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Sample records for phosphorylates histone h2ax

  1. Histone deacetylase inhibitors enhance phosphorylation of histone H2AX after ionizing radiation

    SciTech Connect

    Zhang Yubin; Adachi, Masaaki . E-mail: adachi@sapmed.ac.jp; Zou Huichao; Hareyama, Masato; Imai, Kohzoh; Shinomura, Yasuhisa

    2006-07-01

    Purpose Histone deacetylase (HDAC) inhibitors are believed to be promising radiosensitizers. To explore their effects on ionizing radiation (IR), we examined whether the HDAC inhibitors m-carboxycinnamic acid bis-hydroxamide (CBHA) and depsipeptide FK228 affect H2AX phosphorylation ({gamma}-H2AX), a landmark of DNA double-strand breaks after IR exposure. Methods and Materials We evaluated the effects of the HDAC inhibitors on clonogenic assay in human lung carcinoma A549 cells and progression of A549 xenograft tumors. IR-induced DNA damage was evaluated by histone {gamma}-H2AX. Histone hyperacetylation was induced by overexpression of histone acetyltransferase p300 and evaluated by Western blots. Results M-carboxycinnamic acid bishydroxyamide pretreatment radiosensitized A549 cells and strongly inhibited A549 xenograft tumor progression. CBHA and FK228, but not 5-fluorouracil, enhanced IR-induced {gamma}-H2AX in A549 and other cancer cell lines. Overexpression of p300 similarly augmented IR-induced {gamma}-H2AX. Conclusion The results of this study suggest that HDAC inhibitors enhance IR-induced {gamma}-H2AX, most likely through histone hyperacetylation, and radiosensitize various cancers.

  2. Evaluation of chemical phototoxicity, focusing on phosphorylated histone H2AX

    PubMed Central

    Ibuki, Yuko; Toyooka, Tatsushi

    2015-01-01

    Histone H2AX is a minor component of nuclear histone H2A. The phosphorylation of histone H2AX at Ser 139, termed γ-H2AX, was originally identified as an early event after the direct formation of DNA double-strand breaks (DSBs) by ionizing radiation. Now, the generation of γ-H2AX is also considered to occur in association with secondarily formed DSBs by cellular processing such as DNA replication and repair at the site of the initial damage, including DNA adducts, crosslinks, and UV-induced photolesions. Therefore, γ-H2AX is currently attracting attention as a new biomarker for detecting various genotoxic insults. We have determined the toxic impact of various environmental stresses such as chemicals, light and/or their coexposure using γ-H2AX, and found that the γ-H2AX assay exhibited high sensitivity and a low false-positive rate as a detection system of genotoxic potential. In this review, we introduced our recent findings concerning the evaluation of chemical phototoxicity, focusing on γ-H2AX. PMID:25480829

  3. Phosphorylation of histone H2AX in the mouse brain from development to senescence.

    PubMed

    Barral, Serena; Beltramo, Riccardo; Salio, Chiara; Aimar, Patrizia; Lossi, Laura; Merighi, Adalberto

    2014-01-01

    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. PMID:24451138

  4. Coexposure to silver nanoparticles and ultraviolet A synergistically enhances the phosphorylation of histone H2AX.

    PubMed

    Zhao, Xiaoxu; Takabayashi, Fumiyo; Ibuki, Yuko

    2016-09-01

    Owing to the wide application of silver nanoparticles (AgNPs), the assessment of health risks associated with their use is of great importance. In this study, we revealed that the potential genotoxicity of AgNPs was enhanced by ultraviolet A (UVA) exposure. Three cultured cell lines were treated with AgNPs, followed by exposure to UVA. AgNPs induced phosphorylation of histone H2AX (γ-H2AX) following the formation of DNA double-strand breaks (DSBs), which was synergistically enhanced by UVA exposure. Enhanced γ-H2AX was observed only in cell lines that positively took up AgNPs, and microsized Ag particles, which were difficult to incorporate into cells, showed no γ-H2AX. Incorporation of AgNPs was not increased by UVA exposure. AgNO3 treatment followed by UVA exposure also induced a marked increase in γ-H2AX, indicating that the enhanced γ-H2AX was attributed to Ag ions released from AgNPs. Ag ions reacted with the -SH group of antioxidant molecules, such as glutathione, and induced intracellular oxidative conditions. 8-Hydroxy-2'-deoxyguanosine was formed in the cells treated with AgNPs, which was augmented by UVA irradiation, suggesting that intracellular oxidation caused oxidative DNA damage, leading to the enhanced formation of DSBs and γ-H2AX. Ag has been considered a safe metal; however, our results provide important insights into the influence of sunlight on the genotoxic potency of AgNPs. PMID:27383448

  5. Determination of Histone H2AX Phosphorylation in DT40 Cells.

    PubMed

    Nishihara, Kana; Shahane, Sampada A; Xia, Menghang

    2016-01-01

    Visualization of DNA damage response protein recruitment to DNA damage sites enables measurement of the DNA damage. DNA double-strand breaks (DSBs) and blocked replication forks induce the phosphorylation of H2AX at serine 139 (γH2AX), and accumulate γH2AX which can then be detected as foci. The detection of γH2AX foci by immunostaining with antibodies that recognize γH2AX is an indicator of DSBs presence. This chapter describes the measurement of γH2AX immunostaining using a high-content imaging platform in chicken DT40 B-lymphocyte cell lines. PMID:27518625

  6. Slow elimination of phosphorylated histone {gamma}-H2AX from DNA of terminally differentiated mouse heart cells in situ

    SciTech Connect

    Gavrilov, Boris; Vezhenkova, Irina; Firsanov, Denis; Solovjeva, Liudmila; Svetlova, Maria; Mikhailov, Vyacheslav; Tomilin, Nikolai . E-mail: nvtom@hotmail.com

    2006-09-08

    Phosphorylation of replacement histone H2AX occurs in megabase chromatin domains around double-strand DNA breaks (DSBs) and this modification (called {gamma}-H2AX) may serve as a useful marker of genome damage and repair in terminally differentiated cells. Here using immunohistochemistry we studied kinetics of {gamma}-H2AX formation and elimination in the X-irradiated mouse heart and renal epithelial tissues in situ. Unirradiated tissues have 3-5% {gamma}-H2AX-positive cells and in tissues fixed 1 h after X-irradiation {gamma}-H2AX-positive nuclei are induced in a dose-dependent manner approaching 20-30% after 3 Gy of IR. Analysis of mouse tissues at different times after 3 Gy of IR showed that maximal induction of {gamma}-H2AX in heart is observed 20 min after IR and then is decreased slowly with about half remaining 23 h later. In renal epithelium maximum of the {gamma}-H2AX-positive cells is observed 40 min after IR and then decreases to control values in 23 h. This indicates that there are significant variations between non-proliferating mammalian tissues in the initial H2AX phosphorylation rate as well as in the rate of {gamma}-H2AX elimination after X-irradiation, which should be taken into account in the analysis of radiation responses.

  7. Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

    SciTech Connect

    Koike, Manabu Mashino, Minako; Sugasawa, Jun; Koike, Aki

    2007-11-30

    Histone H2AX undergoes phosphorylation on Ser 139 ({gamma}-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, {gamma}-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, {gamma}-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, {gamma}-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, {gamma}-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.

  8. Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation

    SciTech Connect

    Mahrhofer, Hartmut; Buerger, Susann; Oppitz, Ulrich; Flentje, Michael; Djuzenova, Cholpon S. . E-mail: djuzenova_t@klinik.uni-wuerzburg.de

    2006-02-01

    Purpose: To analyze the radiation-induced levels of {gamma}H2AX and its decay kinetics in 10 human cell lines covering a wide range of cellular radiosensitivity (SF2, 0.06-0.63). Methods and Materials: Five tumor cell lines included Colo-800 melanoma, two glioblastoma (MO59J and MO59K), fibrosarcoma HT 1080, and breast carcinoma MCF7. Five primary skin fibroblasts lines included two normal strains, an ataxia telangiectasia strain, and two fibroblast strains from breast cancer patients with an adverse early skin reaction to radiotherapy. Cellular radiosensitivity was assessed by colony-forming test. Deoxyribonucleic acid damage and repair were analyzed according to nuclear {gamma}H2AX foci intensity, with digital image analysis. Results: The cell lines tested showed a wide degree of variation in the background intensity of immunostained nuclear histone {gamma}H2AX, which was higher for the tumor cell lines compared with the fibroblast strains. It was not possible to predict clonogenic cell survival (SF2) for the 10 cell lines studied from the radiation-induced {gamma}H2AX intensity. In addition, the slopes of the dose-response (0-4 Gy) curves, the rates of {gamma}H2AX disappearance, and its residual expression ({<=}18 h after irradiation) did not correlate with SF2 values. Conclusions: The results from 10 cell lines showed that measurements of immunofluorescence intensity by digital image analysis of phosphorylated histone H2AX as a surrogate marker of DNA double-strand breaks did not allow reliable ranking of cell strains according to their clonogenic survival after irradiation.

  9. Critical role of lysine 134 methylation on histone H2AX for γ-H2AX production and DNA repair

    PubMed Central

    Sone, Kenbun; Piao, Lianhua; Nakakido, Makoto; Ueda, Koji; Jenuwein, Thomas; Nakamura, Yusuke; Hamamoto, Ryuji

    2014-01-01

    The presence of phosphorylated histone H2AX (γ-H2AX) is associated with the local activation of DNA-damage repair pathways. Although γ-H2AX deregulation in cancer has previously been reported, the molecular mechanism involved and its relationship with other histone modifications remain largely unknown. Here we find that the histone methyltransferase SUV39H2 methylates histone H2AX on lysine 134. When H2AX was mutated to abolish K134 methylation, the level of γ-H2AX became significantly reduced. We also found lower γ-H2AX activity following the introduction of double-strand breaks in Suv39h2 knockout cells or on SUV39H2 knockdown. Tissue microarray analyses of clinical lung and bladder tissues also revealed a positive correlation between H2AX K134 methylation and γ-H2AX levels. Furthermore, introduction of K134-substituted histone H2AX enhanced radio- and chemosensitivity of cancer cells. Overall, our results suggest that H2AX methylation plays a role in the regulation of γ-H2AX abundance in cancer. PMID:25487737

  10. DNA Double-Strand Breaks by Cr(VI) Are Targeted to Euchromatin and Cause ATR-Dependent Phosphorylation of Histone H2AX and Its Ubiquitination

    PubMed Central

    DeLoughery, Zachary; Luczak, Michal W.; Ortega-Atienza, Sara; Zhitkovich, Anatoly

    2015-01-01

    Hexavalent chromium is a human respiratory carcinogen that undergoes intracellular activation in vivo primarily via reduction with ascorbate. Replication of Cr-adducted DNA triggers mismatch repair that generates toxic DNA double-strand breaks (DSBs) as secondary lesions. Here, we examined the intranuclear distribution of chromate-induced breaks and a central DSB signaling branch targeting histone H2AX. Using ascorbate-restored cells (H460 human lung epithelial cells, normal human lung and normal mouse embryonic fibroblasts (MEFs)), we found that Cr(VI) produced a typical DSB-associated spectrum of H2AX modifications, including its Ser139-phosphorylated (known as γH2AX) and mono- and diubiquitinated forms. However, whereas canonical DSB signaling relies on ATM, the formation of γH2AX and its ubiquitinated products by Cr(VI) was dependent on ATR kinase. Based on the established mode of ATR activation, this suggests that Cr-induced DSB are not blunt-ended and likely contain single-stranded tails. Confocal imaging with markers of active and inactive chromatin revealed a selective formation of Cr-induced DSB in euchromatin of mouse and human cells. In contrast to DSB, Cr-DNA adducts were produced in both types of chromatin. The euchromatin targeting of Cr-induced DSB makes these lesions particularly dangerous by increasing the probability of deleting active tumor suppressors and producing oncogenic translocations. Accumulation of transcription-inhibiting ubiquitinated forms of γH2AX in euchromatin is expected to contribute to the ability of Cr(VI) to suppress upregulation of inducible genes. PMID:25288669

  11. Tissue-specific DNA-PK-dependent H2AX phosphorylation and {gamma}-H2AX elimination after X-irradiation in vivo

    SciTech Connect

    Koike, Manabu Sugasawa, Jun; Yasuda, Mariko; Koike, Aki

    2008-11-07

    Histone H2AX rapidly undergoes phosphorylation at Ser139 ({gamma}-H2AX) in response to DNA double-strand breaks. Although ATM kinase and DNA-PK phosphorylate Ser139 of H2AX in culture cells, the regulatory mechanism of {gamma}-H2AX level remains unclear in vivo. Here, we detected the phosphorylation of H2AX and the elimination of {gamma}-H2AX in the mouse skin after X-irradiation. Furthermore, following X-irradiation, the level of {gamma}-H2AX also increased in mice lacking either ATM or DNA-PK. Although the elimination after X-irradiation was detected in the skin of these mutant mice, the elimination in DNA-PK-deficient mice was slower than that in C3H and ATM knockout mice, suggesting that a fraction of {gamma}-H2AX in the skin is eliminated in a DNA-PK-dependent manner. Although the DNA-PK-dependent elimination of {gamma}-H2AX was also detected in the liver, kidney, and spleen, the DNA-PK-dependent phosphorylation of H2AX was detected in the spleen only. These results suggest that the regulatory mechanism of {gamma}-H2AX level is tissue-specific.

  12. Actin disruption agents induce phosphorylation of histone H2AX in human breast adenocarcinoma MCF-7 cells.

    PubMed

    Shin, Ik Jae; Ahn, Yong-Tae; Kim, Yongkuk; Kim, Jong-Myoung; An, Won G

    2011-05-01

    Modified actin dynamics are a unique feature of transformed cancer cells and thereby promising targets for cancer chemotherapy. While latrunculin B (LB) and pectenotoxin-2 (PTX-2), both derived from natural sources, inhibit actin polymerization, jasplakinolide (JSP) prevents actin depolymerization. The purpose of this study was to examine the detailed molecular action of actin disruption inducing apoptosis via double strand breaks (DSBs). Actin disruption induced phosphorylation of H2AX, a well known DSB marker leading to G2 arrest and consequently resulted in apoptosis on MCF-7 cancer cells. Cells impaired by actin disruption activated Erk (extracellular signal-related kinase) and p53 protein was involved in DNA damage responses, but did not change the levels of p21Cip1/WAF1 protein in MCF-7 cells. To overcome the DSBs by actin disruption, MCF-7 cells set the repair system through the homologous recombination (HR) pathway. These results indicate that actin is involved in the signaling inducing DSBs and HR repair as well as G2 cell cycle arrest in human cancer. Therefore, the results suggest that actin disruption might be a potential candidate for developing anti-cancer therapies in human breast cancer. PMID:21399880

  13. Histone deacetylase inhibitors valproic acid and depsipeptide sensitize retinoblastoma cells to radiotherapy by increasing H2AX phosphorylation and p53 acetylation-phosphorylation.

    PubMed

    Kawano, Takeshi; Akiyama, Masaharu; Agawa-Ohta, Miyuki; Mikami-Terao, Yoko; Iwase, Satsuki; Yanagisawa, Takaaki; Ida, Hiroyuki; Agata, Naoki; Yamada, Hisashi

    2010-10-01

    Although p53 is intact in most cases of retinoblastoma, it is largely inactivated by the ubiqutin-proteasome system through interaction with murine double minute 2 (MDM2) and murine double minute X (MDMX). The present study showed that the histone deacetylase (HDAC) inhibitors valproic acid (VPA) and depsipeptide (FK228) synergistically enhanced ionizing radiation (IR)-induced apoptosis, associated with activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase in Y79 and WER1-Rb1 human retinoblastoma cells. Both VPA and FK228 enhanced IR-induced phosphorylation of histone H2AX on Ser139 preceding apoptosis. Exposure of cells to IR in the presence of VPA or FK228 induced the accumulation of p53 acetylated at Lys382 and phosphorylated at Ser46 through the reduction of binding affinity with MDM2 and MDMX. These results suggest that acetylation of p53 by HDAC inhibitors is a promising new therapeutic target in refractory retinoblastoma. PMID:20811699

  14. Systematic Identification of Functional Residues in Mammalian Histone H2AX

    PubMed Central

    Chen, Wei-Ta; Alpert, Amir; Leiter, Courtney; Gong, Fade

    2013-01-01

    The histone variant H2AX is a principal component of chromatin involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). H2AX is thought to operate primarily through its C-terminal S139 phosphorylation, which mediates the recruitment of DNA damage response (DDR) factors to chromatin at DSB sites. Here, we describe a comprehensive screen of 67 residues in H2AX to determine their contributions to H2AX functions. Our analysis revealed that H2AX is both sumoylated and ubiquitylated. Individual residues defective for sumoylation, ubiquitylation, and S139 phosphorylation in untreated and damaged cells were identified. Specifically, we identified an acidic triad region in both H2A and H2AX that is required in cis for their ubiquitylation. We also report the characterization of a human H2AX knockout cell line, which exhibits DDR defects, including p53 activation, following DNA damage. Collectively, this work constitutes the first genetic complementation system for a histone in human cells. Finally, our data reveal new roles for several residues in H2AX and define distinct functions for H2AX in human cells. PMID:23109425

  15. Elimination of radiation-induced {gamma}-H2AX foci in mammalian nucleus can occur by histone exchange

    SciTech Connect

    Svetlova, Maria; Solovjeva, Liudmila; Nishi, Kayoko; Nazarov, Igor; Siino, Joseph; Tomilin, Nikolai . E-mail: nvtom@mail.ru

    2007-06-29

    Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX ({gamma}-H2AX) and formation of large nuclear {gamma}-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of {gamma}-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced {gamma}-H2AX foci. Maximal number of {gamma}-H2AX foci is observed one hour after irradiation, when {approx}20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of {gamma}-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of {gamma}-H2AX. This indicates that elimination of {gamma}-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.

  16. Histone H2AX participates the DNA damage-induced ATM activation through interaction with NBS1

    SciTech Connect

    Kobayashi, Junya Tauchi, Hiroshi; Chen, Benjamin; Bruma, Sandeep; Tashiro, Satoshi; Matsuura, Shinya; Tanimoto, Keiji; Chen, David J.; Komatsu, Kenshi

    2009-03-20

    Phosphorylated histone H2AX ({gamma}-H2AX) functions in the recruitment of DNA damage response proteins to DNA double-strand breaks (DSBs) and facilitates DSB repair. ATM also co-localizes with {gamma}-H2AX at DSB sites following its auto-phosphorylation. However, it is unclear whether {gamma}-H2AX has a role in activation of ATM-dependent cell cycle checkpoints. Here, we show that ATM as well as NBS1 is recruited to damaged-chromatin in a {gamma}-H2AX-dependent manner. Foci formation of phosphorylated ATM and ATM-dependent phosphorylation is repressed in H2AX-knockdown cells. Furthermore, anti-{gamma}-H2AX antibody co-immunoprecipitates an ATM-like protein kinase activity in vitro and recombinant H2AX increases in vitro kinase activity of ATM from un-irradiated cells. Moreover, H2AX-deficient cells exhibited a defect in ATM-dependent cell cycle checkpoints. Taken together, {gamma}-H2AX has important role for effective DSB-dependent activation of ATM-related damage responses via NBS1.

  17. 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.

    PubMed

    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. PMID:17111099

  18. MicroRNA-138 modulates DNA damage response by repressing histone H2AX expression

    PubMed Central

    Wang, Yemin; Huang, Jen-Wei; Li, Ming; Cavenee, Webster K.; Mitchell, Patrick S.; Zhou, Xiaofeng; Tewari, Muneesh; Furnari, Frank B.; Taniguchi, Toshiyasu

    2011-01-01

    Precise regulation of DNA damage response is crucial for cellular survival after DNA damage, and its abrogation often results in genomic instability in cancer. Phosphorylated histone H2AXH2AX) forms nuclear foci at sites of DNA damage and facilitates DNA damage response and repair. MicroRNAs are short, non-protein-encoding RNA molecules, which post-transcriptionally regulate gene expression by repressing translation of and/or degrading mRNA. How microRNAs modulate DNA damage response is largely unknown. In this study, we developed a cell-based screening assay utilizing ionizing radiation-induced γH2AX foci formation in a human osteosarcoma cell line, U2OS, as the readout. By screening a library of human microRNA mimics, we identified several microRNAs that inhibited γH2AX foci formation. Among them, miR-138 directly targeted the histone H2AX 3′-UTR, reduced histone H2AX expression and induced chromosomal instability after DNA damage. Overexpression of miR-138 inhibited homologous recombination and enhanced cellular sensitivity to multiple DNA damaging agents (cisplatin, camptothecin, and ionizing radiation). Reintroduction of histone H2AX in miR-138 overexpressing cells attenuated miR-138-mediated sensitization to cisplatin and camptothecin. Our study suggests that miR-138 is an important regulator of genomic stability and a potential therapeutic agent to improve the efficacy of radiotherapy and chemotherapy with DNA damaging agents. PMID:21693595

  19. High LET-induced H2AX phosphorylation around the Bragg curve.

    PubMed

    Desai, N; Durante, M; Lin, Z W; Cucinotta, F; Wu, H

    2005-01-01

    We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (gamma-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent gamma-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced gamma-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of gamma-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects. PMID:15934200

  20. High LET-induced H2AX phosphorylation around the Bragg curve

    NASA Astrophysics Data System (ADS)

    Desai, N.; Durante, M.; Lin, Z. W.; Cucinotta, F.; Wu, H.

    We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (γ-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of γ-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/ y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent γ-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced γ-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of γ-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects.

  1. UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells.

    PubMed

    Oh, Kyu-Seon; Bustin, Michael; Mazur, Sharlyn J; Appella, Ettore; Kraemer, Kenneth H

    2011-01-01

    DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER. PMID:20947453

  2. Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus

    PubMed Central

    Namas, Rajaie; Renauer, Paul; Ognenovski, Mikhail; Tsou, Pei-Suen; Sawalha, Amr H

    2016-01-01

    Objective Defective or inefficient DNA double-strand break (DSB) repair results in failure to preserve genomic integrity leading to apoptotic cell death, a hallmark of systemic lupus erythematosus (SLE). Compelling evidence linked environmental factors that increase oxidative stress with SLE risk and the formation of DSBs. In this study, we sought to further explore genotoxic stress sensitivity in SLE by investigating DSB accumulation as a marker linking the effect of environmental stressors and the chromatin microenvironment. Methods DSBs were quantified in peripheral blood mononuclear cell subsets from patients with SLE, healthy controls, and patients with rheumatoid arthritis (RA) by measuring phosphorylated H2AX (phospho-H2AX) levels with flow cytometry. Phospho-H2AX levels were assessed in G0/G1, S and G2 cell-cycle phases using propidium iodide staining, and after oxidative stress using 0.5 µM hydrogen peroxide exposure for 0, 2, 5, 10, 30 and 60 min. Results DSB levels were significantly increased in CD4+ T cells, CD8+ T cells and monocytes in SLE compared with healthy controls (p=2.16×10−4, 1.68×10−3 and 4.74×10−3, respectively) and RA (p=1.05×10−3, 1.78×10−3 and 2.43×10−2, respectively). This increase in DSBs in SLE was independent of the cell-cycle phase, and correlated with disease activity. In CD4+ T cells, CD8+ T cells and monocytes, oxidative stress exposure induced significantly higher DSB accumulation in SLE compared with healthy controls (60 min; p=1.64×10−6, 8.11×10−7 and 2.04×10−3, respectively). Conclusions Our data indicate that SLE T cells and monocytes have increased baseline DSB levels and an increased sensitivity to acquiring DSBs in response to oxidative stress. Although the mechanism underlying DSB sensitivity in SLE requires further investigation, accumulation of DSB may serve a biomarker for disease activity in SLE and help explain increased apoptotic cell accumulation in this disease. PMID:27158526

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

    PubMed

    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 < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of 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. PMID:24735637

  4. High LET - induced H2AX phosphorylation at sites of DNA double strand breaks

    NASA Astrophysics Data System (ADS)

    Desai, N.; Cucinotta, F.; Wu, H.

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  5. Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions

    PubMed Central

    Turinetto, Valentina; Giachino, Claudia

    2015-01-01

    In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a ‘protagonist’ in different scenarios. This review will present and discuss an up-to-date view regarding the ‘non-canonical’ H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing ‘non-canonical’ functions to be carried out in different cell types. PMID:25712102

  6. Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation

    PubMed Central

    Wu, Xin-pin; Xiong, Min; Xu, Cheng-shan; Duan, Lian-ning; Dong, Ya-qiong; Luo, Yuan; Niu, Tian-hui; Lu, Cheng-rong

    2015-01-01

    Aim: The phosphorylation of histone H2AX, a novel tumor suppressor protein, is involved in regulation of cancer cell apoptosis. The aim of this study was to examine whether H2AX phosphorylation was required for resveratrol-induced apoptosis of human chronic myelogenous leukemia (CML) cells in vitro. Methods: K562 cells were tested. Cell apoptosis was analyzed using flow cytometry, and the phosphorylation of H2AX and other signaling proteins was examined with Western blotting. To analyze the signaling pathways, the cells were transfected with lentiviral vectors encoding H2AX-wt or specific siRNAs. Results: Treatment of K562 cells with resveratrol (20–100 μmol/L) induced apoptosis and phosphorylation of H2AX at Ser139 in time- and dose-dependent manners, but reduced phosphorylation of histone H3 at Ser10. Resveratrol treatment activated two MAPK family members p38 and JNK, and blocked the activation of another MAPK family member ERK. Pretreatment with the p38 inhibitor SB202190 or the JNK inhibitor SP600125 dose-dependently reduced resveratrol-induced phosphorylation of H2AX, which were also observed when the cells were transfected with p38- or JNK-specific siRNAs. Overexpression of H2AX in K562 cells markedly increased resveratrol-induced apoptosis, whereas overexpression of H2AX-139m (Ser139 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. K562 cells transfected with H2AX-specific siRNAs were resistant to resveratrol-induced apoptosis. Conclusion: H2AX phosphorylation at Ser139 in human CML cells, which is regulated by p38 and JNK, is essential for resveratrol-induced apoptosis. PMID:25619392

  7. Co-mutation of histone H2AX S139A with Y142A rescues Y142A-induced ionising radiation sensitivity

    PubMed Central

    Brown, James A.L.; Eykelenboom, John K.; Lowndes, Noel F.

    2012-01-01

    Under normal conditions histone H2AX is constitutively phosphorylated on tyrosine (Y) 142 by Williams–Beuren syndrome transcription factor kinase (WSTF). Following DNA double strand breaks (DSB), Y142 is de-phosphorylated and serine (S) 139 is phosphorylated. Here we explored DSB-dependent cross talk between H2AX residues S139 and Y142. H2axY142A mutation resulted in increased sensitivity to ionising radiation (IR), compared to H2axS139A. Interestingly, co-mutation of S139A and Y142A rescued IR sensitivity. The DSB response proteins 53Bp1 and Rad51 were recruited to IR-induced foci (IRIF) in H2axS139A, H2axY142A and H2axS139A/Y142A cells. Our results suggest that H2axY142A IR sensitivity is dependent upon the C-terminal residue, S139. PMID:23772364

  8. VRK1 chromatin kinase phosphorylates H2AX and is required for foci formation induced by DNA damage.

    PubMed

    Salzano, Marcella; Sanz-García, Marta; Monsalve, Diana M; Moura, David S; Lazo, Pedro A

    2015-01-01

    All types of DNA damage cause a local alteration and relaxation of chromatin structure. Sensing and reacting to this initial chromatin alteration is a necessary trigger for any type of DNA damage response (DDR). In this context, chromatin kinases are likely candidates to participate in detection and reaction to a locally altered chromatin as a consequence of DNA damage and, thus, initiate the appropriate cellular response. In this work, we demonstrate that VRK1 is a nucleosomal chromatin kinase and that its depletion causes loss of histones H3 and H4 acetylation, which are required for chromatin relaxation, both in basal conditions and after DNA damage, independently of ATM. Moreover, VRK1 directly and stably interacts with histones H2AX and H3 in basal conditions. In response to DNA damage induced by ionizing radiation, histone H2AX is phosphorylated in Ser139 by VRK1. The phosphorylation of H2AX and the formation of γH2AX foci induced by ionizing radiation (IR), are prevented by VRK1 depletion and are rescued by kinase-active, but not kinase-dead, VRK1. In conclusion, we found that VRK1 is a novel chromatin component that reacts to its alterations and participates very early in DDR, functioning by itself or in cooperation with ATM. PMID:25923214

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

    PubMed Central

    Broering, Tyler J.; Wang, Yuan-Liang; Pandey, Ram Naresh; Hegde, Rashmi S.; Wang, Shao-Chun; Namekawa, Satoshi H.

    2015-01-01

    ABSTRACT 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. PMID:25979708

  10. γH2AX and cancer

    PubMed Central

    Bonner, William M.; Redon, Christophe E.; Dickey, Jennifer S.; Nakamura, Asako J.; Sedelnikova, Olga A.; Solier, Stéphanie; Pommier, Yves

    2011-01-01

    Histone H2AX phosphorylation on a serine four residues from the carboxyl terminus (producing γH2AX) is a sensitive marker for DNA double-strand breaks (DSBs). DSBs may lead to cancer but, paradoxically, are also used to kill cancer cells. Using γH2AX detection to determine the extent of DSB induction may help to detect precancerous cells, to stage cancers, to monitor the effectiveness of cancer therapies and to develop novel anticancer drugs. PMID:19005492

  11. Unique epigenetic influence of H2AX phosphorylation and H3K56 acetylation on normal stem cell radioresponses

    PubMed Central

    Jacobs, Keith M.; Misri, Sandeep; Meyer, Barbara; Raj, Suyash; Zobel, Cheri L.; Sleckman, Barry P.; Hallahan, Dennis E.; Sharma, Girdhar G.

    2016-01-01

    Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non–stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation–induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylationH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment. PMID:26941327

  12. TIPRL Inhibits Protein Phosphatase 4 Activity and Promotes H2AX Phosphorylation in the DNA Damage Response

    PubMed Central

    Rosales, Kimberly Romero; Reid, Michael A.; Yang, Ying; Tran, Thai Q.; Wang, Wen-I; Lowman, Xazmin; Pan, Min; Kong, Mei

    2015-01-01

    Despite advances in our understanding of protein kinase regulation in the DNA damage response, the mechanism that controls protein phosphatase activity in this pathway is unclear. Unlike kinases, the activity and specificity of serine/threonine phosphatases is governed largely by their associated proteins. Here we show that Tip41-like protein (TIPRL), an evolutionarily conserved binding protein for PP2A-family phosphatases, is a negative regulator of protein phosphatase 4 (PP4). Knockdown of TIPRL resulted in increased PP4 phosphatase activity and formation of the active PP4-C/PP4R2 complex known to dephosphorylate γ-H2AX. Thus, overexpression of TIPRL promotes phosphorylation of H2AX, and increases γ-H2AX positive foci in response to DNA damage, whereas knockdown of TIPRL inhibits γ-H2AX phosphorylation. In correlation with γ-H2AX levels, we found that TIPRL overexpression promotes cell death in response to genotoxic stress, and knockdown of TIPRL protects cells from genotoxic agents. Taken together, these data demonstrate that TIPRL inhibits PP4 activity to allow for H2AX phosphorylation and the subsequent DNA damage response. PMID:26717153

  13. Induction and Rejoining of DNA Double Strand Breaks Assessed by H2AX Phosphorylation in Melanoma Cells Irradiated with Proton and Lithium Beams

    SciTech Connect

    Ibanez, Irene L.; Bracalente, Candelaria; Molinari, Beatriz L.; Palmieri, Monica A.; Policastro, Lucia; Kreiner, Andres J.; Burlon, Alejandro A.; Valda, Alejandro; Navalesi, Daniela; Davidson, Jorge; Davidson, Miguel; Vazquez, Monica; Ozafran, Mabel; Duran, Hebe

    2009-07-15

    Purpose: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. Methods and Materials: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX ({gamma}H2AX) foci at 30 min and 6 h post-irradiation. Results: Survival curves showed the increasing effectiveness of radiation as a function of LET. {gamma}H2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low LET radiation, revealing the repair capacity of DSBs. An increase in the size of {gamma}H2AX foci in cells irradiated with lithium beams was found, as compared with gamma and proton irradiations, which could be attributed to the clusters of DSBs induced by high LET radiation. Foci size increased at 6 h post-irradiation for lithium and proton irradiations in relation with persistent DSBs, showing a correlation with surviving fraction. Conclusions: Our results showed the response of B16-F0 cells to charged particle beams evaluated by the detection of {gamma}H2AX foci. We conclude that {gamma}H2AX foci size is an accurate parameter to correlate the rejoining of DSBs induced by different LET radiations and radiosensitivity.

  14. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast.

    PubMed

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-09-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain-phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  15. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast

    PubMed Central

    Hammet, Andrew; Magill, Christine; Heierhorst, Jörg; Jackson, Stephen P

    2007-01-01

    Phosphorylation of histone H2A or H2AX is an early and sensitive marker of DNA damage in eukaryotic cells, although mutation of the conserved damage-dependent phosphorylation site is well tolerated. Here, we show that H2A phosphorylation is required for cell-cycle arrest in response to DNA damage at the G1/S transition in budding yeast. Furthermore, we show that the tandem BRCT domain of Rad9 interacts directly with phosphorylated H2A in vitro and that a rad9 point mutation that abolishes this interaction results in in vivo phenotypes that are similar to those caused by an H2A phosphorylation site mutation. Remarkably, similar checkpoint defects are also caused by a Rad9 Tudor domain mutation that impairs Rad9 chromatin association already in undamaged cells. These findings indicate that constitutive Tudor domain-mediated and damage-specific BRCT domain–phospho-H2A-dependent interactions of Rad9 with chromatin cooperate to establish G1 checkpoint arrest. PMID:17721446

  16. γ-H2AX and phosphorylated ATM focus formation in cancer cells after laser plasma X irradiation.

    PubMed

    Sato, Katsutoshi; Nishikino, Masaharu; Okano, Yasuaki; Ohshima, Shinsuke; Hasegawa, Noboru; Ishino, Masahiko; Kawachi, Tetsuya; Numasaki, Hodaka; Teshima, Teruki; Nishimura, Hiroaki

    2010-10-01

    The usefulness of laser plasma X-ray pulses for medical and radiation biological studies was investigated, and the effects of laser plasma X rays were compared with those of conventional sources such as a linear accelerator. A cell irradiation system was developed that used copper-Kα (8 keV) lines from an ultrashort high-intensity laser to produce plasma. The absorbed dose of the 8 keV laser plasma X-ray pulse was estimated accurately with Gafchromic® EBT film. When the cells were irradiated with approximately 2 Gy of laser plasma X rays, the circular regions on γ-H2AX-positive cells could be clearly identified. Moreover, the numbers of γ-H2AX and phosphorylated ataxia telangiectasia mutated (ATM) foci induced by 8 keV laser plasma X rays were comparable to those induced by 4 MV X rays. These results indicate that the laser plasma X ray source may be useful for radiation biology studies. PMID:20718602

  17. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    SciTech Connect

    Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa; Komatsu, Kenshi; Oda, Shoji; Mitani, Hiroshi

    2012-12-14

    Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the

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

    PubMed Central

    Tarrade, Stephane; Bhardwaj, Tanya; Flegal, Matthew; Bertrand, Lindsey; Velegzhaninov, Ilya; Moskalev, Alexey; Klokov, Dmitry

    2015-01-01

    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. PMID:26694365

  19. Intense THz pulses cause H2AX phosphorylation and activate DNA damage response in human skin tissue

    PubMed Central

    Titova, Lyubov V.; Ayesheshim, Ayesheshim K.; Golubov, Andrey; Fogen, Dawson; Rodriguez-Juarez, Rocio; Hegmann, Frank A.; Kovalchuk, Olga

    2013-01-01

    Recent emergence and growing use of terahertz (THz) radiation for medical imaging and public security screening raise questions on reasonable levels of exposure and health consequences of this form of electromagnetic radiation. In particular, picosecond-duration THz pulses have shown promise for novel diagnostic imaging techniques. However, the effects of THz pulses on human cells and tissues thus far remain largely unknown. We report on the investigation of the biological effects of pulsed THz radiation on artificial human skin tissues. We observe that exposure to intense THz pulses for ten minutes leads to a significant induction of H2AX phosphorylation, indicating that THz pulse irradiation may cause DNA damage in exposed skin tissue. At the same time, we find a THz-pulse-induced increase in the levels of several proteins responsible for cell-cycle regulation and tumor suppression, suggesting that DNA damage repair mechanisms are quickly activated. Furthermore, we find that the cellular response to pulsed THz radiation is significantly different from that induced by exposure to UVA (400 nm). PMID:23577291

  20. Genome-wide profiles of H2AX and γ-H2AX differentiate endogenous and exogenous DNA damage hotspots in human cells

    PubMed Central

    Seo, Jungmin; Kim, Sang Cheol; Lee, Heun-Sik; Kim, Jung Kyu; Shon, Hye Jin; Salleh, Nur Lina Mohd; Desai, Kartiki Vasant; Lee, Jae Ho; Kang, Eun-Suk; Kim, Jin Sung; Choi, Jung Kyoon

    2012-01-01

    Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy. PMID:22467212

  1. H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown

    PubMed Central

    Dickey, Jennifer S.; Zemp, Franz J.; Altamirano, Alvin; Sedelnikova, Olga A.; Bonner, William M.; Kovalchuk, Olga

    2011-01-01

    Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs. PMID:21183548

  2. Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

    SciTech Connect

    Jung, Eun Joo; Kim, Deok Ryong

    2011-01-21

    Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we established TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.

  3. Thermal response of proteins (histone H2AX, H3.1) by a coarse-grained Monte Carlo simulation with a knowledge-based phenomenological potential

    NASA Astrophysics Data System (ADS)

    Fritsche, Miriam; Heermann, Dieter; Pandey, Ras; Farmer, Barry

    2012-02-01

    Using a coarse-grained bond fluctuating model, we investigate structure and dynamics of two histones, H2AX (143 residues) and H3.1 (136 residues) as a function of temperature (T). A knowledged based contact matrix is used as an input for a phenomenological residue-residue interaction in a generalized Lennard-Jones potential. Metropolis algorithm is used to execute stochastic movement of each residue. A number of local and global physical quantities are analyzed. Despite unique energy and mobility profiles of its residues in a specific sequence, the histone H3.1 appears to undergo a structural transformation from a random coil to a globular conformation on reducing the temperature. The radius of gyration of the histone H2AX, in contrast, exhibits a non-monotonic dependence on temperature with a maximum at a characteristic temperature (Tc) where crossover occurs from a positive (stretching below Tc) to negative (contraction above Tc) thermal response on increasing T. Multi-scale structures of the proteins are examined by a detailed analysis of their structure functions.

  4. Proteomic dissection of cell type-specific H2AX-interacting protein complex associated with hepatocellular carcinoma

    PubMed Central

    Yang, Xiaoli; Zou, Peng; Yao, Jun; Yun, Dong; Bao, Huimin; Du, Ruyun; Long, Jing; Chen, Xian

    2010-01-01

    The replacement histone variant H2AX senses DNA double-strand breaks (DSBs) and recruits characteristic sets of proteins at its phosphorylated (γ-H2AX) foci for concurrent DNA repair. We reasoned that the H2AX interaction network, or interactome formed in the tumor-associated DNA DSB environment such as in hepatocellular carcinoma (HCC) cells, where pre-neoplastic lesions frequently occur, is indicative of HCC pathogenic status. By using an in vivo dual-tagging quantitative proteomic method, we identified 102 H2AX-specific interacting partners in HCC cells that stably expressed FLAG-tagged H2AX at close to the endogenous level. Using bioinformatics tools for data-dependent network analysis, we further found binary relationships among these interactors in defined pathway modules, implicating H2AX in a multi-functional role of coordinating a variety of biological pathways involved in DNA damage recognition and DNA repair, apoptosis, nucleic acid metabolism, Ca2+-binding signaling, cell cycle, etc. Furthermore our observations suggest that these pathways interconnect through key pathway components or H2AX interactors. The physiological accuracy of our quantitative proteomic approach in determining H2AX-specific interactors was evaluated by both co-immunoprecipitation/ immunoblotting and confocal co-localization experiments performed on HCC cells. Due to their involvement in diverse functions, the H2AX interactors involved in different pathway modules, such as Poly(ADP-ribose) polymerase1, 14-3-3ζ, coflin1, and peflin1, were examined for their relative H2AX binding affinities in paired hepatocytes and HCC cells. Treatment with the DSB-inducing agent bleomycin enhanced binding of these proteins to H2AX, suggesting an active role of H2AX in coordinating the functional pathways of each protein in DNA damage recognition and repair. PMID:20000738

  5. Conformational Temperature-Dependent Behavior of a Histone H2AX: A Coarse-Grained Monte Carlo Approach Via Knowledge-Based Interaction Potentials

    PubMed Central

    Fritsche, Miriam; Pandey, Ras B.; Farmer, Barry L.; Heermann, Dieter W.

    2012-01-01

    Histone proteins are not only important due to their vital role in cellular processes such as DNA compaction, replication and repair but also show intriguing structural properties that might be exploited for bioengineering purposes such as the development of nano-materials. Based on their biological and technological implications, it is interesting to investigate the structural properties of proteins as a function of temperature. In this work, we study the spatial response dynamics of the histone H2AX, consisting of 143 residues, by a coarse-grained bond fluctuating model for a broad range of normalized temperatures. A knowledge-based interaction matrix is used as input for the residue-residue Lennard-Jones potential. We find a variety of equilibrium structures including global globular configurations at low normalized temperature (), combination of segmental globules and elongated chains (), predominantly elongated chains (), as well as universal SAW conformations at high normalized temperature (). The radius of gyration of the protein exhibits a non-monotonic temperature dependence with a maximum at a characteristic temperature () where a crossover occurs from a positive (stretching at ) to negative (contraction at ) thermal response on increasing . PMID:22442661

  6. γH2AX and Chk1 phosphorylation as predictive pharmacodynamic biomarkers of Chk1 inhibitor-chemotherapy combination treatments

    PubMed Central

    2014-01-01

    Background Chk1 inhibitors are currently in clinical trials in combination with a range of cytotoxic agents and have the potential to potentiate the clinical activity of a large number of standard of care chemotherapeutic agents. Utilizing pharmacodynamic biomarkers to optimize drug dose and scheduling in these trials could greatly enhance the likelihood of clinical success. Methods In this study, we evaluated the in vitro potentiation of the cytotoxicity of a range of cytotoxic chemotherapeutic drugs by the novel Chk1 inhibitor V158411 in p53 mutant colon cancer cells. Pharmacodynamic biomarkers were evaluated in vitro. Results V158411 potentiated the cytotoxicity of a range of chemotherapeutic agents with distinct mechanisms of action in p53 mutant colon cancer cell lines grown in anchorage dependent or independent culture conditions. Analysis of pharmacodynamic biomarker changes identified dependencies on the chemotherapeutic agent, the concentration of the chemotherapeutic and the duration of time between combination treatment and biomarker analysis. A reduction in total Chk1 and S296/S317/S345 phosphorylation occurred consistently with all cytotoxics in combination with V158411 but did not predict cell line potentiation. Induction of γH2AX levels was chemotherapeutic dependent and correlated closely with potentiation of gemcitabine and camptothecin in p53 mutant colon cancer cells. Conclusions Our results suggest that Chk1 phosphorylation could be a useful biomarker for monitoring inhibition of Chk1 activity in clinical trials involving a range of V158411-chemotherapy combinations and γH2AX induction as a predictor of potentiation in combinations containing gemcitabine or camptothecin. PMID:24996846

  7. Redundant and nonredundant functions of ATM and H2AX in αβ T-lineage lymphocytes.

    PubMed

    Yin, Bu; Lee, Baeck-Seung; Yang-Iott, Katherine S; Sleckman, Barry P; Bassing, Craig H

    2012-08-01

    The ataxia telangiectasia mutated (ATM) kinase and H2AX histone tumor suppressor proteins are each critical for maintenance of cellular genomic stability and suppression of lymphomas harboring clonal translocations. ATM is the predominant kinase that phosphorylates H2AX in chromatin around DNA double-strand breaks, including along lymphocyte Ag receptor loci cleaved during V(D)J recombination. However, combined germline inactivation of Atm and H2ax in mice causes early embryonic lethality associated with substantial cellular genomic instability, indicating that ATM and H2AX exhibit nonredundant functions in embryonic cells. To evaluate potential nonredundant roles of ATM and H2AX in somatic cells, we generated and analyzed Atm-deficient mice with conditional deletion of H2ax in αβ T-lineage lymphocytes. Combined Atm/H2ax inactivation starting in early-stage CD4(-)/CD8(-) thymocytes resulted in lower numbers of later-stage CD4(+)/CD8(+) thymocytes, but led to no discernible V(D)J recombination defect in G1 phase cells beyond that observed in Atm-deficient cells. H2ax deletion in Atm-deficient thymocytes also did not affect the incidence or mortality of mice from thymic lymphomas with clonal chromosome 14 (TCRα/δ) translocations. Yet, in vitro-stimulated Atm/H2ax-deficient splenic αβ T cells exhibited a higher frequency of genomic instability, including radial chromosome translocations and TCRβ translocations, compared with cells lacking Atm or H2ax. Collectively, our data demonstrate that both redundant and nonredundant functions of ATM and H2AX are required for normal recombination of TCR loci, proliferative expansion of developing thymocytes, and maintenance of genomic stability in cycling αβ T-lineage cells. PMID:22730535

  8. High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

    PubMed Central

    Singh, Indrabahadur; Ozturk, Nihan; Cordero, Julio; Mehta, Aditi; Hasan, Diya; Cosentino, Claudia; Sebastian, Carlos; Krüger, Marcus; Looso, Mario; Carraro, Gianni; Bellusci, Saverio; Seeger, Werner; Braun, Thomas; Mostoslavsky, Raul; Barreto, Guillermo

    2015-01-01

    The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome. PMID:26045162

  9. Histone phosphorylation

    PubMed Central

    Rossetto, Dorine; Avvakumov, Nikita; Côté, Jacques

    2012-01-01

    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. PMID:22948226

  10. Genotoxicity screening via the γH2AX by flow assay.

    PubMed

    Smart, D J; Ahmedi, K P; Harvey, J S; Lynch, A M

    2011-10-01

    The measurement of serine139-phosphorylated histone H2AXH2AX) provides a biomarker of DNA double-strand breaks (DSBs) and may identify potential genotoxic activity. In order to evaluate a flow cytometry assay for γH2AX detection (hereafter termed the γH2AX by flow assay), 6 prototypical (3 pro- and 3 proximate) genotoxins, i.e. dimethylbenz[a]anthracene (DMBA), 2-acetylaminofluorene (2-AAF), benzo[a]pyrene (B[a]P), methyl methane sulphonate (MMS), methyl nitrosourea (MNU) and 4-nitroquinoline oxide (4NQO), were selected to define assay evaluation criteria. In addition, 3 non-genotoxic cytotoxins (phthalic anhydride, n-butyl chloride and hexachloroethane) were included to investigate the influence of cytotoxicity on assay performance. At similar cytotoxicity levels (relative cell counts; RCC 75-40%) all prototypical genotoxins induced marked concentration-dependent increases in γH2AX compared with the non-genotoxins. As a result, assay evaluation criteria for a positive effect were defined as >1.5-fold γH2AX @ RCC >25%. Twenty five additional chemicals with diverse structures and genotoxic activity were selected to evaluate the γH2AX by flow assay. Results were compared with Ames bacterial and in vitro mammalian genotoxicity tests (mouse lymphoma assay and/or chromosome aberration assay). γH2AX by flow assay results were highly predictive of Ames (sensitivity 100%; specificity 67%; concordance 82%) and in vitro mammalian genotoxicity tests (sensitivity 91%; specificity 89%; concordance 91%) and provide additional evidence that γH2AX is a biomarker of potential genotoxic activity, underpinned mechanistically by the cellular response to DSBs. Discordant findings were predominately attributed to differences in specificity for some mammalian cell genotoxins that are Ames non-mutagens or for "biologically-irrelevant" positives in the mammalian tests. Simple anilines were classified as genotoxic following rat liver S9-mediated bioactivation, however, effects on

  11. The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts

    SciTech Connect

    Paulsen, Michelle T.; Ljungman, Mats . E-mail: ljungman@umich.edu

    2005-11-15

    Juglone (5-hydroxy-1,4-naphtoquinone) is a natural toxin produced by walnut trees. In this study we show that juglone differentially reduces viability of human cells in culture. Normal fibroblast were found to be especially sensitive to juglone and lost viability primarily through a rapid apoptotic and necrotic response. This response may have been triggered by DNA damage since juglone induced a rapid and strong phosphorylation of H2AX in all phases of the cell cycle. Furthermore, juglone inhibits mRNA synthesis in human fibroblasts in a dose-dependent manner. Surprisingly, juglone caused a drastic reduction of the basal level of p53 in human fibroblasts and this loss could not be fully rescued by proteasome and calpain I inhibitors. However, when cells were pretreated with UV light or ionizing radiation, juglone was not able to reduce the cellular levels of activated p53. Our results show that juglone has multiple effects on cells such as the induction of DNA damage, inhibition of transcription, reduction of p53 protein levels and the induction of cell death.

  12. γH2AX expression in cytological specimens as a biomarker of response to radiotherapy in solid malignancies

    PubMed Central

    Shah, Ketan; Boghozian, Ramon A.; Kartsonaki, Christiana; Shah, Ketan A.

    2015-01-01

    Many anticancer treatments, including radiotherapy, act by damaging DNA and hindering cell function and proliferation. H2AX is a histone protein directly associated with DNA that is phosphorylated to produce γH2AX that accumulates in foci in an early response to DNA double‐strand breaks, the most deleterious lesion caused by anticancer therapy. This study reports a γH2AX detection assay that has the potential to be used as a biomarker of response to guide cancer treatment. γH2AX immunostaining was applied to tumour cell specimens obtained using fine needle aspiration (FNA). Liquid‐based cytology and direct smear cytology methods were evaluated and immunostaining protocols established using FNA samples from five cancer patients. The assay was then applied to three patients before and after radiotherapy. Results demonstrate induction of γH2AX foci following treatment, persisting for as long as one week after therapy. Immunostaining for γH2AX has been successfully applied to FNA samples, providing an opportunity to evaluate γH2AX as a treatment response marker in cancer. Diagn. Cytopathol. 2016;44:141–146. © 2015 The Authors Diagnostic Cytopathology Published by Wiley Periodicals, Inc. PMID:26667983

  13. Age-Related Disease Association of Endogenous γ-H2AX Foci in Mononuclear Cells Derived from Leukapheresis

    PubMed Central

    Schurman, Shepherd H.; Dunn, Christopher A.; Greaves, Rebecca; Yu, Binbing; Ferrucci, Luigi; Croteau, Deborah L.; Seidman, Michael M.; Bohr, Vilhelm A.

    2012-01-01

    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. PMID:23029205

  14. New mechanism of γ-H2AX generation: Surfactant-induced actin disruption causes deoxyribonuclease I translocation to the nucleus and forms DNA double-strand breaks.

    PubMed

    Zhao, Xiaoxu; Yang, Gang; Toyooka, Tatsushi; Ibuki, Yuko

    2015-12-01

    We previously showed that nonionic surfactants, nonylphenol polyethoxylates (NPEOs), induced phosphorylation of histone H2AX, forming γ-H2AX. In this study, we analyzed the mechanism of γ-H2AX generation by an NPEO with 15 ethylene oxide units (NPEO(15)). In MCF-7 breast carcinoma cells, NPEO(15) treatment induced γ-H2AX in a dose-dependent manner. EDTA and ZnCl2, two inhibitors of deoxyribonuclease I (DNase I), inhibited both the γ-H2AX and DNA double-strand breaks induced by NPEO(15). NPEO(15) disrupted filamentous actin and released free DNase I as detected by cell fractionation analysis. Based on immunofluorescence staining of DNase I and monitoring DNase I-GFP localization, DNase I was translocated from the cytosol to the nucleus of cells after treatment with NPEO(15). This translocation did not occur with the common DNA damage inducers ultraviolet B irradiation and hydrogen peroxide. Other surfactants, Tween 20, Triton X-100 and Nonidet P-40, also generated γ-H2AX. These results show that γ-H2AX induction by surfactants including NPEOs, occurs via a new mechanism involving release of free DNase I with actin disruption. This mechanism is distinct from the process of γ-H2AX generation caused by direct chemically induced DNA damage. PMID:26653977

  15. Essential role of autoactivation circuitry on Aurora B-mediated H2AX-pS121 in mitosis

    PubMed Central

    Shimada, Midori; Goshima, Takahiro; Matsuo, Hiromi; Johmura, Yoshikazu; Haruta, Mayumi; Murata, Kazuhiro; Tanaka, Hiromitsu; Ikawa, Masahito; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Proper deposition and activation of Aurora B at the centromere is critical for faithful chromosome segregation in mammals. However, the mechanistic basis for abrupt Aurora B kinase activation at the centromere has not yet been fully understood. We demonstrate here that Aurora B-mediated phosphorylation of histone H2AX at serine 121 (H2AX-pS121) promotes Aurora B autophosphorylation and is essential for proper chromosome segregation. Aurora B-mediated H2AX-pS121 is specifically detected at the centromere during mitosis. H2AX depletion results in a severe defect in activation and deposition of Aurora B at this locus. A phosphomimic mutant of H2AX at S121 interacts with activated Aurora B more efficiently than wild-type in vitro. Taken together, these results propose a model in which Aurora B-mediated H2AX-pS121 probably provide a platform for Aurora B autoactivation circuitry at centromeres and thus play a pivotal role in proper chromosome segregation. PMID:27389782

  16. Essential role of autoactivation circuitry on Aurora B-mediated H2AX-pS121 in mitosis.

    PubMed

    Shimada, Midori; Goshima, Takahiro; Matsuo, Hiromi; Johmura, Yoshikazu; Haruta, Mayumi; Murata, Kazuhiro; Tanaka, Hiromitsu; Ikawa, Masahito; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Proper deposition and activation of Aurora B at the centromere is critical for faithful chromosome segregation in mammals. However, the mechanistic basis for abrupt Aurora B kinase activation at the centromere has not yet been fully understood. We demonstrate here that Aurora B-mediated phosphorylation of histone H2AX at serine 121 (H2AX-pS121) promotes Aurora B autophosphorylation and is essential for proper chromosome segregation. Aurora B-mediated H2AX-pS121 is specifically detected at the centromere during mitosis. H2AX depletion results in a severe defect in activation and deposition of Aurora B at this locus. A phosphomimic mutant of H2AX at S121 interacts with activated Aurora B more efficiently than wild-type in vitro. Taken together, these results propose a model in which Aurora B-mediated H2AX-pS121 probably provide a platform for Aurora B autoactivation circuitry at centromeres and thus play a pivotal role in proper chromosome segregation. PMID:27389782

  17. The γH2AX DNA damage assay from a drop of blood

    PubMed Central

    Heylmann, Daniel; Kaina, Bernd

    2016-01-01

    DNA double-strand breaks (DSB) and blocked replication forks activate the DNA damage response (DDR), a signaling pathway marked by phosphorylation of histone 2AX (H2AX). The phosphorylated form, γH2AX, accumulates at the site of damage and can be detected as foci by immunocytochemistry. Therefore, γH2AX is a sensitive and robust biomarker of DNA damage, notably DSB. Cells from peripheral blood are often used for studies on genotoxic exposure of humans. They are limited, however, by the amount of blood required and the costly blood purification method. Here, we present a method that enables the detection of DNA damage by the analysis of γH2AX foci in a drop of blood. The blood drop method (BDM) is simple, fast, inexpensive and allows large series of blood sampling and storage over time. It can be combined with genotoxic treatment of cells in the collected blood sample for experimental purposes on DNA damage induction and repair. The BDM is suitable for rapid and large-scale screenings of genetic damage in human and animal populations. PMID:26940638

  18. Induction and quantification of gammma-H2AX foci following cx- and gamma-irradiaton

    NASA Technical Reports Server (NTRS)

    Leatherbarrow, E. L.; Cucinotta, F. A.; O'Neill, Peter

    2004-01-01

    Following DNA damage the histone H2AX becomes phosphorylated and can be visualised by immunofluorescence as an indicator of DSBs in individual cells. Using a wild type hamster cell line (V79-4) exposed to either a-particles or to Co-60 gamma-rays to induce DNA DSBs at different doses (20-200OmGy), the dose dependent induction of gamma-H2AX foci were scored both manually (by eye) and using image analysis. A linearly dependence on dose was found for both radiations. The number of DSBs determined by image analysis after a post-irradiation period of 30 minutes at 37 C, is 16.6 foci/cell/Gy for alpha-irradiation and 12.2 foci/cell/Gy for gamma-irradiation; the latter being 3-4 times the levels observed by eye and comparable to gamma-radiation-induced levels of prompt DSBs more recently reported using pulse field gel electrophoresis (approx. 16 DSBs/Gy). The average size of the gamma-H2AX foci induced by alpha-irradiation (0.30 square micrometers) is approximately 1.5 times larger than those induced by gamma-irradiation (0.19 square micrometers). The timescale of induction and removal of DSBs up to 24 hours post-irradiation, was investigated with gamma-H2AX foci levels found to remain significantly higher than controls for 4 or 6 hours in gamma-irradiated samples or alpha irradiated samples, respectively. These results demonstrate that not only gamma radiation but also alpha-radiation induce phosphorylation of the H2AX histone in response to DSBs even at low doses (20mGy for gamma-rays, 1 track/cell on average for alpha-particles) and the variation in size and dephosphorylation of the induced foci is dependent on radiation quality (LET).

  19. ATR, BRCA1 and gammaH2AX localize to unsynapsed chromosomes at the pachytene stage in human oocytes.

    PubMed

    Garcia-Cruz, R; Roig, I; Robles, P; Scherthan, H; Garcia Caldés, M

    2009-01-01

    Asynapsis of homologous chromosomes at the pachytene stage has been associated with gametogenic failure and infertility, but the cellular mechanisms involved are currently unknown in human meiocytes. In mice, the protein encoded by the breast-cancer susceptibility gene Brca1 has been described to direct kinase ATR (ataxia telangiectasia and Rad3 related) to any unpaired DNA at the pachytene stage, where ATR triggers H2AX phosphorylation, resulting in the silencing of those chromosomes. In this study, the distribution of ATR, BRCA1 and the phosphorylated histone gammaH2AX is assessed by immunofluorescence in human oocytes and it is found that they localize at unpaired chromosomes at the pachytene stage. Evidence is shown to propose that BRCA1, ATR and gammaH2AX in the human may be part of a system such as the one previously described in mouse, which signals unsynapsed chromosomes at pachytene and may lead to their silencing. PMID:19146767

  20. Positive immunohistochemical staining of gammaH2AX is associated with tumor progression in gastric cancers from radiation-exposed patients.

    PubMed

    Sentani, Kazuhiro; Oue, Naohide; Sakamoto, Naoya; Nishisaka, Takashi; Fukuhara, Toshiyuki; Matsuura, Hiroo; Yasui, Wataru

    2008-11-01

    To elucidate the mechanism of radiation-induced cancers, molecular analysis of cancers in atomic bomb (A-bomb) exposure is important. DNA double-strand breaks (DSBs) are thought to be caused by the deleterious effects of ionizing radiation, and gammaH2AX (serine 139 phosphorylated form of histone H2AX) is reported to be a significant marker for DSBs. In the present study, we performed immunohistochemical analysis of gammaH2AX in gastric cancers (GCs) from 66 exposed and 47 non-exposed patients who developed GC after the bombing. Of the 47 GCs from non-exposed patients, 6 (13%) cases showed nuclear positive staining for gammaH2AX, whereas of the 66 GCs from exposed patients, 20 (30%) cases were positive (P=0.0405). However, among stage I GC, there was no significant difference in gammaH2AX expression frequency between exposed patients and non-exposed patients. Among exposed patients, stage II-IV cases were more frequently positive for gammaH2AX than stage I cases (P=0.0197). Among GCs from non-exposed patients, gammaH2AX staining showed no significant association with Lauren's classification, depth of invasion, lymph node metastasis or TNM stage. These results suggest that the characteristics of tumor cells differ between GCs from exposed and non-exposed patients. DSBs may be involved in progression of GC in exposed patients. PMID:18949412

  1. DNA DSB measurements and modelling approaches based on gamma-H2AX foci time evolution

    NASA Astrophysics Data System (ADS)

    Esposito, Giuseppe; Campa, Alessandro; Antonelli, Francesca; Mariotti, Luca; Belli, Mauro; Giardullo, Paola; Simone, Giustina; Antonella Tabocchini, Maria; Ottolenghi, Andrea

    DNA double strand breaks (DSBs) induced by ionising radiation are considered the main dam-age related to the deleterious consequences in the cells. Unrepaired or mis-repaired DSBs can cause mutations or loss of chromosome regions which can eventually lead to cell death or neo-plastic transformation. Quantification of the number and complexity of DSBs induced by low doses of radiation remains a complex problem. About ten years ago Rogakou et al. proposed an immunofluorescent technique able to detect even a single DSB per cell. This approach is based on the serine 139 phosphorylation of many molecules (up to 2000) of histone H2AX (γg-H2AX) following the induction of a DSB in the DNA. DSB can be visualized as foci by immunofluores-cence by using phospho-specific antibodies, so that enumeration of foci can be used to measure DSB induction and processing. It is still not completely clear how γ-H2AX dephosphorylation takes place; however it has been related with DSB repair, in particular with the efficiency of DSB repair. In this work we analyse the H2AX phosphorylation-dephosphorylation kinetics after irradiation of primary human fibroblasts (AG1522 cell line) with radiation of differing quality, that is γ-rays and α-particles (125 keV/µm), with the aim of comparing the time evolution of γ-H2AX foci. Our results show that, after a dose of 0.5 Gy, both γ-rays and α-particles induce the maximum number of γ-H2AX foci within 30 minutes from irradiation, that this number depends on the radiation type and is consistent with the number of track traversal in α-irradiated nuclei, that the dephosphorylation kinetics are very different, being the α-induced foci rate of disappearence slower than that of γ-induced foci. In this work a modellistic approach to estimate the number of DSB induced by γ-rays detectable by using the γ-H2AX assay is presented. The competing processes of appearance and disappearance of visible foci will be modeled taking into account the

  2. γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin

    NASA Astrophysics Data System (ADS)

    Redon, Christophe E.; Dickey, Jennifer S.; Bonner, William M.; Sedelnikova, Olga A.

    2009-04-01

    Ionizing radiation (IR) exposure is inevitable in our modern society and can lead to a variety of deleterious effects including cancer and birth defects. A reliable, reproducible and sensitive assessment of exposure to IR and the individual response to that exposure would provide much needed information for the optimal treatment of each donor examined. We have developed a diagnostic test for IR exposure based on detection of the phosphorylated form of variant histone H2AX (γ-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The cell responds to a nascent DSB through the phosphorylation of thousands of H2AX molecules flanking the damaged site. This highly amplified response can be visualized as a γ-H2AX focus in the chromatin that can be detected in situ with the appropriate antibody. Here we assess the usability of γ-H2AX focus formation as a possible biodosimeter for human exposure to IR using peripheral blood lymphocytes irradiated ex vivo and three-dimensional artificial models of human skin biopsies. In both systems, the tissues were exposed to 0.2-5 Gy, doses of IR that might be realistically encountered in various scenarios such as cancer radiotherapies or accidental exposure to radiation. Since the γ-H2AX response is maximal 30 min after exposure and declines over a period of hours as the cells repair the damage, we examined the time limitations of the useful detectability of γ-H2AX foci. We report that a linear response proportional to the initial radiation dose was obtained 48 and 24 h after exposure in blood samples and skin cells respectively. Thus, detection of γ-H2AX formation to monitor DNA damage in minimally invasive blood and skin tests could be useful tools to determine radiation dose exposure and analyze its effects on humans.

  3. Low doses of X-rays induce prolonged and ATM-independent persistence of γH2AX foci in human gingival mesenchymal stem cells.

    PubMed

    Osipov, Andreyan N; Pustovalova, Margarita; Grekhova, Anna; Eremin, Petr; Vorobyova, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry Y; Eremin, Ilya

    2015-09-29

    Diagnostic imaging delivering low doses of radiation often accompany human mesenchymal stem cells (MSCs)-based therapies. However, effects of low dose radiation on MSCs are poorly characterized. Here we examine patterns of phosphorylated histone H2AXH2AX) and phospho-S1981 ATM (pATM) foci formation in human gingiva-derived MSCs exposed to X-rays in time-course and dose-response experiments. Both γH2AX and pATM foci accumulated linearly with dose early after irradiation (5-60 min), with a maximum induction observed at 30-60 min (37 ± 3 and 32 ± 3 foci/cell/Gy for γH2AX and pATM, respectively). The number of γH2AX foci produced by intermediate doses (160 and 250 mGy) significantly decreased (40-60%) between 60 and 240 min post-irradiation, indicating rejoining of DNA double-strand breaks. In contrast, γH2AX foci produced by low doses (20-80 mGy) did not change after 60 min. The number of pATM foci between 60 and 240 min decreased down to control values in a dose-independent manner. Similar kinetics was observed for pATM foci co-localized with γH2AX foci. Collectively, our results suggest differential DNA double-strand break signaling and processing in response to low vs. intermediate doses of X-rays in human MSCs. Furthermore, mechanisms governing the prolonged persistence of γH2AX foci in these cells appear to be ATM-independent. PMID:26314960

  4. Analysis of Lymphocytic DNA Damage in Early Multiple Sclerosis by Automated Gamma-H2AX and 53BP1 Foci Detection: A Case Control Study

    PubMed Central

    Rasche, Ludwig; Heiserich, Lisa; Behrens, Janina Ruth; Lenz, Klaus; Pfuhl, Catherina; Wakonig, Katharina; Gieß, René Markus; Freitag, Erik; Eberle, Caroline; Wuerfel, Jens; Dörr, Jan; Bauer, Peter; Bellmann-Strobl, Judith; Paul, Friedemann; Roggenbuck, Dirk; Ruprecht, Klemens

    2016-01-01

    Background In response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS). Objective To evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection. Methods Immunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls. Results The median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not

  5. Highly Sensitive Automated Method for DNA Damage Assessment: Gamma-H2AX Foci Counting and Cell Cycle Sorting

    PubMed Central

    Hernández, Laia; Terradas, Mariona; Martín, Marta; Tusell, Laura; Genescà, Anna

    2013-01-01

    Phosphorylation of the H2AX protein is an early step in the double strand break (DSB) repair pathway; therefore, phosphorylated histoneH2AX) foci scoring is widely used as a measure for DSBs. Foci scoring is performed either manually or semi-automatically using hand-operated capturing and image analysis software. In general, both techniques are laborious and prone to artifacts associated with manual scoring. While a few fully automated methods have been described in the literature, none of them have been used to quantify γH2AX foci in combination with a cell cycle phase analysis. Adding this feature to a rapid automated γH2AX foci quantification method would reduce the scoring uncertainty that arises from the variations in the background level of the γH2AX signal throughout the cell cycle. The method was set up to measure DNA damage induced in human mammary epithelial cells by irradiation under a mammogram device. We adapted a FISH (fluorescent in situ hybridization) Spot-counting system, which has a slide loader with automatic scanning and cell capture system throughout the thickness of each cell (z-stack), to meet our assay requirements. While scanning the sample, the system classifies the selected nuclei according to the signal patterns previously described by the user. For our purposes, a double staining immunofluorescence was carried out with antibodies to detect γH2AX and pericentrin, an integral component of the centrosome. We could thus distinguish both the number of γH2AX foci per cell and the cell cycle phase. Furthermore, restrictive settings of the program classifier reduced the “touching nuclei” problem described in other image analysis software. The automated scoring was faster than and as sensitive as its manually performed counterpart. This system is a reliable tool for γH2AX radio-induced foci counting and provides essential information about the cell cycle stage. It thus offers a more complete and rapid assessment of DNA damage. PMID

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

    SciTech Connect

    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.

  7. Chronic oxidative stress promotes H2AX protein degradation and enhances chemosensitivity in breast cancer patients.

    PubMed

    Gruosso, Tina; Mieulet, Virginie; Cardon, Melissa; Bourachot, Brigitte; Kieffer, Yann; Devun, Flavien; Dubois, Thierry; Dutreix, Marie; Vincent-Salomon, Anne; Miller, Kyle Malcolm; Mechta-Grigoriou, Fatima

    2016-01-01

    Anti-cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD-/Nrf2-antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly-ubiquitination and promotes its degradation by the proteasome. ROS-mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple-Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2-antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS-mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients. PMID:27006338

  8. Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis.

    PubMed

    Fujitani, Kazuko; Otomo, Asako; Wada, Mikako; Takamatsu, Nobuhiko; Ito, Michihiko

    2016-04-01

    In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AXH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively. PMID:27239441

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

    PubMed Central

    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

  10. The SAGA Deubiquitination Module Promotes DNA Repair and Class Switch Recombination through ATM and DNAPK-Mediated γH2AX Formation.

    PubMed

    Ramachandran, Shaliny; Haddad, Dania; Li, Conglei; Le, Michael X; Ling, Alexanda K; So, Clare C; Nepal, Rajeev M; Gommerman, Jennifer L; Yu, Kefei; Ketela, Troy; Moffat, Jason; Martin, Alberto

    2016-05-17

    Class switch recombination (CSR) requires activation-induced deaminase (AID) to instigate double-stranded DNA breaks at the immunoglobulin locus. DNA breaks activate the DNA damage response (DDR) by inducing phosphorylation of histone H2AX followed by non-homologous end joining (NHEJ) repair. We carried out a genome-wide screen to identify CSR factors. We found that Usp22, Eny2, and Atxn7, members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitination module, are required for deubiquitination of H2BK120ub following DNA damage, are critical for CSR, and function downstream of AID. The SAGA deubiquitinase activity was required for optimal irradiation-induced γH2AX formation, and failure to remove H2BK120ub inhibits ATM- and DNAPK-induced γH2AX formation. Consistent with this effect, these proteins were found to function upstream of various double-stranded DNA repair pathways. This report demonstrates that deubiquitination of histone H2B impacts the early stages of the DDR and is required for the DNA repair phase of CSR. PMID:27160905

  11. TOPORS modulates H2AX discriminating genotoxic stresses.

    PubMed

    Seong, Ki Moon; Nam, Seon Young; Kim, Ji-Young; Yang, Kwang Hee; An, Sungkwan; Jin, Young-Woo; Kim, Cha Soon

    2012-11-01

    H2AX plays an important role in chromatin reorganization implicated in DNA repair and apoptosis under various DNA damaging conditions. In this study, the interaction between TOPORS (topoisomerase I-binding protein) and H2AX was verified using mammalian cell extracts exposed to diverse DNA damaging stresses such as ionizing radiation, doxorubicin, camptothecin, and hydrogen peroxide. In vitro assays for ubiquitination revealed that TOPORS functions as a novel E3 ligase for H2AX ubiquitination. TOPORS was found to be dissociated from H2AX proteins when cells were exposed to oxidative stress, but not replication-inducing DNA damaging stress. The protein stability of H2AX was decreased when TOPORS was ectopically expressed in cells, and oxidative stresses such as hydrogen peroxide and ionizing radiation induced recovery of the H2AX protein level. Therefore, these biochemical data suggest that TOPORS plays a key role in the turnover of H2AX protein, discriminating the type of DNA damaging stress. PMID:22972498

  12. Gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in targeted and bystander human artificial skin models and peripheral blood lymphocytes

    NASA Astrophysics Data System (ADS)

    Redon, Christophe; Dickey, Jennifer; Bonner, William; Sedelnikova, Olga

    Ionizing radiation (IR) exposure is inevitable. In addition to exposure from cosmic rays, the sun and radioactive substances, modern society has created new sources of radiation exposure such as space and high altitude journeys, X-ray diagnostics, radiological treatments and the increasing threat of radiobiological terrorism. For these reasons, a reliable, reproducible and sensitive assessment of dose and time exposure to IR is essential. We developed a minimally invasive diagnostic test for IR exposure based on detection of a phosphorylated variant of histone H2AX (gamma-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs). The phosphorylation of thousands of H2AX molecules forms a gamma-H2AX focus in the chromatin flanking the DSB site that can be detected in situ. We analyzed gamma- H2AX focus formation in both directly irradiated cells as well as in un-irradiated "bystanders" in close contact with irradiated cells. In order to insure minimal invasiveness, we examined commercially available artificial skin models as a surrogate for human skin biopsies as well as peripheral blood lymphocytes. In human skin models, cells in a thin plane were microbeamirradiated and gamma-H2AX formation was measured both in irradiated and in distal bystander cells over time. In irradiated cells DSB formation reached a maximum at 15-30 minutes post- IR and then declined within several hours; all cells were affected. In marked contrast, the incidence of DSBs in bystander cells reached a maximum by 12-48 hours post-irradiation, gradually decreasing over the 7 day time course. At the maxima, 40-60% of bystander cells were affected. Similarly, we analyzed blood samples exposed to IR ex vivo at doses ranging from 0.02 to 3 Gy. The amount of DNA damage was linear in respect to radiation dose and independent of the age or sex of the blood donor. The method is highly reproducible and highly sensitive. In directly irradiated cells, the number of gamma-H2AX foci peaked

  13. Ultrasound-induced DNA damage and signal transductions indicated by gammaH2AX

    NASA Astrophysics Data System (ADS)

    Furusawa, Yukihiro; Fujiwara, Yoshisada; Zhao, Qing-Li; Hassan, Mariame Ali; Ogawa, Ryohei; Tabuchi, Yoshiaki; Takasaki, Ichiro; Takahashi, Akihisa; Ohnishi, Takeo; Kondo, Takashi

    2011-09-01

    Ultrasound (US) has been shown to induce cancer cell death via different forms including apoptosis. Here, we report the potential of low-intensity pulsed US (LIPUS) to induce genomic DNA damage and subsequent DNA damage response. Using the ionizing radiation-induced DNA double-strand breaks (DSBs) as the positive control, we were able to observe the induction of DSBs (as neutral comet tails) and the subsequent formation of gammaH2AX-positive foci (by immunofluorescence detection) in human leukemia cells following exposure to LIPUS. The LIPUS-induced DNA damage arose most likely from the mechanical, but not sonochemical, effect of cavitation, based on our observation that the suppression of inertial cavitation abrogated the gammH2AX foci formation, whereas scavenging of free radical formation (e.g., hydroxyl radical) had no protective effect on it. Treatment with the specific kinase inhibitor of ATM or DNA-PKcs, which can phosphorylate H2AX Ser139, revealed that US-induced gammaH2AX was inhibited more effectively by the DNA-PK inhibitor than ATM kinase inhibitor. Notably, these inhibitor effects were opposite to those with radiation-induced gammH2AX. In conclusion, we report, for the first time that US can induce DNA damage and the DNA damage response as indicated by gammaH2AX was triggered by the cavitational mechanical effects. Thus, it is expected that the data shown here may provide a better understanding of the cellular responses to US.

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

    SciTech Connect

    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.

  15. Microwaves from GSM Mobile Telephones Affect 53BP1 and γ-H2AX Foci in Human Lymphocytes from Hypersensitive and Healthy Persons

    PubMed Central

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

    2005-01-01

    The data on biologic effects of nonthermal microwaves (MWs) from mobile telephones are diverse, and these effects are presently ignored by safety standards of the International Commission for Non-Ionizing Radiation Protection (ICNIRP). In the present study, we investigated effects of MWs of Global System for Mobile Communication (GSM) at different carrier frequencies on human lymphocytes from healthy persons and from persons reporting hypersensitivity to electromagnetic fields (EMFs). We measured the changes in chromatin conformation, which are indicative of stress response and genotoxic effects, by the method of anomalous viscosity time dependence, and we analyzed tumor suppressor p53-binding protein 1 (53BP1) and phosphorylated histone H2AX (γ-H2AX), which have been shown to colocalize in distinct foci with DNA double-strand breaks (DSBs), using immunofluorescence confocal laser microscopy. We found that MWs from GSM mobile telephones affect chromatin conformation and 53BP1/γ-H2AX foci similar to heat shock. For the first time, we report here that effects of MWs from mobile telephones on human lymphocytes are dependent on carrier frequency. On average, the same response was observed in lymphocytes from hypersensitive and healthy subjects. PMID:16140623

  16. ATM Localization and Heterochromatin Repair Depend on Direct Interaction of the 53BP1-BRCT2 Domain with γH2AX

    PubMed Central

    Baldock, Robert A.; Day, Matthew; Wilkinson, Oliver J.; Cloney, Ross; Jeggo, Penelope A.; Oliver, Antony W.; Watts, Felicity Z.; Pearl, Laurence H.

    2015-01-01

    Summary 53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications—H4K20me2 and H2AK13/K15ub—downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1. PMID:26628370

  17. ATM Localization and Heterochromatin Repair Depend on Direct Interaction of the 53BP1-BRCT2 Domain with γH2AX.

    PubMed

    Baldock, Robert A; Day, Matthew; Wilkinson, Oliver J; Cloney, Ross; Jeggo, Penelope A; Oliver, Antony W; Watts, Felicity Z; Pearl, Laurence H

    2015-12-15

    53BP1 plays multiple roles in mammalian DNA damage repair, mediating pathway choice and facilitating DNA double-strand break repair in heterochromatin. Although it possesses a C-terminal BRCT2 domain, commonly involved in phospho-peptide binding in other proteins, initial recruitment of 53BP1 to sites of DNA damage depends on interaction with histone post-translational modifications--H4K20me2 and H2AK13/K15ub--downstream of the early γH2AX phosphorylation mark of DNA damage. We now show that, contrary to current models, the 53BP1-BRCT2 domain binds γH2AX directly, providing a third post-translational mark regulating 53BP1 function. We find that the interaction of 53BP1 with γH2AX is required for sustaining the 53BP1-dependent focal concentration of activated ATM that facilitates repair of DNA double-strand breaks in heterochromatin in G1. PMID:26628370

  18. Evaluation of genotoxicity using automated detection of γH2AX in metabolically competent HepaRG cells.

    PubMed

    Quesnot, Nicolas; Rondel, Karine; Audebert, Marc; Martinais, Sophie; Glaise, Denise; Morel, Fabrice; Loyer, Pascal; Robin, Marie-Anne

    2016-01-01

    The in situ detection of γH2AX was recently reported to be a promising biomarker of genotoxicity. In addition, the human HepaRG hepatoma cells appear to be relevant for investigating hepatic genotoxicity since they express most of drug metabolizing enzymes and a wild type p53. The aim of this study was to determine whether the automated in situ detection of γH2AX positive HepaRG cells could be relevant for evaluation of genotoxicity after single or long-term repeated in vitro exposure compared to micronucleus assay. Metabolically competent HepaRG cells were treated daily with environmental contaminants and genotoxicity was evaluated after 1, 7 and 14 days. Using these cells, we confirmed the genotoxicity of aflatoxin B1 and benzo(a)pyrene and demonstrated that dimethylbenzanthracene, fipronil and endosulfan previously found genotoxic with comet or micronucleus assays also induced γH2AX phosphorylation. Furthermore, we showed that fluoranthene and bisphenol A induced γH2AX while no effect had been previously reported in HepG2 cells. In addition, induction of γH2AX was observed with some compounds only after 7 days, highlighting the importance of studying long-term effects of low doses of contaminants. Together, our data demonstrate that automated γH2AX detection in metabolically competent HepaRG cells is a suitable high-through put genotoxicity screening assay. PMID:26282955

  19. The nuclear γ-H2AX apoptotic ring: implications for cancers and autoimmune diseases.

    PubMed

    Solier, Stéphanie; Pommier, Yves

    2014-06-01

    Apoptosis is a fundamental process for metazoan development. It is also relevant to the pathophysiology of immune diseases and cancers and to the outcome of cancer chemotherapies, as well as being a target for cancer therapies. Apoptosis involves intrinsic pathways typically initiated by DNA damaging agents and engaging mitochondria, and extrinsic pathways typically initiated by "death receptors" and their ligands TRAIL and TNF at the cell surface. Recently, we discovered the apoptotic ring, which microscopically looks like a nuclear annular staining early in apoptosis. This ring is, in three-dimensional space, a thick intranuclear shell consisting of epigenetic modifications including histone H2AX and DNA damage response (DDR) proteins. It excludes the DNA repair factors usually associated with γ-H2AX in the DDR nuclear foci. Here, we summarize our knowledge of the apoptotic ring, and discuss its biological and pathophysiological relevance, as well as its value as a potential pharmacodynamic biomarker for anticancer therapies. PMID:24448903

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

    SciTech Connect

    Yamagata, Kazutsune; Kitabayashi, Issay

    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. Gamma-H2AX foci counting: image processing and control software for high-content screening

    NASA Astrophysics Data System (ADS)

    Barber, P. R.; Locke, R. J.; Pierce, G. P.; Rothkamm, K.; Vojnovic, B.

    2007-02-01

    Phosphorylation of the chromatin protein H2AX (forming γH2AX) is implicated in the repair of DNA double strand breaks (DSB's); a large number of H2AX molecules become phosphorylated at the sites of DSB's. Fluorescent staining of the cell nuclei for γH2AX, via an antibody, visualises the formation of these foci, allowing the quantification of DNA DSB's and forming the basis for a sensitive biological dosimeter of ionising radiation. We describe an automated fluorescence microscopy system, including automated image processing, to count γH2AX foci. The image processing is performed by a Hough transform based algorithm, CHARM, which has wide applicability for the detection and analysis of cells and cell colonies. This algorithm and its applications for cell nucleus and foci detection will be described. The system also relies heavily on robust control software, written using multi-threaded cbased modules in LabWindows/CVI that adapt to the timing requirements of a particular experiment for optimised slide/plate scanning and mosaicing, making use of modern multi-core processors. The system forms the basis of a general purpose high-content screening platform with wide ranging applications in live and fixed cell imaging and tissue micro arrays, that in future, can incorporate spectrally and time-resolved information.

  2. Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction

    PubMed Central

    Jiang, Bo; Zhao, Shengqian; Tao, Zhen; Wen, Jin; Yang, Yancheng; Zheng, Yin; Yan, Hongling; Sheng, Ying; Gao, Aimin

    2016-01-01

    Using an in vitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. In vitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues. PMID:27247425

  3. Controlled bile acid exposure to oesophageal mucosa causes up-regulation of nuclear γ-H2AX possibly via iNOS induction.

    PubMed

    Jiang, Bo; Zhao, Shengqian; Tao, Zhen; Wen, Jin; Yang, Yancheng; Zheng, Yin; Yan, Hongling; Sheng, Ying; Gao, Aimin

    2016-08-01

    Using an in vitro model in which flatmounts of oesophagus was periodically exposed to bile acids, we demonstrate, using multiple methods, that the bile acid receptor TGR5, inducible nitric oxide synthase (iNOS) and γ-histone family 2A variant (γ-H2AX) are up-regulated. This indicates that bile acids cause up-regulation of iNOS, which further causes genotoxic stress as evidenced by increase of the highly sensitive marker, phosphorylated histone. In vitro nitric oxide (NO) assays showed increased production of nitric acid in the oesophageal epithelium exposed to the bile acids. This increase was inhibited in the presence of the nonspecific iNOS inhibitor aminoguanidine (AG). Cumulatively, the results of the present study provide suggestion that not only acid reflux, but also non-acid reflux of bile may cause genotoxic stress. These aspects merit to be tested in wide spectrum of Barrett epithelial tissues. PMID:27247425

  4. Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation

    PubMed Central

    Nakatani, Tsunetoshi; Yamagata, Kazuo; Kimura, Tohru; Oda, Masaaki; Nakashima, Hiroyuki; Hori, Mayuko; Sekita, Yoichi; Arakawa, Tatsuhiko; Nakamura, Toshinobu; Nakano, Toru

    2015-01-01

    In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AXH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis. PMID:25694116

  5. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W.; Weiss, Robert H.; Murray, David

    2015-01-01

    Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21WAF1 (p21) exhibit low numbers of γH2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21−/−) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21−/− and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX. PMID:26006237

  6. Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

    PubMed Central

    2011-01-01

    Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds. PMID:21261999

  7. New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

    PubMed Central

    Dale Rein, Idun; Stokke, Caroline; Jalal, Marwa; Myklebust, June H; Patzke, Sebastian; Stokke, Trond

    2015-01-01

    Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AXH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G1 cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G2. Intriguingly, G2 cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G2H” compartment), to cells that there were almost negative and had about 2 foci (termed “G2L” compartment). EdU-labeling of S phase cells revealed that G2H was directly populated from S phase, while G2L was populated from G2H, but in control cells also directly from S phase. The length of G2H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G2H, and enter mitosis after a 2–3 h delay in G2L. PMID:26317799

  8. Structure of a protein (H2AX): a comparative study with knowledge-based interactions

    NASA Astrophysics Data System (ADS)

    Fritsche, Miriam; Heermann, Dieter; Farmer, Barry; Pandey, Ras

    2013-03-01

    The structural and conformational properties of the histone protein H2AX (with143 residues) is studied by a coarse-grained model as a function of temperature (T). Three knowledge-based phenomenological interactions (MJ, BT, and BFKV) are used as input to a generalized Lennard-Jones potential for residue-residue interactions. Large-scale Monte Carlo simulations are performed to identify similarity and differences in the equilibrium structures with these potentials. Multi-scale structures of the protein are examined by a detailed analysis of their structure functions. We find that the radius of gyration (Rg) of H2AX depends non-monotonically on temperature with a maximum at a characteristic value Tc, a common feature to each interaction. The characteristic temperature and the range of non-monotonic thermal response and decay pattern are, however, sensitive to interactions. A comparison of the structural properties emerging from three potentials will be presented in this talk. This work is supported by Air Force Research Laboratory.

  9. Staining Against Phospho-H2AX (γ-H2AX) as a Marker for DNA Damage and Genomic Instability in Cancer Tissues and Cells.

    PubMed

    Nagelkerke, Anika; Span, Paul N

    2016-01-01

    Phospho-H2AX or γ-H2AX- is a marker of DNA double-stranded breaks and can therefore be used to monitor DNA repair after, for example, irradiation. In addition, positive staining for phospho-H2AX may indicate genomic instability and telomere dysfunction in tumour cells and tissues. Here, we provide a protocol to perform immunostaining for phospho-H2AX on cells, cryosections and formalin-fixed, paraffin-embedded tissues. Crucial steps in the protocol and troubleshooting suggestions are indicated. We also provide suggestions on how to combine staining against γ-H2AX with stainings against components of the tumour microenvironment, such as hypoxia and blood vessels. PMID:27325258

  10. Histone tyrosine phosphorylation comes of age

    PubMed Central

    Singh, Rakesh Kumar

    2011-01-01

    Histones were discovered over a century ago and have since been found to be the most extensively post-translationally modified proteins, although tyrosine phosphorylation of histones had remained elusive until recently. The year 2009 proved to be a landmark year for histone tyrosine (Y) phosphorylation as five research groups independently discovered this modification. Three groups describe phosphorylation of Y142 in the variant histone H2A.X, where it may be involved in the cellular decision making process to either undergo DNA repair or apoptosis in response to DNA damage. Further, one group suggests that phosphorylation of histone H3 on Y99 is crucial for its regulated proteolysis in yeast, while another found that Y41 phosphorylation modulates chromatin architecture and oncogenesis in mammalian cells. These pioneering studies provide the initial conceptual framework for further analyses of the diverse roles of tyrosine phosphorylation on different histones, with far reaching implications for human health and disease. PMID:20935492

  11. Activation of p38 mitogen-activated protein kinase by celecoxib oppositely regulates survivin and gamma-H2AX in human colorectal cancer cells

    SciTech Connect

    Hsiao, P.-W.; Chang, C.-C.; Liu, H.-F.; Tsai, C.-M.; Chiu, Ted H.; Chao, J.-I . E-mail: chaoji@mail.tcu.edu.tw

    2007-07-01

    Cancer cells express survivin that facilitates tumorigenesis. Celecoxib has been shown to reduce human colorectal cancers. However, the role and regulation of survivin by celecoxib in colorectal carcinoma cells remain unclear. Treatment with 40-80 {mu}M celecoxib for 24 h induced cytotoxicity and proliferation inhibition via a concentration-dependent manner in RKO colorectal carcinoma cells. Celecoxib blocked the survivin protein expression and increased the phosphorylation of H2AX at serine-193 ({gamma}-H2AX). The survivin gene knockdown by transfection with a survivin siRNA revealed that the loss of survivin correlated with the expression of {gamma}-H2AX. Meanwhile, celecoxib increased caspase-3 activation and apoptosis. Celecoxib activated the phosphorylation of p38 mitogen-activated protein (MAP) kinase. The phosphorylated proteins of p38 MAP kinase and {gamma}-H2AX were observed in the apoptotic cells. SB203580, a specific p38 MAP kinase inhibitor, protected the survivin protein expression and decreased the levels of {gamma}-H2AX and apoptosis in the celecoxib-exposed cells. The blockade of survivin expression increased the celecoxib-induced cytotoxicity; conversely, overexpression of survivin by transfection with a survivin-expressing vector raised the cancer cell proliferation and resisted the celecoxib-induced cell death. Our results provide for the first time that p38 MAP kinase participates in the down-regulation of survivin and subsequently induces the activation of {gamma}-H2AX for mediating apoptosis following treatment with celecoxib in human colorectal cancer cells.

  12. Accumulation of DSBs in {gamma}-H2AX domains fuel chromosomal aberrations

    SciTech Connect

    Scherthan, H. Hieber, L.; Braselmann, H.; Meineke, V.; Zitzelsberger, H.

    2008-07-11

    DNA double strand breaks (DSBs) pose a severe hazard to the genome as erroneous rejoining of DSBs can lead to mutation and cancer. Here, we have investigated the correlation between X irradiation-induced {gamma}-H2AX foci, theoretically induced DSBs, and the minimal number of mis-rejoined DNA breaks (MNB) in irradiated lymphocytes obtained from two healthy humans by painting of the whole chromosome complement by spectral karyotyping. There were less {gamma}-H2AX foci/dose than theoretically expected, while misrepair, as expressed by MNB/{gamma}-H2AX focus, was similar at 0.5 and 1 Gy but 3.6-fold up at 3 Gy. Hence, our results suggest that X-ray-induced {gamma}-H2AX foci in G0 lymphocyte nuclei contain more than one DSB and that the increasing number of DSBs per {gamma}-H2AX repair factory lead to an increased rate of misrepair.

  13. Induction and inhibition of the pan-nuclear gamma-H2AX response in resting human peripheral blood lymphocytes after X-ray irradiation

    PubMed Central

    Ding, D; Zhang, Y; Wang, J; Zhang, X; Gao, Y; Yin, L; Li, Q; Li, J; Chen, H

    2016-01-01

    Human peripheral blood lymphocytes (HPBLs) are one of the most sensitive cells to ionizing radiation (IR) in the human body, and IR-induced DNA damage and functional impairment of HPBLs are the adverse consequences of IR accidents and major side effects of radiotherapy. Phosphorylated H2AXH2AX) is a sensitive marker for DNA double-strand breaks, but the role and regulation of the pan-nuclear γH2AX response in HPBLs after IR remain unclear. We herein demonstrated that the pan-nuclear γH2AX signals were increased in a time- and dose-dependent manner, colocalized with >94% of TUNEL apoptotic staining, and displayed a typical apoptotic pattern in resting HPBLs after low LET X-ray IR. In addition, the X-irradiation-induced pan-nuclear p-ATM and p-DNA-PKcs responses also occurred in resting HPBLs, and were colocalized with 92–95% of TUNEL staining and 97–98% of the pan-nuclear γH2AX signals, respectively, with a maximum at 6 h post irradiation, but disappeared at 24 h post irradiation. Moreover, ATM/DNA-PKcs inhibitor KU55933, p53 inhibitor PFT-μ and pan-caspase inhibitor ZVAD-fmk significantly decreased X-irradiation-induced pan-nuclear γH2AX signals and TUNEL staining, protected HPBLs from apoptosis, but decreased the proliferative response to mitogen in X-irradiated HPBLs. Notably, whereas both KU55933 and PFT-μ increased the IR-induced chromosome breaks and mis-repair events through inhibiting the formation of p-ATM, p-DNA-PKcs and γH2AX foci in X-irradiated HPBLs, the ZVAD-fmk did not increase the IR-induced chromosomal instability. Taken together, our data indicate that pan-nuclear γH2AX response represents an apoptotic signal that is triggered by the transient pan-nuclear ATM and DNA-PKcs activation, and mediated by p53 and pan-caspases in X-irradiated HPBLs, and that caspase inhibitors are better than ATM/DNA-PKcs inhibitors and p53 inhibitors to block pan-nuclear γH2AX response/apoptosis and protect HPBLs from IR. PMID:27551505

  14. Induction and inhibition of the pan-nuclear gamma-H2AX response in resting human peripheral blood lymphocytes after X-ray irradiation.

    PubMed

    Ding, D; Zhang, Y; Wang, J; Zhang, X; Gao, Y; Yin, L; Li, Q; Li, J; Chen, H

    2016-01-01

    Human peripheral blood lymphocytes (HPBLs) are one of the most sensitive cells to ionizing radiation (IR) in the human body, and IR-induced DNA damage and functional impairment of HPBLs are the adverse consequences of IR accidents and major side effects of radiotherapy. Phosphorylated H2AXH2AX) is a sensitive marker for DNA double-strand breaks, but the role and regulation of the pan-nuclear γH2AX response in HPBLs after IR remain unclear. We herein demonstrated that the pan-nuclear γH2AX signals were increased in a time- and dose-dependent manner, colocalized with >94% of TUNEL apoptotic staining, and displayed a typical apoptotic pattern in resting HPBLs after low LET X-ray IR. In addition, the X-irradiation-induced pan-nuclear p-ATM and p-DNA-PKcs responses also occurred in resting HPBLs, and were colocalized with 92-95% of TUNEL staining and 97-98% of the pan-nuclear γH2AX signals, respectively, with a maximum at 6 h post irradiation, but disappeared at 24 h post irradiation. Moreover, ATM/DNA-PKcs inhibitor KU55933, p53 inhibitor PFT-μ and pan-caspase inhibitor ZVAD-fmk significantly decreased X-irradiation-induced pan-nuclear γH2AX signals and TUNEL staining, protected HPBLs from apoptosis, but decreased the proliferative response to mitogen in X-irradiated HPBLs. Notably, whereas both KU55933 and PFT-μ increased the IR-induced chromosome breaks and mis-repair events through inhibiting the formation of p-ATM, p-DNA-PKcs and γH2AX foci in X-irradiated HPBLs, the ZVAD-fmk did not increase the IR-induced chromosomal instability. Taken together, our data indicate that pan-nuclear γH2AX response represents an apoptotic signal that is triggered by the transient pan-nuclear ATM and DNA-PKcs activation, and mediated by p53 and pan-caspases in X-irradiated HPBLs, and that caspase inhibitors are better than ATM/DNA-PKcs inhibitors and p53 inhibitors to block pan-nuclear γH2AX response/apoptosis and protect HPBLs from IR. PMID:27551505

  15. Relationship between spontaneous γH2AX foci formation and progenitor functions in circulating hematopoietic stem and progenitor cells among atomic-bomb survivors.

    PubMed

    Kajimura, Junko; Kyoizumi, Seishi; Kubo, Yoshiko; Misumi, Munechika; Yoshida, Kengo; Hayashi, Tomonori; Imai, Kazue; Ohishi, Waka; Nakachi, Kei; Weng, Nan-Ping; Young, Lauren F; Shieh, Jae-Hung; Moore, Malcolm A; van den Brink, Marcel R M; Kusunoki, Yoichiro

    2016-05-01

    Accumulated DNA damage in hematopoietic stem cells is a primary mechanism of aging-associated dysfunction in human hematopoiesis. About 70 years ago, atomic-bomb (A-bomb) radiation induced DNA damage and functional decreases in the hematopoietic system of A-bomb survivors in a radiation dose-dependent manner. The peripheral blood cell populations then recovered to a normal range, but accompanying cells derived from hematopoietic stem cells still remain that bear molecular changes possibly caused by past radiation exposure and aging. In the present study, we evaluated radiation-related changes in the frequency of phosphorylated (Ser-139) H2AXH2AX) foci formation in circulating CD34-positive/lineage marker-negative (CD34+Lin-) hematopoietic stem and progenitor cells (HSPCs) among 226Hiroshima A-bomb survivors. An association between the frequency of γH2AX foci formation in HSPCs and the radiation dose was observed, but the γH2AX foci frequency was not significantly elevated by past radiation. We found a negative correlation between the frequency of γH2AX foci formation and the length of granulocyte telomeres. A negative interaction effect between the radiation dose and the frequency of γH2AX foci was suggested in a proportion of a subset of HSPCs as assessed by the cobblestone area-forming cell assay (CAFC), indicating that the self-renewability of HSPCs may decrease in survivors who were exposed to a higher radiation dose and who had more DNA damage in their HSPCs. Thus, although many years after radiation exposure and with advancing age, the effect of DNA damage on the self-renewability of HSPCs may be modified by A-bomb radiation exposure. PMID:27169377

  16. Anacardic acid sensitizes prostate cancer cells to radiation therapy by regulating H2AX expression

    PubMed Central

    Yao, Kun; Jiang, Xianzhen; He, leye; Tang, Yuxin; Yin, Guangming; Zeng, Qing; Jiang, Zhiqiang; Tan, Jing

    2015-01-01

    Anacardic acid (6-pentadecylsalicylic acid, AA), a natural compound isolated from the traditional medicine Amphipterygiumadstringens, has been reported as potential antitumor agents in various cancers including prostate cancer (PC). However, the effects and mechanism of AA on the radiosensitivity of prostate cancer remains unknown. The results indicated that AA exhibited strong antitumor activity in PC cell lines, either as a single agentor in combination with radiation. AA significantly induced the downregulation of H2AX and p-H2AX expression, increase of cell apoptosis and decreasing of cell invasion, which were reversed by overexpressed H2AX. These results suggest that AA sensitize prostate cancer cells to radiation therapy by repressing H2AX expression. PMID:26884865

  17. KAP-1 Promotes Resection of Broken DNA Ends Not Protected by γ-H2AX and 53BP1 in G1-Phase Lymphocytes

    PubMed Central

    Tubbs, Anthony T.; Dorsett, Yair; Chan, Elizabeth; Helmink, Beth; Lee, Baeck-Seung; Hung, Putzer; George, Rosmy; Bredemeyer, Andrea L.; Mittal, Anuradha; Pappu, Rohit V.; Chowdhury, Dipanjan; Mosammaparast, Nima; Krangel, Michael S.

    2014-01-01

    The resection of broken DNA ends is required for DNA double-strand break (DSB) repair by homologous recombination (HR) but can inhibit normal repair by nonhomologous end joining (NHEJ), the main DSB repair pathway in G1-phase cells. Antigen receptor gene assembly proceeds through DNA DSB intermediates generated in G1-phase lymphocytes by the RAG endonuclease. These DSBs activate ATM, which phosphorylates H2AX, forming γ-H2AX in flanking chromatin. γ-H2AX prevents CtIP from initiating resection of RAG DSBs. Whether there are additional proteins required to promote resection of these DNA ends is not known. KRAB-associated protein 1 (KAP-1) (TRIM28) is a transcriptional repressor that modulates chromatin structure and has been implicated in the repair of DNA DSBs in heterochromatin. Here, we show that in murine G1-phase lymphocytes, KAP-1 promotes resection of DSBs that are not protected by H2AX and its downstream effector 53BP1. In these murine cells, KAP-1 activity in DNA end resection is attenuated by a single-amino-acid change that reflects a KAP-1 polymorphism between primates and other mammalian species. These findings establish KAP-1 as a component of the machinery that can resect DNA ends in G1-phase cells and suggest that there may be species-specific features to this activity. PMID:24842905

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

    SciTech Connect

    Bracalente, Candelaria; Ibañez, Irene L.; Molinari, Beatriz; Palmieri, Mónica; Kreiner, Andrés; Valda, Alejandro; 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.

  19. Involvement of ROS-p38-H2AX axis in novel curcumin analogues-induced apoptosis in breast cancer cells.

    PubMed

    Dong, Yinhui; Yin, Shutao; Song, Xinhua; Huo, Yazhen; Fan, Lihong; Ye, Min; Hu, Hongbo

    2016-04-01

    Curcumin-based structural modification for developing more effective curcumin analogues has been drawning increasing attention. As alternative approach, using LC/MS guided purification, we previously obtained a series of novel natural terpene-conjugated curcuminoids from turmeric, and some of them exhibited even more potent anti-cancer activity against multiple types of cancer cells than curcumin. The purpose of this follow-up study was designed to decipher the mechanisms involved in anti-cancer activity of these novel curcumin analogues. Apoptosis was evaluated using sub-G1 analysis by flow cytometry and Cell Death ELISA Kit. Changes of protein expression were analyzed by western blotting. RNA interference was employed to inhibit expression of specific protein. We found that bisabolocurcumin ether (T1) and demethoxybisabolocurcumin ether (T2) were able to trigger much stronger apoptosis induction in multiple types of cancer cells than curcumin, which was attributed to persistent and stronger ROS generation. ROS induction by T1 resulted in activation of p38/H2AX axis and p53. Inhibition of p38/H2AX led to a significant reduction of apoptosis, whereas inactivation of p53 caused a dramatically enhanced H2AX phosphorylation and apoptosis induction, suggesting activation of p38/H2AX contributed to apoptosis induction by T1, whereas p53 activation protected novel curcumins-induced apoptosis via suppression of H2AX activation. Our findings provide mechanistic support for the potential use of terpene-conjugated curcuminoids as a novel class of cancer chemopreventive agents. © 2015 Wiley Periodicals, Inc. PMID:25647442

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

    NASA Astrophysics Data System (ADS)

    Niu, H.; Chang, H. C.; Cho, I. C.; Chen, C. H.; Liu, C. S.; Chou, W. T.

    2014-08-01

    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 repair.

  1. Generation of an alpaca-derived nanobody recognizing γ-H2AX

    PubMed Central

    Rajan, Malini; Mortusewicz, Oliver; Rothbauer, Ulrich; Hastert, Florian D.; Schmidthals, Katrin; Rapp, Alexander; Leonhardt, Heinrich; Cardoso, M. Cristina

    2015-01-01

    Post-translational modifications are difficult to visualize in living cells and are conveniently analyzed using antibodies. Single-chain antibody fragments derived from alpacas and called nanobodies can be expressed and bind to the target antigenic sites in living cells. As a proof of concept, we generated and characterized nanobodies against the commonly used biomarker for DNA double strand breaks γ-H2AX. In vitro and in vivo characterization showed the specificity of the γ-H2AX nanobody. Mammalian cells were transfected with fluorescent fusions called chromobodies and DNA breaks induced by laser microirradiation. We found that alternative epitope recognition and masking of the epitope in living cells compromised the chromobody function. These pitfalls should be considered in the future development and screening of intracellular antibody biomarkers. PMID:26500838

  2. Histone markers identify the mode of action for compounds positive in the TK6 micronucleus assay.

    PubMed

    Cheung, Jennifer R; Dickinson, Donna A; Moss, Jocelyn; Schuler, Maik J; Spellman, Richard A; Heard, Pamela L

    2015-01-01

    The in vitro micronucleus assay with TK6 cells is frequently used as part of the genotoxicity testing battery for pharmaceuticals. Consequently, follow-up testing strategies are needed for positive compounds to determine their mode of action, which would then allow for deployment of appropriate in vivo follow-up strategies. We have chosen 3 micronucleus positive compounds, the clastogen etoposide, the aneugen noscapine and the cytotoxicant tunicamycin to evaluate different approaches to determine their aneugenic or clastogenic properties. Each of the three compounds were evaluated following 4 and 24h of continuous treatment by flow cytometry for micronucleus induction, the aneugenicity markers phosphorylated-histone 3 (p-H3) and polyploidy, the clastogenicity marker γH2AX and the apoptosis marker cleaved caspase 3. They were further evaluated by Western blot for mono-ubiquitinated and γH2AX. Results show that the clastogen etoposide produced a dose related increase in γH2AX and mono-ubiquitinated H2AX and a dose related decrease in p-H3 positive mitotic cells. Conversely, the aneugen produced increases in p-H3 and polyploidy with no significant increases seen in mono-ubiquitinated H2AX or γH2AX. Lastly, the cytotoxicant tunicamycin induced neither an increase in p-H3 nor γH2AX. All three compounds produced dose-related increases in cleaved caspase 3. The results from this study provide evidence that adding clastogenicity and aneugenicity markers to the in vitro micronucleus assay in TK6 cells could help to identify the mode of action of positive compounds. The combination of endpoints suggested here needs to be further evaluated by a broader set of test compounds. PMID:25726170

  3. Calculation of Dose Deposition in Nanovolumes and Simulation of gamma-H2AX Experiments

    NASA Technical Reports Server (NTRS)

    Plante, Ianik

    2010-01-01

    Monte-Carlo track structure simulations can accurately simulate experimental data: a) Frequency of target hits. b) Dose per event. c) Dose per ion. d) Radial dose. The dose is uniform in micrometers sized voxels; at the nanometer scale, the difference in energy deposition between high and low-LET radiations appears. The calculated 3D distribution of dose voxels, combined with chromosomes simulated by random walk is very similar to the distribution of DSB observed with gamma-H2AX experiments. This is further evidenced by applying a visualization threshold on dose.

  4. FociCounter: a freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.

    PubMed

    Jucha, Anna; Wegierek-Ciuk, Aneta; Koza, Zbigniew; Lisowska, Halina; Wojcik, Andrzej; Wojewodzka, Maria; Lankoff, Anna

    2010-02-01

    Gamma-H2AX foci are sensitive and specific indicator for the induction of DNA double-strand breaks (DSBs) and an immunocytochemical assay with antibodies recognizing gamma-H2AX has become the gold standard for the detection of this type of DNA lesion. Quantification of gamma-H2AX foci can be achieved by various methods such as Western blotting, flow cytometry, visual analysis and computational analysis with a fluorescence microscope. The best sensitivity is achieved by computer analysis. Since no freeware programme for the analysis of gamma-H2AX foci exists for a PC platform, the aim of our study was to develop a simple and user-friendly public-domain software. The algorithm applied in our programme allows determination of the number of foci in a single cell, a focus intensity per cell, as well as a cell intensity. Its graphical user interface is based on a GTK+ library and the whole application can be run under a variety of operating systems, including MS Windows and Linux. The programme called FociCounter is publicly available at http://focicounter.sourceforge.net. Application of the programme was tested by analysing gamma-H2AX foci in CHO and MO59K cells irradiated in vitro with X-rays and validated by comparing the results obtained with the outcome of automated image analysis and flow cytometry. PMID:20018253

  5. The proximity ligation assay reveals that at DNA double-strand breaks WRAP53β associates with γH2AX and controls interactions between RNF8 and MDC1

    PubMed Central

    Rassoolzadeh, Hanif; Coucoravas, Christos; Farnebo, Marianne

    2015-01-01

    We recently demonstrated that WRAP53β acts as a key regulator of ubiquitin-dependent repair of DNA double-strand breaks. Here, we applied the proximity ligation assay (PLA) to show that at such breaks WRAP53β accumulates in close proximity to γH2AX and, furthermore as demonstrated by their co-immunoprecipitation (IP) binds to γH2AX, in a manner dependent on the ATM and ATR kinases. Moreover, formation of complexes between MDC1 and both its partners RNF8 and phosphorylated ATM was visualized. The interaction of MDC1 with RNF8, but not with ATM requires WRAP53β, suggesting that WRAP53β facilitates the former interaction without altering phosphorylation of MDC1 by ATM. Furthermore, our findings highlight PLA as a more sensitive method for the analysis of recruitment of repair factors and complex formation at DNA breaks that are difficult to detect using conventional immunofluorescence. PMID:26734725

  6. 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

    PubMed Central

    Tommasino, Francesco; Friedrich, Thomas; Jakob, Burkhard; Meyer, Barbara; Durante, Marco; Scholz, Michael

    2015-01-01

    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, experiments performed

  7. USE OF THE γ-H2AX ASSAY TO MONITOR DNA DAMAGE AND REPAIR IN TRANSLATIONAL CANCER RESEARCH

    PubMed Central

    Ivashkevich, Alesia; Redon, Christophe E.; Nakamura, Asako J.; Martin, Roger F.; Martin, Olga A.

    2011-01-01

    Formation of γ-H2AX in response to DNA double stranded breaks (DSBs) provides the basis for a sensitive assay of DNA damage in human biopsies. The review focuses on the application of γ-H2AX-based methods to translational studies to monitor the clinical response to DNA targeted therapies such as some forms of chemotherapy, external beam radiotherapy, radionuclide therapy or combinations thereof. The escalating attention on radiation biodosimetry has also highlighted the potential of the assay including renewed efforts to assess the radiosensitivity of prospective radiotherapy patients. Finally the γ-H2AX response has been suggested as a basis for an in vivo imaging modality. PMID:22198208

  8. Phosphorylation of histone variant regions in chromatin: unlocking the linker?

    PubMed

    Green, G R

    2001-01-01

    Histone variants illuminate the behavior of chromatin through their unique structures and patterns of postsynthetic modification. This review examines the literature on heteromorphous histone structures in chromatin, structures that are primary targets for histone kinases and phosphatases in vivo. Special attention is paid to certain well-studied experimental systems: mammalian culture cells, chicken erythrocytes, sea urchin sperm, wheat sprouts, Tetrahymena, and budding yeast. A common theme emerges from these studies. Specialized, highly basic structures in histone variants promote chromatin condensation in a variety of developmental situations. Before, and sometimes after condensed chromatin is formed, the chromatin is rendered soluble by phosphorylation of the heteromorphous regions, preventing their interaction with linker DNA. A simple structural model accounting for histone variation and phosphorylation is presented. PMID:11467741

  9. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  10. RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

    PubMed Central

    Mamouni, Kenza; Cristini, Agnese; Guirouilh-Barbat, Josée; Monferran, Sylvie; Lemarié, Anthony; Faye, Jean-Charles; Lopez, Bernard S.

    2014-01-01

    Unlike other Rho GTPases, RhoB is rapidly induced by DNA damage, and its expression level decreases during cancer progression. Because inefficient repair of DNA double-strand breaks (DSBs) can lead to cancer, we investigated whether camptothecin, an anticancer drug that produces DSBs, induces RhoB expression and examined its role in the camptothecin-induced DNA damage response. We show that in camptothecin-treated cells, DSBs induce RhoB expression by a mechanism that depends notably on Chk2 and its substrate HuR, which binds to RhoB mRNA and protects it against degradation. RhoB-deficient cells fail to dephosphorylate γH2AX following camptothecin removal and show reduced efficiency of DSB repair by homologous recombination. These cells also show decreased activity of protein phosphatase 2A (PP2A), a phosphatase for γH2AX and other DNA damage and repair proteins. Thus, we propose that DSBs activate a Chk2-HuR-RhoB pathway that promotes PP2A-mediated dephosphorylation of γH2AX and DSB repair. Finally, we show that RhoB-deficient cells accumulate endogenous γH2AX and chromosomal abnormalities, suggesting that RhoB loss increases DSB-mediated genomic instability and tumor progression. PMID:24912678

  11. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  12. Alterations of Histone H1 Phosphorylation During Bladder Carcinogenesis

    PubMed Central

    Telu, Kelly H.; Abbaoui, Besma; Thomas-Ahner, Jennifer M.; Zynger, Debra L.; Clinton, Steven K.

    2013-01-01

    There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid Chromatography-Mass Spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions. PMID:23675690

  13. Linking Gamma-H2AX Foci and Cancer in Rat Skin Exposed to Heavy Ions and Electron Radiation.

    PubMed

    Burns, Fredric J; Tang, Moon-shong; Wu, Feng; Schmid, Ernst

    2015-08-01

    This study uses acute doses of three test radiations, [Ar ions (L = 125 keVμ), Ne ions (L = 25 keVμ) and electron radiation] to examine a potential quantitative link between rat skin cancer induction and gamma-H2AX foci in rat keratinocytes exposed in vitro to radiations with comparable L values. Theory provided a testable link between cancer yield and gamma-H2AX foci yields: YCa(D,L)rat = (NF)2YAX(D,L)keratinocyte (eqn 1), where YCa(D,L) is cancers(rat) at 1.0 y, YAX(D,L) is in vitro gamma-H2AX foci(keratinocyte) , D is radiation dose, L is linear energy transfer, N is irradiated keratinocytes in vivo, and F is the error rate of end joining. An explicit expression for cancer yield was derived based on cancers arising in the ion track region in proportion to D and L (first term) and independently in proportion to D in the delta ray region in between the ion tracks (second term): YCa(D,L) = CCaLD + BCaD (eqn 1a). Parameters quantified include: CCa = 0.000589 ± 0.000150 cancers-micron[rat(kev)Gy]; BCa = 0.0088 ± 0.0035 cancers(ratGy), F = (8.18 ± 0.91) × 10; N = (8.8 ± 1.2) × 10 and (NF)2 = 0.036 ± 0.006 cancer keratinocyte(rat H2AX foci). Verification of eqns (1) and (1a) and the constancy of F support the hypothesis that end-rejoining errors play a major role in radiation carcinogenesis in rat skin. Cancer yields per rat were consistently predictable based on gamma-H2AX foci yields in keratinocytes in vitro such that 27.8 H2AXfoci(keratinocyte) predicted 1.0 cancer(rat) at 1 y. PMID:26107436

  14. γH2AX and p53 responses in TK6 cells discriminate promutagens and nongenotoxicants in the presence of rat liver S9.

    PubMed

    Bernacki, Derek T; Bryce, Steven M; Bemis, Jeffrey C; Kirkland, David; Dertinger, Stephen D

    2016-08-01

    Previous work with a diverse set of reference chemicals suggests that an in vitro multiplexed flow cytometry-based assay (MultiFlow™ DNA Damage Kit-p53, γH2AX, Phospho-Histone H3) can distinguish direct-acting clastogens and aneugens from nongenotoxicants (Bryce SM et al. []: Environ Mol Mutagen 57:171-189). This work extends this line of investigation to include compounds that require metabolic activation to form reactive electrophiles. For these experiments, TK6 cells were exposed to 11 promutagens and 37 presumed nongenotoxicants in 96 well plates. Unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. Exposure occurred for 4 hr after which time cells were washed to remove S9 and test article. Immediately following the wash and again at 24 hr, cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation, robotic sampling was employed for walk-away flow cytometric data acquisition. Univariate logistic regression analyses indicated that γH2AX induction and p53 activation provide the greatest degree of discrimination between clastogens and nongenotoxicants. Multivariate prediction algorithms that incorporated both of these endpoints, in each combination of time points, were evaluated. The best performing models correctly predicted 9 clastogens out of 11 and 36 nongenotoxicants out of 37. These results are encouraging as they suggest that an efficient and highly scalable multiplexed assay can effectively identify clastogenic chemicals that require bioactivation. More work is planned with a broader range of chemicals, additional cell lines, and other laboratories to further evaluate the merits and limitations of this approach. Environ. Mol. Mutagen. 57:546-558, 2016. © 2016 Wiley Periodicals, Inc. PMID:27364561

  15. γ-H2AX Kinetic Profile in Mouse Lymphocytes Exposed to the Internal Emitters Cesium-137 and Strontium-90

    PubMed Central

    Turner, Helen C.; Shuryak, Igor; Weber, Waylon; Doyle-Eisele, Melanie; Melo, Dunstana; Guilmette, Raymond; Amundson, Sally A.; Brenner, David J.

    2015-01-01

    In the event of a dirty bomb scenario or an industrial nuclear accident, a significant dose of volatile radionuclides such as 137Cs and 90Sr may be dispersed into the atmosphere as a component of fallout and inhaled or ingested by hundreds and thousands of people. To study the effects of prolonged exposure to ingested radionuclides, we have performed long-term (30 day) internal-emitter mouse irradiations using soluble-injected 137CsCl and 90SrCl2 radioisotopes. The effect of ionizing radiation on the induction and repair of DNA double strand breaks (DSBs) in peripheral mouse lymphocytes in vivo was determined using the γ-H2AX biodosimetry marker. Using a serial sacrifice experimental design, whole-body radiation absorbed doses for 137Cs (0 to 10 Gy) and 90Sr (0 to 49 Gy) were delivered over 30 days following exposure to each radionuclide. The committed absorbed doses of the two internal emitters as a function of time post exposure were calculated based on their retention parameters and their derived dose coefficients for each specific sacrifice time. In order to measure the kinetic profile for γ-H2AX, peripheral blood samples were drawn at 5 specific timed dose points over the 30-day study period and the total γ-H2AX nuclear fluorescence per lymphocyte was determined using image analysis software. A key finding was that a significant γ-H2AX signal was observed in vivo several weeks after a single radionuclide exposure. A mechanistically-motivated model was used to analyze the temporal kinetics of γ-H2AX fluorescence. Exposure to either radionuclide showed two peaks of γ-H2AX: one within the first week, which may represent the death of mature, differentiated lymphocytes, and the second at approximately three weeks, which may represent the production of new lymphocytes from damaged progenitor cells. The complexity of the observed responses to internal irradiation is likely caused by the interplay between continual production and repair of DNA damage, cell cycle

  16. The first gamma-H2AX biodosimetry intercomparison exercise of the developing European biodosimetry network RENEB.

    PubMed

    Barnard, S; Ainsbury, E A; Al-hafidh, J; Hadjidekova, V; Hristova, R; Lindholm, C; Monteiro Gil, O; Moquet, J; Moreno, M; Rößler, U; Thierens, H; Vandevoorde, C; Vral, A; Wojewódzka, M; Rothkamm, K

    2015-04-01

    In the event of a mass casualty radiation incident, the gamma-H2AX foci assay could be a useful tool to estimate radiation doses received by individuals. The rapid processing time of blood samples of just a few hours and the potential for batch processing, enabling high throughput, make the assay ideal for early triage categorisation to separate the 'worried well' from the low and critically exposed by quantifying radiation-induced foci in peripheral blood lymphocytes. Within the RENEB framework, 8 European laboratories have taken part in the first European gamma-H2AX biodosimetry exercise, which consisted of a telescoring comparison of 200 circulated foci images taken from 8 samples, and a comparison of 10 fresh blood lymphocyte samples that were shipped overnight to participating labs 4 or 24 h post-exposure. Despite large variations between laboratories in the dose-response relationship for foci induction, the obtained results indicate that the network should be able to use the gamma-H2AX assay for rapidly identifying the most severely exposed individuals within a cohort who could then be prioritised for accurate chromosome dosimetry. PMID:25118318

  17. Linking Gamma-H2AX Foci and Cancer in Rat Skin Exposed to Heavy Ions and Electron Radiation

    PubMed Central

    Burns, Fredric J.; Tang, Moon-shong; Wu, Feng; Schmid, Ernst

    2015-01-01

    Abstract This study uses acute doses of three test radiations, [40Ar ions (L = 125 keVμ−1), 20Ne ions (L = 25 keVμ−1) and electron radiation] to examine a potential quantitative link between rat skin cancer induction and gamma-H2AX foci in rat keratinocytes exposed in vitro to radiations with comparable L values. Theory provided a testable link between cancer yield and gamma-H2AX foci yields: YCa(D,L)rat−1 = (NF)2−1YAX(D,L)keratinocyte−1 (eqn 1), where YCa(D,L) is cancers(rat) −1 at 1.0 y, YAX(D,L) is in vitro gamma-H2AX foci(keratinocyte) −1, D is radiation dose, L is linear energy transfer, N is irradiated keratinocytes in vivo, and F is the error rate of end joining. An explicit expression for cancer yield was derived based on cancers arising in the ion track region in proportion to D and L (first term) and independently in proportion to D2 in the delta ray region in between the ion tracks (second term): YCa(D,L) = CCaLD + BCaD2 (eqn 1a). Parameters quantified include: CCa = 0.000589 ± 0.000150 cancers-micron[rat(kev)Gy]−1; BCa = 0.0088 ± 0.0035 cancers(ratGy2)−1, F = (8.18 ± 0.91) × 10−10; N = (8.8 ± 1.2) × 107 and (NF)2−1 = 0.036 ± 0.006 cancer keratinocyte(rat H2AX foci)−1. Verification of eqns (1) and (1a) and the constancy of F support the hypothesis that end-rejoining errors play a major role in radiation carcinogenesis in rat skin. Cancer yields per rat were consistently predictable based on gamma-H2AX foci yields in keratinocytes in vitro such that 27.8 H2AXfoci(keratinocyte)−1 predicted 1.0 cancer(rat)−1 at 1 y. PMID:26107436

  18. Frequency of gamma H2AX foci in healthy volunteers and health workers occupationally exposed to X-irradiation and its relevance in biological dosimetry.

    PubMed

    Raavi, Venkateswarlu; Basheerudeen, Safa Abdul Syed; Jagannathan, Vijayalakshmi; Joseph, Santosh; Chaudhury, Nabo Kumar; Venkatachalam, Perumal

    2016-08-01

    Gamma-H2AX (γ-H2AX) assay is a marker to measure double-strand breaks in the deoxyribonucleic acid. Variables such as age, oxidative stress, temperature, genetic factors and inter-individual variation have been reported to influence the baseline γ-H2AX focus levels. Therefore, knowledge on baseline frequency of γ-H2AX foci in a targeted population would facilitate reliable radiation triage and dose estimation. The objective of the present study was to establish the baseline data using blood samples from healthy volunteers (n = 130) differing in age, occupation and lifestyle as well as from occupationally exposed health workers (n = 20). The γ-H2AX focus assay was performed using epifluorescence microscopy. In vitro dose-response curve for γ-H2AX foci was constructed in blood samples (n = 3) exposed to X-rays (30 min post-exposure). The mean γ-H2AX focus frequency obtained in healthy volunteers was 0.042 ± 0.001 and showed an age-related increase (p < 0.001). Significantly higher (p < 0.005) focus frequencies were observed in health workers (0.066 ± 0.005) than in healthy volunteers. A sub-group analysis did not show a significant (p > 0.1) difference in γ-H2AX focus frequency among sexes. Blood exposed in vitro to X-rays showed dose-dependent increase in γ-H2AX foci frequency (Y = 0.1902 ± 0.1363 + 2.9020 ± 0.3240 * D). Baseline frequency of γ-H2AX foci obtained from different age groups showed a significant (p < 0.01) influence on the dose-response coefficients. The overall results demonstrated that the γ-H2AX assay can be used as a reliable biomarker for radiation triage and estimating the radiation absorbed dose by considering variables such as age, occupation and lifestyle factors. PMID:27287768

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

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    Yamashita, Sachiko; Tanaka, Masakazu; Sato, Teruaki; Ida, Chieri; Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke; Moss, Joel; Miwa, Masanao

    2016-08-01

    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

  1. Predicting Radiosensitivity with Gamma-H2AX Foci Assay after Single High-Dose-Rate and Pulsed Dose-Rate Ionizing Irradiation.

    PubMed

    van Oorschot, Bregje; Hovingh, Suzanne; Dekker, Annelot; Stalpers, Lukas J; Franken, Nicolaas A P

    2016-02-01

    Gamma-H2AX foci detection is the standard method to quantify DNA double-strand break (DSB) induction and repair. In this study, we investigated the induction and decay of γ-H2AX foci of different tumor cell lines and fibroblasts with known mutations in DNA damage repair genes, including ATM, LigIV, DNA-PKcs, Rad51 and Rad54. A radiation dose of 2.4 Gy was used for either an acute single high-dose-rate (sHDR) exposure or a pulsed dose-rate (pDR) exposure over 24 h. The number of γ-H2AX foci was determined at 30 min and 24 h after sHDR irradiation and directly after pDR irradiation. In a similar manner, γ-H2AX foci were also examined in lymphocytes of patients with differences in normal tissue toxicity after a total radiation dose of 1 Gy. In an initial count of the number of foci 30 min after sHDR irradiation, repair-proficient cell types could not be distinguished from repair-deficient cell types. However at 24 h postirradiation, while we observed a large decrease in foci numbers in NHEJ-proficient cells, the amount of γ-H2AX foci in cell types with mutated NHEJ repair remained at high levels. Except for IRS-1SF cells, HR-deficient cell types eventually did show a moderate decrease in foci number over time, albeit to a lesser extent than their corresponding parentals or repair-proficient control cells. In addition, analysis of γ-H2AX foci after sHDR exposure of patients with different sensitivity status clearly showed individual differences in radiation response. Radiosensitive patients could be distinguished from the more radioresistant patients with γ-H2AX foci decay ratios (initial number of foci divided by residual number of foci). Significantly higher decay ratios were observed in patients without toxicities, indicating more proficient repair compared to patients with radiation-induced side effects. After pDR irradiation, no consistent correlation could be found between foci number and radiosensitivity. In conclusion, γ-H2AX formation is a rapid and

  2. Flow cytometry-assisted quantification of γH2AX expression has potential as a rapid high-throughput biodosimetry tool.

    PubMed

    Achel, Daniel G; Serafin, Antonio M; Akudugu, John M

    2016-08-01

    Large-scale radiological events require immediate and accurate estimates of doses received by victims, and possibly the first responders, to assist in treatment decisions. Although there are numerous efforts worldwide to develop biodosimetric tools to adequately handle triage needs during radiological incidents, such endeavours do not seem to actively involve sub-Saharan Africa which currently has a significant level of nuclear-related activity. To initiate a similar interest in Africa, ex vivo radiation-induced γH2AX expression in peripheral blood lymphocytes from fourteen healthy donors was assessed using flow cytometry. While the technique shows potential for use as a rapid high-throughput biodosimetric tool for radiation absorbed doses up to 5 Gy, significant inter-individual differences in γH2AX expression emerged. Also, female donors exhibited higher levels of γH2AX expression than their male counterparts. To address these shortcomings, gender-based in-house dose-response curves for γH2AX induction in lymphocytes 2, 4, and 6 h after X-ray irradiation are proposed for the South African population. The obtained results show that γH2AX is a good candidate biomarker for biodosimetry, but might need some refinement and validation through further studies involving a larger cohort of donors. PMID:27262315

  3. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  4. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    PubMed

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  5. γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

    PubMed Central

    Fernandez-Palomo, Cristian; Mothersill, Carmel; Bräuer-Krisch, Elke; Laissue, Jean; Seymour, Colin; Schültke, Elisabeth

    2015-01-01

    Objective 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). Methods 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. Results 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. Conclusion 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. PMID:25799425

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

    PubMed Central

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

    2016-01-01

    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. PMID:27445126

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

    PubMed

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

    2016-01-01

    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. PMID:27445126

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles

    PubMed Central

    2012-01-01

    Background Little is known about the cellular effects of exposure to mixed beams of high and low linear energy transfer radiation. So far, the effects of combined exposures have mainly been assessed with clonogenic survival or cytogenetic methods, and the results are contradictory. The gamma-H2AX assay has up to now not been applied in this context, and it is a promising tool for investigating the early cellular response to mixed beam irradiation. Purpose To determine the dose response and repair kinetics of gamma-H2AX ionizing radiation-induced foci in VH10 human fibroblasts exposed to mixed beams of 241Am alpha particles and X-rays. Results VH10 human fibroblasts were irradiated with each radiation type individually or both in combination at 37°C. Foci were scored for repair kinetics 0.5, 1, 3 and 24 h after irradiation (one dose per irradiation type), and for dose response at the 1 h time point. The dose response effect of mixed beam was additive, and the relative biological effectiveness for alpha particles (as compared to X-rays) was of 0.76 ± 0.52 for the total number of foci, and 2.54 ± 1.11 for large foci. The repair kinetics for total number of foci in cells exposed to mixed beam irradiation was intermediate to that of cells exposed to alpha particles and X-rays. However, for mixed beam-irradiated cells the frequency and area of large foci were initially lower than predicted and increased during the first 3 hours of repair (while the predicted number and area did not). Conclusions The repair kinetics of large foci after mixed beam exposure was significantly different from predicted based on the effect of the single dose components. The formation of large foci was delayed and they did not reach their maximum area until 1 h after irradiation. We hypothesize that the presence of low X-ray-induced damage engages the DNA repair machinery leading to a delayed DNA damage response to the more complex DNA damage induced by alpha particles. PMID:23121736

  10. 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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

    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

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

    SciTech Connect

    Audebert, M.; Zeman, F.; Beaudoin, R.; Péry, A.; Cravedi, J.-P.

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

    NASA Astrophysics Data System (ADS)

    Klimovskaia, Ilnaz M.; Young, Clifford; Strømme, Caroline B.; Menard, Patrice; Jasencakova, Zuzana; Mejlvang, Jakob; Ask, Katrine; Ploug, Michael; Nielsen, Michael L.; Jensen, Ole N.; Groth, Anja

    2014-03-01

    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 and dissect how phosphorylation has an impact on human Asf1 function. The divergent C-terminal tail of Asf1a is phosphorylated at several sites, and this is required for timely progression through S phase. Consistent with this, biochemical analysis of wild-type and phospho-mimetic Asf1a shows that phosphorylation enhances binding to histones and the downstream chaperones CAF-1 and HIRA. Moreover, we find that TLK phosphorylation of Asf1a is induced in cells experiencing deficiency of new histones and that TLK interaction with Asf1a involves its histone-binding pocket. We thus propose that TLK signalling promotes histone supply in S phase by targeting histone-free Asf1 and stimulating its ability to shuttle histones to sites of chromatin assembly.

  14. Histone H3 phosphorylation – A versatile chromatin modification for different occasions

    PubMed Central

    Sawicka, Anna; Seiser, Christian

    2012-01-01

    Post-translation modifications of histones modulate the accessibility and transcriptional competence of specific chromatin regions within the eukaryotic genome. Phosphorylation of histone H3 is unique in the sense that it associates on one hand with open chromatin during gene activation and marks on the other hand highly condensed chromatin during mitosis. Phosphorylation of serine residues at histone H3 is a highly dynamic process that creates together with acetylation and methylation marks at neighboring lysine residues specific combinatorial patterns that are read by specific detector proteins. In this review we describe the importance of different histone H3 phosphorylation marks for chromatin condensation during mitosis. In addition, we review the signals that trigger histone H3 phosphorylation and the factors that control this reversible modification during interphase and mediate the biological readout of the signal. Finally, we discuss different models describing the role of histone H3 phosphorylation in the activation of transcription of poised genes or by transient derepression of epigenetically silenced genes. We propose that histone H3 phosphorylation in the context with lysine methylation might temporarily relieve the silencing of specific genes without affecting the epigenetic memory. PMID:22564826

  15. Effect of Dose Rate on Residual γ-H2AX Levels and Frequency of Micronuclei in X-Irradiated Mouse Lymphocytes

    PubMed Central

    Turner, H. C.; Shuryak, I.; Taveras, M.; Bertucci, A.; Perrier, J. R.; Chen, C.; Elliston, C. D.; Johnson, G. W.; Smilenov, L. B.; Amundson, S. A.; Brenner, D. J.

    2015-01-01

    The biological risks associated with low-dose-rate (LDR) radiation exposures are not yet well defined. To assess the risk related to DNA damage, we compared the yields of two established biodosimetry end points, γ-H2AX and micronuclei (MNi), in peripheral mouse blood lymphocytes after prolonged in vivo exposure to LDR X rays (0.31 cGy/min) vs. acute high-dose-rate (HDR) exposure (1.03 Gy/min). C57BL/6 mice were total-body irradiated with 320 kVP X rays with doses of 0, 1.1, 2.2 and 4.45 Gy. Residual levels of total γ-H2AX fluorescence in lymphocytes isolated 24 h after the start of irradiation were assessed using indirect immunofluorescence methods. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptotic cell frequency in lymphocytes sampled at 24 h. Curve fitting analysis suggested that the dose response for γ-H2AX yields after acute exposures could be described by a linear dependence. In contrast, a linear-quadratic dose-response shape was more appropriate for LDR exposure (perhaps reflecting differences in repair time after different LDR doses). Dose-rate sparing effects (P < 0.05) were observed at doses ≤2.2 Gy, such that the acute dose γ-H2AX and TUNEL-positive cell yields were significantly larger than the equivalent LDR yields. At the 4.45 Gy dose there was no difference in γ-H2AX expression between the two dose rates, whereas there was a two- to threefold increase in apoptosis in the LDR samples compared to the equivalent 4.45 Gy acute dose. Micronuclei yields were measured at 24 h and 7 days using the in vitro cytokinesis-blocked micronucleus (CBMN) assay. The results showed that MNi yields increased up to 2.2 Gy with no further increase at 4.45 Gy and with no detectable dose-rate effect across the dose range 24 h or 7 days post exposure. In conclusion, the γ-H2AX biomarker showed higher sensitivity to measure dose-rate effects after low-dose LDR X rays compared to MNi formation; however

  16. ERK/MAPK Regulates Hippocampal Histone Phosphorylation Following Contextual Fear Conditioning

    ERIC Educational Resources Information Center

    Levenson, Jonathan M.; Sweatt, J. David; Chwang, Wilson B.; O'Riordan, Kenneth J.

    2006-01-01

    Long-term memory formation is regulated by many distinct molecular mechanisms that control gene expression. An emerging model for effecting a stable, coordinated pattern of gene transcription involves epigenetic tagging through modifications of histones or DNA. In this study, we investigated the regulation of histone phosphorylation in the…

  17. The Use of Gamma-H2AX as a Biodosimeter for Total-Body Radiation Exposure in Non-Human Primates

    PubMed Central

    Redon, Christophe E.; Nakamura, Asako J.; Gouliaeva, Ksenia; Rahman, Arifur; Blakely, William F.; Bonner, William M.

    2010-01-01

    Background There is a crucial shortage of methods capable of determining the extent of accidental exposures of human beings to ionizing radiation. However, knowledge of individual exposures is essential for early triage during radiological incidents to provide optimum possible life-sparing medical procedures to each person. Methods and Findings We evaluated immunocytofluorescence-based quantitation of γ-H2AX foci as a biodosimeter of total-body radiation exposure (60Co γ-rays) in a rhesus macaque (Macaca mulatta) model. Peripheral blood lymphocytes and plucked hairs were collected from 4 cohorts of macaques receiving total body irradiation doses ranging from 1 Gy to 8.5 Gy. Each cohort consisted of 6 experimental and 2 control animals. Numbers of residual γ-H2AX foci were proportional to initial irradiation doses and statistically significant responses were obtained until 1 day after 1 Gy, 4 days after 3.5 and 6.5 Gy, and 14 days after 8.5 Gy in lymphocytes and until 1 day after 1 Gy, at least 2 days after 3.5 and 6.5 Gy, and 9 days after 8.5 Gy in plucked hairs. Conclusion These findings indicate that quantitation of γ-H2AX foci may make a robust biodosimeter for analyzing total-body exposure to ionizing radiation in humans. This tool would help clinicians prescribe appropriate types of medical intervention for optimal individual outcome. These results also demonstrate that the use of a high throughput γ-H2AX biodosimeter would be useful for days post-exposure in applications like large-scale radiological events or radiation therapy. In addition, this study validates a possibility to use plucked hair in future clinical trials investigating genotoxic effects of drugs and radiation treatments. PMID:21124906

  18. Modulation of 14-3-3 interaction with phosphorylated histone H3 by combinatorial modification patterns

    PubMed Central

    Winter, Stefan; Fischle, Wolfgang; Seiser, Christian

    2011-01-01

    Post-translational modifications of histones are determining factors in the global and local regulation of genome activity. Phosphorylation of histone H3 is globally associated with mitotic chromatin compaction but occurs in a much more restricted manner during interphase transcriptional regulation of a limited subset of genes. In the course of gene regulation, serine 10 phosphorylation at histone H3 is targeted to a very small fraction of nucleosomes that is highly susceptible to additional acetylation events. Recently, we and others have identified 14-3-3 as a binding protein that recognizes both phosphorylated serine 10 and phosphorylated serine 28 on histone H3. In vitro, the affinity of 14-3-3 for phosphoserine 10 is weak but becomes significantly increased by additional acetylation of either lysine 9 or lysine 14 on the same histone tail. In contrast, the histone H3S28 site matches elements of 14-3-3 high affinity consensus motifs. This region mediates an initial stronger interaction that is less susceptible to modulation by “auxiliary” modifications. Here we discuss the binding of 14-3-3 proteins to histone H3 in detail and putative biological implications of these interactions. PMID:18418070

  19. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  20. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    PubMed

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  1. γ-H2AX responds to DNA damage induced by long-term exposure to combined low-dose-rate neutron and γ-ray radiation.

    PubMed

    Zhang, Junlin; He, Ying; Shen, Xianrong; Jiang, Dingwen; Wang, Qingrong; Liu, Qiong; Fang, Wen

    2016-01-01

    Risk estimates for low-dose radiation (LDR) remain controversial. The possible involvement of DNA repair-related genes in long-term low-dose-rate neutron-gamma radiation exposure is poorly understood. In this study, 60 rats were divided into control groups and irradiated groups, which were exposed to low-dose-rate n-γ combined radiation (LDCR) for 15, 30, or 60 days. The effects of different cumulative radiation doses on peripheral blood cell (PBC), subsets of T cells of peripheral blood lymphocytes (PBL) and DNA damage repair were investigated. Real-time PCR and immunoblot analyses were used to detect expression of DNA DSB-repair-related genes involved in the NHEJ pathway, such as Ku70 and Ku80, in PBL. The mRNA level of H2AX and the expression level of γ-H2AX were detected by real-time PCR, immunoblot, and flow cytometry. White blood cells (WBC) and platelets (PLT) of all ionizing radiation (IR) groups decreased significantly, while no difference was seen between the 30 day and 60 day exposure groups. The numbers of CD3(+), CD4(+) T cells and CD4(+)/CD8(+) in the PBL of IR groups were lower than in the control group. In the 30 day and 60 day exposure groups, CD8(+) T cells decreased significantly. Real-time PCR and immunoblot results showed no significant difference in the mRNA and protein expression of Ku70 and Ku80 between the control groups and IR groups. However, the mRNA of H2AX increased significantly, and there was a positive correlation with dose. There was no difference in the protein expression of γ-H2AX between 30 day and 60 day groups, which may help to explain the damage to PBL. In conclusion, PBL damage increased with cumulative dose, suggesting that γ-H2AX, but neither Ku70 nor Ku80, plays an important role in PBL impairment induced by LDCR. PMID:26774665

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

    PubMed Central

    Chatzimichail, E.; Matthaios, D.; Bouros, D.; Karakitsos, P.; Romanidis, K.; Kakolyris, S.; Papashinopoulos, G.; Rigas, A.

    2014-01-01

    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. PMID:24527431

  3. Synchrotron-based imaging of chromium and  γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water

    SciTech Connect

    Thompson, Chad M.; Seiter, Jennifer; Chappell, Mark A.; Tappero, Ryan V.; Proctor, Deborah M.; Suh, Mina; Wolf, Jeffrey C.; Haws, Laurie C.; Vitale, Rock; Mittal, Liz; Kirman, Christopher R.; Hays, Sean M.; Harris, Mark A.

    2014-10-28

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ~2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. Lastly, these findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.

  4. Synchrotron-based imaging of chromium and  γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water

    DOE PAGESBeta

    Thompson, Chad M.; Seiter, Jennifer; Chappell, Mark A.; Tappero, Ryan V.; Proctor, Deborah M.; Suh, Mina; Wolf, Jeffrey C.; Haws, Laurie C.; Vitale, Rock; Mittal, Liz; et al

    2014-10-28

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodenamore » from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ~2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. Lastly, these findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia.« less

  5. Synchrotron-Based Imaging of Chromium and γ-H2AX Immunostaining in the Duodenum Following Repeated Exposure to Cr(VI) in Drinking Water

    PubMed Central

    Thompson, Chad M.; Seiter, Jennifer; Chappell, Mark A.; Tappero, Ryan V.; Proctor, Deborah M.; Suh, Mina; Wolf, Jeffrey C.; Haws, Laurie C.; Vitale, Rock; Mittal, Liz; Kirman, Christopher R.; Hays, Sean M.; Harris, Mark A.

    2015-01-01

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum—the latter evidenced by lengthening of the crypt compartment by ∼2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. These findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia. PMID:25352572

  6. Increased γ-H2AX and Rad51 DNA Repair Biomarker Expression in Human Cell Lines Resistant to the Chemotherapeutic Agents Nitrogen Mustard and Cisplatin.

    PubMed

    Adam-Zahir, Sheba; Plowman, Piers N; Bourton, Emma C; Sharif, Fariha; Parris, Christopher N

    2014-01-01

    Chemotherapeutic anticancer drugs mediate cytotoxicity by a number of mechanisms. However, alkylating agents which induce DNA interstrand crosslinks (ICL) are amongst the most effective anticancer agents and often form the mainstay of many anticancer therapies. The effectiveness of these drugs can be limited by the development of drug resistance in cancer cells and many studies have demonstrated that alterations in DNA repair kinetics are responsible for drug resistance. In this study we developed two cell lines resistant to the alkylating agents nitrogen mustard (HN2) and cisplatin (Pt). To determine if drug resistance was associated with enhanced ICL DNA repair we used immunocytochemistry and imaging flow cytometry to quantitate the number of γ-H2AX and Rad51 foci in the nuclei of cells after drug exposure. γ-H2AX was used to evaluate DNA strand breaks caused by repair incision nucleases and Rad51 was used to measure the activity of homologous recombination in the repair of ICL. In the drug-resistant derivative cell lines there was overall a significant increase in the number and persistence of both γ-H2AX and Rad51 foci in the nuclei of cells over a 72-hour period, when compared to the non-resistant parental cell lines (ANOVA p < 0.0001). In a Pt-resistant ovarian cancer cell line (A2780cis(R)) a similar enhancement of DNA repair was observed when compared to the non-drug-resistant wild-type ovarian cancer cells (A2780) following exposure to HN2. Our data suggest that using DNA repair biomarkers to evaluate mechanisms of resistance in cancer cell lines and human tumours may be of experimental and clinical benefit. We concede, however, that examination of a larger population of cell lines and tumours is required to fully evaluate the validity of this approach. PMID:26138778

  7. Synchrotron-based imaging of chromium and γ-H2AX immunostaining in the duodenum following repeated exposure to Cr(VI) in drinking water.

    PubMed

    Thompson, Chad M; Seiter, Jennifer; Chappell, Mark A; Tappero, Ryan V; Proctor, Deborah M; Suh, Mina; Wolf, Jeffrey C; Haws, Laurie C; Vitale, Rock; Mittal, Liz; Kirman, Christopher R; Hays, Sean M; Harris, Mark A

    2015-01-01

    Current drinking water standards for chromium are for the combined total of both hexavalent and trivalent chromium (Cr(VI) and Cr(III)). However, recent studies have shown that Cr(III) is not carcinogenic to rodents, whereas mice chronically exposed to high levels of Cr(VI) developed duodenal tumors. These findings may suggest the need for environmental standards specific for Cr(VI). Whether the intestinal tumors arose through a mutagenic or non-mutagenic mode of action (MOA) greatly impacts how drinking water standards for Cr(VI) are derived. Herein, X-ray fluorescence (spectro)microscopy (µ-XRF) was used to image the Cr content in the villus and crypt regions of duodena from B6C3F1 mice exposed to 180 mg/l Cr(VI) in drinking water for 13 weeks. DNA damage was also assessed by γ-H2AX immunostaining. Exposure to Cr(VI) induced villus blunting and crypt hyperplasia in the duodenum--the latter evidenced by lengthening of the crypt compartment by ∼2-fold with a concomitant 1.5-fold increase in the number of crypt enterocytes. γ-H2AX immunostaining was elevated in villi, but not in the crypt compartment. µ-XRF maps revealed mean Cr levels >30 times higher in duodenal villi than crypt regions; mean Cr levels in crypt regions were only slightly above background signal. Despite the presence of Cr and elevated γ-H2AX immunoreactivity in villi, no aberrant foci indicative of transformation were evident. These findings do not support a MOA for intestinal carcinogenesis involving direct Cr-DNA interaction in intestinal stem cells, but rather support a non-mutagenic MOA involving chronic wounding of intestinal villi and crypt cell hyperplasia. PMID:25352572

  8. Polo-like kinase-1 triggers histone phosphorylation by Haspin in mitosis

    PubMed Central

    Zhou, Linli; Tian, Xiaoying; Zhu, Cailei; Wang, Fangwei; Higgins, Jonathan MG

    2014-01-01

    Histone modifications coordinate the chromatin localization of key regulatory factors in mitosis. For example, mitotic phosphorylation of Histone H3 threonine-3 (H3T3ph) by Haspin creates a binding site for the chromosomal passenger complex (CPC). However, how these histone modifications are spatiotemporally controlled during the cell cycle is unclear. Here we show that Plk1 binds to Haspin in a Cdk1-phosphorylation-dependent manner. Reducing Plk1 activity decreases the phosphorylation of Haspin and inhibits H3T3ph, particularly in prophase, suggesting that Plk1 is required for initial activation of Haspin in early mitosis. These studies demonstrate that Plk1 can positively regulate CPC recruitment in mitosis. PMID:24413556

  9. The N-terminus of histone H2B, but not that of histone H3 or its phosphorylation, is essential for chromosome condensation

    PubMed Central

    de la Barre, Anne-Elisabeth; Angelov, Dimitri; Molla, Annie; Dimitrov, Stefan

    2001-01-01

    We have studied the role of individual histone N-termini and the phosphorylation of histone H3 in chromosome condensation. Nucleosomes, reconstituted with histone octamers containing different combinations of recombinant full-length and tailless histones, were used as competitors for chromosome assembly in Xenopus egg extracts. Nucleosomes reconstituted with intact octamers inhibited chromosome condensation as efficiently as the native ones, while tailless nucleosomes were unable to affect this process. Importantly, the addition to the extract of particles containing only intact histone H2B strongly interfered with chromosome formation while such an effect was not observed with particles lacking the N-terminal tail of H2B. This demonstrates that the inhibition effect observed in the presence of competitor nucleosomes is mainly due to the N-terminus of this histone, which, therefore, is essential for chromosome condensation. Nucleosomes in which all histones but H3 were tailless did not impede chromosome formation. In addition, when competitor nucleosome particles were reconstituted with full-length H2A, H2B and H4 and histone H3 mutated at the phosphorylable serine 10 or serine 28, their inhibiting efficiency was identical to that of the native particles. Hence, the tail of H3, whether intact or phosphorylated, is not important for chromosome condensation. A novel hypothesis, termed ‘the ready production label’ was suggested to explain the role of histone H3 phosphorylation during cell division. PMID:11707409

  10. Loss of CCDC6, the First Identified RET Partner Gene, Affects pH2AX S139 Levels and Accelerates Mitotic Entry upon DNA Damage

    PubMed Central

    Muller, Mark T.; Pacelli, Roberto; Fusco, Alfredo; Celetti, Angela

    2012-01-01

    CCDC6 was originally identified in chimeric genes caused by chromosomal translocation involving the RET proto-oncogene in some thryoid tumors mostly upon ionizing radiation exposure. Recognised as a pro-apoptotic phosphoprotein that negatively regulates CREB1-dependent transcription, CCDC6 is an ATM substrate that is responsive to genotoxic stress. Here we report that following genotoxic stress, loss or inactivation of CCDC6 in cancers that carry the CCDC6 fusion, accelerates the dephosphorylation of pH2AX S139, resulting in defective G2 arrest and premature mitotic entry. Moreover, we show that CCDC6 depleted cells appear to repair DNA damaged in a shorter time compared to controls, based on reporter assays in cells. High-troughput proteomic screening predicted the interaction between the CCDC6 gene product and the catalytic subunit of Serin–Threonin Protein Phosphatase 4 (PP4c) recently identified as the evolutionarily conserved pH2AX S139 phosphatase that is activated upon DNA Damage. We describe the interaction between CCDC6 and PP4c and we report the modulation of PP4c enzymatic activity in CCDC6 depleted cells. We discuss the functional significance of CCDC6-PP4c interactions and hypothesize that CCDC6 may act in the DNA Damage Response by negatively modulating PP4c activity. Overall, our data suggest that in primary tumours the loss of CCDC6 function could influence genome stability and thereby contribute to carcinogenesis. PMID:22655027

  11. [Slow Formation and Degradation of γH2AX Foci in Human Skin Fibroblasts Exposed to Low-Dose X-Ray Radiation].

    PubMed

    Grekhova, A K; Eremin, P S; Osipov, A N; Eremin, I I; Pustovalova, M V; Ozerov, I V; Smetanina, N M; Lazareva, N L; Vorobyeva, N Yu; Pulin, A A; Maksimova, O A; Gordeev, A V; Bushmanov, A Yu; Kotenko, K V

    2015-01-01

    It was shown that the kinetics of changes of γH2AX foci number (marker of DNA double-strand breaks) in human skin fibroblasts after exposure to low doses of X-ray radiation (20, 40 and 80 mGy) differs from that observed after exposure to medium-low doses (160 and 240 mGy). After exposure to 160 and 240 mGy the highest number of γH2AX foci was detected at 30 min after exposure (first experimental point) and further their decrease was observed. At the same time we observed a fast phase of repair (upto 4 h), in which there was a decrease of the foci amount to ~50-60% and a slow phase of repair (from 4 h to 24 h). After 24 h only ~3-5% of the foci amount observed at 30 min after irradiation was left. After exposure to low doses, the foci number did not decrease during 2 h and even 24 h after exposure their amount was ~25% from that observed at maximum points (1 h after irradiation at 40 and 80 mGy and 2 h after irradiation at 20 mGy). PMID:26601539

  12. Age- and oxidative stress-induced DNA damage in Drosophila intestinal stem cells as marked by Gamma-H2AX.

    PubMed

    Park, Joung-Sun; Lee, Shin-Hae; Na, Hyun-Jin; Pyo, Jung-Hoon; Kim, Young-Shin; Yoo, Mi-Ae

    2012-05-01

    A decline in stem cell function is considered as a major cause of tissue atrophy, organ-system failure, cancer development and aging process. For a better understanding of the mechanism underlying age-related decline of stem cell function, characterization of aged stem cells is required. DNA damage induces epigenetic modifications that are associated with cell dysfunction. In mammals, γH2AX has been shown as DNA damage marker and an adaptor for recruiting chromatin modifying factors. In current study, utilizing a well-accepted Drosophila midgut model for stem-cell biology, we demonstrated aging- and oxidative stress-related accumulation of γH2AvD foci, analogous to mammal γH2AX, in Drosophila intestinal stem cells (ISCs), and obtained evidence that the changes in γH2AvD is closely associated with γ-ray-induced DNA damage in ISCs and age-related accumulation of 8-oxo-2'-deoxyguanosine. The significance of our study is to document the first direct evidence for the accumulation of age-related DNA-damage in ISCs, and to show γH2AvD as a useful biomarker in exploring the molecular mechanisms underlying stem cell aging in the Drosophila midgut. PMID:22387531

  13. Biochemical kinetics model of DSB repair and induction of gamma-H2AX foci by non-homologous end joining.

    PubMed

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

    2008-02-01

    We developed a biochemical kinetics approach to describe the repair of double-strand breaks (DSBs) produced by low-LET radiation by modeling molecular events associated with non-homologous end joining (NHEJ). A system of coupled nonlinear ordinary differential equations describes the induction of DSBs and activation pathways for major NHEJ components including Ku70/80, DNA-PKcs, and the ligase IV-XRCC4 heterodimer. The autophosphorylation of DNA-PKcs and subsequent induction of gamma-H2AX foci observed after ionizing radiation exposure were modeled. A two-step model of regulation of repair by DNA-PKcs was developed with an 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 the second step depends on DSB complexity, with a much slower rate for complex DSBs. The model faithfully reproduced several experimental data sets, including DSB rejoining as measured by pulsed-field gel electrophoresis (PFGE) at 10 min postirradiation or longer and quantification of the induction of gamma-H2AX foci. A process that is independent of DNA-PKcs is required for the model to reproduce experimental data for rejoining before 10 min postirradiation. Predictions are made for the behaviors of NHEJ components at low doses and dose rates, and a steady state is found at dose rates of 0.1 Gy/h or lower. PMID:18220463

  14. Phosphorylation and arginine methylation mark histone H2A prior to deposition during Xenopus laevis development

    PubMed Central

    2014-01-01

    Background Stored, soluble histones in eggs are essential for early development, in particular during the maternally controlled early cell cycles in the absence of transcription. Histone post-translational modifications (PTMs) direct and regulate chromatin-templated transactions, so understanding the nature and function of pre-deposition maternal histones is essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Little is known about histone H2A pre-deposition modifications nor known about the transitions that occur upon the onset of zygotic control of the cell cycle and transcription at the mid-blastula transition (MBT). Results We isolated histones from staged Xenopus laevis oocytes, eggs, embryos, and assembled pronuclei to identify changes in histone H2A modifications prior to deposition and in chromatin. Soluble and chromatin-bound histones from eggs and embryos demonstrated distinct patterns of maternal and zygotic H2A PTMs, with significant pre-deposition quantities of S1ph and R3me1, and R3me2s. We observed the first functional distinction between H2A and H4 S1 phosphorylation, as we showed that H2A and H2A.X-F (also known as H2A.X.3) serine 1 (S1) is phosphorylated concomitant with germinal vesicle breakdown (GVBD) while H4 serine 1 phosphorylation occurs post-MBT. In egg extract H2A/H4 S1 phosphorylation is independent of the cell cycle, chromatin assembly, and DNA replication. H2AS1ph is highly enriched on blastula chromatin during repression of zygotic gene expression while H4S1ph is correlated with the beginning of maternal gene expression and the lengthening of the cell cycle, consistent with distinct biological roles for H2A and H4 S1 phosphorylation. We isolated soluble H2A and H2A.X-F from the egg and chromatin-bound in pronuclei and analyzed them by mass spectrometry analysis to quantitatively determine abundances of S1ph and R3 methylation. We show that H2A and H4 S1ph, R3me1 and R3me2s are

  15. Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    PubMed Central

    North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

    2014-01-01

    Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

  16. Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

    PubMed

    Klingberg, Rebecca; Jost, Jan Oliver; Schümann, Michael; Gelato, Kathy Ann; Fischle, Wolfgang; Krause, Eberhard; Schwarzer, Dirk

    2015-01-16

    Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails. PMID:25330109

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

    SciTech Connect

    Pouliliou, Stamatia E.; Dimitriou, Thespis; Giatromanolaki, Alexandra; Papazoglou, Dimitrios; Pappa, Aglaia; Pistevou, Kyriaki

    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

  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.

    PubMed

    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. PMID:27260225

  19. Investigating chromosome damage and gammaH2AX response in human lymphocytes and lymphocyte subsets as potential biomarkers of radiation sensitivity

    NASA Astrophysics Data System (ADS)

    Beaton, Lindsay A.

    This thesis examines in vitro irradiated blood samples from prostate cancer patients exhibiting late normal tissue damage after receiving radiotherapy, for lymphocyte response. Chromosomal aberrations, translocations and proliferation rate are measured, as well as gammaH2AX response in lymphocytes and lymphocyte subsets. The goal of this thesis is to determine whether the lymphocyte response to in vitro radiation could be used as a marker for radiosensitivity. 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. The samples were irradiated and were analyzed for dicentric chromosomes, excess fragments and proliferation rates (at 6 Gy), translocations, stable and unstable damage (at 4 Gy), and dose response (up to 10 Gy), along with time response after 2 Gy (0 -- 24 h). Chromosome aberrations, excess fragments per cell, translocations per cell and proliferation rates were analyzed by brightfield and fluorescent microscopy, while the gammaH2AX response in lymphocytes and lymphocyte subsets was analyzed by flow cytometry. Both groups were statistically similar for all endpoints at 0 Gy. At 6 Gy, there were statistically significant differences between the radiosensitive and control cohorts for three endpoints; the mean number of dicentric chromosomes per cell, the mean number of excess fragments per cell and the proportion of cells in second metaphase. At 4 Gy, there were statistically significant differences between the two cohorts for three endpoints; the mean number of translocations per cell, the mean number of dicentric chromosomes per cell and the mean number of deletions per cell. There were no significant differences between the gammaH2AX

  20. Calibration of the γ-H2AX DNA Double Strand Break Focus Assay for Internal Radiation Exposure of Blood Lymphocytes

    PubMed Central

    Eberlein, Uta; Peper, Michel; Fernández, Maria; Lassmann, Michael; Scherthan, Harry

    2015-01-01

    DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations. PMID:25853575

  1. Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes.

    PubMed

    Eberlein, Uta; Peper, Michel; Fernández, Maria; Lassmann, Michael; Scherthan, Harry

    2015-01-01

    DNA double strand break (DSB) formation induced by ionizing radiation exposure is indicated by the DSB biomarkers γ-H2AX and 53BP1. Knowledge about DSB foci formation in-vitro after internal irradiation of whole blood samples with radionuclides in solution will help us to gain detailed insights about dose-response relationships in patients after molecular radiotherapy (MRT). Therefore, we studied the induction of radiation-induced co-localizing γ-H2AX and 53BP1 foci as surrogate markers for DSBs in-vitro, and correlated the obtained foci per cell values with the in-vitro absorbed doses to the blood for the two most frequently used radionuclides in MRT (I-131 and Lu-177). This approach led to an in-vitro calibration curve. Overall, 55 blood samples of three healthy volunteers were analyzed. For each experiment several vials containing a mixture of whole blood and radioactive solutions with different concentrations of isotonic NaCl-diluted radionuclides with known activities were prepared. Leukocytes were recovered by density centrifugation after incubation and constant blending for 1 h at 37°C. After ethanol fixation they were subjected to two-color immunofluorescence staining and the average frequencies of the co-localizing γ-H2AX and 53BP1 foci/nucleus were determined using a fluorescence microscope equipped with a red/green double band pass filter. The exact activity was determined in parallel in each blood sample by calibrated germanium detector measurements. The absorbed dose rates to the blood per nuclear disintegrations occurring in 1 ml of blood were calculated for both isotopes by a Monte Carlo simulation. The measured blood doses in our samples ranged from 6 to 95 mGy. A linear relationship was found between the number of DSB-marking foci/nucleus and the absorbed dose to the blood for both radionuclides studied. There were only minor nuclide-specific intra- and inter-subject deviations. PMID:25853575

  2. Twist1 and Slug mediate H2AX-regulated epithelial-mesenchymal transition in breast cells.

    PubMed

    Weyemi, Urbain; Redon, Christophe E; Sethi, Taresh K; Burrell, Allison S; Jailwala, Parthav; Kasoji, Manjula; Abrams, Natalie; Merchant, Anand; Bonner, William M

    2016-09-16

    The epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific. PMID:27315462

  3. 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.

    PubMed

    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

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

    PubMed

    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 50mg/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. PMID:26232259

  5. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch

    PubMed Central

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-01-01

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis. PMID:27515126

  6. Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

    PubMed Central

    Hait, Nitai C; Wise, Laura E; Allegood, Jeremy C; O’Brien, Megan; Avni, Dorit; Reeves, Thomas M; Knapp, Pamela E; Lu, Junyan; Luo, Cheng; Miles, Michael F; Milstien, Sheldon; Lichtman, Aron H; Spiegel, Sarah

    2014-01-01

    FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories. PMID:24859201

  7. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    PubMed Central

    Le, Ly-Thuy-Tram; Vu, Hong-Lien; Nguyen, Chi-Hung; Molla, Annie

    2013-01-01

    Summary Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors. PMID:23616922

  8. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

    PubMed Central

    Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

    2013-01-01

    Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

  9. Mitotic Phosphorylation of Histone H3: Spatio-Temporal Regulation by Mammalian Aurora Kinases

    PubMed Central

    Crosio, Claudia; Fimia, Gian Maria; Loury, Romain; Kimura, Masashi; Okano, Yukio; Zhou, Hongyi; Sen, Subrata; Allis, C. David; Sassone-Corsi, Paolo

    2002-01-01

    Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G2 phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Mutation of Ser-10 is essential in Tetrahymena, since it results in abnormal chromosome segregation and extensive chromosome loss during mitosis and meiosis, establishing a strong link between signaling and chromosome dynamics. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. Here we show that the expression of Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G2/M transition. During the G2 phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, our findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system. PMID:11784863

  10. A cdc2-like kinase phosphorylates histone H1 in the amitotic macronucleus of Tetrahymena.

    PubMed Central

    Roth, S Y; Collini, M P; Draetta, G; Beach, D; Allis, C D

    1991-01-01

    Genetic and biochemical studies have shown that cdc2 protein kinase plays a pivotal role in a highly conserved mechanism controlling the entry of cells into mitosis. It is generally believed that one function of cdc2 kinase is to phosphorylate histone H1 which in turn promotes mitotic chromosome condensation. However, direct evidence linking H1 phosphorylation to mitotic chromatin condensation is limited and the exact cellular function(s) of H1 phosphorylation remains unclear. In this study, we show that mammalian cdc2 kinase phosphorylates H1 from the amitotic macronucleus of Tetrahymena with remarkable fidelity. Furthermore, we demonstrate that macronuclei from Tetrahymena contain a growth-associated H1 kinase activity which closely resembles cdc2 kinase from other eukaryotes. Using polyclonal antibodies raised against yeast p34cdc2, we have detected a 36 kd immunoactive polypeptide in macronuclei which binds to Suc1 (p13)-coated beads and closely follows H1 kinase activity. Since macronuclei divide without mitotic chromosome condensation, these data demonstrate that H1 phosphorylation by cdc2 kinase may be necessary, but is not sufficient to promote mitotic chromatin condensation. The fact that an activity which strongly resembles mammalian cdc2 kinase is active during cell growth in a nucleus which does not undergo mitosis and chromosome condensation suggests that other factors are needed for a true mitotic division to occur. These data also reinforce the notion that H1 phosphorylation has important functions outside mitosis both in Tetrahymena and in mammalian cells. Images PMID:2065655

  11. Histone deacetylase 2 is phosphorylated, ubiquitinated, and degraded by cigarette smoke.

    PubMed

    Adenuga, David; Yao, Hongwei; March, Thomas H; Seagrave, Jeanclare; Rahman, Irfan

    2009-04-01

    Cigarette smoke (CS)-induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome-dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke. PMID:18927347

  12. Ciprofloxacin increases survival after ionizing irradiation combined injury: γ-H2AX formation, cytokine/chemokine, and red blood cells.

    PubMed

    Kiang, Juliann G; Fukumoto, Risaku

    2014-06-01

    Exposure to ionizing radiation alone (radiation injury, RI) or combined with traumatic tissue injury (radiation combined injury, CI) is a crucial life-threatening factor in nuclear and radiological accidents. It is well documented that RI and CI occur at the molecular, cellular, tissue, and system levels. However, their mechanisms remain largely unclear. It has been observed in dogs, pigs, rats, guinea pigs, and mice that radiation exposure combined with burns, wounds, or bacterial infection results in greater mortality than radiation exposure alone. In this laboratory, the authors found that B6D2F1/J female mice exposed to 9.75 Gy ⁶⁰Co-γ photon radiation followed by 15% total body surface area wounds experienced 50% higher mortality (over a 30-d observation period) compared to irradiation alone. CI enhanced DNA damages, amplified iNOS activation, induced massive release of pro-inflammatory cytokines, overexpressed MMPs and TLRs, and aggravated sepsis that led to cell death. In the present study, B6D2F1/J mice that received CI were treated with ciprofloxacin (CIP, 90 mg/kg p.o., q.d. within 2 h after CI through day 21). At day 1, CIP treatment reduced CI-induced γ-H2AX formation significantly. At day 10, CIP treatment not only reduced cytokine/chemokine concentrations significantly, including IL-6 and KC (i.e., IL-8 in humans), but also enhanced IL-3 production compared to vehicle-treated controls. CIP also elevated red blood cell counts, hemoglobin levels, and hematocrits. At day 30, CIP treatment increased 45% survival after CI (i.e., 2.3-fold increase over vehicle treatment). The results suggest that CIP may prove to be an effective therapeutic drug for CI. PMID:24776905

  13. Imaging flow cytometry as a sensitive tool to detect low-dose-induced DNA damage by analyzing 53BP1 and γH2AX foci in human lymphocytes.

    PubMed

    Durdik, Matus; Kosik, Pavol; Gursky, Jan; Vokalova, Lenka; Markova, Eva; Belyaev, Igor

    2015-12-01

    Ionizing radiation induced foci (IRIF) are considered the most sensitive indicator for DNA double-strand break (DSB) detection. Monitoring DSB induction by low doses of ionizing radiation is important due to the increasing exposure in the general population. γH2AX and 53BP1 are commonly used molecular markers for in situ IRIF assessment. Imaging flow cytometry (IFC) via ImageStream system provides a new opportunity in this field. We analyzed the formation of 53BP1, γH2AX foci and their co-localization induced by γ-rays (2, 5, 10, 50, 200 cGy) in human lymphocytes using ImageStream and the automated microscopic system Metafer. We observed very similar sensitivity of both systems for the detection of endogenous and low-dose-induced IRIF. Statistically significant induction of γH2AX foci was found at doses of 2 and 10 cGy using ImageStream and Metafer, respectively. Statistically significant induction of 53BP1 foci was evident at doses ≥ 5 cGy when analyzed by IFC. Analysis of the co-localizing foci by ImageStream and Metafer showed statistical significance at doses ≥ 2 cGy, suggesting that foci co-localization is a sensitive parameter for DSB quantification. Assessment of γH2AX, 53BP1 foci and their co-localization by Metafer and ImageStream showed similar linear dose responses in the low-dose range up to 10 cGy, although IFC showed slightly better resolution for IRIF in this dose range. At higher doses, IFC underestimated IRIF numbers. Using the imaging ability of ImageStream, we introduced an optimized assay by gating γH2AX foci positive (with 1 or more γH2AX foci) and negative (cells without foci) cells. This assay resulted in statistically significant IRIF induction at doses ≥ 5cGy and a linear dose response up to 50 cGy. In conclusion, we provide evidence for the use of IFC as an accurate high throughput assay for the prompt detection and enumeration of endogenous and low-dose induced IRIF. PMID:26243567

  14. Importance of protamine phosphorylation to histone displacement in spermatids: can the disruption of this process be used for male contraception

    SciTech Connect

    Balhorn, R.; Hud, N.V.; Corzett, M.; Mazrimas, J.

    1995-06-01

    Protamine is a small protein that packages DNA in the sperm of most vertebrates. Shortly after its synthesis, the serine and threonine residues in each protamine are phosphorylated and the modified proteins are deposited onto DNA, displacing the histones and other chromatin proteins. We have hypothesized that the phosphorylation of protamine 1 induces protamine dimerization and these dimers are required for efficient histone displacement. Histone displacement by protamines in late-step spermatids appears to be essential for the production of fertile sperm in man and other mammals, and the disruption of this process could provide a new approach for male contraception. As a first step towards testing this theory, we have initiated a set of in vitro experiments to determine whether of not protamine phosphorylation is essential for histone displacement. Thee results of these experiments, although incomplete, confirm that unphosphorylated protamine cannot effectively displace histone from DNA. Polyarginine molecules twice the size of a protamine molecule and salmine dimer were found to be more effective. These results are consistent with the theory that the disruption of protamine phosphorylation may prove to be a useful new approach for male contraception if it can be shown to facilitate or induce protamine dimerization.

  15. γH2AX formation kinetics in PBMCs of rabbits exposed to acute and fractionated radiation and attenuation of focus frequency through preadministration of a combination of podophyllotoxin and rutin hydrate.

    PubMed

    Yashavarddhan, M H; Shukla, Sandeep K; Srivastava, Nitya N; Suar, Mrutyunjay; Dutta, Sangeeta; Kalita, Bhargab; Ranjan, Rajiv; Singh, Abhinav; Bajaj, Sania; Gupta, Manju L

    2016-07-01

    DNA damage can be assessed by the quantitation of γH2AX foci that form at DSB sites. This study examines the generation and persistence of γH2AX foci, variability in foci size after acute and fractionated radiation exposure, and the effect of pretreatment with a safe radioprotective formulation termed G-003M on foci generation and persistence. G-003M contains a combination of podophyllotoxin and rutin hydrate, and was administered intramuscularly to rabbits 1 hr prior to Co(60) gamma irradiation. Rabbits were assigned to one of the following treatment groups: untreated, G-003M alone, irradiated (single dose 8 Gy, fractionated 2 Gy/day for 4 days or single dose 2 Gy) or G-003M preadministration followed by radiation exposure. Foci continuously persisted for a week in peripheral blood mononuclear cells of rabbits exposed to a single 8 Gy dose. However, the number of foci gradually decreased after reaching a maximum at 1 h. In rabbits exposed to fractionated radiation, foci detected 1 hr after the final exposure were significantly larger (P < 0.001) than in rabbits exposed to a single 8 Gy dose, but disappeared completely after 24 h. In both groups, foci reappeared on days 11-15 in terminally ill animals. G-003M pretreatment significantly (P < 0.05) attenuated the formation of γH2AX foci in all irradiated rabbits. This study reveals that γH2AX focus assessment could be used to confirm radiation exposure, that focus size reflects the type of radiation exposure (acute or fractionated), that the re-appearance of foci is a strong indicator of imminent death in animals, and that G-003M provides protection against radiation. Environ. Mol. Mutagen. 57:455-468, 2016. © 2016 Wiley Periodicals, Inc. PMID:27338557

  16. The influence of x-ray contrast agents in computed tomography on the induction of dicentrics and γ-H2AX foci in lymphocytes of human blood samples

    NASA Astrophysics Data System (ADS)

    Jost, G.; Golfier, S.; Pietsch, H.; Lengsfeld, P.; Voth, M.; Schmid, T. E.; Eckardt-Schupp, F.; Schmid, E.

    2009-10-01

    The aim of this study was to investigate and quantify two biomarkers for radiation exposure (dicentrics and γ-H2AX foci) in human lymphocytes after CT scans in the presence of an iodinated contrast agent. Blood samples from a healthy donor were exposed to CT scans in the absence or presence of iotrolan 300 at iodine concentrations of 5 or 50 mg ml-1 blood. The samples were exposed to 0.025, 0.05, 0.1 and 1 Gy in a tissue equivalent body phantom. Chromosome aberration scoring and automated microscopic analysis of γ-H2AX foci were performed in parts of the same samples. The theoretical physical dose enhancement factor (DEF) was calculated on the basis of the mass energy-absorption coefficients of iodine and blood and the photon energy spectrum of the CT tube. No significant differences in the yields of dicentrics and γ-H2AX foci were observed in the absence or presence of 5 mg iodine ml-1 blood up to 0.1 Gy, whereas at 1 Gy the yields were elevated for both biomarkers. At an iodine concentration of 50 mg ml-1 serving as a positive control, a biological DEF of 9.5 ± 1.4 and 2.3 ± 0.5 was determined for dicentrics and γ-H2AX foci, respectively. A physical DEF of 1.56 and 6.30 was calculated for 5 and 50 mg iodine ml-1, respectively. Thus, it can be concluded that in the diagnostic dose range (radiation and contrast dose), no relevant biological dose-enhancing effect could be detected, whereas a clear biological dose-enhancing effect could be found for a contrast dose well outside the diagnostic CT range for the complete radiation dose range with both methods.

  17. In vivo efficacy of a novel histone deacetylase inhibitor in combination with radiation for the treatment of gliomas1

    PubMed Central

    Entin-Meer, Michal; Yang, Xiaodong; VandenBerg, Scott R.; Lamborn, Kathleen R.; Nudelman, Abraham; Rephaeli, Ada; Haas-Kogan, Daphne Adele

    2007-01-01

    Histone modification has emerged as a promising approach to cancer therapy. We explored the in vivo efficacy of a butyric acid derivative, pivaloyloxymethyl butyrate (AN-9), for the treatment of gliomas. Relative to control and single-modality treatments, the combination of AN-9 and radiation significantly inhibited tumor growth and prolonged time to failure in mice bearing glioma xenografts. The enhanced response to radiation was accompanied by inhibition of cellular proliferation and by increased phosphorylation of H2AX, implicating DNA double-strand breaks in the antineoplastic effects of AN-9 and radiation. The data suggest that AN-9 in combination with radiation may be an effective therapy for malignant gliomas. PMID:17347490

  18. In vivo efficacy of a novel histone deacetylase inhibitor in combination with radiation for the treatment of gliomas.

    PubMed

    Entin-Meer, Michal; Yang, Xiaodong; VandenBerg, Scott R; Lamborn, Kathleen R; Nudelman, Abraham; Rephaeli, Ada; Haas-Kogan, Daphne Adele

    2007-04-01

    Histone modification has emerged as a promising approach to cancer therapy. We explored the in vivo efficacy of a butyric acid derivative, pivaloyloxymethyl butyrate (AN-9), for the treatment of gliomas. Relative to control and single-modality treatments, the combination of AN-9 and radiation significantly inhibited tumor growth and prolonged time to failure in mice bearing glioma xenografts. The enhanced response to radiation was accompanied by inhibition of cellular proliferation and by increased phosphorylation of H2AX, implicating DNA double-strand breaks in the antineoplastic effects of AN-9 and radiation. The data suggest that AN-9 in combination with radiation may be an effective therapy for malignant gliomas. PMID:17347490

  19. DNA Double-Strand Break Analysis by {gamma}-H2AX Foci: A Useful Method for Determining the Overreactors to Radiation-Induced Acute Reactions Among Head-and-Neck Cancer Patients

    SciTech Connect

    Goutham, Hassan Venkatesh; Mumbrekar, Kamalesh Dattaram; Vadhiraja, Bejadi Manjunath; Fernandes, Donald Jerard; Sharan, Krishna; Kanive Parashiva, Guruprasad; Kapaettu, Satyamoorthy; Bola Sadashiva, Satish Rao

    2012-12-01

    Purpose: Interindividual variability in normal tissue toxicity during radiation therapy is a limiting factor for successful treatment. Predicting the risk of developing acute reactions before initiation of radiation therapy may have the benefit of opting for altered radiation therapy regimens to achieve minimal adverse effects with improved tumor cure. Methods and Materials: DNA double-strand break (DSB) induction and its repair kinetics in lymphocytes of head-and-neck cancer patients undergoing chemoradiation therapy was analyzed by counting {gamma}-H2AX foci, neutral comet assay, and a modified version of neutral filter elution assay. Acute normal tissue reactions were assessed by Radiation Therapy Oncology Group criteria. Results: The correlation between residual DSBs and the severity of acute reactions demonstrated that residual {gamma}-H2AX foci in head-and-neck cancer patients increased with the severity of oral mucositis and skin reaction. Conclusions: Our results suggest that {gamma}-H2AX analysis may have predictive implications for identifying the overreactors to mucositis and skin reactions among head-and-neck cancer patients prior to initiation of radiation therapy.

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

    PubMed

    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. PMID:25690891

  1. HIPK2 controls cytokinesis and prevents tetraploidization by phosphorylating histone H2B at the midbody.

    PubMed

    Rinaldo, Cinzia; Moncada, Alice; Gradi, Alessandra; Ciuffini, Laura; D'Eliseo, Donatella; Siepi, Francesca; Prodosmo, Andrea; Giorgi, Alessandra; Pierantoni, Giovanna M; Trapasso, Francesco; Guarguaglini, Giulia; Bartolazzi, Armando; Cundari, Enrico; Schininà, M Eugenia; Fusco, Alfredo; Soddu, Silvia

    2012-07-13

    Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization. PMID:22658722

  2. Control of histone H3 phosphorylation by CaMKIIδ in response to haemodynamic cardiac stress

    PubMed Central

    Awad, Salma; Al-Haffar, Kamar Mohamed Adib; Marashly, Qussay; Quijada, Pearl; Kunhi, Muhammad; Al-Yacoub, Nadya; Wade, Fallou S; Mohammed, Shamayel Faheem; Al-Dayel, Fouad; Sutherland, George; Assiri, Abdullah; Sussman, Mark; Bers, Donald; Al-Habeeb, Waleed; Poizat, Coralie

    2015-01-01

    Heart failure is associated with the reactivation of a fetal cardiac gene programme that has become a hallmark of cardiac hypertrophy and maladaptive ventricular remodelling, yet the mechanisms that regulate this transcriptional reprogramming are not fully understood. Using mice with genetic ablation of calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ), which are resistant to pathological cardiac stress, we show that CaMKIIδ regulates the phosphorylation of histone H3 at serine-10 during pressure overload hypertrophy. H3 S10 phosphorylation is strongly increased in the adult mouse heart in the early phase of cardiac hypertrophy and remains detectable during cardiac decompensation. This response correlates with up-regulation of CaMKIIδ and increased expression of transcriptional drivers of pathological cardiac hypertrophy and of fetal cardiac genes. Similar changes are detected in patients with end-stage heart failure, where CaMKIIδ specifically interacts with phospho-H3. Robust H3 phosphorylation is detected in both adult ventricular myocytes and in non-cardiac cells in the stressed myocardium, and these signals are abolished in CaMKIIδ-deficient mice after pressure overload. Mechanistically, fetal cardiac genes are activated by increased recruitment of CaMKIIδ and enhanced H3 phosphorylation at hypertrophic promoter regions, both in mice and in human failing hearts, and this response is blunted in CaMKIIδ-deficient mice under stress. We also document that the chaperone protein 14–3–3 binds phosphorylated H3 in response to stress, allowing proper elongation of fetal cardiac genes by RNA polymerase II (RNAPII), as well as elongation of transcription factors regulating cardiac hypertrophy. These processes are impaired in CaMKIIδ-KO mice after pathological stress. The findings reveal a novel in vivo function of CaMKIIδ in regulating H3 phosphorylation and suggest a novel epigenetic mechanism by which CaMKIIδ controls cardiac hypertrophy.

  3. Quantitative Mass Spectrometry Reveals that Intact Histone H1 Phosphorylations are Variant Specific and Exhibit Single Molecule Hierarchical Dependence.

    PubMed

    Chen, Yu; Hoover, Michael E; Dang, Xibei; Shomo, Alan A; Guan, Xiaoyan; Marshall, Alan G; Freitas, Michael A; Young, Nicolas L

    2016-03-01

    Breast cancer was the second leading cause of cancer related mortality for females in 2014. Recent studies suggest histone H1 phosphorylation may be useful as a clinical biomarker of breast and other cancers because of its ability to recognize proliferative cell populations. Although monitoring a single phosphorylated H1 residue is adequate to stratify high-grade breast tumors, expanding our knowledge of how H1 is phosphorylated through the cell cycle is paramount to understanding its role in carcinogenesis. H1 analysis by bottom-up MS is challenging because of the presence of highly homologous sequence variants expressed by most cells. These highly basic proteins are difficult to analyze by LC-MS/MS because of the small, hydrophilic nature of peptides produced by tryptic digestion. Although bottom-up methods permit identification of several H1 phosphorylation events, these peptides are not useful for observing the combinatorial post-translational modification (PTM) patterns on the protein of interest. To complement the information provided by bottom-up MS, we utilized a top-down MS/MS workflow to permit identification and quantitation of H1 proteoforms related to the progression of breast cells through the cell cycle. Histones H1.2 and H1.4 were observed in MDA-MB-231 metastatic breast cells, whereas an additional histone variant, histone H1.3, was identified only in nonneoplastic MCF-10A cells. Progressive phosphorylation of histone H1.4 was identified in both cell lines at mitosis (M phase). Phosphorylation occurred first at S172 followed successively by S187, T18, T146, and T154. Notably, phosphorylation at S173 of histone H1.2 and S172, S187, T18, T146, and T154 of H1.4 significantly increases during M phase relative to S phase, suggesting that these events are cell cycle-dependent and may serve as markers for proliferation. Finally, we report the observation of the H1.2 SNP variant A18V in MCF-10A cells. PMID:26209608

  4. Characterisation of an aerosol exposure system to evaluate the genotoxicity of whole mainstream cigarette smoke using the in vitro γH2AX assay by high content screening

    PubMed Central

    2014-01-01

    Background The genotoxic effect of cigarette smoke is routinely measured by treating cells with cigarette Particulate Matter (PM) at different dose levels in submerged cell cultures. However, PM exposure cannot be considered as a complete exposure as it does not contain the gas phase component of the cigarette smoke. The in vitro γH2AX assay by High Content Screening (HCS) has been suggested as a complementary tool to the standard battery of genotoxicity assays as it detects DNA double strand breaks in a high-throughput fashion. The aim of this study was to further optimise the in vitro γH2AX assay by HCS to enable aerosol exposure of human bronchial epithelial BEAS-2B cells at the air-liquid interface (ALI). Methods Whole mainstream cigarette smoke (WMCS) from two reference cigarettes (3R4F and M4A) were assessed for their genotoxic potential. During the study, a further characterisation of the Borgwaldt RM20S® aerosol exposure system to include single dilution assessment with a reference gas was also carried out. Results The results of the optimisation showed that both reference cigarettes produced a positive genotoxic response at all dilutions tested. However, the correlation between dose and response was low for both 3R4F and M4A (Pearson coefficient, r = -0.53 and -0.44 respectively). During the additional characterisation of the exposure system, it was observed that several pre-programmed dilutions did not perform as expected. Conclusions Overall, the in vitro γH2AX assay by HCS could be used to evaluate WMCS in cell cultures at the ALI. Additionally, the extended characterisation of the exposure system indicates that assessing the performance of the dilutions could improve the existing routine QC checks. PMID:25056295

  5. Artemis deficiency confers a DNA double-strand break repair defect and Artemis phosphorylation status is altered by DNA damage and cell cycle progression.

    PubMed

    Wang, Junhua; Pluth, Janice M; Cooper, Priscilla K; Cowan, Morton J; Chen, David J; Yannone, Steven M

    2005-05-01

    Mutations in the Artemis gene are causative in a subset of human severe combined immunodeficiencies (SCIDs) and Artemis-deficient cells exhibit radiation sensitivity and defective V(D)J recombination, implicating Artemis function in non-homologous end joining (NHEJ). Here we show that Artemis-deficient cells from Athabascan-speaking Native American SCID patients (SCIDA) display significantly elevated sensitivity to ionizing radiation (IR) but only a very subtle defect in DNA double-strand (DSB) break repair in contrast to the severe DSB repair defect of NHEJ-deficient cells. Primary human SCIDA fibroblasts accumulate and exhibit persistent arrest at both the G1/S and G2/M boundaries in response to IR, consistent with the presence of persistent DNA damage. Artemis protein is phosphorylated in a PI3-like kinase-dependent manner after either IR or a number of other DNA damaging treatments including etoposide, but SCIDA cells are not hypersensitive to treatment with etoposide. Inhibitor studies with various DNA damaging agents establish multiple phosphorylation states and suggest multiple kinases function in Artemis phosphorylation. We observe that Artemis phosphorylation occurs rapidly after irradiation like that of histone H2AX. However, unlike H2AX, Artemis de-phosphorylation is uncoupled from overall DNA repair and correlates instead with cell cycle progression to or through mitosis. Our results implicate a direct and non-redundant function of Artemis in the repair of a small subset of DNA double-strand breaks, possibly those with hairpin termini, which may account for the pronounced radiation sensitivity observed in Artemis-deficient cells. PMID:15811628

  6. Increased Histone H3 Phosphorylation in Neurons in Specific Brain Structures after Induction of Status Epilepticus in Mice

    PubMed Central

    Mori, Tetsuji; Wakabayashi, Taketoshi; Ogawa, Haruyuki; Hirahara, Yukie; Koike, Taro; Yamada, Hisao

    2013-01-01

    Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3+ (PH3+) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation. PMID:24147063

  7. Three distinct patterns of histone H3Y41 phosphorylation mark active genes.

    PubMed

    Dawson, Mark A; Foster, Samuel D; Bannister, Andrew J; Robson, Samuel C; Hannah, Rebecca; Wang, Xiaonan; Xhemalce, Blerta; Wood, Andrew D; Green, Anthony R; Göttgens, Berthold; Kouzarides, Tony

    2012-09-27

    The JAK2 tyrosine kinase is a critical mediator of cytokine-induced signaling. It plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). We used chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq) to define the genome-wide pattern of H3Y41ph in human erythroid leukemia cells. Our results indicate that H3Y41ph is located at three distinct sites: (1) at a subset of active promoters, where it overlaps with H3K4me3, (2) at distal cis-regulatory elements, where it coincides with the binding of STAT5, and (3) throughout the transcribed regions of active, tissue-specific hematopoietic genes. Together, these data extend our understanding of this conserved and essential signaling pathway and provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription. PMID:22999934

  8. Three Distinct Patterns of Histone H3Y41 Phosphorylation Mark Active Genes

    PubMed Central

    Dawson, Mark A.; Foster, Samuel D.; Bannister, Andrew J.; Robson, Samuel C.; Hannah, Rebecca; Wang, Xiaonan; Xhemalce, Blerta; Wood, Andrew D.; Green, Anthony R.; Göttgens, Berthold; Kouzarides, Tony

    2012-01-01

    Summary The JAK2 tyrosine kinase is a critical mediator of cytokine-induced signaling. It plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). We used chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq) to define the genome-wide pattern of H3Y41ph in human erythroid leukemia cells. Our results indicate that H3Y41ph is located at three distinct sites: (1) at a subset of active promoters, where it overlaps with H3K4me3, (2) at distal cis-regulatory elements, where it coincides with the binding of STAT5, and (3) throughout the transcribed regions of active, tissue-specific hematopoietic genes. Together, these data extend our understanding of this conserved and essential signaling pathway and provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription. PMID:22999934

  9. Contribution of the Serine 129 of Histone H2A to Chromatin Structure▿

    PubMed Central

    Fink, Michel; Imholz, Daniela; Thoma, Fritz

    2007-01-01

    Phosphorylation of a yeast histone H2A at C-terminal serine 129 has a central role in double-strand break repair. Mimicking H2A phosphorylation by replacement of serine 129 with glutamic acid (hta1-S129E) suggested that phosphorylation destabilizes chromatin structures and thereby facilitates the access of repair proteins. Here we have tested chromatin structures in hta1-S129 mutants and in a C-terminal tail deletion strain. We show that hta1-S129E affects neither nucleosome positioning in minichromosomes and genomic loci nor supercoiling of minichromosomes. Moreover, hta1-S129E has no effect on chromatin stability measured by conventional nuclease digestion, nor does it affect DNA accessibility and repair of UV-induced DNA lesions by nucleotide excision repair and photolyase in vivo. Similarly, deletion of the C-terminal tail has no effect on nucleosome positioning and stability. These data argue against a general role for the C-terminal tail in chromatin organization and suggest that phosphorylated H2A, γ-H2AX in higher eukaryotes, acts by recruitment of repair components rather than by destabilizing chromatin structures. PMID:17353265

  10. Multifaceted Histone H3 Methylation and Phosphorylation Readout by the Plant Homeodomain Finger of Human Nuclear Antigen Sp100C.

    PubMed

    Zhang, Xiaojie; Zhao, Dan; Xiong, Xiaozhe; He, Zhimin; Li, Haitao

    2016-06-10

    The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout. Interestingly, reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation. PMID:27129259

  11. Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana

    PubMed Central

    Wang, Zhen; Casas-Mollano, Juan Armando; Xu, Jianping; Riethoven, Jean-Jack M.; Zhang, Chi; Cerutti, Heriberto

    2015-01-01

    Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5′ genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s). PMID:26100864

  12. Histone deacetylase 1 phosphorylation at S421 and S423 is constitutive in vivo, but dispensable in vitro

    SciTech Connect

    Karwowska-Desaulniers, Paulina; Ketko, Anastasia; Kamath, Nayana; Pflum, Mary Kay H. . E-mail: pflum@chem.wayne.edu

    2007-09-21

    Histone Deacetylase 1 (HDAC1) is a transcriptional regulator associated with proliferation, apoptosis, and tumorigenesis, although its precise cellular role is unclear. HDAC1 was previously characterized as a phosphoprotein where mutation of phosphorylated S421 and S423 resulted in a loss of deacetylase activity and protein association. Here, the role of phosphorylation in regulating HDAC1 function was examined using phospho-specific antibodies. The antibody studies revealed that phosphorylation at S421 and S423 is constant during the cell cycle, under stress conditions, or in the presence of kinase or phosphatase inhibitors. Further, phosphorylation is dispensable for catalysis or protein association in vitro, as revealed by phosphatase studies. Truncation mutants of HDAC1 demonstrated that binding to Sin3A is promoted by S421 and S423 phosphorylation, while interaction with RbAp48 is not. Taken together, the data are consistent with constitutive phosphorylation of HDAC1 at S421 and S423 in vivo, which is dispensable for activity in vitro.

  13. Anti-phosphorylated histone H2AThr120: a universal microscopic marker for centromeric chromatin of mono- and holocentric plant species.

    PubMed

    Demidov, D; Schubert, V; Kumke, K; Weiss, O; Karimi-Ashtiyani, R; Buttlar, J; Heckmann, S; Wanner, G; Dong, Q; Han, F; Houben, A

    2014-01-01

    Based on the analysis of 20 different monocot and eudicot species, we propose that the centromeric distribution of the phosphorylated histone H2AThr120 is evolutionary highly conserved across species with mono- and holocentric chromosomes. Therefore, antibodies recognizing the phosphorylated threonine 120 of the histone H2A can serve as a universal marker for the cytological detection of centromeres of mono- and holokinetic plant species. In addition, super resolution microscopy of signals specific to the centromere-specific histone H3 variant CENH3 and to H2AThr120ph revealed that these histone variants are incorporated into different nucleosomes, which form distinct, partly intermingled chromatin domains. This specific arrangement of both histone variants suggests different centromeric functions during the cell cycle. PMID:24642790

  14. Histone H3 Phosphorylation in Human Skin Histoculture as a Tool to Evaluate Patient’s Response to Antiproliferative Drugs

    PubMed Central

    Ugarte, Fernando; Porth, Katherine; Sadekova, Svetlana

    2015-01-01

    Evaluation of patient’s response to chemotherapeutic drugs is often difficult and time consuming. Skin punch biopsies are easily accessible material that can be used for the evaluation of surrogate biomarkers of a patient’s response to a drug. In this study, we hypothesized that assessment of phosphorylated histone H3 in human skin punch biopsies could be used as a pharmacodynamics biomarker of patient’s response to the kinesin spindle protein inhibitor SCH2047069. To test this hypothesis, we used a human skin histoculture technique that allows culturing intact human skin in the presence of the drug. Human melanoma and skin histocultures were treated with SCH2047069, and the effect of the drug was assessed by increasing histone H3 phosphorylation using immunohistochemistry. Our results demonstrate that SCH2047069 has a significant effect on cell proliferation in human melanoma and skin histoculture and justify using human skin punch biopsies for evaluation of the pharmacodynamic changes induced by SCH2047069. ACRONYMS Histone subunit H3 (H3), Kinesin spindle protein (KSP), 5-ethynyl-2′-deoxyuridine (EDU), Dimethyl sulfoxide (DMSO), Formalin-fixed paraffin embedded (FFPE). PMID:26917945

  15. Molecular mechanisms for the regulation of histone mRNA stem-loop-binding protein by phosphorylation

    SciTech Connect

    Zhang, Jun; Tan, Dazhi; DeRose, Eugene F.; Perera, Lalith; Dominski, Zbigniew; Marzluff, William F.; Tong, Liang; Tanaka Hall, Traci M.

    2014-08-06

    Replication-dependent histone mRNAs end with a conserved stem loop that is recognized by stem-loop–binding protein (SLBP). The minimal RNA-processing domain of SLBP is phosphorylated at an internal threonine, and Drosophila SLBP (dSLBP) also is phosphorylated at four serines in its 18-aa C-terminal tail. We show that phosphorylation of dSLBP increases RNA-binding affinity dramatically, and we use structural and biophysical analyses of dSLBP and a crystal structure of human SLBP phosphorylated on the internal threonine to understand the striking improvement in RNA binding. Together these results suggest that, although the C-terminal tail of dSLBP does not contact the RNA, phosphorylation of the tail promotes SLBP conformations competent for RNA binding and thereby appears to reduce the entropic penalty for the association. Increased negative charge in this C-terminal tail balances positively charged residues, allowing a more compact ensemble of structures in the absence of RNA.

  16. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis.

    PubMed

    Yoshida, Makoto M; Ting, Lily; Gygi, Steven P; Azuma, Yoshiaki

    2016-06-20

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  17. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    SciTech Connect

    Cooper, E.; Spaulding, S.W.

    1983-05-01

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with /sup 32/Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of /sup 32/P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of /sup 32/P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the /sup 32/P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, (/sup 32/P) HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

  18. 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

    NASA Astrophysics Data System (ADS)

    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

  19. Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

    PubMed Central

    Sur, Swastika; Swier, Vicki J.; Radwan, Mohamed M.; Agrawal, Devendra K.

    2016-01-01

    Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia. PMID:26820885

  20. Coordinated regulation of Nrf2 and histone H3 serine 10 phosphorylation in arsenite-activated transcription of the human heme oxygenase-1 gene.

    PubMed

    Ray, Paul D; Huang, Bo-Wen; Tsuji, Yoshiaki

    2015-10-01

    Expression of the antioxidant gene heme oxygenase-1 (HO-1) is primarily induced through NF-E2-related factor 2 (Nrf2)-mediated activation of the antioxidant response element (ARE). Gene transcription is coordinately regulated by transcription factor activity at enhancer elements and epigenetic alterations such as the posttranslational modification of histone proteins. However, the role of histone modifications in the Nrf2-ARE axis remains largely uncharacterized. The environmental contaminant arsenite is a potent inducer of both HO-1 expression and phosphorylation of histone H3 serine 10 (H3S10); therefore, we investigated the relationships between Nrf2 and H3S10 phosphorylation in arsenite-induced, ARE-dependent, transcriptional activation of the human HO-1 gene. Arsenite increased phosphorylation of H3S10 both globally and at the HO-1 promoter concomitantly with HO-1 transcription in human HaCaT keratinocytes. Conversely, arsenite-induced H3S10 phosphorylation and HO-1 expression were blocked by N-acetylcysteine (NAC), the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and JNK knockdown (siJNK). Interestingly, ablation of arsenite-induced H3S10 phosphorylation by SP600125 or siJNK did not inhibit Nrf2 nuclear accumulation nor ARE binding, despite inhibiting HO-1 expression. In response to arsenite, binding of Nrf2 to the HO-1 ARE preceded phosphorylation of H3S10 at the HO-1 ARE. Furthermore, arsenite-mediated occupancy of phosphorylated H3S10 at the HO-1 ARE was decreased in Nrf2-deficient mouse embryonic fibroblasts. These results suggest the involvement of H3S10 phosphorylation in the Nrf2-ARE axis by proposing that Nrf2 may influence H3S10 phosphorylation at the HO-1 ARE and additional promoter regions. Our data highlights the complex interplay between Nrf2 and H3S10 phosphorylation in arsenite-activated HO-1 transcription. PMID:26291278

  1. The temporal and spatial pattern of histone H3 phosphorylation at serine 28 and serine 10 is similar in plants but differs between mono- and polycentric chromosomes.

    PubMed

    Gernand, D; Demidov, D; Houben, A

    2003-01-01

    Immunolabeling using site-specific antibodies against phosphorylated histone H3 at serine 10 or serine 28 revealed in plants an almost similar temporal and spatial pattern of both post-translational modification sites at mitosis and meiosis. During the first meiotic division the entire chromosomes are highly H3 phosphorylated. In the second meiotic division, like in mitosis, the chromosomes contain high phosphorylation levels in the pericentromeric region and very little H3 phosphorylation along the arms of monocentric species. In the polycentric plant Luzula luzuloides phosphorylation at both serine positions occurs along the whole chromosomes, whereas in monocentric species, only the pericentromeric regions showed strong signals from mitotic prophase to telophase. No phosphorylated serine 10 or serine 28 was detectable on single chromatids at anaphase II resulting from equational segregation of rye B chromosome univalents during the preceding anaphase I. In addition, we found a high level of serine 28 as well as of serine 10 phosphorylation along the entire mitotic monocentric chromosomes after treatment of mitotic cells using the phosphatase inhibitor cantharidin. These observations suggest that histone H3 phosphorylation at serine 10 and 28 is an evolutionarily conserved event and both sites are likely to be involved in the same process, such as sister chromatid cohesion. PMID:14610360

  2. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

    PubMed Central

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.11402.001 PMID:26878753

  3. Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis.

    PubMed

    Wike, Candice L; Graves, Hillary K; Hawkins, Reva; Gibson, Matthew D; Ferdinand, Michelle B; Zhang, Tao; Chen, Zhihong; Hudson, Damien F; Ottesen, Jennifer J; Poirier, Michael G; Schumacher, Jill; Tyler, Jessica K

    2016-01-01

    Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. PMID:26878753

  4. Comparison of phosphorylation kinetics in DNA repair proteins after exposure to high and low LET radiations

    NASA Astrophysics Data System (ADS)

    Okayasu, R.; Okabe, A.; Takakura, K.

    We irradiated plateau phase normal human fibroblasts with 2 Gy X-rays 70 keV um carbon 290MeV n and 200 keV um iron ions 500 MeV n and observed the kinetics of phosphorylation in various proteins associated with DNA double strand break DSB repair GammaH2AX foci a marker for DSBs were detected immediately after irradiation and the peak of phosphorylation was seen 30 to 60 min post-irradiation for three kinds of radiations Disappearance of gamma-H2AX foci was much faster for X-irradiated samples than that for heavy ion irradiated samples the phosphorylation kinetics for carbon and iron ions are similar for gamma-H2AX foci In contrast phosphorylation of an NHEJ protein DNA-PKcs threonine 2609 was significantly delayed in carbon and iron irradiated cells when compared to X-irradiated cells Disappearance of DNA-PKcs sites was much faster in X-irradiated samples than carbon and iron samples which showed a similar pattern as in the case of gamma-H2AX Furthermore in the case of ATM protein phosphorylation serine 1981 iron irradiation alone caused a significant initial delay but the kinetics of disappearance is similar for iron and carbon samples with much higher remaining number of foci in iron samples than those for X-rays and carbon ions These results suggest that 1 high LET irradiation induces complex and or severe DNA DSB damage which affects the function of DSB repair proteins 2 Both ATM and DNA-PKcs may recognize the complexity of DSBs but ATM may be more sensitive to detecting the complexity of DSB damage 3 gamma-H2AX may

  5. Histone H3 phosphorylation in GBM: a new rational to guide the use of kinase inhibitors in anti-GBM therapy.

    PubMed

    Pacaud, Romain; Cheray, Mathilde; Nadaradjane, Arulraj; Vallette, François M; Cartron, Pierre-François

    2015-01-01

    Histones post-translational modifications (PTMs) are crucial components of diverse processes that modulate chromatin. Among the histones PTMs, the histones phosphorylation appears such crucial since it plays a significant role into DNA repair structure, transcription and chromatin compaction during cell division and apoptosis. However, little is known about the prognostic value of the histone phosphorylation in human cancer. This point could be considerate such as an important gap in anti-cancer therapy since the use of adequate kinase inhibitors could remedy to the aberrant histone phosphorylation associated with a poor prognosis factor. To remedy at this situation, we analyzed the phosphorylation level of histone H3 at the residues T3, T6, S10, S28, Y41 and T45 in a collection of 42 glioblastoma multiformes (GBM). Our data indicated that the high level of pH3T6, pH3S10 and pH3Y41 are signatures associated with a poor prognosis of overall survival (OS) of GBM treated with the "temozolomide and irradiation standard" treatment of GBM (named TMZ+Irad treatment). Our data also showed that these signatures are correlated with the high activity of kinases already described as writers of the pH3T6, pH3S10 and pH3Y41 i.e. the PKC, Aurora-B and JAK2, respectively. Finally, our analysis revealed that the use of Enzastaurin, AZD1152, and AZD1480 abrogated the high level of pH3T6, pH3S10 and pH3Y41 while increasing the sensitivity to the "temozolomide and irradiation"-induced cell death. To conclude, it appears that this work provides biomarkers for patient stratification for a therapy including kinase inhibitors. PMID:25553095

  6. Histone H3 Phosphorylation in GBM: a New Rational to Guide the Use of Kinase Inhibitors in anti-GBM Therapy

    PubMed Central

    Pacaud, Romain; Cheray, Mathilde; Nadaradjane, Arulraj; Vallette, François M.; Cartron, Pierre-François

    2015-01-01

    Histones post-translational modifications (PTMs) are crucial components of diverse processes that modulate chromatin. Among the histones PTMs, the histones phosphorylation appears such crucial since it plays a significant role into DNA repair structure, transcription and chromatin compaction during cell division and apoptosis. However, little is known about the prognostic value of the histone phosphorylation in human cancer. This point could be considerate such as an important gap in anti-cancer therapy since the use of adequate kinase inhibitors could remedy to the aberrant histone phosphorylation associated with a poor prognosis factor. To remedy at this situation, we analyzed the phosphorylation level of histone H3 at the residues T3, T6, S10, S28, Y41 and T45 in a collection of 42 glioblastoma multiformes (GBM). Our data indicated that the high level of pH3T6, pH3S10 and pH3Y41 are signatures associated with a poor prognosis of overall survival (OS) of GBM treated with the "temozolomide and irradiation standard" treatment of GBM (named TMZ+Irad treatment). Our data also showed that these signatures are correlated with the high activity of kinases already described as writers of the pH3T6, pH3S10 and pH3Y41 i.e. the PKC, Aurora-B and JAK2, respectively. Finally, our analysis revealed that the use of Enzastaurin, AZD1152, and AZD1480 abrogated the high level of pH3T6, pH3S10 and pH3Y41 while increasing the sensitivity to the “temozolomide and irradiation”-induced cell death. To conclude, it appears that this work provides biomarkers for patient stratification for a therapy including kinase inhibitors. PMID:25553095

  7. Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

    SciTech Connect

    Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

    2009-12-15

    Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

  8. Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

    PubMed Central

    Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

    2015-01-01

    Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

  9. Kaposi΄s sarcoma-associated herpesvirus ORF36 protein induces chromosome condensation and phosphorylation of histone H3.

    PubMed

    Kim, Sunmi; Cha, Seho; Jang, Jun Hyeong; Kim, Yejin; Seo, Taegun

    2013-01-01

    Kaposi΄s sarcoma-associated herpesvirus (KSHV) has been known as an agent causing Kaposi΄s sarcoma, primary effusion lymphoma, and multicentric Castleman΄s disease. In the lytic phase of the virus cycle, various viral genes are expressed, which causes host cell dysregulation. Among the lytic genes, viral protein kinase (vPK) encoded by ORF36 is a member of serine/threonine protein kinase (CHPK) family, which is involved in viral gene expression, viral DNA replication and encapsidation, and nuclear egress of virions. Recent studies have shown that the BGLF4 protein of Epstein-Barr virus (EBV), a member of the CHPK family, alters the host cell chromatin structure through phosphorylation of its key regulators. The role of KSHV ORF36 in cellular mitotic events, however, is not yet understood. In the current study, we showed that KSHV ORF36 induced chromosome condensation and phosphorylation of histone H3 on Ser 10, which are known as cellular mitosis markers. These processes have occurred in a kinase activity-dependent manner. PMID:23530827

  10. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine

    PubMed Central

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-01-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. PMID:26951077

  11. Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

    PubMed

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2016-06-01

    The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds. PMID:26951077

  12. Nuclear c-Abl-mediated tyrosine phosphorylation induces chromatin structural changes through histone modifications that include H4K16 hypoacetylation

    SciTech Connect

    Aoyama, Kazumasa; Fukumoto, Yasunori; Ishibashi, Kenichi; Kubota, Sho; Morinaga, Takao; Horiike, Yasuyoshi; Yuki, Ryuzaburo; Takahashi, Akinori; Nakayama, Yuji; Yamaguchi, Naoto

    2011-12-10

    c-Abl tyrosine kinase, which is ubiquitously expressed, has three nuclear localization signals and one nuclear export signal and can shuttle between the nucleus and the cytoplasm. c-Abl plays important roles in cell proliferation, adhesion, migration, and apoptosis. Recently, we developed a pixel imaging method for quantitating the level of chromatin structural changes and showed that nuclear Src-family tyrosine kinases are involved in chromatin structural changes upon growth factor stimulation. Using this method, we show here that nuclear c-Abl induces chromatin structural changes in a manner dependent on the tyrosine kinase activity. Expression of nuclear-targeted c-Abl drastically increases the levels of chromatin structural changes, compared with that of c-Abl. Intriguingly, nuclear-targeted c-Abl induces heterochromatic profiles of histone methylation and acetylation, including hypoacetylation of histone H4 acetylated on lysine 16 (H4K16Ac). The level of heterochromatic histone modifications correlates with that of chromatin structural changes. Adriamycin-induced DNA damage stimulates translocation of c-Abl into the nucleus and induces chromatin structural changes together with H4K16 hypoacetylation. Treatment with trichostatin A, a histone deacetylase inhibitor, blocks chromatin structural changes but not nuclear tyrosine phosphorylation by c-Abl. These results suggest that nuclear c-Abl plays an important role in chromatin dynamics through nuclear tyrosine phosphorylation-induced heterochromatic histone modifications.

  13. Ataxia telangiectasia mutated (ATM) is dispensable for endonuclease I-SceI-induced homologous recombination in mouse embryonic stem cells.

    PubMed

    Rass, Emilie; Chandramouly, Gurushankar; Zha, Shan; Alt, Frederick W; Xie, Anyong

    2013-03-01

    Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency. PMID:23355489

  14. Histone Deacetylation Critically Determines T-cell Subset Radiosensitivity1

    PubMed Central

    Pugh, Jason L.; Sukhina, Alona S.; Seed, Thomas M.; Manley, Nancy R.; Sempowski, Gregory A.; van den Brink, Marcel R.M.; Smithey, Megan J.; Nikolich-Zugich, Janko

    2014-01-01

    Lymphocytes are sensitive to ionizing radiation and naïve lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naïve (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice, and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and : (i) levels of pro- and anti-apoptotic Bcl-2 family members; or (ii) the H2-AX content and maximal γH2-AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2-AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment, improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells, but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences. PMID:24990082

  15. Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes

    PubMed Central

    Nguyen, Alexandra L.; Gentilello, Amanda S.; Balboula, Ahmed Z.; Shrivastava, Vibha; Ohring, Jacob; Schindler, Karen

    2014-01-01

    ABSTRACT Meiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochore–microtubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis. PMID:25315835

  16. Extended string-like binding of the phosphorylated HP1α N-terminal tail to the lysine 9-methylated histone H3 tail

    PubMed Central

    Shimojo, Hideaki; Kawaguchi, Ayumi; Oda, Takashi; Hashiguchi, Nobuto; Omori, Satoshi; Moritsugu, Kei; Kidera, Akinori; Hiragami-Hamada, Kyoko; Nakayama, Jun-ichi; Sato, Mamoru; Nishimura, Yoshifumi

    2016-01-01

    The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α’s N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α’s chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α’s N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α’s N-terminal tail underlies the enhancement in H3 binding due to phosphorylation. PMID:26934956

  17. p53 Ser15 phosphorylation and histone modifications contribute to IR-induced miR-34a transcription in mammary epithelial cells

    PubMed Central

    Wang, Bo; Li, Dongping

    2013-01-01

    Previous studies have demonstrated that miR-34a is a direct transcriptional target of tumor suppressor p53 and plays a crucial role in p53-mediated biological processes, such as cell cycle arrest, apoptosis and senescence. However, the role of p53 phosphorylation at Ser15 and histone modifications in ionizing radiation (IR)-induced miR-34a transcription in human mammary epithelial cells remains unknown. The present study showed that IR triggers miR-34a induction in rat mammary gland tissue and human mammary epithelial cells in a dose- and time-dependent fashion. Gene copy number and CpG methylation exhibit no effect on IR-inducible miR-34a expression, while the levels of phosphorylated p53 at Ser15 are markedly elevated in human mammary epithelial cells 96 h post-IR, which correlates with IR-inducible miR-34a transcription and the p38 MAPK pathway. Conversely, suppression of p38 MAPK with SB239063 inhibits IR-induced p53 phosphorylation at Ser15 and miR-34a expression in a dose-dependent manner. Our study found that wild-type p53 is enriched at miR-34a promoter, and luciferase activity of miR-34a promoter reporter is attenuated by either mutant p53 (Ser15Ala) or mutant miR-34a promoter. Furthermore, IR also triggers phosphorylation, tri-methylation and acetylation of histone H3 and acetylation of histone H4, which correlates with IR-inducible miR-34a transcription, while SAHA potentiates IR-inducible miR-34a expression. Moreover, acetyl-histone H3 is significantly enriched at miR-34a promoter in IR-exposed HMEC cells. Yet, we show that there is no correlation between IR-inducible miR-34a expression and IR-induced rapid and transient G2/M arrest. In sum, our novel data for the first time demonstrate that IR-induced p53 Ser15 phosphorylation via p38 MAPK is essential for its functional regulation of IR-inducible miR-34a transcription in human mammary epithelial cells, and that histone modifications may also play a key role in IR-inducible miR-34a expression. PMID

  18. Phosphatase Rtr1 Regulates Global Levels of Serine 5 RNA Polymerase II C-Terminal Domain Phosphorylation and Cotranscriptional Histone Methylation.

    PubMed

    Hunter, Gerald O; Fox, Melanie J; Smith-Kinnaman, Whitney R; Gogol, Madelaine; Fleharty, Brian; Mosley, Amber L

    2016-09-01

    In eukaryotes, the C-terminal domain (CTD) of Rpb1 contains a heptapeptide repeat sequence of (Y1S2P3T4S5P6S7)n that undergoes reversible phosphorylation through the opposing action of kinases and phosphatases. Rtr1 is a conserved protein that colocalizes with RNA polymerase II (RNAPII) and has been shown to be important for the transition from elongation to termination during transcription by removing RNAPII CTD serine 5 phosphorylation (Ser5-P) at a selection of target genes. In this study, we show that Rtr1 is a global regulator of the CTD code with deletion of RTR1 causing genome-wide changes in Ser5-P CTD phosphorylation and cotranscriptional histone H3 lysine 36 trimethylation (H3K36me3). Using chromatin immunoprecipitation and high-resolution microarrays, we show that RTR1 deletion results in global changes in RNAPII Ser5-P levels on genes with different lengths and transcription rates consistent with its role as a CTD phosphatase. Although Ser5-P levels increase, the overall occupancy of RNAPII either decreases or stays the same in the absence of RTR1 Additionally, the loss of Rtr1 in vivo leads to increases in H3K36me3 levels genome-wide, while total histone H3 levels remain relatively constant within coding regions. Overall, these findings suggest that Rtr1 regulates H3K36me3 levels through changes in the number of binding sites for the histone methyltransferase Set2, thereby influencing both the CTD and histone codes. PMID:27247267

  19. Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

    PubMed Central

    Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

    2015-01-01

    Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

  20. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    SciTech Connect

    Eto, Masumi; Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi; Kim, Jee In

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  1. Differential regulation of the phosphorylation of Trimethyl-lysine27 histone H3 at serine 28 in distinct populations of striatal projection neurons.

    PubMed

    Bonito-Oliva, Alessandra; Södersten, Erik; Spigolon, Giada; Hu, Xiaochen; Hellysaz, Arash; Falconi, Anastasia; Gomes, Ana-Luisa; Broberger, Christian; Hansen, Klaus; Fisone, Gilberto

    2016-08-01

    Phosphorylation of histone H3 (H3) on serine 28 (S28) at genomic regions marked by trimethylation of lysine 27 (H3K27me3) often correlates with increased expression of genes normally repressed by Polycomb group proteins (PcG). We show that amphetamine, an addictive psychostimulant, and haloperidol, a typical antipsychotic drug, increase the phosphorylation of H3 at S28 and that this effect occurs in the context of H3K27me3. The increases in H3K27me3S28p occur in distinct populations of projection neurons located in the striatum, the major component of the basal ganglia. Genetic inactivation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), reduces the phosphorylation of H3K27me3S28 produced by amphetamine and haloperidol. In contrast, knockout of the mitogen- and stress activated kinase 1 (MSK1), which is implicated in the phosphorylation of histone H3, decreases the effect of amphetamine, but not that of haloperidol. Chromatin immunoprecipitation analysis shows that amphetamine and haloperidol increase the phosphorylation of H3K27me3S28 at the promoter regions of Atf3, Npas4 and Lipg, three genes repressed by PcG. These results identify H3K27me3S28p as a potential mediator of the effects exerted by amphetamine and haloperidol, and suggest that these drugs may act by re-activating PcG repressed target genes. PMID:26947946

  2. Effect of SPL (Spent Pot Liner) and its main components on root growth, mitotic activity and phosphorylation of Histone H3 in Lactuca sativa L.

    PubMed

    Freitas, Aline Silva; Fontes Cunha, Isabela Martinez; Andrade-Vieira, Larissa Fonseca; Techio, Vânia Helena

    2016-02-01

    Spent Pot Liner (SPL) is a solid waste from the aluminum industry frequently disposed of in industrial landfills; it can be leached and contaminate the soil, sources of drinking water and plantations, and thus may pose a risk to human health and to ecosystems. Its composition is high variable, including cyanide, fluoride and aluminum salts, which are highly toxic and environmental pollutants. This study evaluated the effect of SPL and its main components on root growth and the mitosis of Lactuca sativa, by investigating the mechanisms of cellular and chromosomal alterations with the aid of immunolocalization. To this end, newly emerged roots of L. sativa were exposed to SPL and its main components (solutions of cyanide, fluoride and aluminum) and to calcium chloride (control) for 48h. After this, root length was measured and cell cycle was examined by means of conventional cytogenetics and immunolocalization. Root growth was inhibited in the treatments with SPL and aluminum; chromosomal and nuclear alterations were observed in all treatments. The immunolocalization evidenced normal dividing cells with regular temporal and spatial distribution of histone H3 phosphorylation at serine 10 (H3S10ph). However, SPL and its main components inhibited the phosphorylation of histone H3 at serine 10, inactivated pericentromeric regions and affected the cohesion of sister chromatids, thus affecting the arrangement of chromosomes in the metaphase plate and separation of chromatids in anaphase. In addition, these substances induced breaks in pericentromeric regions, characterized as fragile sites. PMID:26615478

  3. Cancer-preventive peptide lunasin from Solanum nigrum L. inhibits acetylation of core histones H3 and H4 and phosphorylation of retinoblastoma protein (Rb).

    PubMed

    Jeong, Jin Boo; Jeong, Hyung Jin; Park, Jae Ho; Lee, Sun Hee; Lee, Jeong Rak; Lee, Hee Kyeong; Chung, Gyu Young; Choi, Jeong Doo; de Lumen, Ben O

    2007-12-26

    Lunasin, a unique 43 amino acid, 4.8 kDa cancer-chemopreventive peptide initially reported in soybean and now found in barley and wheat, has been shown to be cancer-chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. To identify bioactive components in traditional herbal medicines and in search for new sources of lunasin, we report here the properties of lunasin from Solanum nigrum L. (SNL), a plant indigenous to northeast Asia. Lunasin was screened in the crude extracts of five varieties of the medicinal plants of Solanaceae origin and seven other major herbal plants. An in vitro digestion stability assay for measuring bioavailability was carried out on SNL crude protein and autoclaved SNL using pepsin and pancreatin. A nonradioactive histone acetyltransferase (HAT) assay and HAT activity colorimetric assay were used to measure the inhibition of core histone acetylation. The inhibitory effect of lunasin on the phosphorylation of retinoblastoma protein (Rb) was determined by immunoblotting against phospho-Rb. Lunasin isolated from autoclaved SNL inhibited core histone H3 and H4 acetylation, the activities of the HATs, and the phosphorylation of the Rb protein. Lunasin in the crude protein and in the autoclaved crude protein was very stable to pepsin and pancreatin in vitro digestion, while the synthetic pure lunasin was digested at 2 min after the reaction. We conclude that lunasin is a bioactive and bioavailable component in SNL and that consumption of SNL may play an important role in cancer prevention. PMID:18038993

  4. The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells

    PubMed Central

    Cellai, C; Balliu, M; Laurenzana, A; Guandalini, L; Matucci, R; Miniati, D; Torre, E; Nebbioso, A; Carafa, V; Altucci, L; Romanelli, M N; Paoletti, F

    2012-01-01

    Abstract Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn++-chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4– but not (R)-2 – caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy. PMID:22004558

  5. The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells.

    PubMed

    Cellai, C; Balliu, M; Laurenzana, A; Guandalini, L; Matucci, R; Miniati, D; Torre, E; Nebbioso, A; Carafa, V; Altucci, L; Romanelli, M N; Paoletti, F

    2012-08-01

    Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy. PMID:22004558

  6. Loss of centromeric histone H2AT120 phosphorylation accompanies somatic chromosomes inactivation in the aberrant spermatocytes of Acricotopus lucidus (Diptera, Chironomidae).

    PubMed

    Staiber, Wolfgang

    2016-01-01

    In the germ line of the chironomid Acricotopus lucidus, two cells with quite different chromosome constitutions result from the last unequal gonial mitosis. In the male, the future primary spermatocyte receives all the germ line-limited chromosomes (=Ks) together with somatic chromosomes (=Ss), and later on undergoes meiotic divisions, while the connected aberrant spermatocyte gets only Ss and remains undivided with chromosomes inactivated in a metaphase-like condensed state. This raises the question whether the centromeres of the permanently condensed Ss of the aberrant spermatocyte remain active during meiosis of the connected regular spermatocyte. Active centromeres exhibit an epigenetic phosphorylation mark at threonine 120 of histone H2A. To visualise the centromeric H2A phosphorylation of the Ss in the aberrant spermatocyte, meiotic stages were immunostained with different anti-phospho histone H2AT120 antibodies. Clear H2AT120ph signals appear at the centromeres of the Ss during prophase, persist on the metaphase-like condensed Ss during meiosis I of the connected primary spermatocyte and disappear during transition to meiosis II. The centromeres of the Ss and Ks of the regular spermatocytes display H2AT120ph signals from prophase I to anaphase II. The loss of the H2AT120 phosphorylation detected on the centromeres of the Ss of the aberrant spermatocyte indicating their deactivation supports the idea of a programmed inactivation of the Ss to block the entry of the germ line-derived aberrant spermatocyte, lacking Ks, into meiosis, and thus to prevent the generation of sperms possessing only Ss. This mechanism would ensure the presence of the Ks in the germ line. PMID:25820679

  7. Chromosome missegregation during anaphase triggers p53 cell cycle arrest through histone H3.3 Ser31 phosphorylation.

    PubMed

    Hinchcliffe, Edward H; Day, Charles A; Karanjeet, Kul B; Fadness, Sela; Langfald, Alyssa; Vaughan, Kevin T; Dong, Zigang

    2016-06-01

    Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed. PMID:27136267

  8. Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

    PubMed Central

    Ho, Catherine M. K.; Donovan-Banfield, I’ah Z.; Tan, Li; Zhang, Tinghu; Gray, Nathanael S.; Strang, Blair L.

    2016-01-01

    Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. PMID:26930276

  9. Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells.

    PubMed

    Ho, Catherine M K; Donovan-Banfield, I'ah Z; Tan, Li; Zhang, Tinghu; Gray, Nathanael S; Strang, Blair L

    2016-01-01

    Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα. PMID:26930276

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

    PubMed

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

    2016-01-01

    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. PMID:26598521

  11. PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3

    PubMed Central

    Zhang, Hui; Wang, Zhiquan; Zhang, Zhiguo

    2013-01-01

    Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells. PMID:23828041

  12. Phosphorylation of DNA damage-recognizing proteins at heavy-ion track

    NASA Astrophysics Data System (ADS)

    Ohnishi, T.; Takahashi, A.; Nojima, K.; Furusawa, Y.; Ohnishi, K.

    To identify the repair dynamics for high LET-radiation-induced DNA damage we analyzed the focus formation after exposure to iron-ion beams 500 MeV u 200 KeV um using immunocytochemical methods Since the focus formation of phospho-H2AX gamma-H2AX which is well understood to be activated at radiation-induced double strand breaks DSBs we performed the visualization of the tracks spatial distribution of lesions from an aspect of dose dependency The number of this track induced by iron-ion beams was well corresponded with the value of a calculation well In addition we demonstrate that DNA damage-recognizing proteins such as phospho-serine 1981 of ATM phospho-threonine 2609 of DNA-PKcs phospho-serine 343 of NBS1 and phospho-threonine 68 of Chk2 co-localized with gamma-H2AX at high LET-radiation-induced portion These findings suggest that iron-ion beams were quite effective for detection of DNA damages of DSBs recognized with DNA repair enzymes used here after phosphorylation of them because iron-ion beams can be used to generate extremely localized at DNA damages within restricted regions of the nuclei

  13. The phosphorylated prodrug FTY720 is a histone deacetylase inhibitor that reactivates ERα expression and enhances hormonal therapy for breast cancer

    PubMed Central

    Hait, N C; Avni, D; Yamada, A; Nagahashi, M; Aoyagi, T; Aoki, H; Dumur, C I; Zelenko, Z; Gallagher, E J; Leroith, D; Milstien, S; Takabe, K; Spiegel, S

    2015-01-01

    Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and does not respond to conventional hormonal therapies. Strategies that lead to re-expression of ERα could sensitize ERα-negative breast cancers to selective ER modulators. FTY720 (fingolimod, Gilenya), a sphingosine analog, is the Food and Drug Administration (FDA)-approved prodrug for treatment of multiple sclerosis that also has anticancer actions that are not yet well understood. We found that FTY720 is phosphorylated in breast cancer cells by nuclear sphingosine kinase 2 and accumulates there. Nuclear FTY720-P is a potent inhibitor of class I histone deacetylases (HDACs) that enhances histone acetylations and regulates expression of a restricted set of genes independently of its known effects on canonical signaling through sphingosine-1-phosphate receptors. High-fat diet (HFD) and obesity, which is now endemic, increase breast cancer risk and have been associated with worse prognosis. HFD accelerated the onset of tumors with more advanced lesions and increased triple-negative spontaneous breast tumors and HDAC activity in MMTV-PyMT transgenic mice. Oral administration of clinically relevant doses of FTY720 suppressed development, progression and aggressiveness of spontaneous breast tumors in these mice, reduced HDAC activity and strikingly reversed HFD-induced loss of estrogen and progesterone receptors in advanced carcinoma. In ERα-negative human and murine breast cancer cells, FTY720 reactivated expression of silenced ERα and sensitized them to tamoxifen. Moreover, treatment with FTY720 also re-expressed ERα and increased therapeutic sensitivity of ERα-negative syngeneic breast tumors to tamoxifen in vivo more potently than a known HDAC inhibitor. Our work suggests that a multipronged attack with FTY720 is a novel combination approach for effective treatment of both conventional hormonal therapy-resistant breast cancer and triple-negative breast cancer. PMID:26053034

  14. Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.

    PubMed Central

    Kardalinou, E; Zhelev, N; Hazzalin, C A; Mahadevan, L C

    1994-01-01

    Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus. Images PMID:8289787

  15. Shot-gun proteomic analysis of mitochondrial D-loop DNA binding proteins: identification of mitochondrial histones.

    PubMed

    Choi, Yon-Sik; Hoon Jeong, Jae; Min, Hye-Ki; Jung, Hee-Jung; Hwang, Daehee; Lee, Sang-Won; Kim Pak, Youngmi

    2011-05-01

    Transcription and replication of mitochondrial DNA (mtDNA) are regulated by nuclear DNA-encoded proteins that are targeted into mitochondria. A decrease in mtDNA copy number results in mitochondrial dysfunction, which may lead to insulin resistance and metabolic syndromes. We analyzed mitochondrial proteins that physically bind to human mitochondrial D-loop DNA using a shot-gun proteomics approach following protein enrichment by D-loop DNA-linked affinity chromatography. A total of 152 D-loop DNA binding proteins were identified by peptide sequencing using ultra high pressure capillary reverse-phase liquid chromatography/tandem mass spectrometry. Bioinformatic analysis showed that 68 were mitochondrial proteins, 96 were DNA/RNA/protein binding proteins and 114 proteins might form a complex via protein-protein interactions. Histone family members of H1, H2A, H2B, H3, and H4, were detected in abundance among them. In particular, histones H2A and H2B were present in the mitochondrial membrane as integral membrane proteins and not bound directly to mtDNA inside the organelle. Histones H1.2, H3 and H4 were associated with the outer mitochondrial membrane. Silencing of H2AX expression inhibited mitochondrial protein transport. Our data suggests that many mitochondrial proteins may reside in multiple subcellular compartments like H2AX and exert multiple functions. PMID:21359316

  16. S6 kinase 2 is bound to chromatin-nuclear matrix cellular fractions and is able to phosphorylate histone H3 at threonine 45 in vitro and in vivo.

    PubMed

    Ismail, Heba M S; Hurd, Paul J; Khalil, Mahmoud I M; Kouzarides, Tony; Bannister, Andrew; Gout, Ivan

    2014-06-01

    The activity of S6 kinases (S6K) is highly induced in cancer cells highlighting an essential role in carcinogenesis. The S6K family has two members: S6K1 and S6K2 which bear common as well as distinct features. In an attempt to identify S6K2 unique sequence features compared to S6K1, we applied extensive bioinformatic analysis and motif search approaches. Interestingly, we identified 14 unique protein signatures which are present in proteins directly connected to chromatin and/or involved in transcription regulation. Using chromatin binding assay, we biochemically showed that S6K2 is bound to chromatin as well as nuclear matrix cellular fractions in HEK293 cells. The presence of S6K2 in chromatin fractions raised the possibility that it may be in close proximity to a number of chromatin substrates. For that, we then searched for S6K phosphorylation consensus sites RXRXXT/S in mammalian proteins using the SWISS-PROT database. Interestingly, we identified some potential phosphorylation sites in histone H3 (Thr45). Using in vitro kinase assays and siRNA-based knockdown strategy; we confirmed that S6K2 but not S6K1 or AKT is essential for histone H3-Thr45 phosphorylation in HEK293 cells. Furthermore, we show that the nuclear localisation sequence in the S6K2 C-terminus is essential for this modification. We have found that, H3-Thr45 phosphorylation correlates to S6K activation in response to mitogens and TPA-induced cell differentiation of leukaemic cell lines U937, HL60 and THP1. Overall, we demonstrate that S6K2 is a novel kinase that can phosphorylate histone H3 at position Thr45, which may play a role during cell proliferation and/or differentiation. PMID:23564320

  17. Role of phosphorylated histone H3 serine 10 in DEN-induced deregulation of Pol III genes and cell proliferation and transformation

    PubMed Central

    Zhong, Shuping

    2013-01-01

    The products of Pol III genes (RNA polymerase III-dependent genes), such as tRNAs and 5S rRNA, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. An increase in Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB, augments Pol III gene transcription and is sufficient for cell transformation and tumor formation. We have demonstrated that enhancement of Brf1 and Pol III gene expression is associated with the occurrences of hepatocellular carcinoma (HCC) in mice. This suggests that Brf1 may be a key molecule during HCC development. Diethylnitrosamine (DEN), a chemical carcinogen, has been used to induce HCC in rodents. To determine the role of Brf1 and the epigenetic-regulating events in cell proliferation and transformation, hepatocytes were treated with DEN. The results indicate that DEN increases proliferation and transformation of AML-12 cells. DEN enhanced Brf1 expression and tRNALeu and 5S rRNA transcription, as well as H3S10ph (phosphorylation of histone H3 serine 10). Interestingly, DEN-induced Pol III gene transcription and H3S10ph in tumor cells of liver are significantly higher than in non-tumor cells. Inhibition of H3S10ph by H3S10A attenuates the induction of Brf1 and Pol III genes. Further analysis indicates that H3S10ph occupies the promoters of Brf1 and Pol III genes to modulate their expression. Blocking H3S10ph represses cell proliferation and transformation. These results demonstrate that DEN induces H3S10ph, which mediate Brf1 expression, including but not limited Brf1-dependent genes, to upregulate Pol III gene transcription, resulting in an increase in cell proliferation and transformation. PMID:23774401

  18. RNF20-SNF2H Pathway of Chromatin Relaxation in DNA Double-Strand Break Repair

    PubMed Central

    Kato, Akihiro; Komatsu, Kenshi

    2015-01-01

    Rapid progress in the study on the association of histone modifications with chromatin remodeling factors has broadened our understanding of chromatin dynamics in DNA transactions. In DNA double-strand break (DSB) repair, the well-known mark of histones is the phosphorylation of the H2A variant, H2AX, which has been used as a surrogate marker of DSBs. The ubiquitylation of histone H2B by RNF20 E3 ligase was recently found to be a DNA damage-induced histone modification. This modification is required for DSB repair and regulated by a distinctive pathway from that of histone H2AX phosphorylation. Moreover, the connection between H2B ubiquitylation and the chromatin remodeling activity of SNF2H has been elucidated. In this review, we summarize the current knowledge of RNF20-mediated processes and the molecular link to H2AX-mediated processes during DSB repair. PMID:26184323

  19. Expression and functionality of histone H2A variants in cancer

    PubMed Central

    Monteiro, Fátima Liliana; Baptista, Tiago; Amado, Francisco; Vitorino, Rui; Jerónimo, Carmen; Helguero, Luisa A.

    2014-01-01

    Regulation of gene expression includes the replacement of canonical histones for non-allelic histone variants, as well as their multiple targeting by postranslational modifications. H2A variants are highly conserved between species suggesting they execute important functions that cannot be accomplished by canonical histones. Altered expression of many H2A variants is associated to cancer. MacroH2A variants are enriched in heterocromatic foci and are necessary for chromatin condensation. MacroH2A1.1 and macroH2A1.2 are two mutually exclusive isoforms. MacroH2A1.1 and macroH2A2 inhibit proliferation and are associated with better cancer prognosis; while macroH2A1.2 is associated to cancer progression. H2AX variant functions as a sensor of DNA damage and defines the cellular response towards DNA repair or apoptosis; therefore, screening approaches and therapeutic options targeting H2AX have been proposed. H2A.Z is enriched in euchromatin, acting as a proto-oncogene with established roles in hormone responsive cancers and overexpressed in endocrine-resistant disease. Other H2A family members have also been found altered in cancer, but their function remains unknown. Substantial progress has been made to understand histone H2A variants, their contribution to normal cellular function and to cancer development and progression. Yet, implementation of high resolution mass spectrometry is needed to further our knowledge on highly homologous H2A variants expression and function. PMID:25003966

  20. Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4(+) T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb.

    PubMed

    Jamaluddin, Md Saha; Hu, Pei-Wen; Jan, Yih; Siwak, Edward B; Rice, Andrew P

    2016-02-01

    Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4(+) T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4(+) cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4(+) T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4(+) T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation. PMID:26727990

  1. Simvastatin enhances NMDA receptor GluN2B expression and phosphorylation of GluN2B and GluN2A through increased histone acetylation and Src signaling in hippocampal CA1 neurons.

    PubMed

    Chen, Tingting; Zhang, Baofeng; Li, Guoxi; Chen, Lei; Chen, Ling

    2016-08-01

    Simvastatin (SV) can improve cognitive deficits in Alzheimer's disease patients and mice. Herein, we report that the administration of SV (20 mg/kg) for 5 days in mice (SV-mice) or the treatment of slices with SV (10 μM) for 4 h (SV-slices) could increase the density of NMDA-evoked inward currents (INMDA) in hippocampal CA1 pyramidal cells, which were blocked by farnesol (FOH) that converts farnesyl pyrophosphate (FPP), but not geranylgeraniol (GGOH) that increases geranylgeranylpyrophosphate (GGPP). Sensitivity of INMDA to ifenprodil in SV-mice or SV-slices was significantly increased. The levels of hippocampal GluN2B and GluN2A or Src phosphorylation in SV-mice or SV-slices were higher than controls, which were sensitive to FOH. The Src inhibitor PP2 could inhibit the SV-enhanced phosphorylation of GluN2B and GluN2A and SV-augmented INMDA, but PI3K inhibitor LY294002 did not. The levels of GluN2B mRNA and protein were elevated in SV-mice, which was abolished by FOH, but not by GGOH or PP2. Furthermore, the histone H3K9 and H3K27 acetylation of GluN2B promoter was increased in SV-mice, which was suppressed by FOH rather than GGOH or PP2. In control mice and slices, the reduction of FPP by farnesyl transferase inhibitor could increase the levels of GluN2B expression, the histone H3K9 and H3K27 acetylation and enhance the phosphorylation of GluN2B, GluN2A and Src. The findings indicate that the administration of SV can enhance GluN2B expression and GluN2B and GluN2A phosphorylation leading to augmentation of NMDAR activity through reducing FPP to increase histone acetylation of GluN2B and Src signaling. PMID:27016018

  2. Nanoparticle formulations of histone deacetylase inhibitors for effective chemoradiotherapy in solid tumors

    PubMed Central

    Wang, Edina C.; Min, Yuanzeng; Palm, Robert C.; Fiordalisi, James J.; Wagner, Kyle T.; Hyder, Nabeel; Cox, Adrienne D.; Caster, Joseph; Tian, Xi; Wang, Andrew Z.

    2015-01-01

    Histone deacetylase inhibitors (HDACIs) represent a class of promising agents that can improve radiotherapy in cancer treatment. However, the full therapeutic potential of HDACIs as radiosensitizers has been restricted by limited efficacy in solid malignancies. In this study, we report the development of nanoparticle (NP) formulations of HDACIs that overcome these limitations, illustrating their utility to improve the therapeutic ratio of the clinically established first generation HDACI vorinostat and a novel second generation HDACI quisinostat. We demonstrate that NP HDACIs are potent radiosensitizers in vitro and are more effective as radiosensitizers than small molecule HDACIs in vivo using mouse xenograft models of colorectal and prostate carcinomas. We found that NP HDACIs enhance the response of tumor cells to radiation through the prolongation of γ-H2AX foci. Our work illustrates an effective method for improving cancer radiotherapy treatment. PMID:25771011

  3. The ATM substrate KAP1 controls DNA repair in heterochromatin: Regulation by HP1 proteins and Serine 473/824 phosphorylation

    PubMed Central

    White, D. E.; Rafalska-Metcalf, I. U.; Ivanov, A. V.; Corsinotti, A.; Peng, H.; Lee, S. C.; Trono, D.; Janicki, S. M.; Rauscher, F. J.

    2016-01-01

    The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor which binds to HP1 during gene silencing, but is also robustly phosphorylated by ATM at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells which lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at Serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin. PMID:22205726

  4. Immunocytochemical and immunogold analyses of histone H4 acetylation during Chara vulgaris spermiogenesis.

    PubMed

    Wojtczak, Agnieszka

    2016-03-01

    Histone acetylation is one of the epigenetic modifications which play a significant role in chromatin remodeling during spermiogenesis. Acetylation of the histone H4 makes the exchange of nucleoproteins easy. Research on mouse spermatogenesis showed that H4 histone acetylated at Lys 12 (H4K12ac) was specific only to spermatids. Immunocytochemical studies of Chara vulgaris spermatids with the use of antibodies against the histone H4K12ac revealed positive reactions in spermatid nuclei at stages I-VII. This reaction, connected with nuclear condensation, was much stronger at the early stages of spermiogenesis than later on. Moreover, it showed that at the stages V-VII in spermatid nuclei the presence of the histone H4K12ac corresponded with DNA double-strand breaks. Electron microscopy studies with the use of immunogold technique revealed an almost twofold difference between the mean total numbers of gold grains in the examined chromatin in both stages. This study showed nearly equal distribution of gold grains on condensed and non-condensed chromatin of spermatids at the stage III/IV (48.11% and 51.89%, respectively). In the later stage-VI, when chromatin condensation proceeded, labeling of condensed chromatin reached 57.27%, while in the case of non-condensed chromatin it dropped to 42.73%. The percentage analysis also revealed an increase (above 9%) in condensed chromatin labeling in relation to the stage III/IV. Intensive acetylation of histone H4 at the early stages is correlated with DNA DSBs and transcriptional activity. It facilitates chromatin loosening, which enables the correct course of chromatin remodeling at a later stage. Histone γH2AX also influences chromatin structure in many biological processes in different cell types. Current studies reveal other similarities regarding histone H4 acetylation, not only between Chara and mammals but between invertebrates (molluscs) and vertebrates (bony fishes) as well. PMID:26774747

  5. The development of a monoclonal antibody recognizing the Drosophila melanogaster phosphorylated histone H2A variant (γ-H2AV).

    PubMed

    Lake, Cathleen M; Holsclaw, Julie Korda; Bellendir, Stephanie P; Sekelsky, Jeff; Hawley, R Scott

    2013-09-01

    The recognition of DNA double-strand breaks (DSBs) using a phospho-specific antibody to the histone 2A variant has become the gold standard assay for DNA damage detection. Here we report on the development of the first monoclonal antibody to the phospho-specific form of Drosophila H2AV and characterize the specificity of this antibody to programmed DSBs in oocytes and rereplication sites in endocycling cells by immunofluorescence assays and to DSBs resulting from irradiation in both cell culture and whole tissue by Western blot assays. These studies show that the antibody derived in the study is highly specific for this modification that occurs at DSB sites, and therefore will be a new useful tool within the Drosophila community for the study of DNA damage response, DSB repair, meiotic recombination and chemical agents that cause DNA damage. PMID:23833215

  6. Coordinated Regulation of TIP60 and Poly(ADP-Ribose) Polymerase 1 in Damaged-Chromatin Dynamics.

    PubMed

    Ikura, Masae; Furuya, Kanji; Fukuto, Atsuhiko; Matsuda, Ryo; Adachi, Jun; Matsuda, Tomonari; Kakizuka, Akira; Ikura, Tsuyoshi

    2016-05-15

    The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses. PMID:26976643

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

    PubMed

    Bulat, Tanja; Keta, Otilija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-03-01

    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. PMID:26959322

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

    PubMed

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin; Ding, Nan; Qi, Yongmei; Zhang, Yingmei; Wang, Jufang; Huang, Dejun

    2015-09-01

    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 (1-8h 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. PMID:26201248

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

    PubMed Central

    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, 111In, 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 111In 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

  10. Histone Deacetylases

    PubMed Central

    Parbin, Sabnam; Kar, Swayamsiddha; Shilpi, Arunima; Sengupta, Dipta; Deb, Moonmoon; Rath, Sandip Kumar

    2014-01-01

    In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer. PMID:24051359

  11. Targeting post-translational modifications of histones for cancer therapy.

    PubMed

    Hsu, Y-C; Hsieh, Y-H; Liao, C-C; Chong, L-W; Lee, C-Y; Yu, Y-L; Chou, R-H

    2015-01-01

    Post-translational modifications (PTMs) on histones including acetylation, methylation, phosphorylation, citrullination, ubiquitination, ADP ribosylation, and sumoylation, play important roles in different biological events including chromatin dynamics, DNA replication, and transcriptional regulation. Aberrant histones PTMs leads to abnormal gene expression and uncontrolled cell proliferation, followed by development of cancers. Therefore, targeting the enzymes required for specific histone PTMs holds a lot of potential for cancer treatment. In this review article, we retrospect the latest studies in the regulations of acetylation, methylation, and phosphorylation of histones. We also summarize inhibitors/drugs that target these modifications for cancer treatment. PMID:26518898

  12. Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro.

    PubMed

    Petrillo, Stephanie K; Desmeules, Patrice; Truong, To-Quyen; Devine, Patrick J

    2011-06-01

    Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥3μM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylationH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1μM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24h after exposure to 3-10μMPM. Numbers of oocytes with ≥5 γH2AX foci were significantly increased both 1 and 8days after exposure to ≥1μMPM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥30μM) and increased γH2AX foci (≥3μM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring. PMID:21439308

  13. Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro

    SciTech Connect

    Petrillo, Stephanie K.; Desmeules, Patrice; Truong, To-Quyen; Devine, Patrick J.

    2011-06-01

    Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM ({>=} 3 {mu}M) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation ({gamma}H2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1 {mu}M PM induced significant losses of primordial and small primary follicles, respectively. PM-induced {gamma}H2AX was observed predominantly in oocytes, in which foci of {gamma}H2AX staining increased in a concentration-dependent manner and peaked 18-24 h after exposure to 3-10 {mu}M PM. Numbers of oocytes with {>=} 5 {gamma}H2AX foci were significantly increased both 1 and 8 days after exposure to {>=} 1 {mu}M PM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers ({>=} 30 {mu}M) and increased {gamma}H2AX foci ({>=} 3 {mu}M) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

  14. Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

    SciTech Connect

    Bauerschmidt, Christina; Helleday, Thomas

    2011-02-01

    Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

  15. Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

    PubMed Central

    Cross, Margaret E.; Ord, Margery G.

    1971-01-01

    1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) at concentrations of 0.01–0.1μm. The pattern of incorporation of label from [5-3H]uridine and [6-3H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1μm) prevented the stimulation of [5-3H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular Pi pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate 32P from [32P]Pi. 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase. PMID:4331256

  16. Imaging Histone Methylations in Living Animals.

    PubMed

    Sekar, Thillai V; Paulmurugan, Ramasamy

    2016-01-01

    Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions. Since histone methylation is dysregulated in various cellular diseases, it has been considered a potential therapeutic target for drug development. Currently there is no simple method available to screen and preclinically evaluate drugs modulating this cellular process, we recently developed two different methods by adopting reporter gene technology to screen drugs and to preclinically evaluate them in living animals. Method detects and quantitatively monitors the level of histone methylations in intact cells, is of a prerequisite to screen small molecules that modulate histone lysine methylation. Here, we describe two independent optical imaging sensors developed to image histone methylations in cells and in living animals. Since we used standard PCR-based cloning strategies to construct different plasmid vectors shown in this chapter, we are not providing any details regarding the construction methods, instead, we focus on detailing various methods used for measuring histone methylation-assisted luciferase quantitation in cells and imaging in living animals. PMID:27424907

  17. Polycomb Protein SCML2 Associates with USP7 and Counteracts Histone H2A Ubiquitination in the XY Chromatin during Male Meiosis

    PubMed Central

    Luo, Mengcheng; Zhou, Jian; Leu, N. Adrian; Abreu, Carla M.; Wang, Jianle; Anguera, Montserrat C.; de Rooij, Dirk G.; Jasin, Maria; Wang, P. Jeremy

    2015-01-01

    Polycomb group proteins mediate transcriptional silencing in diverse developmental processes. Sex chromosomes undergo chromosome-wide transcription silencing during male meiosis. Here we report that mouse SCML2 (Sex comb on midleg-like 2), an X chromosome-encoded polycomb protein, is specifically expressed in germ cells, including spermatogonia, spermatocytes, and round spermatids. SCML2 associates with phosphorylated H2AX and localizes to the XY body in spermatocytes. Loss of SCML2 in mice causes defective spermatogenesis, resulting in sharply reduced sperm production. SCML2 interacts with and recruits a deubiquitinase, USP7, to the XY body in spermatocytes. In the absence of SCML2, USP7 fails to accumulate on the XY body, whereas H2A monoubiquitination is dramatically augmented in the XY chromatin. Our results demonstrate that the SCML2/USP7 complex constitutes a novel molecular pathway in modulating the epigenetic state of sex chromosomes during male meiosis. PMID:25634095

  18. Linker histones in hormonal gene regulation.

    PubMed

    Vicent, G P; Wright, R H G; Beato, M

    2016-03-01

    In the present review, we summarize advances in our knowledge on the role of the histone H1 family of proteins in breast cancer cells, focusing on their response to progestins. Histone H1 plays a dual role in gene regulation by hormones, both as a structural component of chromatin and as a dynamic modulator of transcription. It contributes to hormonal regulation of the MMTV promoter by stabilizing a homogeneous nucleosome positioning, which reduces basal transcription whereas at the same time promoting progesterone receptor binding and nucleosome remodeling. These combined effects enhance hormone dependent gene transcription, which eventually requires H1 phosphorylation and displacement. Various isoforms of histone H1 have specific functions in differentiated breast cancer cells and compact nucleosomal arrays to different extents in vitro. Genome-wide studies show that histone H1 has a key role in chromatin dynamics of hormone regulated genes. A complex sequence of enzymatic events, including phosphorylation by CDK2, PARylation by PARP1 and the ATP-dependent activity of NURF, are required for H1 displacement and gene de-repression, as a prerequisite for further nucleosome remodeling. Similarly, during hormone-dependent gene repression a dedicated enzymatic mechanism controls H1 deposition at promoters by a complex containing HP1γ, LSD1 and BRG1, the ATPase of the BAF complex. Thus, a broader vision of the histone code should include histone H1, as the linker histone variants actively participate in the regulation of the chromatin structure. How modifications of the core histones tails affect H1 modifications and vice versa is one of the many questions that remains to be addressed to provide a more comprehensive view of the histone cross-talk mechanisms. PMID:26518266

  19. S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response

    SciTech Connect

    Lee, Haemi; Kwak, Hee-Jin; Cho, Il-taeg; Park, Seok Hee; Lee, Chang-Hun

    2009-01-02

    53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and {gamma}H2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.

  20. Immunofluorescent Detection of DNA Double Strand Breaks induced by High-LET Radiation

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wu, Honglu; Desai, Nirav

    2004-01-01

    Within cell nuclei, traversing charged heavy ion particles lead to the accumulation of proteins related to DNA lesions and repair along the ion trajectories. Irradiation using a standard geometric setup with the beam path perpendicular to the cell monolayer generates discrete foci of several proteins known to localize at sites of DNA double strand breaks (DSBs). One such molecule is the histone protein H2AX (gamma-H2AX), which gets rapidly phosphorylated in response to ionizing radiation. Here we present data obtained with a modified irradiation geometry characterized by a beam path parallel to a monolayer of human fibroblast cells. This new irradiation geometry leads to the formation of gamma-H2AX aggregates in the shape of streaks stretching over several micrometers in the x/y plane, thus enabling the analysis of the fluorescence distributions along the particle trajectories. Qualitative analysis of these distributions presented insights into the DNA repair kinetics along the primary track structure and visualization of possible chromatin movement. We also present evidence of colocalization of gamma-H2AX with several other proteins in responses to ionizing radiation exposure. Analysis of gamma-H2AX has the potential to provide useful information on human cell responses to high LET radiation after exposure to space-like radiation.

  1. Histones in protistan evolution.

    PubMed

    Rizzo, P J

    1985-01-01

    The potential of comparative studies on histones for use in protistan evolution is discussed, using algal histones as specific examples. A basic premise for the importance of histones in protistan evolution is the observation that these proteins are completely absent in prokaryotes (and cytoplasmic organelles), but with few exceptions, the same five major histone types are found in all higher plants and animals. Since the histone content of the algae and other protists is not constant, some of these organisms may represent transition forms between the prokaryotic and eukaryotic modes of packaging the genetic material. Comparative studies of protistan histones may thus be of help in determining evolutionary relationships. However, several problems are encounter with protistan histones, including difficulties in isolating nuclei, proteolytic degradation, anomalous gel migration of histones, and difficulties in histone identification. Because of the above problems, and the observed variability in protistan histones, it is suggested that several criteria be employed for histone identification in protists. PMID:3910133

  2. The heat shock protein 90 inhibitor SNX5422 has a synergistic activity with histone deacetylase inhibitors in induction of death of anaplastic thyroid carcinoma cells.

    PubMed

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2016-02-01

    The influence of the heat shock protein 90 (hsp90) inhibitor SNX5422 alone or in combination with the histone deacetylase (HDAC) inhibitors PXD101, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA) on survival of anaplastic thyroid carcinoma (ATC) cells was investigated. In 8505C and CAL62 cells, SNX5422 caused cell death with concomitant changes in the expression of hsp90 client proteins. After treatment of both SNX5422 and PXD101, SAHA and TSA, compared with treatment of SNX5422 alone, cell viability was diminished, whereas inhibition rate and cytotoxic activity were enhanced. All of the combination index values were lower than 1.0, suggesting the synergism between SNX5422 and PXD101, SAHA and TSA in induction of cell death. In cells treated with both SNX5422 and PXD101, SAHA and TSA, compared with cells treated with SNX5422 alone, the protein levels of Akt, phospho-4EBP1, phospho-S6 K, and survivin were diminished, while those of γH2AX, acetyl. histone H3, acetyl. histone H4, cleaved PARP, and cleaved caspase-3 were enhanced. In conclusion, these results demonstrate that SNX5422 has a cytotoxic activity in conjunction with alterations in the expression of hsp90 client proteins in ATC cells. Moreover, SNX5422 synergizes with HDAC inhibitors in induction of cytotoxicity accompanied by the suppression of PI3K/Akt/mTOR signaling and survivin, and the overexpression of DNA damage-related proteins in ATC cells. PMID:26219406

  3. Double-strand DNA breaks recruit the centromeric histone CENP-A

    PubMed Central

    Zeitlin, Samantha G.; Baker, Norman M.; Chapados, Brian R.; Soutoglou, Evi; Wang, Jean Y. J.; Berns, Michael W.; Cleveland, Don W.

    2009-01-01

    The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair. PMID:19717431

  4. Isotype-Specific Inhibition of Histone Deacetylases: Identification of Optimal Targets for Radiosensitization

    PubMed Central

    Kim, Jin Ho; Moon, Sung Ho; No, Mina; Kim, Jae Jin; Choi, Eun Jung; Cho, Bong Jun; Kim, Jae Sung; Kim, Il Han; Kim, In Ah

    2016-01-01

    Purpose Histone deacetylase (HDAC) inhibitors radiosensitize tumor cells. To elucidate mechanisms underlying radiosensitization by HDAC inhibition, understanding of differential contributions of HDAC isotypes is needed. The aim of this study was to investigate involvement of known HDAC isotypes in modulation of cellular radiosensitivity. Materials and Methods Because pharmacologic HDAC inhibitors lack isotype-specificity, RNA interference against 11 HDAC isotypes was used to inhibit HDAC in an isotype-specific manner. Radiation cell survival was evaluated using a clonogenic assay in SQ20B cells transfected with small interfering RNA specifically targeting HDAC isotypes. Immunocytochemistry was performed for detection of γH2AX foci. Protein expression was measured using Western blotting. Results Among 11 HDAC isotypes tested, specific inhibition of 7 isotypes (HDAC1, HDAC3, HDAC4, HDAC6, HDAC7, HDAC10, and HDAC11) enhanced radiation lethality in SQ20B cells. Radiosensitization by inhibition of these HDAC isotypes was accompanied by delay of DNA double strand break repair. Radiosensitivity of SQ20B cells was not altered by selective inhibition of the remaining four isotypes (HDAC2, HDAC5, HDAC8, and HDAC9). Inhibition of HDAC isotypes resulted in downregulation of various proteins involved in pro-survival and DNA damage repair pathways. Conclusion Isotype-specificity exists in HDAC inhibition-induced radiosensitization. Different HDAC isotypes are differentially involved in modulation of cellular radiosensitivity. PMID:26582395

  5. WIP1, a homeostatic regulator of the DNA damage response, is targeted by HIPK2 for phosphorylation and degradation.

    PubMed

    Choi, Dong Wook; Na, Wooju; Kabir, Mohammad Humayun; Yi, Eunbi; Kwon, Seonjeong; Yeom, Jeonghun; Ahn, Jang-Won; Choi, Hee-Hyun; Lee, Youngha; Seo, Kyoung Wan; Shin, Min Kyoo; Park, Se-Ho; Yoo, Hae Yong; Isono, Kyo-Ichi; Koseki, Haruhiko; Kim, Seong-Tae; Lee, Cheolju; Kwon, Yunhee Kim; Choi, Cheol Yong

    2013-08-01

    WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR. PMID:23871434

  6. Histone deacetylases: Targets for antifungal drug development

    PubMed Central

    Kmetzsch, Livia

    2015-01-01

    The interaction of pathogens and its hosts causes a drastic change in the transcriptional landscape in both cells. Among the several mechanisms of gene regulation, transcriptional initiation is probably the main point. In such scenario, the access of transcriptional machinery to promoter is highly regulated by post-translational modification of histones, such as acetylation, phosphorylation and others. Inhibition of histone deacetylases is able to reduce fungal pathogens fitness during infection and, therefore, is currently being considered for the development of new antifungal therapy strategies. PMID:26151486

  7. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett’s Esophageal Adenocarcinoma Cells

    PubMed Central

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett’s patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

  8. Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

    PubMed

    Li, Dan; Cao, Weibiao

    2016-01-01

    The mechanisms whereby bile acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett's patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA. PMID:27511066

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

    SciTech Connect

    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.

  10. Histone H1--DNA interaction. On the mechanism of DNA strands crosslinking by histone H1.

    PubMed Central

    Glotov, B O; Nikolaev, L G; Severin, E S

    1978-01-01

    Crosslinking of DNA fibers by histone H1 or phosphorylated on Ser-37 histone H1, and by the individual fragments of the H1 polypeptide chain was studied by the method of turbidimetry. The dependence of the turbidity of DNA-protein complexes on the ionic strength in solution suggests that the condensation of H1.DNA complexes in vitro is apparently due to both specific histone-DNA interactions with the contribution of hydrogen and/or hydrophobic bonds and the formation of polycationic "bridges" fastening the DNA fibers. The effectiveness of the condensation is postulated to be a function of a proportion between the two mechanisms which in turn can be controlled by slight changes in ionic surroundings. The sharp dependence of shrinkage of H1.DNA complexes on ionic strength at "physiological" salt concentrations could provide a mechanism to regulate density and consequently the total activity of chromatin in the cell nuclei. The phosphorylation of histone H1 on Ser-37 by a specific histone kinase does not noticeably affect the pattern of DNA crosslinking by the H1. Images PMID:27766

  11. An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts

    PubMed Central

    Pinazza, M; Borga, C; Agnusdei, V; Minuzzo, S; Fossati, G; Paganin, M; Michielotto, B; De Paoli, A; Basso, G; Amadori, A; te Kronnie, G; Indraccolo, S

    2016-01-01

    Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia. PMID:26764573

  12. An immediate transcriptional signature associated with response to the histone deacetylase inhibitor Givinostat in T acute lymphoblastic leukemia xenografts.

    PubMed

    Pinazza, M; Borga, C; Agnusdei, V; Minuzzo, S; Fossati, G; Paganin, M; Michielotto, B; De Paoli, A; Basso, G; Amadori, A; te Kronnie, G; Indraccolo, S

    2016-01-01

    Despite some success with certain hematological malignancies and in contrast with the strong pro-apoptotic effects measured in vitro, the overall response rate of acute lymphoblastic leukemia (ALL) to histone deacetylase inhibitors (HDACis) is low. With the aim to improve the understanding of how HDACis work in vivo, we investigated the therapeutic efficacy of the clinically approved HDACi Givinostat in a collection of nine pediatric human T-ALL engrafted systemically in NOD/SCID mice. We observed highly heterogeneous antileukemia responses to Givinostat, associated with reduction of the percentage of infiltrating blasts in target organs, induction of apoptosis and differentiation. These effects were not associated with the T-ALL cytogenetic subgroup. Transcriptome analysis disclosed an immediate transcriptional signature enriched in genes involved in cell-cycle regulation and DNA repair, which was validated by quantitative RT-PCR and was associated with in vivo response to this HDACi. Increased phospho-H2AX levels, a marker of DNA damage, were measured in T-ALL cells from Givinostat responders. These results indicate that the induction of the DNA damage response could be an early biomarker of the therapeutic effects of Givinostat in T-ALL models. This information should be considered in the design of future clinical trials with HDACis in acute leukemia. PMID:26764573

  13. Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

    PubMed

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

    2016-05-01

    Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential

  14. Quantitative Phosphoproteomics Reveals Crosstalk Between Phosphorylation and O-GlcNAc in the DNA Damage Response Pathway

    PubMed Central

    Zhong, Jun; Martinez, Marissa; Sengupta, Srona; Lee, Albert; Wu, Xinyan; Chaerkady, Raghothama; Chatterjee, Aditi; O’Meally, Robert N.; Cole, Robert N.; Pandey, Akhilesh; Zachara, Natasha E.

    2015-01-01

    The modification of intracellular proteins by monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification of metazoans. The addition and removal of O-GlcNAc is catalyzed by the O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. One mechanism by which O-GlcNAc is thought to mediate proteins is by regulating phosphorylation. To provide insight into the pathways regulated by O-GlcNAc, we have utilized stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics to carry out comparisons of site-specific phosphorylation in OGT wild-type (WT) and Null cells. Quantitation of the phosphoproteome demonstrated that out of 5,529 phosphoserine, phosphothreonine and phosphotyrosine sites, 232 phosphosites were upregulated and 133 downregulated in the absence of O-GlcNAc. Collectively, these data suggest that deletion of OGT has a profound effect on the phosphorylation of cell cycle and DNA damage response proteins. Key events were confirmed by biochemical analyses and demonstrate a increase in the activating autophosphorylation event on ATM (Ser1987) and on ATM’s downstream targets p53, H2AX and Chk2. Together, these data support widespread changes in the phosphoproteome upon removal of O-GlcNAc, suggesting that O-GlcNAc regulates processes such as the cell cycle, genomic stability, and lysosomal biogenesis. PMID:25263469

  15. Cancer Chemoprotection Through Nutrient-mediated Histone Modifications

    PubMed Central

    Gao, Yifeng; Tollefsbol, Trygve O.

    2016-01-01

    Epigenetics, the study of heritable changes in gene expression without modifying the nucleotide sequence, is among the most important topics in medicinal chemistry and cancer chemoprotection. Among those changes, DNA methylation and histone modification have been shown to be associated with various types of cancers in a number of ways, many of which are regulated by dietary components that are mostly found in plants. Although, mechanisms of nutrient components affecting histone acetylation/deacetylation in cancer are widely studied, how those natural compounds affect cancer through other histone modifications, such as methylation, phosphorylation and ubiquitylation, is rarely reviewed. Thus, this review article discusses impacts recently studied on histone acetylation as well as other histone modifications by dietary components, such as genistein, resveratrol, curcumin, epigallocatechin-3-gallate (EGCG), 3,3′-diindolylmethane (DIM), diallyl disulfide, garcinol, procyanidin B3, quercetin, sulforaphane and other isothiocyanates, in various types of cancer. PMID:25891109

  16. Inhibition of mitotic-specific histone phophorylation by sodium arsenite

    SciTech Connect

    Cobo, J.M.; Valdez, J.G.; Gurley, L.R.

    1994-10-01

    Synchronized cultures of Chinese hamster cells (line CHO) were used to measure the effects of 10{mu}M sodium arsenite on histone phosphorylation. This treatment caused cell proliferation to be temporarily arrested, after which the cells spontaneously resumed cell proliferation in a radiomimetric manner. Immediately following treatment, it was found that sodium arsenite affected only mitotic-specific HI and H3 phosphorylations. Neither interphase, nor mitotic, H2A and H4 phosphorylations were affected, nor was interphase HI Phosphorylation affected. The phosphorylation of HI was inhibited only in mitosis, reducing HI phosphorylation to 38.1% of control levels, which was the level of interphase HI phosphorylation. The phosphorylation of both H3 variants was inhibited in mitosis, the less hydrophobic H3 to 19% and the more hydrophobic H3 to 24% of control levels. These results suggest that sodium arsenite may inhibite cell proliferation by interfering with the cyclin B/p34{sup cdc2} histone kinase activity which is thought to play a key role in regulating the cell cycle. It has been proposed by our laboratory that HI and H3 phosphorylations play a role in restructuring interphase chromatin into metaphase chromosomes. Interference of this process by sodium arsenite may lead to structurally damaged chromosomes resulting in the increased cancer risks known to be produced by arsenic exposure from the environment.

  17. Genome-wide analysis of regulation of gene expression and H3K9me2 distribution by JIL-1 kinase mediated histone H3S10 phosphorylation in Drosophila

    PubMed Central

    Cai, Weili; Wang, Chao; Li, Yeran; Yao, Changfu; Shen, Lu; Liu, Sanzhen; Bao, Xiaomin; Schnable, Patrick S.; Girton, Jack; Johansen, Jørgen; Johansen, Kristen M.

    2014-01-01

    In this study we have determined the genome-wide relationship of JIL-1 kinase mediated H3S10 phosphorylation with gene expression and the distribution of the epigenetic H3K9me2 mark. We show in wild-type salivary gland cells that the H3S10ph mark is predominantly enriched at active genes whereas the H3K9me2 mark is largely associated with inactive genes. Comparison of global transcription profiles in salivary glands from wild-type and JIL-1 null mutant larvae revealed that the expression levels of 1539 genes changed at least 2-fold in the mutant and that a substantial number (49%) of these genes were upregulated whereas 51% were downregulated. Furthermore, the results showed that downregulation of genes in the mutant was correlated with higher levels or acquisition of the H3K9me2 mark whereas upregulation of a gene was correlated with loss of or diminished H3K9 dimethylation. These results are compatible with a model where gene expression levels are modulated by the levels of the H3K9me2 mark independent of the state of the H3S10ph mark, which is not required for either transcription or gene activation to occur. Rather, H3S10 phosphorylation functions to indirectly maintain active transcription by counteracting H3K9 dimethylation and gene silencing. PMID:24598257

  18. Histone Modifiers in Cancer

    PubMed Central

    Cohen, Idan; Poręba, Elżbieta; Kamieniarz, Kinga; Schneider, Robert

    2011-01-01

    Covalent modifications of histones can regulate all DNA-dependent processes. In the last few years, it has become more and more evident that histone modifications are key players in the regulation of chromatin states and dynamics as well as in gene expression. Therefore, histone modifications and the enzymatic machineries that set them are crucial regulators that can control cellular proliferation, differentiation, plasticity, and malignancy processes. This review discusses the biology and biochemistry of covalent histone posttranslational modifications (PTMs) and evaluates the dual role of their modifiers in cancer: as oncogenes that can initiate and amplify tumorigenesis or as tumor suppressors. PMID:21941619

  19. Histone variants and epigenetics.

    PubMed

    Henikoff, Steven; Smith, M Mitchell

    2015-01-01

    Histones package and compact DNA by assembling into nucleosome core particles. Most histones are synthesized at S phase for rapid deposition behind replication forks. In addition, the replacement of histones deposited during S phase by variants that can be deposited independently of replication provide the most fundamental level of chromatin differentiation. Alternative mechanisms for depositing different variants can potentially establish and maintain epigenetic states. Variants have also evolved crucial roles in chromosome segregation, transcriptional regulation, DNA repair, and other processes. Investigations into the evolution, structure, and metabolism of histone variants provide a foundation for understanding the participation of chromatin in important cellular processes and in epigenetic memory. PMID:25561719

  20. Disruption of DNA Damage-Response by Propyl Gallate and 9-Aminoacridine.

    PubMed

    Matsuda, Shun; Matsuda, Yoko; Yanagisawa, Shin-Ya; Ikura, Masae; Ikura, Tsuyoshi; Matsuda, Tomonari

    2016-06-01

    The DNA-damage response (DDR) protects the genome from various types of endogenous and exogenous DNA damage, and can itself be a target of certain chemicals that give rise to chromosomal aberrations. Here, we developed a screening method to detect inhibition of Mediator of DNA damage Checkpoint 1 (MDC1) foci formation (the Enhanced Green Fluorescent Protein (EGFP)-MDC1 foci formation-inhibition assay) using EGFP-MDC1-expressing human cells. The assay identified propyl gallate (PG) and 9-aminoacridine (9-AA) as inhibitors of camptothecin (CPT)-induced MDC1 foci formation. We demonstrated that the inhibition of CPT-induced MDC1 foci formation by PG was caused by the direct suppression of histone H2AX phosphorylation at Ser139 (γH2AX), which is required for MDC1 foci formation, by quantifying γH2AX in cells and in vitro 9-AA also directly suppressed H2AX Ser139-phosphorylation in vitro but the concentration was much higher than that required to suppress CPT-induced MDC1 foci formation in cells. Consistent with these findings, PG and 9-AA both suppressed CPT-induced G2/M cell-cycle arrest and increased the number of abnormal nuclei. Our results suggest that early DDR-inhibitory effects of PG and 9-AA contribute to their chromosome-damaging potential, and that the EGFP-MDC1 foci formation-inhibition assay is useful for detection of and screening for H2AX Ser139-phosphorylation-inhibitory effects of chemicals. PMID:26928355

  1. Transcriptional Alterations of ET-1 Axis and DNA Damage in Lung Tissue of a Rat Obesity Model

    PubMed Central

    Cabiati, Manuela; Salvadori, Costanza; Guiducci, Letizia; Caselli, Chiara; Prescimone, Tommaso; Facioni, Maria Sole; Azzarà, Alessia; Chiaramonte, Anna; Mazzoni, Stefano; Bruschi, Fabrizio; Giannessi, Daniela

    2015-01-01

    Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (COfc-Ofc) or acute hyperglycemia (COAH-OAH). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in OAH rats were significantly higher (p=0.019) than in the corresponding CO and COAH rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection. PMID:25517973

  2. Mutagenic Effects of Perfluorooctanesulfonic Acid in gpt Delta Transgenic System Are Mediated by Hydrogen Peroxide.

    PubMed

    Wang, Yichen; Zhang, Xuefeng; Wang, Meimei; Cao, Yiyi; Wang, Xinan; Liu, Yun; Wang, Juan; Wang, Jing; Wu, Lijun; Hei, Tom K; Luan, Yang; Xu, An

    2015-05-19

    Perfluorooctane sulfate (PFOS), a persistent organic pollutant, has recently been closely linked with an increased risk of tumorigenesis. However, PFOS has yielded negative results in various tests of genotoxicity. The present study aimed to investigate the mutagenic response to PFOS in the gpt delta transgenic mouse mutation system and to illustrate the contribution of hydrogen peroxide (H2O2) to PFOS genotoxicity. Mutations at the redBA/gam loci were determined by Spi(-) assay both in vitro and in vivo. DNA damage was measured by phosphorylated histone H2AX (γ-H2AX) and mouse bone marrow micronucleus (MN) testing. Our data showed that PFOS induced concentration-dependent increases in γ-H2AX foci and in mutation frequencies at redBA/gam loci in transgenic mouse embryonic fibroblast cells, which were confirmed by the formation of MNs in the bone marrow and the observations of mutation induction in the livers of gpt delta transgenic mice. Concurrent treatment with catalase, an efficient H2O2 scavenger, significantly decreased the formation of γ-H2AX foci and the mutation yields induced by PFOS. In addition, the generation of H2O2 was found to be closely related to the abnormal peroxisomal β-oxidation caused by PFOS. These finding might provide new mechanistical information about genotoxic effects of PFOS. PMID:25875360

  3. Biological Response of Positron Emission Tomography Scan Exposure and Adaptive Response in Humans

    PubMed Central

    Schnarr, Kara; Carter, Timothy F.; Gillis, Daniel; Webber, Colin; Dayes, Ian; Dolling, Joanna A.; Gulenchyn, Karen; Boreham, Douglas R.

    2015-01-01

    The biological effects of exposure to radioactive fluorodeoxyglucose (18F-FDG) were investigated in the lymphocytes of patients undergoing positron emission tomography (PET) procedures. Low-dose, radiation-induced cellular responses were measured using 3 different end points: (1) apoptosis; (2) chromosome aberrations; and (3) γH2AX foci formation. The results showed no significant change in lymphocyte apoptosis, or chromosome aberrations, as a result of in vivo 18F-FDG exposure, and there was no evidence the PET scan modified the apoptotic response of lymphocytes to a subsequent 2 Gy in vitro challenge irradiation. However, lymphocytes sampled from patients following a PET scan showed an average of 22.86% fewer chromosome breaks and 39.16% fewer dicentrics after a subsequent 2 Gy in vitro challenge irradiation. The effect of 18F-FDG exposure on phosphorylation of histone H2AXH2AX) in lymphocytes of patients showed a varied response between individuals. The relationship between γH2AX foci formation and increasing activity of 18F-FDG was not directly proportional to dose. This variation is most likely attributed to differences in the factors that combine to constitute an individual’s radiation response. In summary, the results of this study indicate18F-FDG PET scans may not be detrimental but can elicit variable responses between individuals and can modify cellular response to subsequent radiation exposures. PMID:26740810

  4. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  5. Interplay between histone H1 structure and function.

    PubMed

    Roque, Alicia; Ponte, Inma; Suau, Pedro

    2016-03-01

    H1 linker histones are involved both in the maintenance of higher-order chromatin structure and in gene regulation. Histone H1 exists in multiple isoforms, is evolutionarily variable and undergoes a large variety of post-translational modifications. We review recent progress in the understanding of the folding and structure of histone H1 domains with an emphasis on the interactions with DNA. The importance of intrinsic disorder and hydrophobic interactions in the folding and function of the carboxy-terminal domain (CTD) is discussed. The induction of a molten globule-state in the CTD by macromolecular crowding is also considered. The effects of phosphorylation by cyclin-dependent kinases on the structure of the CTD, as well as on chromatin condensation and oligomerization, are described. We also address the extranuclear functions of histone H1, including the interaction with the β-amyloid peptide. PMID:26415976

  6. Histone modifications and mitosis: countermarks, landmarks, and bookmarks.

    PubMed

    Wang, Fangwei; Higgins, Jonathan M G

    2013-04-01

    The roles of post-translational histone modifications in regulating transcription and DNA damage have been widely studied and discussed. Although mitotic histone marks, particularly phosphorylation, were discovered four decades ago, their roles in mitosis have been outlined only in the past few years. Here we aim to provide an integrated view of how histone modifications act as 'countermarks', 'landmarks', and 'bookmarks' to displace, recruit, and 'remember' the location of regulatory proteins during and shortly after mitosis. These capabilities allow histone marks to help downregulate interphase functions such as transcription during mitosis, to facilitate chromatin events required to accomplish chromosome segregation, and to contribute to the maintenance of epigenetic states through mitosis. PMID:23246430

  7. Brownian dynamics simulation of the effect of histone modification on nucleosome structure

    NASA Astrophysics Data System (ADS)

    Li, Wei; Dou, Shuo-Xing; Xie, Ping; Wang, Peng-Ye

    2007-05-01

    Using Brownian dynamics we simulate the effect of histone modification, such as phosphorylation, acetylation, and methylation, on nucleosome structure by varying the interaction force between DNA and the histone octamer. The simulation shows that the structural stability of nucleosome is very sensitive to the interaction force, and the DNA unwrapping from the modified histone octamer usually occurs turn by turn. Furthermore, the effects of temperature and DNA break as well as the competition between modified and normal histone octamers are investigated, with the simulation results being in agreement with the experimental observation that phosphorylated nucleosomes near DNA breaks are more easily depleted. Though the simulation study may only give a coarse grained view of the DNA unwrapping process for the modified histone octamer, it may provide insight into the mechanism of DNA repair.

  8. BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage.

    PubMed

    Fabbro, Megan; Savage, Kienan; Hobson, Karen; Deans, Andrew J; Powell, Simon N; McArthur, Grant A; Khanna, Kum Kum

    2004-07-23

    BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53(Ser-15) following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53(Ser-15) efficiently. Phosphorylation of p53(Ser-15) is necessary for an IR-induced G(1)/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53(Ser-15) phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G(1)/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G(1)/S cell cycle progression after DNA damage. PMID:15159397

  9. Chromatin Proteomics Reveals Variable Histone Modifications during the Life Cycle of Trypanosoma cruzi.

    PubMed

    de Jesus, Teresa Cristina Leandro; Nunes, Vinícius Santana; Lopes, Mariana de Camargo; Martil, Daiana Evelin; Iwai, Leo Kei; Moretti, Nilmar Silvio; Machado, Fabrício Castro; de Lima-Stein, Mariana L; Thiemann, Otavio Henrique; Elias, Maria Carolina; Janzen, Christian; Schenkman, Sergio; da Cunha, Julia Pinheiro Chagas

    2016-06-01

    Histones are well-conserved proteins that form the basic structure of chromatin in eukaryotes and undergo several post-translational modifications, which are important for the control of transcription, replication, DNA damage repair, and chromosome condensation. In early branched organisms, histones are less conserved and appear to contain alternative sites for modifications, which could reveal evolutionary unique functions of histone modifications in gene expression and other chromatin-based processes. Here, by using high-resolution mass spectrometry, we identified and quantified histone post-translational modifications in two life cycle stages of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. We detected 44 new modifications, namely: 18 acetylations, seven monomethylations, seven dimethylations, seven trimethylations, and four phosphorylations. We found that replicative (epimastigote stage) contains more histone modifications than nonreplicative and infective parasites (trypomastigote stage). Acetylations of lysines at the C-terminus of histone H2A and methylations of lysine 23 of histone H3 were found to be enriched in trypomastigotes. In contrast, phosphorylation in serine 23 of H2B and methylations of lysine 76 of histone H3 predominates in proliferative states. The presence of one or two methylations in the lysine 76 was found in cells undergoing mitosis and cytokinesis, typical of proliferating parasites. Our findings provide new insights into the role of histone modifications related to the control of gene expression and cell-cycle regulation in an early divergent organism. PMID:27108550

  10. Ciprofloxacin-induced G2 arrest and apoptosis in TK6 lymphoblastoid cells is not dependent on DNA double-strand break formation

    PubMed Central

    Smart, Daniel J.; Halicka, H. Dorota; Traganos, Frank; Darzynkiewicz, Zbigniew; Williams, Gary M.

    2008-01-01

    Drugs developed for the treatment of conditions other than neoplasia can also show promise as potential antitumor agents. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells, and is thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition and stabilized cleavage complex (SCC) formation. DSBs trigger Ser-139 phosphorylation of histone H2AXH2AX) by PI-3-like kinases including ATM; γH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated lymphocytes (Lymphs). Treatment of TK6 cells (wild-type p53) with 100 µg/ml CPFX for 2-10 h produced no increase in γH2AX; to the contrary, its level in S phase cells was reduced at 10 h compared to controls. Nevertheless, stabilization of topo IIα, ATM Ser-1981 phosphorylation and G2 arrest was observed in TK6 cells exposed to CPFX for ≥4 h. However, following 24 h treatment, γH2AX was dramatically increased in a sub-population of cells indicating the onset of apoptosis (confirmed by presence of activated caspase 3). CPFX had a similar lack of effect on induction of γH2AX at early time points in WTK1 and NH32 cells (devoid of functional p53) and proliferating Lymphs, however, induction of apoptosis was less pronounced than in TK6 cells. Formation of SCC and activation of ATM (but lack of γH2AX induction) indicates topo II-mediated chromatin or DNA changes in the absence of DSBs; ATM activation apparently triggers the G2M checkpoint leading to G2 arrest. The subsequent induction of apoptosis appears to be facilitated by functional p53. CPFX may therefore have a potential use as a chemotherapeutic agent in the treatment of lymphoblast-derived cancer. PMID:18059176

  11. Local generation of fumarate promotes DNA repair through inhibition of histone H3 demethylation

    PubMed Central

    Jiang, Yuhui; Qian, Xu; Shen, Jianfeng; Wang, Yugang; Li, Xinjian; Liu, Rui; Xia, Yan; Chen, Qianming; Peng, Guang; Lin, Shiaw-Yih; Lu, Zhimin

    2016-01-01

    Histone methylation regulates DNA repair. However, the mechanisms that underlie the regulation of histone methylation during this repair remain to be further defined. Here, we show that ionizing radiation (IR) induces DNA-PK-dependent phosphorylation of nuclear fumarase at T236, which leads to an interaction between fumarase and the histone variant H2A.Z at DNA double-strand break (DSB) regions. Locally generated fumarate inhibits KDM2B histone demethylase activity, resulting in enhanced dimethylation of histone H3 K36; in turn, this increases the accumulation of the Ku70-containing DNA-PK at DSB regions for non-homologous end joining (NHEJ) DNA repair and cell survival. These findings reveal a feedback mechanism that underlies DNA-PK regulation by chromatin-associated fumarase and an instrumental function of fumarase in regulating histone H3 methylation and DNA repair. PMID:26237645

  12. Baculoviruses Modulate a Proapoptotic DNA Damage Response To Promote Virus Multiplication

    PubMed Central

    Mitchell, Jonathan K.

    2012-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) initiates apoptosis in diverse insects through events triggered by virus DNA (vDNA) replication. To define the proapoptotic pathway and its role in antivirus defense, we investigated the link between the host's DNA damage response (DDR) and apoptosis. We report here that AcMNPV elicits a DDR in the model insect Drosophila melanogaster. Replication of vDNA activated DDR kinases, as evidenced by ATM-driven phosphorylation of the Drosophila histone H2AX homolog (H2Av), a critical regulator of the DDR. Ablation or inhibition of ATM repressed H2Av phosphorylation and blocked virus-induced apoptosis. The DDR kinase inhibitors caffeine and KU55933 also prevented virus-induced apoptosis in cells derived from the permissive AcMNPV host, Spodoptera frugiperda. This block occurred at a step upstream of virus-mediated depletion of the cellular inhibitor-of-apoptosis protein, an event that initiates apoptosis in Spodoptera and Drosophila. Thus, the DDR is a conserved, proapoptotic response to baculovirus infection. DDR inhibition also repressed vDNA replication and reduced virus yields 100,000-fold, demonstrating that the DDR contributes to virus production, despite its recognized antivirus role. In contrast to virus-induced phosphorylation of Drosophila H2Av, AcMNPV blocked phosphorylation of the Spodoptera H2AX homolog (SfH2AX). Remarkably, AcMNPV also suppressed SfH2AX phosphorylation following pharmacologically induced DNA damage. These findings indicate that AcMNPV alters canonical DDR signaling in permissive cells. We conclude that AcMNPV triggers a proapoptotic DDR that is subsequently modified, presumably to stimulate vDNA replication. Thus, manipulation of the DDR to facilitate multiplication is an evolutionarily conserved strategy among DNA viruses of insects and mammals. PMID:23035220

  13. Induction and persistence of radiation-induced DNA damage is more pronounced in young animals than in old animals

    PubMed Central

    Hudson, Darryl; Kovalchuk, Igor; Koturbash, Igor; Kolb, Bryan; Martin, Olga A.; Kovalchuk, Olga

    2011-01-01

    Younger individuals are more prone to develop cancer upon ionizing radiation (IR) exposure. Radiation-induced tumors are associated with inefficient repair of IR-induced DNA damage and genome instability. Phosphorylation of histone H2AX (γ-H2AX) is the initial event in repair of IR-induced DNA damage on the chromatin flanking the DNA strand breaks. This step is crucially important for the repair of DNA strand breaks and for the maintenance of genome stability. We studied the molecular underpinnings of the age-related IR effects using an animal model. By assaying for IR-induced γ-H2AX foci we analyzed the induction and repair of the DNA strand breaks in spleen, thymus, liver, lung, kidney, cerebellum, hippocampus, frontal cortex and olfactory bulb of 7, 14, 24, 30 and 45 days old male and female mice as a function of age. We demonstrate that tissues of younger animals are much more susceptible to IR-induced DNA damage. Younger animals exhibited higher levels of γ-H2AX formation which partially correlated with cellular proliferation and expression of DNA repair proteins. Induction and persistence of γ-H2AX foci was the highest in lymphoid organs (thymus and spleen) of 7 and 14 day old mice. The lowest focal induction was seen in lung and brain of young animals. The mechanisms of cell and tissue-specificity of in vivo IR responses need to be further dissected. This study provides a roadmap for the future analyses of DNA damage and repair induction in young individuals. PMID:21685513

  14. AMPK/Snf1 signaling regulates histone acetylation: Impact on gene expression and epigenetic functions.

    PubMed

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2016-08-01

    AMP-activated protein kinase (AMPK) and its yeast homolog, Snf1, are critical regulators in the maintenance of energy metabolic balance not only stimulating energy production but also inhibiting energy-consuming processes. The AMPK/Snf1 signaling controls energy metabolism by specific phosphorylation of many metabolic enzymes and transcription factors, enhancing or suppressing their functions. The AMPK/Snf1 complexes can be translocated from cytoplasm into nuclei where they are involved in the regulation of transcription. Recent studies have indicated that AMPK/Snf1 activation can control histone acetylation through different mechanisms affecting not only gene transcription but also many other epigenetic functions. For instance, AMPK/Snf1 enzymes can phosphorylate the histone H3S10 (yeast) and H2BS36 (mammalian) sites which activate specific histone acetyltransferases (HAT), consequently enhancing histone acetylation. Moreover, nuclear AMPK can phosphorylate type 2A histone deacetylases (HDAC), e.g. HDAC4 and HDAC5, triggering their export from nuclei thus promoting histone acetylation reactions. AMPK activation can also increase the level of acetyl CoA, e.g. by inhibiting fatty acid and cholesterol syntheses. Acetyl CoA is a substrate for HATs, thus increasing their capacity for histone acetylation. On the other hand, AMPK can stimulate the activity of nicotinamide phosphoribosyltransferase (NAMPT) which increases the level of NAD(+). NAD(+) is a substrate for nuclear sirtuins, especially for SIRT1 and SIRT6, which deacetylate histones and transcription factors, e.g. those regulating ribosome synthesis and circadian clocks. Histone acetylation is an important epigenetic modification which subsequently can affect chromatin remodeling, e.g. via bromodomain proteins. We will review the signaling mechanisms of AMPK/Snf1 in the control of histone acetylation and subsequently clarify their role in the epigenetic regulation of ribosome synthesis and circadian clocks

  15. A Phosphotyrosine Switch Controls the Association of Histone Mark Readers with Methylated Proteins.

    PubMed

    Irving-Hooper, Bronwyn Kate; Binda, Olivier

    2016-03-22

    Although histone post-translational modifications play a paramount role in controlling access to genetic information, our understanding of the precise mechanisms regulating chromatin signaling remains superficial. For instance, histone H3 trimethylated on lysine 9 (H3K9(me3)) favors the association of chromodomain proteins such as heterochromatin protein 1α (HP1α) with chromatin. However, HP1α and other such chromatin proteins are not covering all specific histone marks at all times. Thus, how are these reader-histone interactions regulated? We propose tyrosine phosphorylation within the aromatic cage of histone mark readers as a molecular switch that can either turn ON or OFF and even alter the specificity of reader-histone interactions. We have identified tyrosine phosphorylation events on the chromatin proteins HP1α and M-phase phosphoprotein 8 that regulate their association with methylated histones in vitro (synthetic peptides, calf thymus purified histones, and nucleosomes), but also in cells, thus controlling access to genetic information. PMID:26562627

  16. Synthesis, Biological Evaluation, and Structure–Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks

    PubMed Central

    2012-01-01

    Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AXH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents. PMID:22694057

  17. Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells

    NASA Astrophysics Data System (ADS)

    Hable, V.; Dollinger, G.; Greubel, C.; Hauptner, A.; Krücken, R.; Dietzel, S.; Cremer, T.; Drexler, G. A.; Friedl, A. A.; Löwe, R.

    2006-04-01

    Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone γ-H2AX are studied. For this purpose cells are irradiated in line patterns. The γ-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

  18. Adamantanyl-Histone Deacetylase Inhibitor H6CAHA Exhibits Favorable Pharmacokinetics and Augments Prostate Cancer Radiation Sensitivity

    SciTech Connect

    Konsoula, Zacharoula; Cao Hong; Velena, Alfredo; Jung, Mira

    2011-04-01

    Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to {gamma}-radiation in vitro and in vivo. Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice. Results: H6CAHA demonstrated good solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 {+-} 0.91 {mu}M x h, and its half-life was 11.17 {+-} 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of {gamma}H2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with {gamma}-radiation (10 x 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days. Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation.

  19. Assaying Pharmacodynamic Endpoints with Targeted Therapy: Flavopiridol and 17AAG Induced Dephosphorylation of Histone H1.5 in Acute Myeloid Leukemia

    PubMed Central

    Wang, Liwen; Harshman, Sean W.; Liu, Shujun; Ren, Chen; Xu, Hua; Sallans, Larry; Grever, Michael; Byrd, John C.; Marcucci, Guido; Freitas, Michael A.

    2011-01-01

    Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this report we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 Acute Myeloid Leukemia (AML) cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. Liquid chromatography mass spectrometry profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin dependent kinase inhibitor, flavopiridol, and the Hsp90 inhibitor, 17AAG (17-(Allylamino)-17-demethoxygeldanamycin). In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation. PMID:21110323

  20. The human actin-related protein hArp5: Nucleo-cytoplasmic shuttling and involvement in DNA repair

    SciTech Connect

    Kitayama, Kumiko; Kamo, Mariko; Oma, Yukako; Matsuda, Ryo; Uchida, Takafumi; Ikura, Tsuyoshi; Tashiro, Satoshi; Ohyama, Takashi; Winsor, Barbara; Harata, Masahiko

    2009-01-15

    Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5{delta} yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX ({gamma}-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced {gamma}-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

  1. Biological function and regulation of histone and non-histone lysine methylation in response to DNA damage.

    PubMed

    Chen, Yongcan; Zhu, Wei-Guo

    2016-07-01

    DNA damage response (DDR) signaling network is initiated to protect cells from various exogenous and endogenous damage resources. Timely and accurate regulation of DDR proteins is required for distinct DNA damage repair pathways. Post-translational modifications of histone and non-histone proteins play a vital role in the DDR factor foci formation and signaling pathway. Phosphorylation, ubiquitylation, SUMOylation, neddylation, poly(ADP-ribosyl)ation, acetylation, and methylation are all involved in the spatial-temporal regulation of DDR, among which phosphorylation and ubiquitylation are well studied. Studies in the past decade also revealed extensive roles of lysine methylation in response to DNA damage. Lysine methylation is finely regulated by plenty of lysine methyltransferases, lysine demethylases, and can be recognized by proteins with chromodomain, plant homeodomain, Tudor domain, malignant brain tumor domain, or proline-tryptophan-tryptophan-proline domain. In this review, we outline the dynamics and regulation of histone lysine methylation at canonical (H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20) and non-canonical sites after DNA damage, and discuss their context-specific functions in DDR protein recruitment or extraction, chromatin environment establishment, and transcriptional regulation. We also present the emerging advances of lysine methylation in non-histone proteins during DDR. PMID:27217472

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

    PubMed Central

    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

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

    PubMed

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

    2015-03-30

    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

  4. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients

    PubMed Central

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing. PMID:26237679

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

    PubMed Central

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

    2015-01-01

    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. PMID:25999347

  6. Histone Modifications and Cancer.

    PubMed

    Audia, James E; Campbell, Robert M

    2016-01-01

    SUMMARYHistone posttranslational modifications represent a versatile set of epigenetic marks involved not only in dynamic cellular processes, such as transcription and DNA repair, but also in the stable maintenance of repressive chromatin. In this article, we review many of the key and newly identified histone modifications known to be deregulated in cancer and how this impacts function. The latter part of the article addresses the challenges and current status of the epigenetic drug development process as it applies to cancer therapeutics. PMID:27037415

  7. FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.

    PubMed

    Deng, Qiudong; Holler, Christopher J; Taylor, Georgia; Hudson, Kathryn F; Watkins, William; Gearing, Marla; Ito, Daisuke; Murray, Melissa E; Dickson, Dennis W; Seyfried, Nicholas T; Kukar, Thomas

    2014-06-01

    Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS. PMID:24899704

  8. BRCC36 is essential for ionizing radiation-induced BRCA1 phosphorylation and nuclear foci formation.

    PubMed

    Chen, Xiaowei; Arciero, Cletus A; Wang, Chunrong; Broccoli, Dominique; Godwin, Andrew K

    2006-05-15

    We have previously reported the identification and characterization of a novel BRCA1/2 interacting protein complex, BRCC (BRCA1/2-containing complex). BRCC36, one of the proteins in BRCC, directly interacts with BRCA1, and regulates the ubiquitin E3 ligase activity of BRCC. Importantly, BRCC36 is aberrantly expressed in the vast majority of breast tumors, indicating a potential role in the pathogenesis of this disease. To further elucidate the functional consequence of abnormal BRCC36 expression in breast cancer, we have done in vivo silencing studies using small interfering RNAs targeting BRCC36 in breast cancer cell lines, i.e., MCF-7, ZR-75-1, and T47D. Knock-down of BRCC36 alone does not affect cell growth, but when combined with ionizing radiation (IR) exposure, it leads to an increase in the percentage of cells undergoing apoptosis when compared with the small interfering RNA control group in breast cancer cells. Immunoblot analysis shows that inhibition of BRCC36 has no effect on the activation of ATM, expression of p21 and p53, or BRCA1-BARD1 interaction following IR exposure. Importantly, BRCC36 depletion disrupts IR-induced phosphorylation of BRCA1. Immunofluorescent staining of BRCA1 and gamma-H2AX indicates that BRCC36 depletion prevents the formation of BRCA1 nuclear foci in response to DNA damage in breast cancer cells. These results show that down-regulation of BRCC36 expression impairs the DNA repair pathway activated in response to IR by inhibiting BRCA1 activation, thereby sensitizing breast cancer cells to IR-induced apoptosis. PMID:16707425

  9. Global chromatin fibre compaction in response to DNA damage

    SciTech Connect

    Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. Black-Right-Pointing-Pointer DNA repair foci are found in soluble chromatin. Black-Right-Pointing-Pointer Biophysical analysis reveals global chromatin fibre compaction after DNA damage. Black-Right-Pointing-Pointer DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation ({gamma}H2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and {gamma}H2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by

  10. Histone mimics: digging down under

    PubMed Central

    LIN, Yiwei; ZHOU, Binhua P.

    2014-01-01

    Epigenetic deregulation is intimately associated with the development of human diseases. Intensive studies are currently underway to clarify the mechanism for the sake of achieving ideal diagnostic and therapeutic goals. It has been demonstrated that enzymes with histone-modifying activities can also target non-histone proteins, with the underlying mechanism remaining obscure. In this review, we focus on a novel histone mimicry strategy that may be wildly adapted during the non-histone substrate recognition process. Its potential clinical implications are also discussed. PMID:24966873

  11. A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful dna repair in cancer cell lines

    PubMed Central

    Schellenbauer, Amelie; Biard, Denis; Paget, Vincent; Morel-Altmeyer, Sandrine; Guipaud, Olivier; Chambon, Christophe; Salles, Bernard; Maloum, Karim; Merle-Béral, Hélène; Chevillard, Sylvie; Delic, Jozo

    2015-01-01

    Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells. PMID:26337656

  12. Gamma irradiation induces DNA double-strand breaks in fibroblasts: a model study for the development of biodosimetry

    NASA Astrophysics Data System (ADS)

    Uttayarat, P.; Tangtong, T.; Sukapirom, K.; Boonsirichai, K.

    2015-05-01

    Double-strand breaks (DSBs) of DNAs induced by ionizing radiation can pose detrimental damages on organisms which include genetic instability and cell death. It is necessary to be able to assess health risks associated with irradiation from both accidental and therapeutic exposures in a timely manner for proper medical treatments. This present study showed the first attempt to develop a biodosimetric measure in Thailand based on the quantification of phosphorylated histone H2AX (γ-H2AX) formed at DSB sites with an aim to establish a dose response curve using a two-dimensional (2D) cell culture model. Human dermal fibroblasts were grown into monolayers before irradiated by gamma rays from a Co-60 source in a custom-made lead chamber at doses 0, 0.2, 1, 2 and 4 Gy and a dose rate of 0.21 Gy/min. After 30 min post exposure, γ-H2AX proteins were immunofluorescently labelled for evaluation by confocal microscopy and flow cytometry. The accumulation of phosphorylated γ-H2AX proteins at DSBs appeared as nuclear foci with the most prominent intensity at 4 Gy. Linear regression analysis of flow cytometric data showed a linear response (R2 = 0.9862) of foci intensity in proportion to irradiation dose. In addition, the fraction of cell viability was shown to decrease at higher doses. This technique can be further developed as a quick assessment tool to identify individuals subjected to accidental radiation in parallel to other established biodosimetric measures.

  13. Histone deacetylases and atherosclerosis.

    PubMed

    Zheng, Xia-xia; Zhou, Tian; Wang, Xin-An; Tong, Xiao-hong; Ding, Jia-wang

    2015-06-01

    Atherosclerosis is the most common pathological process that leads to cardiovascular diseases, a disease of large- and medium-sized arteries that is characterized by a formation of atherosclerotic plaques consisting of necrotic cores, calcified regions, accumulated modified lipids, smooth muscle cells (SMCs), endothelial cells, leukocytes, and foam cells. Recently, the question about how to suppress the occurrence of atherosclerosis and alleviate the progress of cardiovascular disease becomes the hot topic. Accumulating evidence suggests that histone deacetylases(HDACs) play crucial roles in arteriosclerosis. This review summarizes the effect of HDACs and HDAC inhibitors(HDACi) on the progress of atherosclerosis. PMID:25875381

  14. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  15. Poly(ADP-ribose) polymerase 1 modulates the lethality of CHK1 inhibitors in carcinoma cells.

    PubMed

    Mitchell, Clint; Park, Margaret; Eulitt, Patrick; Yang, Chen; Yacoub, Adly; Dent, Paul

    2010-11-01

    Prior studies have demonstrated that inhibition of CHK1 can promote the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphorylation of histone H2AX and that inhibition of poly(ADP-ribose) polymerase 1 (PARP1) can affect growth factor-induced ERK1/2 activation. The present studies were initiated to determine whether CHK1 inhibitors interacted with PARP1 inhibition to facilitate apoptosis. Transient expression of dominant-negative CHK1 raised basal ERK1/2 activity and prevented CHK1 inhibitors from activating ERK1/2. CHK1 inhibitors modestly increased the levels of PARP1 ADP ribosylation and molecular or small-molecule inhibition of PARP1 blocked CHK1 inhibitor-stimulated histone H2AX phosphorylation and activation of ERK1/2. Stimulated histone H2AX phosphorylation was ataxia telangiectasia-mutated protein-dependent. Multiple CHK1 inhibitors interacted in a greater than additive fashion with multiple PARP1 inhibitors to cause transformed cell-killing in short-term viability assays and synergistically killed tumor cells in colony-formation assays. Overexpression of BCL-xL or loss of BAX/BAK function, but not the function of BID, suppressed CHK1 inhibitor + PARP1 inhibitor lethality. Inhibition of BCL-2 family protein function enhanced CHK1 inhibitor + PARP1 inhibitor lethality and restored drug-induced cell-killing in cells overexpressing BCL-xL. Thus, PARP1 plays an important role in regulating the ability of CHK1 inhibitors to activate ERK1/2 and the DNA damage response. An inability of PARP1 to modulate this response results in transformed cell death mediated through the intrinsic apoptosis pathway. PMID:20696794

  16. The C Terminus of the Histone Chaperone Asf1 Cross-Links to Histone H3 in Yeast and Promotes Interaction with Histones H3 and H4

    PubMed Central

    Dennehey, Briana K.; Noone, Seth; Liu, Wallace H.; Smith, Luke

    2013-01-01

    The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing. PMID:23184661

  17. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    SciTech Connect

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  18. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    PubMed Central

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly

    2015-01-01

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA-protein crosslinks (DPC) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. PMID:25817892

  19. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

    PubMed Central

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A.; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  20. Histone and Non-Histone Targets of Dietary Deacetylase Inhibitors.

    PubMed

    Kim, Eunah; Bisson, William H; Löhr, Christiane V; Williams, David E; Ho, Emily; Dashwood, Roderick H; Rajendran, Praveen

    2016-01-01

    Acetylation is an important, reversible post-translational modification affecting histone and non-histone proteins with critical roles in gene transcription, DNA replication, DNA repair, and cell cycle progression. Key regulatory enzymes include histone deacetylase (HDACs) and histone acetyltransferases (HATs). Overexpressed HDACs have been identified in many human cancers, resulting in repressed chromatin states that interfere with vital tumor suppressor functions. Inhibition of HDAC activity has been pursued as a mechanism for re-activating repressed genes in cancers, with some HDAC inhibitors showing promise in the clinical setting. Dietary compounds and their metabolites also have been shown to modulate HDAC activity or expression. Out of this body of research, attention increasingly has shifted towards non-histone targets of HDACs and HATs, such as transcriptions factors, hormone receptors, DNA repair proteins, and cytoskeletal components. These aspects are covered in present review, along with the possible clinical significance. Where such data are available, examples are cited from the literature of studies with short chain fatty acids, polyphenols, isoflavones, indoles, organosulfur compounds, organoselenium compounds, sesquiterpene lactones, isoflavones, and various miscellaneous agents. By virtue of their effects on both histone and non-histone proteins, dietary chemopreventive agents modulate the cellular acetylome in ways that are only now becoming apparent. A better understanding of the molecular mechanisms will likely enhance the potential to more effectively combat diseases harboring altered epigenetic landscapes and dysregulated protein signaling. PMID:26303421

  1. Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase

    PubMed Central

    2011-01-01

    Background Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear. Results Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth. Conclusions Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development. PMID:21527018

  2. Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

    PubMed

    Faustino, Luciana R; Carvalho, Adeline A; Silva, Cleidson M G; Rossetto, Rafael; Lopes, Cláudio A P; van Tilburg, Maurício F; Carneiro, Pedro B M; Báo, Sônia N; Moura, Arlindo A A; Bordignon, Vilceu; Figueiredo, José R; Rodrigues, Ana Paula R

    2015-03-01

    Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AXH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues. PMID:25481978

  3. A study of the synergistic effect of folate-decorated polymeric micelles incorporating Hydroxycamptothecin with radiotherapy on xenografted human cervical carcinoma.

    PubMed

    You, Hong; Fu, ShaoZhi; Qin, XingHu; Yu, YanXin; Yang, Bo; Zhang, GuangPeng; Sun, XiaoYang; Feng, Yue; Chen, Yue; Wu, JingBo

    2016-04-01

    In this study, Hydroxycamptothecin (HCPT)-loaded micelles were formed in water by the self-assembly of folate (FA)-decorated amphiphilic block copolymer, methoxy poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL), and achieved a hydrodynamic diameter about of 132 nm. HCPT release from the micelles exhibited no initial burst but showed a sustained release profile. The cytotoxicity and targeting ability of FA conjugated polymeric micelles was investigated by using methylthiazoletetrazolium (MTT) and fluorescence microscopy. We found that FA-conjugated micelles had superior cytotoxicity against HeLa cells compared to non-conjugated micelles, and that they exerted this effect by folate receptor (FR)-mediated endocytosis. In addition, HeLa cells were xenografted into nude mice and subjected to radiotherapy (RT) and/or HCPT-loaded micelle treatment. The antitumor efficacy was detected by analysis of tumor growth delay (TGD) and median survival time. Micro fluorine-18-deoxyglucose PET/computed tomography ((18)F-FDG PET/CT) was performed to assess early tumor response to HCPT-loaded micelles in combination with RT. Analysis of cell cycle redistribution, apoptosis and expression of histone H2AX phosphorylation (λ-H2AX) was used to evaluate the mechanism by which HCPT loaded micelles led to radiosensitization. Taken together, the results showed that HCPT-loaded FA decorated micelles efficiently sensitized xenografts in mice to RT, and induced G2/M phase arrest, apoptosis and expression of λ-H2AX. PMID:26752212

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

    NASA Astrophysics Data System (ADS)

    Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

    2007-07-01

    DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX 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 γH2AX 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 3N 4 window showed a homogenous Hsp70 expression pattern.

  5. Interaction of heavy ions with nuclear chromatin: Spatiotemporal investigations of biological responses in a cellular environment

    NASA Astrophysics Data System (ADS)

    Jakob, B.; Taucher-Scholz, G.

    2006-04-01

    Ion beams offer the possibility to generate strictly localized DNA lesions within subregions of a cell nucleus. The distribution of the ion-induced damage can be indirectly visualized by immunocytochemical detection of repair-related proteins as radiation-induced foci. The proteins analyzed here were the double-strand break marker γ-H2AX, the excision repair and replication protein PCNA and the cell cycle regulator CDKN1A. A newly developed adjustable sample holder is now used to apply an irradiation geometry characterized by a small angle between the plane of the cellular monolayer and the incoming ion beam. This allows the spatial analysis of protein accumulations along ion trajectories, revealing an unexpected clustering after irradiation with low-energy zinc ions. The patterns of protein aggregation observed show considerable intrinsic variability, but similar patterns of protein clustering were obtained for functionally different proteins irrespective of the type of ion beam applied, confirming previous observations for lower and higher LET beams. Foci sizes within ion tracks were found to be larger for γ-H2AX foci in comparison to CDKN1A foci, in agreement with the known histone H2AX phosphorylation response. The results suggest that not the pattern of dose deposition but the underlying chromatin structure determines the distribution of protein clusters along tracks. Therefore, the requirement of time-lapse studies using live cells is emphasized for future studies on chromatin movement as a potential component of the DNA damage response.

  6. Structure and function of histone methylation-binding proteins in plants.

    PubMed

    Liu, Yanli; Min, Jinrong

    2016-06-15

    Post-translational modifications of histones play important roles in modulating many essential biological processes in both animals and plants. These covalent modifications, including methylation, acetylation, phosphorylation, ubiquitination, SUMOylation and so on, are laid out and erased by histone-modifying enzymes and read out by effector proteins. Recent studies have revealed that a number of developmental processes in plants are under the control of histone post-translational modifications, such as floral transition, seed germination, organogenesis and morphogenesis. Therefore, it is critical to identify those protein domains, which could specifically recognize these post-translational modifications to modulate chromatin structure and regulate gene expression. In the present review, we discuss the recent progress in understanding the structure and function of the histone methylation readers in plants, by focusing on Arabidopsis thaliana proteins. PMID:27288029

  7. Dynamics of histone H2A, H4 and HS1ph during spermatogenesis with a focus on chromatin condensation and maturity of spermatozoa

    PubMed Central

    Zhang, Zhao-Hui; Mu, Shu-Mei; Guo, Ming-Shen; Wu, Jiang-li; Li, Yan-qin; Zhang, Han; Wang, Ying; Kang, Xian-Jiang

    2016-01-01

    Histones and histone phosphorylation play vital roles during animal spermatogenesis and spermatozoa maturation. The dynamic distribution of histones H2A and H4 and phosphorylated H2A and H4 at serine 1 (HS1ph) was explored in mammalian and Decapoda germ cells, with a special focus on the distribution of H2A, H4 and HS1ph between mouse condensed spermatozoa chromatin and crab non-condensed spermatozoa chromatin. The distribution of histone marks was also analysed in mature spermatozoa with different chromatin structures. Histone H2A and H4 marks were closely associated with the relatively loose chromatin structure in crab spermatozoa. The significant decrease in the HS1ph signal during spermatogenesis suggests that eliminating most of these epigenetic marks in the nucleusis closely associated with spermatozoa maturity. PMID:27121047

  8. DNA Damage Focus Analysis in Blood Samples of Minipigs Reveals Acute Partial Body Irradiation

    PubMed Central

    Lamkowski, Andreas; Forcheron, Fabien; Agay, Diane; Ahmed, Emad A.; Drouet, Michel; Meineke, Viktor; Scherthan, Harry

    2014-01-01

    Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (±6%) Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1–8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after

  9. DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.

    PubMed

    Lamkowski, Andreas; Forcheron, Fabien; Agay, Diane; Ahmed, Emad A; Drouet, Michel; Meineke, Viktor; Scherthan, Harry

    2014-01-01

    Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR) induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs). The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI) with 49 Gy (± 6%) Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL) of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available early after IR

  10. Enhanced top-down characterization of histone post-translational modifications

    SciTech Connect

    Tian, Zhixin; Tolić, Nikola; Zhao, Rui; Moore, Ronald J.; Hengel, Shawna M.; Robinson, Errol W.; Stenoien, David L.; Wu, Si; Smith, Richard D.; Paša-Tolić, Ljiljana

    2012-01-01

    Background: Multiple post-translational modifications (PTMs) on core histones often work synergistically to fine tune chromatin structure and functions, generating a “histone code” that can be interpreted by a variety of chromatin interacting proteins. Although previous bottom-up and middle-down proteomic approaches have been developed for limited characterization of PTMs on histone N-terminal tails, high-throughput methods for comprehensive identification of PTMs distributed along the entire primary amino acid sequence are yet to be implemented. Results: Here we report a novel online two-dimensional liquid chromatography - tandem mass spectrometry (2D LC–MS/MS) platform for high-throughput and sensitive characterization of histone PTMs at the intact protein level. The metal-free LC system with reverse phase separation followed by weak cation exchange – hydrophilic interaction chromatography (WCX-HILIC) and online Orbitrap Velos tandem mass spectrometry allowed for unambiguous identification of over 700 histone isoforms from a single 2D LC–MS/MS analysis of 7.5 µg of purified core histones. In comparison with previous offline top-down analysis of H4, this online study identified 100 additional isoforms from 100-fold less sample. This platform enabled comprehensive characterization of histone modifications, including those beyond tail regions, with dramatically improved throughput and sensitivity compared to more traditional platforms. Isoforms identified included those with combinatorial PTMs extending well beyond the N-terminal tail regions as well as a large number of phosphorylated isoforms.

  11. The Role of Dietary Histone Deacetylases (HDACs) Inhibitors in Health and Disease

    PubMed Central

    Bassett, Shalome A.; Barnett, Matthew P. G.

    2014-01-01

    Modification of the histone proteins associated with DNA is an important process in the epigenetic regulation of DNA structure and function. There are several known modifications to histones, including methylation, acetylation, and phosphorylation, and a range of factors influence each of these. Histone deacetylases (HDACs) remove the acetyl group from lysine residues within a range of proteins, including transcription factors and histones. Whilst this means that their influence on cellular processes is more complex and far-reaching than histone modifications alone, their predominant function appears to relate to histones; through deacetylation of lysine residues they can influence expression of genes encoded by DNA linked to the histone molecule. HDAC inhibitors in turn regulate the activity of HDACs, and have been widely used as therapeutics in psychiatry and neurology, in which a number of adverse outcomes are associated with aberrant HDAC function. More recently, dietary HDAC inhibitors have been shown to have a regulatory effect similar to that of pharmacological HDAC inhibitors without the possible side-effects. Here, we discuss a number of dietary HDAC inhibitors, and how they may have therapeutic potential in the context of a whole food. PMID:25322459

  12. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  13. CBP histone acetyltransferase activity regulates embryonic neural differentiation in the normal and Rubinstein-Taybi syndrome brain.

    PubMed

    Wang, Jing; Weaver, Ian C G; Gauthier-Fisher, Andrée; Wang, Haoran; He, Ling; Yeomans, John; Wondisford, Frederic; Kaplan, David R; Miller, Freda D

    2010-01-19

    Increasing evidence indicates that epigenetic changes regulate cell genesis. Here, we ask about neural precursors, focusing on CREB binding protein (CBP), a histone acetyltransferase that, when haploinsufficient, causes Rubinstein-Taybi syndrome (RTS), a genetic disorder with cognitive dysfunction. We show that neonatal cbp(+/-) mice are behaviorally impaired, displaying perturbed vocalization behavior. cbp haploinsufficiency or genetic knockdown with siRNAs inhibited differentiation of embryonic cortical precursors into all three neural lineages, coincident with decreased CBP binding and histone acetylation at promoters of neuronal and glial genes. Inhibition of histone deacetylation rescued these deficits. Moreover, CBP phosphorylation by atypical protein kinase C zeta was necessary for histone acetylation at neural gene promoters and appropriate differentiation. These data support a model in which environmental cues regulate CBP activity and histone acetylation to control neural precursor competency to differentiate, and indicate that cbp haploinsufficiency disrupts this mechanism, thereby likely causing cognitive dysfunction in RTS. PMID:20152182

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

    PubMed Central

    Ganesan, Shanthi; Nteeba, Jackson; Keating, Aileen F.

    2014-01-01

    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 (1mg/kg; intraperitoneal injection) at 18 wks 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. PMID:25448685

  15. Spatio-temporal radiation biology with conventionally or laser-accelerated particles for ELIMED

    SciTech Connect

    Ristić-Fira, A.; Bulat, T.; Keta, O.; Petrović, I.; Romano, F.; Cirrone, P.; Cuttone, G.

    2013-07-26

    The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumor cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (γ-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (γ-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of γ-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between γ-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the data base that might promote pulsed sources for medical treatments of malignant growths.

  16. Imaging Features that Discriminate between Foci Induced by High-and Low-LET Radiation in Human Fibroblasts

    SciTech Connect

    Costes, Sylvain V.; Boissiere, Arnaud; Ravani, Shraddha; Romano,Raquel; Parvin, Bahram; Barcellos-Hoff, Mary Helen

    2006-10-08

    In this study, we investigated the formation ofradiation-induced foci in normal human fibroblasts exposed to X rays or130 keV/mum nitrogen ions using antibodies to phosphorylated proteinkinase ataxia telangiectasia mutated (ATMp) and histone H2AX(gamma-H2AX). High-content automatic image analysis was used to quantifythe immunofluorescence of radiation-induced foci. The size ofradiation-induced foci increased for both proteins over a 2-h periodafter nitrogen-ion irradiation, while the size of radiation-induced focidid not change after exposure to low-LET radiation. The number ofradiation-induced ATMp foci showed a more rapid rise and greaterfrequency after X-ray exposure and was resolved more rapidly such thatthe frequency of radiation-induced foci decreased by 90 percent comparedto 60 percent after exposure to high-LET radiation 2 h after 30 cGy. Incontrast, the kinetics of radiation-induced gamma-H2AX focus formationwas similar for high- and low-LET radiation in that it reached a plateauearly and remained constant for up to 2 h. High-resolution 3D images ofradiation-induced gamma-H2AX foci and dosimetry computation suggest thatmultiple double-strand breaks from nitrogen ions are encompassed withinlarge nuclear domains of 4.4 Mbp. Our work shows that the size andfrequency of radiation-induced foci vary as a function of radiationquality, dose, time and protein target. Thus, even though double-strandbreaks and radiation-induced foci are correlated, the dynamic nature ofboth contradicts their accepted equivalence for low doses of differentradiation qualities.

  17. Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Cariveau, Mickael J.

    2005-07-01

    Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

  18. Spatio-temporal radiation biology with conventionally or laser-accelerated particles for ELIMED

    NASA Astrophysics Data System (ADS)

    Ristić-Fira, A.; Bulat, T.; Keta, O.; Romano, F.; Cirrone, P.; Cuttone, G.; Petrović, I.

    2013-07-01

    The aim of this study is to investigate the behavior of radio-resistant human malignant cells, thus enabling better understanding of radiobiological effects of ions in such a case. Radiation sources such as accelerated continuous ion beams and laser technology-based ultra short radiation sources with energy of around 10 MeV will be used. The HTB140 melanoma cells are chosen since it has been shown that they represent the limit case of cellular radio-resistance among the studied tumor cell lines. These cells are particularly interesting as they provide data on the very edge of inactivation capacity of each beam line that is tested. After exposing the cell monolayers to continuous radiations of low (γ-rays) and high (protons) linear energy transfer, the kinetics of disappearance of the phosphorylated histone H2AX (γ-H2AX) foci per cell will be determined. The same procedure will be performed with the pulsed high dose rate protons. Detection and quantification of γ-H2AX foci will be performed by immunohistochemical 3D time-dependent imaging analyses using laser scanning confocal microscopy. Immunoblotting will enable the follow-up of the relation between γ-H2AX and cell cycle arrest via the p53/p21 pathway. In such a way the spatio-temporal changes on sub-cellular level will be visualized, quantified and compared. These results will show whether there is a difference in the effects on cells between continuous and pulsed irradiation mode. Therefore, they will contribute to the data base that might promote pulsed sources for medical treatments of malignant growths.

  19. Critical Function of γH2A in S-Phase

    PubMed Central

    Mejia-Ramirez, Eva; Limbo, Oliver; Langerak, Petra; Russell, Paul

    2015-01-01

    Abstract Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AXH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis. PMID:26368543

  20. Critical Function of γH2A in S-Phase.

    PubMed

    Mejia-Ramirez, Eva; Limbo, Oliver; Langerak, Petra; Russell, Paul

    2015-09-01

    Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AXH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis. PMID:26368543

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

    SciTech Connect

    Ganesan, Shanthi Nteeba, Jackson Keating, Aileen F.

    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.

  2. Large-scale analysis of phosphorylated proteins in maize leaf.

    PubMed

    Bi, Ying-Dong; Wang, Hong-Xia; Lu, Tian-Cong; Li, Xiao-Hui; Shen, Zhuo; Chen, Yi-Bo; Wang, Bai-Chen

    2011-02-01

    Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation. PMID:21053013

  3. Class I Histone Deacetylase Thd1p Promotes Global Chromatin Condensation in Tetrahymena thermophila▿

    PubMed Central

    Parker, Kathryn; Maxson, Julia; Mooney, Alissa; Wiley, Emily A.

    2007-01-01

    Class I histone deacetylases (HDACs) regulate DNA-templated processes such as transcription. They act both at specific loci and more generally across global chromatin, contributing to acetylation patterns that may underlie large-scale chromatin dynamics. Although hypoacetylation is correlated with highly condensed chromatin, little is known about the contribution of individual HDACs to chromatin condensation mechanisms. Using the ciliated protozoan Tetrahymena thermophila, we investigated the role of a specific class I HDAC, Τhd1p, in the reversible condensation of global chromatin. In this system, the normal physiological response to cell starvation includes the widespread condensation of the macronuclear chromatin and general repression of gene transcription. We show that the chromatin in Thd1p-deficient cells failed to condense during starvation. The condensation failure correlated with aberrant hyperphosphorylation of histone H1 and the overexpression of CDC2, encoding the major histone H1 kinase. Changes in the rate of acetate turnover on core histones and in the distribution of acetylated lysines 9 and 23/27 on histone H3 isoforms that were found to correlate with normal chromatin condensation were absent from Thd1p mutant cells. These results point to a role for a class I HDAC in the formation of reversible higher-order chromatin structures and global genome compaction through mechanisms involving the regulation of H1 phosphorylation and core histone acetylation/deacetylation kinetics. PMID:17715364

  4. Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method

    PubMed Central

    Maile, Tobias M.; Izrael-Tomasevic, Anita; Cheung, Tommy; Guler, Gulfem D.; Tindell, Charles; Masselot, Alexandre; Liang, Jun; Zhao, Feng; Trojer, Patrick; Classon, Marie; Arnott, David

    2015-01-01

    Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5. PMID:25680960

  5. Phosphorylation and RLK signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genomes encode hundreds of receptor-like kinases (RLKs) with an organization of functional domains similar to that of animal receptor kinases. Ligand-dependent phosphorylation has now been demonstrated for several plant RLKs and identification of specific phosphorylation sites followed by thei...

  6. Functional Role of G9a Histone Methyltransferase in Cancer

    PubMed Central

    Casciello, Francesco; Windloch, Karolina; Gannon, Frank; Lee, Jason S.

    2015-01-01

    Post-translational modifications of DNA and histones are epigenetic mechanisms, which affect the chromatin structure, ultimately leading to gene expression changes. A number of different epigenetic enzymes are actively involved in the addition or the removal of various covalent modifications, which include acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Deregulation of these processes is a hallmark of cancer. For instance, G9a, a histone methyltransferase responsible for histone H3 lysine 9 (H3K9) mono- and dimethylation, has been observed to be upregulated in different types of cancer and its overexpression has been associated with poor prognosis. Key roles played by these enzymes in various diseases have led to the hypothesis that these molecules represent valuable targets for future therapies. Several small molecule inhibitors have been developed to specifically block the epigenetic activity of these enzymes, representing promising therapeutic tools in the treatment of human malignancies, such as cancer. In this review, the role of one of these epigenetic enzymes, G9a, is discussed, focusing on its functional role in regulating gene expression as well as its implications in cancer initiation and progression. We also discuss important findings from recent studies using epigenetic inhibitors in cell systems in vitro as well as experimental tumor growth and metastasis assays in vivo. PMID:26441991

  7. Effect of protein kinase P on phosphorylations catalyzed by the epidermal growth factor.

    PubMed Central

    Abdel-Ghany, M; Kole, H K; Racker, E

    1987-01-01

    Protein kinase P (PK-P) activated by histones or certain other basic compounds has been purified previously from yeast [Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N. & Racker, E. (1987) Proc. Natl. Acad. Sci. USA 84, 925-929]. It is shown here that PK-P is present in solubilized membranes of A-431 carcinoma cells where it changes the epidermal growth factor (EGF) receptor kinase activity. Polylysine, a weak PK-P activator, inhibited the autophosphorylation of the EGF receptor both in the absence and presence of EGF. Increased PK-P activity induced by histone 1, a potent activator, gave rise to increased autophosphorylation of the EGF receptor as well as phosphorylation at tyrosine residues of numerous other endogenous membrane components. The stimulation by histone was particularly striking in the presence of EGF. A similar stimulation was achieved with polylysine and EGF on addition of yeast PK-P. However, addition of yeast PK-P in the presence of histone 1 markedly inhibited the EGF-stimulated phosphorylation of endogenous membrane proteins. We conclude from these results that the effect of PK-P on the EGF receptor takes place in three phases: at low levels PK-P inhibits the autophosphorylation, at intermediate levels it stimulates the autophosphorylation as well as the EGF-dependent phosphorylation of numerous other membrane proteins, and at high levels it inhibits the phosphorylation of these proteins. Images PMID:3501120

  8. Elevated histone expression promotes life span extension.

    PubMed

    Feser, Jason; Truong, David; Das, Chandrima; Carson, Joshua J; Kieft, Jeffrey; Harkness, Troy; Tyler, Jessica K

    2010-09-10

    Changes to the chromatin structure accompany aging, but the molecular mechanisms underlying aging and the accompanying changes to the chromatin are unclear. Here, we report a mechanism whereby altering chromatin structure regulates life span. We show that normal aging is accompanied by a profound loss of histone proteins from the genome. Indeed, yeast lacking the histone chaperone Asf1 or acetylation of histone H3 on lysine 56 are short lived, and this appears to be at least partly due to their having decreased histone levels. Conversely, increasing the histone supply by inactivation of the histone information regulator (Hir) complex or overexpression of histones dramatically extends life span via a pathway that is distinct from previously known pathways of life span extension. This study indicates that maintenance of the fundamental chromatin structure is critical for slowing down the aging process and reveals that increasing the histone supply extends life span. PMID:20832724

  9. Histone variants: emerging players in cancer biology

    PubMed Central

    Vardabasso, Chiara; Hasson, Dan; Ratnakumar, Kajan; Chung, Chi-Yeh; Duarte, Luis F.

    2014-01-01

    Histone variants are key players in shaping chromatin structure, and, thus, in regulating fundamental cellular processes such as chromosome segregation and gene expression. Emerging evidence points towards a role for histone variants in contributing to tumor progression, and, recently, the first cancer-associated mutation in a histone variant-encoding gene was reported. In addition, genetic alterations of the histone chaperones that specifically regulate chromatin incorporation of histone variants are rapidly being uncovered in numerous cancers. Collectively, these findings implicate histone variants as potential drivers of cancer initiation and/or progression, and, therefore, targeting histone deposition or the chromatin remodeling machinery may be of therapeutic value. Here, we review the mammalian histone variants of the H2A and H3 families in their respective cellular functions, and their involvement in tumor biology. PMID:23652611

  10. Inhibition by calmodulin of calcium/phospholipid-dependent protein phosphorylation.

    PubMed Central

    Albert, K A; Wu, W C; Nairn, A C; Greengard, P

    1984-01-01

    Calmodulin was previously found to inhibit the Ca2+/phospholipid-dependent phosphorylation of an endogenous substrate, called the 87-kilodalton protein, in a crude extract prepared from rat brain synaptosomal cytosol. We investigated the mechanism of this inhibition, using Ca2+/phospholipid-dependent protein kinase and the 87-kilodalton protein, both of which had been purified to homogeneity from bovine brain. Rabbit brain calmodulin and some other Ca2+-binding proteins inhibited the phosphorylation of the 87-kilodalton protein by this kinase in the purified system. Calmodulin also inhibited the Ca2+/phospholipid-dependent phosphorylation of H1 histone, synapsin I, and the delta subunit of the acetylcholine receptor, with use of purified components. These results suggest that calmodulin may be a physiological regulator of Ca2+/phospholipid-dependent protein kinase. Images PMID:6233611

  11. Diversity and Divergence of Dinoflagellate Histone Proteins

    PubMed Central

    Marinov, Georgi K.; Lynch, Michael

    2015-01-01

    Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed. PMID:26646152

  12. Beyond Histone and Deacetylase: An Overview of Cytoplasmic Histone Deacetylases and Their Nonhistone Substrates

    PubMed Central

    Yao, Ya-Li; Yang, Wen-Ming

    2011-01-01

    Acetylation of lysines is a prominent form of modification in mammalian proteins. Deacetylation of proteins is catalyzed by histone deacetylases, traditionally named after their role in histone deacetylation, transcriptional modulation, and epigenetic regulation. Despite the link between histone deacetylases and chromatin structure, some of the histone deacetylases reside in various compartments in the cytoplasm. Here, we review how these cytoplasmic histone deacetylases are regulated, the identification of nonhistone substrates, and the functional implications of their nondeacetylase enzymatic activities. PMID:21234400

  13. Crude phenolic extracts from extra virgin olive oil circumvent de novo breast cancer resistance to HER1/HER2-targeting drugs by inducing GADD45-sensed cellular stress, G2/M arrest and hyperacetylation of Histone H3.

    PubMed

    Oliveras-Ferraros, Cristina; Fernández-Arroyo, Salvador; Vazquez-Martin, Alejandro; Lozano-Sánchez, Jesús; Cufí, Sílvia; Joven, Jorge; Micol, Vicente; Fernández-Gutiérrez, Alberto; Segura-Carretero, Antonio; Menendez, Javier A

    2011-06-01

    Characterization of the molecular function of complex phenols naturally present in extra virgin olive oil (EVOO) against the HER2-gene amplified JIMT-1 cell line, a unique breast cancer model that inherently exhibits cross-resistance to multiple HER1/2-targeted drugs including trastuzumab, gefitinib, erlotinib and lapatinib, may underscore innovative cancer molecules with novel therapeutic targets because they should efficiently circumvent de novo resistance to HER1/2 inhibitors in order to elicit tumoricidal effects. We identified pivotal signaling pathways associated with the efficacy of crude phenolic extracts (PEs) obtained from 14 monovarietals of Spanish EVOOs. i) MTT-based cell viability and HPLC coupled to time-of-flight (TOF) mass spectrometry assays revealed that anti-cancer activity of EVOO PEs positively correlated with the phenolic index (i.e., total content of phenolics) and with a higher presence of the complex polyphenols secoiridoids instead of lignans. ii) Genome-wide analyses using 44 K Agilent's whole human arrays followed by Gene Set Enrichment Analysis (GSEA)-based screening of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database revealed a differential modulation of the JIMT-1 transcriptome at the level of the cell cycle and p53 pathways. EVOO PEs differentially impacted the expression status of stress-sensing, G2-M check-point-related GADD45 genes and of p53-related CDKN1A, CDKN1C and PMAIP-1 genes. iii) Cell cycle and fluorescence microscopy analyses confirmed that secoiridoid-rich EVOO PE inhibited mitosis to promote G2-M cell cycle arrest. This was accompanied with the appearance of diffuse, even DNA staining with γH2AX and pan-nuclear hyperacetylation of Histone H3 at Lysine 18. iv) Semi-quantitative Signaling Node Multi-Target ELISAs determined that secoiridoid-rich EVOO PE drastically activated the mitogen-activated protein kinases MEK1 and p38 MAPK, a GADD45-related kinase involved in Histone H3 acetylation

  14. Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest

    SciTech Connect

    Suzuki, Masatoshi; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami . E-mail: nabe@rri.kyoto-u.ac.jp

    2006-02-03

    Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and {gamma}-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation.

  15. Interstitial chromatin alteration causes persistent p53 activation involved in the radiation-induced senescence-like growth arrest.

    PubMed

    Suzuki, Masatoshi; Suzuki, Keiji; Kodama, Seiji; Watanabe, Masami

    2006-02-01

    Various stresses including ionizing radiation give normal human fibroblasts a phenotype of senescence-like growth arrest (SLGA), manifested by p53-dependent irreversible G1 arrest. To determine the mechanism of persistent activation of p53, we examined phosphorylated Ataxia telangiectasia mutated (ATM) and phosphorylated histone H2AX foci formation after X-irradiation. Although the multiple tiny foci, detected soon after (<30 min) irradiation, gradually disappeared, some of these foci changed to large foci and persisted for 5 days. Large foci containing phosphorylated ATM and gamma-H2AX co-localized and foci with p53 phosphorylated at serine 15 also showed the same distribution. Interestingly, the signals obtained by telomere fluorescence in situ hybridization (FISH) assay did not co-localize with 90% of the large foci. Our results indicate that chromatin alteration in interstitial chromosomal regions is the most likely cause of continuous activation of p53, which results in the induction of SLGA by ionizing radiation. PMID:16360120

  16. Epigenetic Modifications and Accumulation of DNA Double-Strand Breaks in Oral Lichen Planus Lesions Presenting Poor Response to Therapy.

    PubMed

    Dillenburg, Caroline S; Martins, Marco A T; Almeida, Luciana O; Meurer, Luise; Squarize, Cristiane H; Martins, Manoela D; Castilho, Rogerio M

    2015-07-01

    Epigenetics refers to changes in cell characteristics that occur independently of modifications to the deoxyribonucleic acid (DNA) sequence. Alterations mediated by epigenetic mechanisms are important factors in cancer progression. Although an exciting prospect, the identification of early epigenetic markers associated with clinical outcome in premalignant and malignant disorders remains elusive. We examined alterations in chromatin acetylation in oral lichen planus (OLP) with distinct clinical behavior and compared the alterations to the levels of DNA double-strand breaks (DSBs). We analyzed 42 OLP patients, who had different responses to therapy, for acetyl-histone H3 at lys9 (H3K9ac), which is associated with enhanced transcription and nuclear decondensation, and the presence of DSBs, as determined by accumulation of phosphorylated γH2AX foci. Patients with high levels of H3K9ac acetylation failed to respond to therapy or experienced disease recurrence shortly after therapy. Similar to H3K9ac, patients who responded poorly to therapy had increased accumulation of DNA DSB, indicating genomic instability. These findings suggest that histone modifications occur in OLP, and H3K9ac and γH2AX histones may serve as epigenetic markers for OLP recurrence. PMID:26222871

  17. Epigenetic Modifications and Accumulation of DNA Double-Strand Breaks in Oral Lichen Planus Lesions Presenting Poor Response to Therapy

    PubMed Central

    Dillenburg, Caroline S.; Martins, Marco A.T.; Almeida, Luciana O.; Meurer, Luise; Squarize, Cristiane H.; Martins, Manoela D.; Castilho, Rogerio M.

    2015-01-01

    Abstract Epigenetics refers to changes in cell characteristics that occur independently of modifications to the deoxyribonucleic acid (DNA) sequence. Alterations mediated by epigenetic mechanisms are important factors in cancer progression. Although an exciting prospect, the identification of early epigenetic markers associated with clinical outcome in premalignant and malignant disorders remains elusive. We examined alterations in chromatin acetylation in oral lichen planus (OLP) with distinct clinical behavior and compared the alterations to the levels of DNA double-strand breaks (DSBs). We analyzed 42 OLP patients, who had different responses to therapy, for acetyl-histone H3 at lys9 (H3K9ac), which is associated with enhanced transcription and nuclear decondensation, and the presence of DSBs, as determined by accumulation of phosphorylated γH2AX foci. Patients with high levels of H3K9ac acetylation failed to respond to therapy or experienced disease recurrence shortly after therapy. Similar to H3K9ac, patients who responded poorly to therapy had increased accumulation of DNA DSB, indicating genomic instability. These findings suggest that histone modifications occur in OLP, and H3K9ac and γH2AX histones may serve as epigenetic markers for OLP recurrence. PMID:26222871

  18. Selenite activates the ATM kinase-dependent DNA repair pathway in human osteosarcoma cells with mitochondrial dysfunction.

    PubMed

    Wojewoda, Marta; Walczak, Jarosław; Duszyński, Jerzy; Szczepanowska, Joanna

    2015-06-01

    Mitochondrial dysfunction and reactive oxygen species (ROS) induced oxidative damage are implicated in the pathogenesis of several human diseases. Based on our previous findings that ROS level was higher in human osteosarcoma cybrids--Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) and was reduced by selenite treatment, this study was designed to elucidate the effects of selenite administration on oxidative and nitrosative damage to lipids, proteins and DNA. Oxidative and nitrosative damage to lipids and proteins was not increased in NARP cybrids or mitochondrial DNA-lacking Rho0 cells (displaying mitochondrial dysfunction) when compared with control WT cells. However, we found the enhanced formation of DNA double-strand breaks based on the level of histone γH2AX (phosphorylated at Ser 139), which is known to be phosphorylated by ATM (Ataxia Telangiectasia Mutated) kinase in response to DNA damage. Selenite increased the activity of ATM kinase in NARP cybrids and Rho0 cells without concomitant increase in levels of histone γH2AX. Activation of the ATM kinase-dependent DNA repair pathway triggered by selenite could not be associated with enhanced DNA damage but might rather result from selenite-induced activation of ATM-dependent DNA repair mechanisms which could account for protective effects of this agent. PMID:25862479

  19. Antitumor activity of a small-molecule inhibitor of the histone kinase Haspin

    PubMed Central

    Huertas, D; Soler, M; Moreto, J; Villanueva, A; Martinez, A; Vidal, A; Charlton, M; Moffat, D; Patel, S; McDermott, J; Owen, J; Brotherton, D; Krige, D; Cuthill, S; Esteller, M

    2012-01-01

    The approval of histone deacetylase inhibitors for treatment of lymphoma subtypes has positioned histone modifications as potential targets for the development of new classes of anticancer drugs. Histones also undergo phosphorylation events, and Haspin is a protein kinase the only known target of which is phosphorylation of histone H3 at Thr3 residue (H3T3ph), which is necessary for mitosis progression. Mitotic kinases can be blocked by small drugs and several clinical trials are underway with these agents. As occurs with Aurora kinase inhibitors, Haspin might be an optimal candidate for the pharmacological development of these compounds. A high-throughput screening for Haspin inhibitors identified the CHR-6494 compound as being one promising such agent. We demonstrate that CHR-6494 reduces H3T3ph levels in a dose-dependent manner and causes a mitotic catastrophe characterized by metaphase misalignment, spindle abnormalities and centrosome amplification. From the cellular standpoint, the identified small-molecule Haspin inhibitor causes arrest in G2/M and subsequently apoptosis. Importantly, ex vivo assays also demonstrate its anti-angiogenetic features; in vivo, it shows antitumor potential in xenografted nude mice without any observed toxicity. Thus, CHR-6494 is a first-in-class Haspin inhibitor with a wide spectrum of anticancer effects that merits further preclinical research as a new member of the family of mitotic kinase inhibitors. PMID:21804608

  20. Mining Conditional Phosphorylation Motifs.

    PubMed

    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/. PMID:26356863

  1. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening

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

    SciTech Connect

    Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan; Kakadiya, Rajesh B.; Su, Tsann-Long; Yih, Ling-Huei

    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.

  3. Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells.

    PubMed

    Stuhldreier, Fabian; Kassel, Stefanie; Schumacher, Lena; Wesselborg, Sebastian; Proksch, Peter; Fritz, Gerhard

    2015-05-28

    We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10-20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AXH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy. PMID:25697484

  4. Folate deficiency affects histone methylation.

    PubMed

    Garcia, Benjamin A; Luka, Zigmund; Loukachevitch, Lioudmila V; Bhanu, Natarajan V; Wagner, Conrad

    2016-03-01

    Formaldehyde is extremely toxic reacting with proteins to crosslinks peptide chains. Formaldehyde is a metabolic product in many enzymatic reactions and the question of how these enzymes are protected from the formaldehyde that is generated has largely remained unanswered. Early experiments from our laboratory showed that two liver mitochondrial enzymes, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH) catalyze oxidative demethylation reactions (sarcosine is a common name for monomethylglycine). The enzymatic products of these enzymes were the demethylated substrates and formaldehyde, produced from the removed methyl group. Both DMGDH and SDH contain FAD and both have tightly bound tetrahydrofolate (THF), a folate coenzyme. THF binds reversibly with formaldehyde to form 5,10-methylene-THF. At that time we showed that purified DMGDH, with tightly bound THF, reacted with formaldehyde generated during the reaction to form 5,10-methylene-THF. This effectively scavenged the formaldehyde to protect the enzyme. Recently, post-translational modifications on histone tails have been shown to be responsible for epigenetic regulation of gene expression. One of these modifications is methylation of lysine residues. The first enzyme discovered to accomplish demethylation of these modified histones was histone lysine demethylase (LSD1). LSD1 specifically removes methyl groups from di- and mono-methylated lysines at position 4 of histone 3. This enzyme contained tightly bound FAD and the products of the reaction were the demethylated lysine residue and formaldehyde. The mechanism of LSD1 demethylation is analogous to the mechanism previously postulated for DMGDH, i.e. oxidation of the N-methyl bond to the methylene imine followed by hydrolysis to generate formaldehyde. This suggested that THF might also be involved in the LSD1 reaction to scavenge the formaldehyde produced. Our hypotheses are that THF is bound to native LSD1 by analogy to DMGDH and SDH and

  5. NUCLEOPHOSMIN/B23 NEGATIVELY REGULATES GCN5-DEPENDENT HISTONE ACETYLATION AND TRANSACTIVATION

    SciTech Connect

    Zou, Yonglong; Wu, Jun; Giannone, Richard J; Boucher, Lorrie; Du, Hansen; Huang, Ying; Johnson, Dabney K; Liu, Yie; Wang, Yisong

    2007-01-01

    Nucleophosmin/B23 is a multifunctional phosphoprotein that is overexpressed in cancer cells and has been shown to be involved in both positive and negative regulation of transcription. In this study, we first identified GCN5 acetyltransferase as a B23-interacting protein by mass spectrometry, which was then confirmed by in vivo co-immunoprecipitation. In vitro assay demonstrated that B23 bound the PCAF-N domain of GCN5 and inhibited GCN5-mediated acetylation of both free and mononucleosomal histones, probably through interfering with GCN5 and masking histones from being acetylated. Mitotic B23 exhibited higher inhibitory activity on GCN5-mediated histone acetylation than interphase B23. Immunodepletion experiments of mitotic extracts revealed that phosphorylation of B23 at Thr199 enhanced the inhibition of GCN5-mediated histone acetylation. Moreover, luciferase reporter and microarray analyses suggested that B23 attenuated GCN5-mediated transactivation in vivo. Taken together, our studies suggest a molecular mechanism of B23 in the mitotic inhibition of GCN5-mediated histone acetylation and transactivation.

  6. Mitotic Activation of a Novel Histone Deacetylase 3-Linker Histone H1.3 Protein Complex by Protein Kinase CK2*

    PubMed Central

    Patil, Hemangi; Wilks, Carrie; Gonzalez, Rhiannon W.; Dhanireddy, Sudheer; Conrad-Webb, Heather; Bergel, Michael

    2016-01-01

    Histone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis and the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II, or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (N-CoR) that accumulated in synchronized HeLa cells in late G2 phase and mitosis. Nonetheless, the deacetylation activity by HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated with H1.3 was highly phosphorylated on Ser-424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late G2 phase cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2) activated the HDAC3 in vitro. In vivo, CK2α and CK2α' double knockdown cells demonstrated a significant decrease in HDAC3 Ser-424 phosphorylation during mitosis. HDAC3 and H1.3 co-localized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially co-localized with chromatin during prophase and interphase. H1 has been reported previously to associate with microtubules and, therefore, could potentially function in targeting HDAC3 to the microtubules. We suggest that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin and regulation of polar microtubules dynamic instability. PMID:26663086

  7. Inhibition of Bcr serine kinase by tyrosine phosphorylation.

    PubMed Central

    Liu, J; Wu, Y; Ma, G Z; Lu, D; Haataja, L; Heisterkamp, N; Groffen, J; Arlinghaus, R B

    1996-01-01

    The first exon of the BCR gene encodes a new serine/threonine protein kinase. Abnormal fusion of the BCR and ABL genes, resulting from the formation of the Philadelphia chromosome (Ph), is the hallmark of Ph-positive leukemia. We have previously demonstrated that the Bcr protein is tyrosine phosphorylated within first-exon sequences by the Bcr-Abl oncoprotein. Here we report that in addition to tyrose 177 (Y-177), Y-360 and Y283 are phosphorylated in Bcr-Abl proteins in vitro. Moreover, Bcr tyrosine 360 is phosphorylated in vivo within both Bcr-Abl and Bcr. Bcr mutant Y177F had a greatly reduced ability to transphosphorylate casein and histone H1, whereas Bcr mutants Y177F and Y283F had wild-type activities. In contrast, the Y360F mutation had little effect on Bcr's autophosphorylation activity. Tyrosine-phosphorylated Bcr, phosphorylated in vitro by Bcr-Abl, was greatly inhibited in its serine/threonine kinase activity, impairing both auto- and transkinase activities of Bcr. Similarly, the isolation of Bcr from cells expressing Bcr-Abl under conditions that preserve phosphotyrosine residues also reduced Bcr's kinase activity. These results indicate that tyrosine 360 of Bcr is critical for the transphosphorylation activity of Bcr and that in Ph-positive leukemia, Bcr serine/threonine kinase activity is seriously impaired. PMID:8622703

  8. Release and activity of histone in diseases.

    PubMed

    Chen, R; Kang, R; Fan, X-G; Tang, D

    2014-01-01

    Histones and their post-translational modifications have key roles in chromatin remodeling and gene transcription. Besides intranuclear functions, histones act as damage-associated molecular pattern molecules when they are released into the extracellular space. Administration of exogenous histones to animals leads to systemic inflammatory and toxic responses through activating Toll-like receptors and inflammasome pathways. Anti-histone treatment (e.g., neutralizing antibodies, activated protein C, recombinant thrombomodulin, and heparin) protect mice against lethal endotoxemia, sepsis, ischemia/reperfusion injury, trauma, pancreatitis, peritonitis, stroke, coagulation, and thrombosis. In addition, elevated serum histone and nucleosome levels have been implicated in multiple pathophysiological processes and progression of diseases including autoimmune diseases, inflammatory diseases, and cancer. Therefore, extracellular histones could serve as biomarkers and novel therapeutic targets in human diseases. PMID:25118930

  9. Histone Acetylation in Fungal Pathogens of Plants

    PubMed Central

    Jeon, Junhyun; Kwon, Seomun; Lee, Yong-Hwan

    2014-01-01

    Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed. PMID:25288980

  10. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

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

    SciTech Connect

    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. - 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.

  12. Phosphorylation of epigenetic "readers, writers and erasers": Implications for developmental reprogramming and the epigenetic basis for health and disease.

    PubMed

    Treviño, Lindsey S; Wang, Quan; Walker, Cheryl L

    2015-07-01

    Epigenetic reprogramming that occurs during critical periods of development can increase the susceptibility to many diseases in adulthood. Programming of the epigenome during development occurs via the activity of a variety of epigenetic modifiers, including "readers, writers and erasers" of histone methyl marks. Posttranslational modification of these programmers can alter their activity, resulting in global or gene-specific changes in histone methylation and gene transcription. This review summarizes what is currently known about phosphorylation of histone methyltransferases ("writers"), demethylases ("erasers") and effector proteins ("readers) that program the epigenome, and the impact of this posttranslational modification on their activity. Understanding how the activity of these epigenetic programmers is perturbed by environmental exposures via changes in phosphorylation is key to understanding mechanisms of developmental reprogramming and the epigenetic basis of health and disease. PMID:25841987

  13. Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

    PubMed Central

    van Attikum, Haico; Fritsch, Olivier; Gasser, Susan M

    2007-01-01

    INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (γH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, γH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation. PMID:17762868

  14. The interplay of histone modifications – writers that read

    PubMed Central

    Zhang, Tianyi; Cooper, Sarah; Brockdorff, Neil

    2015-01-01

    Histones are subject to a vast array of posttranslational modifications including acetylation, methylation, phosphorylation, and ubiquitylation. The writers of these modifications play important roles in normal development and their mutation or misregulation is linked with both genetic disorders and various cancers. Readers of these marks contain protein domains that allow their recruitment to chromatin. Interestingly, writers often contain domains which can read chromatin marks, allowing the reinforcement of modifications through a positive feedback loop or inhibition of their activity by other modifications. We discuss how such positive reinforcement can result in chromatin states that are robust and can be epigenetically maintained through cell division. We describe the implications of these regulatory systems in relation to modifications including H3K4me3, H3K79me3, and H3K36me3 that are associated with active genes and H3K27me3 and H3K9me3 that have been linked to transcriptional repression. We also review the crosstalk between active and repressive modifications, illustrated by the interplay between the Polycomb and Trithorax histone-modifying proteins, and discuss how this may be important in defining gene expression states during development. PMID:26474904

  15. Influence of Arsenic on Global Levels of Histone Posttranslational Modifications: a Review of the Literature and Challenges in the Field.

    PubMed

    Howe, Caitlin G; Gamble, Mary V

    2016-09-01

    Arsenic is a human carcinogen and also increases the risk for non-cancer outcomes. Arsenic-induced epigenetic dysregulation may contribute to arsenic toxicity. Although there are several reviews on arsenic and epigenetics, these have largely focused on DNA methylation. Here, we review investigations of the effects of arsenic on global levels of histone posttranslational modifications (PTMs). Multiple studies have observed that arsenic induces higher levels of H3 lysine 9 dimethylation (H3K9me2) and also higher levels of H3 serine 10 phosphorylation (H3S10ph), which regulate chromosome segregation. In contrast, arsenic causes a global loss of H4K16ac, a histone PTM that is a hallmark of human cancers. Although the findings for other histone PTMs have not been entirely consistent across studies, we discuss biological factors which may contribute to these inconsistencies, including differences in the dose, duration, and type of arsenic species examined; the tissue or cell line evaluated; differences by sex; and exposure timing. We also discuss two important considerations for the measurement of histone PTMs: proteolytic cleavage of histones and arsenic-induced alterations in histone expression. PMID:27352015

  16. Mapping Post-translational Modifications of Histones H2A, H2B and H4 in Schizosaccharomyces pombe.

    PubMed

    Xiong, Lei; Wang, Yinsheng

    2011-03-30

    Core histones are known to carry a variety of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation and ubiquitination, which play important roles in the epigenetic control of gene expression. The nature and biological functions of these PTMs in histones from plants, animals and budding yeast have been extensively investigated. In contrast, the corresponding studies for fission yeast were mainly focused on histone H3. In the present study, we applied LC-nano-ESI-MS/MS, coupled with multiple protease digestion, to identify PTMs in histones H2A, H2B and H4 from Schizosaccharomyces pombe (S. pombe), the typical model organism of fission yeast. Various protease digestions provided high sequence coverage for PTM mapping, and accurate mass measurement of fragment ions allowed for unambiguous differentiation of acetylation from tri-methylation. Many modification sites conserved in other organisms were identified in S. pombe. In addition, some unique modification sites, including N-terminal acetylation in H2A and H2B as well as K123 acetylation in H2A.β, were observed. Our results provide a comprehensive picture of the PTMs of histones H2A, H2B and H4 in S. pombe, which serves as a foundation for future investigations on the regulation and functions of histone modifications in this important model organism. PMID:21516229

  17. Histone exchange and histone modifications during transcription and aging☆

    PubMed Central

    Das, Chandrima; Tyler, Jessica K.

    2014-01-01

    The organization of the eukaryotic genome into chromatin enables DNA to fit inside the nucleus while also regulating the access of proteins to the DNA to facilitate genomic functions such as transcription, replication and repair. The basic repeating unit of chromatin is the nucleosome, which includes 147 bp of DNA wrapped 1.65 times around an octamer of core histone proteins comprising two molecules each of H2A, H2B, H3 and H4 [1]. Each nucleosome is a highly stable unit, being maintained by over 120 direct protein–DNA interactions and several hundred water mediated ones [1]. Accordingly, there is considerable interest in understanding how processive enzymes such as RNA polymerases manage to pass along the coding regions of our genes that are tightly packaged into arrays of nucleosomes. Here we present the current mechanistic understanding of this process and the evidence for profound changes in chromatin dynamics during aging. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly. PMID:24459735

  18. Investigating Histone Acetylation Stoichiometry and Turnover Rate.

    PubMed

    Fan, J; Baeza, J; Denu, J M

    2016-01-01

    Histone acetylation is a dynamic epigenetic modification that functions in the regulation of DNA-templated reactions, such as transcription. This lysine modification is reversibly controlled by histone (lysine) acetyltransferases and deacetylases. Here, we present methods employing isotopic labeling and mass spectrometry (MS) to comprehensively investigate histone acetylation dynamics. Turnover rates of histone acetylation are determined by measuring the kinetics of labeling from (13)C-labeled precursors of acetyl-CoA, which incorporates (13)C-carbon onto histones via the acetyltransferase reaction. Overall histone acetylation states are assessed from complete protease digestion to single amino acids, which is followed by MS analysis. Determination of site-specific acetylation stoichiometry is achieved by chemically acetylating endogenous histones with isotopic acetic anhydride, followed by trypsin digestion and LC-MS analysis. Combining metabolic labeling with stoichiometric analysis permits determination of both acetylation level and acetylation dynamics. When comparing genetic, diet, or environmental perturbations, these methods permit both a global and site-specific evaluation of how histone acetylation is dynamically regulated. PMID:27423860

  19. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  20. Acetaldehyde alters MAP kinase signalling and epigenetic histone modifications in hepatocytes.

    PubMed

    Shukla, Shivendra D; Lee, Youn Ju; Park, Pil-hoon; Aroor, Annayya R

    2007-01-01

    Although both oxidative and non-oxidative metabolites of ethanol are involved in generating ethanol matabolic stress (Emess), the oxidative metabolite acetaldehyde plays a critical role in the cellular actions of ethanol. We have investigated the effects of acetaldehyde on p42/44 MAP kinase, p46/p54 c-jun N-terminal kinase (JNK1/JNK2) and p38 MAP kinase in hepatocytes. Acetaldehyde caused temporal activation of p42/44 MAPK followed by JNK, but the activation of the p42/44 MAPK was not a prerequisite for the JNK activation. Activation ofJNK1 by acetaldehyde was greater than JNK2. Ethanol and acetaldehyde activatedJNK have opposing roles; ethanol-induced JNK activation increased apoptosis whereas that by acetaldehyde decreased apoptosis. Acetaldehyde also caused histone H3 acetylation at Lys9 and phosphorylation of histone H3 at Serl0 and 28, the latter being dependent on p38 MAP kinase. Phosphorylation at Ser28 was higher than at Serl0. Thus acetaldehyde distinctively alters MAP kinase signalling and histone modifications, processes involved in transcriptional activation. PMID:17590997

  1. The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation

    PubMed Central

    Schaft, Daniel; Roguev, Assen; Kotovic, Kimberly M.; Shevchenko, Anna; Sarov, Mihail; Shevchenko, Andrej; Neugebauer, Karla M.; Francis Stewart, A.

    2003-01-01

    Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor. Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation. First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2. Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II. Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3. Fourth, in the absence of ssn3, mRNA production from gal1 required SET2. Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription. Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions. Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation. PMID:12736296

  2. The histone 3 lysine 36 methyltransferase, SET2, is involved in transcriptional elongation.

    PubMed

    Schaft, Daniel; Roguev, Assen; Kotovic, Kimberly M; Shevchenko, Anna; Sarov, Mihail; Shevchenko, Andrej; Neugebauer, Karla M; Stewart, A Francis

    2003-05-15

    Existing evidence indicates that SET2, the histone 3 lysine 36 methyltransferase of Saccharomyces cerevisiae, is a transcriptional repressor. Here we show by five main lines of evidence that SET2 is involved in transcriptional elongation. First, most, if not all, subunits of the RNAP II holoenzyme co-purify with SET2. Second, all of the co-purifying RNAP II subunit, RPO21, was phosphorylated at serines 5 and 2 of the C-terminal domain (CTD) tail, indicating that the SET2 association is specific to either the elongating or SSN3 repressed forms (or both) of RNAP II. Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3. Fourth, in the absence of ssn3, mRNA production from gal1 required SET2. Fifth, SET2 was detected on gal1 by in vivo crosslinking after, but not before, the induction of transcription. Similarly, SET2 physically associated with the transcribed region of pdr5 but was not detected on gal1 or pdr5 promoter regions. Since SET2 is also a histone methyltransferase, these results suggest a role for histone 3 lysine 36 methylation in transcriptional elongation. PMID:12736296

  3. Regulation of cilia assembly, disassembly, and length by protein phosphorylation.

    PubMed

    Cao, Muqing; Li, Guihua; Pan, Junmin

    2009-01-01

    The exact mechanism by which cells are able to assemble, regulate, and disassemble cilia or flagella is not yet completely understood. Recent studies in several model systems, including Chlamydomonas, Tetrahymena, Leishmania, Caenorhabditis elegans, and mammals, provide increasing biochemical and genetic evidence that phosphorylation of multiple protein kinases plays a key role in cilia assembly, disassembly, and length regulation. Members of several protein kinase families--including aurora kinases, never in mitosis A (NIMA)-related protein kinases, mitogen-activated protein (MAP) kinases, and a novel cyclin-dependent protein kinase--are involved in the ciliary regulation process. Among the newly identified protein kinase substrates are Chlamydomonas kinesin-13 (CrKinesin13), a microtubule depolymerizer, and histone deacetylase 6 (HDAC6), a microtubule deacetylase. Chlamydomonas aurora/Ipl1p-like protein kinase (CALK) and CrKinesin13 are two proteins that undergo phosphorylation changes correlated with flagellar assembly or disassembly. CALK becomes phosphorylated when flagella are lost, whereas CrKinesin13 is phosphorylated when new flagella are assembled. Conversely, suppressing CrKinesin13 expression results in cells with shorter flagella. PMID:20362099

  4. AFM of self-assembled lambda DNA-histone networks.

    PubMed

    Liu, YuYing; Guthold, Martin; Snyder, Matthew J; Lu, HongFeng

    2015-10-01

    Atomic force microscopy (AFM) was used to investigate the self-assembly behavior of λ-DNA and histones at varying histone:DNA ratios. Without histones and at the lowest histone:DNA ratio (less than one histone per 1000 base pairs of DNA), the DNA appeared as individual (uncomplexed), double-stranded DNA molecules. At increasing histone concentrations (one histone per 500, 250 and 167 base pairs of DNA), the DNA molecules started to form extensive polygonal networks of mostly pentagons and hexagons. The observed networks might be one of the naturally occurring, stable DNA-histone structures. The condensing effects of the divalent cations Mg(2+) and Ca(2+) on the DNA-histone complexes were also investigated. The networks persisted at high Mg(2+) concentration (20mM) and the highest histone concentration. At high Ca(2+) concentration and the highest histone concentration, the polygonal network disappeared and, instead, individual, tightly condensed aggregates were formed. PMID:26141439

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

    PubMed

    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. PMID:25818601

  6. Hydroxyurea-induced replication stress causes poly(ADP-ribose) polymerase-2 accumulation and changes its intranuclear location in root meristems of Vicia faba.

    PubMed

    Rybaczek, Dorota

    2016-07-01

    Replication stress induced by 24 and 48h exposure to 2.5mM hydroxyurea (HU) increased the activity of poly(ADP-ribose) polymerase-2 (PARP-2; EC 2.4.2.30) in root meristem cells of Vicia faba. An increase in the number of PARP-2 foci was accompanied by their delocalization from peripheral areas to the interior of the nucleus. Our results indicate that the increase in PARP-2 was connected with an increase in S139-phosphorylated H2AX histones. The findings suggest the possible role of PARP-2 in replication stress. We also confirm that the intranuclear location of PARP-2 depends on the duration of HU-induced replication stress, confirming the role of PARP-2 as an indicator of stress intensity. Finally, we conclude that the more intense the HU-mediated replication stress, the greater the probability of PARP-2 activation or H2AXS139 phosphorylation, but also the greater the chance of increasing the efficiency of repair processes and a return to normal cell cycle progression. PMID:27155387

  7. Chk1 phosphorylated at serine345 is a predictor of early local recurrence and radio-resistance in breast cancer.

    PubMed

    Alsubhi, Nouf; Middleton, Fiona; Abdel-Fatah, Tarek M A; Stephens, Peter; Doherty, Rachel; Arora, Arvind; Moseley, Paul M; Chan, Stephen Y T; Aleskandarany, Mohammed A; Green, Andrew R; Rakha, Emad A; Ellis, Ian O; Martin, Stewart G; Curtin, Nicola J; Madhusudan, Srinivasan

    2016-02-01

    Radiation-induced DNA damage activates the DNA damage response (DDR). DDR up-regulation may predict radio-resistance and increase the risk of early local recurrence despite radiotherapy in early stage breast cancers. In 1755 early stage breast cancers, DDR signalling [ATM, ATR, total Ckh1, Chk1 phosphorylated at serine(345) (pChk1), Chk2, p53], base excision repair [PARP1, POLβ, XRCC1, FEN1, SMUG1], non-homologous end joining (Ku70/Ku80, DNA-PKcs) and homologous recombination [RAD51, BRCA1, γH2AX, BLM, WRN, RECQL5, PTEN] protein expression was correlated to time to early local recurrence. Pre-clinically, radio-sensitization by inhibition of Chk1 activation by ATR inhibitor (VE-821) and inhibition of Chk1 (V158411) were investigated in MDA-MB-231 (p53 mutant) and MCF-7 (p53 wild-type) breast cancer cells. In the whole cohort, 208/1755 patients (11.9%) developed local recurrence of which 126 (61%) developed local recurrence within 5 years of initiation of primary therapy. Of the 20 markers tested, only pChk1 and p53 significantly associated with early local recurrence (p value = 0.015 and 0.010, respectively). When analysed together, high cytoplasmic pChk1-nuclear pChk1 (p = 0.039), high cytoplasmic pChk1-p53 (p = 0.004) and high nuclear pChk1-p53 (p = 0.029) co-expression remain significantly linked to early local recurrence. In multivariate analysis, cytoplasmic pChk1 level independently predicted early local recurrence (p = 0.025). In patients who received adjuvant local radiotherapy (n = 949), p53 (p = 0.014) and high cytoplasmic pChk1-p53 (p = 0.017) remain associated with early local recurrence. Pre-clinically, radio-sensitisation by VE-821 or V158411 was observed in both MCF-7 and MDA-MB-231 cells and was more pronounced in MCF-7 cells. We conclude that pChk1 is a predictive biomarker of radiotherapy resistance and early local recurrence. PMID:26459098

  8. Aluminum interaction with human brain tau protein phosphorylation by various kinases

    SciTech Connect

    El-Sebae, A.H.; Zeid, M.M.A.; Saleh, M.A. . Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. ); Blancato, J. . Environmental Monitoring Systems Lab.)

    1993-01-01

    Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 uM range activated in-vitro [[gamma]-[sup 32]P] ATP phosphorylation of the brain (tau) [Tau] protein in both normal human or E. coli expressed [Tau] forms; in the presence of the kinases P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 uM to 500 uM range induced non-enzymatic phosphorylation of [Tau] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GTP, and [alpha]-GTP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Tau] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GTP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Tau] by Al[sup 3+] was accompanied by molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the longterm neurological effect of Al[sup 3+] in human brain leading to the formation of the neurofibrillary tangles related to Alzeheimer's disease.

  9. Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression.

    PubMed

    Borgal, Lori; Rinschen, Markus M; Dafinger, Claudia; Liebrecht, Valérie I; Abken, Hinrich; Benzing, Thomas; Schermer, Bernhard

    2016-04-17

    The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair. PMID:26919559

  10. HistoneDB 2.0: a histone database with variants--an integrated resource to explore histones and their variants.

    PubMed

    Draizen, Eli J; Shaytan, Alexey K; Mariño-Ramírez, Leonardo; Talbert, Paul B; Landsman, David; Panchenko, Anna R

    2016-01-01

    Compaction of DNA into chromatin is a characteristic feature of eukaryotic organisms. The core (H2A, H2B, H3, H4) and linker (H1) histone proteins are responsible for this compaction through the formation of nucleosomes and higher order chromatin aggregates. Moreover, histones are intricately involved in chromatin functioning and provide a means for genome dynamic regulation through specific histone variants and histone post-translational modifications. 'HistoneDB 2.0--with variants' is a comprehensive database of histone protein sequences, classified by histone types and variants. All entries in the database are supplemented by rich sequence and structural annotations with many interactive tools to explore and compare sequences of different variants from various organisms. The core of the database is a manually curated set of histone sequences grouped into 30 different variant subsets with variant-specific annotations. The curated set is supplemented by an automatically extracted set of histone sequences from the non-redundant protein database using algorithms trained on the curated set. The interactive web site supports various searching strategies in both datasets: browsing of phylogenetic trees; on-demand generation of multiple sequence alignments with feature annotations; classification of histone-like sequences and browsing of the taxonomic diversity for every histone variant. HistoneDB 2.0 is a resource for the interactive comparative analysis of histone protein sequences and their implications for chromatin function. Database URL: http://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0. PMID:26989147

  11. Synaptic plasticity and phosphorylation

    PubMed Central

    Lee, Hey-Kyoung

    2009-01-01

    A number of neuronal functions, including synaptic plasticity, depend on proper regulation of synaptic proteins, many of which can be rapidly regulated by phosphorylation. Neuronal activity controls the function of these synaptic proteins by exquisitely regulating the balance of various protein kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms underscores important roles that these synaptic phosphoproteins play in regulating both pre- and post-synaptic functions. This review will focus on key postsynaptic phosphoproteins that have been implicated to play a role in synaptic plasticity. PMID:16904750

  12. Epigenetic disruptions of histone signatures for the trophectoderm and inner cell mass in mouse parthenogenetic embryos.

    PubMed

    Chen, Yi-Hui; Yu, John

    2015-03-01

    Epigenetic asymmetry has been shown to be associated with the first lineage allocation event in preimplantation development, that is, the formation of the trophectoderm (TE) and inner cell mass (ICM) lineages in the blastocyst. Since parthenogenesis causes aberrant segregation between the TE and ICM lineages, we examined several development-associated histone modifications in parthenotes, including those involved in (i) transcriptional activation [acetylated histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac), trimethylated histone H3 lysine 4 (H3K4Me3), and dimethylated histone H3 arginine 26 (H3R26Me2)] and (ii) transcriptional repression [trimethylated histone H3 lysine 9 (H3K9Me3) and lysine 27 (H3K27Me3), and mono-ubiquitinated histone H2A lysine 119 (H2AK119u1)]. Here, we report that in parthenotes, H3R26Me2 expression decreased from the morula stage, while expression patterns and levels of H3K9Ac, H3K27Me3, and H2AK119u1 were unchanged until the blastocyst stage; whereas H3K14Ac, H3K4Me3, and H3K9Me3 showed normal patterns and levels of expressions. Relative to the decrease of H3K9Ac in the ICM and increase in the TE of parthenotes, we detected reduced expression of TAT-interactive protein 60 acetyltransferase and histone deacetylase 1 deacetylase in the ICM and TE of parthenotes, respectively. Relative to the decrease of H3R26Me2, we also observed decreased expression of coactivator-associated arginine methyltransferase 1 methyltransferase and increased expression of the Wnt effector transcription factor 7L2 and miR-181c microRNA in parthenotes. Furthermore, relative to the decrease in H3K27Me3 and H2AK119u1, we found increased phosphorylation of Akt1 and enhancer of zeste homolog 2 in parthenogenetic TE. Therefore, our findings that histone signatures are impaired in parthenotes provide a mechanistic explanation for aberrant lineage segregation and TE defects. PMID:25315067

  13. Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode.

    PubMed

    Prihandoko, Rudi; Bradley, Sophie J; Tobin, Andrew B; Butcher, Adrian J

    2015-01-01

    G protein-coupled receptors (GPCRs) are rapidly phosphorylated following agonist occupation in a process that mediates receptor uncoupling from its cognate G protein, a process referred to as desensitization. In addition, this process provides a mechanism by which receptors can engage with arrestin adaptor molecules and couple to downstream signaling pathways. The importance of this regulatory process has been highlighted recently by the understanding that ligands can direct receptor signaling along one pathway in preference to another, the phenomenon of signaling bias that is partly mediated by the phosphorylation status or phosphorylation barcode of the receptor. Methods to determine the phosphorylation status of a GPCR in vitro and in vivo are necessary to understand not only the physiological mechanisms involved in GPCR signaling, but also to fully examine the signaling properties of GPCR ligands. This unit describes detailed methods for determining the overall phosphorylation pattern on a receptor (the phosphorylation barcode), as well as mass spectrometry approaches that can define the precise sites that become phosphorylated. These techniques, coupled with the generation and characterization of receptor phosphorylation-specific antibodies, provide a full palate of techniques necessary to determine the phosphorylation status of any given GPCR subtype. PMID:26344213

  14. UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation.

    PubMed

    Matsunuma, Ryoichi; Niida, Hiroyuki; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

    2016-02-01

    Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4(DDB2). Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

  15. Melatonin protects the integrity of granulosa cells by reducing oxidative stress in nuclei, mitochondria, and plasma membranes in mice

    PubMed Central

    TANABE, Manabu; TAMURA, Hiroshi; TAKETANI, Toshiaki; OKADA, Maki; LEE, Lifa; TAMURA, Isao; MAEKAWA, Ryo; ASADA, Hiromi; YAMAGATA, Yoshiaki; SUGINO, Norihiro

    2014-01-01

    Melatonin protects luteinized granulosa cells (GCs) from oxidative stress in the follicle during ovulation. However, it is unclear in which cellular components (e.g., nuclei, mitochondria, or plasma membranes) melatonin works as an antioxidant. GCs from immature (3 wks) ICR mice were incubated with hydrogen peroxide (H2O2; 0.01, 0.1, 1, 10 mM) in the presence or absence of melatonin (100 μg/ml) for 2 h. DNA damage was assessed by fluorescence-based immunocytochemistry using specific antibodies for 8-hydroxydeoxyguanosine (8-OHdG), an indicator of oxidative guanine base damage in DNA, and for histone H2AX phosphorylationH2AX), a marker of double-strand breaks of DNA. Mitochondrial function was assessed by the fluorescence intensity of MitoTracker Red probes, which diffuse across the membrane and accumulate in mitochondria with active membrane potentials. Lipid peroxidation of plasma membranes was analyzed by measuring hexanoyl-lysine (HEL), a oxidative stress marker for lipid peroxidation. Apoptosis of GCs was assessed by nuclear fragmentation using DAPI staining, and apoptotic activities were evaluated by caspase-3/7 activities. H2O2 treatment significantly increased the fluorescence intensities of 8-OHdG and γH2AX, reduced the intensity of MitoTracker Red in the mitochondria, increased HEL concentrations in GCs, and enhanced the number of apoptotic cells and caspase-3/7 activities. All these changes were significantly decreased by melatonin treatment. Melatonin reduced oxidative stress-induced DNA damage, mitochondrial dysfunction, lipid peroxidation, and apoptosis in GCs, suggesting that melatonin protects GCs by reducing oxidative stress of cellular components including nuclei, mitochondria, and plasma membranes. Melatonin helps to maintain the integrity of GCs as an antioxidant in the preovulatory follicle. PMID:25366368

  16. Elevated Expression of Hepatoma Up-Regulated Protein Inhibits γ-Irradiation-Induced Apoptosis of Prostate Cancer Cells.

    PubMed

    Hassan, Mohamed; El Khattouti, Abdelouahid; Ejaeidi, Ahmed; Ma, Tangeng; Day, William A; Espinoza, Ingrid; Vijayakumar, Srinivasan; Gomez, Christian R

    2016-06-01

    Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ-irradiation-induced apoptosis. γ-irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ-irradiation-induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ-irradiation-induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP. J. Cell. Biochem. 117: 1308-1318, 2016. © 2015 Wiley Periodicals, Inc. PMID:26505164

  17. Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation.

    PubMed

    Flegal, Matthew; Blimkie, Melinda S; Wyatt, Heather; Bugden, Michelle; Surette, Joel; Klokov, Dmitry

    2015-01-01

    Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of γ- and β-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called γH2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled γH2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors. PMID:26168333

  18. Radiation Response of Cancer Stem-Like Cells From Established Human Cell Lines After Sorting for Surface Markers

    SciTech Connect

    Al-Assar, Osama; Muschel, Ruth J.; Mantoni, Tine S.; McKenna, W. Gillies; Brunner, Thomas B.

    2009-11-15

    Purpose: A subpopulation of cancer stem-like cells (CSLC) is hypothesized to exist in different cancer cell lines and to mediate radioresistance in solid tumors. Methods and Materials: Cells were stained for CSLC markers and sorted (fluorescence-activated cell sorter/magnetic beads) to compare foci and radiosensitivity of phosphorylated histone H2AX at Ser 139 (gamma-H2AX) in sorted vs. unsorted populations in eight cell lines from different organs. CSLC properties were examined using anchorage-independent growth and levels of activated Notch1. Validation consisted of testing tumorigenicity and postirradiation enrichment of CSLC in xenograft tumors. Results: The quantity of CSLC was generally in good agreement with primary tumors. CSLC from MDA-MB-231 (breast) and Panc-1 and PSN-1 (both pancreatic) cells had fewer residual gamma-H2AX foci than unsorted cells, pointing to radioresistance of CSLC. However, only MDA-MB-231 CSLC were more radioresistant than unsorted cells. Furthermore, MDA-MB-231 CSLC showed enhanced anchorage-independent growth and overexpression of activated Notch1 protein. The expression of cancer stem cell surface markers in the MDA-MB-231 xenograft model was increased after exposure to fractionated radiation. In contrast to PSN-1 cells, a growth advantage for MDA-MB-231 CSLC xenograft tumors was found compared to tumors arising from unsorted cells. Conclusions: CSLC subpopulations showed no general radioresistant phenotype, despite the quantities of CSLC subpopulations shown to correspond relatively well in other reports. Likewise, CSLC characteristics were found in some but not all of the tested cell lines. The reported problems in testing for CSLC in cell lines may be overcome by additional techniques, beyond sorting for markers.

  19. Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

    PubMed Central

    Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

    2008-01-01

    Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AXH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

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

    PubMed Central

    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. PMID:23550213

  1. BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1

    PubMed Central

    Ge, Chunmin; Che, Lixiao; Ren, Jinyu; Pandita, Raj K.; Lu, Jing; Li, Kaiyi; Pandita, Tej K.; Du, Chunying

    2015-01-01

    The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI–SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1–SWI–SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability. PMID:25733871

  2. Microbial Pathogens Trigger Host DNA Double-Strand Breaks Whose Abundance Is Reduced by Plant Defense Responses

    PubMed Central

    Song, Junqi; Bent, Andrew F.

    2014-01-01

    Immune responses and DNA damage repair are two fundamental processes that have been characterized extensively, but the links between them remain largely unknown. We report that multiple bacterial, fungal and oomycete plant pathogen species induce double-strand breaks (DSBs) in host plant DNA. DNA damage detected by histone γ-H2AX abundance or DNA comet assays arose hours before the disease-associated necrosis caused by virulent Pseudomonas syringae pv. tomato. Necrosis-inducing paraquat did not cause detectable DSBs at similar stages after application. Non-pathogenic E. coli and Pseudomonas fluorescens bacteria also did not induce DSBs. Elevation of reactive oxygen species (ROS) is common during plant immune responses, ROS are known DNA damaging agents, and the infection-induced host ROS burst has been implicated as a cause of host DNA damage in animal studies. However, we found that DSB formation in Arabidopsis in response to P. syringae infection still occurs in the absence of the infection-associated oxidative burst mediated by AtrbohD and AtrbohF. Plant MAMP receptor stimulation or application of defense-activating salicylic acid or jasmonic acid failed to induce a detectable level of DSBs in the absence of introduced pathogens, further suggesting that pathogen activities beyond host defense activation cause infection-induced DNA damage. The abundance of infection-induced DSBs was reduced by salicylic acid and NPR1-mediated defenses, and by certain R gene-mediated defenses. Infection-induced formation of γ-H2AX still occurred in Arabidopsis atr/atm double mutants, suggesting the presence of an alternative mediator of pathogen-induced H2AX phosphorylation. In summary, pathogenic microorganisms can induce plant DNA damage. Plant defense mechanisms help to suppress rather than promote this damage, thereby contributing to the maintenance of genome integrity in somatic tissues. PMID:24699527

  3. Oxidative Glial Cell Damage Associated with White Matter Lesions in the Aging Human Brain

    PubMed Central

    Al-Mashhadi, Sufana; Simpson, Julie E.; Heath, Paul R.; Dickman, Mark; Forster, Gillian; Matthews, Fiona E.; Brayne, Carol; Ince, Paul G.; Wharton, Stephen B.

    2016-01-01

    White matter lesions (WML) are common in brain aging and are associated with dementia. We aimed to investigate whether oxidative DNA damage and occur in WML and in apparently normal white matter in cases with lesions. Tissue from WML and control white matter from brains with lesions (controls lesional) and without lesions (controls non-lesional) were obtained, using post-mortem magnetic resonance imaging-guided sampling, from the Medical Research Council Cognitive Function and Ageing Study. Oxidative damage was assessed by immunohistochemistry to 8-hydroxy-2′-deoxoguanosine (8-OHdG) and Western blotting for malondialdehyde. DNA response was assessed by phosphorylated histone H2AXH2AX), p53, senescence markers and by quantitative Reverse transcription polymerase chain reaction (RT-PCR) panel for candidate DNA damage-associated genes. 8-OHdG was expressed in glia and endothelium, with increased expression in both WML and controls lesional compared with controls non-lesional (P < 0.001). γH2Ax showed a similar, although attenuated difference among groups (P = 0.03). Expression of senescence-associated β-galactosidase and p16 suggested induction of senescence mechanisms in glia. Oxidative DNA damage and a DNA damage response are features of WML pathogenesis and suggest candidate mechanisms for glial dysfunction. Their expression in apparently normal white matter in cases with WML suggests that white matter dysfunction is not restricted to lesions. The role of this field-effect lesion pathogenesis and cognitive impairment are areas to be defined. PMID:25311358

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the 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 the effects of spaceflight on the cellular response 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 induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the 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 Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

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

    NASA Technical Reports Server (NTRS)

    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

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

    NASA Technical Reports Server (NTRS)

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

    2016-01-01

    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 µg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by foci and pattern counting of phosphorylated histone protein H2AX (?-H2AX). The cells on the ISS showed modestly increased average foci 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 profile 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 ?-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect

  7. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair.

    PubMed

    Galanty, Yaron; Belotserkovskaya, Rimma; Coates, Julia; Jackson, Stephen P

    2012-06-01

    Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylationH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems. PMID:22661229

  8. Uncouplers of oxidative phosphorylation.

    PubMed

    Terada, H

    1990-07-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  9. Uncouplers of oxidative phosphorylation.

    PubMed Central

    Terada, H

    1990-01-01

    Uncouplers of oxidative phosphorylation in mitochondria inhibit the coupling between the electron transport and phosphorylation reactions and thus inhibit ATP synthesis without affecting the respiratory chain and ATP synthase (H(+)-ATPase). Miscellaneous compounds are known to be uncouplers, but weakly acidic uncouplers are representative because they show very potent activities. The most potent uncouplers discovered so far are the hindered phenol SF 6847, and hydrophobic salicylanilide S-13, which are active in vitro at concentrations in the 10 nM range. For induction of uncoupling, an acid dissociable group, bulky hydrophobic moiety and strong electron-withdrawing group are required. Weakly acidic uncouplers are considered to produce uncoupling by their protonophoric action in the H(+)-impermeable mitochondrial membrane. For exerting these effects, the stability of the respective uncoupler anions in the hydrophobic membrane is very important. High stability is achieved by delocalization of the polar ionic charge through uncoupler (chemical)-specific mechanisms. Such an action of weakly acidic uncouplers is characteristic of the highly efficient membrane targeting action of a nonsite-specific type of bioactive compound. PMID:2176586

  10. Nucleosome Dancing at the Tempo of Histone Tail Acetylation

    PubMed Central

    Galvani, Angélique; Thiriet, Christophe

    2015-01-01

    The impact of histone acetylation on transcription was revealed over 50 years ago by Allfrey and colleagues. However, it took decades for an understanding of the fine mechanism by which this posttranslational modification affects chromatin structure and promotes transcription. Here, we review breakthroughs linking histone tail acetylation, histone dynamics, and transcription. We also discuss the histone exchange during transcription and highlight the important function of a pool of non-chromatinized histones in chromatin dynamics. PMID:26184324

  11. Quantitative Proteomic Approaches to Studying Histone Modifications

    PubMed Central

    Zee, Barry M; Young, Nicolas L; Garcia, Benjamin A

    2011-01-01

    Histone post-translational modifications (PTMs) positively and negatively regulate gene expression, and are consequently a vital influence on the genomic profile of all eukaryotic species. The study of histone PTMs using classical methods in molecular biology, such as immunofluorescence and Western blotting, is challenging given the technical issues of the approaches, and chemical diversity and combinatorial patterns of the modifications. In light of these many technical limitations, mass spectrometry (MS) is emerging as the most unbiased and rigorous experimental platform to identify and quantify histone PTMs in a high-throughput manner. This review covers the latest developments in mass spectrometry for the analysis of histone PTMs, with the hope of inspiring the continued integration of proteomic, genomic and epigenetic research. PMID:21966350

  12. High-performance capillary electrophoresis of histones

    SciTech Connect

    Gurley, L.R.; London, J.E.; Valdez, J.G.

    1991-01-01

    A high performance capillary electrophoresis (HPCE) system has been developed for the fractionation of histones. This system involves electroinjection of the sample and electrophoresis in a 0.1M phosphate buffer at pH 2.5 in a 50 {mu}m {times} 35 cm coated capillary. Electrophoresis was accomplished in 9 minutes separating a whole histone preparation into its components in the following order of decreasing mobility; (MHP) H3, H1 (major variant), H1 (minor variant), (LHP) H3, (MHP) H2A (major variant), (LHP) H2A, H4, H2B, (MHP) H2A (minor variant) where MHP is the more hydrophobic component and LHP is the less hydrophobic component. This order of separation is very different from that found in acid-urea polyacrylamide gel electrophoresis and in reversed-phase HPLC and, thus, brings the histone biochemist a new dimension for the qualitative analysis of histone samples. 27 refs., 8 figs.

  13. ATRX tolerates activity-dependent histone H3 methyl/phos switching to maintain repetitive element silencing in neurons

    PubMed Central

    Noh, Kyung-Min; Zhao, Dan; Xiang, Bin; Wenderski, Wendy; Lewis, Peter W.; Shen, Li; Li, Haitao; Allis, C. David

    2015-01-01

    ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. PMID:25538301

  14. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  15. Phosphorylation site prediction in plants.

    PubMed

    Yao, Qiuming; Schulze, Waltraud X; Xu, Dong

    2015-01-01

    Protein phosphorylation events on serine, threonine, and tyrosine residues are the most pervasive protein covalent bond modifications in plant signaling. Both low and high throughput studies reveal the importance of phosphorylation in plant molecular biology. Although becoming more and more common, the proteome-wide screening on phosphorylation by experiments remains time consuming and costly. Therefore, in silico prediction methods are proposed as a complementary analysis tool to enhance the phosphorylation site identification, develop biological hypothesis, or help experimental design. These methods build statistical models based on the experimental data, and they do not have some of the technical-specific bias, which may have advantage in proteome-wide analysis. More importantly computational methods are very fast and cheap to run, which makes large-scale phosphorylation identifications very practical for any types of biological study. Thus, the phosphorylation prediction tools become more and more popular. In this chapter, we will focus on plant specific phosphorylation site prediction tools, with essential illustration of technical details and application guidelines. We will use Musite, PhosPhAt and PlantPhos as the representative tools. We will present the results on the prediction of the Arabidopsis protein phosphorylation events to give users a general idea of the performance range of the three tools, together with their strengths and limitations. We believe these prediction tools will contribute more and more to the plant phosphorylation research community. PMID:25930706

  16. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  17. Western Analysis of Histone Modifications (Aspergillus nidulans)

    PubMed Central

    Soukup, Alexandra; Keller, Nancy P.

    2016-01-01

    Western blotting allows for the specific detection of proteins and/or modifications of proteins by an antibody of interest. This protocol utilizes a crude nuclei extraction protocol for Aspergillus nidulans to enrich for histones and other nuclear proteins prior to gel electrophoresis. Post translational modifications of histones may then be easily detected. After electrophoresis, the selected antibodies are used to detect and quantify levels of the modifications of interest.

  18. Countercurrent-distribution studies on histones

    PubMed Central

    Butler, J. A. V.; Power, D. F.; Palau, J.

    1967-01-01

    1. The possibilities of fractionating histones and histone fractions by means of countercurrent distribution between two phases formed by water and butan-2-ol, in the presence of various concentrations of trichloroacetic acid, have been examined. 2. Although the principal histone fractions differ considerably in their partition ratios, a satisfactory resolution of the principal histone fractions from the whole histone has not been achieved. 3. The histone fractions obtained by other methods can be resolved with suitable concentrations of trichloroacetic acid. Besides the main peak several subsidiary peaks are obtained in most cases, the composition of which corresponds with others of the main fractions. 4. The method is therefore capable of removing from the principal fractions as previously prepared contamination by other fractions. 5. Except in one case, no fraction with composition unlike other fractions has been obtained. In several cases the material isolated from the principal peak behaves as a single component on running again. In two cases fractions with similar compositions were distinguished by countercurrent distribution. PMID:6029613

  19. Inhibition of Histone Acetyltransferase by Glycosaminoglycans

    PubMed Central

    Buczek-Thomas, Jo Ann; Hsia, Edward; Rich, Celeste B.; Foster, Judith A.; Nugent, Matthew A.

    2008-01-01

    Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (> 8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an ~50-fold increase in the apparent Km of pCAF for histone H4. Heparan sulfate proteoglycans isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous glycosaminoglycans might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an ~50% reduction of histone H3 acetylation after 6 hours of treatment. In addition, Chinese hamster ovary cells deficient in glycosaminoglycan synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. Glycosaminoglycans represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation. PMID:18459114

  20. Histone modification and chromatin remodeling during NER.

    PubMed

    Waters, Raymond; van Eijk, Patrick; Reed, Simon

    2015-12-01

    Here we review our developments of and results with high resolution studies on global genome nucleotide excision repair (GG-NER) in Saccharomyces cerevisiae. Technologies were developed to examine NER at nucleotide resolution in yeast sequences of choice and to determine how these related to local changes in chromatin. We focused on how GG-NER relates to histone acetylation for its functioning and we identified the histone acetyltransferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. Factors influencing this Gcn5-mediated event are considered which include Rad16, a GG-NER specific SWI/SNF factor and the yeast histone variant of H2AZ (Htz1). We describe results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then consider the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the NER of UV-induced cyclobutane pyrimidine dimers throughout entire yeast genome. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage before it is returned to its pre-damaged status to maintain epigenetic codes. PMID:26422133

  1. Interpreting thelanguage of histone and DNA modifications

    PubMed Central

    Rothbart, Scott B.; Strahl, Brian D.

    2014-01-01

    A major mechanism regulating the accessibility and function of eukaryotic genomes are the covalent modifications to DNA and histone proteins that dependably package our genetic information inside the nucleus of every cell. Formally postulated over a decade ago, it is becoming increasingly clear that post-translational modifications (PTMs) on histones act singly and in combination to form a language or ‘code’ that is read by specialized proteins to facilitate downstream functions in chromatin. Underappreciated at the time was the level of complexity harbored both within histone PTMs and their combinations, as well as within the proteins that read and interpret the language. In addition to histone PTMs, newly-identified DNA modifications that can recruit specific effector proteins has raised further awareness that histone PTMs operate within a broader language of epigenetic modifications to orchestrate the dynamic functions associated with chromatin. Here, we highlight key recent advances in our understanding of the epigenetic language encompassing histone and DNA modifications and foreshadow challenges that lie ahead as we continue our quest to decipher the fundamental mechanisms of chromatin regulation. PMID:24631868

  2. Histone Chaperone Asf1 Plays an Essential Role in Maintaining Genomic Stability in Fission Yeast

    PubMed Central

    Tanae, Katsuhiro; Horiuchi, Tomitaka; Matsuo, Yuzy; Katayama, Satoshi; Kawamukai, Makoto

    2012-01-01

    The histone H3-H4 chaperone Asf1 is involved in chromatin assembly (or disassembly), histone exchange, regulation of transcription, and chromatin silencing in several organisms. To investigate the essential functions of Asf1 in Schizosaccharomyces pombe, asf1-ts mutants were constructed by random mutagenesis using PCR. One mutant (asf1-33(ts)) was mated with mutants in 77 different kinase genes to identify synthetic lethal combinations. The asf1-33 mutant required the DNA damage checkpoint factors Chk1 and Rad3 for its survival at the restrictive temperature. Chk1, but not Cds1, was phosphorylated in the asf1-33 mutant at the restrictive temperature, indicating that the DNA damage checkpoint was activated in the asf1-33 mutant. DNA damage occured in the asf1-33 mutant, with degradation of the chromosomal DNA observed through pulse-field gel electrophoresis and the formation of Rad22 foci. Sensitivity to micrococcal nuclease in the asf1-33 mutant was increased compared to the asf1+ strain at the restrictive temperature, suggesting that asf1 mutations also caused a defect in overall chromatin structure. The Asf1-33 mutant protein was mislocalized and incapable of binding histones. Furthermore, histone H3 levels at the centromeric outer repeat region were decreased in the asf1-33 mutant and heterochromatin structure was impaired. Finally, sim3, which encodes a CenH3 histone chaperone, was identified as a strong suppressor of the asf1-33 mutant. Taken together, these results clearly indicate that Asf1 plays an essential role in maintaining genomic stability in S. pombe. PMID:22291963

  3. Phosphorylated. beta. -dicarbonyl compounds

    SciTech Connect

    Liorber, B.G.; Tarzivolova, T.A.; Pavlov, V.A.; Zykova, T.V.; Kisilev, V.V.; Tumasheva, N.A.; Slizkii, A.Yu.; Shagvaleev, F.S.

    1987-08-20

    The reaction of trialkyl phosphites with alkyl malonyl chlorides leads to alkyl 3-dialkoxyphosphoryl-3-oxopropionates, which exist in the stable E-enol form. Depending on the basicities of the bases, the reactions of alkyl 3-dialkoxyphosphoryl-3-oxopropionates with nitrogen bases proceed with retention of the C-P