulators. We argue that these promiscuous kinases connect several signal transduction pathways and serve the role of signal integration....

  13. Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation.

    Science.gov (United States)

    Mendez, Gina; Cilli, Domenica; Berardinelli, Francesco; Viganotti, Mara; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio; di Masi, Alessandra

    2012-10-01

    The Nijmegen breakage syndrome (NBS) is a genetic disorder caused by mutations in NBN gene and characterized by chromosomal instability and hypersensitivity to ionizing radiations (IR). The N-terminus of nibrin (NBN) contains a tandem breast cancer 1 (BRCA1) carboxy-terminal (BRCT) domain that represents one of the major mediators of phosphorylation-dependent protein-protein interactions in processes related to cell cycle checkpoint and DNA repair functions. Patients with NBS compound heterozygous for the 657del5 hypomorphic mutation and for the Arg215Trp missense mutation (corresponding to the 643C>T gene mutation) display a clinical phenotype more severe than that of patients homozygous for the 657del5 mutation. Here, we show that both the 657del5 and Arg215Trp mutations, occurring within the tandem BRCT domains of NBN, although not altering the assembly of the MRE11/RAD50/NBN (MRN) complex, affect the MRE11 IR-induced nuclear foci (IRIF) formation and the DNA double-strand break (DSB) signaling via the phosphorylation of both ataxia-telangiectasia-mutated (ATM) kinase and ATM downstream targets (e.g., SMC1 and p53). Remarkably, data obtained indicate that the cleavage of the BRCT tandem domains of NBN by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation. Indeed, the 70-kDa NBN fragment, arising from the 657del5 mutation, maintains the capability to interact with MRE11 and γ-H2AX and to form IRIF. Altogether, the role of the tandem BRCT domains of NBN in the localization of the MRN complex at the DNA DSB and in the activation of the damage response is highlighted. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  14. Regulation of BRCA1 Function by DNA Damage-Induced Site-Specific Phosphorylation

    Science.gov (United States)

    2007-06-01

    concentration of Mg 2. Interestingly, mammalian cell extracts deficient in Fanconi anemia proteins had a 3–9-fold reduction in DNA end-joining...Mavinakere, M., and Campbell, C. Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts. J. Biol. Chem., 276: 9543–9549...suppressive properties of BRCA1 de - rive, at least in part, from its response to tissue-specific DNA damage. In this regard, certain oxidative

  15. Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage

    Directory of Open Access Journals (Sweden)

    Lek Mun Leong

    2016-01-01

    Full Text Available The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action.

  16. Genetic and environmental influence on DNA strand break repair: a twin study

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    Accumulation of DNA damage deriving from exogenous and endogenous sources has significant consequences for cellular survival, and is implicated in aging, cancer, and neurological diseases. Different DNA repair pathways have evolved in order to maintain genomic stability. Genetic and environmental......-strand breaks), and some of the most hazardous lesions (DNA double-strand breaks). DNA damage signaling response (Gamma-H2AX signaling), relative amount of endogenous damage, and DNA-strand break repair capacities were studied in peripheral blood mononuclear cells from 198 twins (94 monozygotic and 104...

  17. HER2 Signaling Drives DNA Anabolism and Proliferation through SRC-3 Phosphorylation and E2F1-Regulated Genes.

    Science.gov (United States)

    Nikolai, Bryan C; Lanz, Rainer B; York, Brian; Dasgupta, Subhamoy; Mitsiades, Nicholas; Creighton, Chad J; Tsimelzon, Anna; Hilsenbeck, Susan G; Lonard, David M; Smith, Carolyn L; O'Malley, Bert W

    2016-03-15

    Approximately 20% of early-stage breast cancers display amplification or overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance to endocrine therapy. Targeting HER2(+) tumors with trastuzumab or the receptor tyrosine kinase (RTK) inhibitor lapatinib significantly improves survival, yet tumor resistance and progression of metastatic disease still develop over time. Although the mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components and gene regulatory networks that bestow therapeutic resistance and limitless proliferative potential are incompletely understood. Here, we use biochemical and bioinformatic approaches to identify effectors and targets of HER2 transcriptional signaling in human breast cancer. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is reduced upon HER2 inhibition, and recruitment of SRC-3 to regulatory elements of endogenous genes is impaired. Transcripts regulated by HER2 signaling are highly enriched with E2F1 binding sites and define a gene signature associated with proliferative breast tumor subtypes, cell-cycle progression, and DNA replication. We show that HER2 signaling promotes breast cancer cell proliferation through regulation of E2F1-driven DNA metabolism and replication genes together with phosphorylation and activity of the transcriptional coactivator SRC-3. Furthermore, our analyses identified a cyclin-dependent kinase (CDK) signaling node that, when targeted using the CDK4/6 inhibitor palbociclib, defines overlap and divergence of adjuvant pharmacologic targeting. Importantly, lapatinib and palbociclib strictly block de novo synthesis of DNA, mostly through disruption of E2F1 and its target genes. These results have implications for rational discovery of pharmacologic combinations in preclinical models of adjuvant treatment and therapeutic resistance. ©2016 American Association for Cancer Research.

  18. JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks

    Directory of Open Access Journals (Sweden)

    Michael Van Meter

    2016-09-01

    Full Text Available The accumulation of damage caused by oxidative stress has been linked to aging and to the etiology of numerous age-related diseases. The longevity gene, sirtuin 6 (SIRT6, promotes genome stability by facilitating DNA repair, especially under oxidative stress conditions. Here we uncover the mechanism by which SIRT6 is activated by oxidative stress to promote DNA double-strand break (DSB repair. We show that the stress-activated protein kinase, c-Jun N-terminal kinase (JNK, phosphorylates SIRT6 on serine 10 in response to oxidative stress. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites and is required for efficient recruitment of poly (ADP-ribose polymerase 1 (PARP1 to DNA break sites and for efficient repair of DSBs. Our results demonstrate a post-translational mechanism regulating SIRT6, and they provide the link between oxidative stress signaling and DNA repair pathways that may be critical for hormetic response and longevity assurance.

  19. Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT in DNA repair, apoptosis and necrosis after cisplatin

    Directory of Open Access Journals (Sweden)

    Calkins Anne S

    2011-06-01

    Full Text Available Abstract Background Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK binds to DNA double strand breaks (DSBs through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. Results Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1, Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT. The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. Conclusions DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and

  20. Sulfur dioxide inhibits expression of mitochondrial oxidative phosphorylation genes encoded by both nuclear DNA and mitochondrial DNA in rat lungs.

    Science.gov (United States)

    Qin, Guohua; Wang, Jiaoxia; Sang, Nan

    2017-01-01

    Epidemiological studies show that sulfur dioxide (SO2), a major air pollutant, is associated with the morbidity and mortality of respiratory tract diseases. The aim of the present study was to determine the effects of SO2 on mitochondria and the corresponding molecular characterization in the lung. Male Wistar rats were exposed to 0, 3.5, 7, and 14 mg/m3 SO2 (4 h/day, 30 days). Mitochondrial dysfunction including decreases of cytochrome c oxidase (COX) activity and mitochondrial membrane potential (MMP) was observed in the lungs of rats after SO2 inhalation. We showed that total mitochondrial DNA (mtDNA) content was significantly decreased in the lungs from rats exposed to SO2. Furthermore, SO2 repressed the expression of complex IV and V subunits encoded by both nuclear DNA (nDNA) and mtDNA. Moreover, such changes were accompanied by depressions of three regulatory factors: peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM). The findings suggest that SO2 exposure induced mitochondrial dysfunction in rat lungs. Both nDNA and mtDNA are involved in SO2-induced depression of mitochondrial biogenesis in the lungs. There might be a tissue-specific response of mitochondrial biosynthesis to SO2 inhalation. Such impairment may lead to cellular dysfunction and eventually lung diseases.

  1. Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families.

    Science.gov (United States)

    Brown, M D; Zhadanov, S; Allen, J C; Hosseini, S; Newman, N J; Atamonov, V V; Mikhailovskaya, I E; Sukernik, R I; Wallace, D C

    2001-07-01

    Leber's hereditary optic neuropathy (LHON) is characterized by maternally transmitted, bilateral, central vision loss in young adults. It is caused by mutations in the mitochondrial DNA (mtDNA) encoded genes that contribute polypeptides to NADH dehydrogenase or complex I. Four mtDNA variants, the nucleotide pair (np) 3460A, 11778A, 14484C, and 14459A mutations, are known as "primary" LHON mutations and are found in most, but not all, of the LHON families reported to date. Here, we report the extensive genetic and biochemical analysis of five Russian families from the Novosibirsk region of Siberia manifesting maternally transmitted optic atrophy consistent with LHON. Three of the five families harbor known LHON primary mutations. Complete sequence analysis of proband mtDNA in the other two families has revealed novel complex I mutations at nps 3635A and 4640C, respectively. These mutations are homoplasmic and have not been reported in the literature. Biochemical analysis of complex I in patient lymphoblasts and transmitochondrial cybrids demonstrated a respiration defect with complex-I-linked substrates, although the specific activity of complex I was not reduced. Overall, our data suggests that the spectrum of mtDNA mutations associated with LHON in Russia is similar to that in Europe and North America and that the np 3635A and 4640C mutations may be additional mtDNA complex I mutations contributing to LHON expression.

  2. The Roles of P53 and its Family Proteins, P63 and P73, in the DNA Damage Stress Response in Organogenesis Stage Mouse Embryos.

    Science.gov (United States)

    El Husseini, Nazem; Hales, Barbara F

    2017-12-07

    Members of the P53 transcription factor family, P53, P63 and P73, play important roles in normal development and in regulating the expression of genes that control apoptosis and cell cycle progression in response to genotoxic stress. P53 is involved in the DNA damage response pathway that is activated by hydroxyurea in organogenesis-stage murine embryos. The extent to which P63 and P73 contribute to this stress response is not known. To address this question, we examined the roles of P53, P63 and P73 in mediating the response of Trp53-positive and Trp53-deficient murine embryos to a single dose of hydroxyurea (400 mg/kg) on gestational day 9. Hydroxyurea treatment downregulated the expression of Trp63 and upregulated Trp73 in the absence of effects on the levels of Trp53 transcripts; Trp73 upregulation was P53-dependent. At the protein level, hydroxyurea treatment increased the levels and phosphorylation of P53 in the absence of effects on P63 and P73. Upregulation of the expression of genes that regulate cell cycle arrest and apoptosis, Cdkn1a, Rb1, Fas, Trp53inp1 and Pmaip1, was P53-dependent in hydroxyurea-treated embryos. The increase in cleaved caspase-3 and cleaved mammalian sterile-20 like-1 (MST-1) kinase levels induced by hydroxyurea was also P53-dependent; in contrast, the increase in phosphorylated H2AX, a marker of DNA double strand breaks, in response to hydroxyurea treatment was only partially P53 dependent. Together, our data show that P53 is the principal P53 family member that is activated in the embryonic DNA damage response. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  3. Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway.

    Science.gov (United States)

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Watrin, Claude; Samba-Louaka, Ascel; Oswald, Eric

    2006-12-01

    The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.

  4. Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

    Science.gov (United States)

    Dutta, Sujoy; Warshall, Case; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

    2014-01-01

    Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment. PMID:24831807

  5. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiang-Yuan; Wang, Zhen [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Bei [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Ying-Jian, E-mail: yjzhang111@aliyun.com [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Ying-Yi, E-mail: liyingyi@fudan.edu.cn [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

    2016-04-22

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevated the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human pancreatic

  6. Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells

    OpenAIRE

    Dodson, Helen; Morrison, Ciaran G.

    2009-01-01

    The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of ?-H2AX foci...

  7. Changes in the Number of Double-Strand DNA Breaks in Chinese Hamster V79 Cells Exposed to γ-Radiation with Different Dose Rates

    Directory of Open Access Journals (Sweden)

    Andreyan N. Osipov

    2013-07-01

    Full Text Available A comparative investigation of the induction of double-strand DNA breaks (DSBs in the Chinese hamster V79 cells by γ-radiation at dose rates of 1, 10 and 400 mGy/min (doses ranged from 0.36 to 4.32 Gy was performed. The acute radiation exposure at a dose rate of 400 mGy/min resulted in the linear dose-dependent increase of the γ-H2AX foci formation. The dose-response curve for the acute exposure was well described by a linear function y = 1.22 + 19.7x, where “y” is an average number of γ-H2AX foci per a cell and “x” is the absorbed dose (Gy. The dose rate reduction down to 10 mGy/min lead to a decreased number of γ-H2AX foci, as well as to a change of the dose-response relationship. Thus, the foci number up to 1.44 Gy increased and reached the “plateau” area between 1.44 and 4.32 Gy. There was only a slight increase of the γ-H2AX foci number (up to 7 in cells after the protracted exposure (up to 72 h to ionizing radiation at a dose rate of 1 mGy/min. Similar effects of the varying dose rates were obtained when DNA damage was assessed using the comet assay. In general, our results show that the reduction of the radiation dose rate resulted in a significant decrease of DSBs per cell per an absorbed dose.

  8. A mitotic phosphorylation feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2/M DNA damage checkpoint.

    Directory of Open Access Journals (Sweden)

    Marcel A T M van Vugt

    2010-01-01

    Full Text Available DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1. We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.

  9. Structure-function study of deinococcal serine/threonine protein kinase implicates its kinase activity and DNA repair protein phosphorylation roles in radioresistance of Deinococcus radiodurans.

    Science.gov (United States)

    Rajpurohit, Yogendra S; Misra, Hari S

    2013-11-01

    The DR2518 (RqkA) a eukaryotic type serine/threonine protein kinase in Deinococcus radiodurans was characterized for its role in bacterial response to oxidative stress and DNA damage. The K42A, S162A, T169A and S171A mutation in RqkA differentially affected its kinase activity and functional complementation for γ radiation resistance in Δdr2518 mutant. For example, K42A mutant was completely inactive and showed no complementation while S171A, T169A and T169A/S171A mutants were less active and complemented proportionally to different levels as compared to wild type. Amongst, different DNA binding proteins that purified RqkA could phosphorylate, PprA a DNA repair protein, phosphorylation had improved its affinity to DNA by 4 fold and could enhance its supportive role in intermolecular ligation by T4 DNA ligase. RqkA phosphorylates PprA at threonine 72 (T72), serine 112 (S112) and threonine 144 (T144) in vitro with the majority of it goes to T72 site. Unlike wild type PprA and single mutants of T72, S112 and T144 residues, the T72AS112A double and T72AS112AT144A triple mutant derivatives of PprA did not phosphorylate in vivo and also failed to complement PprA loss in D. radiodurans. Deletion of rqkA in pprA::cat background enhanced radiosensitivity of pprA mutant, which became nearly similar to ΔrqkA resistance to γ radiation. These results suggested that K42 of RqkA is essential for catalytic functions and the kinase activity of RqkA as well as phosphorylation of PprA have roles in γ radiation resistance of D. radiodurans. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

    Science.gov (United States)

    Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L; Sauer, Sascha; Sperling, Silke R

    2016-04-07

    DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.

    Science.gov (United States)

    Leong, Wan Yee; Guo, Hong; Ma, Ou; Huang, Hui; Cantor, Alan B; Friedman, Alan D

    2016-01-08

    Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Titanium dioxide nanoparticles induce DNA damage and genetic instability in vivo in mice.

    Science.gov (United States)

    Trouiller, Benedicte; Reliene, Ramune; Westbrook, Aya; Solaimani, Parrisa; Schiestl, Robert H

    2009-11-15

    Titanium dioxide (TiO(2)) nanoparticles are manufactured worldwide in large quantities for use in a wide range of applications including pigment and cosmetic manufacturing. Although TiO(2) is chemically inert, TiO(2) nanoparticles can cause negative health effects, such as respiratory tract cancer in rats. However, the mechanisms involved in TiO(2)-induced genotoxicity and carcinogenicity have not been clearly defined and are poorly studied in vivo. The present study investigates TiO(2) nanoparticles-induced genotoxicity, oxidative DNA damage, and inflammation in a mice model. We treated wild-type mice with TiO(2) nanoparticles in drinking water and determined the extent of DNA damage using the comet assay, the micronuclei assay, and the gamma-H2AX immunostaining assay and by measuring 8-hydroxy-2'-deoxyguanosine levels and, as a genetic instability endpoint, DNA deletions. We also determined mRNA levels of inflammatory cytokines in the peripheral blood. Our results show that TiO(2) nanoparticles induced 8-hydroxy-2'-deoxyguanosine, gamma-H2AX foci, micronuclei, and DNA deletions. The formation of gamma-H2AX foci, indicative of DNA double-strand breaks, was the most sensitive parameter. Inflammation was also present as characterized by a moderate inflammatory response. Together, these results describe the first comprehensive study of TiO(2) nanoparticles-induced genotoxicity in vivo in mice possibly caused by a secondary genotoxic mechanism associated with inflammation and/or oxidative stress. Given the growing use of TiO(2) nanoparticles, these findings raise concern about potential health hazards associated with TiO(2) nanoparticles exposure.

  13. Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, Paul F.; Nham, Peter B.; Urbin, Salustra S.; Hinz, John M.; Jones, Irene M. [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States); Thompson, Larry H., E-mail: thompson14@llnl.gov [Biosciences and Biotechnology Division, PO Box 808, L-452, Lawrence Livermore National Laboratory, Livermore, CA, 94551-0808 (United States)

    2010-01-05

    DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of {sup 137}Cs {gamma}-rays. Quiescent G{sub 0}/G{sub 1}-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24 h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV {approx} 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV {approx} 0.6; mean {+-} SD of 1.00 {+-} 0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24 h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24 h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.

  14. Mitotic instability in wheat x Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern.

    Science.gov (United States)

    Brasileiro-Vidal, A C; Brammer, S; Puertas, M J; Zanatta, A C; Prestes, A; Moraes-Fernandes, M I B; Guerra, M

    2005-06-01

    To evaluate the mitotic stability of Triticum aestivum x Thinopyrum ponticum derivatives (BC(2)F(7) and BC(2)F(5) doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring' and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.

  15. The Role of DNA Methylation Changes in Radiation-Induced Bystander Effects in cranial irradiated Mice

    Science.gov (United States)

    Zhang, Meng; Sun, Yeqing; Xue, Bei; Wang, Xinwen; Wang, Jiawen

    2016-07-01

    Heavy-ion radiation could lead to bystander effect in neighboring non-hit cells by signals released from directly-irradiated cells. The exact mechanisms of radiation-induced bystander effect in distant organ remain obscure, yet accumulating evidence points to the role of DNA methylation changes in bystander effect. To identify the molecular mechanism that underlies bystander effects of heavy-ion radiation, the male Balb/c and C57BL mice were cranial exposed to 40, 200, 2000mGy dose of carbon heavy-ion radiation, while the rest of the animal body was shielded. The γH2AX foci as the DNA damage biomarker in directly irradiation organ ear and the distant organ liver were detected on 0, 1, 2, 6, 12 and 24h after radiation, respectively. Methylation-sensitive amplifcation polymorphism (MSAP) was used to monitor the level of polymorphic genomic DNA methylation changed with dose and time effects. The results show that cranial irradiated mice could induce the γH2AX foci and genomic DNA methylation changes significantly in both the directly irradiation organ ear and the distant organ liver. The percent of DNA methylation changes were time-dependent and tissue-specific. Demethylation polymorphism rate were highest separately at 1 h in 200 mGy and 6 h in 2000 mGy after irradiation in ear. The global DNA methylation changes tended to occur in the CG sites. We also found that the numbers of γH2AX foci and the genomic methylation changes of heavy-ion radiation-induced bystander effect in liver could be obvious 1 h after radiation and achieved the maximum at 6 h, while the changes could recover gradually at 12 h. The results suggest that mice head exposed to heavy-ion radiation can induce damage and methylation pattern changed in both directly radiation organ ear and distant organ liver. Moreover, our findings are important to understand the molecular mechanism of radiation induced bystander effects in vivo. Keywords: Heavy-ion radiation; Bystander effect; DNA methylation; γH2

  16. Human embryos commonly form abnormal nuclei during development: a mechanism of DNA damage, embryonic aneuploidy, and developmental arrest.

    Science.gov (United States)

    Kort, Daniel H; Chia, Gloryn; Treff, Nathan R; Tanaka, Akemi J; Xing, Tongji; Vensand, Lauren Bauer; Micucci, Stephanie; Prosser, Robert; Lobo, Roger A; Sauer, Mark V; Egli, Dieter

    2016-02-01

    damage was significantly higher in cells with microscopic nuclear abnormalities (γH2AX (phosphorylated (Ser139) histone H2A.X): 87.1%, 74/85; replication protein A: 72.9%, 62/85) relative to cells with normal nuclear morphology (γH2AX: 9.3%, 60/642; RPA: 5.6%, 36/642) (P < 0.05). Blastomeres containing nuclear abnormalities were strongly associated with aneuploidy (Fisher exact test, two-tailed, P < 0.01). The embryos used were de-identified, and the clinical and IVF history was unknown. This study explores a mechanism of abnormal embryonic development post-fertilization. While most of the current data have explored abnormal meiotic chromosome segregation in oocytes as a primary mechanism of reproductive failure, abnormal nuclear formation during early mitotic cell division in IVF embryos also plays a significant role. The detection of abnormal nuclear formation may have clinical application in noninvasive embryo selection during IVF. The study was supported by Columbia University and the New York Stem Cell Foundation. Authors declare no competing interest. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  17. Induction of DNA double-strand breaks by monochlorophenol isomers and ChKM in human gingival fibroblasts.

    Science.gov (United States)

    Shehata, M; Durner, J; Thiessen, D; Shirin, M; Lottner, S; Van Landuyt, K; Furche, S; Hickel, R; Reichl, F X

    2012-09-01

    Phenol has been traditionally used in dental treatment as a sedative for the pulp or as disinfectant for carious cavity and root canal. However, phenol is regarded as a mutagenic and carcinogenic agent and its use in dental practice is now therefore restricted. Monochlorophenols are derivatives of phenol, which are still used clinically as root canal disinfectants, they are even more active antiseptics/disinfectants than phenol, and the so-called Walkhoff (ChKM) solution makes use of monochlorophenol for root canal disinfection. Ingredients in the ChKM solution are the monochlorophenol compound 4-chlorophenol (4-CP), camphor, and menthol. In literature, the use of the ChKM solution is controversial because of a possible DNA toxicity of the ingredient 4-CP. However, it is unknown whether ChKM can really induce DNA damage in human oral cells. In this study, the induction of DNA double-strand breaks (DSBs) by ChKM and monochlorophenol compounds (2-chlorophenol, 2-CP; 3-chlorophenol, 3-CP; and 4-chlorophenol, 4-CP) was tested in human gingival fibroblasts (HGFs). DNA DSBs (foci) induced in HGFs unexposed and exposed to monochlorophenols or ChKM solution were investigated using the γ-H2AX DNA focus assay, which is a direct marker for DSBs. DSBs result in the ATM-dependent phosphorylation of the histone H2AX. When cells were exposed to medium or medium + DMSO (1 %) (negative controls), an average of 3 foci per cell were found. In positive control cells (H₂O₂ + medium, or H₂O₂ + medium + DMSO (1 %), an average of 35 foci each were found. About 20 DSB foci per cell were found, when HGFs were exposed to 2-CP (4 mM), 3-CP (2.3 mM), 4-CP (2.1 mM), or ChKM (corresponding to 1.5 mM 4-CP). Our results show increasing DNA toxicities in the order of 2-CP toxicity was found for 4-CP in combination with camphor in the ChKM solution, compared to the 4-CP alone. No significant differences regarding multi-foci cells (cells that contain more than 40 foci) were found when HGFs

  18. DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age.

    Science.gov (United States)

    Kim, Ha-Neui; Chang, Jianhui; Shao, Lijian; Han, Li; Iyer, Srividhya; Manolagas, Stavros C; O'Brien, Charles A; Jilka, Robert L; Zhou, Daohong; Almeida, Maria

    2017-08-01

    Age-related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age-dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1-Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP-Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP-Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21CIP1 levels, as well as increased levels of GATA4 and activation of NF-κB - two major stimulators of the senescence-associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro-osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  19. Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis

    Directory of Open Access Journals (Sweden)

    Rodrigo eMartínez-Espinoza

    2015-08-01

    Full Text Available The control of Giardia duodenalis infections is carried out mainly by drugs, among these albendazole (ABZ is commonly used. Although the cytotoxic effect of ABZ usually involves binding to β-tubulin, it has been suggested that oxidative stress may also play a role in its parasiticidal mechanism. In this work the effect of ABZ in Giardia clones that are susceptible or resistant to different concentrations (1.35, 8 and 250 μM of this drug was analyzed. Reactive oxygen species (ROS were induced by ABZ in susceptible clones and this was associated with a decrease in growth that was alleviated by cysteine supplementation. Remarkably, ABZ-resistant clones exhibited partial cross-resistance to H2O2, whereas a Giardia H2O2-resistant strain can grow in the presence of ABZ. Lipid oxidation and protein carbonylation in ABZ-treated parasites did not show significant differences as compared to untreated parasites; however ABZ induced the formation of 8OHdG adducts and DNA degradation, indicating nucleic acid oxidative damage. This was supported by observations of histone H2AX phosphorylation in ABZ-susceptible trophozoites treated with 250 μM ABZ. Flow cytometry analysis showed that ABZ partially arrested cell cycle in drug-susceptible clones at G2/M phase at the expense of cells in G1 phase. Also, ABZ treatment resulted in phosphatidylserine exposure on the parasite surface, an event related to apoptosis. All together these data suggest that ROS induced by ABZ affect Giardia genetic material through oxidative stress mechanisms and subsequent induction of apoptotic-like events.

  20. Interplay with the Mre11-Rad50-Nbs1 complex and phosphorylation by GSK3β implicate human B-Myb in DNA-damage signaling.

    Science.gov (United States)

    Henrich, Sarah Marie; Usadel, Clemens; Werwein, Eugen; Burdova, Kamila; Janscak, Pavel; Ferrari, Stefano; Hess, Daniel; Klempnauer, Karl-Heinz

    2017-01-27

    B-Myb, a highly conserved member of the Myb transcription factor family, is expressed ubiquitously in proliferating cells and controls the cell cycle dependent transcription of G2/M-phase genes. Deregulation of B-Myb has been implicated in oncogenesis and loss of genomic stability. We have identified B-Myb as a novel interaction partner of the Mre11-Rad50-Nbs1 (MRN) complex, a key player in the repair of DNA double strand breaks. We show that B-Myb directly interacts with the Nbs1 subunit of the MRN complex and is recruited transiently to DNA-damage sites. In response to DNA-damage B-Myb is phosphorylated by protein kinase GSK3β and released from the MRN complex. A B-Myb mutant that cannot be phosphorylated by GSK3β disturbs the regulation of pro-mitotic B-Myb target genes and leads to inappropriate mitotic entry in response to DNA-damage. Overall, our work suggests a novel function of B-Myb in the cellular DNA-damage signalling.

  1. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    Science.gov (United States)

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  2. Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    López-Camarillo César

    2008-04-01

    Full Text Available Abstract Background In eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. RAD52 epistasis group genes involved in recombinational DNA repair, including mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 and rad59 genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in Entamoeba histolytica the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown. Results In this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote E. histolytica using UV-C (150 J/m2 irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In E. histolytica genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the E. histolytica RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear foci-like structures in E. histolytica trophozoites. Purified recombinant EhRAD51 exhibited DNA binding

  3. DNA double strand breaks as predictor of efficacy of the alpha-particle emitter Ac-225 and the electron emitter Lu-177 for somatostatin receptor targeted radiotherapy.

    Directory of Open Access Journals (Sweden)

    Franziska Graf

    Full Text Available RATIONALE: Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to the beta-particle emitter Lutetium-177 labeled somatostatin-analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of γH2AX-foci formation. METHODS: To determine the relative biological effectiveness (RBE between the two isotopes (as - biological consequence of different ionisation-densities along a particle-track, somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks (DSB were quantified by immunofluorescence staining of γH2AX-foci. Cell cycle was analyzed by flow cytometry. In vivo uptake of both radiolabeled somatostatin-analogues into subcutaneously growing AR42J tumors and the number of cells displaying γH2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities estimated from in vitro cytotoxicity. RESULTS: Ac-225-DOTATOC resulted in ED50 values of 14 kBq/ml after 48 h, whereas Lu-177-DOTATOC displayed ED50 values of 10 MBq/ml. The number of DSB grew with increasing concentration of Ac-225-DOTATOC and similarly with Lu-177-DOTATOC when applying a factor of 700-fold higher activity compared to Ac-225. Already 24 h after incubation with 2.5-10 kBq/ml, Ac-225-DOTATOC cell-cycle studies showed up to a 60% increase in the percentage of tumor cells in G2/M phase. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical (7.5%ID/g, though the overall number of cells with γH2AX-foci was higher in tumors treated with 48 kBq Ac-225-DOTATOC compared to tumors treated with 30 MBq Lu-177-DOTATOC (35% vs. 21%. Tumors with a volume of 0.34 ml reached delayed exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC and after 21 days (34 MBq Lu-177-DOTATOC. CONCLUSION: γH2AX-foci formation, triggered

  4. Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells.

    Science.gov (United States)

    Hsieh, Yi-Hsien; Lee, Chien-Hsing; Chen, Hsiao-Yun; Hsieh, Shu-Ching; Lin, Chia-Liang; Tsai, Jen-Pi

    2015-09-01

    Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dose-dependent manner as determined by comet assay and measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide double-stained assay. The activities of caspase-3, -9, and poly ADP-ribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsic-related proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC.

  5. Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism.

    Science.gov (United States)

    Rehmani, Nida; Zafar, Atif; Arif, Hussain; Hadi, Sheikh Mumtaz; Wani, Altaf A

    2017-04-01

    Oxidative DNA damage has been implicated in the pathogenesis of neurological disorders, cancer and ageing. Owing to the established link between labile copper concentrations and neurological diseases, it is critical to explore the interactions of neurotransmitters and drug supplements with copper. Herein, we investigate the pro-oxidant DNA damage induced by the interaction of L-DOPA and dopamine (DA) with copper. The DNA binding affinity order of the compounds has been determined by in silico molecular docking. Agarose gel electrophoresis reveals that L-DOPA and DA are able to induce strand scission in plasmid pcDNA3.1 (+/-) in a copper dependent reaction. These metabolites also cause cellular DNA breakage in human lymphocytes by mobilizing endogenous copper, as assessed by comet assay. Further, L-DOPA and DA-mediated DNA breaks were detected by the appearance of post-DNA damage sensitive marker γH2AX in cancer cell lines accumulating high copper. Immunofluorescence demonstrated the co-localization of downstream repair factor 53BP1 at the damaged induced γH2AX foci in cancer cells. The present study corroborates and provides a mechanism to the hypothesis that suggests metal-mediated oxidation of catecholamines contributes to the pathogenesis of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Increased sister chromatid cohesion and DNA damage response factor localization at an enzyme-induced DNA double-strand break in vertebrate cells.

    LENUS (Irish Health Repository)

    Dodson, Helen

    2009-10-01

    The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM target in the DSB response. These data show that the same genetic pathways control how cells respond to single DSBs and to multiple lesions induced by whole-cell DNA damage.

  7. Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation.

    Science.gov (United States)

    Yang, Lina; Liu, Yuanyuan; Sun, Chao; Yang, Xinrui; Yang, Zhen; Ran, Juntao; Zhang, Qiuning; Zhang, Hong; Wang, Xinyu; Wang, Xiaohu

    2015-11-01

    Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G2/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5-50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of

  8. Poly(ADP-ribose) binds to the splicing factor ASF/SF2 and regulates its phosphorylation by DNA topoisomerase I.

    Science.gov (United States)

    Malanga, Maria; Czubaty, Alicja; Girstun, Agnieszka; Staron, Krzysztof; Althaus, Felix R

    2008-07-18

    Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

  9. N-acetyl cysteine protects against ionizing radiation-induced DNA damage but not against cell killing in yeast and mammals

    Energy Technology Data Exchange (ETDEWEB)

    Reliene, Ramune [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Medicine, Center for Human Nutrition, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Pollard, Julianne M. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Sobol, Zhanna; Trouiller, Benedicte [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Gatti, Richard A. [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Schiestl, Robert H., E-mail: rschiestl@mednet.ucla.edu [Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Biomedical Physics Interdepartmental Program, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Radiation Oncology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095 (United States); Department of Environmental Health Sciences, School of Public Health, University of California Los Angeles, Los Angeles, CA 90095 (United States)

    2009-06-01

    Ionizing radiation (IR) induces DNA strand breaks leading to cell death or deleterious genome rearrangements. In the present study, we examined the role of N-acetyl-L-cysteine (NAC), a clinically proven safe agent, for it's ability to protect against {gamma}-ray-induced DNA strand breaks and/or DNA deletions in yeast and mammals. In the yeast Saccharomyces cerevisiae, DNA deletions were scored by reversion to histidine prototrophy. Human lymphoblastoid cells were examined for the frequency of {gamma}-H2AX foci formation, indicative of DNA double strand break formation. DNA strand breaks were also measured in mouse peripheral blood by the alkaline comet assay. In yeast, NAC reduced the frequency of IR-induced DNA deletions. However, NAC did not protect against cell death. NAC also reduced {gamma}-H2AX foci formation in human lymphoblastoid cells but had no protective effect in the colony survival assay. NAC administration via drinking water fully protected against DNA strand breaks in mice whole-body irradiated with 1 Gy but not with 4 Gy. NAC treatment in the absence of irradiation was not genotoxic. These data suggest that, given the safety and efficacy of NAC in humans, NAC may be useful in radiation therapy to prevent radiation-mediated genotoxicity, but does not interfere with efficient cancer cell killing.

  10. Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

    Science.gov (United States)

    Horvath, Beatrix M; Kourova, Hana; Nagy, Szilvia; Nemeth, Edit; Magyar, Zoltan; Papdi, Csaba; Ahmad, Zaki; Sanchez-Perez, Gabino F; Perilli, Serena; Blilou, Ikram; Pettkó-Szandtner, Aladár; Darula, Zsuzsanna; Meszaros, Tamas; Binarova, Pavla; Bogre, Laszlo; Scheres, Ben

    2017-05-02

    The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  11. Defining the Contribution of MC1R Physiological Ligands to ATR Phosphorylation at Ser435, a Predictor of DNA Repair in Melanocytes.

    Science.gov (United States)

    Jarrett, Stuart G; Wolf Horrell, Erin M; Boulanger, Mary C; D'Orazio, John A

    2015-12-01

    The melanocortin 1 receptor (MC1R), a GS-coupled receptor that signals through cAMP and protein kinase A (PKA), regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions, and assess NER function. We report that melanocyte-stimulating hormone (α-MSH) or ACTH induce ATR-pS435, enhance XPA's association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human β-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction, and reduce DNA repair. Although ASIP and HBD3 each blocked α-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.

  12. DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy.

    Science.gov (United States)

    Ronchetti, Livia; Melucci, Elisa; De Nicola, Francesca; Goeman, Frauke; Casini, Beatrice; Sperati, Francesca; Pallocca, Matteo; Terrenato, Irene; Pizzuti, Laura; Vici, Patrizia; Sergi, Domenico; Di Lauro, Luigi; Amoreo, Carla Azzurra; Gallo, Enzo; Diodoro, Maria Grazia; Pescarmona, Edoardo; Vitale, Ilio; Barba, Maddalena; Buglioni, Simonetta; Mottolese, Marcella; Fanciulli, Maurizio; De Maria, Ruggero; Maugeri-Saccà, Marcello

    2017-06-01

    The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AX(high) /pATM(high) ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AX(high) /pATM(high) model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AX(high) /pATM(high) model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance. © 2017 UICC.

  13. An ImageJ-based algorithm for a semi-automated method for microscopic image enhancement and DNA repair foci counting

    Energy Technology Data Exchange (ETDEWEB)

    Klokov, D., E-mail: dmitry.klokov@cnl.ca [Canadian Nuclear Laboratories, Chalk River, Ontario (Canada); Suppiah, R. [Queen' s Univ., Dept. of Biomedical and Molecular Sciences, Kingston, Ontario (Canada)

    2015-06-15

    Proper evaluation of the health risks of low-dose ionizing radiation exposure heavily relies on the ability to accurately measure very low levels of DNA damage in cells. One of the most sensitive methods for measuring DNA damage levels is the quantification of DNA repair foci that consist of macromolecular aggregates of DNA repair proteins, such as γH2AX and 53BP1, forming around individual DNA double-strand breaks. They can be quantified using immunofluorescence microscopy and are widely used as markers of DNA double-strand breaks. However this quantification, if performed manually, may be very tedious and prone to inter-individual bias. Low-dose radiation studies are especially sensitive to this potential bias due to a very low magnitude of the effects anticipated. Therefore, we designed and validated an algorithm for the semi-automated processing of microscopic images and quantification of DNA repair foci. The algorithm uses ImageJ, a freely available image analysis software that is customizable to individual cellular properties or experimental conditions. We validated the algorithm using immunolabeled 53BP1 and γH2AX in normal human fibroblast AG01522 cells under both normal and irradiated conditions. This method is easy to learn, can be used by nontrained personnel, and can help avoiding discrepancies in inter-laboratory comparison studies examining the effects of low-dose radiation. (author)

  14. Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

    Science.gov (United States)

    Aguilar-Quesada, Rocío; Muñoz-Gámez, José Antonio; Martín-Oliva, David; Peralta, Andreína; Valenzuela, Ma Teresa; Matínez-Romero, Rubén; Quiles-Pérez, Rosa; Murcia, Josiane Menissier-de; de Murcia, Gilbert; de Almodóvar, Mariano Ruiz; Oliver, F Javier

    2007-01-01

    ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase. PMID:17459151

  15. Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

    Directory of Open Access Journals (Sweden)

    de Murcia Gilbert

    2007-04-01

    Full Text Available Abstract ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway and activates ATM kinase.

  16. Age and gender effects on DNA strand break repair in peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Garm, Christian; Moreno-Villanueva, Maria; Bürkle, Alexander

    2013-01-01

    Exogenous and endogenous damage to DNA is constantly challenging the stability of our genome. This DNA damage increase the frequency of errors in DNA replication, thus causing point mutations or chromosomal rearrangements and has been implicated in aging, cancer, and neurodegenerative diseases....... Therefore, efficient DNA repair is vital for the maintenance of genome stability. The general notion has been that DNA repair capacity decreases with age although there are conflicting results. Here, we focused on potential age-associated changes in DNA damage response and the capacities of repairing DNA...... single-strand breaks (SSBs) and double-strand breaks (DSBs) in human peripheral blood mononuclear cells (PBMCs). Of these lesions, DSBs are the least frequent but the most dangerous for cells. We have measured the level of endogenous SSBs, SSB repair capacity, γ-H2AX response, and DSB repair capacity...

  17. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

    Science.gov (United States)

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

    2016-01-01

    Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Analysis of Flow Cytometry DNA Damage Response Protein Activation Kinetics Following X-rays and High Energy Iron Nuclei Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Universities Space Research Association; Chappell, Lori J.; Whalen, Mary K.; Gurai, Sheena; Ponomarev, Artem; Cucinotta, Francis A.; Pluth, Janice M.

    2010-12-15

    We developed a mathematical method to analyze flow cytometry data to describe the kinetics of {gamma}H2AX and pATF2 phosphorylations ensuing various qualities of low dose radiation in normal human fibroblast cells. Previously reported flow cytometry kinetic results for these DSB repair phospho-proteins revealed that distributions of intensity were highly skewed, severely limiting the detection of differences in the very low dose range. Distributional analysis reveals significant differences between control and low dose samples when distributions are compared using the Kolmogorov-Smirnov test. Radiation quality differences are found in the distribution shapes and when a nonlinear model is used to relate dose and time to the decay of the mean ratio of phosphoprotein intensities of irradiated samples to controls. We analyzed cell cycle phase and radiation quality dependent characteristic repair times and residual phospho-protein levels with these methods. Characteristic repair times for {gamma}H2AX were higher following Fe nuclei as compared to X-rays in G1 cells (4.5 {+-} 0.46 h vs 3.26 {+-} 0.76 h, respectively), and in S/G2 cells (5.51 {+-} 2.94 h vs 2.87 {+-} 0.45 h, respectively). The RBE in G1 cells for Fe nuclei relative to X-rays for {gamma}H2AX was 2.05 {+-} 0.61 and 5.02 {+-} 3.47, at 2 h and 24-h postirradiation, respectively. For pATF2, a saturation effect is observed with reduced expression at high doses, especially for Fe nuclei, with much slower characteristic repair times (>7 h) compared to X-rays. RBEs for pATF2 were 0.66 {+-} 0.13 and 1.66 {+-} 0.46 at 2 h and 24 h, respectively. Significant differences in {gamma}H2AX and pATF2 levels comparing irradiated samples to control were noted even at the lowest dose analyzed (0.05 Gy) using these methods of analysis. These results reveal that mathematical models can be applied to flow cytometry data to uncover important and subtle differences following exposure to various qualities of low dose radiation.

  19. 53BP1 facilitates long-range DNA end-joining during V(D)J recombination.

    Science.gov (United States)

    Difilippantonio, Simone; Gapud, Eric; Wong, Nancy; Huang, Ching-Yu; Mahowald, Grace; Chen, Hua Tang; Kruhlak, Michael J; Callen, Elsa; Livak, Ferenc; Nussenzweig, Michel C; Sleckman, Barry P; Nussenzweig, André

    2008-11-27

    Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.

  20. Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

    Science.gov (United States)

    Mata-Garrido, Jorge; Casafont, Iñigo; Tapia, Olga; Berciano, Maria T; Lafarga, Miguel

    2016-04-22

    There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of

  1. Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

    Science.gov (United States)

    Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

    2018-01-05

    The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

  2. MRE11-RAD50-NBS1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    OpenAIRE

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Kawata, Tetsuya; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Nishikawa, Ryo; Shigematsu, Naoyuki

    2014-01-01

    BACKGROUND: (blind field) METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylationH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cel...

  3. Mobile phone radiofrequency exposure has no effect on DNA double strand breaks (DSB) in human lymphocytes.

    Science.gov (United States)

    Danese, Elisa; Lippi, Giuseppe; Buonocore, Ruggero; Benati, Marco; Bovo, Chiara; Bonaguri, Chiara; Salvagno, Gian Luca; Brocco, Giorgio; Roggenbuck, Dirk; Montagnana, Martina

    2017-07-01

    The use of mobile phones has been associated with an increased risk of developing certain type of cancer, especially in long term users. Therefore, this study was aimed to investigate the potential genotoxic effect of mobile phone radiofrequency exposure on human peripheral blood mononuclear cells in vitro. The study population consisted in 14 healthy volunteers. After collection of two whole blood samples, the former was placed in a plastic rack, 1 cm from the chassis of a commercial mobile phone (900 MHz carrier frequency), which was activated by a 30-min call. The second blood sample was instead maintained far from mobile phones or other RF sources. The influence of mobile phone RF on DNA integrity was assessed by analyzing γ-H2AX foci in lymphocytes using immunofluorescence staining kit on AKLIDES. No measure of γ-H2AX foci was significantly influenced by mobile phone RF exposure, nor mobile phone exposure was associated with significant risk of genetic damages in vitro (odds ratio comprised between 0.27 and 1.00). The results of this experimental study demonstrate that exposure of human lymphocytes to a conventional 900 MHz RF emitted by a commercial mobile phone for 30 min does not significantly impact DNA integrity.

  4. Prostate cancer stem-like cells proliferate slowly and resist etoposide-induced cytotoxicity via enhancing DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Judy [Division of Nephrology, Department of Medicine, McMaster University, Juravinski Innovation Tower, Room T3310, St. Joseph' s Hospital, 50 Charlton Ave East, Hamilton, Ontario, Canada L8S 4L8 (Canada); Father Sean O' Sullivan Research Institute, Hamilton, Ontario, Canada L8N 4A6 (Canada); The Hamilton Centre for Kidney Research (HCKR), St. Joseph' s Hamilton Healthcare, Hamilton, Ontario, Canada L8N 4A6 (Canada); Tang, Damu, E-mail: damut@mcmaster.ca [Division of Nephrology, Department of Medicine, McMaster University, Juravinski Innovation Tower, Room T3310, St. Joseph' s Hospital, 50 Charlton Ave East, Hamilton, Ontario, Canada L8S 4L8 (Canada); Father Sean O' Sullivan Research Institute, Hamilton, Ontario, Canada L8N 4A6 (Canada); The Hamilton Centre for Kidney Research (HCKR), St. Joseph' s Hamilton Healthcare, Hamilton, Ontario, Canada L8N 4A6 (Canada)

    2014-10-15

    Despite the development of chemoresistance as a major concern in prostate cancer therapy, the underlying mechanisms remain elusive. In this report, we demonstrate that DU145-derived prostate cancer stem cells (PCSCs) progress slowly with more cells accumulating in the G1 phase in comparison to DU145 non-PCSCs. Consistent with the important role of the AKT pathway in promoting G1 progression, DU145 PCSCs were less sensitive to growth factor-induced activation of AKT in comparison to non-PCSCs. In response to etoposide (one of the most commonly used chemotherapeutic drugs), DU145 PCSCs survived significantly better than non-PCSCs. In addition to etoposide, PCSCs demonstrated increased resistance to docetaxel, a taxane drug that is commonly used to treat castration-resistant prostate cancer. Etoposide produced elevated levels of γH2AX and triggered a robust G2/M arrest along with a coordinated reduction of the G1 population in PCSCs compared to non-PCSCs, suggesting that elevated γH2AX plays a role in the resistance of PCSCs to etoposide-induced cytotoxicity. We have generated xenograft tumors from DU145 PCSCs and non-PCSCs. Consistent with the knowledge that PCSCs produce xenograft tumors with more advanced features, we were able to demonstrate that PCSC-derived xenograft tumors displayed higher levels of γH2AX and p-CHK1 compared to non-PCSC-produced xenograft tumors. Collectively, our research suggests that the elevation of DNA damage response contributes to PCSC-associated resistance to genotoxic reagents. - Highlights: • Increased survival in DU145 PCSCs following etoposide-induced cytotoxicity. • PCSCs exhibit increased sensitivity to etoposide-induced DDR. • Resistance to cytotoxicity may be due to slower proliferation in PCSCs. • Reduced kinetics to growth factor induced activation of AKT in PCSCs.

  5. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

    Science.gov (United States)

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

    2017-11-03

    We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated with pathogenic mitochondrial DNA mutations affecting complexes I and III.

    Science.gov (United States)

    Bonora, Elena; Porcelli, Anna Maria; Gasparre, Giuseppe; Biondi, Annalisa; Ghelli, Anna; Carelli, Valerio; Baracca, Alessandra; Tallini, Giovanni; Martinuzzi, Andrea; Lenaz, Giorgio; Rugolo, Michela; Romeo, Giovanni

    2006-06-15

    Oncocytic tumors are characterized by cells with an aberrant accumulation of mitochondria. To assess mitochondrial function in neoplastic oncocytic cells, we studied the thyroid oncocytic cell line XTC.UC1 and compared it with other thyroid non-oncocytic cell lines. Only XTC.UC1 cells were unable to survive in galactose, a condition forcing cells to rely solely on mitochondria for energy production. The rate of respiration and mitochondrial ATP synthesis driven by complex I substrates was severely reduced in XTC.UC1 cells. Furthermore, the enzymatic activity of complexes I and III was dramatically decreased in these cells compared with controls, in conjunction with a strongly enhanced production of reactive oxygen species. Osteosarcoma-derived transmitochondrial cell hybrids (cybrids) carrying XTC.UC1 mitochondrial DNA (mtDNA) were generated to discriminate whether the energetic failure depended on mitochondrial or nuclear DNA mutations. In galactose medium, XTC.UC1 cybrid clones showed reduced viability and ATP content, similarly to the parental XTC.UC1, clearly pointing to the existence of mtDNA alterations. Sequencing of XTC.UC1 mtDNA identified a frameshift mutation in ND1 and a nonconservative substitution in cytochrome b, two mutations with a clear pathogenic potential. In conclusion, this is the first demonstration that mitochondrial dysfunction of XTC.UC1 is due to a combined complex I/III defect associated with mtDNA mutations, as proven by the transfer of the defective energetic phenotype with the mitochondrial genome into the cybrids.

  7. Analysis of flow cytometry DNA damage response protein activation kinetics after exposure to x rays and high-energy iron nuclei.

    Science.gov (United States)

    Chappell, Lori J; Whalen, Mary K; Gurai, Sheena; Ponomarev, Artem; Cucinotta, Francis A; Pluth, Janice M

    2010-12-01

    We developed a mathematical method to analyze flow cytometry data to describe the kinetics of γ-H2AX and pATF2 phosphorylation in normal human fibroblast cells after exposure to various qualities of low-dose radiation. Previously reported flow cytometry kinetics for these DSB repair phospho-proteins revealed that distributions of intensity were highly skewed, severely limiting the detection of differences in the very low-dose range. Distributional analysis revealed significant differences between control and low-dose samples when distributions were compared using the Kolmogorov-Smirnov test. Differences in radiation quality were found in the distribution shapes and when a nonlinear model was used to relate dose and time to the decay of the mean ratio of phospho-protein intensities of irradiated samples to controls. We analyzed cell cycle phase- and radiation quality-dependent characteristic repair times and residual phospho-protein levels with these methods. Characteristic repair times for γ-H2AX were higher after exposure to iron nuclei compared to X rays in G(1) cells and in S/G(2) cells. The RBE in G(1) cells for iron nuclei relative to X rays for γ-H2AX was 2.1 ± 0.6 and 5.0 ± 3.5 at 2 and 24 h after irradiation, respectively. For pATF2, a saturation effect was observed with reduced expression at high doses, especially for iron nuclei, with much slower characteristic repair times (>7 h) compared to X rays. RBEs for pATF2 were 0.7 ± 0.1 and 1.7 ± 0.5 at 2 and 24 h, respectively. Significant differences in γ-H2AX and pATF2 levels when irradiated samples were compared to controls were noted even at the lowest dose analyzed (0.05 Gy). These results show that mathematical models can be applied to flow cytometry data to identify important and subtle differences after exposure to various qualities of low-dose radiation.

  8. Inhibition of DNA Binding by the Phosphorylation of Poly ADP-Ribose Polymerase Protein Catalyzed by Protein Kinase C

    Science.gov (United States)

    1993-04-21

    glycohydrolase and ADP-ribose polymerase (3). Besides enzymatic activities, ADPRT possesses significant colligative properties towards DNA termini and certain...differentiation of particular cell types (3). The biochemical role of ADPRT in living cells in most probably related to both catalytic and colligative properties

  9. A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

    Directory of Open Access Journals (Sweden)

    Stougaard Magnus

    2007-08-01

    Full Text Available Abstract Background The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes. Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules. Results Chemically synthesized oligonucleotides with hairpin structures were acquired from our standard supplier. The stem of the hairpin contained recognition sequences for the Nt. Alw I nicking enzyme and the Mly I restriction enzyme. These double stranded regions were positioned in a way to allow self-templated circularization of the oligonucleotide. Following ligation, tandem repeats of the complementary sequence of the circular oligonucleotide could be produced through rolling circle DNA synthesis. By running successive rounds of ligation, rolling circle DNA synthesis, and nicking, the original oligonucleotide could be amplified as either the (+-strand or the (--strand. Alternatively, the hairpin structure could be removed by cleavage with the Mly I restriction enzyme, thereby releasing the oligonucleotide sequence contained within the hairpin structure from the hairpin. Conclusion We present here a method for the enzymatic production through DNA amplification of oligonucleotides with freely designable 5'-ends and 3'-ends, using hairpin-containing self-templating oligonucleotides. The hairpin comprises recognition sequences for a nicking enzyme and a restriction enzyme. The oligonucleotides are amplified by successive rounds of ligation, rolling circle DNA synthesis and nicking. Furthermore, the

  10. Cigarette toxicity triggers Leber's hereditary optic neuropathy by affecting mtDNA copy number, oxidative phosphorylation and ROS detoxification pathways

    Science.gov (United States)

    Giordano, L; Deceglie, S; d'Adamo, P; Valentino, M L; La Morgia, C; Fracasso, F; Roberti, M; Cappellari, M; Petrosillo, G; Ciaravolo, S; Parente, D; Giordano, C; Maresca, A; Iommarini, L; Del Dotto, V; Ghelli, A M; Salomao, S R; Berezovsky, A; Belfort, R; Sadun, A A; Carelli, V; Loguercio Polosa, P; Cantatore, P

    2015-01-01

    Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disease, is associated with mitochondrial DNA (mtDNA) point mutations affecting Complex I subunits, usually homoplasmic. This blinding disorder is characterized by incomplete penetrance, possibly related to several genetic modifying factors. We recently reported that increased mitochondrial biogenesis in unaffected mutation carriers is a compensatory mechanism, which reduces penetrance. Also, environmental factors such as cigarette smoking have been implicated as disease triggers. To investigate this issue further, we first assessed the relationship between cigarette smoke and mtDNA copy number in blood cells from large cohorts of LHON families, finding that smoking was significantly associated with the lowest mtDNA content in affected individuals. To unwrap the mechanism of tobacco toxicity in LHON, we exposed fibroblasts from affected individuals, unaffected mutation carriers and controls to cigarette smoke condensate (CSC). CSC decreased mtDNA copy number in all cells; moreover, it caused significant reduction of ATP level only in mutated cells including carriers. This implies that the bioenergetic compensation in carriers is hampered by exposure to smoke derivatives. We also observed that in untreated cells the level of carbonylated proteins was highest in affected individuals, whereas the level of several detoxifying enzymes was highest in carriers. Thus, carriers are particularly successful in reactive oxygen species (ROS) scavenging capacity. After CSC exposure, the amount of detoxifying enzymes increased in all cells, but carbonylated proteins increased only in LHON mutant cells, mostly from affected individuals. All considered, it appears that exposure to smoke derivatives has a more deleterious effect in affected individuals, whereas carriers are the most efficient in mitigating ROS rather than recovering bioenergetics. Therefore, the identification of genetic modifiers that

  11. Radiation-induced DNA Double Strand Breaks and Their Modulations by Treatments with Moringa oleifera Lam. Leaf Extracts: A Cancer Cell Culture Model

    Directory of Open Access Journals (Sweden)

    K. Boonsirichai

    2014-04-01

    Full Text Available Gamma radiation brings deleterious effects upon human cells by inducing oxidative stress and DNA damages. Antioxidants have been shown to confer protective effects on irradiated normal cells. Moringa oleifera Lam. is a widely used nutritional supplement with antioxidant activities. This report showed that antioxidant-containing supplements, in addition to protecting normal cells, could protect cancer cells against genotoxic effects of gamma radiation. -H2AX immunofluorescent foci were utilized as an indicator of radiation-induced DNA double strand breaks. MCF-7 human breast adenocarcinoma cells were irradiated with 2-8 Gy gamma radiation. A linear relationship between the formation of -H2AX foci and radiation dose was observed with an average of 10 foci per cell per Gy. A 30-minute pretreatment of the cells with either the aqueous or the ethanolic extract of M. oleifera leaves could partially protect the cells from radiation-induced DNA double strand breaks. A pretreatment with 500 µg/mL aqueous extract reduced the number of foci formed by 15% when assayed at 30 minutes post-irradiation. The ethanolic extract was more effective; 500 µg/mL of its concentration reduced the number of foci among irradiated cells by 30%. The results indicated that irradiated cancer cells responded similarly to nutritional supplements containing antioxidants as irradiated normal cells. These natural antioxidants could confer protective effects upon cancer cells against gamma radiation

  12. Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

    Science.gov (United States)

    Conic, Sascha; Desplancq, Dominique; Ferrand, Alexia; Fischer, Veronique; Heyer, Vincent; Reina San Martin, Bernardo; Pontabry, Julien; Oulad-Abdelghani, Mustapha; Babu N, Kishore; Wright, Graham D; Molina, Nacho; Weiss, Etienne; Tora, László

    2018-02-12

    Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells. © 2018 Conic et al.

  13. DNA-PK-mediated phosphorylation of EZH2 regulates the DNA damage-induced apoptosis to maintain T-cell genomic integrity

    OpenAIRE

    Wang, Y.; Sun, H.; Wang, J; Wang, H; L. Meng; Xu, C; Jin, M; Wang, B.; Zhang, Y.; Zhu, T.

    2016-01-01

    EZH2 is a histone methyltransferase whose functions in stem cells and tumor cells are well established. Accumulating evidence shows that EZH2 has critical roles in T cells and could be a promising therapeutic target for several immune diseases. To further reveal the novel functions of EZH2 in human T cells, protein co-immunoprecipitation combined mass spectrometry was conducted and several previous unknown EZH2-interacting proteins were identified. Of them, we focused on a DNA damage responsi...

  14. NPRL2 sensitizes human non-small cell lung cancer (NSCLC cells to cisplatin treatment by regulating key components in the DNA repair pathway.

    Directory of Open Access Journals (Sweden)

    Gitanjali Jayachandran

    2010-08-01

    Full Text Available NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2 yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM kinase activated by cisplatin and promote downstream gamma-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular-weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy.

  15. NPRL2 Sensitizes Human Non-Small Cell Lung Cancer (NSCLC) Cells to Cisplatin Treatment by Regulating Key Components in the DNA Repair Pathway

    Science.gov (United States)

    Wang, Bingbing; Roth, Jack A.; Ji, Lin

    2010-01-01

    NPRL2, one of the tumor suppressor genes residing in a 120-kb homozygous deletion region of human chromosome band 3p21.3, has a high degree of amino acid sequence homology with the nitrogen permease regulator 2 (NPR2) yeast gene, and mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Previously, we showed that restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment in vitro and in vivo. In this study, we show that sensitization of non-small cell lung cancer (NSCLC) cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway. NPRL2 can phosphorylate ataxia telangiectasia mutated (ATM) kinase activated by cisplatin and promote downstream γ-H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular-weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Activated Chk1 and Chk2 increase the expression of cell cycle checkpoint proteins, including Cdc25A and Cdc25C, leading to higher levels of G2/M arrest in tumor cells treated with NPRL2 and cisplatin than in tumor cells treated with cisplatin only. Our results therefore suggest that ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2/M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy. PMID:20700484

  16. The involvement of human RECQL4 in DNA double-strand break repair

    DEFF Research Database (Denmark)

    Singh, Dharmendra Kumar; Karmakar, Parimal; Aamann, Maria Diget

    2010-01-01

    Rothmund-Thomson syndrome (RTS) is an autosomal recessive hereditary disorder associated with mutation in RECQL4 gene, a member of the human RecQ helicases. The disease is characterized by genomic instability, skeletal abnormalities and predisposition to malignant tumors, especially osteosarcomas....... The precise role of RECQL4 in cellular pathways is largely unknown; however, recent evidence suggests its involvement in multiple DNA metabolic pathways. This study investigates the roles of RECQL4 in DNA double-strand break (DSB) repair. The results show that RECQL4-deficient fibroblasts are moderately......-induced DSBs and remains for a shorter duration than WRN and BLM, indicating its distinct role in repair of DSBs. Endogenous RECQL4 also colocalizes with gammaH2AX at the site of DSBs. The RECQL4 domain responsible for its DNA damage localization has been mapped to the unique N-terminus domain between amino...

  17. Sp1 phosphorylation by cyclin-dependent kinase 1/cyclin B1 represses its DNA-binding activity during mitosis in cancer cells.

    Science.gov (United States)

    Chuang, J-Y; Wang, S-A; Yang, W-B; Yang, H-C; Hung, C-Y; Su, T-P; Chang, W-C; Hung, J-J

    2012-11-22

    Sp1 is important for the transcription of many genes. Our previous studies have shown that Sp1 is degraded in normal cell, but it is preserved in cancer cells during mitosis and exists a priori in the daughter cells, ready to engage in gene transcription and thereby contributes to the proliferation and survival of cancer cells. The mechanism by which Sp1 is preserved in cancer cells during mitosis remains unknown. In this study, we observed that Sp1 strongly colocalized with cyclin-dependent kinase 1 (CDK1)/cyclin B1 during mitosis. Moreover, we showed that Sp1 is a novel mitotic substrate of CDK1/cyclin B1 and is phosphorylated by it at Thr 739 before the onset of mitosis. Phospho-Sp1 reduced its DNA-binding ability and facilitated the chromatin condensation process during mitosis. Mutation of Thr739 to alanine resulted in Sp1 remaining in the chromosomes, delayed cell-cycle progression, and eventually led to apoptosis. Screening of Sp1-associated proteins during mitosis by using liquid chromatography/mass spectrometry indicated the tethering of Sp1 to myosin/F-actin. Furthermore, phospho-Sp1 and myosin/F-actin appeared to exist as a congregated ring at the periphery of the chromosome. However, at the end of mitosis and the beginning of interphase, Sp1 was dephosphorylated by PP2A and returned to the chromatin. These results indicate that cancer cells use CDK1 and PP2A to regulate the movement of Sp1 in and out of the chromosomes during cell-cycle progression, which may benefit cancer-cell proliferation.

  18. The Quiescent Cellular State is Arf/p53-Dependent and Associated with H2AX Downregulation and Genome Stability

    Directory of Open Access Journals (Sweden)

    Mitsuko Masutani

    2012-05-01

    Full Text Available Cancer is a disease associated with genomic instability and mutations. Excluding some tumors with specific chromosomal translocations, most cancers that develop at an advanced age are characterized by either chromosomal or microsatellite instability. However, it is still unclear how genomic instability and mutations are generated during the process of cellular transformation and how the development of genomic instability contributes to cellular transformation. Recent studies of cellular regulation and tetraploidy development have provided insights into the factors triggering cellular transformation and the regulatory mechanisms that protect chromosomes from genomic instability.

  19. MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden); Glas, Rickard, E-mail: rickard.glas@ki.se [Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet, Karolinska University Hospital, 171 76 Stockholm (Sweden)

    2010-08-27

    Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.

  20. Replication stress, DNA damage signalling, and cytomegalovirus infection in human medulloblastomas

    DEFF Research Database (Denmark)

    Bartek, Jiri; Fornara, Olesja; Merchut-Maya, Joanna Maria

    2017-01-01

    of the clinical specimens also showed expression of HCMV immediate early and late proteins, in comparative analyses using three immunohistochemical protocols. Cell culture experiments validated the chronic endogenous replication stress in medulloblastoma cell lines and showed sharply differential, intriguing...... eight established immunohistochemical markers to assess the status of the DDR machinery, we found pronounced endogenous DNA damage signalling (γH2AX marker) and robust constitutive activation of both the ATM-Chk2 and ATR-Chk1 DNA damage checkpoint kinase cascades, yet unexpectedly modest p53 tumour...... responses of normal cells and medulloblastoma cells to HCMV infection, including differential subcellular mislocalization and enhancement of replication stress-related 53BP1 body formation, the latter in cell-non-autonomous manner. Overall, our results strongly indicate that in human medulloblastomas...

  1. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage.

    Directory of Open Access Journals (Sweden)

    María-Alexandra Cañas

    Full Text Available Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether

  2. Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43*

    Science.gov (United States)

    Nonaka, Takashi; Suzuki, Genjiro; Tanaka, Yoshinori; Kametani, Fuyuki; Hirai, Shinobu; Okado, Haruo; Miyashita, Tomoyuki; Saitoe, Minoru; Akiyama, Haruhiko; Masai, Hisao; Hasegawa, Masato

    2016-01-01

    Intracellular aggregates of phosphorylated TDP-43 are a major component of ubiquitin-positive inclusions in the brains of patients with frontotemporal lobar degeneration and ALS and are considered a pathological hallmark. Here, to gain insight into the mechanism of intracellular TDP-43 accumulation, we examined the relationship between phosphorylation and aggregation of TDP-43. We found that expression of a hyperactive form of casein kinase 1 δ (CK1δ1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 (ubiquitin- and p62-positive) in cultured neuroblastoma SH-SY5Y cells. Insoluble phosphorylated TDP-43 prepared from cells co-expressing TDP-43 and CK1δ1-317 functioned as seeds for TDP-43 aggregation in cultured cells, indicating that CK1δ1-317-induced aggregated TDP-43 has prion-like properties. A striking toxicity and alterations of TDP-43 were also observed in yeast expressing TDP-43 and CK1δ1-317. Therefore, abnormal activation of CK1δ causes phosphorylation of TDP-43, leading to the formation of cytoplasmic TDP-43 aggregates, which, in turn, may trigger neurodegeneration. PMID:26769969

  3. A small-molecule inhibitor of human DNA polymerase eta potentiates the effects of cisplatin in tumor cells.

    Science.gov (United States)

    Zafar, Maroof K; Maddukuri, Leena; Ketkar, Amit; Penthala, Narsimha; Reed, Megan R; Eddy, Sarah D; Crooks, Peter A; Eoff, Robert L

    2018-01-18

    Translesion DNA synthesis (TLS) performed by human DNA polymerase eta (hpol η) allows tolerance of damage from cis-diamminedichloroplatinum(II) (CDDP or cisplatin). We have developed hpol η inhibitors derived from N-aryl-substituted indole barbituric acid (IBA), indole thiobarbituric acid (ITBA), and indole quinuclidine scaffolds and identified 5-((5-chloro-1-(naphthalen-2-ylmethyl)-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (PNR-7-02), an ITBA derivative that inhibited hpol η activity with an IC50 value of 8 μM and exhibited five- to ten-fold specificity for hpol η over replicative pols. We conclude from kinetic analyses, chemical footprinting assays, and molecular docking that PNR-7-02 binds to a site on the little finger domain and interferes with the proper orientation of template DNA to inhibit hpol η. A synergistic increase in CDDP toxicity was observed in hpol η-proficient cells co-treated with PNR-7-02 (combination index values = 0.4-0.6). Increased γH2AX formation accompanied treatment of hpol η-proficient cells with CDDP and PNR-7-02, indicative of increased DNA damage from CDDP through inhibition of hpol η TLS activity. Importantly, PNR-7-02 did not impact the effect of CDDP on cell viability or γH2AX in hpol η-deficient cells. In summary, we observed hpol η-dependent effects on DNA damage/replication stress and sensitivity to CDDP in cells treated with PNR-7-02. The ability to employ a small-molecule inhibitor of hpol η to improve the cytotoxic effect of CDDP may aid in the development of more effective chemotherapeutic strategies.

  4. DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells.

    LENUS (Irish Health Repository)

    Martin, Lynn M

    2013-07-10

    DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2 Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2 h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24 h after 0.2 Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.

  5. Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

    Science.gov (United States)

    Wu, Yuliang; Sommers, Joshua A.; Suhasini, Avvaru N.; Leonard, Thomas; Deakyne, Julianna S.; Mazin, Alexander V.; Shin-ya, Kazuo; Kitao, Hiroyuki

    2010-01-01

    Fanconi anemia (FA) is a genetic disease characterized by congenital abnormalities, bone marrow failure, and susceptibility to leukemia and other cancers. FANCJ, one of 13 genes linked to FA, encodes a DNA helicase proposed to operate in homologous recombination repair and replicational stress response. The pathogenic FANCJ-A349P amino acid substitution resides immediately adjacent to a highly conserved cysteine of the iron-sulfur domain. Given the genetic linkage of the FANCJ-A349P allele to FA, we investigated the effect of this particular mutation on the biochemical and cellular functions of the FANCJ protein. Purified recombinant FANCJ-A349P protein had reduced iron and was defective in coupling adenosine triphosphate (ATP) hydrolysis and translocase activity to unwinding forked duplex or G-quadruplex DNA substrates or disrupting protein-DNA complexes. The FANCJ-A349P allele failed to rescue cisplatin or telomestatin sensitivity of a FA-J null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, expression of FANCJ-A349P in a wild-type background exerted a dominant-negative effect, indicating that the mutant protein interferes with normal DNA metabolism. The ability of FANCJ to use the energy from ATP hydrolysis to produce the force required to unwind DNA or destabilize protein bound to DNA is required for its role in DNA repair. PMID:20639400

  6. Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

    Science.gov (United States)

    Ershova, E S; Sergeeva, V A; Chausheva, A I; Zheglo, D G; Nikitina, V A; Smirnova, T D; Kameneva, L V; Porokhovnik, L N; Kutsev, S I; Troshin, P A; Voronov, I I; Khakina, E A; Veiko, N N; Kostyuk, S V

    2016-07-01

    Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation

    Science.gov (United States)

    Mittra, Indraneel; Samant, Urmila; Sharma, Suvarna; Raghuram, Gorantla V; Saha, Tannistha; Tidke, Pritishkumar; Pancholi, Namrata; Gupta, Deepika; Prasannan, Preeti; Gaikwad, Ashwini; Gardi, Nilesh; Chaubal, Rohan; Upadhyay, Pawan; Pal, Kavita; Rane, Bhagyeshri; Shaikh, Alfina; Salunkhe, Sameer; Dutt, Shilpee; Mishra, Pradyumna K; Khare, Naveen K; Nair, Naveen K; Dutt, Amit

    2017-01-01

    Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities. PMID:28580170

  8. Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation.

    Science.gov (United States)

    Mittra, Indraneel; Samant, Urmila; Sharma, Suvarna; Raghuram, Gorantla V; Saha, Tannistha; Tidke, Pritishkumar; Pancholi, Namrata; Gupta, Deepika; Prasannan, Preeti; Gaikwad, Ashwini; Gardi, Nilesh; Chaubal, Rohan; Upadhyay, Pawan; Pal, Kavita; Rane, Bhagyeshri; Shaikh, Alfina; Salunkhe, Sameer; Dutt, Shilpee; Mishra, Pradyumna K; Khare, Naveen K; Nair, Naveen K; Dutt, Amit

    2017-01-01

    Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell-cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities.

  9. Mouse zygotes respond to severe sperm DNA damage by delaying paternal DNA replication and embryonic development.

    Directory of Open Access Journals (Sweden)

    Joanna E Gawecka

    Full Text Available Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF. SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B followed by irreversible DNA degradation by a nuclease(s. Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.

  10. Relative biological effectiveness of high linear energy transfer α-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells.

    Science.gov (United States)

    Franken, Nicolaas A P; Hovingh, Suzanne; Ten Cate, Rosemarie; Krawczyk, Przemek; Stap, Jan; Hoebe, Ron; Aten, Jacob; Barendsen, Gerrit W

    2012-03-01

    Ionizing radiation-induced foci (IRIF) of DNA repair-related proteins accumulated at DNA double-strand break (DSB) sites have been suggested to be a powerful biodosimetric tool. However, the relationship between IRIF induction and biologically relevant endpoints, such as cell death and formation of chromosome rearrangements is less clear, especially for high linear energy transfer (LET) radiation. It is thus not sufficiently established whether IRIF are valid indicators of biological effectiveness of the various radiation types. This question is more significant in light of the recent advancements in light ion-beam and radionuclide therapy. Dose-effect relationships were determined for the induction of DNA-DSBs, chromosome aberrations and reproductive cell death in cultured SW-1573 cells irradiated with γ-rays from a Cs-137 source or with α-particles from an Am-241 source. Values of relative biological effectiveness (RBE) of the high LET α-particles were derived for these effects. DNA-DSB were detected by scoring of γ-H2AX foci, chromosome aberrations by fragments and translocations using premature chromosome condensation and cell survival by colony formation. Analysis of dose-effect relations was based on the linear-quadratic model. Except for the survival curves, for other effects no significant contribution was derived of the quadratic term in the range of doses up to 2 Gy of γ-rays. Calculated RBE values derived for the linear component of dose-effect relations for γ-H2AX foci, cell reproductive death, chromosome fragments and colour junctions are 1.0±0.3, 14.7±5.1, 15.3±5.9 and 13.3±6.0, respectively. RBE values calculated at a certain biological effect level are 1, 4, 13 and 13, respectively. The RBE values derived from the LQ model are preferred as they are based on clinically relevant doses. The results show that with low LET radiation only a small fraction of the numerous DNA-DSBs yield chromosome damage and reproductive cell death. It is

  11. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Sun Xiaonan [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Yang Chunying [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Liu Hai; Wang Qi [Department of Radiation Oncology, Sir Run Run Shaw Hospital, Sir Run Run Shaw Institute of Clinical Medicine of Zhejiang University, Hangzhou (China); Wu Shixiu [Department of Radiation Oncology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou (China); Li Xia; Xie Tian [Research Center of Biomedicine and Health, Hangzhou Normal University, Hangzhou (China); Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Xu Bo, E-mail: bxu@tmhs.org [Department of Radiation Oncology, Methodist Hospital Research Institute, Weill Cornell Medical College, Houston, TX (United States); Zheng, Shu, E-mail: zhengshu@zju.edu.cn [Cancer Institute, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou (China)

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  12. Signalization and repair of the DNA double-strand breaks of in the cerebral tumors: modulation of the radiation response with the chemotherapy treatments; Signalization et reparation des cassures double-brin de l'ADN dans les gliomes: modulation de la reponse aux traitements chimio-radiotherapeutiques

    Energy Technology Data Exchange (ETDEWEB)

    Marcinkova-Bencokova, Z

    2007-07-15

    There are about 6000 new cases of nervous system tumours each year in France. However, the current radio chemotherapeutic approaches against brain tumours remain still insufficient to produce a satisfactory therapeutic index. In parallel, the knowledge of the early radiobiological events has considerably progressed in the last few years. This thesis aims to provide new insights in the molecular and cellular response of brain tumours to radio chemotherapy. This thesis was divided into four stages. Stage 1: a novel DNA double-strand breaks repair pathway depending on the MRE11 protein but independent of the phosphorylation of H2AX emerged from the study of artefacts of the immunofluorescence technique and a systematic analysis of the radiosensitivity of human cells. Stage 2: the radiobiological features of 3 rodent models of glioma among the most used in preclinical trials and of 7 human glioma cell lines were investigated. Functional impairments of the BRCA1 protein in response to radiation and/or cisplatin were observed in the majority of the models tested, raising the question of the role of this protein in the anti-glioma treatments and in glioma genesis. Stage 3: in order to extend our approach to genetic syndromes associated with cerebral tumours predisposition, the radiobiological characteristics of the fibroblasts resulting from patients suffering from neurofibromatosis type 1 (NF1), a pathology associated with a strong incidence of peripheral nervous system tumours, were investigated. NF1 appeared to be a syndrome with moderated radiosensitivity, associated with a weak deficiency of DNA end-joining repair but with a strong activity of MRE11. These results enabled us to propose a preliminary model involving both proteins BRCA1 and NF1. Stage 4: considering the role of BRCA1 in the inhibition of some tyrosine kinase activity and in the response to cisplatin, we tested the radiobiological effects of treatments combining radiation, cisplatin and tyrosine kinase

  13. Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches.

    Directory of Open Access Journals (Sweden)

    Alba Martínez

    Full Text Available Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D form of eIF4E, but not phospho-dead (S209A eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin, starvation (glucose+glutamine withdrawal, and oxidative stress (arsenite. De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1. We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.

  14. RF-EMF exposure at 1800 MHz did not elicit DNA damage or abnormal cellular behaviors in different neurogenic cells.

    Science.gov (United States)

    Su, Liling; Wei, Xiaoxia; Xu, Zhengping; Chen, Guangdi

    2017-04-01

    Despite many years of studies, the debate on genotoxic effects of radiofrequency electromagnetic fields (RF-EMF) continues. To systematically evaluate genotoxicity of RF-EMF, this study examined effects of RF-EMF on DNA damage and cellular behavior in different neurogenic cells. Neurogenic A172, U251, and SH-SY5Y cells were intermittently (5 min on/10 min off) exposed to 1800 MHz RF-EMF at an average specific absorption rate (SAR) of 4.0 W/kg for 1, 6, or 24 h. DNA damage was evaluated by quantification of γH2AX foci, an early marker of DNA double-strand breaks. Cell cycle progression, cell proliferation, and cell viability were examined by flow cytometry, hemocytometer, and cell counting kit-8 assay, respectively. Results showed that exposure to RF-EMF at an SAR of 4.0 W/kg neither significantly induced γH2AX foci formation in A172, U251, or SH-SY5Y cells, nor resulted in abnormal cell cycle progression, cell proliferation, or cell viability. Furthermore, prolonged incubation of these cells for up to 48 h after exposure did not significantly affect cellular behavior. Our data suggest that 1800 MHz RF-EMF exposure at 4.0 W/kg is unlikely to elicit DNA damage or abnormal cellular behaviors in neurogenic cells. Bioelectromagnetics. 38:175-185, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Influence of different iodinated contrast media on the induction of DNA double-strand breaks after in vitro X-ray irradiation.

    Science.gov (United States)

    Deinzer, Christoph K W; Danova, Daniela; Kleb, Beate; Klose, Klaus J; Heverhagen, Johannes T

    2014-01-01

    The objective of this work was to examine differences in DNA double-strand break induction in peripheral blood lymphocytes after in vitro X-ray irradiation between iodinated contrast agents. Four different iodinated X-ray contrast agents--three of them with two different iodine concentrations--and mannitol (negative control; concentration of 150 mg mannitol per ml blood) were pipetted into blood samples so that there was a concentration of 0, 7.5 or 15 mg of iodine per ml blood in the samples. Negative controls without contrast medium (0 mg of iodine per ml blood) were also processed for every irradiation dose. The tubes were exposed to 0, 20 or 500 mGy in vitro X-ray irradiation. After that, the lymphocytes were separated by using density-gradient centrifugation. Fluorescence microscopy was applied to determine the average number of γH2AX-foci per lymphocyte in the presence or absence of different contrast media or mannitol. Differences in the number of γH2AX-foci were statistically analysed by one-way ANOVA and post-hoc Tukey's honestly significant difference test. Iodinated contrast agents led to a statistically significant increase in DNA double-strand breaks after in vitro irradiation. This effect increased statistically significant with rising radiation dose and appeared independent of the contrast agent used (iopromid, iodixanol, iomeprol, iopamidol). A statistically significant difference in DNA damage between the different tested contrast agents was not found. Therefore, the increase in DNA double-strand breaks depends solely on the amount of iodine applied. For evaluation of clinical consequences, our findings could be tested in further animal studies. Copyright © 2014 John Wiley & Sons, Ltd.

  16. Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Noda, Taichi [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Takahashi, Akihisa [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Kondo, Natsuko [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Mori, Eiichiro [Department of Biology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Okamoto, Noritomo [Department of Otorhinolaryngology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Nakagawa, Yosuke [Department of Oral and Maxillofacial Surgery, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); Ohnishi, Ken [Department of Biology, Ibaraki Prefectual University of Health Sciences, 4669-2 Ami, Ami-mati, Inasiki-gun, Ibaraki 300-0394 (Japan); Zdzienicka, Malgorzata Z. [Department of Molecular Cell Genetics, Collegium Medicum in Bydgoszcz, Nicolaus-Copernicus-University in Torun, ul. Sklodowskiej-Curie 9, 85-094 Bydgoszcz (Poland); Thompson, Larry H. [Biosciences and Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808 (United States); Helleday, Thomas [Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford, OX3 7DQ (United Kingdom); Department of Genetics, Microbiology and Toxicology Stockholm University, SE-106 91 Stockholm (Sweden); Asada, Hideo [Department of Dermatology, School of Medicine, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521 (Japan); and others

    2011-01-07

    The role of the Fanconi anemia (FA) repair pathway for DNA damage induced by formaldehyde was examined in the work described here. The following cell types were used: mouse embryonic fibroblast cell lines FANCA{sup -/-}, FANCC{sup -/-}, FANCA{sup -/-}C{sup -/-}, FANCD2{sup -/-} and their parental cells, the Chinese hamster cell lines FANCD1 mutant (mt), FANCGmt, their revertant cells, and the corresponding wild-type (wt) cells. Cell survival rates were determined with colony formation assays after formaldehyde treatment. DNA double strand breaks (DSBs) were detected with an immunocytochemical {gamma}H2AX-staining assay. Although the sensitivity of FANCA{sup -/-}, FANCC{sup -/-} and FANCA{sup -/-}C{sup -/-} cells to formaldehyde was comparable to that of proficient cells, FANCD1mt, FANCGmt and FANCD2{sup -/-} cells were more sensitive to formaldehyde than the corresponding proficient cells. It was found that homologous recombination (HR) repair was induced by formaldehyde. In addition, {gamma}H2AX foci in FANCD1mt cells persisted for longer times than in FANCD1wt cells. These findings suggest that formaldehyde-induced DSBs are repaired by HR through the FA repair pathway which is independent of the FA nuclear core complex. -- Research highlights: {yields} We examined to clarify the repair pathways of formaldehyde-induced DNA damage. Formaldehyde induces DNA double strand breaks (DSBs). {yields} DSBs are repaired through the Fanconi anemia (FA) repair pathway. {yields} This pathway is independent of the FA nuclear core complex. {yields} We also found that homologous recombination repair was induced by formaldehyde.

  17. Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2017-02-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 °C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37 °C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  18. Detection of DNA Damage by Space Radiation in Human Fibroblasts Flown on the International Space Station

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; hide

    2017-01-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 degrees Centigrade in space for 14 days before being fixed for analysis of DNA damages with the gamma-H2AX assay. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate gamma rays at 37 degrees Centigrade. Cells exposed to chronic gamma rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET (Linear Energy Transfer) protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  19. Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks

    Directory of Open Access Journals (Sweden)

    Pierre Caron

    2015-11-01

    Full Text Available DNA double-strand breaks (DSBs elicit the so-called DNA damage response (DDR, largely relying on ataxia telangiectasia mutated (ATM and DNA-dependent protein kinase (DNA-PKcs, two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within “repair foci.” Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.

  20. ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair.

    Science.gov (United States)

    Leung, Justin W C; Makharashvili, Nodar; Agarwal, Poonam; Chiu, Li-Ya; Pourpre, Renaud; Cammarata, Michael B; Cannon, Joe R; Sherker, Alana; Durocher, Daniel; Brodbelt, Jennifer S; Paull, Tanya T; Miller, Kyle M

    2017-02-01

    Chromatin connects DNA damage response factors to sites of damaged DNA to promote the signaling and repair of DNA lesions. The histone H2A variants H2AX, H2AZ, and macroH2A represent key chromatin constituents that facilitate DNA repair. Through proteomic screening of these variants, we identified ZMYM3 (zinc finger, myeloproliferative, and mental retardation-type 3) as a chromatin-interacting protein that promotes DNA repair by homologous recombination (HR). ZMYM3 is recruited to DNA double-strand breaks through bivalent interactions with both histone and DNA components of the nucleosome. We show that ZMYM3 links the HR factor BRCA1 to damaged chromatin through specific interactions with components of the BRCA1-A subcomplex, including ABRA1 and RAP80. By regulating ABRA1 recruitment to damaged chromatin, ZMYM3 facilitates the fine-tuning of BRCA1 interactions with DNA damage sites and chromatin. Consistent with a role in regulating BRCA1 function, ZMYM3 deficiency results in impaired HR repair and genome instability. Thus, our work identifies a critical chromatin-binding DNA damage response factor, ZMYM3, which modulates BRCA1 functions within chromatin to ensure the maintenance of genome integrity. © 2017 Leung et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Bottini, N.

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... on protein-tyrosine phosphorylation in this gram-positive model organism. With its two kinases, two kinase modulators, three phosphatases and at least four different tyrosine-phosphorylated substrates, B. subtilis is the bacterium with the highest number of presently known participants in the global network...

  2. Elucidaton of DNA methylation changes in response to ionizng radiation induced double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Herrlitz, Maren Linda

    2014-07-04

    would be an effect of overexpression or be indicative of a possible function in these nuclear subcompartments is yet to be elucidated. Additionally, by using flow cytometry analysis, exposure to IR and concomitant overexpression of TET2CD-GFP strongly induced 5hmC formation, therefore suggesting a function of TET2 in response to irradiation. Recruitment analysis showed that the TET2 catalytic domain was recruited to UV laser-induced but not X-rays- or heavy ion-induced damage sites. Endogenous TET2, which was analyzed in high TET2 expressing human fibroblasts, was recruited to damage sites after irradiation with heavy ions or X-rays. As 5hmC is the direct product of the catalytic activity of TET enzymes, local 5hmC formation and abundance at damage sites was investigated. It was observed that 5hmC accumulated at heavy ion- as well as X-ray-induced DNA double strand breaks (DSBs). In addition, investigating 5hmC foci over time after irradiation with X-rays revealed that 5hmC formation and kinetics is similar to that of γH2AX foci, whereby every 5hmC focus co-localized with γH2AX. However, this did not hold true for all γH2AX foci, whose total number was always higher than that of 5hmC. Furthermore, 5hmC (and γH2AX) foci formation was almost unaffected by the inhibition of DNA-PKcs' enzymatic activity. Conversely, 5hmC and γH2AX foci persistence was significantly delayed after DNA-PKcs inhibition. Results obtained in this thesis show that DNA methylation changes (5hmC formation) take place within the time frame of one replication cycle after exposure to IR and that these changes can be observed at sites of DSBs. 5hmC at DSBs might be formed by the oxidative function of TET2, which was shown to be recruited to DSBs. However, involvement of the other TET enzymes in 5hmC production cannot be excluded. Therefore, these results suggest a role of 5hmC in the response to IR induced DSBs, whereby the here presented data suggest that the fast, radiation induced

  3. Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Dimitra T Stefanou

    Full Text Available Ovarian carcinoma (OC is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780 and one resistant (A2780/C30 to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs from OC patients, sensitive (n = 7 or resistant (n = 4 to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9 were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05. Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03. Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05. We conclude

  4. Histone H3 lysine 9 acetylation pattern suggests that X and B chromosomes are silenced during entire male meiosis in a grasshopper.

    Science.gov (United States)

    Cabrero, J; Teruel, M; Carmona, F D; Jiménez, R; Camacho, J P M

    2007-01-01

    The facultative heterochromatic X chromosome in leptotene spermatocytes of the grasshopper Eyprepocnemis plorans showed marked hypoacetylation for lysine 9 in the H3 histone (H3-K9) with no sign of histone H2AX phosphorylation. Since H3-K9 hypoacetylation precedes the meiotic appearance of phosphorylated H2AX (gamma-H2AX), which marks the beginning of recombinational DNA double-strand breaks (DSBs), it seems that meiotic sex-chromosome inactivation (MSCI) in this grasshopper occurs prior to the beginning of recombination and hence synapsis (which in this species begins later than recombination). In addition, all constitutively heterochromatic chromosome regions harbouring a 180-bp tandem-repeat DNA and rDNA (B chromosomes and pericentromeric regions of A chromosomes) were H3-K9 hypoacetylated at early leptotene even though they will synapse at subsequent stages. This also suggests that meiotic silencing in this grasshopper might be independent of synapsis. The H3-K9 hypoacetylated state of facultative and constitutive heterochromatin persisted during subsequent meiotic stages and was even apparent in round spermatids. Finally, the fact that B chromosomes are differentially hypoacetylated in testis and embryo interphase cells suggests that they might be silenced early in development and remain this way for most (or all) life-cycle stages. Copyright (c) 2007 S. Karger AG, Basel.

  5. Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity

    Science.gov (United States)

    Dickey, Jennifer S.; Baird, Brandon J.; Redon, Christophe E.; Avdoshina, Valeriya; Palchik, Guillermo; Wu, Junfang; Kondratyev, Alexei; Bonner, William M.; Martin, Olga A.

    2012-01-01

    Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo. PMID:22941641

  6. DNA Damage Response Pathway and Replication Fork Stress During Oligonucleotide Directed Gene Editing

    Directory of Open Access Journals (Sweden)

    Melissa Bonner

    2012-01-01

    Full Text Available Single-stranded DNA oligonucleotides (ODNs can be used to direct the exchange of nucleotides in the genome of mammalian cells in a process known as gene editing. Once refined, gene editing should become a viable option for gene therapy and molecular medicine. Gene editing is regulated by a number of DNA recombination and repair pathways whose natural activities often lead to single- and double-stranded DNA breaks. It has been previously shown that introduction of a phosphorotioated ODN, designed to direct a gene-editing event, into cells results in the activation of γH2AX, a well-recognized protein biomarker for double-stranded DNA breakage. Using a single copy, integrated mutant enhanced green fluorescent protein (eGFP gene as our target, we now demonstrate that several types of ODNs, capable of directing gene editing, also activate the DNA damage response and the post-translational modification of proliferating cell nuclear antigen (PCNA, a signature modification of replication stress. We find that the gene editing reaction itself leads to transient DNA breakage, perhaps through replication fork collapse. Unmodified specific ODNs elicit a lesser degree of replication stress than their chemically modified counterparts, but are also less active in gene editing. Modified phosphothioate oligonucleotides (PTOs are detrimental irrespective of the DNA sequence. Such collateral damage may prove problematic for proliferation of human cells genetically modified by gene editing.

  7. Combination treatment of renal cell carcinoma with belinostat and 5-fluorouracil: a role for oxidative stress induced DNA damage and HSP90 regulated thymidine synthase.

    Science.gov (United States)

    Kim, Mi Joung; Lee, Jee Suk; Park, Sang Eun; Yi, Hye-Jin; Jeong, In Gab; Kang, Jong Soon; Yun, Jieun; Lee, Joo-Yong; Ro, Seonggu; Lee, Jung Shin; Choi, Eun Kyung; Hwang, Jung Jin; Kim, Choung-Soo

    2015-05-01

    Despite several therapeutic options renal cell carcinoma is associated with a poor clinical outcome. Therefore, we investigated whether combining 5-fluorouracil with the histone deacetylase inhibitor belinostat would exert a synergistic effect on renal cell carcinoma cells in vitro and in vivo. We used SN12C cells treated with 5-fluorouracil and/or belinostat in vitro and in xenograft experiments in vivo. Cell viability and death mechanisms were assessed by MTS assay and Western blot. To investigate the role of reactive oxygen species we used H2DCF-DA, reactive oxygen species scavengers and the roGFP2 construct. Belinostat potentiated the anticancer effect of 5-fluorouracil. It synergistically induced apoptosis by activating caspases and increasing the subG1 cell population. Effects on reactive oxygen species mediated DNA damage included decreased thioredoxin expression and increased levels of TBP-2, γ-H2AX and Ac-H3. Furthermore, belinostat attenuated the 5-fluorouracil mediated induction of thymidylate synthase via HSP90 hyperacetylation. Co-administration of 5-fluorouracil with belinostat similarly reduced tumor volume and weight, and increased γ-H2AX and Ac-H3 levels in the SN12C xenograft model. In combination with 5-fluorouracil the targeted inhibitor of histone deacetylase synergistically inhibited renal cancer cell growth by the blockade of thymidylate synthase induction and the induction of reactive oxygen species mediated DNA damage in vitro and in vivo. Our results suggest that combined treatment with belinostat and 5-fluorouracil may represent a promising new approach to renal cancer. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  8. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  9. Recruitment of Oct4 protein to UV-damaged chromatin in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Eva Bártová

    Full Text Available Oct4 is a specific marker of embryonic stem cell (ESC pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine.We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm, induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylationH2AX. Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin.Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

  10. Histone phosphorylation: a chromatin modification involved in diverse nuclear events.

    Science.gov (United States)

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-10-01

    Histone posttranslational modifications are key components of diverse processes that modulate chromatin structure. These marks function as signals during various chromatin-based events, and act as platforms for recruitment, assembly or retention of chromatin-associated factors. The best-known function of histone phosphorylation takes place during cellular response to DNA damage, when phosphorylated histone H2A(X) demarcates large chromatin domains around the site of DNA breakage. However, multiple studies have also shown that histone phosphorylation plays crucial roles in chromatin remodeling linked to other nuclear processes. In this review, we summarize the current knowledge of histone phosphorylation and describe the many kinases and phosphatases that regulate it. We discuss the key roles played by this histone mark in DNA repair, transcription and chromatin compaction during cell division and apoptosis. Additionally, we describe the intricate crosstalk that occurs between phosphorylation and other histone modifications and allows for sophisticated control over the chromatin remodeling processes.

  11. The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

    Science.gov (United States)

    Xu, Ye; Sun, Yingli; Jiang, Xiaofeng; Ayrapetov, Marina K.; Moskwa, Patryk; Yang, Shenghong; Weinstock, David M.

    2010-01-01

    The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage. PMID:20876283

  12. Alpha, beta-unsaturated lactones 2-furanone and 2-pyrone induce cellular DNA damage, formation of topoisomerase I- and II-DNA complexes and cancer cell death.

    Science.gov (United States)

    Calderón-Montaño, José Manuel; Burgos-Morón, Estefanía; Orta, Manuel Luis; Pastor, Nuria; Austin, Caroline A; Mateos, Santiago; López-Lázaro, Miguel

    2013-09-12

    The alpha, beta-unsaturated lactones 2-furanone and 2-pyrone are part of the chemical structure of a variety of naturally occurring compounds (e.g., cardenolides, bufadienolides, acetogenins, coumarins, and food-flavoring furanones), some of which have shown anticancer activity and/or DNA damaging effects. Here we report that 2-furanone and 2-pyrone induce cellular DNA damage (assessed by the comet assay and the gamma-H2AX focus assay) and the formation of topoisomerase I- and topoisomerase II-DNA complexes in cells (visualized and quantified in situ by the TARDIS assay). Cells mutated in BRCA2 (deficient in homologous recombination repair) were significantly hypersensitive to the cytotoxic activity of 2-pyrone, therefore suggesting that BRCA2 plays an important role in the repair of DNA damage induced by this lactone. Both lactones were cytotoxic in A549 lung cancer cells at lower concentrations than in MRC5 non-malignant lung fibroblasts. The possible involvement of 2-furanone and 2-pyrone in the anticancer and DNA-damaging activities of compounds containing these lactones is discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. MRE11-RAD50-NBS1 COMPLEX INHIBITOR MIRIN ENHANCES RADIOSENSITIVITY IN HUMAN GLIOBLASTOMA CELLS

    Science.gov (United States)

    Mishima, Kazuhiko; Mishima-Kaneko, Masayo; Kawata, Tetsuya; Saya, Hideyuki; Ishimaru, Naozumi; Yamada, Kouichi; Nishikawa, Ryo; Shigematsu, Naoyuki

    2014-01-01

    BACKGROUND: (blind field) METHODS: Glioma cell lines (U251, LN229 and LN428) were irradiated with and without Mirin and then clonogenicity, apoptosis, and cell cycle change were examined. Western blot analysis was performed to determine the relative potency of Mirin to inhibit the radioresistance, through the signaling activity of AKT. We also examined the levels of H2AX phosphorylationH2AX), which is a marker of DNA double-strand breaks (DSBs) using Western blot. RESULTS: Glioblastoma cells pretreated with Mirin demonstrated an enhanced sensitivity to radiation. FACS analysis revealed that Mirin and radiation caused the glioma cells to accumulate in the G2/M-phase of the cell cycle and the combination of these two treatments further increased the G2/M fraction of the glioma cells. Mirin significantly enhanced radiation-induced apoptotic cell death. Also, Mirin blocked the basal and increase of radiation-induced AKT phosphorylation. We observed that the combination of Mirin and radiation increased persistence of γH2AX at 24 h suggesting the inhibition of DNA DSBs repair. CONCLUSIONS: These results indicate that Mirin can effectively enhance glioma cell radiosensitivity. It suggests that Mirin is a potent radiosensitizer for treating glioma cells. SECONDARY CATEGORY: n/a.

  14. 53BP1 mediates the fusion of mammalian telomeres rendered dysfunctional by DNA-PKcs loss or inhibition.

    Directory of Open Access Journals (Sweden)

    Ivana Rybanska-Spaeder

    Full Text Available Telomere dysfunction promotes genomic instability and carcinogenesis via inappropriate end-to-end chromosomal rearrangements, or telomere fusions. Previous work indicates that the DNA Damage Response (DDR factor 53BP1 promotes the fusion of telomeres rendered dysfunctional by loss of TRF2, but is dispensable for the fusion of telomeres lacking Pot1 or critically shortened (in telomerase-deficient mice. Here, we examine a role for 53BP1 at telomeres rendered dysfunctional by loss or catalytic inhibition of DNA-PKcs. Using mouse embryonic fibroblasts lacking 53BP1 and/or DNA-PKcs, we show that 53BP1 deficiency suppresses G1-generated telomere fusions that normally accumulate in DNA-PKcs-deficient fibroblasts with passage. Likewise, we find that 53BP1 promotes telomere fusions during the replicative phases of the cell cycle in cells treated with the specific DNA-PKcs inhibitor NU7026. However, telomere fusions are not fully abrogated in DNA-PKcs-inhibited 53BP1-deficient cells, but occur with a frequency approximately 10-fold lower than in control 53BP1-proficient cells. Treatment with PARP inhibitors or PARP1 depletion abrogates residual fusions, while Ligase IV depletion has no measurable effect, suggesting that PARP1-dependent alternative end-joining operates at low efficiency at 53BP1-deficient, DNA-PKcs-inhibited telomeres. Finally, we have also examined the requirement for DDR factors ATM, MDC1 or H2AX in this context. We find that ATM loss or inhibition has no measurable effect on the frequency of NU7026-induced fusions in wild-type MEFs. Moreover, analysis of MEFs lacking both ATM and 53BP1 indicates that ATM is also dispensable for telomere fusions via PARP-dependent end-joining. In contrast, loss of either MDC1 or H2AX abrogates telomere fusions in response to DNA-PKcs inhibition, suggesting that these factors operate upstream of both 53BP1-dependent and -independent telomere rejoining. Together, these experiments define a novel

  15. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xuejiao [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000 (China); Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Wang, Shou-Lin, E-mail: wangshl@njmu.edu.cn [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China)

    2013-07-15

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  16. Disruption of PCNA-lamins A/C interactions by prelamin A induces DNA replication fork stalling.

    Science.gov (United States)

    Cobb, Andrew M; Murray, Thomas V; Warren, Derek T; Liu, Yiwen; Shanahan, Catherine M

    2016-09-02

    The accumulation of prelamin A is linked to disruption of cellular homeostasis, tissue degeneration and aging. Its expression is implicated in compromised genome stability and increased levels of DNA damage, but to date there is no complete explanation for how prelamin A exerts its toxic effects. As the nuclear lamina is important for DNA replication we wanted to investigate the relationship between prelamin A expression and DNA replication fork stability. In this study we report that the expression of prelamin A in U2OS cells induced both mono-ubiquitination of proliferating cell nuclear antigen (PCNA) and subsequent induction of Pol η, two hallmarks of DNA replication fork stalling. Immunofluorescence microscopy revealed that cells expressing prelamin A presented with high levels of colocalisation between PCNA and γH2AX, indicating collapse of stalled DNA replication forks into DNA double-strand breaks. Subsequent protein-protein interaction assays showed prelamin A interacted with PCNA and that its presence mitigated interactions between PCNA and the mature nuclear lamina. Thus, we propose that the cytotoxicity of prelamin A arises in part, from it actively competing against mature lamin A to bind PCNA and that this destabilises DNA replication to induce fork stalling which in turn contributes to genomic instability.

  17. Regulation of DNA replication and chromosomal polyploidy by the MLL-WDR5-RBBP5 methyltransferases

    Directory of Open Access Journals (Sweden)

    Fei Lu

    2016-10-01

    Full Text Available DNA replication licensing occurs on chromatin, but how the chromatin template is regulated for replication remains mostly unclear. Here, we have analyzed the requirement of histone methyltransferases for a specific type of replication: the DNA re-replication induced by the downregulation of either Geminin, an inhibitor of replication licensing protein CDT1, or the CRL4CDT2 ubiquitin E3 ligase. We found that siRNA-mediated reduction of essential components of the MLL-WDR5-RBBP5 methyltransferase complexes including WDR5 or RBBP5, which transfer methyl groups to histone H3 at K4 (H3K4, suppressed DNA re-replication and chromosomal polyploidy. Reduction of WDR5/RBBP5 also prevented the activation of H2AX checkpoint caused by re-replication, but not by ultraviolet or X-ray irradiation; and the components of MLL complexes co-localized with the origin recognition complex (ORC and MCM2-7 replicative helicase complexes at replication origins to control the levels of methylated H3K4. Downregulation of WDR5 or RBBP5 reduced the methylated H3K4 and suppressed the recruitment of MCM2-7 complexes onto replication origins. Our studies indicate that the MLL complexes and H3K4 methylation are required for DNA replication but not for DNA damage repair.

  18. Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.

    Science.gov (United States)

    Gao, Hong; Prasad, G L; Zacharias, Wolfgang

    2014-05-01

    The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Protein phosphorylation during Plasmodium berghei gametogenesis.

    Science.gov (United States)

    Alonso-Morales, Alberto; González-López, Lorena; Cázares-Raga, Febe Elena; Cortés-Martínez, Leticia; Torres-Monzón, Jorge Aurelio; Gallegos-Pérez, José Luis; Rodríguez, Mario Henry; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

    2015-09-01

    Plasmodium gametogenesis within the mosquito midgut is a complex differentiation process involving signaling mediated by phosphorylation, which modulate metabolic routes and protein synthesis required to complete this development. However, the mechanisms leading to gametogenesis activation are poorly understood. We analyzed protein phosphorylation during Plasmodium berghei gametogenesis in vitro in serum-free medium using bidimensional electrophoresis (2-DE) combined with immunoblotting (IB) and antibodies specific to phosphorylated serine, threonine and tyrosine. Approximately 75 protein exhibited phosphorylation changes, of which 23 were identified by mass spectrometry. These included components of the cytoskeleton, heat shock proteins, and proteins involved in DNA synthesis and signaling pathways among others. Novel phosphorylation events support a role for these proteins during gametogenesis. The phosphorylation sites of six of the identified proteins, HSP70, WD40 repeat protein msi1, enolase, actin-1 and two isoforms of large subunit of ribonucleoside reductase were investigated using TiO2 phosphopeptides enrichment and tandem mass spectrometry. In addition, transient exposure to hydroxyurea, an inhibitor of ribonucleoside reductase, impaired male gametocytes exflagellation in a dose-dependent manner, and provides a resource for functional studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Mining Conditional Phosphorylation Motifs.

    Science.gov (United States)

    Liu, Xiaoqing; Wu, Jun; Gong, Haipeng; Deng, Shengchun; He, Zengyou

    2014-01-01

    Phosphorylation motifs represent position-specific amino acid patterns around the phosphorylation sites in the set of phosphopeptides. Several algorithms have been proposed to uncover phosphorylation motifs, whereas the problem of efficiently discovering a set of significant motifs with sufficiently high coverage and non-redundancy still remains unsolved. Here we present a novel notion called conditional phosphorylation motifs. Through this new concept, the motifs whose over-expressiveness mainly benefits from its constituting parts can be filtered out effectively. To discover conditional phosphorylation motifs, we propose an algorithm called C-Motif for a non-redundant identification of significant phosphorylation motifs. C-Motif is implemented under the Apriori framework, and it tests the statistical significance together with the frequency of candidate motifs in a single stage. Experiments demonstrate that C-Motif outperforms some current algorithms such as MMFPh and Motif-All in terms of coverage and non-redundancy of the results and efficiency of the execution. The source code of C-Motif is available at: https://sourceforge. net/projects/cmotif/.

  1. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  2. Phosphorylation of p300 by ATM controls the stability of NBS1

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Eun Ryoung [Department of Molecular Science and Technology, College of Natural Sciences, Ajou University, Suwon 443-749 (Korea, Republic of); Choi, Jae Duk [Department of Molecular Science and Technology, College of Natural Sciences, Ajou University, Suwon 443-749 (Korea, Republic of); School of Biological Sciences, Seoul National University, Seoul 151 (Korea, Republic of); Jeong, Gajin [School of Biological Sciences, Seoul National University, Seoul 151 (Korea, Republic of); Lee, Jong-Soo, E-mail: jsjlee@mail.ajou.ac.kr [Department of Molecular Science and Technology, College of Natural Sciences, Ajou University, Suwon 443-749 (Korea, Republic of)

    2010-07-09

    Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon {gamma}-irradiation in ATM{sup +} cells but not ATM{sup -} cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

  3. Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Kalpana Mujoo

    2017-11-01

    Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

  4. A comparison of DNA damage probes in two HMEC lines withX-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Wisnewski, Christy L.; Bjornstad, Kathleen A.; Rosen, ChristoperJ.; Chang, Polly Y.; Blakely, Eleanor A.

    2007-01-19

    In this study, we investigated {gamma}H2AX{sup ser139} and 53BP1{sup ser25}, DNA damage pathway markers, to observe responses to radiation insult. Two Human Mammary Epithelial Cell (HMEC) lines were utilized to research the role of immortalization in DNA damage marker expression, HMEC HMT-3522 (S1) with an infinite lifespan, and a subtype of HMEC 184 (184V) with a finite lifespan. Cells were irradiated with 50 cGy X-rays, fixed with 4% paraformaldehyde after 1 hour repair at 37 C, and processed through immunofluorescence. Cells were visualized with a fluorescent microscope and images were digitally captured using Image-Pro Plus software. The 184V irradiated cells exhibited a more positive punctate response within the nucleus for both DNA damage markers compared to the S1 irradiated cells. We will expand the dose and time course in future studies to augment the preliminary data from this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process proteins of HMECs in order to understand what is 'normal' and to evaluate the usefulness of cell lines as experimental models.

  5. Phosphorylation in hydrogen bacteria.

    Science.gov (United States)

    Bongers, L

    1967-05-01

    The electron-transport system of cell-free extracts obtained from Hydrogenomonas H-20 has been studied with particular reference to phosphorylation associated with the oxyhydrogen reaction. Cell-free preparations of this organism exhibit oxidative phosphorylation with hydrogen and succinate as electron donors. This activity could be uncoupled with a number of agents. Ratios of phosphorylative activity to oxidative activity observed varied from 0.2 to 0.7. Factors affecting the efficiency of phosphorylation were examined. Inhibitor and spectrophotometric studies indicated that phosphorylation with hydrogen as electron donor occurs exclusively at a site in an abbreviated electron transport chain between H(2) and cytochrome b. The possible occurrence of a cytochrome b oxidase and the requirement for a quinone are discussed, as well as the correlation between the abbreviated pathway and the energy generation by the cell. Evidence is presented which indicates that nicotinamide adenine dinucleotide does not participate in the hydrogen oxidation path which is coupled to adenosine triphosphate formation.

  6. New DNA-binding radioprotectors

    Science.gov (United States)

    Martin, Roger

    The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is

  7. Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Patrick Naumann

    Full Text Available Sulforaphane (SFN, an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT.In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT.Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4.Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

  8. Regulation of DNA Damage Response by Estrogen Receptor β-Mediated Inhibition of Breast Cancer Associated Gene 2

    Directory of Open Access Journals (Sweden)

    Yuan-Hao Lee

    2015-04-01

    Full Text Available Accumulating evidence suggests that ubiquitin E3 ligases are involved in cancer development as their mutations correlate with genomic instability and genetic susceptibility to cancer. Despite significant findings of cancer-driving mutations in the BRCA1 gene, estrogen receptor (ER-positive breast cancers progress upon treatment with DNA damaging-cytotoxic therapies. In order to understand the underlying mechanism by which ER-positive breast cancer cells develop resistance to DNA damaging agents, we employed an estrogen receptor agonist, Erb-041, to increase the activity of ERβ and negatively regulate the expression and function of the estrogen receptor α (ERα in MCF-7 breast cancer cells. Upon Erb-041-mediated ERα down-regulation, the transcription of an ERα downstream effector, BCA2 (Breast Cancer Associated gene 2, correspondingly decreased. The ubiquitination of chromatin-bound BCA2 was induced by ultraviolet C (UVC irradiation but suppressed by Erb-041 pretreatment, resulting in a blunted DNA damage response. Upon BCA2 silencing, DNA double-stranded breaks increased with Rad51 up-regulation and ataxia telangiectasia mutated (ATM activation. Mechanistically, UV-induced BCA2 ubiquitination and chromatin binding were found to promote DNA damage response and repair via the interaction of BCA2 with ATM, γH2AX and Rad51. Taken together, this study suggests that Erb-041 potentiates BCA2 dissociation from chromatin and co-localization with Rad51, resulting in inhibition of homologous recombination repair.

  9. gamma H2AX/53BP1 foci as a potential pre-treatment marker of HNSCC tumors radiosensitivity preliminary methodological study and discussion

    Czech Academy of Sciences Publication Activity Database

    Falk, Martin; Hořáková, Z.; Svobodová, M.; Masařík, M.; Kopečná, Olga; Gumulec, J.; Raudenská, M.; Depeš, Daniel; Bačíková, Alena; Falková, Iva; Binkova, H.

    2017-01-01

    Roč. 71, č. 9 (2017), č. článku 241. ISSN 1434-6079 R&D Projects: GA ČR(CZ) GA16-12454S Institutional support: RVO:68081707 Keywords : squamous-cell carcinoma * cancer -associated fibroblasts Subject RIV: EB - Genetics ; Molecular Biology

  10. DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available PURPOSE: In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. METHODS AND MATERIALS: In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. RESULTS: Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. CONCLUSIONS: Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

  11. DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations.

    Science.gov (United States)

    Saha, Bidisha; Zitnik, Galynn; Johnson, Simon; Nguyen, Quyen; Risques, Rosa A; Martin, George M; Oshima, Junko

    2013-01-01

    Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT (human telomerase reverse transcriptase), did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, γ-H2AX foci (reflecting double strand DNA damage) in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening.

  12. DNA Damage Accumulation and TRF2 Degradation in Atypical Werner Syndrome Fibroblasts with LMNA mutations

    Directory of Open Access Journals (Sweden)

    Bidisha eSaha

    2013-07-01

    Full Text Available Segmental progeroid syndromes are groups of disorders with multiple features suggestive of accelerated aging. One subset of adult-onset progeroid syndromes, referred to as atypical Werner syndrome (AWS, is caused by mutations in the LMNA gene, which encodes a class of nuclear intermediate filaments, lamin A/C. We previously described rapid telomere attrition and accelerated replicative senescence in cultured fibroblasts overexpressing mutant lamin A. In this study, we investigated the cellular phenotypes associated with accelerated telomere shortening in LMNA mutant primary fibroblasts. In early passage primary fibroblasts with R133L or L140R LMNA mutations, shelterin protein components were already reduced while cells still retained telomere lengths comparable to those of controls. There was a significant inverse correlation between the degree of abnormal nuclear morphology and the level of TRF2, a shelterin subunit, suggesting a potential causal relationship. Stabilization of the telomeres via the introduction of the catalytic subunit of human telomerase, hTERT, did not prevent degradation of shelterin components, indicating that reduced TRF2 in LMNA mutants is not mediated by short telomeres. Interestingly, -H2AX foci (reflecting double strand DNA damage in early passage LMNA mutant primary fibroblasts and LMNA mutant hTERT fibroblasts were markedly increased in non-telomeric regions of DNA. Our results raise the possibility that mutant lamin A/C causes global genomic instability with accumulation of non-telomeric DNA damage as an early event, followed by TRF2 degradation and telomere shortening.

  13. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

    Science.gov (United States)

    Murakawa, Yasuhiro; Sonoda, Eiichiro; Barber, Louise J; Zeng, Weihua; Yokomori, Kyoko; Kimura, Hiroshi; Niimi, Atsuko; Lehmann, Alan; Zhao, Guang Yu; Hochegger, Helfrid; Boulton, Simon J; Takeda, Shunichi

    2007-09-15

    Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

  14. Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Qianzheng; Battu, Aruna; Ray, Alo; Wani, Gulzar; Qian, Jiang; He, Jinshan; Wang, Qi-en [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Wani, Altaf A., E-mail: wani.2@osu.edu [Department of Radiology, The Ohio State University, Columbus, OH 43210 (United States); Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, OH 43210 (United States); James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 (United States)

    2015-06-15

    Highlights: • HDAC1 and HDAC2 co-localize with UV radiation-induced DNA damage sites. • HDAC1 translocation to chromatin is dependent on DDB2 function. • HDAC1 and HDAC2 are involved in H3K56Ac deacetylation. • H3K56Ac deacetylation requires DDB1 and DDB2 but not XPA or XPC functions. • HDAC1/2 depletion decreases XPC ubiquitination and local γH2AX accumulation. - Abstract: Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.

  15. Validation of JCountPro software for efficient assessment of ionizing radiation-induced foci in human lymphocytes.

    Science.gov (United States)

    Jakl, Lukáš; Lobachevsky, Pavel; Vokálová, Lenka; Durdík, Matúš; Marková, Eva; Belyaev, Igor

    2016-12-01

    Ionizing radiation-induced foci (IRIF) known also as DNA repair foci represent the most sensitive and specific assay for assessing DNA double-strand break (DSB). IRIF are usually visualized and enumerated with the aid of fluorescence microscopy using antibodies to phosphorylated γH2AX and 53BP1. Although several approaches and software packages were developed for quantification of IRIF, not one of them was commonly accepted and inter-laboratory variability in the outputs was reported. In this study, JCountPro software was validated for IRIF enumeration in two independent laboratories. Human lymphocytes were γ-irradiated at doses of 0, 2, 5, 10 and 50 cGy. The cells were fixed, permeabilized and IRIF were immunostained using appropriate antibodies. Cell images were acquired with automatic Metafer system. Endogenous and radiation-induced γH2AX and 53BP1 foci were enumerated using JCountPro. This analysis was performed from the same cell galleries by the researchers from two laboratories. Yield of foci was analyzed by either arithmetic mean (AM) value (foci/cell) or principal average (PA) derived from the approximation of foci distribution with Poisson statistics. Statistical analysis was performed using factorial ANOVA. Enumeration of 53BP1, γH2AX and co-localized 53BP1/γH2AX foci by JCountPro was essentially the same between laboratories. IRIF were detected at all doses and linear dose response was obtained in the studied dose range. PA values from Poisson distribution fitted the data better as compared to AM values and were more powerful and sensitive for IRIF analysis than the AM values. All JCountPro data were confirmed by visual focus enumeration. We concluded that the JCountPro software was efficient in objectively enumerating IRIF regardless of an individual researcher's bias and has a potential for usage in clinics and molecular epidemiology.

  16. Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

    Science.gov (United States)

    She, Hua; Mao, Zixu

    2017-01-01

    The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

  17. Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid

    Science.gov (United States)

    Burgos-Morón, Estefanía; Calderón-Montaño, José Manuel; Orta, Manuel Luis; Guillén-Mancina, Emilio; Mateos, Santiago; López-Lázaro, Miguel

    2016-01-01

    Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee. PMID:27399778

  18. A missense mutation in Rev7 disrupts formation of Polζ, impairing mouse development and repair of genotoxic agent-induced DNA lesions.

    Science.gov (United States)

    Khalaj, Maryam; Abbasi, Abdolrahim; Yamanishi, Hiroshi; Akiyama, Kouyou; Wakitani, Shuso; Kikuchi, Sotaro; Hirose, Michiko; Yuzuriha, Misako; Magari, Masaki; Degheidy, Heba A; Abe, Kuniya; Ogura, Atsuo; Hashimoto, Hiroshi; Kunieda, Tetsuo

    2014-02-07

    Repro22 is a mutant mouse produced via N-ethyl-N-nitrosourea-induced mutagenesis that shows sterility with germ cell depletion caused by defective proliferation of primordial germ cells, decreased body weight, and partial lethality during embryonic development. Using a positional cloning strategy, we identified a missense mutation in Rev7/Mad2l2 (Rev7(C70R)) and confirmed that the mutation is the cause of the defects in repro22 mice through transgenic rescue with normal Rev7. Rev7/Mad2l2 encodes a subunit of DNA polymerase ζ (Polζ), 1 of 10 translesion DNA synthesis polymerases known in mammals. The mutant REV7 did not interact with REV3, the catalytic subunit of Polζ. Rev7(C70R/C70R) cells showed decreased proliferation, increased apoptosis, and arrest in S phase with extensive γH2AX foci in nuclei that indicated accumulation of DNA damage after treatment with the genotoxic agent mitomycin C. The Rev7(C70R) mutation does not affect the mitotic spindle assembly checkpoint. These results demonstrated that Rev7 is essential in resolving the replication stalls caused by DNA damage during S phase. We concluded that Rev7 is required for primordial germ cell proliferation and embryonic viability and development through the translesion DNA synthesis activity of Polζ preserving DNA integrity during cell proliferation, which is required in highly proliferating embryonic cells.

  19. AZD1775 induces toxicity through double-stranded DNA breaks independently of chemotherapeutic agents in p53-mutated colorectal cancer cells.

    Science.gov (United States)

    Webster, Peter John; Littlejohns, Anna Tiffany; Gaunt, Hannah Jane; Prasad, K Raj; Beech, David John; Burke, Dermot Anthony

    2017-01-01

    AZD1775 is a small molecule WEE1 inhibitor used in combination with DNA-damaging agents to cause premature mitosis and cell death in p53-mutated cancer cells. Here we sought to determine the mechanism of action of AZD1775 in combination with chemotherapeutic agents in light of recent findings that AZD1775 can cause double-stranded DNA (DS-DNA) breaks. AZD1775 significantly improved the cytotoxicity of 5-FU in a p53-mutated colorectal cancer cell line (HT29 cells), decreasing the IC 50 from 9.3 μM to 3.5 μM. Flow cytometry showed a significant increase in the mitotic marker pHH3 (3.4% vs. 56.2%) and DS-DNA break marker γH2AX (5.1% vs. 50.7%) for combination therapy compared with 5-FU alone. Combination therapy also increased the amount of caspase-3 dependent apoptosis compared with 5-FU alone (4% vs. 13%). The addition of exogenous nucleosides to combination therapy significantly rescued the increased DS-DNA breaks and caspase-3 dependent apoptosis almost to the levels of 5-FU monotherapy. In conclusion, AZD1775 enhances 5-FU cytotoxicity through increased DS-DNA breaks, not premature mitosis, in p53-mutated colorectal cancer cells. This finding is important for designers of future clinical trials when considering the optimal timing and duration of AZD1775 treatment.

  20. Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field.

    LENUS (Irish Health Repository)

    Shields, L

    2014-10-31

    Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells are not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA