Science.gov

Sample records for checkpoint silencing mechanism

  1. Thyroid Hormone Receptor Interacting Protein 13 (TRIP13) AAA-ATPase Is a Novel Mitotic Checkpoint-silencing Protein*

    PubMed Central

    Wang, Kexi; Sturt-Gillespie, Brianne; Hittle, James C.; Macdonald, Dawn; Chan, Gordon K.; Yen, Tim J.; Liu, Song-Tao

    2014-01-01

    The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochore-microtubule attachment. The target of the checkpoint is the E3 ubiquitin ligase anaphase-promoting complex/cyclosome. Once all chromosomes are properly attached and bioriented at the metaphase plate, the checkpoint needs to be silenced. Previously, we and others have reported that TRIP13 AAA-ATPase binds to the mitotic checkpoint-silencing protein p31comet. Here we show that endogenous TRIP13 localizes to kinetochores. TRIP13 knockdown delays metaphase-to-anaphase transition. The delay is caused by prolonged presence of the effector for the checkpoint, the mitotic checkpoint complex, and its association and inhibition of the anaphase-promoting complex/cyclosome. These results suggest that TRIP13 is a novel mitotic checkpoint-silencing protein. The ATPase activity of TRIP13 is essential for its checkpoint function, and interference with TRIP13 abolished p31comet-mediated mitotic checkpoint silencing. TRIP13 overexpression is a hallmark of cancer cells showing chromosomal instability, particularly in certain breast cancers with poor prognosis. We suggest that premature mitotic checkpoint silencing triggered by TRIP13 overexpression may promote cancer development. PMID:25012665

  2. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  3. HORMAD1-dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes.

    PubMed

    Kogo, Hiroshi; Tsutsumi, Makiko; Ohye, Tamae; Inagaki, Hidehito; Abe, Takaya; Kurahashi, Hiroki

    2012-06-01

    Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis.

  4. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    SciTech Connect

    Nakadate, Yusuke; Kodera, Yasuo; Kitamura, Yuka; Tachibana, Taro; Tamura, Tomohide; Koizumi, Fumiaki

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  5. Cell cycle control, checkpoint mechanisms, and genotoxic stress.

    PubMed Central

    Shackelford, R E; Kaufmann, W K; Paules, R S

    1999-01-01

    The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle

  6. The Dynamical Mechanisms of the Cell Cycle Size Checkpoint

    NASA Astrophysics Data System (ADS)

    Feng, Shi-Fu; Yan, Jie; Liu, Zeng-Rong; Yang, Ling

    2012-10-01

    Cell division must be tightly coupled to cell growth in order to maintain cell size, whereas the mechanisms of how initialization of mitosis is regulated by cell size remain to be elucidated. We develop a mathematical model of the cell cycle, which incorporates cell growth to investigate the dynamical properties of the size checkpoint in embryos of Xenopus laevis. We show that the size checkpoint is naturally raised from a saddle-node bifurcation, and in a mutant case, the cell loses its size control ability due to the loss of this saddle-node point.

  7. Roles of different pools of the mitotic checkpoint complex and the mechanisms of their disassembly

    PubMed Central

    Eytan, Esther; Sitry-Shevah, Danielle; Teichner, Adar; Hershko, Avram

    2013-01-01

    The mitotic (or spindle assembly) checkpoint system prevents premature separation of sister chromatids in mitosis. When the checkpoint is turned on, the mitotic checkpoint complex (MCC) inhibits the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). MCC is composed of the checkpoint proteins BubR1, Bub3, and Mad2 associated with the APC/C activator Cdc20. The mechanisms of the assembly of MCC when the checkpoint is turned on, and of its disassembly when the checkpoint is inactivated, are not sufficiently understood. Previous reports indicated that APC/C-mediated polyubiquitylation of Cdc20 in MCC is required for the dissociation of APC/C-associated MCC, but not of free MCC. The pool of free MCC is disassembled by an ATP-dependent process stimulated by the Mad2-binding protein p31comet. It remained unknown whether free MCC is the precursor or the dissociation product of APC/C-bound MCC. By characterizing the mechanisms of the disassembly of APC/C-bound MCC in a purified system, we find that it cannot be the source of free MCC, because it is bound at high affinity and is released only in ubiquitylated or partially disassembled forms. By the use of a cell-free system from Xenopus eggs that reproduces the mitotic checkpoint, we show that MCC can be assembled in the absence of APC/C in a checkpoint-dependent manner. We propose that when the checkpoint is turned on, free MCC is the precursor of APC/C-bound MCC. When the mitotic checkpoint is extinguished, both APC/C-bound and free MCC pools have to be disassembled to release APC/C from inhibition. PMID:23754430

  8. On the Mechanism of Gene Silencing in Saccharomyces cerevisiae

    PubMed Central

    Steakley, David Lee; Rine, Jasper

    2015-01-01

    Multiple mechanisms have been proposed for gene silencing in Saccharomyces cerevisiae, ranging from steric occlusion of DNA binding proteins from their recognition sequences in silenced chromatin to a specific block in the formation of the preinitiation complex to a block in transcriptional elongation. This study provided strong support for the steric occlusion mechanism by the discovery that RNA polymerase of bacteriophage T7 could be substantially blocked from transcribing from its cognate promoter when embedded in silenced chromatin. Moreover, unlike previous suggestions, we found no evidence for stalled RNA polymerase II within silenced chromatin. The effectiveness of the Sir protein–based silencing mechanism to block transcription activated by Gal4 at promoters in the domain of silenced chromatin was marginal, yet it improved when tested against mutant forms of the Gal4 protein, highlighting a role for specific activators in their sensitivity to gene silencing. PMID:26082137

  9. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy

    PubMed Central

    Topalian, Suzanne L.; Taube, Janis M.; Anders, Robert A.; Pardoll, Drew M.

    2017-01-01

    With recent approvals for multiple therapeutic antibodies that block cytotoxic T lymphocyte associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) in melanoma, non-small-cell lung cancer and kidney cancer, and additional immune checkpoints being targeted clinically, many questions still remain regarding the optimal use of drugs that block these checkpoint pathways. Defining biomarkers that predict therapeutic effects and adverse events is a crucial mandate, highlighted by recent approvals for two PDL1 diagnostic tests. Here, we discuss biomarkers for anti-PD1 therapy based on immunological, genetic and virological criteria. The unique biology of the CTLA4 immune checkpoint, compared with PD1, requires a different approach to biomarker development. Mechanism-based insights from such studies may guide the design of synergistic treatment combinations based on immune checkpoint blockade. PMID:27079802

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

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

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

  11. Subverting the adaptive immune resistance mechanism to improve clinical responses to immune checkpoint blockade therapy

    PubMed Central

    Kim, Young J

    2015-01-01

    The correlation between tumor-infiltrating lymphocyte (TIL)-expression of programmed cell death ligand 1 (PD-L1) and clinical responsiveness to the PD-1 blocking antibody nivolumab implicates adaptive immune evasion mechanisms in cancer. We review our findings that tumor cell PD-L1 expression is induced by interferon γ (IFNγ) producing TILs. We provide a mechanistic rationale for combining IFNγ+ T helper type 1 (Th1)-inducing cancer vaccines with PD-1 immune checkpoint blockade. PMID:25964860

  12. Structure and Gene-Silencing Mechanisms of Small Noncoding RNAs

    NASA Astrophysics Data System (ADS)

    Chu, Chia-Ying; Rana, Tariq M.

    Small (19-31-nucleotides) noncoding RNAs were identified in the past 10 years for their distinct function in gene silencing. The best known gene-silencing phenomenon, RNA interference (RNAi), is triggered in a sequence-specific manner by endogenously produced or exogenously introduced small doubled-stranded RNAs. As knowledge of the structure and function of the RNAi machinery has expanded, this phenomenon has become a powerful tool for biochemical research; it has enormous potential for therapeutics. This chapter summarizes significant aspects of three major classes of small noncoding, regulatory RNAs: small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Here, we focus on the biogenesis of these small RNAs, their structural features and coupled effectors as well as the mechanisms of each small regulatory RNA pathway which reveal fascinating ways by which gene silencing is controlled and fine-tuned at an epigenetic level.

  13. Checkpointing filesystem

    DOEpatents

    Gara, Alan G.; Giampapa, Mark E.; Steinmacher-Burow, Burkhard D.

    2005-05-17

    The present in invention is directed to a checkpointing filesystem of a distributed-memory parallel supercomputer comprising a node that accesses user data on the filesystem, the filesystem comprising an interface that is associated with a disk for storing the user data. The checkpointing filesystem provides for taking and checkpoint of the filesystem and rolling back to a previously taken checkpoint, as well as for writing user data to and deleting user data from the checkpointing filesystem. The checkpointing filesystem provides a recently written file allocation table (WFAT) for maintaining information regarding the user data written since a previously taken checkpoint and a recently deleted file allocation table (DFAT) for maintaining information regarding user data deleted from since the previously taken checkpoint, both of which are utilized by the checkpointing filesystem to take a checkpoint of the filesystem and rollback the filesystem to a previously taken checkpoint, as well as to write and delete user data from the checkpointing filesystem.

  14. The interplay among chromatin dynamics, cell cycle checkpoints and repair mechanisms modulates the cellular response to DNA damage.

    PubMed

    Lazzaro, Federico; Giannattasio, Michele; Muzi-Falconi, Marco; Plevani, Paolo

    2007-06-01

    Cells are continuously under the assault of endogenous and exogenous genotoxic stress that challenges the integrity of DNA. To cope with such a formidable task cells have evolved surveillance mechanisms, known as checkpoints, and a variety of DNA repair systems responding to different types of DNA lesions. These lesions occur in the context of the chromatin structure and, as expected for all DNA transactions, the cellular response to DNA damage is going to be influenced by the chromatin enviroment. In this review, we will discuss recent studies implicating chromatin remodelling factors and histone modifications in the response to DNA double-strand breaks (DSBs) and in checkpoint activation in response to UV lesions.

  15. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    SciTech Connect

    Iida, Tetsushi; Iida, Naoko; Tsutsui, Yasuhiro; Yamao, Fumiaki; Kobayashi, Takehiko

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  16. Caenorhabditis elegans histone methyltransferase MET-2 shields the male X chromosome from checkpoint machinery and mediates meiotic sex chromosome inactivation.

    PubMed

    Checchi, Paula M; Engebrecht, JoAnne

    2011-09-01

    Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meiosis with unsynapsed regions and are not recognized by checkpoint machinery. We conducted a directed RNAi screen in Caenorhabditis elegans to identify regulatory factors that prevent recognition of heteromorphic sex chromosomes as unpaired and uncovered a role for the SET domain histone H3 lysine 9 histone methyltransferase (HMTase) MET-2 and two additional HMTases in shielding the male X from checkpoint machinery. We found that MET-2 also mediates the transcriptional silencing program of meiotic sex chromosome inactivation (MSCI) but not meiotic silencing of unsynapsed chromatin (MSUC), suggesting that these processes are distinct. Further, MSCI and checkpoint shielding can be uncoupled, as double-strand breaks targeted to an unpaired, transcriptionally silenced extra-chromosomal array induce checkpoint activation in germ lines depleted for met-2. In summary, our data uncover a mechanism by which repressive chromatin architecture enables checkpoint proteins to distinguish between the partnerless male X chromosome and asynapsed chromosomes thereby shielding the lone X from inappropriate activation of an apoptotic program.

  17. Mechanical Checkpoint For Persistent Cell Polarization In Adhesion-Naive Fibroblasts

    PubMed Central

    Bun, Philippe; Liu, JunJun; Turlier, Hervé; Liu, ZengZhen; Uriot, Karen; Joanny, Jean-François; Coppey-Moisan, Maïté

    2014-01-01

    Cell polarization is a fundamental biological process implicated in nearly every aspect of multicellular development. The role of cell-extracellular matrix contacts in the establishment and the orientation of cell polarity have been extensively studied. However, the respective contributions of substrate mechanics and biochemistry remain unclear. Here we propose a believed novel single-cell approach to assess the minimal polarization trigger. Using nonadhered round fibroblast cells, we show that stiffness sensing through single localized integrin-mediated cues are necessary and sufficient to trigger and direct a shape polarization. In addition, the traction force developed by cells has to reach a minimal threshold of 56 ± 1.6 pN for persistent polarization. The polarization kinetics increases with the stiffness of the cue. The polarized state is characterized by cortical actomyosin redistribution together with cell shape change. We develop a physical model supporting the idea that a local and persistent inhibition of actin polymerization and/or myosin activity is sufficient to trigger and sustain the polarized state. Finally, the cortical polarity propagates to an intracellular polarity, evidenced by the reorientation of the centrosome. Our results define the minimal adhesive requirements and quantify the mechanical checkpoint for persistent cell shape and organelle polarization, which are critical regulators of tissue and cell development. PMID:25028874

  18. Epigenetic regulation of immune checkpoints: another target for cancer immunotherapy?

    PubMed

    Ali, Mahmoud A; Matboli, Marwa; Tarek, Marwa; Reda, Maged; Kamal, Kamal M; Nouh, Mahmoud; Ashry, Ahmed M; El-Bab, Ahmed Fath; Mesalam, Hend A; Shafei, Ayman El-Sayed; Abdel-Rahman, Omar

    2017-01-01

    Epigenetic changes in oncogenes and tumor-suppressor genes contribute to carcinogenesis. Understanding the epigenetic and genetic components of tumor immune evasion is crucial. Few cancer genetic mutations have been linked to direct correlations with immune evasion. Studies on the epigenetic modulation of the immune checkpoints have revealed a critical interaction between epigenetic and immune modulation. Epigenetic modifiers can activate many silenced genes. Some of them are immune checkpoints regulators that turn on immune responses and others turn them off resulting in immune evasion. Many forms of epigenetic inheritance mechanisms may play a role in regulation of immune checkpoints including: covalent modifications, noncoding RNA and histone modifications. In this review, we will show how the potential interaction between epigenetic and immune modulation may lead to new approaches for specific epigenome/immunome-targeted therapies for cancer.

  19. Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana

    PubMed Central

    He, Chongsheng; Huang, Hai; Xu, Lin

    2013-01-01

    Polycomb group (PcG) proteins act in an evolutionarily conserved epigenetic pathway that regulates chromatin structures in plants and animals, repressing many developmentally important genes by modifying histones. PcG proteins can form at least two multiprotein complexes: Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2, respectively). The functions of Arabidopsis thaliana PRCs have been characterized in multiple stages of development and have diverse roles in response to environmental stimuli. Recently, the mechanism that precisely regulates Arabidopsis PcG activity was extensively studied. In this review, we summarize recent discoveries in the regulations of PcG at the three different layers: the recruitment of PRCs to specific target loci, the polyubiquitination and degradation of PRC2, and the antagonism of PRC2 activity by the Trithorax group proteins. Current knowledge indicates that the powerful activity of the PcG pathway is strictly controlled for specific silencing of target genes during plant development and in response to environmental stimuli. PMID:24312106

  20. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  1. Compiler-assisted static checkpoint insertion

    NASA Technical Reports Server (NTRS)

    Long, Junsheng; Fuchs, W. K.; Abraham, Jacob A.

    1992-01-01

    This paper describes a compiler-assisted approach for static checkpoint insertion. Instead of fixing the checkpoint location before program execution, a compiler enhanced polling mechanism is utilized to maintain both the desired checkpoint intervals and reproducible checkpoint 1ocations. The technique has been implemented in a GNU CC compiler for Sun 3 and Sun 4 (Sparc) processors. Experiments demonstrate that the approach provides for stable checkpoint intervals and reproducible checkpoint placements with performance overhead comparable to a previously presented compiler assisted dynamic scheme (CATCH) utilizing the system clock.

  2. Recovery from the DNA Replication Checkpoint

    PubMed Central

    Chaudhury, Indrajit; Koepp, Deanna M.

    2016-01-01

    Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

  3. Ataxia-telangiectasia mutated (ATM) silencing promotes neuroblastoma progression through a MYCN independent mechanism.

    PubMed

    Mandriota, Stefano J; Valentijn, Linda J; Lesne, Laurence; Betts, David R; Marino, Denis; Boudal-Khoshbeen, Mary; London, Wendy B; Rougemont, Anne-Laure; Attiyeh, Edward F; Maris, John M; Hogarty, Michael D; Koster, Jan; Molenaar, Jan J; Versteeg, Rogier; Ansari, Marc; Gumy-Pause, Fabienne

    2015-07-30

    Neuroblastoma, a childhood cancer with highly heterogeneous biology and clinical behavior, is characterized by genomic aberrations including amplification of MYCN. Hemizygous deletion of chromosome 11q is a well-established, independent marker of poor prognosis. While 11q22-q23 is the most frequently deleted region, the neuroblastoma tumor suppressor in this region remains to be identified. Chromosome bands 11q22-q23 contain ATM, a cell cycle checkpoint kinase and tumor suppressor playing a pivotal role in the DNA damage response. Here, we report that haploinsufficiency of ATM in neuroblastoma correlates with lower ATM expression, event-free survival, and overall survival. ATM loss occurs in high stage neuroblastoma without MYCN amplification. In SK-N-SH, CLB-Ga and GI-ME-N human neuroblastoma cells, stable ATM silencing promotes neuroblastoma progression in soft agar assays, and in subcutaneous xenografts in nude mice. This effect is dependent on the extent of ATM silencing and does not appear to involve MYCN. Our findings identify ATM as a potential haploinsufficient neuroblastoma tumor suppressor, whose inactivation mirrors the increased aggressiveness associated with 11q deletion in neuroblastoma.

  4. Upregulated Op18/stathmin activity causes chromosomal instability through a mechanism that evades the spindle assembly checkpoint

    SciTech Connect

    Holmfeldt, Per; Sellin, Mikael E.; Gullberg, Martin

    2010-07-15

    Op18/stathmin (Op18) is a microtubule-destabilizing protein that is phosphorylation-inactivated during mitosis and its normal function is to govern tubulin subunit partitioning during interphase. Human tumors frequently overexpress Op18 and a tumor-associated Q18{yields}E mutation has been identified that confers hyperactivity, destabilizes spindle microtubules, and causes mitotic aberrancies, polyploidization, and chromosome loss in K562 leukemia cells. Here we determined whether wild-type and mutant Op18 have the potential to cause chromosomal instability by some means other than interference with spindle assembly, and thereby bypassing the spindle assembly checkpoint. Our approach was based on Op18 derivatives with distinct temporal order of activity during mitosis, conferred either by differential phosphorylation inactivation or by anaphase-specific degradation through fusion with the destruction box of cyclin B1. We present evidence that excessive Op18 activity generates chromosomal instability through interference occurring subsequent to the metaphase-to-anaphase transition, which reduces the fidelity of chromosome segregation to spindle poles during anaphase. Similar to uncorrected merotelic attachment, this mechanism evades detection by the spindle assembly checkpoint and thus provides an additional route to chromosomal instability.

  5. Checkpointing in speculative versioning caches

    DOEpatents

    Eichenberger, Alexandre E; Gara, Alan; Gschwind, Michael K; Ohmacht, Martin

    2013-08-27

    Mechanisms for generating checkpoints in a speculative versioning cache of a data processing system are provided. The mechanisms execute code within the data processing system, wherein the code accesses cache lines in the speculative versioning cache. The mechanisms further determine whether a first condition occurs indicating a need to generate a checkpoint in the speculative versioning cache. The checkpoint is a speculative cache line which is made non-speculative in response to a second condition occurring that requires a roll-back of changes to a cache line corresponding to the speculative cache line. The mechanisms also generate the checkpoint in the speculative versioning cache in response to a determination that the first condition has occurred.

  6. Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

    PubMed Central

    Mauri, Marta; Kirchner, Marieluise; Aharoni, Reuven; Ciolli Mattioli, Camilla; van den Bruck, David; Gutkovitch, Nadya; Modepalli, Vengamanaidu; Selbach, Matthias; Moran, Yehu; Chekulaeva, Marina

    2017-01-01

    Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. PMID:27604873

  7. Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution.

    PubMed

    Mauri, Marta; Kirchner, Marieluise; Aharoni, Reuven; Ciolli Mattioli, Camilla; van den Bruck, David; Gutkovitch, Nadya; Modepalli, Vengamanaidu; Selbach, Matthias; Moran, Yehu; Chekulaeva, Marina

    2017-01-25

    Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.

  8. Genome Reactivation after the Silence in Mitosis: Recapitulating Mechanisms of Development?

    PubMed Central

    Zaret, Kenneth S.

    2014-01-01

    Transcription is silenced during mitosis and re-activated at mitotic exit. The dynamics and identities of “bookmarking” transcription factors and chromatin marks that mediate reactivation often recapitulate that observed during cell identity establishment in development. Thus, features of post-mitotic gene re-activation can provide insights into mechanisms of developmental cell fate establishment. PMID:24780732

  9. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

    In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

  10. DNA damage checkpoint recovery and cancer development

    SciTech Connect

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong; Legerski, Randy J.

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  11. The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome.

    PubMed

    Kumari, Daman; Usdin, Karen

    2010-12-01

    Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and the most common known cause of autism. Most cases of FXS result from the expansion of a CGG·CCG repeat in the 5' UTR of the FMR1 gene that leads to gene silencing. It has previously been shown that silenced alleles are associated with histone H3 dimethylated at lysine 9 (H3K9Me2) and H3 trimethylated at lysine 27 (H3K27Me3), modified histones typical of developmentally repressed genes. We show here that these alleles are also associated with elevated levels of histone H3 trimethylated at lysine 9 (H3K9Me3) and histone H4 trimethylated at lysine 20 (H4K20Me3). All four of these modified histones are present on exon 1 of silenced alleles at levels comparable to that seen on pericentric heterochromatin. The two groups of histone modifications show a different distribution on fragile X alleles: H3K9Me2 and H3K27Me3 have a broad distribution, whereas H3K9Me3 and H4K20Me3 have a more focal distribution with the highest level of these marks being present in the vicinity of the repeat. This suggests that the trigger for gene silencing may be local to the repeat itself and perhaps involves a mechanism similar to that involved in the formation of pericentric heterochromatin.

  12. Cell Cycle Regulation by Checkpoints

    PubMed Central

    Barnum, Kevin J.; O’Connell, Matthew J.

    2016-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate. PMID:24906307

  13. Cell cycle regulation by checkpoints.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2014-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate.

  14. Strong host resistance targeted against a viral suppressor of the plant gene silencing defence mechanism.

    PubMed Central

    Li, H W; Lucy, A P; Guo, H S; Li, W X; Ji, L H; Wong, S M; Ding, S W

    1999-01-01

    The 2b protein encoded by cucumber mosaic cucumovirus (Cmv2b) acts as an important virulence determinant by suppressing post-transcriptional gene silencing (PTGS), a natural plant defence mechanism against viruses. We report here that the tomato aspermy cucumovirus 2b protein (Tav2b), when expressed from the unrelated tobacco mosaic tobamovirus (TMV) RNA genome, activates strong host resistance responses to TMV in tobacco which are typical of the gene-for-gene disease resistance mechanism. Domain swapping between Cmv2b, which does not elicit these responses, and Tav2b, revealed functional domains in Tav2b critical for triggering virus resistance and hypersensitive cell death. Furthermore, substitution of two amino acids from Tav2b by those found at the same positions in Cmv2b, Lys21-->Val and Arg28-->Ser, abolished the ability to induce hypersensitive cell death and virus resistance. However, in Nicotiana benthamiana, a species related to tobacco, Tav2b functions as a virulence determinant and suppresses PTGS. Thus, a viral suppressor of the host gene silencing defence mechanism is the target of another independent host resistance mechanism. Our results provide new insights into the complex molecular strategies employed by viruses and their hosts for defence, counter-defence and counter counter-defence. PMID:10329615

  15. Mechanism of Action of 2-Aminobenzamide HDAC Inhibitors in Reversing Gene Silencing in Friedreich's Ataxia.

    PubMed

    Soragni, Elisabetta; Chou, C James; Rusche, James R; Gottesfeld, Joel M

    2015-01-01

    The genetic defect in Friedreich's ataxia (FRDA) is the hyperexpansion of a GAA•TTC triplet in the first intron of the FXN gene, encoding the essential mitochondrial protein frataxin. Histone post-translational modifications near the expanded repeats are consistent with heterochromatin formation and consequent FXN gene silencing. Using a newly developed human neuronal cell model, derived from patient-induced pluripotent stem cells, we find that 2-aminobenzamide histone deacetylase (HDAC) inhibitors increase FXN mRNA levels and frataxin protein in FRDA neuronal cells. However, only compounds targeting the class I HDACs 1 and 3 are active in increasing FXN mRNA in these cells. Structural analogs of the active HDAC inhibitors that selectively target either HDAC1 or HDAC3 do not show similar increases in FXN mRNA levels. To understand the mechanism of action of these compounds, we probed the kinetic properties of the active and inactive inhibitors, and found that only compounds that target HDACs 1 and 3 exhibited a slow-on/slow-off mechanism of action for the HDAC enzymes. HDAC1- and HDAC3-selective compounds did not show this activity. Using siRNA methods in the FRDA neuronal cells, we show increases in FXN mRNA upon silencing of either HDACs 1 or 3, suggesting the possibility that inhibition of each of these class I HDACs is necessary for activation of FXN mRNA synthesis, as there appears to be redundancy in the silencing mechanism caused by the GAA•TTC repeats. Moreover, inhibitors must have a long residence time on their target enzymes for this activity. By interrogating microarray data from neuronal cells treated with inhibitors of different specificity, we selected two genes encoding histone macroH2A (H2AFY2) and Polycomb group ring finger 2 (PCGF2) that were specifically down-regulated by the inhibitors targeting HDACs1 and 3 versus the more selective inhibitors for further investigation. Both genes are involved in transcriptional repression and we speculate

  16. ATR acts stage specifically to regulate multiple aspects of mammalian meiotic silencing.

    PubMed

    Royo, Hélène; Prosser, Haydn; Ruzankina, Yaroslava; Mahadevaiah, Shantha K; Cloutier, Jeffrey M; Baumann, Marek; Fukuda, Tomoyuki; Höög, Christer; Tóth, Attila; de Rooij, Dirk G; Bradley, Allan; Brown, Eric J; Turner, James M A

    2013-07-01

    In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.

  17. Noncoding RNAs and immune checkpoints-clinical implications as cancer therapeutics.

    PubMed

    Smolle, Maria A; Calin, Horatiu N; Pichler, Martin; Calin, George A

    2017-01-28

    A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice.

  18. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

    PubMed Central

    Lakatos, Lóránt; Szittya, György; Silhavy, Dániel; Burgyán, József

    2004-01-01

    RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19–siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing. PMID:14976549

  19. On the robustness of SAC silencing in closed mitosis

    NASA Astrophysics Data System (ADS)

    Ruth, Donovan; Liu, Jian

    Mitosis equally partitions sister chromatids to two daughter cells. This is achieved by properly attaching these chromatids via their kinetochores to microtubules that emanate from the spindle poles. Once the last kinetochore is properly attached, the spindle microtubules pull the sister chromatids apart. Due to the dynamic nature of microtubules, however, kinetochore-microtubule attachment often goes wrong. When this erroneous attachment occurs, it locally activates an ensemble of proteins, called the spindle assembly checkpoint proteins (SAC), which halts the mitotic progression until all the kinetochores are properly attached by spindle microtubules. The timing of SAC silencing thus determines the fidelity of chromosome segregation. We previously established a spatiotemporal model that addresses the robustness of SAC silencing in open mitosis for the first time. Here, we focus on closed mitosis by examining yeast mitosis as a model system. Though much experimental work has been done to study the SAC in cells undergoing closed mitosis, the processes responsible are not well understood. We leverage and extend our previous model to study SAC silencing mechanism in closed mitosis. We show that a robust signal of the SAC protein accumulation at the spindle pole body can be achieved. This signal is a nonlinear increasing function of number of kinetochore-microtubule attachments, and can thus serve as a robust trigger to time the SAC silencing. Together, our mechanism provides a unified framework across species that ensures robust SAC silencing and fidelity of chromosome segregation in mitosis. Intramural research program in NHLBI at NIH.

  20. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  1. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  2. Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

    PubMed

    Aqil, Madeeha; Deliu, Zane; Elseth, Kim M; Shen, Grace; Xue, Jiaping; Radosevich, James A

    2014-03-01

    Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher

  3. Epigenetics meets immune checkpoints.

    PubMed

    Covre, Alessia; Coral, Sandra; Di Giacomo, Anna Maria; Taverna, Pietro; Azab, Mohammad; Maio, Michele

    2015-06-01

    Epigenetic alterations play a pivotal role in cancer development and progression. Pharmacologic reversion of such alterations is feasible, and second generation "epigenetic drugs" are in development and have been demonstrated to possess significant immunomodulatory properties. This knowledge, together with the availability of new and highly effective immunotherapeutic agents including immune checkpoint(s) blocking monoclonal antibodies, allows us to plan for highly innovative proof-of-principle combination studies that will likely open the path to more effective anticancer therapies.

  4. Efficient Checkpointing of Virtual Machines using Virtual Machine Introspection

    SciTech Connect

    Aderholdt, Ferrol; Han, Fang; Scott, Stephen L; Naughton, III, Thomas J

    2014-01-01

    Cloud Computing environments rely heavily on system-level virtualization. This is due to the inherent benefits of virtualization including fault tolerance through checkpoint/restart (C/R) mechanisms. Because clouds are the abstraction of large data centers and large data centers have a higher potential for failure, it is imperative that a C/R mechanism for such an environment provide minimal latency as well as a small checkpoint file size. Recently, there has been much research into C/R with respect to virtual machines (VM) providing excellent solutions to reduce either checkpoint latency or checkpoint file size. However, these approaches do not provide both. This paper presents a method of checkpointing VMs by utilizing virtual machine introspection (VMI). Through the usage of VMI, we are able to determine which pages of memory within the guest are used or free and are better able to reduce the amount of pages written to disk during a checkpoint. We have validated this work by using various benchmarks to measure the latency along with the checkpoint size. With respect to checkpoint file size, our approach results in file sizes within 24% or less of the actual used memory within the guest. Additionally, the checkpoint latency of our approach is up to 52% faster than KVM s default method.

  5. Immune checkpoint inhibitors for cancer treatment.

    PubMed

    Park, Junsik; Kwon, Minsuk; Shin, Eui-Cheol

    2016-11-01

    During immune responses antigen-specific T cells are regulated by several mechanisms, including through inhibitory receptors and regulatory T cells, to avoid excessive or persistent immune responses. These regulatory mechanisms, which are called 'immune checkpoints', suppress T cell responses, particularly in patients with chronic viral infections and cancer where viral antigens or tumor antigens persist for a long time and contribute to T cell exhaustion. Among these regulatory mechanisms, cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed cell death 1 (PD-1) are the most well-known receptors and both have been targeted for drug development. As a result, anti-CTLA-4 and anti-PD-1 (or anti-PD-L1) antibodies were recently developed as immune checkpoint inhibitors for use in cancer treatments. In this review we describe several receptors that function as immunological checkpoints as well as the pharmaceuticals that target them.

  6. The fail-safe mechanism of post-transcriptional silencing of unspliced HAC1 mRNA

    PubMed Central

    Di Santo, Rachael; Aboulhouda, Soufiane; Weinberg, David E

    2016-01-01

    HAC1 encodes a transcription factor that is the central effector of the unfolded protein response (UPR) in budding yeast. When the UPR is inactive, HAC1 mRNA is stored as an unspliced isoform in the cytoplasm and no Hac1 protein is detectable. Intron removal is both necessary and sufficient to relieve the post-transcriptional silencing of HAC1 mRNA, yet the precise mechanism by which the intron prevents Hac1 protein accumulation has remained elusive. Here, we show that a combination of inhibited translation initiation and accelerated protein degradation—both dependent on the intron—prevents the accumulation of Hac1 protein when the UPR is inactive. Functionally, both components of this fail-safe silencing mechanism are required to prevent ectopic production of Hac1 protein and concomitant activation of the UPR. Our results provide a mechanistic understanding of HAC1 regulation and reveal a novel strategy for complete post-transcriptional silencing of a cytoplasmic mRNA. DOI: http://dx.doi.org/10.7554/eLife.20069.001 PMID:27692069

  7. Preferential skeletal muscle myosin loss in response to mechanical silencing in a novel rat intensive care unit model: underlying mechanisms

    PubMed Central

    Ochala, Julien; Gustafson, Ann-Marie; Diez, Monica Llano; Renaud, Guillaume; Li, Meishan; Aare, Sudhakar; Qaisar, Rizwan; Banduseela, Varuna C; Hedström, Yvette; Tang, Xiaorui; Dworkin, Barry; Ford, G Charles; Nair, K Sreekumaran; Perera, Sue; Gautel, Mathias; Larsson, Lars

    2011-01-01

    Abstract The muscle wasting and impaired muscle function in critically ill intensive care unit (ICU) patients delay recovery from the primary disease, and have debilitating consequences that can persist for years after hospital discharge. It is likely that, in addition to pernicious effects of the primary disease, the basic life support procedures of long-term ICU treatment contribute directly to the progressive impairment of muscle function. This study aims at improving our understanding of the mechanisms underlying muscle wasting in ICU patients by using a unique experimental rat ICU model where animals are mechanically ventilated, sedated and pharmacologically paralysed for duration varying between 6 h and 14 days. Results show that the ICU intervention induces a phenotype resembling the severe muscle wasting and paralysis associated with the acute quadriplegic myopathy (AQM) observed in ICU patients, i.e. a preferential loss of myosin, transcriptional down-regulation of myosin synthesis, muscle atrophy and a dramatic decrease in muscle fibre force generation capacity. Detailed analyses of protein degradation pathways show that the ubiquitin proteasome pathway is highly involved in this process. A sequential change in localisation of muscle-specific RING finger proteins 1/2 (MuRF1/2) observed during the experimental period is suggested to play an instrumental role in both transcriptional regulation and protein degradation. We propose that, for those critically ill patients who develop AQM, complete mechanical silencing, due to pharmacological paralysis or sedation, is a critical factor underlying the preferential loss of the molecular motor protein myosin that leads to impaired muscle function or persisting paralysis. PMID:21320889

  8. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons

    PubMed Central

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-01-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline. PMID:21764773

  9. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons.

    PubMed

    Shpiz, Sergey; Olovnikov, Ivan; Sergeeva, Anna; Lavrov, Sergey; Abramov, Yuri; Savitsky, Mikhail; Kalmykova, Alla

    2011-11-01

    In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

  10. Diverse gene-silencing mechanisms with distinct requirements for RNA polymerase subunits in Zea mays.

    PubMed

    Sloan, Amy E; Sidorenko, Lyudmila; McGinnis, Karen M

    2014-11-01

    In Zea mays, transcriptional regulation of the b1 (booster1) gene requires a distal enhancer and MEDIATOR OF PARAMUTATION1 (MOP1), MOP2, and MOP3 proteins orthologous to Arabidopsis components of the RNA-dependent DNA methylation pathway. We compared the genetic requirements for MOP1, MOP2, and MOP3 for endogenous gene silencing by two hairpin transgenes with inverted repeats of the a1 (anthocyaninless1) gene promoter (a1pIR) and the b1 gene enhancer (b1IR), respectively. The a1pIR transgene induced silencing of endogenous A1 in mop1-1 and mop3-1, but not in Mop2-1 homozygous plants. This finding suggests that transgene-derived small interfering RNAs (siRNAs) circumvented the requirement for MOP1, a predicted RNA-dependent RNA polymerase, and MOP3, the predicted largest subunit of RNA polymerase IV (Pol IV). Because the Arabidopsis protein orthologous to MOP2 is the second largest subunit of Pol IV and V, our results may indicate that hairpin-induced siRNAs cannot bypass the requirement for the predicted scaffolding activity of Pol V. In contrast to a1pIR, the b1IR transgene silenced endogenous B1 in all three homozygous mutant genotypes--mop1-1, Mop2-1, and mop3-1--suggesting that transgene mediated b1 silencing did not involve MOP2-containing Pol V complexes. Based on the combined results for a1, b1, and three previously described loci, we propose a speculative hypothesis of locus-specific deployment of Pol II, MOP2-containing Pol V, or alternative versions of Pol V with second largest subunits other than MOP2 to explain the mechanistic differences in silencing at specific loci, including one example associated with paramutation.

  11. Silencing MaxiK Activity in Corporal Smooth Muscle Cells Initiates Compensatory Mechanisms to Maintain Calcium Homeostasis

    PubMed Central

    Calenda, Giulia; Suadicani, Sylvia Ottilie; Iglesias, Rodolfo; Spray, David Conover; Melman, Arnold; Davies, Kelvin Paul

    2010-01-01

    Introduction The MaxiK potassium channel is regulated by voltage and intracellular calcium, and plays a critical role in regulating intracellular calcium concentration ([Ca2+]i), which is the ultimate determinant of smooth muscle tone. Tight control of corpus cavernosum smooth muscle (CCSM) tone is critically important and misregulation can result in erectile dysfunction. Aim Because of the tight functional linkage of MaxiK and calcium channel activity, the aim of this study was to determine the effects of silencing and pharmacological inhibition of MaxiK on calcium homeostasis and intercellular calcium signaling in CCSM cells. Methods We compared changes in the basal intracellular [Ca2+]i and parameters defining intercellular calcium wave (ICW) spread in 48 hours MaxiK silenced CCSM cells vs. acute blockade of the channel with iberiotoxin. To analyze changes occurring in gene expression we performed micro-array analysis following MaxiK silencing for 48 hours. Main Outcome Measures Changes in Fura-2 fluorescence intensities were measured to evaluate basal [Ca2+]i levels and ICW parameters. Microarray analysis of global gene expression was performed. Results Forty-eight hours after MaxiK silencing the basal [Ca2+]i, the ICW amplitude and spread among CCSM cells were not markedly different in silenced compared to mock transfected controls, whereas short-term blockade significantly increased basal [Ca2+]i level and amplified Ca2+ signaling among CCSM cells. Micro-array analysis showed that several genes within Ca2+ homeostasis and smooth muscle tone regulation pathways had significantly altered expression. Conclusions Our results indicate that while short-term blockade of the MaxiK channel is associated with an increase in basal [Ca2+]i, Ca2+ homeostasis is restored during the 48 hours period following silencing. We hypothesize that the different pathways regulating [Ca2+]i and CCSM tone are linked through molecular crosstalk and that their coordinated regulation is

  12. Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

    PubMed

    Rincón-Arano, Héctor; Guerrero, Georgina; Valdes-Quezada, Christian; Recillas-Targa, Félix

    2009-10-15

    Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

  13. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  14. A novel mechanism underlying the susceptibility of neuronal cells to nitric oxide: the occurrence and regulation of protein S-nitrosylation is the checkpoint.

    PubMed

    He, J; Wang, T; Wang, P; Han, P; Yin, Q; Chen, C

    2007-09-01

    The susceptibility of neuronal cells to nitric oxide (NO) is a key issue in NO-mediated neurotoxicity. However, the underlying mechanism remains unclear. As a cyclic guanosine monophosphate (cGMP)-independent NO signaling pathway, S-nitrosylation (or S-nitrosation) has been suggested to occur as a post-translational modification in parallel with O-phosphorylation. The underlying mechanism of the involvement of protein S-nitrosylation in the susceptibility of neuronal cells to NO has been little investigated. In this study, we focused on the role of S-nitrosothiols (RSNO) in the susceptibility of a cerebellar cell line R2 to NO. Our results showed the following: (i) S-nitrosoglutathione (GSNO) induced a burst of RSNO in GSH-depleted R2 cells, the majority of which were primarily contributed by the S-nitrosylation of proteins (Pro-SNOs), and was followed by severe neuronal necrosis; (ii) the elevation in the level of Pro-SNOs resulted from a dysfunction of S-nitroglutathione reductase (GSNOR) as a result of its substrate, GSNO, being unavailable in GSH-depleted cells. In the meantime, the suppression of GSNOR increased NO-mediated neurotoxicity in R2 cells, as well as in cerebellar granule neurons; (iii) Our results also demonstrate that the burst of RSNO is the "checkpoint" of cell fate: if RSNO can be reduced to free thiol proteins, cells will survive; if they are further oxidized, cells will die; and (iv) GSH-ethyl ester and Vitamin C protected R2 cells against GSNO neurotoxicity through two distinct mechanisms: by inhibiting the elevation of Pro-SNOs and by reducing Pro-SNOs to free thiol proteins, respectively. A novel mechanism underlying the susceptibility of neuronal cells to NO is proposed and some potential strategies to prevent the NO-mediated neurotoxicity are discussed.

  15. Toward a systems-level view of mitotic checkpoints.

    PubMed

    Ibrahim, Bashar

    2015-03-01

    Reproduction and natural selection are the key elements of life. In order to reproduce, the genetic material must be doubled, separated and placed into two new daughter cells, each containing a complete set of chromosomes and organelles. In mitosis, transition from one process to the next is guided by intricate surveillance mechanisms, known as the mitotic checkpoints. Dis-regulation of cell division through checkpoint malfunction can lead to developmental defects and contribute to the development or progression of tumors. This review approaches two important mitotic checkpoints, the spindle assembly checkpoint (SAC) and the spindle position checkpoint (SPOC). The highly conserved spindle assembly checkpoint (SAC) controls the onset of anaphase by preventing premature segregation of the sister chromatids of the duplicated genome, to the spindle poles. In contrast, the spindle position checkpoint (SPOC), in the budding yeast Saccharomyces cerevisiae, ensures that during asymmetric cell division mitotic exit does not occur until the spindle is properly aligned with the cell polarity axis. Although there are no known homologs, there is indication that functionally similar checkpoints exist also in animal cells. This review can be regarded as an "executable model", which could be easily translated into various quantitative concrete models like Petri nets, ODEs, PDEs, or stochastic particle simulations. It can also function as a base for developing quantitative models explaining the interplay of the various components and proteins controlling mitosis.

  16. Overlapped checkpointing with hardware assist

    SciTech Connect

    Mitchell, Christopher J; Nunez, James A; Wang, Jun

    2009-01-01

    We present a new approach to handling the demanding I/O workload incurred during checkpoint writes encountered in High Performance Computing. Prior efforts to improve performance have been primarily bound by mechanical limitations of the hard drive. Our research surpasses this limitation by providing a method to: (1) write checkpoint data to a high-speed, non-volatile buffer, and (2) asynchronously write this data to permanent storage while resuming computation. This removes the hard drive from the critical data path because our I/O node based buffers isolate the compute nodes from the storage servers. This solution is feasible because of industry declines in cost for high-capacity, non-volatile storage technologies. Testing was conducted on a small-scale cluster to prove the design, and then scaled at Los Alamos National Laboratory. Results show a definitive speedup factor for select workloads over writing directly to a typical global parallel file system; the Panasas ActiveScale File System.

  17. Hypothyroidism in Cancer Patients on Immune Checkpoint Inhibitors with anti-PD1 Agents: Insights on Underlying Mechanisms.

    PubMed

    Alhusseini, M; Samantray, J

    2017-01-10

    Background: Immune therapy using monoclonal antibodies against cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death 1 receptor (PD-1) for various cancers have been reported to cause thyroid dysfunction. Little is known, however, about the underlying pathogenic mechanisms and the course of hypothyroidism that subsequently develops. In this report, we use the change in thyroglobulin and thyroid antibody levels in patients on immune therapy who develop hypothyroidism to better understand its pathogenesis as well as examine the status of hypothyroidism in the long term. Methods: We report a case series of 10 patients who developed hypothyroidism after initiation of immune therapy (either anti-PD-1 alone or in combination with anti-CTLA-4). Available thyroid antibodies including anti-thyroglobulin (anti-Tg), anti-thyroid peroxidase (anti-TPO), and thyroid stimulating immunoglobulin (TSI) were noted during the initial thyroiditis phase as well as the hypothyroid phase. Persistence or remission of hypothyroidism was noted at 6 months. Summary: During the thyroiditis phase, 50% of the patients had elevated Tg titers, 40% had elevated anti-Tg, and 40% had elevated TSI. All of these titers decreased during the hypothyroid phase. Permanent hypothyroidism was noted in 80% of the cases. Conclusion: Hypothyroidism following initiation of immune therapy has immunologic and non-immunologic mediated mechanisms and is likely to be persistent.

  18. MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

    PubMed Central

    Heair, Hannah M.; Kemper, Austin G.; Roy, Bhaskar; Lopes, Helena B.; Rashid, Harunur; Clarke, John C.; Afreen, Lubana K.; Ferraz, Emanuela P.; Kim, Eddy; Javed, Amjad; Beloti, Marcio M.; MacDougall, Mary

    2015-01-01

    Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. PMID:26124283

  19. The Schizosaccharomyces pombe rad3 checkpoint gene.

    PubMed Central

    Bentley, N J; Holtzman, D A; Flaggs, G; Keegan, K S; DeMaggio, A; Ford, J C; Hoekstra, M; Carr, A M

    1996-01-01

    The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution. Images PMID:8978690

  20. Cooperative binding at a distance by even-skipped protein correlates with repression and suggests a mechanism of silencing.

    PubMed

    TenHarmsel, A; Austin, R J; Savenelli, N; Biggin, M D

    1993-05-01

    In this study, we examined how the Drosophila developmental control gene even-skipped (eve) represses transcription. Tissue culture cells were used to show that eve contains domains which inhibit transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Different portions of eve were fused to a heterologous DNA binding domain to show that three adjacent regions of eve contribute to silencing. There appear to be two mechanisms by which eve protein represses transcription. In this study, we used in vitro transcription and DNA binding experiments to provide evidence for one of these mechanisms. Repression in vitro correlates with binding of eve protein to two low-affinity sites in the Ubx proximal promoter. Occupancy of these low-affinity sites is dependent upon cooperative binding of other eve molecules to a separate high-affinity site. Some of these sites are separated by over 150 bp of DNA, and the data suggest that this intervening DNA is bent to form a looped structure similar to those caused by prokaryotic repressors. One of the low-affinity sites overlaps an activator element bound by the zeste transcription factor. Binding of eve protein is shown to exclude binding by zeste protein. These data suggest a mechanism for silencing whereby a repressor protein would be targeted to DNA by a high-affinity element, which itself does not overlap activator elements. Cooperative binding of further repressor molecules to distant low-affinity sites, and competition with activators bound at these sites lead to repression at a distance.

  1. Using the Sirocco File System for high-bandwidth checkpoints.

    SciTech Connect

    Klundt, Ruth Ann; Curry, Matthew L.; Ward, H. Lee

    2012-02-01

    The Sirocco File System, a file system for exascale under active development, is designed to allow the storage software to maximize quality of service through increased flexibility and local decision-making. By allowing the storage system to manage a range of storage targets that have varying speeds and capacities, the system can increase the speed and surety of storage to the application. We instrument CTH to use a group of RAM-based Sirocco storage servers allocated within the job as a high-performance storage tier to accept checkpoints, allowing computation to potentially continue asynchronously of checkpoint migration to slower, more permanent storage. The result is a 10-60x speedup in constructing and moving checkpoint data from the compute nodes. This demonstration of early Sirocco functionality shows a significant benefit for a real I/O workload, checkpointing, in a real application, CTH. By running Sirocco storage servers within a job as RAM-only stores, CTH was able to store checkpoints 10-60x faster than storing to PanFS, allowing the job to continue computing sooner. While this prototype did not include automatic data migration, the checkpoint was available to be pushed or pulled to disk-based storage as needed after the compute nodes continued computing. Future developments include the ability to dynamically spawn Sirocco nodes to absorb checkpoints, expanding this mechanism to other fast tiers of storage like flash memory, and sharing of dynamic Sirocco nodes between multiple jobs as needed.

  2. Keeping checkpoint/restart viable for exascale systems.

    SciTech Connect

    Riesen, Rolf E.; Bridges, Patrick G.; Stearley, Jon R.; Laros, James H., III; Oldfield, Ron A.; Arnold, Dorian; Pedretti, Kevin Thomas Tauke; Ferreira, Kurt Brian; Brightwell, Ronald Brian

    2011-09-01

    Next-generation exascale systems, those capable of performing a quintillion (10{sup 18}) operations per second, are expected to be delivered in the next 8-10 years. These systems, which will be 1,000 times faster than current systems, will be of unprecedented scale. As these systems continue to grow in size, faults will become increasingly common, even over the course of small calculations. Therefore, issues such as fault tolerance and reliability will limit application scalability. Current techniques to ensure progress across faults like checkpoint/restart, the dominant fault tolerance mechanism for the last 25 years, are increasingly problematic at the scales of future systems due to their excessive overheads. In this work, we evaluate a number of techniques to decrease the overhead of checkpoint/restart and keep this method viable for future exascale systems. More specifically, this work evaluates state-machine replication to dramatically increase the checkpoint interval (the time between successive checkpoint) and hash-based, probabilistic incremental checkpointing using graphics processing units to decrease the checkpoint commit time (the time to save one checkpoint). Using a combination of empirical analysis, modeling, and simulation, we study the costs and benefits of these approaches on a wide range of parameters. These results, which cover of number of high-performance computing capability workloads, different failure distributions, hardware mean time to failures, and I/O bandwidths, show the potential benefits of these techniques for meeting the reliability demands of future exascale platforms.

  3. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    PubMed Central

    Najor, Nicole A.; Weatherford, Layne; Brush, George S.

    2016-01-01

    In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control. PMID:27678521

  4. C. elegans RNA-dependent RNA polymerases rrf-1 and ego-1 silence Drosophila transgenes by differing mechanisms.

    PubMed

    Duan, Guowen; Saint, Robert B; Helliwell, Chris A; Behm, Carolyn A; Wang, Ming-Bo; Waterhouse, Peter M; Gordon, Karl H J

    2013-04-01

    Drosophila possesses the core gene silencing machinery but, like all insects, lacks the canonical RNA-dependent RNA polymerases (RdRps) that in C. elegans either trigger or enhance two major small RNA-dependent gene silencing pathways. Introduction of two different nematode RdRps into Drosophila showed them to be functional, resulting in differing silencing activities. While RRF-1 enhanced transitive dsRNA-dependent silencing, EGO-1 triggered dsRNA-independent silencing, specifically of transgenes. The strain w; da-Gal4; UAST-ego-1, constitutively expressing ego-1, is capable of silencing transgene including dsRNA hairpin upon a single cross, which created a powerful tool for research in Drosophila. In C. elegans, EGO-1 is involved in transcriptional gene silencing (TGS) of chromosome regions that are unpaired during meiosis. There was no opportunity for meiotic interactions involving EGO-1 in Drosophila that would explain the observed transgene silencing. Transgene DNA is, however, unpaired during the pairing of chromosomes in embryonic mitosis that is an unusual characteristic of Diptera, suggesting that in Drosophila, EGO-1 triggers transcriptional silencing of unpaired DNA during embryonic mitosis.

  5. Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco.

    PubMed

    Alamillo, Josefa M; Saénz, Pilar; García, Juan Antonio

    2006-10-01

    Plum pox virus (PPV) is able to replicate in inoculated leaves of Nicotiana tabacum, but is defective in systemic movement in this host. However, PPV produces a systemic infection in transgenic tobacco expressing the silencing suppressor P1/HC-Pro from tobacco etch virus (TEV). In this work we show that PPV is able to move to upper non-inoculated leaves of tobacco plants expressing bacterial salicylate hydroxylase (NahG) that degrades salicylic acid (SA). Replication and accumulation of PPV is higher in the locally infected leaves of plants deficient in SA or expressing TEV P1/HC-Pro silencing suppressor. Accumulation of viral derived small RNAs was reduced in the NahG transgenic plants, suggesting that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco. Besides, expression of SA-mediated defense transcripts, such as those of pathogenesis-related (PR) proteins PR-1 and PR-2 or alternative oxidase-1, as well as that of the putative RNA-dependent RNA polymerase NtRDR1, is induced in response to PPV infection, and the expression patterns of these defense transcripts are altered in the TEV P1/HC-Pro transgenic plants. Long-distance movement of PPV is highly enhanced in NahG x P1/HC-Pro double-transgenic plants and systemic symptoms in these plants reveal that the expression of an RNA-silencing suppressor and the lack of SA produce additive but distinct effects. Our results suggest that SA might act as an enhancer of the RNA-silencing antiviral defense in tobacco, and that silencing suppressors, such as P1/HC-Pro, also alter the SA-mediated defense. Both an RNA-silencing and an SA-mediated defense mechanism could act together to limit PPV infection.

  6. Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924.

    PubMed

    Blank, Jonathan L; Liu, Xiaozhen J; Cosmopoulos, Katherine; Bouck, David C; Garcia, Khristofer; Bernard, Hugues; Tayber, Olga; Hather, Greg; Liu, Ray; Narayanan, Usha; Milhollen, Michael A; Lightcap, Eric S

    2013-01-01

    MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

  7. Targeting immune checkpoints in malignant glioma

    PubMed Central

    Li, Tete; Liu, Yong-Jun; Chen, Wei; Chen, Jingtao

    2017-01-01

    Malignant glioma is the most common and a highly aggressive cancer in the central nervous system (CNS). Cancer immunotherapy, strategies to boost the bodys anti-cancer immune responses instead of directly targeting tumor cells, recently achieved great success in treating several human solid tumors. Although once considered immune privileged and devoid of normal immunological functions, CNS is now considered a promising target for cancer immunotherapy, featuring the recent progresses in neurobiology and neuroimmunology and a highly immunosuppressive state in malignant glioma. In this review, we focus on immune checkpoint inhibitors, specifically, antagonizing monoclonal antibodies for programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and indoleamine 2,3-dioxygenase (IDO). We discuss advances in the working mechanisms of these immune checkpoint molecules, their status in malignant glioma, and current preclinical and clinical trials targeting these molecules in malignant glioma. PMID:27756892

  8. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  9. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  10. Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

    PubMed Central

    Wang, Sidney H.; Elgin, Sarah C. R.

    2011-01-01

    Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line–specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi. PMID:22160707

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

    PubMed

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

    2015-05-08

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

  12. Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

    PubMed

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Pertíñez, Samuel Núñez; Busto, Jon V; Martín, César; Alagia, Adele; Goñi, Félix M; Eritja, Ramón; Alkorta, Itziar

    2017-01-01

    The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

  13. Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats.

    PubMed

    Starlard-Davenport, Athena; Tryndyak, Volodymyr P; James, Smitha R; Karpf, Adam R; Latendresse, John R; Beland, Frederick A; Pogribny, Igor P

    2010-03-01

    Breast cancer, the most common malignancy in women, emerges through a multistep process, encompassing the progressive sequential evolution of morphologically distinct stages from a normal cell to hyperplasia (with and without atypia), carcinoma in situ, invasive carcinoma and metastasis. The success of treatment of breast cancer could be greatly improved by the detection at early stages of cancer. In the present study, we investigated the underlying molecular mechanisms involved in breast carcinogenesis in Augustus and Copenhagen-Irish female rats, a cross between the ACI strains, induced by continuous exposure to 17beta-estradiol. The results of our study demonstrate that early stages of estrogen-induced breast carcinogenesis are characterized by altered global DNA methylation, aberrant expression of proteins responsible for the proper maintenance of DNA methylation pattern and epigenetic silencing of the critical Rassf1a (Ras-association domain family 1, isoform A) tumor suppressor gene. Interestingly, transcriptional repression of the Rassf1a gene in mammary glands during early stages of breast carcinogenesis was associated with an increase in trimethylation of histones H3 lysine 9 and H3 lysine 27 and de novo CpG island methylation and at the Rassf1a promoter and first exon. In conclusion, we demonstrate that epigenetic alterations precede formation of preneoplastic lesions indicating the significance of epigenetic events in induction of oncogenic pathways in early stages of carcinogenesis.

  14. New checkpoints in cancer immunotherapy.

    PubMed

    Ni, Ling; Dong, Chen

    2017-03-01

    Immune responses must be fine-tuned to allow effective clearance of invading pathogens, while maintain tolerance to self-antigens. T cells are the major effector cells for fighting and killing tumor cells. Immune checkpoints play a pivotal role in T cell activation, and determine the functional outcome of T cell receptor (TCR) signaling. The blockade of immune checkpoints CTLA-4 and PD-1 has already been one of the most successful cancer immunotherapies. In this review, we will focus on three novel inhibitory B7 family checkpoint molecules, B7-H3, B7S1 and VISTA. The aim of this article is to summarize their expressions in tumors as well as their roles in controlling and suppressing T cell immune responses and anti-tumor immunity. These pathways may be explored in future cancer immunotherapy.

  15. Network support for system initiated checkpoints

    DOEpatents

    Chen, Dong; Heidelberger, Philip

    2013-01-29

    A system, method and computer program product for supporting system initiated checkpoints in parallel computing systems. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity.

  16. Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery*

    PubMed Central

    Peng, Aimin; Wang, Ling; Fisher, Laura A.

    2011-01-01

    Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G2/M DNA damage checkpoint and efficient checkpoint recovery. PMID:21708943

  17. p31comet promotes disassembly of the mitotic checkpoint complex in an ATP-dependent process

    PubMed Central

    Teichner, Adar; Eytan, Esther; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Dumin, Elena; Gromis, Jonathan; Hershko, Avram

    2011-01-01

    Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31comet in this process. It is known that p31comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC. PMID:21300909

  18. Immune Checkpoint Inhibitors and Prostate Cancer: A New Frontier?

    PubMed Central

    Modena, Alessandra; Ciccarese, Chiara; Iacovelli, Roberto; Brunelli, Matteo; Montironi, Rodolfo; Fiorentino, Michelangelo; Tortora, Giampaolo; Massari, Francesco

    2016-01-01

    Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (mCRPC), agents that provide durable disease control and long-term survival are still needed. It is a fact that a tumor-induced immunosuppressive status (mediated by aberrant activation of inhibitory immune checkpoint pathways as a mechanism to evade host immune surveillance) plays a crucial role in the pathogenesis of cancer, including prostate cancer (PC), making CRPC patients suitable candidates for immunotherapy. Therefore, growing interest of anticancer research aims at blocking immune checkpoints (mainly targeting CTLA-4 and PD1/PD-L1 pathways) to restore and enhance cellular-mediated antitumor immunity and achieve durable tumor regression. In this review, we describe the current knowledge regarding the role of immune checkpoints in mediating PC progression, focusing on CTLA-4 and PD1 pathways. We also provide current clinical data available, an update on ongoing trials of immune checkpoint inhibitors in PC. Finally, we discuss the necessity to identify prognostic and predictive biomarkers of immune activity, and we analyze new immune checkpoints with a role as promising targets for PC therapy. PMID:27471580

  19. Kinetochore localization of spindle checkpoint proteins: who controls whom?

    PubMed

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-10-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other.

  20. Analysis of the Functionality of the Mitotic Checkpoints.

    PubMed

    Fraschini, Roberta

    2017-01-01

    During cell division the main goal of the cell is to produce two daughter cells with the same genome as the mother, i.e., maintain its genetic stability. Since this issue is essential to preserve the cell ability to proliferate properly, all eukaryotic cells have developed several pathways, called mitotic checkpoints, that regulate mitotic entry, progression, and exit in response to different cellular signals. Given the evolutive conservation of mechanisms and proteins involved in the cell cycle control from yeast to humans, the budding yeast S. cerevisiae has been very helpful to gain insight in these complex regulations. Here, we describe how the checkpoint can be activated and which cellular phenotypes can be used as markers of checkpoint activation.

  1. Non-volatile memory for checkpoint storage

    DOEpatents

    Blumrich, Matthias A.; Chen, Dong; Cipolla, Thomas M.; Coteus, Paul W.; Gara, Alan; Heidelberger, Philip; Jeanson, Mark J.; Kopcsay, Gerard V.; Ohmacht, Martin; Takken, Todd E.

    2014-07-22

    A system, method and computer program product for supporting system initiated checkpoints in high performance parallel computing systems and storing of checkpoint data to a non-volatile memory storage device. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity. In one embodiment, the non-volatile memory is a pluggable flash memory card.

  2. Checkpointing for a hybrid computing node

    DOEpatents

    Cher, Chen-Yong

    2016-03-08

    According to an aspect, a method for checkpointing in a hybrid computing node includes executing a task in a processing accelerator of the hybrid computing node. A checkpoint is created in a local memory of the processing accelerator. The checkpoint includes state data to restart execution of the task in the processing accelerator upon a restart operation. Execution of the task is resumed in the processing accelerator after creating the checkpoint. The state data of the checkpoint are transferred from the processing accelerator to a main processor of the hybrid computing node while the processing accelerator is executing the task.

  3. Antiviral silencing in animals.

    PubMed

    Li, Hong-Wei; Ding, Shou-Wei

    2005-10-31

    RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.

  4. MDC1 directs chromosome-wide silencing of the sex chromosomes in male germ cells.

    PubMed

    Ichijima, Yosuke; Ichijima, Misako; Lou, Zhenkun; Nussenzweig, André; Camerini-Otero, R Daniel; Chen, Junjie; Andreassen, Paul R; Namekawa, Satoshi H

    2011-05-01

    Chromosome-wide inactivation is an epigenetic signature of sex chromosomes. The mechanism by which the chromosome-wide domain is recognized and gene silencing is induced remains unclear. Here we identify an essential mechanism underlying the recognition of the chromosome-wide domain in the male germline. We show that mediator of DNA damage checkpoint 1 (MDC1), a binding partner of phosphorylated histone H2AX (γH2AX), defines the chromosome-wide domain, initiates meiotic sex chromosome inactivation (MSCI), and leads to XY body formation. Importantly, MSCI consists of two genetically separable steps. The first step is the MDC1-independent recognition of the unsynapsed axis by DNA damage response (DDR) factors such as ataxia telangiectasia and Rad3-related (ATR), TOPBP1, and γH2AX. The second step is the MDC1-dependent chromosome-wide spreading of DDR factors to the entire chromatin. Furthermore, we demonstrate that, in somatic cells, MDC1-dependent amplification of the γH2AX signal occurs following replicative stress and is associated with transcriptional silencing. We propose that a common DDR pathway underlies both MSCI and the response of somatic cells to replicative stress. These results establish that the DDR pathway centered on MDC1 triggers epigenetic silencing of sex chromosomes in germ cells.

  5. Space Reclamation for Uncoordinated Checkpointing in Message-Passing Systems. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min

    1993-01-01

    Checkpointing and rollback recovery are techniques that can provide efficient recovery from transient process failures. In a message-passing system, the rollback of a message sender may cause the rollback of the corresponding receiver, and the system needs to roll back to a consistent set of checkpoints called recovery line. If the processes are allowed to take uncoordinated checkpoints, the above rollback propagation may result in the domino effect which prevents recovery line progression. Traditionally, only obsolete checkpoints before the global recovery line can be discarded, and the necessary and sufficient condition for identifying all garbage checkpoints has remained an open problem. A necessary and sufficient condition for achieving optimal garbage collection is derived and it is proved that the number of useful checkpoints is bounded by N(N+1)/2, where N is the number of processes. The approach is based on the maximum-sized antichain model of consistent global checkpoints and the technique of recovery line transformation and decomposition. It is also shown that, for systems requiring message logging to record in-transit messages, the same approach can be used to achieve optimal message log reclamation. As a final topic, a unifying framework is described by considering checkpoint coordination and exploiting piecewise determinism as mechanisms for bounding rollback propagation, and the applicability of the optimal garbage collection algorithm to domino-free recovery protocols is demonstrated.

  6. Phenotypic checkpoints regulate neuronal development.

    PubMed

    Ben-Ari, Yehezkel; Spitzer, Nicholas C

    2010-11-01

    Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and constitute presymptomatic signatures of neurological disorders when they go awry.

  7. Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

    PubMed Central

    Forbes, K C; Humphrey, T; Enoch, T

    1998-01-01

    Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA. PMID:9832516

  8. The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis.

    PubMed

    Venkei, Zsolt G; Yamashita, Yukiko M

    2015-01-01

    Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. To our knowledge, the COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. Here, by establishing colcemid-induced microtubule depolymerization as a sensitive assay, we examined the characteristics of COC activity and find that it functions uniquely in GSCs but not in their differentiating progeny. We show that the COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types.

  9. Practising Silence in Teaching

    ERIC Educational Resources Information Center

    Forrest, Michelle

    2013-01-01

    The concept "silence" has diametrically opposed meanings; it connotes peace and contemplation as well as death and oblivion. Silence can also be considered a practice. There is keeping the rule of silence to still the mind and find inner truth, as well as forcibly silencing in the sense of subjugating another to one's own purposes.…

  10. NONO regulates the intra-S-phase checkpoint in response to UV radiation.

    PubMed

    Alfano, L; Costa, C; Caporaso, A; Altieri, A; Indovina, P; Macaluso, M; Giordano, A; Pentimalli, F

    2016-02-04

    The main risk factor for skin cancer is ultraviolet (UV) exposure, which causes DNA damage. Cells respond to UV-induced DNA damage by activating the intra-S-phase checkpoint, which prevents replication fork collapse, late origin firing and stabilizes fragile sites. Recently, the 54-kDa multifunctional protein NONO was found to be involved in the non-homologous end-joining DNA repair process and in poly ADP-ribose polymerase 1 activation. Interestingly, NONO is mutated in several tumour types and emerged as a crucial factor underlying both melanoma development and progression. Therefore, we set out to evaluate whether NONO could be involved in the DNA-damage response to UV radiations. We generated NONO-silenced HeLa cell clones and found that lack of NONO decreased cell growth rate. Then, we challenged NONO-silenced cells with exposure to UV radiations and found that NONO-silenced cells, compared with control cells, continued to synthesize DNA, failed to block new origin firing and impaired CHK1S345 phosphorylation showing a defective checkpoint activation. Consistently, NONO is present at the sites of UV-induced DNA damage where it localizes to RAD9 foci. To position NONO in the DNA-damage response cascade, we analysed the loading onto chromatin of various intra-S-phase checkpoint mediators and found that NONO favours the loading of topoisomerase II-binding protein 1 acting upstream of the ATM and Rad3-related kinase activity. Strikingly, re-expression of NONO, through an sh-resistant mRNA, rescued CHK1S345 phosphorylation in NONO-silenced cells. Interestingly, NONO silencing affected cell response to UV radiations also in a melanoma cell line. Overall, our data uncover a new role for NONO in mediating the cellular response to UV-induced DNA damage.

  11. Immune Checkpoint Modulators: An Emerging Antiglioma Armamentarium

    PubMed Central

    Kim, Eileen S.; Kim, Jennifer E.; Patel, Mira A.; Mangraviti, Antonella; Ruzevick, Jacob; Lim, Michael

    2016-01-01

    Immune checkpoints have come to the forefront of cancer therapies as a powerful and promising strategy to stimulate antitumor T cell activity. Results from recent preclinical and clinical studies demonstrate how checkpoint inhibition can be utilized to prevent tumor immune evasion and both local and systemic immune suppression. This review encompasses the key immune checkpoints that have been found to play a role in tumorigenesis and, more specifically, gliomagenesis. The review will provide an overview of the existing preclinical and clinical data, antitumor efficacy, and clinical applications for each checkpoint with respect to GBM, as well as a summary of combination therapies with chemotherapy and radiation. PMID:26881264

  12. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa.

    PubMed

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions.

  13. RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa

    PubMed Central

    Chicas, Agustin; Cogoni, Carlo; Macino, Giuseppe

    2004-01-01

    RNA interference (RNAi) can silence genes at the transcriptional level by targeting locus-specific Lys9H3 methylation or at the post-transcriptional level by targeting mRNA degradation. Here we have cloned and sequenced genomic regions methylated in Lys9H3 in Neurospora crassa to test the requirements for components of the RNAi pathway in this modification. We find that 90% of clones map to repeated sequences and relics of transposons that have undergone repeat-induced point mutations (RIP). We find siRNAs derived from transposon relics indicating that the RNAi machinery targets these regions. This is confirmed by the fact that the presence of these siRNAs depends on components of the RNAi pathway such as the RdRP (QDE-1), the putative RecQ helicase (QDE-3) and the two Dicer enzymes. We show that Lys9H3 methylation of RIP sequences is not affected in mutants of the RNAi pathway indicating that the RNAi machinery is not involved in transcriptional gene silencing in Neurospora. We find that RIP regions are transcribed and that the transcript level increases in the mutants of the RNAi pathway. These data suggest that the biological function of the Neurospora RNAi machinery is to control transposon relics and repeated sequences by targeting degradation of transcripts derived from these regions. PMID:15302921

  14. A conserved checkpoint monitors meiotic chromosome synapsis inCaenorhabditis elegans

    SciTech Connect

    Bhalla, Needhi; Dernburg, Abby F.

    2005-07-14

    We report the discovery of a checkpoint that monitorssynapsis between homologous chromosomes to ensure accurate meioticsegregation. Oocytes containing unsynapsed chromosomes selectivelyundergo apoptosis even if agermline DNA damage checkpoint is inactivated.This culling mechanism isspecifically activated by unsynapsed pairingcenters, cis-acting chromosomesites that are also required to promotesynapsis in Caenorhabditis elegans. Apoptosis due to synaptic failurealso requires the C. elegans homolog of PCH2,a budding yeast pachytenecheckpoint gene, which suggests that this surveillance mechanism iswidely conserved.

  15. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  16. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia

    PubMed Central

    Knaus, Hanna A.; Kanakry, Christopher G.; Luznik, Leo; Gojo, Ivana

    2016-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body’s own immune system to fight leukemic cells. PMID:25981611

  17. Immunomodulatory Drugs: Immune Checkpoint Agents in Acute Leukemia.

    PubMed

    Knaus, Hanna A; Kanakry, Christopher G; Luznik, Leo; Gojo, Ivana

    2017-01-01

    Intrinsic immune responses to acute leukemia are inhibited by a variety of mechanisms, such as aberrant antigen expression by leukemia cells, secretion of immunosuppressive cytokines and expression of inhibitory enzymes in the tumor microenvironment, expansion of immunoregulatory cells, and activation of immune checkpoint pathways, all leading to T cell dysfunction and/or exhaustion. Leukemic cells, similar to other tumor cells, hijack these inhibitory pathways to evade immune recognition and destruction by cytotoxic T lymphocytes. Thus, blockade of immune checkpoints has emerged as a highly promising approach to augment innate anti-tumor immunity in order to treat malignancies. Most evidence for the clinical efficacy of this immunotherapeutic strategy has been seen in patients with metastatic melanoma, where anti-CTLA-4 and anti-PD-1 antibodies have recently revolutionized treatment of this lethal disease with otherwise limited treatment options. To meet the high demand for new treatment strategies in acute leukemia, clinical testing of these promising therapies is commencing. Herein, we review the biology of multiple inhibitory checkpoints (including CTLA-4, PD-1, TIM-3, LAG-3, BTLA, and CD200R) and their contribution to immune evasion by acute leukemias. In addition, we discuss the current state of preclinical and clinical studies of immune checkpoint inhibition in acute leukemia, which seek to harness the body's own immune system to fight leukemic cells.

  18. Impact of histone H4K16 acetylation on the meiotic recombination checkpoint in Saccharomyces cerevisiae

    PubMed Central

    Cavero, Santiago; Herruzo, Esther; Ontoso, David; San-Segundo, Pedro A.

    2016-01-01

    In meiotic cells, the pachytene checkpoint or meiotic recombination checkpoint is a surveillance mechanism that monitors critical processes, such as recombination and chromosome synapsis, which are essential for proper distribution of chromosomes to the meiotic progeny. Failures in these processes lead to the formation of aneuploid gametes. Meiotic recombination occurs in the context of chromatin; in fact, the histone methyltransferase Dot1 and the histone deacetylase Sir2 are known regulators of the pachytene checkpoint in Saccharomyces cerevisiae. We report here that Sas2-mediated acetylation of histone H4 at lysine 16 (H4K16ac), one of the Sir2 targets, modulates meiotic checkpoint activity in response to synaptonemal complex defects. We show that, like sir2, the H4-K16Q mutation, mimicking constitutive acetylation of H4K16, eliminates the delay in meiotic cell cycle progression imposed by the checkpoint in the synapsis-defective zip1 mutant. We also demonstrate that, like in dot1, zip1-induced phosphorylation of the Hop1 checkpoint adaptor at threonine 318 and the ensuing Mek1 activation are impaired in H4-K16 mutants. However, in contrast to sir2 and dot1, the H4-K16R and H4-K16Q mutations have only a minor effect in checkpoint activation and localization of the nucleolar Pch2 checkpoint factor in ndt80-prophase-arrested cells. We also provide evidence for a cross-talk between Dot1-dependent H3K79 methylation and H4K16ac and show that Sir2 excludes H4K16ac from the rDNA region on meiotic chromosomes. Our results reveal that proper levels of H4K16ac orchestrate this meiotic quality control mechanism and that Sir2 impinges on additional targets to fully activate the checkpoint. PMID:28357333

  19. Intellectual property issues of immune checkpoint inhibitors

    PubMed Central

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  20. Traffic into silence: endomembranes and post-transcriptional RNA silencing

    PubMed Central

    Kim, Yun Ju; Maizel, Alexis; Chen, Xuemei

    2014-01-01

    microRNAs (miRNAs) and small interfering RNAs (siRNAs) are small RNAs that repress gene expression at the post-transcriptional level in plants and animals. Small RNAs guide Argonaute-containing RNA-induced silencing complexes to target RNAs in a sequence-specific manner, resulting in mRNA deadenylation followed by exonucleolytic decay, mRNA endonucleolytic cleavage, or translational inhibition. Although our knowledge of small RNA biogenesis, turnover, and mechanisms of action has dramatically expanded in the past decade, the subcellular location of small RNA-mediated RNA silencing still needs to be defined. In contrast to the prevalent presumption that RNA silencing occurs in the cytosol, emerging evidence reveals connections between the endomembrane system and small RNA activities in plants and animals. Here, we summarize the work that uncovered this link between small RNAs and endomembrane compartments and present an overview of the involvement of the endomembrane system in various aspects of RNA silencing. We propose that the endomembrane system is an integral component of RNA silencing that has been long overlooked and predict that a marriage between cell biology and RNA biology holds the key to a full understanding of post-transcriptional gene regulation by small RNAs. PMID:24668229

  1. Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

    PubMed Central

    Braunstein, Ilana; Miniowitz, Shirly; Moshe, Yakir; Hershko, Avram

    2007-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint [Sudakin V, Chan GKT, Yen TJ (2001) J Cell Biol 154:925–936]. We examined this problem by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5′-(γ-thio)triphosphate (ATP[γS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, we isolated inhibitory factors associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, we find that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration. PMID:17360335

  2. Regulation of AURORA B function by mitotic checkpoint protein MAD2.

    PubMed

    Shandilya, Jayasha; Medler, Kathryn F; Roberts, Stefan G E

    2016-08-17

    Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.

  3. RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

    PubMed Central

    Sifuentes-Romero, Itzel; Merchant-Larios, Horacio; Milton, Sarah L.; Moreno-Mendoza, Norma; Díaz-Hernández, Verónica; García-Gasca, Alejandra

    2013-01-01

    The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination. PMID:24705165

  4. Abscission checkpoint control: stuck in the middle with Aurora B.

    PubMed

    Carmena, Mar

    2012-07-01

    At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges 'stuck' in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.

  5. Hitchcock's Melodramatic Silence.

    ERIC Educational Resources Information Center

    Hemmeter, Thomas

    1996-01-01

    Argues that the filmwork of Alfred Hitchcock shows his manipulation of melodramatic silence in that his films demonstrate a link between silence and truth. Concludes that in the simultaneous longing for and denial of the power of film silence lies the modernist complexity of Hitchcock's films that suggests the uses of melodramatic language in a…

  6. The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

    PubMed

    Pérez-Hidalgo, Livia; Moreno, Sergio; San-Segundo, Pedro A

    2008-12-01

    In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1(+) as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

  7. Integrated Circuits: How Transcriptional Silencing and Counter-Silencing Facilitate Bacterial Evolution

    PubMed Central

    Will, W. Ryan; Navarre, William W.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer is a major contributor to bacterial evolution and diversity. For a bacterial cell to utilize newly-acquired traits such as virulence and antibiotic resistance, new genes must be integrated into the existing regulatory circuitry to allow appropriate expression. Xenogeneic silencing of horizontally-acquired genes by H-NS or other nucleoid-associated proteins avoids adventitious expression and can be relieved by other DNA-binding counter-silencing proteins in an environmentally- and physiologically-responsive manner. Biochemical and genetic analyses have recently demonstrated that counter-silencing can occur at a variety of promoter architectures, in contrast to classical transcriptional activation. Disruption of H-NS nucleoprotein filaments by DNA bending is a suggested mechanism by which silencing can be relieved. This review discusses recent advances in our understanding of the mechanisms and importance of xenogeneic silencing and counter-silencing in the successful integration of horizontally-acquired genes into regulatory networks. PMID:25461567

  8. Checkpoint inhibitors in Hodgkin's lymphoma.

    PubMed

    Jezeršek Novaković, Barbara

    2016-04-01

    Hodgkin's lymphoma is unusual among cancers in that it consists of a small number of malignant Hodgkin/Reed-Sternberg cells in a sea of immune system cells, including T cells. Most of these T cells are reversibly inactivated in different ways and their reactivation may induce a very strong immune response to cancer cells. One way of reactivation of T cells is with antibodies blocking the CTLA-4 and especially with antibodies directed against PD-1 or the PD-L1 ligand thereby reversing the tumor-induced downregulation of T-cell function and augmenting antitumor immune activity at the priming (CTLA-4) or tissue effector (PD-1) phase. Immune checkpoint inhibitors have been evidenced as an additional treatment option with substantial effectiveness and acceptable toxicity in heavily pretreated patients with Hodgkin's lymphoma. Particularly, PD-1 blockade with nivolumab and pembrolizumab has demonstrated significant single-agent activity in this select population.

  9. Immune-Checkpoint Inhibitors in the Era of Precision Medicine: What Radiologists Should Know

    PubMed Central

    Tirumani, Sree Harsha; Hodi, Frank Stephen; Nishino, Mizuki

    2017-01-01

    Over the past five years immune-checkpoint inhibitors have dramatically changed the therapeutic landscape of advanced solid and hematologic malignancies. The currently approved immune-checkpoint inhibitors include antibodies to cytotoxic T-lymphocyte antigen-4, programmed cell death (PD-1), and programmed cell death ligand (PD-L1 and PD-L2). Response to immune-checkpoint inhibitors is evaluated on imaging using the immune-related response criteria. Activation of immune system results in a unique toxicity profile termed immune-related adverse events. This article will review the molecular mechanism, clinical applications, imaging of immune-related response patterns and adverse events associated with immune-checkpoint inhibitors. PMID:28096717

  10. Immune-Checkpoint Inhibitors in the Era of Precision Medicine: What Radiologists Should Know.

    PubMed

    Braschi-Amirfarzan, Marta; Tirumani, Sree Harsha; Hodi, Frank Stephen; Nishino, Mizuki

    2017-01-01

    Over the past five years immune-checkpoint inhibitors have dramatically changed the therapeutic landscape of advanced solid and hematologic malignancies. The currently approved immune-checkpoint inhibitors include antibodies to cytotoxic T-lymphocyte antigen-4, programmed cell death (PD-1), and programmed cell death ligand (PD-L1 and PD-L2). Response to immune-checkpoint inhibitors is evaluated on imaging using the immune-related response criteria. Activation of immune system results in a unique toxicity profile termed immune-related adverse events. This article will review the molecular mechanism, clinical applications, imaging of immune-related response patterns and adverse events associated with immune-checkpoint inhibitors.

  11. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

    PubMed Central

    Kitagawa, Mayumi; Caldez, Matias J.; Gunaratne, Jayantha; Lee, Sang Hyun

    2016-01-01

    The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. PMID:27631493

  12. Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells.

    PubMed

    Forn, Marta; Muñoz, Mar; Tauriello, Daniele V F; Merlos-Suárez, Anna; Rodilla, Verónica; Bigas, Anna; Batlle, Eduard; Jordà, Mireia; Peinado, Miguel A

    2013-12-01

    DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.

  13. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions

    PubMed Central

    Ólafsson, Guðjón; Thorpe, Peter H.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  14. Checkpoint inhibitors in lung cancer: latest developments and clinical potential

    PubMed Central

    Schvartsman, Gustavo; Ferrarotto, Renata; Massarelli, Erminia

    2016-01-01

    Lung cancer is the leading cause of cancer death in the United States. The vast majority of patients are diagnosed with metastatic disease with a 5-year survival rate of less than 5%. After first-line chemotherapy or biomarker-matched targeted therapy, only suitable for a small group of patients, further systemic therapy options rendered very limited, if any, benefit until recently. Checkpoint inhibitors have significantly improved outcomes in patients with metastatic non-small cell lung cancer (NSCLC) and are currently an established second-line therapeutic option. In this manuscript, we review the mechanism of action of checkpoint inhibitors, present the available data with approved and experimental agents, discuss the progress that has already been made in the field, as well as toxicity awareness, and future perspectives. PMID:27800034

  15. Update on immune checkpoint inhibitors in gynecological cancers

    PubMed Central

    2017-01-01

    In recent years, progress in our understanding of immune-modulatory signaling pathways in immune cells and the tumor microenvironment (TME) has led to rejuvenated interest in cancer immunotherapy. In particular, immunotherapy targeting the immune checkpoint receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell-death 1 (PD-1), and programmed cell-death ligand 1 (PD-L1) have demonstrated clinical activity in a wide variety of tumors, including gynecological cancers. This review will focus on the emerging clinical data on the therapeutic role of immune checkpoint inhibitors, and potential strategies to enhance the efficacy of this class of compounds, in the context of gynecological cancers. It is anticipated that future biomarker-directed clinical trials will provide further insights into the mechanisms underlying response and resistance to immunotherapy, and help guide our approach to designing therapeutic combinations that have the potential to enhance the benefit of immunotherapy in patients with gynecologic cancers. PMID:28028993

  16. Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation

    PubMed Central

    Johmura, Yoshikazu; Yamashita, Emiri; Shimada, Midori; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients. PMID:27507734

  17. Immune checkpoint therapy for pancreatic cancer

    PubMed Central

    Johansson, Henrik; Andersson, Roland; Bauden, Monika; Hammes, Sarah; Holdenrieder, Stefan; Ansari, Daniel

    2016-01-01

    Novel treatment modalities are necessary for pancreatic cancer. Immunotherapy with immune checkpoint inhibition has shown effect in other solid tumors, and could have a place in pancreatic cancer treatment. Most available clinical studies on immune checkpoint inhibitors for pancreatic cancer are not yet completed and are still recruiting patients. Among the completed trials, there have been findings of a preliminary nature such as delayed disease progression and enhanced overall survival after treatment with immune checkpoint inhibitors in mono- or combination therapy. However, due to small sample sizes, major results are not yet identifiable. The present article provides a clinical overview of immune checkpoint inhibition in pancreatic cancer. PubMed, ClinicalTrials.gov and American Society of Clinical Oncology’s meeting abstracts were systematically searched for relevant clinical studies. Four articles, five abstracts and 25 clinical trials were identified and analyzed in detail. PMID:27920468

  18. Optimal message log reclamation for uncoordinated checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. K.

    1994-01-01

    Uncoordinated checkpointing for message-passing systems allows maximum process autonomy and general nondeterministic execution, but suffers from potential domino effect and the large space overhead for maintaining checkpoints and message logs. Traditionally, it has been assumed that only obsolete checkpoints and message logs before the global recovery line can be garbage-collected. Recently, an approach to identifying all garbage checkpoints based on recovery line transformation and decomposition has been developed. We show in this paper that the same approach can be applied to the problem of identifying all garbage message logs for systems requiring message logging to record in-transit messages. Communication trace-driven simulation for several parallel programs is used to evaluate the proposed algorithm.

  19. Characteristics of post-transcriptional gene silencing.

    PubMed

    Chicas, A; Macino, G

    2001-11-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins.

  20. Characteristics of post-transcriptional gene silencing

    PubMed Central

    Chicas, Agustin; Macino, Giuseppe

    2001-01-01

    A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins. PMID:11713190

  1. Disassembly of mitotic checkpoint complexes by the joint action of the AAA-ATPase TRIP13 and p31comet

    PubMed Central

    Eytan, Esther; Wang, Kexi; Miniowitz-Shemtov, Shirly; Sitry-Shevah, Danielle; Kaisari, Sharon; Yen, Tim J.; Liu, Song-Tao; Hershko, Avram

    2014-01-01

    The mitotic (or spindle assembly) checkpoint system delays anaphase until all chromosomes are correctly attached to the mitotic spindle. When the checkpoint is active, a Mitotic Checkpoint Complex (MCC) assembles and inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C). MCC is composed of the checkpoint proteins Mad2, BubR1, and Bub3 associated with the APC/C activator Cdc20. When the checkpoint signal is turned off, MCC is disassembled and the checkpoint is inactivated. The mechanisms of the disassembly of MCC are not sufficiently understood. We have previously observed that ATP hydrolysis is required for the action of the Mad2-binding protein p31comet to disassemble MCC. We now show that HeLa cell extracts contain a factor that promotes ATP- and p31comet-dependent disassembly of a Cdc20–Mad2 subcomplex and identify it as Thyroid Receptor Interacting Protein 13 (TRIP13), an AAA-ATPase known to interact with p31comet. The joint action of TRIP13 and p31comet also promotes the release of Mad2 from MCC, participates in the complete disassembly of MCC and abrogates checkpoint inhibition of APC/C. We propose that TRIP13 plays centrally important roles in the sequence of events leading to MCC disassembly and checkpoint inactivation. PMID:25092294

  2. Plant RNA silencing in viral defence.

    PubMed

    Pantaleo, Vitantonio

    2011-01-01

    RNA silencing is described in plants and insects as a defence mechanism against foreign nucleic acids, such as invading viruses. The RNA silencing-based antiviral defence involves the production of virus-derived small interfering RNAs and their association to effector proteins, which together drive the sequence specific inactivation of viruses. The entire process of antiviral defence 'borrows' several plant factors involved in other specialized RNA silencing endogenous pathways. Different viruses use variable strategies to infect different host plants, which render the antiviral RNA silencing a complex phenomenon far to be completely clarified. This chapter reports current advances in understanding the main steps of the plant's RNA-silencing response to viral invasion and discusses some of the key questions still to be answered.

  3. Structural basis of checkpoint blockade by monoclonal antibodies in cancer immunotherapy

    PubMed Central

    Lee, Ju Yeon; Lee, Hyun Tae; Shin, Woori; Chae, Jongseok; Choi, Jaemo; Kim, Sung Hyun; Lim, Heejin; Won Heo, Tae; Park, Kyeong Young; Lee, Yeon Ji; Ryu, Seong Eon; Son, Ji Young; Lee, Jee Un; Heo, Yong-Seok

    2016-01-01

    Cancer cells express tumour-specific antigens derived via genetic and epigenetic alterations, which may be targeted by T-cell-mediated immune responses. However, cancer cells can avoid immune surveillance by suppressing immunity through activation of specific inhibitory signalling pathways, referred to as immune checkpoints. In recent years, the blockade of checkpoint molecules such as PD-1, PD-L1 and CTLA-4, with monoclonal antibodies has enabled the development of breakthrough therapies in oncology, and four therapeutic antibodies targeting these checkpoint molecules have been approved by the FDA for the treatment of several types of cancer. Here, we report the crystal structures of checkpoint molecules in complex with the Fab fragments of therapeutic antibodies, including PD-1/pembrolizumab, PD-1/nivolumab, PD-L1/BMS-936559 and CTLA-4/tremelimumab. These complex structures elucidate the precise epitopes of the antibodies and the molecular mechanisms underlying checkpoint blockade, providing useful information for the improvement of monoclonal antibodies capable of attenuating checkpoint signalling for the treatment of cancer. PMID:27796306

  4. Silence Amenity Engineering

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime

    Engineering civilization brought convenient and comfortable life to us. However, some environmental problems such as various pollutions have also been developed with it. Acoustical noise is one of the major problems in modern life. Noise is generated from a noise source and propagates through transmitting medium such as the air and eventually reaches a receiver, usually a human being. The noise problem can be avoided, therefore, if one of those three elements in the noise problem is removed completely. In actual case, engineers are looking for most efficient way combining the controls for these three elements. In this article, basic characteristics of noise is reviewed briefly at first, then sound field analysis to predict sound transmission is discussed Aerodynamic noise is one of the major problems in silence amenity engineering today. Basic concept of the aerodynamic noise generation mechanism is discussed in detail with applications to turbo-machinery and high speed train noise control technology.

  5. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    DOE PAGES

    Di, Sheng; Robert, Yves; Vivien, Frederic; ...

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2more » deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.« less

  6. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    SciTech Connect

    Di, Sheng; Robert, Yves; Vivien, Frederic; Cappello, Franck

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2 deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.

  7. Emerging role of checkpoint blockade therapy in lymphoma

    PubMed Central

    Galanina, Natalie; Kline, Justin; Bishop, Michael R.

    2017-01-01

    Following the successful application of immune checkpoint blockade therapy (CBT) in refractory solid tumors, it has recently gained momentum as a promising modality in the treatment of relapsed lymphoma. This significant therapeutic advance stems from decades of research that elucidated the role of immune regulation pathways and the mechanisms by which tumors can engage these critical pathways to escape immune detection. To date, two main pathways, the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1), have emerged as key targets of CBT demonstrating unprecedented activity particularly in heavily pretreated relapsed/refractory Hodgkin lymphoma and some forms of non-Hodgkin disease. Herein we provide a brief discussion of checkpoint blockade in the context of lymphoma biology with a specific focus on novel checkpoint inhibitors and their therapeutic activity. We discuss current clinical trials and the landscape of CBT to underscore both the remarkable progress and foreseeable limitations of this novel treatment strategy. In particular, we build upon state-of-the-art knowledge and clinical insights gained from the early trials to review potential approaches to how CBT may be integrated with other treatment modalities, including chemoimmunotherapy to improve patient outcomes in the future. Finally, as the role of CBT evolves to potentially become a cornerstone of therapy in refractory/relapsed lymphoma, we briefly emphasize the importance of predictive biomarkers in an effort to select appropriate patients who are most likely to derive benefit from CBT. PMID:28203344

  8. Immune Checkpoint Blockade and Hematopoietic Stem Cell Transplant.

    PubMed

    Merryman, Reid W; Armand, Philippe

    2017-02-01

    Allogeneic hematopoietic stem cell transplant (HSCT) relies primarily upon graft-versus-tumor activity for cancer eradication. Relapse remains the principal cause of treatment failure after HSCT, implying frequent immune escape, which in at least some cases, appears to be mediated by increased expression of inhibitory immune checkpoints. In an attempt to restore anti-tumor immunity, checkpoint blockade therapy (CBT) targeting PD-1 and CLTA-4 has been used in conjunction with both allogeneic and autologous HSCT. Clinical experience in this setting is limited to several small clinical trials and case series, but together they suggest that treatment with CBT can effectively amplify anti-tumor immune responses. However, intrinsic to its mechanism is also the risk that CBT in the HSCT setting may also cause significant immune toxicity. Fatal immune-related adverse events and graft-versus-host disease have been observed, but in most cases, immune side effects appear to be reversible with steroids and CBT discontinuation. As clinical investigation continues, improved understanding of immune checkpoint biology will be critical to optimize safe and efficacious treatment strategies.

  9. Checkpoint inhibition: new treatment options in urologic cancer.

    PubMed

    De Maeseneer, Daan Joost; Delafontaine, Brant; Rottey, Sylvie

    2017-02-01

    Both urothelial (UC) and renal cell cancer (RCC) are highly immunogenic tumours. Recent advances in cellular immunity understanding have resulted in a successful new class of therapeutic agents. Interaction between the programmed cell death 1 (PD1) on regulatory T-cells (Treg) and programmed cell death 1 ligand (PDL1) on cancer cells inhibits an effective immune response and is an important mechanism for cancer cells to evade the immune system. Monoclonal anti-PD1 and anti-PDL1 antibodies inhibit this interaction and are called checkpoint inhibitors. As in non-small lung cancer and melanoma, these agents have shown to be highly active agents in UC and RCC. In metastatic or inoperable UC, no good second line therapy exists and checkpoint inhibitors have shown to be active. Multiple Randomized Clinical Trials (RCT) are underway both in second line and adjuvant settings which could change daily practice. In RCC, an anti-PD1 antibody, Nivolumab, has already proven to be effective in prolonging overall survival, and is included in current guidelines in metastatic RCC in second and third line. RCTs with other anti-PD1 and anti-PDL1 antibodies are ongoing both in metastatic and adjuvant setting. Checkpoint inhibitors have breathed new life into immunotherapy in urologic cancers and are rapidly being included in practice guidelines.

  10. Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

    PubMed

    Ratsima, Hery; Serrano, Diego; Pascariu, Mirela; D'Amours, Damien

    2016-02-16

    Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.

  11. Choreography of the 9-1-1 checkpoint complex: DDK puts a check on the checkpoints.

    PubMed

    Paek, Andrew L; Weinert, Ted

    2010-11-24

    Checkpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.

  12. Monitoring spindle orientation: Spindle position checkpoint in charge.

    PubMed

    Caydasi, Ayse K; Ibrahim, Bashar; Pereira, Gislene

    2010-12-11

    Every cell division in budding yeast is inherently asymmetric and counts on the correct positioning of the mitotic spindle along the mother-daughter polarity axis for faithful chromosome segregation. A surveillance mechanism named the spindle position checkpoint (SPOC), monitors the orientation of the mitotic spindle and prevents cells from exiting mitosis when the spindle fails to align along the mother-daughter axis. SPOC is essential for maintenance of ploidy in budding yeast and similar mechanisms might exist in higher eukaryotes to ensure faithful asymmetric cell division. Here, we review the current model of SPOC activation and highlight the importance of protein localization and phosphorylation for SPOC function.

  13. Asynchronous Checkpoint Migration with MRNet in the Scalable Checkpoint / Restart Library

    SciTech Connect

    Mohror, K; Moody, A; de Supinski, B R

    2012-03-20

    Applications running on today's supercomputers tolerate failures by periodically saving their state in checkpoint files on stable storage, such as a parallel file system. Although this approach is simple, the overhead of writing the checkpoints can be prohibitive, especially for large-scale jobs. In this paper, we present initial results of an enhancement to our Scalable Checkpoint/Restart Library (SCR). We employ MRNet, a tree-based overlay network library, to transfer checkpoints from the compute nodes to the parallel file system asynchronously. This enhancement increases application efficiency by removing the need for an application to block while checkpoints are transferred to the parallel file system. We show that the integration of SCR with MRNet can reduce the time spent in I/O operations by as much as 15x. However, our experiments exposed new scalability issues with our initial implementation. We discuss the sources of the scalability problems and our plans to address them.

  14. ZO-2 silencing induces renal hypertrophy through a cell cycle mechanism and the activation of YAP and the mTOR pathway

    PubMed Central

    Domínguez-Calderón, Alaide; Ávila-Flores, Antonia; Ponce, Arturo; López-Bayghen, Esther; Calderón-Salinas, José-Víctor; Luis Reyes, José; Chávez-Munguía, Bibiana; Segovia, José; Angulo, Carla; Ramírez, Leticia; Gallego-Gutiérrez, Helios; Alarcón, Lourdes; Martín-Tapia, Dolores; Bautista-García, Pablo; González-Mariscal, Lorenza

    2016-01-01

    Renal compensatory hypertrophy (RCH) restores normal kidney function after disease or loss of kidney tissue and is characterized by an increase in organ size due to cell enlargement and not to cell proliferation. In MDCK renal epithelial cells, silencing of the tight junction protein zona occludens 2 (ZO-2 KD) induces cell hypertrophy by two mechanisms: prolonging the time that cells spend at the G1 phase of the cell cycle due to an increase in cyclin D1 level, and augmenting the rate of protein synthesis. The latter is triggered by the nuclear accumulation and increased transcriptional activity of Yes-associated protein (YAP), the main target of the Hippo pathway, which results in decreased expression of phosphatase and tensin homologue. This in turn increased the level of phosphatidylinositol (3,4,5)-triphosphate, which transactivates the Akt/mammalian target of rapamycin pathway, leading to activation of the kinase S6K1 and increased synthesis of proteins and cell size. In agreement, in a rat model of uninephrectomy, RCH is accompanied by decreased expression of ZO-2 and nuclear expression of YAP. Our results reveal a novel role of ZO-2 as a modulator of cell size. PMID:27009203

  15. Mechanism of fiber carcinogenesis: from reactive radical species to silencing of the beta igH3 gene.

    PubMed

    Hei, Tom K; Xu, An; Huang, Sarah X; Zhao, Yongliang

    2006-11-01

    Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.

  16. CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

    PubMed

    Kashima, Lisa; Idogawa, Masashi; Mita, Hiroaki; Shitashige, Miki; Yamada, Tesshi; Ogi, Kazuhiro; Suzuki, Hiromu; Toyota, Minoru; Ariga, Hiroyoshi; Sasaki, Yasushi; Tokino, Takashi

    2012-04-13

    The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

  17. Checkpoint triggering in a computer system

    SciTech Connect

    Cher, Chen-Yong

    2016-09-06

    According to an aspect, a method for triggering creation of a checkpoint in a computer system includes executing a task in a processing node of the computer system and determining whether it is time to read a monitor associated with a metric of the task. The monitor is read to determine a value of the metric based on determining that it is time to read the monitor. A threshold for triggering creation of the checkpoint is determined based on the value of the metric. Based on determining that the value of the metric has crossed the threshold, the checkpoint including state data of the task is created to enable restarting execution of the task upon a restart operation.

  18. A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

    PubMed Central

    DeMoe, Joanna H.; Santaguida, Stefano; Daum, John R.; Musacchio, Andrea; Gorbsky, Gary J.

    2008-01-01

    In mitosis the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate (1). The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Since complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. PMID:19190331

  19. Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing[OPEN

    PubMed Central

    Fultz, Dalen

    2017-01-01

    The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here, we revealed two broad mechanisms that can initiate silencing independently: identity-based and expression-dependent silencing. We found that identity-based silencing is targeted by 21- to 22-nucleotide or 24-nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome. By transforming exogenous TEs into Arabidopsis thaliana, we circumvented identity-based silencing, allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We found that several siRNA-generating mechanisms all trigger de novo expression-dependent RNA-directed DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed by introgression into the wild type does not result in the same level of silencing as direct transformation into the wild type. This demonstrates that the plant genotype during a narrow window of time at TE insertion (or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing. PMID:28193737

  20. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

  1. Optimal message log reclamation for independent checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1993-01-01

    Independent (uncoordinated) check pointing for parallel and distributed systems allows maximum process autonomy but suffers from possible domino effects and the associated storage space overhead for maintaining multiple checkpoints and message logs. In most research on check pointing and recovery, it was assumed that only the checkpoints and message logs older than the global recovery line can be discarded. It is shown how recovery line transformation and decomposition can be applied to the problem of efficiently identifying all discardable message logs, thereby achieving optimal garbage collection. Communication trace-driven simulation for several parallel programs is used to show the benefits of the proposed algorithm for message log reclamation.

  2. Transcriptional gene silencing in humans

    PubMed Central

    Weinberg, Marc S.; Morris, Kevin V.

    2016-01-01

    It has been over a decade since the first observation that small non-coding RNAs can functionally modulate epigenetic states in human cells to achieve functional transcriptional gene silencing (TGS). TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway. TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division, whereas RNAi does not. Early studies of TGS have been largely overlooked, overshadowed by subsequent discoveries of small RNA-directed post-TGS and RNAi. A reappraisal of early work has been brought about by recent findings in human cells where endogenous long non-coding RNAs function to regulate the epigenome. There are distinct and common overlaps between the proteins involved in small and long non-coding RNA transcriptional regulatory mechanisms, suggesting that the early studies using small non-coding RNAs to modulate transcription were making use of a previously unrecognized endogenous mechanism of RNA-directed gene regulation. Here we review how non-coding RNA plays a role in regulation of transcription and epigenetic gene silencing in human cells by revisiting these earlier studies and the mechanistic insights gained to date. We also provide a list of mammalian genes that have been shown to be transcriptionally regulated by non-coding RNAs. Lastly, we explore how TGS may serve as the basis for development of future therapeutic agents. PMID:27060137

  3. Nickel and Epigenetic Gene Silencing

    PubMed Central

    Sun, Hong; Shamy, Magdy; Costa, Max

    2013-01-01

    Insoluble nickel compounds are well-established human carcinogens. Occupational exposure to these compounds leads to increased incidence of lung and nasal cancer in nickel refinery workers. Apart from its weak mutagenic activity and hypoxia mimicking effect there is mounting experimental evidence indicating that epigenetic alteration plays an important role in nickel-induced carcinogenesis. Multiple epigenetic mechanisms have been identified to mediate nickel-induced gene silencing. Nickel ion is able to induce heterochromatinization by binding to DNA-histone complexes and initiating chromatin condensation. The enzymes required for establishing or removing epigenetic marks can be targeted by nickel, leading to altered DNA methylation and histone modification landscapes. The current review will focus on the epigenetic changes that contribute to nickel-induced gene silencing. PMID:24705264

  4. The Mitotic Checkpoint Gene, SIL is Regulated by E2F1

    PubMed Central

    Erez, Ayelet; Chaussepied, Marie; Tina, Colaizzo-Anas; Aplan, Peter; Ginsberg, Doron; Izraeli, Shai

    2009-01-01

    The SIL gene expression is increased in multiple cancers and correlates with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. SIL regulates mitotic entry, organization of the mitotic spindle and cell survival. The E2F transcription factors regulate cell cycle progression by controlling the expression of genes mediating the G1/S transition. More recently E2F has been shown to regulate mitotic spindle checkpoint genes as well. As SIL expression correlates with mitotic checkpoint genes we hypothesized that SIL is regulated by E2F. We mined raw data of published experiments and performed new experiments by modification of E2F expression in cell lines, reporter assays and chromatin immunoprecipitation. Ectopic expression or endogenous activation of E2F induced the expression of SIL, while knockdown of E2F by shRNA, downregulated SIL expression. E2F activated SIL promoter by reporter assay and bound to SIL promoter in-vivo. Taken together these data demonstrate that SIL is regulated by E2F. As SIL is essential for mitotic entry, E2F may regulate G2/M transition through the induction of SIL. Furthermore, as silencing of SIL cause apoptosis in cancer cells, these finding may have therapeutic relevance in tumors with constitutive activation of E2F. PMID:18649360

  5. Aberrantly Silenced Promoters Retain a Persistent Memory of the Silenced State After Long-Term Reactivation

    PubMed Central

    Oyer, Jon A.; Yates, Phillip A.; Godsey, Sarah; Turker, Mitchell S.

    2010-01-01

    A hallmark of aberrant DNA methylation-associated silencing is reversibility. However, long-term stability of reactivated promoters has not been explored. To examine this issue, spontaneous reactivant clones were isolated from mouse embryonal carcinoma cells bearing aberrantly silenced Aprt alleles and re-silencing frequencies were determined as long as three months after reactivation occurred. Despite continuous selection for expression of the reactivated Aprt alleles, exceptionally high spontaneous re-silencing frequencies were observed. A DNA methylation analysis demonstrated retention of sporadic methylation of CpG sites in a protected region of the Aprt promoter in many reactivant alleles suggesting a role for these methylated sites in the re-silencing process. In contrast, a chromatin immunoprecipitation (ChIP) analysis for methyl-H3K4, acetyl-H3K9, and dimethyl-H3K9 levels failed to reveal a specific histone modification that could explain high frequency re-silencing. These results demonstrate that aberrantly silenced and reactivated promoters retain a persistent memory of having undergone the silencing process and suggest the failure to eliminate all CpG methylation as a potential contributing mechanism. PMID:21035468

  6. Checkpointing and Recovery in Distributed and Database Systems

    ERIC Educational Resources Information Center

    Wu, Jiang

    2011-01-01

    A transaction-consistent global checkpoint of a database records a state of the database which reflects the effect of only completed transactions and not the results of any partially executed transactions. This thesis establishes the necessary and sufficient conditions for a checkpoint of a data item (or the checkpoints of a set of data items) to…

  7. Kinetochore Localization of Spindle Checkpoint Proteins: Who Controls Whom?D⃞

    PubMed Central

    Vigneron, Suzanne; Prieto, Susana; Bernis, Cyril; Labbé, Jean-Claude; Castro, Anna; Lorca, Thierry

    2004-01-01

    The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, we have studied the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway. Our results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3, and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other. PMID:15269280

  8. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control.

    PubMed

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K; O'Hanlon, Karen; Quaas, Marianne; Larsen, Brian D; Rolland, Baptiste; Rösner, Heike I; Walter, David; Kousholt, Arne Nedergaard; Menzel, Tobias; Lees, Michael; Johansen, Jens Vilstrup; Rappsilber, Juri; Engeland, Kurt; Sørensen, Claus Storgaard

    2015-01-05

    Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

  9. Transcriptional pausing at the translation start site operates as a critical checkpoint for riboswitch regulation

    PubMed Central

    Chauvier, Adrien; Picard-Jean, Frédéric; Berger-Dancause, Jean-Christophe; Bastet, Laurène; Naghdi, Mohammad Reza; Dubé, Audrey; Turcotte, Pierre; Perreault, Jonathan; Lafontaine, Daniel A.

    2017-01-01

    On the basis of nascent transcript sequencing, it has been postulated but never demonstrated that transcriptional pausing at translation start sites is important for gene regulation. Here we show that the Escherichia coli thiamin pyrophosphate (TPP) thiC riboswitch contains a regulatory pause site in the translation initiation region that acts as a checkpoint for thiC expression. By biochemically probing nascent transcription complexes halted at defined positions, we find a narrow transcriptional window for metabolite binding, in which the downstream boundary is delimited by the checkpoint. We show that transcription complexes at the regulatory pause site favour the formation of a riboswitch intramolecular lock that strongly prevents TPP binding. In contrast, cotranscriptional metabolite binding increases RNA polymerase pausing and induces Rho-dependent transcription termination at the checkpoint. Early transcriptional pausing may provide a general mechanism, whereby transient transcriptional windows directly coordinate the sensing of environmental cues and bacterial mRNA regulation. PMID:28071751

  10. Modulation of DNA damage checkpoint; patenting and possible application for cancer medicine.

    PubMed

    Ishikawa, Kazuhiro; Ishii, Hideshi; Ichimura, Keiichi

    2008-01-01

    Eukaryote cells survey genomic integrity for DNA damage or incomplete replication. Aberrant structures being detected, checkpoint mechanisms are activated to slow down or arrest cell cycle progression, which allow the DNA damage to be repaired and the replication to be completed. In cancer development, precancerous cells overcome selective pressure to escape from blocked cell cycle progression, induced by checkpoint responses to DNA damage. Medical applications targeting the process of DNA damage would lead to efficient repairs of DNA damage or induction of cell death, which contributes to cancer detection, diagnosis and therapeutic approaches. In this article, the recent progress of our knowledge and patenting in modulation of DNA damage checkpoint especially by Rad9-Chk1 pathway is noted and possible application for cancer medicine is discussed.

  11. Hyper-methylation of the upstream CpG island shore is a likely mechanism of GPER1 silencing in breast cancer cells.

    PubMed

    Manjegowda, Mohan C; Gupta, Paridhi Singhal; Limaye, Anil M

    2017-05-30

    potential mechanism of GPER1 silencing in breast tumors.

  12. Defining Effective Combinations of Immune Checkpoint Blockade and Oncolytic Virotherapy

    PubMed Central

    Rojas, Juan J; Sampath, Padma; Hou, Weizhou; Thorne, Steve H

    2015-01-01

    Purpose Recent data from randomized clinical trials with oncolytic viral therapies and with cancer immunotherapies have finally recapitulated the promise these platforms demonstrated in pre-clinical models. Perhaps the greatest advance with oncolytic virotherapy has been the appreciation of the importance of activation of the immune response in therapeutic activity. Meanwhile, the understanding that blockade of immune checkpoints (with antibodies that block the binding of PD1 to PDL1 or CTLA4 to B7-2) is critical for an effective anti-tumor immune response has revitalized the field of immunotherapy. The combination of immune activation using an oncolytic virus and blockade of immune checkpoints is therefore a logical next step. Experimental Design Here we explore such combinations and demonstrate their potential to produce enhanced responses in mouse tumor models. Different combinations and regimens were explored in immunocompetent mouse models of renal and colorectal cancer. Bioluminescence imaging and immune assays were used to determine the mechanisms mediating synergistic or antagonistic combinations. Results Interaction between immune checkpoint inhibitors and oncolytic virotherapy was found to be complex, with correct selection of viral strain, antibody and timing of the combination being critical for synergistic effects. Indeed, some combinations produced antagonistic effects and loss of therapeutic activity. A period of oncolytic viral replication and directed targeting of the immune response against the tumor were required for the most beneficial effects, with CD8+ and NK, but not CD4+ cells mediating the effects. Conclusions These considerations will be critical in the design of the inevitable clinical translation of these combination approaches. PMID:26187615

  13. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  14. Detailed Modeling and Evaluation of a Scalable Multilevel Checkpointing System

    SciTech Connect

    Mohror, Kathryn; Moody, Adam; Bronevetsky, Greg; de Supinski, Bronis R.

    2014-09-01

    High-performance computing (HPC) systems are growing more powerful by utilizing more components. As the system mean time before failure correspondingly drops, applications must checkpoint frequently to make progress. But, at scale, the cost of checkpointing becomes prohibitive. A solution to this problem is multilevel checkpointing, which employs multiple types of checkpoints in a single run. Moreover, lightweight checkpoints can handle the most common failure modes, while more expensive checkpoints can handle severe failures. We designed a multilevel checkpointing library, the Scalable Checkpoint/Restart (SCR) library, that writes lightweight checkpoints to node-local storage in addition to the parallel file system. We present probabilistic Markov models of SCR's performance. We show that on future large-scale systems, SCR can lead to a gain in machine efficiency of up to 35 percent, and reduce the load on the parallel file system by a factor of two. In addition, we predict that checkpoint scavenging, or only writing checkpoints to the parallel file system on application termination, can reduce the load on the parallel file system by 20 × on today's systems and still maintain high application efficiency.

  15. Spindle assembly checkpoint and its regulators in meiosis.

    PubMed

    Sun, Shao-Chen; Kim, Nam-Hyung

    2012-01-01

    BACKGROUND Meiosis is a unique form of cell division in which cells divide twice but DNA is duplicated only once. Errors in chromosome segregation during meiosis will result in aneuploidy, followed by loss of the conceptus during pregnancy or birth defects. During mitosis, cells utilize a mechanism called the spindle assembly checkpoint (SAC) to ensure faithful chromosome segregation. A similar mechanism has been uncovered for meiosis in the last decade, especially in the past several years. METHODS For this review, we included data and relevant information obtained through a PubMed database search for all articles published in English from 1991 through 2011 which included the term 'meiosis', 'spindle assembly checkpoint', or 'SAC'. RESULTS There are 91 studies included. Evidence for the existence of SAC functions in meiosis is provided by studies on the SAC proteins mitotic-arrest deficient-1 (Mad1), Mad2, budding uninhibited by benzimidazole-1 (Bub1), Bub3, BubR1 and Mps1; microtubule-kinetochore attachment regulators Ndc80 complex, chromosomal passenger complex, mitotic centromere-associated kinesin (MCAK), kinetochore null 1 (KNL1) and Mis12 complex and spindle stability regulators. CONCLUSIONS SAC and its regulators exist and function in meiosis, and their malfunctions may cause germ cell aneuploidy. However, species and sexual differences exist. Moreover, interaction of SAC components with other regulators is still poorly understood, which needs further study.

  16. The spindle checkpoint and chromosome segregation in meiosis.

    PubMed

    Gorbsky, Gary J

    2015-07-01

    The spindle checkpoint is a key regulator of chromosome segregation in mitosis and meiosis. Its function is to prevent precocious anaphase onset before chromosomes have achieved bipolar attachment to the spindle. The spindle checkpoint comprises a complex set of signaling pathways that integrate microtubule dynamics, biomechanical forces at the kinetochores, and intricate regulation of protein interactions and post-translational modifications. Historically, many key observations that gave rise to the initial concepts of the spindle checkpoint were made in meiotic systems. In contrast with mitosis, the two distinct chromosome segregation events of meiosis present a special challenge for the regulation of checkpoint signaling. Preservation of fidelity in chromosome segregation in meiosis, controlled by the spindle checkpoint, also has a significant impact in human health. This review highlights the contributions from meiotic systems in understanding the spindle checkpoint as well as the role of checkpoint signaling in controlling the complex divisions of meiosis.

  17. Compiler-Enhanced Incremental Checkpointing for OpenMP Applications

    SciTech Connect

    Bronevetsky, G; Marques, D; Pingali, K; McKee, S; Rugina, R

    2009-02-18

    As modern supercomputing systems reach the peta-flop performance range, they grow in both size and complexity. This makes them increasingly vulnerable to failures from a variety of causes. Checkpointing is a popular technique for tolerating such failures, enabling applications to periodically save their state and restart computation after a failure. Although a variety of automated system-level checkpointing solutions are currently available to HPC users, manual application-level checkpointing remains more popular due to its superior performance. This paper improves performance of automated checkpointing via a compiler analysis for incremental checkpointing. This analysis, which works with both sequential and OpenMP applications, significantly reduces checkpoint sizes and enables asynchronous checkpointing.

  18. RNAi induced gene silencing in crop improvement.

    PubMed

    Sinha, Subodh Kumar

    2010-12-01

    The RNA silencing is one of the innovative and efficient molecular biology tools to harness the down-regulation of expression of gene(s) specifically. To accomplish such selective modification of gene expression of a particular trait, homology dependent gene silencing uses a stunning variety of gene silencing viz. co-suppression, post-transcriptional gene silencing, virus-induced gene silencing etc. This family of diverse molecular phenomena has a common exciting feature of gene silencing which is collectively called RNA interference abbreviated to as RNAi. This molecular phenomenon has become a focal point of plant biology and medical research throughout the world. As a result, this technology has turned out to be a powerful tool in understanding the function of individual gene and has ultimately led to the tremendous use in crop improvement. This review article illustrates the application of RNAi in a broad area of crop improvement where this technology has been successfully used. It also provides historical perspective of RNAi discovery and its contemporary phenomena, mechanism of RNAi pathway.

  19. Titration and hysteresis in epigenetic chromatin silencing

    NASA Astrophysics Data System (ADS)

    Dayarian, Adel; Sengupta, Anirvan M.

    2013-06-01

    Epigenetic mechanisms of silencing via heritable chromatin modifications play a major role in gene regulation and cell fate specification. We consider a model of epigenetic chromatin silencing in budding yeast and study the bifurcation diagram and characterize the bistable and the monostable regimes. The main focus of this paper is to examine how the perturbations altering the activity of histone modifying enzymes affect the epigenetic states. We analyze the implications of having the total number of silencing proteins, given by the sum of proteins bound to the nucleosomes and the ones available in the ambient, to be constant. This constraint couples different regions of chromatin through the shared reservoir of ambient silencing proteins. We show that the response of the system to perturbations depends dramatically on the titration effect caused by the above constraint. In particular, for a certain range of overall abundance of silencing proteins, the hysteresis loop changes qualitatively with certain jump replaced by continuous merger of different states. In addition, we find a nonmonotonic dependence of gene expression on the rate of histone deacetylation activity of Sir2. We discuss how these qualitative predictions of our model could be compared with experimental studies of the yeast system under anti-silencing drugs.

  20. An electronically tunable duct silencer using dielectric elastomer actuators.

    PubMed

    Lu, Zhenbo; Godaba, Hareesh; Cui, Yongdong; Foo, Choon Chiang; Debiasi, Marco; Zhu, Jian

    2015-09-01

    A duct silencer with tunable acoustic characteristics is presented in this paper. Dielectric elastomer, a smart material with lightweight, high elastic energy density and large deformation under high direct current/alternating current voltages, was used to fabricate this duct silencer. The acoustic performances and tunable mechanisms of this duct silencer were experimentally investigated. It was found that all the resonance peaks of this duct silencer could be adjusted using external control signals without any additional mechanical part. The physics of the tunable mechanism is further discussed based on the electro-mechanical interactions using finite element analysis. The present promising results also provide insight into the appropriateness of the duct silencer for possible use as next generation acoustic treatment device to replace the traditional acoustic treatment.

  1. Contribution of Growth and Cell Cycle Checkpoints to Radiation Survival in Drosophila

    PubMed Central

    Jaklevic, Burnley; Uyetake, Lyle; Lemstra, Willy; Chang, Julia; Leary, William; Edwards, Anthony; Vidwans, Smruti; Sibon, Ody; Tin Su, Tin

    2006-01-01

    Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy. PMID:17028317

  2. [Cancer immunotherapy by immuno-checkpoint blockade].

    PubMed

    Kawakami, Yutaka

    2015-10-01

    As cancer immunotherapies utilizing anti-tumor T-cell responses, immuno-checkpoint blockade and adoptive T-cell immunotherapy have recently achieved durable responses even in advanced cancer patients with metastases. Administration of antibodies on the T-cell surface, CTLA-4 and PD-1 (or PD-1 ligand PD-L1), resulted in tumor regression of not only melanoma and renal cell cancer which were known to be relatively sensitive to immunotherapy, but also various malignancies including lung, bladder, ovarian, gastric, and head and neck cancers, as well as hematological malignancies such as Hodgkin and B-cell malignant lymphomas. These findings have changed the status of immunotherapy in the development of cancer treatments. Currently, development of combinations employing cancer immunotherapy with immuno-checkpoint blockade, as well as personalized cancer immunotherapy based on the evaluation of pretreatment immune status, are in progress.

  3. Perfect Rainbow Tradeoff with Checkpoints Revisited

    PubMed Central

    2016-01-01

    The rainbow tradeoff is an algorithm for inverting one-way functions that is widely used in practice to recover passwords from unsalted password hashes. An auxiliary technique referred to as checkpoints can be applied to the rainbow tradeoff to reduce the time taken for these inversions. Working out a rigorous theory that can explain and predict the effects of this technique involves delicate manipulations of the random function and is thus a challenging task. In this work, we compare three existing theoretical analyses of the checkpoint technique. We first demonstrate that the claims made by the three works are incompatible with each other. We then carry out experiments designed to highlight these incompatibilities, obtaining experimental evidences that show just one of the three analyses to be correct. Finally, we discuss the obscure theoretical errors made by the two inadequate analyses. PMID:27855190

  4. Renal effects of immune checkpoint inhibitors.

    PubMed

    Izzedine, Hassan; Mateus, Christine; Boutros, Céline; Robert, Caroline; Rouvier, Philippe; Amoura, Zahir; Mathian, Alexis

    2016-12-26

    Recent advances in immune checkpoint inhibitor (ICPI) development have led to major improvements in oncology patient outcomes. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) are two essential immune checkpoint receptors. Ipilimumab and tremelimumab (anti-CTLA-4-blocking antibodies) and pembrolizumab and nivolumab (antibodies targeting PD-1 receptors) have already been approved by US Food and Drug Administration in several malignancies. Two different forms of ICPI-induced renal damage have been identified, including acute (granulomatous) tubulointerstitial nephritis and immune complex glomerulonephritis. The observed acute renal damage can be reversed upon ICPI drug discontinuation and renal function can recover back to normal following the introduction of systemic corticosteroid treatment. Any delay in treating this complication could result in definitive and irreversible renal injury.

  5. Immunotherapy comes of age: Immune aging & checkpoint inhibitors.

    PubMed

    Elias, Rawad; Karantanos, Theodoros; Sira, Elizabeth; Hartshorn, Kevan L

    2017-02-17

    Immune checkpoint inhibitors (ICIs) are based on the understanding that there are multilayered checks and balances which can be manipulated to unleash already existing, but paralyzed, immune responses to cancer. These agents are safer and more efficacious than classic cytotoxic drugs making them a very attractive therapeutic option, especially in older adults. Current available data do not suggest significant age-associated differences in the clinical profile of ICIs. It must be noted, however, that there is still relatively little information on the use of ICIs in adults over 75years of age and aging is associated with a decline in the immune system or "immunosenescence" which theoretically can reduce the efficacy of these immune based therapies. In this paper, we review the mechanism of action of ICIs, current clinical data on their use in older adults, and age-associated immune changes that might have a direct impact on their activity in this population. We chose to focus on mainly adaptive cellular immunity, and especially on components of the immune system that are implicated directly in the immune checkpoint process.

  6. Optimal viral strategies for bypassing RNA silencing.

    PubMed

    Rodrigo, Guillermo; Carrera, Javier; Jaramillo, Alfonso; Elena, Santiago F

    2011-02-06

    The RNA silencing pathway constitutes a defence mechanism highly conserved in eukaryotes, especially in plants, where the underlying working principle relies on the repressive action triggered by the intracellular presence of double-stranded RNAs. This immune system performs a post-transcriptional suppression of aberrant mRNAs or viral RNAs by small interfering RNAs (siRNAs) that are directed towards their target in a sequence-specific manner. However, viruses have evolved strategies to escape from silencing surveillance while promoting their own replication. Several viruses encode suppressor proteins that interact with different elements of the RNA silencing pathway and block it. The different suppressors are not phylogenetically nor structurally related and also differ in their mechanism of action. Here, we adopt a model-driven forward-engineering approach to understand the evolution of suppressor proteins and, in particular, why viral suppressors preferentially target some components of the silencing pathway. We analysed three strategies characterized by different design principles: replication in the absence of a suppressor, suppressors targeting the first protein component of the pathway and suppressors targeting the siRNAs. Our results shed light on the question of whether a virus must opt for devoting more time into transcription or into translation and on which would be the optimal step of the silencing pathway to be targeted by suppressors. In addition, we discussed the evolutionary implications of such designing principles.

  7. Programmed death 1 immune checkpoint inhibitors.

    PubMed

    Trivedi, Meghna S; Hoffner, Brianna; Winkelmann, Jennifer L; Abbott, Maura E; Hamid, Omid; Carvajal, Richard D

    2015-12-01

    Programmed death 1 (PD-1) is an immune checkpoint that provides inhibitory signals to the immune system in order to modulate the activity of T cells in peripheral tissues and maintain self-tolerance in the setting of infection and inflammation. In cancer, the immune checkpoints are exploited so that the tumor cells are able to evade the immune system. Immune checkpoint inhibitors are a type of cancer immunotherapy that targets pathways such as PD-1 in order to reinvigorate and enhance the immune response against tumor cells. The US Food and Drug Administration (FDA) has approved 2 PD-1 inhibitors, nivolumab and pembrolizumab, and several others are under investigation. Although PD-1 inhibitors have demonstrated activity in many different types of malignancies, FDA approval has been granted only in melanoma and in non-small cell lung cancer (NSCLC). Identifying biomarkers that can predict response to PD-1 inhibitors is critical to maximizing the benefit of these agents. Future directions for PD-1 inhibitors include investigation of combination therapies, use in malignancies other than melanoma and NSCLC, and refinement of biomarkers.

  8. Targeting the spindle assembly checkpoint for breast cancer treatment.

    PubMed

    Marques, Sandra; Fonseca, Joana; Silva, Patrícia M A; Bousbaa, Hassan

    2015-01-01

    Breast cancer is the most common malignancy in women worldwide and the second leading cause of cancer deaths after lung cancer. As in other malignancies, aneuploidy is a common feature of breast cancer and influences its behavior. Aneuploidy has been linked to inappropriate activity of the spindle assembly checkpoint (SAC), a surveillance mechanism that, in normal cells, prevents anaphase onset until correct alignment of all chromosomes at the metaphase is achieved. Interestingly, the widely used anti-microtubule drugs, vinca alkaloids and taxanes, kill cancer cells through chronic arrest in mitosis as a consequence of chronic SAC activation. Deregulated SAC has been reported in breast cancer in many reports and presents an attractive therapeutic strategy. We present here a review of the current knowledge on the SAC defects and the underlying molecular mechanisms in breast cancer, and discuss the potential of SAC components as targets for breast cancer therapies.

  9. The pre-B-cell receptor checkpoint in acute lymphoblastic leukaemia.

    PubMed

    Eswaran, J; Sinclair, P; Heidenreich, O; Irving, J; Russell, L J; Hall, A; Calado, D P; Harrison, C J; Vormoor, J

    2015-08-01

    The B-cell receptor (BCR) and its immature form, the precursor-BCR (pre-BCR), have a central role in the control of B-cell development, which is dependent on a sequence of cell-fate decisions at specific antigen-independent checkpoints. Pre-BCR expression provides the first checkpoint, which controls differentiation of pre-B to immature B-cells in normal haemopoiesis. Pre-BCR signalling regulates and co-ordinates diverse processes within the pre-B cell, including clonal selection, proliferation and subsequent maturation. In B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), B-cell development is arrested at this checkpoint. Moreover, malignant blasts avoid clonal extinction by hijacking pre-BCR signalling in favour of the development of BCP-ALL. Here, we discuss three mechanisms that occur in different subtypes of BCP-ALL: (i) blocking pre-BCR expression; (ii) activating pre-BCR-mediated pro-survival and pro-proliferative signalling, while inhibiting cell cycle arrest and maturation; and (iii) bypassing the pre-BCR checkpoint and activating pro-survival signalling through pre-BCR independent alternative mechanisms. A complete understanding of the BCP-ALL-specific signalling networks will highlight their application in BCP-ALL therapy.

  10. Template based parallel checkpointing in a massively parallel computer system

    DOEpatents

    Archer, Charles Jens; Inglett, Todd Alan

    2009-01-13

    A method and apparatus for a template based parallel checkpoint save for a massively parallel super computer system using a parallel variation of the rsync protocol, and network broadcast. In preferred embodiments, the checkpoint data for each node is compared to a template checkpoint file that resides in the storage and that was previously produced. Embodiments herein greatly decrease the amount of data that must be transmitted and stored for faster checkpointing and increased efficiency of the computer system. Embodiments are directed to a parallel computer system with nodes arranged in a cluster with a high speed interconnect that can perform broadcast communication. The checkpoint contains a set of actual small data blocks with their corresponding checksums from all nodes in the system. The data blocks may be compressed using conventional non-lossy data compression algorithms to further reduce the overall checkpoint size.

  11. The Gift of Silence

    ERIC Educational Resources Information Center

    Haskins, Cathleen

    2011-01-01

    Slowing down, quieting the mind and body, and experiencing silence nourishes the spirit. Montessori educators are mandated to cultivate not just the intellect but the whole child. They recognize that nurturing the spirit of the child is part of what makes this form of education work so well. This article discusses the benefits of stillness and…

  12. On Observing Student Silence

    ERIC Educational Resources Information Center

    Amundrud, Thomas

    2011-01-01

    This article uses conversation analysis (CA) to look at how students in an advanced EGAP (English for general academic purposes) course discussion test create and manage the silence of a group member during the 7-min session. This is combined with a personal narrative inquiry, coinspired by autoethnography, on the author's participation in the…

  13. Programmed death-1 immune checkpoint blockade in the treatment of hematological malignancies.

    PubMed

    Tsirigotis, Panagiotis; Savani, Bipin N; Nagler, Arnon

    2016-09-01

    The use of tumor-specific monoclonal antibodies (MAbs) has revolutionize the field of cancer immunotherapy. Although treatment of malignant diseases with MAbs is promising, many patients fail to respond or relapse after an initial response. Both solid tumors and hematological malignancies develop mechanisms that enable them to evade the host immune system by usurping immune checkpoint pathways such as PD-1, PD-2, PDL-1, or PDL-2 (programmed cell death protein-1 or 2 and PD-Ligand 1 or 2), which are expressed on activated T cells and on T-regulatory, B cells, natural killers, monocytes, and dendritic cells. One of the most exciting anticancer development in recent years has been the immune checkpoint blockade therapy by using MAbs against immune checkpoint receptor and/or ligands. Anti-PD1 antibodies have been tested in clinical studies that included patients with hematological malignancies and showed remarkable efficacy in Hodgkin lymphoma (HL). In our review, we will focus on the effect of PD-1 activation on hematological malignancies and its role as a therapeutic target. Key messages The programmed death 1 (PD1) immune checkpoint is an important homeostatic mechanism of the immune system that helps in preventing autoimmunity and uncontrolled inflammation in cases of chronic infections. However, PD1 pathway is also operated by a wide variety of malignancies and represents one of the most important mechanisms by which tumor cells escape from the surveillance of the immune system. Blocking of immune checkpoints by the use of monoclonal antibodies opened a new era in the field of cancer immunotherapy. Results from clinical trials are promising, and currently, this approach has been proven effective and safe in patients with solid tumors and hematological malignancies.

  14. Xenopus oocyte meiosis lacks spindle assembly checkpoint control

    PubMed Central

    Shao, Hua; Ma, Chunqi; Chen, Eric

    2013-01-01

    The spindle assembly checkpoint (SAC) functions as a surveillance mechanism to detect chromosome misalignment and to delay anaphase until the errors are corrected. The SAC is thought to control mitosis and meiosis, including meiosis in mammalian eggs. However, it remains unknown if meiosis in the eggs of nonmammalian vertebrate species is also regulated by SAC. Using a novel karyotyping technique, we demonstrate that complete disruption of spindle microtubules in Xenopus laevis oocytes did not affect the bivalent-to-dyad transition at the time oocytes are undergoing anaphase I. These oocytes also acquired the ability to respond to parthenogenetic activation, which indicates proper metaphase II arrest. Similarly, oocytes exhibiting monopolar spindles, via inhibition of aurora B or Eg5 kinesin, underwent monopolar anaphase on time and without additional intervention. Therefore, the metaphase-to-anaphase transition in frog oocytes is not regulated by SAC. PMID:23569212

  15. Checkpoint immunotherapy for cancer: superior survival, unaccustomed toxicities.

    PubMed

    Gedye, C; van der Westhuizen, A; John, T

    2015-07-01

    Novel cancer immunotherapy antibodies are moving from clinical trials into routine practice, delivering sustained benefits and prolonged survival to patients with melanoma, lung, kidney and other cancers. These immunostimulatory antibodies non-specifically activate the patient's own immune system by inhibiting immune system checkpoint proteins. This mechanism of action is entirely different to traditional cancer treatments, such as chemotherapy. While there are virtually no immediate toxicities, serious life-threatening autoimmune side-effects such as colitis, dermatitis, hypophysitis, pneumonitis and hepatitis can occur, sometimes starting long after the treatment has been given. Recognition, referral and prompt treatment with immunosuppressive drugs like corticosteroids can control these immune-related side-effects without compromising efficacy. This exciting new class of drugs is defining a new paradigm in cancer therapy.

  16. Two different mitotic checkpoint inhibitors of the anaphase-promoting complex/cyclosome antagonize the action of the activator Cdc20

    PubMed Central

    Eytan, Esther; Braunstein, Ilana; Ganoth, Dvora; Teichner, Adar; Hittle, James C.; Yen, Tim J.; Hershko, Avram

    2008-01-01

    The mitotic checkpoint system ensures the fidelity of chromosome segregation by preventing the completion of mitosis in the presence of any misaligned chromosome. When activated, it blocks the initiation of anaphase by inhibiting the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). Little is known about the biochemical mechanisms by which this system inhibits APC/C, except for the existence of a mitotic checkpoint complex (MCC) inhibitor of APC/C composed of the APC/C activator Cdc20 associated with the checkpoint proteins Mad2, BubR1, and Bub3. We have been studying the mechanisms of the mitotic checkpoint system in extracts that reproduce its downstream events. We found that inhibitory factors are associated with APC/C in the checkpoint-arrested state, which can be recovered from immunoprecipitates. Only a part of the inhibitory activity was caused by MCC [Braunstein I, Miniowitz S, Moshe Y, Hershko A (2007) Proc Natl Acad Sci USA 104:4870–4875]. Here, we show that during exit from checkpoint, rapid disassembly of MCC takes place while APC/C is still inactive. This observation suggested the possible involvement of multiple factors in the regulation of APC/C by the mitotic checkpoint. We have separated a previously unknown inhibitor of APC/C from MCC. This inhibitor, called mitotic checkpoint factor 2 (MCF2), is associated with APC/C only in the checkpoint-arrested state. The inhibition of APC/C by both MCF2 and MCC was decreased at high concentrations of Cdc20. We propose that both MCF2 and MCC inhibit APC/C by antagonizing Cdc20, possibly by interaction with the Cdc20-binding site of APC/C. PMID:18591651

  17. Polycomb proteins control proliferation and transformation independently of cell cycle checkpoints by regulating DNA replication.

    PubMed

    Piunti, Andrea; Rossi, Alessandra; Cerutti, Aurora; Albert, Mareike; Jammula, Sriganesh; Scelfo, Andrea; Cedrone, Laura; Fragola, Giulia; Olsson, Linda; Koseki, Haruhiko; Testa, Giuseppe; Casola, Stefano; Helin, Kristian; d'Adda di Fagagna, Fabrizio; Pasini, Diego

    2014-04-14

    The ability of PRC1 and PRC2 to promote proliferation is a main feature that links polycomb (PcG) activity to cancer. PcGs silence the expression of the tumour suppressor locus Ink4a/Arf, whose products positively regulate pRb and p53 functions. Enhanced PcG activity is a frequent feature of human tumours, and PcG inhibition has been proposed as a strategy for cancer treatment. However, the recurrent inactivation of pRb/p53 responses in human cancers raises a question regarding the ability of PcG proteins to affect cellular proliferation independently from this checkpoint. Here we demonstrate that PRCs regulate cellular proliferation and transformation independently of the Ink4a/Arf-pRb-p53 pathway. We provide evidence that PRCs localize at replication forks, and that loss of their function directly affects the progression and symmetry of DNA replication forks. Thus, we have identified a novel activity by which PcGs can regulate cell proliferation independently of major cell cycle restriction checkpoints.

  18. Molecular basis of APC/C regulation by the spindle assembly checkpoint

    PubMed Central

    Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-01-01

    In the dividing eukaryotic cell the spindle assembly checkpoint (SAC) ensures each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), a multimeric assembly that inhibits the APC/C, delaying chromosome segregation. Here, using cryo-electron microscopy we determined the near-atomic resolution structure of an APC/C-MCC complex (APC/CMCC). We reveal how degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit (Cdc20APC/C) responsible for substrate interactions. BubR1 also obstructs binding of UbcH10 (APC/C’s initiating E2) to repress APC/C ubiquitination activity. Conformational variability of the complex allows for UbcH10 association, and we show from a structure of APC/CMCC in complex with UbcH10 how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced. PMID:27509861

  19. High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.

    PubMed

    Bin Zhang; Xiubin Gu; Uppalapati, Uma; Ashwell, Mark A; Leggett, David S; Li, Chiang J

    2008-07-01

    Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

  20. Contrasting roles of checkpoint proteins as recombination modulators at Fob1-Ter complexes with or without fork arrest.

    PubMed

    Mohanty, Bidyut K; Bairwa, Narendra K; Bastia, Deepak

    2009-04-01

    The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes. The checkpoint adapter proteins Tof1 and Csm3 either positively or negatively controlled recombination depending on whether it was provoked by polar fork arrest or by transcription, respectively. The absolute requirements for these proteins for inducing recombination at an active replication terminus most likely masked their negative modulatory role at a later step of the process. Other checkpoint proteins of the checkpoint adapter/mediator class such as Mrc1 and Rad9, which channel signals from the sensor to the effector kinase, tended to suppress recombination at Fob1-Ter complexes regardless of how it was initiated. We have also discovered that the checkpoint sensor kinase Mec1 and the effector Rad53 were positive modulators of recombination initiated by transcription but had little effect on recombination at Ter. The work also showed that the two pathways were Rad52 dependent but Rad51 independent. Since Ter sites occur in the intergenic spacer of rDNA from yeast to humans, the mechanism is likely to be of widespread occurrence.

  1. The Saccharomyces cerevisiae suppressor of choline sensitivity (SCS2) gene is a multicopy Suppressor of mec1 telomeric silencing defects.

    PubMed Central

    Craven, R J; Petes, T D

    2001-01-01

    Mec1p is a cell cycle checkpoint protein related to the ATM protein kinase family. Certain mec1 mutations or overexpression of Mec1p lead to shortened telomeres and loss of telomeric silencing. We conducted a multicopy suppressor screen for genes that suppress the loss of silencing in strains overexpressing Mec1p. We identified SCS2 (suppressor of choline sensitivity), a gene previously isolated as a suppressor of defects in inositol synthesis. Deletion of SCS2 resulted in decreased telomeric silencing, and the scs2 mutation increased the rate of cellular senescence observed for mec1-21 tel1 double mutant cells. Genetic analysis revealed that Scs2p probably acts through a different telomeric silencing pathway from that affected by Mec1p. PMID:11333225

  2. Tactical Checkpoint: Hail/Warn Suppress/Stop (Poster)

    DTIC Science & Technology

    2010-11-15

    distractor , optical suppression , human behavior, checkpoint, ambient light, driver suppression , human experimentation, light, paintball, obscuration...HAIL/WARN AND - SUPPRESS /STOP Poster Presented at the 2010 Directed Energies Professional Society Meeting, 15-19 November 2010. 5a. CONTRACT NUMBER...warning to a driver that is approaching a checkpoint. The laser, MCNC light, and the windshield obscuration were evaluated for their suppression

  3. [Adverse events of immune checkpoint inhibitors].

    PubMed

    Foller, S; Oppel-Heuchel, H; Fetter, I; Winkler, Y; Grimm, M-O

    2017-04-01

    After immune checkpoint inhibitor therapy was approved for renal cell carcinoma last year, this new immune therapy has spread to urology. Further approvals (atezolizumab, nivolumab, pembrolizumab) are expected in 2017 for metastatic urothelial carcinoma that has progressed following treatment with platinum-based chemotherapy. With expanding use of immune checkpoint inhibitors, experience in diagnosing and managing immune-mediated adverse events increases. Although of low incidence, grade 3/4 toxicities play a central role. Organs most common for immune-mediated adverse events are skin, liver (hepatitis), kidneys (nephritis), gastrointestinal tract (diarrhea and colitis), lungs (pneumonitis), and endocrine organs (hyper-, hypothyroidism and hypophysitis). Diagnostic workup includes routine laboratory tests (including liver function tests) and may be supplemented with hormone values. In cases of pneumonitis or hypophysitis, imaging (high-resolution CT, MRI) can confirm diagnoses. Immune-mediated toxicities are treated with therapy interruption and administration of corticosteroids (and in individual cases additional immunosuppression). Dose modification is not intended!

  4. Mammalian oocyte development: checkpoints for competence.

    PubMed

    Fair, Trudee

    2010-01-01

    During the lifespan of the female, biochemical changes occur in the ovarian environment. These changes are brought about by numerous endogenous and exogenous factors, including husbandry practices, production demands and disease, and can have a profound effect on ovarian oocyte quality and subsequent embryo development. Despite many investigations, there is no consensus regarding the time or period of follicular oocyte development that is particularly sensitive to insult. Here, the key molecular and morphological events that occur during oocyte and follicle growth are reviewed, with a specific focus on identifying critical checkpoints in oocyte development. The secondary follicle stage appears to be a key phase in follicular oocyte development because major events such as activation of the oocyte transcriptome, sequestration of the zona pellucida, establishment of bidirectional communication between the granulosa cells and the oocyte and cortical granule synthesis occur during this period of development. Several months later, the periovulatory period is also characterised by the occurrence of critical events, including appropriate degradation or polyadenylation of mRNA transcripts, resumption of meiosis, spindle formation, chromosome alignment and segregation, and so should also be considered as a potential checkpoint of oocyte development.

  5. A Fhit-ing role in the DNA damage checkpoint response.

    PubMed

    Ishii, Hideshi; Wang, Ya; Huebner, Kay

    2007-05-02

    The FHIT gene encompasses the most active common fragile site of the human genome and is thus exquisitely sensitive to intragenic alterations by DNA damaging agents, alterations that can lead to FHIT allele loss very early in the preneoplastic phase of cancer development, before or coincident with activation of the DNA damage checkpoint. Fhit protein expression is lost or reduced in many preneoplastic lesions and in >50% of cancers, Fhit knockout mice are highly susceptible to carcinogen induction of tumors and Fhit replacement in these mice by gene therapy induces apoptosis and significantly reduces tumor burden. But learning how Fhit induces apoptosis and suppresses tumors has been a challenge because interacting proteins, effectors of Fhit signals, have not been discovered. Nevertheless, the study of Fhit deficient mouse and human tissue-derived and cancer-derived cells in vitro has led to several important conclusions: repair protein-deficient cancers are more likely to be Fhit-deficient; Fhit-deficient cells show enhanced resistance to UVC, mitomycin C, camptothecin and ionizing radiation-induced cell killing, possibly due to strong activation of the ATR pathway following DNA damage; Fhit-deficient cells show higher efficiency of homologous recombination repair, a double-strand break repair pathway in mammalian cells; Fhit protein indirectly affects S-phase checkpoint and DNA repair. Finally, results of a recent study have suggested that the DNA damage-susceptible FRA3B/FHIT chromosome fragile region, paradoxically, encodes a protein, Fhit, that is necessary for protecting cells from accumulation of DNA damage, through modulation of checkpoint proteins Hus1 and phosphoChk1. Thus, inactivation of Fhit contributes to accumulation of abnormal checkpoint phenotypes in cancer development. It will be very important to determine mechanisms employed by Fhit in modulating checkpoint pathways, and to define consequences of Fhit loss in specific preneoplastic and

  6. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  7. An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer

    PubMed Central

    Teixeira, José Henrique; Silva, Patrícia Manuela; Reis, Rita Margarida; Moura, Inês Moranguinho; Marques, Sandra; Fonseca, Joana; Monteiro, Luís Silva; Bousbaa, Hassan

    2014-01-01

    Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20. PMID:24995269

  8. Systematic analysis in Caenorhabditis elegans reveals that the spindle checkpoint is composed of two largely independent branches.

    PubMed

    Essex, Anthony; Dammermann, Alexander; Lewellyn, Lindsay; Oegema, Karen; Desai, Arshad

    2009-02-01

    Kinetochores use the spindle checkpoint to delay anaphase onset until all chromosomes have formed bipolar attachments to spindle microtubules. Here, we use controlled monopolar spindle formation to systematically define the requirements for spindle checkpoint signaling in the Caenorhabditis elegans embryo. The results, when interpreted in light of kinetochore assembly epistasis analysis, indicate that checkpoint activation is coordinately directed by the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB-1-three components independently targeted to the outer kinetochore by the scaffold protein KNL-1. These components orchestrate the integration of a core Mad1(MDF-1)/Mad2(MDF-2)-based signal, with a largely independent Mad3(SAN-1)/BUB-3 pathway. Evidence for independence comes from the fact that subtly elevating Mad2(MDF-2) levels bypasses the requirement for BUB-3 and Mad3(SAN-1) in kinetochore-dependent checkpoint activation. Mad3(SAN-1) does not accumulate at unattached kinetochores and BUB-3 kinetochore localization is independent of Mad2(MDF-2). We discuss the rationale for a bipartite checkpoint mechanism in which a core Mad1(MDF-1)/Mad2(MDF-2) signal generated at kinetochores is integrated with a separate cytoplasmic Mad3(SAN-1)/BUB-3-based pathway.

  9. Rethinking the Day of Silence

    ERIC Educational Resources Information Center

    Murphy, Adriana

    2013-01-01

    Back in 2006, 7th and 8th graders at Green Acres, the K-8 independent school where the author taught in suburban Maryland, participated in the Day of Silence. The Day of Silence is a national event: Students across the country take a one-day pledge of silence to show that they want to make schools safe for all students, regardless of their sexual…

  10. Telomeric Trans-Silencing: An Epigenetic Repression Combining RNA Silencing and Heterochromatin Formation

    PubMed Central

    Josse, Thibaut; Teysset, Laure; Todeschini, Anne-Laure; Sidor, Clara M; Anxolabéhère, Dominique; Ronsseray, Stéphane

    2007-01-01

    The study of P-element repression in Drosophila melanogaster led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. Here, we report that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. We show that this silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3–7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway. PMID:17941712

  11. Applying gene silencing technology to contraception

    PubMed Central

    Dissen, Gregory A.; Lomniczi, Alejandro; Boudreau, Ryan L.; Chen, Yong Hong; Davidson, Beverly L.; Ojeda, Sergio R.

    2013-01-01

    Contents Population control of feral animals is often difficult, as it can be dangerous for the animals, labor intensive, and expensive. Therefore, a useful tool for control of animal populations would be a nonsurgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to noninvasively sterilize cats and dogs. For this approach to succeed it has to meet several conditions: The target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include: a) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction, and b) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:23279544

  12. Applying gene silencing technology to contraception.

    PubMed

    Dissen, G A; Lomniczi, A; Boudreau, R L; Chen, Y H; Davidson, B L; Ojeda, S R

    2012-12-01

    Population control of feral animals is often difficult, as it can be dangerous for the animals, labour intensive and expensive. Therefore, a useful tool for control of animal populations would be a non-surgical method to induce sterility. Our laboratories utilize methods aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A useful framework for design of a new approach will be the combination of these methods with the intended goal to produce a technique that can be used to non-invasively sterilize cats and dogs. For this approach to succeed, it has to meet several conditions: the target gene must be essential for fertility; the method must include a mechanism to effectively and specifically silence the gene of interest; the method of delivering the silencing agent must be minimally invasive, and finally, the silencing effect must be sustained for the lifespan of the target species, so that expansion of the population can be effectively prevented. In this article, we discuss our work to develop gene silencing technology to induce sterility; we will use examples of our previous studies demonstrating that this approach is viable. These studies include (i) the use of viral vectors able to disrupt reproductive cyclicity when delivered to the regions of the brain involved in the control of reproduction and (ii) experiments with viral vectors that are able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy.

  13. "Listening Silence" and Its Discursive Effects

    ERIC Educational Resources Information Center

    Applebaum, Barbara

    2016-01-01

    While researchers have studied how white silence protects white innocence and white ignorance, in this essay Barbara Applebaum explores a form of white silence that she refers to as "listening silence" in which silence protects white innocence but does not necessarily promote resistance to learning. White listening silence can appear to…

  14. A tissue checkpoint regulates type 2 immunity

    PubMed Central

    Van Dyken, Steven J.; Nussbaum, Jesse C.; Lee, Jinwoo; Molofsky, Ari B.; Liang, Hong-Erh; Pollack, Joshua L.; Gate, Rachel E.; Haliburton, Genevieve E.; Ye, Chun J.; Marson, Alexander; Erle, David J.; Locksley, Richard M.

    2017-01-01

    Group 2 innate lymphoid cells (ILC2s) and CD4+ T helper type 2 (Th2) cells are defined by their similar effector cytokines, which together mediate the features of allergic immunity. Here, we show that tissue ILC2s and Th2 cells differentiate independently but share overlapping effector function programs mediated by exposure to the tissue-derived cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Loss of these three tissue signals does not affect lymph node priming but abrogates terminal differentiation of effector Th2 cells and adaptive lung inflammation in a T cell-intrinsic manner. Our findings suggest how diverse perturbations activate type 2 immunity, uncovering a shared local tissue-elicited checkpoint that can be exploited to control both innate and adaptive allergic inflammation. PMID:27749840

  15. The Intra-S Checkpoint Responses to DNA Damage

    PubMed Central

    Iyer, Divya Ramalingam; Rhind, Nicholas

    2017-01-01

    Faithful duplication of the genome is a challenge because DNA is susceptible to damage by a number of intrinsic and extrinsic genotoxins, such as free radicals and UV light. Cells activate the intra-S checkpoint in response to damage during S phase to protect genomic integrity and ensure replication fidelity. The checkpoint prevents genomic instability mainly by regulating origin firing, fork progression, and transcription of G1/S genes in response to DNA damage. Several studies hint that regulation of forks is perhaps the most critical function of the intra-S checkpoint. However, the exact role of the checkpoint at replication forks has remained elusive and controversial. Is the checkpoint required for fork stability, or fork restart, or to prevent fork reversal or fork collapse, or activate repair at replication forks? What are the factors that the checkpoint targets at stalled replication forks? In this review, we will discuss the various pathways activated by the intra-S checkpoint in response to damage to prevent genomic instability. PMID:28218681

  16. Edwin Hubble's Silence

    NASA Astrophysics Data System (ADS)

    Lago, D.

    2013-04-01

    In late 1928 Edwin Hubble was right in the middle of using V. M. Slipher's redshift data to prove that the universe is expanding, when Hubble's boss, George Hale, directed him to drop everything and rush to the Grand Canyon and test it as a possible site for Hale's planned 200-inch telescope. On his way, Hubble stopped at Lowell Observatory and met with V. M. Slipher. The letters both men wrote about this visit suggest that Hubble never said a word about his being in the middle of using Slipher's research to transform the universe. At the least, this silence is symbolic of the silence with which astronomical history has often treated Slipher's work. A survey of the historical literature suggests several reasons for this. Theorists and observers in astronomy (and other sciences) have long had different perspectives about how science works, and those who place more importance on theory have tended to credit the idea of the expanding universe to the theorists. Also, many sources indicate that Edwin Hubble was not a modest man or generous about sharing credit.

  17. Resistance to PD1/PDL1 checkpoint inhibition.

    PubMed

    O'Donnell, Jake S; Long, Georgina V; Scolyer, Richard A; Teng, Michele W L; Smyth, Mark J

    2017-01-01

    For the first time in decades, patients with difficult-to-treat cancers such as advanced stage metastatic melanoma are being offered a glimpse of hope in the form of immunotherapies. By targeting factors that foster the development and maintenance of an immunosuppressive microenvironment within tumors, these therapies release the brakes on the host's own immune system; allowing cure of disease. Indeed, phase III clinical trials have revealed that therapies such as ipilimumab and pembrolizumab which target the CTLA4 and PD-1 immune checkpoints, respectively, have raised the three-year survival of patients with melanoma to ∼70%, and overall survival (>5years) to ∼30%. Despite this unprecedented efficacy, many patients fail to respond, and more concerning, some patients who demonstrate encouraging initial responses to immunotherapy, can acquire resistance over time. There is now an urgent need to identify mechanisms of resistance, to predict outcome and to identify targets for combination therapy. Here, with the aim of guiding future combination trials that target specific resistance mechanisms to immunotherapies, we have summarised and discussed the current understanding of mechanisms promoting resistance to anti-PD1/PDL1 therapies, and how combination strategies which target these pathways might yield better outcomes for patients.

  18. A divergent role of the SIRT1-TopBP1 axis in regulating metabolic checkpoint and DNA damage checkpoint.

    PubMed

    Liu, Tongzheng; Lin, Yi-Hui; Leng, Wenchuan; Jung, Sung Yun; Zhang, Haoxing; Deng, Min; Evans, Debra; Li, Yunhui; Luo, Kuntian; Qin, Bo; Qin, Jun; Yuan, Jian; Lou, Zhenkun

    2014-12-04

    DNA replication is executed only when cells have sufficient metabolic resources and undamaged DNA. Nutrient limitation and DNA damage cause a metabolic checkpoint and DNA damage checkpoint, respectively. Although SIRT1 activity is regulated by metabolic stress and DNA damage, its function in these stress-mediated checkpoints remains elusive. Here we report that the SIRT1-TopBP1 axis functions as a switch for both checkpoints. With glucose deprivation, SIRT1 is activated and deacetylates TopBP1, resulting in TopBP1-Treslin disassociation and DNA replication inhibition. Conversely, SIRT1 activity is inhibited under genotoxic stress, resulting in increased TopBP1 acetylation that is important for the TopBP1-Rad9 interaction and activation of the ATR-Chk1 pathway. Mechanistically, we showed that acetylation of TopBP1 changes the conformation of TopBP1, thereby facilitating its interaction with distinct partners in DNA replication and checkpoint activation. Taken together, our studies identify the SIRT1-TopBP1 axis as a key signaling mode in the regulation of the metabolic checkpoint and the DNA damage checkpoint.

  19. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism.

  20. Organizational Silence in Sports Employees

    ERIC Educational Resources Information Center

    Bastug, Gulsum; Pala, Adem; Yilmaz, Taner; Duyan, Mehdi; Gunel, Ilker

    2016-01-01

    Organizational silence can be defined as a way of behaviour belonging to men and women employees in the organization exhibited without reflecting their feelings, ideas, concerns and suggestions related with their workplaces, works for which they are responsible or other activities of the organization. In the period of organizational silence,…

  1. DNA damage checkpoint adaptation genes are required for division of cells harbouring eroded telomeres

    PubMed Central

    Mersaoui, Sofiane Y.; Gravel, Serge; Karpov, Victor; Wellinger, Raymund J.

    2015-01-01

    In budding yeast, telomerase and the Cdc13p protein are two key players acting to ensure telomere stability. In the absence of telomerase, cells eventually enter a growth arrest which only few can overcome via a conserved process; such cells are called survivors. Survivors rely on homologous recombination-dependent mechanisms for telomeric repeat addition. Previously, we showed that such survivor cells also manage to bypass the loss of the essential Cdc13p protein to give rise to Cdc13-independent (or cap-independent) strains. Here we show that Cdc13-independent cells grow with persistently recognized DNA damage, which does not however result in a checkpoint activation; thus no defect in cell cycle progression is detectable. The absence of checkpoint signalling rather is due to the accumulation of mutations in checkpoint genes such as RAD24 or MEC1. Importantly, our results also show that cells that have lost the ability to adapt to persistent DNA damage, also are very much impaired in generating cap-independent cells. Altogether, these results show that while the capping process can be flexible, it takes a very specific genetic setup to allow a change from canonical capping to alternative capping. We hypothesize that in the alternative capping mode, genome integrity mechanisms are abrogated, which could cause increased mutation frequencies. These results from yeast have clear parallels in transformed human cancer cells and offer deeper insights into processes operating in pre-cancerous human cells that harbour eroded telomeres.

  2. DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD

    PubMed Central

    Lim, Jong-Won; Snider, Lauren; Yao, Zizhen; Tawil, Rabi; Van Der Maarel, Silvère M.; Rigo, Frank; Bennett, C. Frank; Filippova, Galina N.; Tapscott, Stephen J.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the DUX4 transcription factor in skeletal muscle. The DUX4 retrogene is encoded in the D4Z4 macrosatellite repeat array, and smaller array size or a mutation in the SMCHD1 gene results in inefficient epigenetic repression of DUX4 in skeletal muscle, causing FSHD1 and FSHD2, respectively. Previously we showed that the entire D4Z4 repeat is bi-directionally transcribed with the generation of small si- or miRNA-like fragments and suggested that these might suppress DUX4 expression through the endogenous RNAi pathway. Here we show that exogenous siRNA targeting the region upstream of the DUX4 transcription start site suppressed DUX4 mRNA expression and increased both H3K9 methylation and AGO2 recruitment. In contrast, similarly targeted MOE-gapmer antisense oligonucleotides that degrade RNA but do not engage the RNAi pathway did not repress DUX4 expression. In addition, knockdown of DICER or AGO2 using either siRNA or MOE-gapmer chemistries resulted in the induction of DUX4 expression in control muscle cells that normally do not express DUX4, indicating that the endogenous RNAi pathway is necessary to maintain repression of DUX4 in control muscle cells. Together these data demonstrate a role of the endogenous RNAi pathway in repeat-mediated epigenetic repression of the D4Z4 macrosatellite repeat, and show that enhancing the activity of this pathway by supplying exogenous siRNA oligonucleotides represents a potential therapeutic approach to silencing DUX4 in FSHD. PMID:26041815

  3. The eerie silence

    NASA Astrophysics Data System (ADS)

    Davies, Paul

    2010-03-01

    Whether or not we are alone in the universe is one of the great outstanding questions of existence. For thousands of years it was restricted to the realm of philosophy and theology, but 50 years ago it became part of science. In April 1960 a young US astronomer, Frank Drake, began using a radio telescope to investigate whether signals from an extraterrestrial community might be coming our way. Known as the Search for Extraterrestrial Intelligence, or SETI, it has grown into a major international enterprise, involving scientific institutions in several countries. Apart from a few oddities, however, all that the radio astronomers have encountered is an eerie silence. So is humankind the only technological civilization in the universe after all? Or might we be looking for the wrong thing in the wrong place at the wrong time?

  4. Geminiviruses and RNA silencing.

    PubMed

    Vanitharani, Ramachandran; Chellappan, Padmanabhan; Fauquet, Claude M

    2005-03-01

    Geminiviruses are single-stranded circular DNA viruses that cause economically significant diseases in a wide range of crop plants worldwide. In plants, post-transcriptional gene silencing (PTGS) acts as a natural anti-viral defense system and plays a role in genome maintenance and development. During the past decade there has been considerable evidence of PTGS suppression by viruses, which is often required to establish infection in plants. In particular, nuclear-replicating geminiviruses, which have no double-stranded RNA phase in their replication cycle, can induce and suppress the PTGS and become targets for PTGS. Here, we summarize recent developments in determining how these viruses trigger PTGS and how they suppress the induced PTGS, as well as how we can use the system to control these viruses in plants better and manipulate the system to study functional genomics in crop plants.

  5. Immunogenic chemotherapy sensitizes tumors to checkpoint blockade therapy

    PubMed Central

    Pfirschke, Christina; Engblom, Camilla; Rickelt, Steffen; Cortez-Retamozo, Virna; Garris, Christopher; Pucci, Ferdinando; Yamazaki, Takahiro; Colame, Vichnou Poirier; Newton, Andita; Redouane, Younes; Lin, Yi-Jang; Wojtkiewicz, Gregory; Iwamoto, Yoshiko; Mino-Kenudson, Mari; Huynh, Tiffany G.; Hynes, Richard O.; Freeman, Gordon J.; Kroemer, Guido; Zitvogel, Laurence; Weissleder, Ralph; Pittet, Mikael J.

    2016-01-01

    SUMMARY Checkpoint blockade immunotherapies can be extraordinarily effective, but may benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when using appropriately selected immunogenic drugs (e.g. oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53). The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8+ T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs. PMID:26872698

  6. Radiotherapy and immune checkpoint blockades: a snapshot in 2016

    PubMed Central

    Koo, Taeryool; Kim, In Ah

    2016-01-01

    Immune checkpoint blockades including monoclonal antibodies (mAbs) of cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) have been emerged as a promising anticancer therapy. Several immune checkpoint blockades have been approved by US Food and Drug Administration (FDA), and have shown notable success in clinical trials for patients with advanced melanoma and non-small cell lung cancer. Radiotherapy is a promising combination partner of immune checkpoint blockades due to its potent pro-immune effect. This review will cover the current issue and the future perspectives for combined with radiotherapy and immune checkpoint blockades based upon the available preclinical and clinical data. PMID:28030901

  7. ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint

    PubMed Central

    Miniowitz-Shemtov, Shirly; Teichner, Adar; Sitry-Shevah, Danielle; Hershko, Avram

    2010-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate segregation of chromosomes by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. This system acts by inhibiting the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase to target securin for degradation. APC/C is inhibited by a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Mad2, and Cdc20. The molecular mechanisms of the inactivation of the mitotic checkpoint, including the release of APC/C from inhibition, remain obscure. It has been reported that polyubiquitylation by the APC/C is required for the inactivation of the mitotic checkpoint [Reddy SK, Rape M, Margansky WA, Kirschner MW (2007) Nature, 446:921–924]. We confirmed the involvement of polyubiquitylation, but found that another process, which requires ATP cleavage at the β–γ position (as opposed to α–β bond scission involved in ubiquitylation), is essential for the release of APC/C from checkpoint inhibition. ATP (β–γ) cleavage is required both for the dissociation of MCC components from APC/C and for the disassembly of free MCC, whereas polyubiquitylation is involved only in the former process. We find that the requirement for ATP (β–γ) cleavage is not due to the involvement of the 26S proteasome and that the phenomena observed are not due to sustained activity of protein kinase Cdk1/cyclin B, caused by inhibition of the degradation of cyclin B. Thus, some other energy-consuming process is needed for the inactivation of the mitotic checkpoint. PMID:20212161

  8. Tactical Checkpoint: Hail/Warn Suppress/Stop (Briefing Charts)

    DTIC Science & Technology

    2010-11-15

    U.S. Army Research, Development and Engineering Command Tactical Checkpoint: Hail/Warn Suppress /Stop Target Behavioral Response Laboratory... SUPPRESS /STOP Presented at the 2010 Directed Energies Professional Society Meeting, 15-19 November 2010. 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c...driver that is approaching a checkpoint. The laser, MCNC light, and the windshield obscuration were evaluated for their suppression capabilities (ability

  9. A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1.

    PubMed

    García-Rodríguez, Luis J; De Piccoli, Giacomo; Marchesi, Vanessa; Jones, Richard C; Edmondson, Ricky D; Labib, Karim

    2015-10-15

    Defects during chromosome replication in eukaryotes activate a signaling pathway called the S-phase checkpoint, which produces a multifaceted response that preserves genome integrity at stalled DNA replication forks. Work with budding yeast showed that the 'alternative clamp loader' known as Ctf18-RFC acts by an unknown mechanism to activate the checkpoint kinase Rad53, which then mediates much of the checkpoint response. Here we show that budding yeast Ctf18-RFC associates with DNA polymerase epsilon, via an evolutionarily conserved 'Pol ϵ binding module' in Ctf18-RFC that is produced by interaction of the carboxyl terminus of Ctf18 with the Ctf8 and Dcc1 subunits. Mutations at the end of Ctf18 disrupt the integrity of the Pol ϵ binding module and block the S-phase checkpoint pathway, downstream of the Mec1 kinase that is the budding yeast orthologue of mammalian ATR. Similar defects in checkpoint activation are produced by mutations that displace Pol ϵ from the replisome. These findings indicate that the association of Ctf18-RFC with Pol ϵ at defective replication forks is a key step in activation of the S-phase checkpoint.

  10. Molecular checkpoints controlling natural killer cell activation and their modulation for cancer immunotherapy

    PubMed Central

    Kwon, Hyung-Joon; Kim, Nayoung; Kim, Hun Sik

    2017-01-01

    Natural killer (NK) cells have gained considerable attention as promising therapeutic tools for cancer therapy due to their innate selectivity against cancer cells over normal healthy cells. With an array of receptors evolved to sense cellular alterations, NK cells provide early protection against cancer cells by producing cytokines and chemokines and exerting direct cytolytic activity. These effector functions are governed by signals transmitted through multiple receptor–ligand interactions but are not achieved by engaging a single activating receptor on resting NK cells. Rather, they require the co-engagement of different activating receptors that use distinct signaling modules, due to a cell-intrinsic inhibition mechanism. The redundancy of synergizing receptors and the inhibition of NK cell function by a single class of inhibitory receptor suggest the presence of common checkpoints to control NK cell activation through different receptors. These molecular checkpoints would be therapeutically targeted to harness the power of NK cells against diverse cancer cells that express heterogeneous ligands for NK cell receptors. Recent advances in understanding the activation of NK cells have revealed promising candidates in this category. Targeting such molecular checkpoints will facilitate NK cell activation by lowering activation thresholds, thereby providing therapeutic strategies that optimize NK cell reactivity against cancer. PMID:28360428

  11. Combining Immune Checkpoint Inhibitors and Kinase-Inhibiting Supramolecular Therapeutics for Enhanced Anticancer Efficacy.

    PubMed

    Kulkarni, Ashish; Natarajan, Siva Kumar; Chandrasekar, Vineethkrishna; Pandey, Prithvi Raj; Sengupta, Shiladitya

    2016-09-29

    A major limitation of immune checkpoint inhibitors is that only a small subset of patients achieve durable clinical responses. This necessitates the development of combinatorial regimens with immunotherapy. However, some combinations, such as MEK- or PI3K-inhibitors with a PD1-PDL1 checkpoint inhibitor, are pharmacologically challenging to implement. We rationalized that such combinations can be enabled using nanoscale supramolecular targeted therapeutics, which spatially home into tumors and exert temporally sustained inhibition of the target. Here we describe two case studies where nanoscale MEK- and PI3K-targeting supramolecular therapeutics were engineered using a quantum mechanical all-atomistic simulation-based approach. The combinations of nanoscale MEK- and PI3K-targeting supramolecular therapeutics with checkpoint PDL1 and PD1 inhibitors exert enhanced antitumor outcome in melanoma and breast cancers in vivo, respectively. Additionally, the temporal sequence of administration impacts the outcome. The combination of supramolecular therapeutics and immunotherapy could emerge as a paradigm shift in the treatment of cancer.

  12. Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints.

    PubMed

    Koyama, Shohei; Akbay, Esra A; Li, Yvonne Y; Herter-Sprie, Grit S; Buczkowski, Kevin A; Richards, William G; Gandhi, Leena; Redig, Amanda J; Rodig, Scott J; Asahina, Hajime; Jones, Robert E; Kulkarni, Meghana M; Kuraguchi, Mari; Palakurthi, Sangeetha; Fecci, Peter E; Johnson, Bruce E; Janne, Pasi A; Engelman, Jeffrey A; Gangadharan, Sidharta P; Costa, Daniel B; Freeman, Gordon J; Bueno, Raphael; Hodi, F Stephen; Dranoff, Glenn; Wong, Kwok-Kin; Hammerman, Peter S

    2016-02-17

    Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade.

  13. Regulation of zygotic genome activation and DNA damage checkpoint acquisition at the mid-blastula transition

    PubMed Central

    Zhang, Maomao; Kothari, Priyanka; Mullins, Mary; Lampson, Michael A.

    2014-01-01

    Following fertilization, oviparous embryos undergo rapid, mostly transcriptionally silent cleavage divisions until the mid-blastula transition (MBT), when large-scale developmental changes occur, including zygotic genome activation (ZGA) and cell cycle remodeling, via lengthening and checkpoint acquisition. Despite their concomitant appearance, whether these changes are co-regulated is unclear. Three models have been proposed to account for the timing of (ZGA). One model implicates a threshold nuclear to cytoplasmic (N:C) ratio, another stresses the importance cell cycle elongation, while the third model invokes a timer mechanism. We show that precocious Chk1 activity in pre-MBT zebrafish embryos elongates cleavage cycles, thereby slowing the increase in the N:C ratio. We find that cell cycle elongation does not lead to transcriptional activation. Rather, ZGA slows in parallel with the N:C ratio. We show further that the DNA damage checkpoint program is maternally supplied and independent of ZGA. Although pre-MBT embryos detect damage and activate Chk2 after induction of DNA double-strand breaks, the Chk1 arm of the DNA damage response is not activated, and the checkpoint is nonfunctional. Our results are consistent with the N:C ratio model for ZGA. Moreover, the ability of precocious Chk1 activity to delay pre-MBT cell cycles indicate that lack of Chk1 activity limits checkpoint function during cleavage cycles. We propose that Chk1 gain-of-function at the MBT underlies cell cycle remodeling, whereas ZGA is regulated independently by the N:C ratio. PMID:25558827

  14. Angiopoietin-2 as a Biomarker and Target for Immune Checkpoint Therapy.

    PubMed

    Wu, Xinqi; Giobbie-Hurder, Anita; Liao, Xiaoyun; Connelly, Courtney; Connolly, Erin M; Li, Jingjing; Manos, Michael P; Lawrence, Donald; McDermott, David; Severgnini, Mariano; Zhou, Jun; Gjini, Evisa; Lako, Ana; Lipschitz, Mikel; Pak, Christine J; Abdelrahman, Sara; Rodig, Scott; Hodi, F Stephen

    2017-01-01

    Immune checkpoint therapies targeting CTLA-4 and PD-1 have proven effective in cancer treatment. However, the identification of biomarkers for predicting clinical outcomes and mechanisms to overcome resistance remain as critical needs. Angiogenesis is increasingly appreciated as an immune modulator with potential for combinatorial use with checkpoint blockade. Angiopoietin-2 (ANGPT2) is an immune target in patients and is involved in resistance to anti-VEGF treatment with the monoclonal antibody bevacizumab. We investigated the predictive and prognostic value of circulating ANGPT2 in metastatic melanoma patients receiving immune checkpoint therapy. High pretreatment serum ANGPT2 was associated with reduced overall survival in CTLA-4 and PD-1 blockade-treated patients. These treatments also increased serum ANGPT2 in many patients early after treatment initiation, whereas ipilimumab plus bevacizumab treatment decreased serum concentrations. ANGPT2 increases were associated with reduced response and/or overall survival. Ipilimumab increased, and ipilimumab plus bevacizumab decreased, tumor vascular ANGPT2 expression in a subset of patients, which was associated with increased and decreased tumor infiltration by CD68(+) and CD163(+) macrophages, respectively. In vitro, bevacizumab blocked VEGF-induced ANGPT2 expression in tumor-associated endothelial cells, whereas ANGPT2 increased PD-L1 expression on M2-polarized macrophages. Treatments elicited long-lasting and functional antibody responses to ANGPT2 in a subset of patients receiving clinical benefit. Our findings suggest that serum ANGPT2 may be considered as a predictive and prognostic biomarker for immune checkpoint therapy and may contribute to treatment resistance via increasing proangiogenic and immunosuppressive activities in the tumor microenvironment. Targeting ANGPT2 provides a rational combinatorial approach to improve the efficacy of immune therapy. Cancer Immunol Res; 5(1); 17-28. ©2016 AACR.

  15. Induction of a caffeine-sensitive S-phase cell cycle checkpoint by psoralen plus ultraviolet A radiation.

    PubMed

    Joerges, Christoph; Kuntze, Inge; Herzinger, Thomas; Herzinge, Thomas

    2003-09-18

    Induction of interstrand crosslinks (ICLs) in chromosomal DNA is considered a major reason for the antiproliferative effect of psoralen plus ultraviolet A (PUVA). It is unclear as to whether PUVA-induced cell cycle arrest is caused by ICLs mechanically stalling replication forks or by triggering cell cycle checkpoints. Cell cycle checkpoints serve to maintain genomic stability by halting cell cycle progression to prevent replication of damaged DNA templates or segregation of broken chromosomes. Here, we show that HaCaT keratinocytes treated with PUVA arrest with S-phase DNA content. Cells that had completed DNA replication were not perturbed by PUVA and passed through mitosis. Cells treated with PUVA during G1-phase continued traversing G1 until arresting in early S-phase. PUVA induced rapid phosphorylation of the Chk1 checkpoint kinase at Ser345 and a concomitant decrease in Cdc25A levels. Chk1 phosphorylation, decrease of Cdc25 A levels and S-phase arrest were abolished by caffeine, demonstrating that active checkpoint signaling rather than passive mechanical blockage by ICLs causes the PUVA-induced replication arrest. Overexpression of Cdc25A only partially overrode the S-phase arrest, suggesting that additional signaling events implement PUVA-induced S-phase arrest.

  16. Evolutionary expansion of a regulatory network by counter-silencing

    PubMed Central

    Will, William R.; Bale, Denise H.; Reid, Philip J.; Libby, Stephen J.; Fang, Ferric C.

    2014-01-01

    Horizontal gene transfer plays a major role in bacterial evolution. Successful acquisition of new genes requires their incorporation into existing regulatory networks. This study compares the regulation of conserved genes in the PhoPQ regulon of Salmonella enterica serovar Typhimurium with that of PhoPQ-regulated horizontally-acquired genes, which are silenced by the histone-like protein H-NS. We demonstrate that PhoP up-regulates conserved and horizontally-acquired genes by distinct mechanisms. Conserved genes are regulated by classical PhoP-mediated activation and are invariant in promoter architecture, whereas horizontally-acquired genes exhibit variable promoter architecture and are regulated by PhoP-mediated counter-silencing. Biochemical analyses show that a horizontally-acquired promoter adopts different structures in the silenced and counter-silenced states, implicating the remodeling of the H-NS nucleoprotein filament and the subsequent restoration of open complex formation as the central mechanism of counter-silencing. Our results indicate that counter-silencing is favored in the regulatory integration of newly-acquired genes because it is able to accommodate multiple promoter architectures. PMID:25348042

  17. Fission Yeast Apc15 Stabilizes MCC-Cdc20-APC/C Complexes, Ensuring Efficient Cdc20 Ubiquitination and Checkpoint Arrest.

    PubMed

    May, Karen M; Paldi, Flora; Hardwick, Kevin G

    2017-03-28

    During mitosis, cells must segregate the replicated copies of their genome to their daughter cells with extremely high fidelity. Segregation errors lead to an abnormal chromosome number (aneuploidy), which typically results in disease or cell death [1]. Chromosome segregation and anaphase onset are initiated through the action of the multi-subunit E3 ubiquitin ligase known as the anaphase-promoting complex or cyclosome (APC/C [2]). The APC/C is inhibited by the spindle checkpoint in the presence of kinetochore attachment defects [3, 4]. Here we demonstrate that two non-essential APC/C subunits (Apc14 and Apc15) regulate association of spindle checkpoint proteins, in the form of the mitotic checkpoint complex (MCC), with the APC/C. apc14Δ mutants display increased MCC association with the APC/C and are unable to silence the checkpoint efficiently. Conversely, apc15Δ mutants display reduced association between the MCC and APC/C, are defective in poly-ubiquitination of Cdc20, and are checkpoint defective. In vitro reconstitution studies have shown that human MCC-APC/C can contain two molecules of Cdc20 [5-7]. Using a yeast strain expressing two Cdc20 genes with different epitope tags, we show by co-immunoprecipitation that this is true in vivo. MCC binding to the second molecule of Cdc20 is mediated via the C-terminal KEN box in Mad3. Somewhat surprisingly, complexes containing both molecules of Cdc20 accumulate in apc15Δ cells, and the implications of this observation are discussed.

  18. Retinoids regulate a developmental checkpoint for tissue regeneration in Drosophila

    PubMed Central

    Halme, Adrian; Cheng, Michelle; Hariharan, Iswar K.

    2010-01-01

    Summary Drosophila melanogaster larvae have a remarkable capacity for regenerative growth: Damage to their imaginal discs, the larval precursors of adult structures, elicits a robust proliferative response from the surviving tissue [1–4]. However, as in other organisms, developmental progression and differentiation can restrict regenerative capacity of Drosophila tissues. Experiments in Drosophila and other holometabolous insects have demonstrated that either damage to imaginal tissues [5, 6] or transplantation of a damaged imaginal disc [7, 8] delays the onset of metamorphosis, a time when the imaginal discs undergo morphogenesis and differentiation into their adult structures. Therefore, in Drosophila there appears to be a mechanism that senses tissue damage and extends the larval phase to coordinate tissue regeneration with the overall developmental program of the organism. However, how such a pathway functions remains unknown. Here we demonstrate that a developmental checkpoint extends larval growth after imaginal disc damage by inhibiting the transcription of the gene encoding PTTH, a neuropeptide that promotes the release of the steroid hormone ecdysone. Using a genetic screen, we identify a previously unsuspected role for retinoid biosynthesis in regulating PTTH expression and delaying development in response to tissue damage. Retinoid signaling plays an important, but poorly defined role in several vertebrate regeneration models [9–11]. Our findings demonstrate that retinoid biosynthesis in Drosophila is important for the maintenance of a permissive condition for regenerative growth. PMID:20189388

  19. Immunotherapy for Gastric Cancer: A Focus on Immune Checkpoints.

    PubMed

    Alsina, Maria; Moehler, Markus; Hierro, Cinta; Guardeño, Raquel; Tabernero, Josep

    2016-08-01

    Gastric cancer (GC) is a major world-wide health problem. It is the third leading cause of death from cancer. The treatment of advanced GC by chemotherapy has limited efficacy. The addition of some targeted therapies like trastuzumab and ramucirumab have added a modest benefit, but only in human epidermal growth factor receptor 2 (ERBB2 or HER2)-positive patients and in the second-line setting, respectively. The development of new and effective therapeutic strategies must consider the genetic complexity and heterogeneity of GC; prognostic and predictive biomarkers should be identified for clinical implementation. Immune deregulation has been associated with some GC subtypes, especially those that are associated with virus infection and those with a high mutational rate. Different mechanisms to prevent immunologic escape have been characterized during the last years; in particular the PD-1/PD-L1 inhibitors pembrolizumab, avelumab, durvalumab and atezolizumab have shown early sign of efficacy. Therefore, immunotherapeutic strategies may provide new opportunities for GC patients. This review will discuss (1) the main characteristics of GC treatment, (2) the immune response in GC, and (3) the current status of immune-related strategies in clinical development in GC patients, focusing on immune checkpoints therapies.

  20. A dynamical model of the spindle position checkpoint.

    PubMed

    Caydasi, Ayse Koca; Lohel, Maiko; Grünert, Gerd; Dittrich, Peter; Pereira, Gislene; Ibrahim, Bashar

    2012-05-08

    The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother-to-daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase-activating protein (GAP) complex Bfa1-Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1-Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1-Bub2 and Tem1 localization at the SPBs. Based on the measured SPB-bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.

  1. Silence and the Notion of the Commons.

    ERIC Educational Resources Information Center

    Franklin, Ursula

    1994-01-01

    Stresses the value of silence, the right to have silence, and how technology has manipulated the sound environment and therefore taken silence out of common availability. Discusses noise pollution and the manipulative use of sound for private gain. Suggests taking action to restore the right to silence. (LP)

  2. Viral suppressors of RNA silencing in Wheat mosaic virus (WMoV)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA silencing is the most effective antiviral adaptive defense mechanism mounted in higher plants to combat viral infection and proliferation. However, viruses have developed a variety of efficient counter-defense mechanisms by suppression of RNA silencing (VSR) in order to successfully impede the h...

  3. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome

    NASA Astrophysics Data System (ADS)

    Lu, Rui; Folimonov, Alexey; Shintaku, Michael; Li, Wan-Xiang; Falk, Bryce W.; Dawson, William O.; Ding, Shou-Wei

    2004-11-01

    Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense. Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the 20-kb plus-strand RNA genome of citrus tristeza virus (CTV). When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene. Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F1 plants expressing p23 and not from the CP- or p20-expressing F1 plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23. Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels. Notably, CP suppresses intercellular silencing without interfering with intracellular silencing. The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene. Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. RNA interference | citrus tristeza virus | virus synergy | antiviral immunity

  4. Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2 ▿

    PubMed Central

    Kile, Andrew C.; Koepp, Deanna M.

    2010-01-01

    A stable genome is critical to cell viability and proliferation. During DNA replication, the S-phase checkpoint pathway responds to replication stress. In budding yeast, the chromatin-bound F-box protein Dia2 is required to maintain genomic stability and may help replication complexes overcome sites of damaged DNA and natural fragile regions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases. We show here that Dia2 is itself targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint pathway inhibits Dia2 protein degradation. S-phase checkpoint mutants fail to stabilize Dia2 in response to replication stress. Deletion of DIA2 from these checkpoint mutants exacerbates their sensitivity to hydroxyurea, suggesting that stabilization of Dia2 contributes to the replication stress response. Unlike the case for other F-box proteins, deletion of the F-box domain in Dia2 does not stabilize the protein. Rather, an N-terminal domain that is also required for nuclear localization is necessary for degradation. When a strong nuclear localization signal (NLS) is added to dia2 mutants lacking this domain, the Dia2 protein is both stable and nuclear. Together, our results suggest that Dia2 protein turnover does not involve an autocatalytic mechanism and that Dia2 proteolysis is inhibited by activation of the replication stress response. PMID:19858292

  5. WWOX modulates the ATR-mediated DNA damage checkpoint response.

    PubMed

    Abu-Odeh, Mohammad; Hereema, Nyla A; Aqeilan, Rami I

    2016-01-26

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability.

  6. WWOX modulates the ATR-mediated DNA damage checkpoint response

    PubMed Central

    Abu-Odeh, Mohammad; Hereema, Nyla A.; Aqeilan, Rami I.

    2016-01-01

    For many decades genomic instability is considered one of the hallmarks of cancer. Role of the tumor suppressor WWOX (WW domain-containing oxidoreductase) in DNA damage response upon double strand breaks has been recently revealed. Here we demonstrate unforeseen functions for WWOX upon DNA single strand breaks (SSBs) checkpoint activation. We found that WWOX levels are induced following SSBs and accumulate in the nucleus. WWOX deficiency is associated with reduced activation of ataxia telangiectasia and Rad3-related protein (ATR) checkpoint proteins and increased chromosomal breaks. At the molecular level, we show that upon SSBs WWOX is modified at lysine 274 by ubiquitination mediated by the ubiquitin E3 ligase ITCH and interacts with ataxia telangiectasia-mutated (ATM). Interestingly, ATM inhibition was associated with reduced activation of ATR checkpoint proteins suggesting that WWOX manipulation of ATR checkpoint proteins is ATM-dependent. Taken together, the present findings indicate that WWOX plays a key role in ATR checkpoint activation, while its absence might facilitate genomic instability. PMID:26675548

  7. PKCɛ switches Aurora B specificity to exit the abscission checkpoint

    PubMed Central

    Pike, Tanya; Brownlow, Nicola; Kjaer, Svend; Carlton, Jeremy; Parker, Peter J.

    2016-01-01

    The ‘NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ–Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division. PMID:28004745

  8. PKCɛ switches Aurora B specificity to exit the abscission checkpoint.

    PubMed

    Pike, Tanya; Brownlow, Nicola; Kjaer, Svend; Carlton, Jeremy; Parker, Peter J

    2016-12-22

    The 'NoCut', or Aurora B abscission checkpoint can be activated if DNA is retained in the cleavage furrow after completion of anaphase. Checkpoint failure leads to incomplete abscission and a binucleate outcome. These phenotypes are also observed after loss of PKCɛ in transformed cell models. Here we show that PKCɛ directly modulates the Aurora B-dependent abscission checkpoint by phosphorylating Aurora B at S227. This phosphorylation invokes a switch in Aurora B specificity, with increased phosphorylation of a subset of target substrates, including the CPC subunit Borealin. This switch is essential for abscission checkpoint exit. Preventing the phosphorylation of Borealin leads to abscission failure, as does expression of a non-phosphorylatable Aurora B S227A mutant. Further, depletion of the ESCRT-III component and Aurora B substrate CHMP4C enables abscission, bypassing the PKCɛ-Aurora B exit pathway. Thus, we demonstrate that PKCɛ signals through Aurora B to exit the abscission checkpoint and complete cell division.

  9. The emerging role of immune checkpoint inhibition in malignant lymphoma

    PubMed Central

    Hude, Ida; Sasse, Stephanie; Engert, Andreas; Bröckelmann, Paul J.

    2017-01-01

    To evade elimination by the host immune system, tumor cells commonly exploit physiological immune checkpoint pathways, restraining efficient anti-tumor immune cell function. Growing understanding of the complex dialog between tumor cells and their microenvironment contributed to the development of immune checkpoint inhibitors. This innovative strategy has demonstrated paradigm-shifting clinical activity in various malignancies. Antibodies targeting programmed death 1 and cytotoxic T-lymphocyte-associated protein-4 are also being investigated in lymphoid malignancies with varying levels of activity and a favorable toxicity profile. To date, evaluated only in the setting of relapsed or refractory disease, anti-programmed death 1 antibodies such as nivolumab and pembrolizumab show encouraging response rates particularly in classical Hodgkin lymphoma but also in follicular lymphoma and diffuse-large B-cell lymphoma. As the first immune checkpoint inhibitor in lymphoma, nivolumab was approved for the treatment of relapsed or refractory classical Hodgkin lymphoma by the Food and Drug Administration in May 2016. In this review, we assess the role of the pathways involved and potential rationale of checkpoint inhibition in various lymphoid malignancies. In addition to data from current clinical trials, immune-related side effects, potential limitations and future perspectives including promising combinatory approaches with immune checkpoint inhibition are discussed. PMID:27884973

  10. Message Efficient Checkpointing and Rollback Recovery in Heterogeneous Mobile Networks

    NASA Astrophysics Data System (ADS)

    Jaggi, Parmeet Kaur; Singh, Awadhesh Kumar

    2016-06-01

    Heterogeneous networks provide an appealing way of expanding the computing capability of mobile networks by combining infrastructure-less mobile ad-hoc networks with the infrastructure-based cellular mobile networks. The nodes in such a network range from low-power nodes to macro base stations and thus, vary greatly in their capabilities such as computation power and battery power. The nodes are susceptible to different types of transient and permanent failures and therefore, the algorithms designed for such networks need to be fault-tolerant. The article presents a checkpointing algorithm for the rollback recovery of mobile hosts in a heterogeneous mobile network. Checkpointing is a well established approach to provide fault tolerance in static and cellular mobile distributed systems. However, the use of checkpointing for fault tolerance in a heterogeneous environment remains to be explored. The proposed protocol is based on the results of zigzag paths and zigzag cycles by Netzer-Xu. Considering the heterogeneity prevalent in the network, an uncoordinated checkpointing technique is employed. Yet, useless checkpoints are avoided without causing a high message overhead.

  11. Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

    PubMed

    Jiang, Xiujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Lian, Weiguang; Yi, Li; Zhang, Donghui; Yang, Haiyan; Liu, Jianghui; Zhang, Xianghong

    2017-01-01

    Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis

  12. Aortic endothelial cells regulate proliferation of human monocytes in vitro via a mechanism synergistic with macrophage colony-stimulating factor. Convergence at the cyclin E/p27(Kip1) regulatory checkpoint.

    PubMed

    Antonov, A S; Munn, D H; Kolodgie, F D; Virmani, R; Gerrity, R G

    1997-06-15

    Monocyte-derived macrophages (Mphis) are pivotal participants in the pathogenesis of atherosclerosis. Evidence from both animal and human plaques indicates that local proliferation may contribute to accumulation of lesion Mphis, and the major Mphi growth factor, macrophage colony stimulating factor (MCSF), is present in atherosclerotic plaques. However, most in vitro studies have failed to demonstrate that human monocytes/Mphis possess significant proliferative capacity. We now report that, although human monocytes cultured in isolation showed only limited MCSF-induced proliferation, monocytes cocultured with aortic endothelial cells at identical MCSF concentrations underwent enhanced (up to 40-fold) and prolonged (21 d) proliferation. In contrast with monocytes in isolation, this was optimal at low seeding densities, required endothelial cell contact, and could not be reproduced by coculture with smooth muscle cells. Intimal Mphi isolated from human aortas likewise showed endothelial cell contact-dependent, MCSF-induced proliferation. Consistent with a two-signal mechanism governing Mphi proliferation, the cell cycle regulatory protein, cyclin E, was rapidly upregulated by endothelial cell contact in an MCSFindependent fashion, but MCSF was required for successful downregulation of the cell cycle inhibitory protein p27(Kip1) before cell cycling. Thus endothelial cells and MCSF differentially and synergistically regulate two Mphi genes critical for progression through the cell cycle.

  13. Detailed Structural-Functional Analysis of the Krüppel-like Factor 16 (KLF16) Transcription Factor Reveals Novel Mechanisms for Silencing Sp/KLF Sites Involved in Metabolism and Endocrinology*

    PubMed Central

    Daftary, Gaurang S.; Lomberk, Gwen A.; Buttar, Navtej S.; Allen, Thomas W.; Grzenda, Adrienne; Zhang, Jinsan; Zheng, Ye; Mathison, Angela J.; Gada, Ravi P.; Calvo, Ezequiel; Iovanna, Juan L.; Billadeau, Daniel D.; Prendergast, Franklyn G.; Urrutia, Raul

    2012-01-01

    Krüppel-like factor (KLF) proteins have elicited significant attention due to their emerging key role in metabolic and endocrine diseases. Here, we extend this knowledge through the biochemical characterization of KLF16, unveiling novel mechanisms regulating expression of genes involved in reproductive endocrinology. We found that KLF16 selectively binds three distinct KLF-binding sites (GC, CA, and BTE boxes). KLF16 also regulated the expression of several genes essential for metabolic and endocrine processes in sex steroid-sensitive uterine cells. Mechanistically, we determined that KLF16 possesses an activation domain that couples to histone acetyltransferase-mediated pathways, as well as a repression domain that interacts with the histone deacetylase chromatin-remodeling system via all three Sin3 isoforms, suggesting a higher level of plasticity in chromatin cofactor selection. Molecular modeling combined with molecular dynamic simulations of the Sin3a-KLF16 complex revealed important insights into how this interaction occurs at an atomic resolution level, predicting that phosphorylation of Tyr-10 may modulate KLF16 function. Phosphorylation of KLF16 was confirmed by in vivo 32P incorporation and controlled by a Y10F site-directed mutant. Inhibition of Src-type tyrosine kinase signaling as well as the nonphosphorylatable Y10F mutation disrupted KLF16-mediated gene silencing, demonstrating that its function is regulatable rather than constitutive. Subcellular localization studies revealed that signal-induced nuclear translocation and euchromatic compartmentalization constitute an additional mechanism for regulating KLF16 function. Thus, this study lends insights on key biochemical mechanisms for regulating KLF sites involved in reproductive biology. These data also contribute to the new functional information that is applicable to understanding KLF16 and other highly related KLF proteins. PMID:22203677

  14. Immune checkpoint and inflammation as therapeutic targets in pancreatic carcinoma

    PubMed Central

    Kimbara, Shiro; Kondo, Shunsuke

    2016-01-01

    Pancreatic adenocarcinoma (PAC) is one of the most deadly malignant neoplasms, and the efficacy of conventional cytotoxic chemotherapy is far from satisfactory. Recent research studies have revealed that immunosuppression and inflammation are associated with oncogenesis, as well as tumor development, invasion, and metastasis in PAC. Thus, immunosuppression-related signaling, especially that involving immune checkpoint and inflammation, has emerged as novel treatment targets for PAC. However, PAC is an immune-resistant tumor, and it is still unclear whether immune checkpoint or anti-inflammation therapies would be an ideal strategy. In this article, we will review immune checkpoint and inflammation as potential targets, as well as clinical trials and the prospects for immunotherapy in PAC. PMID:27672267

  15. The Abscission Checkpoint: Making It to the Final Cut.

    PubMed

    Nähse, Viola; Christ, Liliane; Stenmark, Harald; Campsteijn, Coen

    2017-01-01

    Cytokinesis is the final stage of cell division and is concluded by abscission of the intercellular bridge to physically separate the daughter cells. Timing of cytokinetic abscission is monitored by a molecular machinery termed the abscission checkpoint. This machinery delays abscission in cells with persistent chromatin in the intercellular bridge. Recent work has also uncovered its response to high membrane tension, nuclear pore defects, and DNA replication stress. Although it is known that the abscission checkpoint depends on persistent activity of the Aurora B protein kinase, we have only recently begun to understand its molecular basis. We propose here a molecular framework for abscission checkpoint signaling and we discuss outstanding questions relating to its function and physiological relevance.

  16. Combination Approaches with Immune-Checkpoint Blockade in Cancer Therapy.

    PubMed

    Swart, Maarten; Verbrugge, Inge; Beltman, Joost B

    2016-01-01

    In healthy individuals, immune-checkpoint molecules prevent autoimmune responses and limit immune cell-mediated tissue damage. Tumors frequently exploit these molecules to evade eradication by the immune system. Over the past years, immune-checkpoint blockade of cytotoxic T lymphocyte antigen-4 and programed death-1 emerged as promising strategies to activate antitumor cytotoxic T cell responses. Although complete regression and long-term survival is achieved in some patients, not all patients respond. This review describes promising, novel combination approaches involving immune-checkpoint blockade in the context of the cancer-immunity cycle, aimed at increasing response rates to the single treatments. Specifically, we discuss combinations that promote antigen release and presentation, that further amplify T cell activation, that inhibit trafficking of regulatory T cells or MSDCs, that stimulate intratumoral T cell infiltration, that increase cancer recognition by T cells, and that stimulate tumor killing.

  17. Combination Approaches with Immune-Checkpoint Blockade in Cancer Therapy

    PubMed Central

    Swart, Maarten; Verbrugge, Inge; Beltman, Joost B.

    2016-01-01

    In healthy individuals, immune-checkpoint molecules prevent autoimmune responses and limit immune cell-mediated tissue damage. Tumors frequently exploit these molecules to evade eradication by the immune system. Over the past years, immune-checkpoint blockade of cytotoxic T lymphocyte antigen-4 and programed death-1 emerged as promising strategies to activate antitumor cytotoxic T cell responses. Although complete regression and long-term survival is achieved in some patients, not all patients respond. This review describes promising, novel combination approaches involving immune-checkpoint blockade in the context of the cancer-immunity cycle, aimed at increasing response rates to the single treatments. Specifically, we discuss combinations that promote antigen release and presentation, that further amplify T cell activation, that inhibit trafficking of regulatory T cells or MSDCs, that stimulate intratumoral T cell infiltration, that increase cancer recognition by T cells, and that stimulate tumor killing. PMID:27847783

  18. Inhibiting Immune Checkpoints for the Treatment of Bladder Cancer

    PubMed Central

    Bidnur, S.; Savdie, R.; Black, P.C.

    2016-01-01

    Background: Increasing evidence supporting the role of immune checkpoint blockade in cancer management has been bolstered by recent reports demonstrating significant and durable clinical responses across multiple tumour types, including metastatic urothelial carcinoma (mUC). The majority of these results are achieved via blockade of the programmed death (PD) axis, which like CTLA-4 blockade permits T-cell activation and immune-mediated anti-tumour activity- essentially harnessing the patient’s own immune system to mount an anti-neoplastic response. However, while clinical responses can be striking, our understanding of the biology of immune checkpoint blockade is only beginning to shed light on how to maximize and even improve patient outcomes with immune checkpoint blockade, especially in UC. Methods: We performed a literature review for immune checkpoint blockade with a focus on rationale for checkpoint therapy and outcomes in UC. We also highlight the advances made in other tumour types, with a focus on the recent 2015 meeting of the American Society for Clinical Oncology. Results: In heavily pre-treated UC, trials are suggesting objective response rates above 30% . These impressive results are seen across multiple different tumour types, especially those with high burden of DNA level mutations. Identification of prognostic biomarkers is currently under investigation, in order to improve patient selection. Interestingly, response to PD-1 directed therapy is seen even in patients with no evidence of PD-1 positivity on immunohistochemistry. This has led to the development of enhanced biomarkers including assessing DNA mutation rates and immune gene signatures, to improve patient selection. Conclusions: Immune checkpoint blockade is an exciting cancer treatment modality which is demonstrating impressive clinical results across multiple tumour types. For UC, anti-PD directed therapy represents a much needed treatment in the metastatic, post chemotherapy context

  19. RNA-guided genetic silencing systems in bacteria and archaea.

    PubMed

    Wiedenheft, Blake; Sternberg, Samuel H; Doudna, Jennifer A

    2012-02-15

    Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based bacterial immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.

  20. Antiviral RNA silencing suppression activity of Tomato spotted wilt virus NSs protein.

    PubMed

    Ocampo Ocampo, T; Gabriel Peralta, S M; Bacheller, N; Uiterwaal, S; Knapp, A; Hennen, A; Ochoa-Martinez, D L; Garcia-Ruiz, H

    2016-06-17

    In addition to regulating gene expression, RNA silencing is an essential antiviral defense system in plants. Triggered by double-stranded RNA, silencing results in degradation or translational repression of target transcripts. Viruses are inducers and targets of RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressors that interfere with this process, such as the Tomato spotted wilt virus (TSWV) NSs protein. The mechanism by which NSs suppresses RNA silencing and its role in viral infection and movement remain to be determined. We cloned NSs from the Hawaii isolate of TSWV and using two independent assays show for the first time that this protein restored pathogenicity and supported the formation of local infection foci by suppressor-deficient Turnip mosaic virus and Turnip crinkle virus. Demonstrating the suppression of RNA silencing directed against heterologous viruses establishes the foundation to determine the means used by NSs to block this antiviral process.

  1. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence.

    PubMed

    Pumplin, Nathan; Voinnet, Olivier

    2013-11-01

    RNA silencing is a central regulator of gene expression in most eukaryotes and acts both at the transcriptional level through DNA methylation and at the post-transcriptional level through direct mRNA interference mediated by small RNAs. In plants and invertebrates, the same pathways also function directly in host defence against viruses by targeting viral RNA for degradation. Successful viruses have consequently evolved diverse mechanisms to avoid silencing, most notably through the expression of viral suppressors of RNA silencing. RNA silencing suppressors have also been recently identified in plant pathogenic bacteria and oomycetes, suggesting that disruption of host silencing is a general virulence strategy across several kingdoms of plant pathogens. There is also increasing evidence that plants have evolved specific defences against RNA-silencing suppression by pathogens, providing yet another illustration of the never-ending molecular arms race between plant pathogens and their hosts.

  2. Kcnq1ot1/Lit1 Noncoding RNA Mediates Transcriptional Silencing by Targeting to the Perinucleolar Region ▿ †

    PubMed Central

    Mohammad, Faizaan; Pandey, Radha Raman; Nagano, Takashi; Chakalova, Lyubomira; Mondal, Tanmoy; Fraser, Peter; Kanduri, Chandrasekhar

    2008-01-01

    The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic. Here we characterize a domain at the 5′ end of the Kcnq1ot1 RNA that carries out transcriptional silencing of linked genes using an episomal vector system. The bidirectional silencing property of Kcnq1ot1 maps to a highly conserved repeat motif within the silencing domain, which directs transcriptional silencing by interaction with chromatin, resulting in histone H3 lysine 9 trimethylation. Intriguingly, the silencing domain is also required to target the episomal vector to the perinucleolar compartment during mid-S phase. Collectively, our data unfold a novel mechanism by which an antisense RNA mediates transcriptional gene silencing of chromosomal domains by targeting them to distinct nuclear compartments known to be rich in heterochromatic machinery. PMID:18299392

  3. Aurora B prevents premature removal of spindle assembly checkpoint proteins from the kinetochore: A key role for Aurora B in mitosis.

    PubMed

    Gurden, Mark D; Anderhub, Simon J; Faisal, Amir; Linardopoulos, Spiros

    2016-07-18

    Accurate chromosome segregation is dependent on the spindle assembly checkpoint (SAC). In current models, the key direct role of Aurora B in the SAC has been suggested to be to promote rapid kinetochore localisation of MPS1, allowing MPS1 to generate the checkpoint signal. However, Aurora B is also thought to play an indirect role in the SAC through the destabilisation of kinetochore-microtubule (KT-MT) attachments. Here, we demonstrate that Aurora B activity is not required for the kinetochore recruitment of the majority of SAC proteins. More importantly, we show that the primary role of Aurora B in the SAC is to prevent the premature removal of SAC proteins from the kinetochore, which is strictly dependent on KT-MT interactions. Moreover, in the presence of KT-MT interactions, Aurora B inhibition silences a persistent SAC induced by tethering MPS1 to the kinetochore. This explains the highly synergistic interaction between Aurora B and MPS1 inhibitors to override the SAC, which is lost when cells are pre-arrested in nocodazole. Furthermore, we show that Aurora B and MPS1 inhibitors synergistically kill a panel of breast and colon cancer cell lines, including cells that are otherwise insensitive to Aurora B inhibitors alone. These data demonstrate that the major role of Aurora B in SAC is to prevent the removal of SAC proteins from tensionless kinetochores, thus inhibiting premature SAC silencing, and highlights a therapeutic strategy through combination of Aurora B and MPS1 inhibitors.

  4. Functional Diversity of Silencers in Budding Yeasts

    PubMed Central

    Sjöstrand, Jimmy O. O.; Kegel, Andreas; Åström, Stefan U.

    2002-01-01

    We studied the silencing of the cryptic mating-type loci HMLα and HMRa in the budding yeast Kluyveromyces lactis. A 102-bp minimal silencer fragment was defined that was both necessary and sufficient for silencing of HMLα. Mutagenesis of the silencer revealed three distinct regions (A, B, and C) that were important for silencing. Recombinant K. lactis ribosomal DNA enhancer binding protein 1 (Reb1p) could bind the silencer in vitro, and point mutations in the B box abolished both Reb1p binding and silencer function. Furthermore, strains carrying temperature-sensitive alleles of the REB1 gene derepressed the transcription of the HMLα1 gene at the nonpermissive temperature. A functional silencer element from the K. lactis cryptic HMRa locus was also identified, which contained both Reb1p binding sites and A boxes, strongly suggesting a general role for these sequences in K. lactis silencing. Our data indicate that different proteins bind to Kluyveromyces silencers than to Saccharomyces silencers. We suggest that the evolution of silencers is rapid in budding yeasts and discuss the similarities and differences between silencers in Saccharomyces and Kluyveromyces. PMID:12456003

  5. Mechanisms and Components of the DNA Damage Checkpoint

    DTIC Science & Technology

    2002-09-01

    survival of UV irradiation. that loss of RAD9 decreases the viability of cells chal- Survival of replication block due to hydroxyurea (HU) lenged with...radiation. Oncogene 19, nized with a factor. Cells were washed into prewarmed YPAD and 6185-6193. grown at 37°C, 70% ethanol fixed samples were sonicated...Schizosaccharomyces pombe . Genes Dev. myces cerevisiae: a possible function of G2 arrest upon UV irradia- 12, 382-395. tion. Mol. Cell. Biol. 10, 4678-4684

  6. Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae

    PubMed Central

    Rossodivita, Alyssa A.; Boudoures, Anna L.; Mecoli, Jonathan P.; Steenkiste, Elizabeth M.; Karl, Andrea L.; Vines, Eudora M.; Cole, Arron M.; Ansbro, Megan R.; Thompson, Jeffrey S.

    2014-01-01

    Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways. PMID:24748660

  7. A quantitative systems view of the spindle assembly checkpoint

    PubMed Central

    Ciliberto, Andrea; Shah, Jagesh V

    2009-01-01

    The idle assembly checkpoint acts to delay chromosome segregation until all duplicated sister chromatids are captured by the mitotic spindle. This pathway ensures that each daughter cell receives a complete copy of the genome. The high fidelity and robustness of this process have made it a subject of intense study in both the experimental and computational realms. A significant number of checkpoint proteins have been identified but how they orchestrate the communication between local spindle attachment and global cytoplasmic signalling to delay segregation is not yet understood. Here, we propose a systems view of the spindle assembly checkpoint to focus attention on the key regulators of the dynamics of this pathway. These regulators in turn have been the subject of detailed cellular measurements and computational modelling to connect molecular function to the dynamics of spindle assembly checkpoint signalling. A review of these efforts reveals the insights provided by such approaches and underscores the need for further interdisciplinary studies to reveal in full the quantitative underpinnings of this cellular control pathway. PMID:19629044

  8. [Immune Checkpoint for a Kidney Cancer and Other Cancers].

    PubMed

    Kageyama, Shunichiro; Yamaguchi, Shigeo; Miura, Kayo; Kato, Shunsuke

    2016-06-01

    Immune checkpoint inhibitors have been getting increasing attention in the field of cancer treatment, resulting in the investigation of numerous drugs and target cancers. Clinical trials of immune checkpoint inhibitors have focused on malignant melanomas and non-small cell lung cancer;however, recently, clinical trials have been carried out for other cancers. To date, 31 phase III clinical trials have been conducted for 13 types of cancer. Recently, the results of the CheckMate025 kidney cancer and CheckMate141 head and neck cancer trials have been reported. These reports showed that nivolumab significantly enhanced overall survival in comparison to that associated with an existing second-line treatment drug. Based on these results, the approval of nivolumab for use in renal cell cancer and head and neck cancer is expected in the near future. Furthermore, the results of 20 phase III clinical trials will be submitted from 2017 to 2019, expanding the approval of immune checkpoint inhibitors. However, many issues such as biomarker searches, the evaluation of antitumor effects, and the impact on medical economy remain to be resolved. In this report, we outline clinical trial trends and the future prospects for immune checkpoint inhibitors.

  9. Breaking the Code of Silence.

    ERIC Educational Resources Information Center

    Halbig, Wolfgang W.

    2000-01-01

    Schools and communities must break the adolescent code of silence concerning threats of violence. Schools need character education stressing courage, caring, and responsibility; regular discussions of the school discipline code; formal security discussions with parents; 24-hour hotlines; and protocols for handling reports of potential violence.…

  10. Fueling the engine and releasing the break: combinational therapy of cancer vaccines and immune checkpoint inhibitors

    PubMed Central

    Kleponis, Jennifer; Skelton, Richard; Zheng, Lei

    2015-01-01

    Immune checkpoint inhibitors are increasingly drawing much attention in the therapeutic development for cancer treatment. However, many cancer patients do not respond to treatments with immune checkpoint inhibitors, partly because of the lack of tumor-infiltrating effector T cells. Cancer vaccines may prime patients for treatments with immune checkpoint inhibitors by inducing effector T-cell infiltration into the tumors and immune checkpoint signals. The combination of cancer vaccine and an immune checkpoint inhibitor may function synergistically to induce more effective antitumor immune responses, and clinical trials to test the combination are currently ongoing. PMID:26487965

  11. Down-Regulation of Gene Expression by RNA-Induced Gene Silencing

    NASA Astrophysics Data System (ADS)

    Travella, Silvia; Keller, Beat

    Down-regulation of endogenous genes via post-transcriptional gene silencing (PTGS) is a key to the characterization of gene function in plants. Many RNA-based silencing mechanisms such as post-transcriptional gene silencing, co-suppression, quelling, and RNA interference (RNAi) have been discovered among species of different kingdoms (plants, fungi, and animals). One of the most interesting discoveries was RNAi, a sequence-specific gene-silencing mechanism initiated by the introduction of double-stranded RNA (dsRNA), homologous in sequence to the silenced gene, which triggers degradation of mRNA. Infection of plants with modified viruses can also induce RNA silencing and is referred to as virus-induced gene silencing (VIGS). In contrast to insertional mutagenesis, these emerging new reverse genetic approaches represent a powerful tool for exploring gene function and for manipulating gene expression experimentally in cereal species such as barley and wheat. We examined how RNAi and VIGS have been used to assess gene function in barley and wheat, including molecular mechanisms involved in the process and available methodological elements, such as vectors, inoculation procedures, and analysis of silenced phenotypes.

  12. Herpes Simplex Virus Type 1 Suppresses RNA-Induced Gene Silencing in Mammalian Cells▿

    PubMed Central

    Wu, Zetang; Zhu, Yali; Bisaro, David M.; Parris, Deborah S.

    2009-01-01

    RNA-induced silencing is a potent innate antiviral defense strategy in plants, and suppression of silencing is a hallmark of pathogenic plant viruses. However, the impact of silencing as a mammalian antiviral defense mechanism and the ability of mammalian viruses to suppress silencing in natural host cells have remained controversial. The ability of herpes simplex virus type 1 (HSV-1) to suppress silencing was examined in a transient expression system that employed an imperfect hairpin to target degradation of transcripts encoding enhanced green fluorescent protein (EGFP). HSV-1 infection suppressed EGFP-specific silencing as demonstrated by increased EGFP mRNA levels and an increase in the EGFP mRNA half-life. The increase in EGFP mRNA stability occurred despite the well-characterized host macromolecular shutoff functions of HSV-1 that globally destabilize mRNAs. Moreover, mutant viruses defective in these functions increased the stability of EGFP mRNA even more than did the wild-type virus in silenced cells compared to results in control cells. The importance of RNA silencing to HSV-1 replication was confirmed by a significantly enhanced virus burst size in cells in which silencing was knocked down with small inhibitory RNAs directed to Argonaute 2, an integral component of the silencing complex. Given that HSV-1 encodes several microRNAs, it is possible that a dynamic equilibrium exists between silencing and silencing suppression that is capable of modulating viral gene expression to promote replication, to evade host defenses, and/or to promote latency. PMID:19369325

  13. Is silence killing your company?

    PubMed

    Perlow, Leslie; Williams, Stephanie

    2003-05-01

    Many times, often with the best of intentions, people at work decide it's more productive to remain silent about their differences than to air them. There's no time, they think, or no point in going against what the boss says. But as new research by the authors shows, silencing doesn't smooth things over or make people more productive. It merely pushes differences beneath the surface and can set in motion powerfully destructive forces. When people stay silent about important disagreements, they can begin to fill with anxiety, anger, and resentment. As long as the conflict is unresolved, their repressed feelings remain potent, making them increasingly distrustful, self-protective, and all the more fearful that if they speak up they will be embarrassed or rejected. Their sense of insecurity grows, leading to further acts of silence, more defensiveness, and more distrust, thereby setting into motion a destructive "spiral of silence." Sooner or later, they mentally opt out--sometimes merely doing what they're told but contributing nothing of their own, sometimes spreading discontent and frustration throughout the workplace that can lead them, and others, to leave without thinking it through. These vicious spirals of silence can be replaced with virtuous spirals of communication, but that requires individuals to find the courage to act differently and executives to create the conditions in which people will value the expression of differences. All too often, behind failed products, broken processes, and mistaken career decisions are people who chose to hold their tongues. Breaking the silence can bring an outpouring of fresh ideas from all levels of an organization--ideas that might just raise the organization's performance to a whole new level.

  14. Virus-induced gene silencing (VIGS) in barley seedling leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is one of the most potent reverse genetics technologies for gene functional characterization. This method exploits a dsRNA-mediated antiviral defense mechanism in plants. Using this method allows researchers to generate rapid phenotypic data in a relatively rapid ...

  15. RNA editing regulates transposon-mediated heterochromatic gene silencing.

    PubMed

    Savva, Yiannis A; Jepson, James E C; Chang, Yao-Jen; Whitaker, Rachel; Jones, Brian C; St Laurent, Georges; Tackett, Michael R; Kapranov, Philipp; Jiang, Nan; Du, Guyu; Helfand, Stephen L; Reenan, Robert A

    2013-01-01

    Heterochromatin formation drives epigenetic mechanisms associated with silenced gene expression. Repressive heterochromatin is established through the RNA interference pathway, triggered by double-stranded RNAs (dsRNAs) that can be modified via RNA editing. However, the biological consequences of such modifications remain enigmatic. Here we show that RNA editing regulates heterochromatic gene silencing in Drosophila. We utilize the binding activity of an RNA-editing enzyme to visualize the in vivo production of a long dsRNA trigger mediated by Hoppel transposable elements. Using homologous recombination, we delete this trigger, dramatically altering heterochromatic gene silencing and chromatin architecture. Furthermore, we show that the trigger RNA is edited and that dADAR serves as a key regulator of chromatin state. Additionally, dADAR auto-editing generates a natural suppressor of gene silencing. Lastly, systemic differences in RNA editing activity generates interindividual variation in silencing state within a population. Our data reveal a global role for RNA editing in regulating gene expression.

  16. Intergenic transcription through a polycomb group response element counteracts silencing.

    PubMed

    Schmitt, Sabine; Prestel, Matthias; Paro, Renato

    2005-03-15

    Polycomb group response elements (PREs) mediate the mitotic inheritance of gene expression programs and thus maintain determined cell fates. By default, PREs silence associated genes via the targeting of Polycomb group (PcG) complexes. Upon an activating signal, however, PREs recruit counteracting trithorax group (trxG) proteins, which in turn maintain target genes in a transcriptionally active state. Using a transgenic reporter system, we show that the switch from the silenced to the activated state of a PRE requires noncoding transcription. Continuous transcription through the PRE induced by an actin promoter prevents the establishment of PcG-mediated silencing. The maintenance of epigenetic activation requires transcription through the PRE to proceed at least until embryogenesis is completed. At the homeotic bithorax complex of Drosophila, intergenic PRE transcripts can be detected not only during embryogenesis, but also at late larval stages, suggesting that transcription through endogenous PREs is required continuously as an anti-silencing mechanism to prevent the access of repressive PcG complexes to the chromatin. Furthermore, all other PREs outside the homeotic complex we tested were found to be transcribed in the same tissue as the mRNA of the corresponding target gene, suggesting that anti-silencing by transcription is a fundamental aspect of the cellular memory system.

  17. In vivo chromatin accessibility correlates with gene silencing in Drosophila.

    PubMed Central

    Boivin, A; Dura, J M

    1998-01-01

    Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure. PMID:9832530

  18. Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

    PubMed Central

    Sagi, Amir; Manor, Rivka; Ventura, Tomer

    2013-01-01

    Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice. PMID:24705266

  19. Immunosuppressive networks and checkpoints controlling antitumor immunity and their blockade in the development of cancer immunotherapeutics and vaccines.

    PubMed

    Butt, A Q; Mills, K H G

    2014-09-18

    Vaccines that promote protective adaptive immune responses have been successfully developed against a range of infectious diseases, and these are normally administered prior to exposure with the relevant virus or bacteria. Adaptive immunity also plays a critical role in the control of tumors. Immunotherapeutics and vaccines that promote effector T cell responses have the potential to eliminate tumors when used in a therapeutic setting. However, the induction of protective antitumor immunity is compromised by innate immunosuppressive mechanisms and regulatory cells that often dominate the tumor microenvironment. Recent studies have shown that blocking these suppressor cells and immune checkpoints to allow induction of antitumor immunity is a successful immunotherapeutic modality for the treatment of cancer. Furthermore, stimulation of innate and consequently adaptive immune responses with concomitant inhibition of immune suppression, especially that mediated by regulatory T (Treg) cells, is emerging as a promising approach to enhance the efficacy of therapeutic vaccines against cancer. This review describes the immunosuppressive mechanisms controlling antitumor immunity and the novel strategies being employed to design effective immunotherapeutics against tumors based on inhibition of suppressor cells or blockade of immune checkpoints to allow induction of more potent effector T cell responses. This review also discusses the potential of using a combination of adjuvants with inhibition of immune checkpoint or suppressor cells for therapeutic vaccines and the translation of pre-clinical studies to the next-generation vaccines against cancer in humans.

  20. A Cell Biological Perspective on Past, Present and Future Investigations of the Spindle Assembly Checkpoint

    PubMed Central

    Joglekar, Ajit P.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a quality control mechanism that ensures accurate chromosome segregation during cell division. It consists of a mechanochemical signal transduction mechanism that senses the attachment of chromosomes to the spindle, and a signaling cascade that inhibits cell division if one or more chromosomes are not attached. Extensive investigations of both these component systems of the SAC have synthesized a comprehensive understanding of the underlying molecular mechanisms. This review recounts the milestone results that elucidated the SAC, compiles a simple model of the complex molecular machinery underlying the SAC, and highlights poorly understood facets of the biochemical design and cell biological operation of the SAC that will drive research forward in the near future. PMID:27869759

  1. Replication, checkpoint suppression and structure of centromeric DNA.

    PubMed

    Romeo, Francesco; Falbo, Lucia; Costanzo, Vincenzo

    2016-11-01

    Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells.

  2. A review of adverse events caused by immune checkpoint inhibitors.

    PubMed

    Fukushima, Satoshi

    2016-01-01

      There has been no effective therapy in the unresectable melanoma for more than 40 years. Anti-PD-1 antibody and anti-CTLA-4 antibody have totally changed the situation. They have clearly shown the survival benefits of the patients with metastatic melanoma. However, immune checkpoint inhibitors sometimes induce various kinds of immune-related adverse events (irAEs). It is very important for the clinicians to know the reported cases of irAEs and to keep in mind the symptoms of irAEs for the early detection. This review describes the previously reported irAEs and adequate managements for irAEs induced by immune checkpoint inhibitors.

  3. Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma.

    PubMed

    Beug, Shawn T; Beauregard, Caroline E; Healy, Cristin; Sanda, Tarun; St-Jean, Martine; Chabot, Janelle; Walker, Danielle E; Mohan, Aditya; Earl, Nathalie; Lun, Xueqing; Senger, Donna L; Robbins, Stephen M; Staeheli, Peter; Forsyth, Peter A; Alain, Tommy; LaCasse, Eric C; Korneluk, Robert G

    2017-02-15

    Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells.

  4. ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

    PubMed Central

    Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

    2016-01-01

    To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

  5. Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma

    PubMed Central

    Beug, Shawn T.; Beauregard, Caroline E.; Healy, Cristin; Sanda, Tarun; St-Jean, Martine; Chabot, Janelle; Walker, Danielle E.; Mohan, Aditya; Earl, Nathalie; Lun, Xueqing; Senger, Donna L.; Robbins, Stephen M.; Staeheli, Peter; Forsyth, Peter A.; Alain, Tommy; LaCasse, Eric C.; Korneluk, Robert G.

    2017-01-01

    Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer cells. PMID:28198370

  6. nab-Paclitaxel as a potential partner with checkpoint inhibitors in solid tumors

    PubMed Central

    Soliman, Hatem H

    2017-01-01

    Tumors recognized by the host immune system are associated with better survival. However, the immune system is often suppressed in patients with established tumor burden. Stimulating the immune system to detect and kill tumor cells has been a challenge in cancer therapy for some time. Recently, novel cancer immunotherapies, such as immune checkpoint inhibitors, monoclonal antibodies, and vaccine therapies, have emerged as promising therapeutic approaches for many solid tumors. However, for some tumors, immunotherapy alone has not provided significant benefits, and some may even be fully resistant to immunotherapy. It has been suggested that the immune system may require “priming” before an immunotherapy can elicit an immune response. Although chemotherapies are believed to be immunosuppressive, when given at the right dose and sequence these agents may provide this “priming” effect for the immune system. In addition to direct cytotoxic killing of tumor cells, standard chemotherapeutic agents can elicit immunogenicity through various mechanisms. This review highlights the general immunomodulatory properties of chemotherapy agents. It also provides a rationale for combined therapy with nab-paclitaxel and immune checkpoint inhibitors. Recent clinical trial data with these combination regimens in solid tumors are presented, along with a summary of ongoing trials. PMID:28053544

  7. Genetic Control of the Trigger for the G2/M Checkpoint

    SciTech Connect

    Hall, Eric J.; Smilenov, Lubomir B.; Young, Erik F.

    2013-10-01

    The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest prior to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture

  8. Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein.

    PubMed

    Vogler, Hannes; Akbergenov, Rashid; Shivaprasad, Padubidri V; Dang, Vy; Fasler, Monika; Kwon, Myoung-Ok; Zhanybekova, Saule; Hohn, Thomas; Heinlein, Manfred

    2007-10-01

    Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

  9. Central Tolerance Blockade to Augment Checkpoint Immunotherapy in Melanoma

    DTIC Science & Technology

    2016-09-01

    needed to bring anti-RANKL antibody to the clinic for treating advanced melanoma patients. To date, our findings include: RANKL is expressed at high...promising therapy for enhancing checkpoint inhibitor efficacy in advanced melanoma. This observation has immediate clinical relevance given the FDA...What was the impact on the development of the principal discipline(s) of the project? Based on our findings, we are planning a Phase 2 clinical

  10. Checkpointing Shared Memory Programs at the Application-level

    SciTech Connect

    Bronevetsky, G; Schulz, M; Szwed, P; Marques, D; Pingali, K

    2004-09-08

    Trends in high-performance computing are making it necessary for long-running applications to tolerate hardware faults. The most commonly used approach is checkpoint and restart(CPR)-the state of the computation is saved periodically on disk, and when a failure occurs, the computation is restarted from the last saved state. At present, it is the responsibility of the programmer to instrument applications for CPR. Our group is investigating the use of compiler technology to instrument codes to make them self-checkpointing and self-restarting, thereby providing an automatic solution to the problem of making long-running scientific applications resilient to hardware faults. Our previous work focused on message-passing programs. In this paper, we describe such a system for shared-memory programs running on symmetric multiprocessors. The system has two components: (i)a pre-compiler for source-to-source modification of applications, and (ii) a runtime system that implements a protocol for coordinating CPR among the threads of the parallel application. For the sake of concreteness, we focus on a non-trivial subset of OpenMP that includes barriers and locks. One of the advantages of this approach is that the ability to tolerate faults becomes embedded within the application itself, so applications become self-checkpointing and self-restarting on any platform. We demonstrate this by showing that our transformed benchmarks can checkpoint and restart on three different platforms (Windows/x86, Linux/x86, and Tru64/Alpha). Our experiments show that the overhead introduced by this approach is usually quite small; they also suggest ways in which the current implementation can be tuned to reduced overheads further.

  11. A Combinatorial Code for Splicing Silencing: UAGG and GGGG Motifs

    PubMed Central

    An, Ping; Burge, Christopher B

    2005-01-01

    Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5′-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes. PMID:15828859

  12. Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

    PubMed Central

    Rieke, Damian T.; Ochsenreither, Sebastian; Klinghammer, Konrad; Seiwert, Tanguy Y.; Klauschen, Frederick; Tinhofer, Inge; Keilholz, Ulrich

    2016-01-01

    Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression. A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma. A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC. Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition. PMID:27683114

  13. Targeting lung cancer through inhibition of checkpoint kinases

    PubMed Central

    Syljuåsen, Randi G.; Hasvold, Grete; Hauge, Sissel; Helland, Åslaug

    2015-01-01

    Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin, or radiation. Altogether, inhibitors of ATR, Chk1, and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways. PMID:25774168

  14. Managing immune checkpoint-blocking antibody side effects.

    PubMed

    Postow, Michael A

    2015-01-01

    Immune checkpoint-blocking antibodies that enhance the immune system's ability to fight cancer are becoming important components of treatment for patients with a variety of malignancies. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) was the first immune checkpoint to be clinically targeted, and ipilimumab, an inhibitor of CTLA-4, was approved by the U.S. Food and Drug Administration (FDA) for patients with advanced melanoma. The programmed cell death-1 (PD-1) receptor and one of its ligands, PD-L1, more recently have shown great promise as therapeutic targets in a variety of malignancies. Nivolumab and pembrolizumab recently have been FDA- approved for patients with melanoma and additional approvals within this therapeutic class are expected. The use of anti-CTLA-4 and anti-PD-1/PD-L1 antibodies is associated with side effects known as immune-related adverse events (irAEs). Immune-related adverse events affect the dermatologic, gastrointestinal, hepatic, endocrine, and other organ systems. Temporary immunosuppression with corticosteroids, tumor necrosis factor-alpha antagonists, mycophenolate mofetil, or other agents can be effective treatment. This article describes the side-effect profile of the checkpoint-blocking antibodies that target CTLA-4 and PD-1/PD-L1 and provides suggestions on how to manage specific irAEs.

  15. Immune checkpoint modulation for non-small cell lung cancer.

    PubMed

    Soria, Jean-Charles; Marabelle, Aurélien; Brahmer, Julie R; Gettinger, Scott

    2015-05-15

    Therapies targeting immune checkpoints have recently shown encouraging activity in patients with heavily pretreated advanced non-small cell lung cancer (NSCLC), independently of NSCLC histology or mutational status, with low toxicity profiles when used as monotherapy. Objective response rates of approximately 20% have been reported in patients with advanced NSCLC treated with antagonist antibodies targeting the immune checkpoint, programmed death 1 (PD-1) on activated T cells, or its primary ligand, programmed death ligand 1 (PD-L1) expressed within the tumor microenvironment. Response rates appear to be higher in patients with tumor PD-L1 expression documented by immunohistochemistry, although responses have been appreciated in patients with reportedly PD-L1-negative tumor specimens. Antibodies directed against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), another immunosuppressive T-cell signaling molecule, are also being evaluated in clinical trials, with one randomized phase II trial demonstrating improved immune-related progression-free survival in lung cancer patients when added to standard chemotherapy. Additional clinical trials are combining anti-CTLA-4 antibodies with either anti-PD-1 or anti-PD-L1 antibodies. Combinations of other immune checkpoint antagonists or agonist antibodies with anti-PD-1 or anti-PD-L1 antibodies are also being pursued.

  16. RAD51-dependent break-induced replication differs in kinetics and checkpoint responses from RAD51-mediated gene conversion.

    PubMed

    Malkova, Anna; Naylor, Maria L; Yamaguchi, Miyuki; Ira, Grzegorz; Haber, James E

    2005-02-01

    Diploid Saccharomyces cells experiencing a double-strand break (DSB) on one homologous chromosome repair the break by RAD51-mediated gene conversion >98% of the time. However, when extensive homologous sequences are restricted to one side of the DSB, repair can occur by both RAD51-dependent and RAD51-independent break-induced replication (BIR) mechanisms. Here we characterize the kinetics and checkpoint dependence of RAD51-dependent BIR when the DSB is created within a chromosome. Gene conversion products appear within 2 h, and there is little, if any, induction of the DNA damage checkpoint; however, RAD51-dependent BIR occurs with a further delay of 2 to 4 h and cells arrest in response to the G(2)/M DNA damage checkpoint. RAD51-dependent BIR does not require special facilitating sequences that are required for a less efficient RAD51-independent process. RAD51-dependent BIR occurs efficiently in G(2)-arrested cells. Once repair is initiated, the rate of repair replication during BIR is comparable to that of normal DNA replication, as copying of >100 kb is completed less than 30 min after repair DNA synthesis is detected close to the DSB.

  17. Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

    PubMed Central

    Kontra, Levente; Tavazza, Mario; Lucioli, Alessandra; Tavazza, Raffaela; Moxon, Simon; Medzihradszky, Anna; Burgyán, József

    2016-01-01

    RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. PMID:27711201

  18. Sobriety checkpoints in Thailand: a review of effectiveness and developments over time.

    PubMed

    Ditsuwan, Vallop; Veerman, J Lennert; Bertram, Melanie; Vos, Theo

    2015-03-01

    This review describes the legal basis for and implementation of sobriety checkpoints in Thailand and identifies factors that influenced their historical development and effectiveness. The first alcohol and traffic injury control law in Thailand was implemented in 1934. The 0.05 g/100 mL blood alcohol concentration limit was set in 1994. Currently, 3 types of sobriety checkpoints are used: general police checkpoints, selective breath testing, and special event sobriety checkpoints. The authors found few reports on the strategies, frequencies, and outcomes for any of these types of checkpoints, despite Thailand having devoted many resources to their implementation. In Thailand and other low-middle income countries, it is necessary to address the country-specific barriers to successful enforcement (including political and logistical issues, lack of equipment, and absence of other supportive alcohol harm reduction measures) before sobriety checkpoints can be expected to be as effective as reported in high-income countries.

  19. Emodnet Med Sea Check-Point - Indicators for decision- maker

    NASA Astrophysics Data System (ADS)

    Besnard, Sophie; Claverie, Vincent; Blanc, Frédérique

    2015-04-01

    The Emodnet Checkpoint projects aim is to assess the cost-effectiveness, reliability and utility of the existing monitoring at the sea basin level. This involves the development of monitoring system indicators and a GIS Platform to perform the assessment and make it available. Assessment or production of Check-Point information is made by developing targeted products based on the monitoring data and determining whether the products are meeting the needs of industry and public authorities. Check-point users are the research community, the 'institutional' policy makers for IMP and MSFD implementation, the 'intermediate users', i.e., users capable to understand basic raw data but that benefit from seeing the Checkpoint targeted products and the assessment of the fitness for purpose. We define assessment criteria aimed to characterize/depict the input datasets in terms of 3 territories capable to show performance and gaps of the present monitoring system, appropriateness, availability and fitness for purpose. • Appropriateness: What is made available to users? What motivate/decide them to select this observation rather than this one. • Availability: How this is made available to the user? Place to understand the readiness and service performance of the EU infrastructure • Fitness for use / fitness for purpose: Ability for non-expert user to appreciate the data exploitability (feedback on efficiency & reliability of marine data) For each territory (appropriateness, Availability and Fitness for purpose / for use), we define several indicators. For example, for Availability we define Visibility, Accessibility and Performance. And Visibility is itself defined by "Easily found" and "EU service". So these indicators can be classified according to their territory and sub-territory as seen above, but also according to the complexity to build them. Indicators are built from raw descriptors in 3 stages:  Stage 1: to give a neutral and basic status directly computed from

  20. [E. M. Jellinek's silenced and silencing transgenerational story].

    PubMed

    Kelemen, Gábor; Márk, Mónika

    2013-01-01

    Jellinek is a kind of archetypal character for future generations in the field of addiction studies. His implosion in the arena of alcoholism around the age of 50 was an unexpected challenge to medical science. We know very little about his own role models giving an intellectual and moral compass to his pragmatic creativity. More than 30 years has passed since Jellinek's death when an American sociologist Ron Roizen started unearthing his silent story. Roizen discerned that there are a lot of unsaid and muted issues in his personal Hungarian past. Our paper, based on the authors' research in Hungarian archives and other sources reveals that not just Jellinek's personal but his transgenerational narrative has been not-yet-said. This silenced and silencing history appears an unfinished business of acculturation of the family, which started prior to four generations. Authors have been concluding that the issue of religious conversion is a critical point in the process of acculturation. They examine the counter move of loyalty to family values and driving force of assimilation making their story unspeakable.

  1. Silence

    NASA Astrophysics Data System (ADS)

    Cogswell, J.

    2011-06-01

    On the occasion of the International Year of Astronomy, I was commissioned to create a mural for the University of Michigan Department of Astronomy, responding to an array of scientific images based on astronomical research, with special focus on the work of University of Michigan astronomers carried out within the building. My paper illustrates the development of this and several subsequent projects, explaining the implications for my artistic practice of entering into this conversation with astronomers and their work.

  2. Two-Level Incremental Checkpoint Recovery Scheme for Reducing System Total Overheads

    PubMed Central

    Li, Huixian; Pang, Liaojun; Wang, Zhangquan

    2014-01-01

    Long-running applications are often subject to failures. Once failures occur, it will lead to unacceptable system overheads. The checkpoint technology is used to reduce the losses in the event of a failure. For the two-level checkpoint recovery scheme used in the long-running tasks, it is unavoidable for the system to periodically transfer huge memory context to a remote stable storage. Therefore, the overheads of setting checkpoints and the re-computing time become a critical issue which directly impacts the system total overheads. Motivated by these concerns, this paper presents a new model by introducing i-checkpoints into the existing two-level checkpoint recovery scheme to deal with the more probable failures with the smaller cost and the faster speed. The proposed scheme is independent of the specific failure distribution type and can be applied to different failure distribution types. We respectively make analyses between the two-level incremental and two-level checkpoint recovery schemes with the Weibull distribution and exponential distribution, both of which fit with the actual failure distribution best. The comparison results show that the total overheads of setting checkpoints, the total re-computing time and the system total overheads in the two-level incremental checkpoint recovery scheme are all significantly smaller than those in the two-level checkpoint recovery scheme. At last, limitations of our study are discussed, and at the same time, open questions and possible future work are given. PMID:25111048

  3. McrEngine: A Scalable Checkpointing System Using Data-Aware Aggregation and Compression

    DOE PAGES

    Islam, Tanzima Zerin; Mohror, Kathryn; Bagchi, Saurabh; ...

    2013-01-01

    High performance computing (HPC) systems use checkpoint-restart to tolerate failures. Typically, applications store their states in checkpoints on a parallel file system (PFS). As applications scale up, checkpoint-restart incurs high overheads due to contention for PFS resources. The high overheads force large-scale applications to reduce checkpoint frequency, which means more compute time is lost in the event of failure. We alleviate this problem through a scalable checkpoint-restart system, mcrEngine. McrEngine aggregates checkpoints from multiple application processes with knowledge of the data semantics available through widely-used I/O libraries, e.g., HDF5 and netCDF, and compresses them. Our novel scheme improves compressibility ofmore » checkpoints up to 115% over simple concatenation and compression. Our evaluation with large-scale application checkpoints show that mcrEngine reduces checkpointing overhead by up to 87% and restart overhead by up to 62% over a baseline with no aggregation or compression.« less

  4. Tissue homogeneity requires inhibition of unequal gene silencing during development

    PubMed Central

    Le, Hai H.; Looney, Monika; Strauss, Benjamin; Bloodgood, Michael

    2016-01-01

    Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans. These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity. PMID:27458132

  5. G2 Checkpoint Responses in Arabidopsis

    SciTech Connect

    Britt, Anne

    2013-03-18

    This project focused on the mechanism and biological significance of the G2 arrest response to replication stress in plants. We employed both forward and reverse genetic approaches to identify genes required for this response. A total of 3 different postdocs, 5 undergraduates, and 2 graduate students participated in the project. We identified several genes required for damage response in plants, including homologs of genes previously identified in animals (ATM and ATR), novel, a plant-specific genes (SOG1) and a gene known in animals but previously thought to be missing from the Arabidopsis genome (ATRIP). We characterized the transcriptome of gamma-irradiated plants, and found that plants, unlike animals, express a robust transcriptional response to damage, involving genes that regulate the cell cycle and DNA metabolism. This response requires both ATM and the transcription factor SOG1. We found that both ATM and ATR play a role in meiosis in plants. We also found that plants have a cell-type-specific programmed cell death response to ionizing radiation and UV light, and that this response requires ATR, ATM, and SOG1. These results were published in a series of 5 papers.

  6. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA.

    PubMed

    Decker, Logan M; Xiao, Hua; Boone, Erin C; Vierling, Michael M; Shanker, Benjamin S; Kingston, Shanika L; Boone, Shannon F; Haynes, Jackson B; Shiu, Patrick K T

    2017-02-07

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus to combat these repetitive elements. One of these defense mechanisms is known as meiotic silencing by unpaired DNA (MSUD), which is an RNA silencing system that identifies and silences unpaired genes during meiosis. Utilizing common RNAi proteins such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified another silencing component known as the cap-binding complex (CBC). Made up of CBP20 and CBP80 (cap-binding proteins 20 and 80), CBC associates with the 5' cap of nascent mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. Similar to animals but unlike plants, CBP20's robust nuclear re-entry is shown to be dependent on CBP80. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors.

  7. Gene silencing triggered by non-LTR retrotransposons in the female germline of Drosophila melanogaster.

    PubMed Central

    Robin, Stéphanie; Chambeyron, Séverine; Bucheton, Alain; Busseau, Isabelle

    2003-01-01

    Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent gene-silencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing. PMID:12807773

  8. "The Silence Itself Is Enough of a Statement": The Day of Silence and LGBTQ Awareness Raising

    ERIC Educational Resources Information Center

    Woolley, Susan W.

    2012-01-01

    This ethnographic study of a high school gay-straight alliance club examines unintended consequences of silence during the Day of Silence, a day of action aimed at addressing anti-LGBTQ bias in schools. While this strategy calls for students to engage in intentional silences to raise awareness of anti-LGBTQ bias, it does not necessarily lead…

  9. Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints.

    PubMed

    Wang, Hui-Min; Cheng, Kuo-Chen; Lin, Cheng-Jung; Hsu, Shu-Wei; Fang, Wei-Cheng; Hsu, Tai-Feng; Chiu, Chien-Chih; Chang, Hsueh-Wei; Hsu, Chun-Hua; Lee, Alan Yueh-Luen

    2010-12-01

    Several compounds from Cinnamomum kotoense show anticancer activities. However, the detailed mechanisms of most compounds from C. kotoense remain unknown. In this study, we investigated the anticancer activity of obtusilactone A (OA) and (-)-sesamin in lung cancer. Our results show that human Lon is upregulated in non-small-cell lung cancer (NSCLC) cell lines, and downregulation of Lon triggers caspase-3 mediated apoptosis. Through enzyme-based screening, we identified two small-molecule compounds, obtusilactone A (OA) and (-)-sesamin from C. kotoense, as potent Lon protease inhibitors. Obtusilactone A and (-)-sesamin interact with Ser855 and Lys898 residues in the active site of the Lon protease according to molecular docking analysis. Thus, we suggest that cancer cytotoxicity of the compounds is partly due to the inhibitory effects on Lon protease. In addition, the compounds are able to cause DNA double-strand breaks and activate checkpoints. Treatment with OA and (-)-sesamin induced p53-independent DNA damage responses in NSCLC cells, including G(1) /S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G(1) accumulation. In conclusion, OA and (-)-sesamin act as both inhibitors of human mitochondrial Lon protease and DNA damage agents to activate the DNA damage checkpoints as well induce apoptosis in NSCLC cells. These dual functions open a bright avenue to develop more selective chemotherapy agents to overcome chemoresistance and sensitize cancer cells to other chemotherapeutics.

  10. How Can Plant DNA Viruses Evade siRNA-Directed DNA Methylation and Silencing?

    PubMed Central

    Pooggin, Mikhail M.

    2013-01-01

    Plants infected with DNA viruses produce massive quantities of virus-derived, 24-nucleotide short interfering RNAs (siRNAs), which can potentially direct viral DNA methylation and transcriptional silencing. However, growing evidence indicates that the circular double-stranded DNA accumulating in the nucleus for Pol II-mediated transcription of viral genes is not methylated. Hence, DNA viruses most likely evade or suppress RNA-directed DNA methylation. This review describes the specialized mechanisms of replication and silencing evasion evolved by geminiviruses and pararetoviruses, which rescue viral DNA from repressive methylation and interfere with transcriptional and post-transcriptional silencing of viral genes. PMID:23887650

  11. The Aurora B Kinase in Chromosome Bi-Orientation and Spindle Checkpoint Signaling

    PubMed Central

    Krenn, Veronica; Musacchio, Andrea

    2015-01-01

    Aurora B, a member of the Aurora family of serine/threonine protein kinases, is a key player in chromosome segregation. As part of a macromolecular complex known as the chromosome passenger complex, Aurora B concentrates early during mitosis in the proximity of centromeres and kinetochores, the sites of attachment of chromosomes to spindle microtubules. There, it contributes to a number of processes that impart fidelity to cell division, including kinetochore stabilization, kinetochore–microtubule attachment, and the regulation of a surveillance mechanism named the spindle assembly checkpoint. In the regulation of these processes, Aurora B is the fulcrum of a remarkably complex network of interactions that feed back on its localization and activation state. In this review, we discuss the multiple roles of Aurora B during mitosis, focusing in particular on its role at centromeres and kinetochores. Many details of the network of interactions at these locations remain poorly understood, and we focus here on several crucial outstanding questions. PMID:26528436

  12. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I.L.; Hofmann, Astrid; Ibrahim, Bashar

    2010-01-01

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. PMID:20855503

  13. Elm1 kinase activates the spindle position checkpoint kinase Kin4.

    PubMed

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I L; Hofmann, Astrid; Ibrahim, Bashar; Pereira, Gislene

    2010-09-20

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

  14. Novel Checkpoints and Cosignaling Molecules in Cancer Immunotherapy.

    PubMed

    Giuroiu, Iulia; Weber, Jeffrey

    The recent demonstration of the antitumor efficacy of checkpoint protein inhibition has resulted in the approval of blocking antibodies against the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway in multiple different histologic findings. Therapeutic successes with PD-1/PD-L1 antibodies in melanoma and lung cancer have been followed by approvals in bladder, renal, and head and neck cancers and Hodgkin lymphoma, with others undoubtedly to come. However, PD-1 is only one of many checkpoints and agonistic regulatory molecules expressed on T cells by which maintenance of the balance between costimulatory and coinhibitory signaling pathways is perturbed in cancer. The manipulation of many of these molecules in cancer patients might be associated with clinical benefit. The majority of the T-cell cosignaling receptors belong to either the immunoglobulin superfamily or the tumor necrosis factor receptor superfamily. A total of 29 immunoglobulin superfamily and 26 tumor necrosis factor receptor superfamily cosignaling receptors have been identified that are expressed on T cells, providing fertile ground for development of inhibitory or agonistic antibodies and small molecules as cancer therapeutics. In the current work, we focus on some of the most promising new checkpoints and agonistic or cosignaling molecules that are in early clinical development as single agents or in combinations with PD-1/PD-L1, cytotoxic T-lymphocyte-associated protein 4 blockade, or chemotherapy with an emphasis on those that have reached the clinic and on important targets that are in late preclinical development.

  15. Do telomeres ask checkpoint proteins: "gimme shelter-in"?

    PubMed

    Weinert, Ted

    2005-12-01

    Telomeres are complicated structures designed to allow one thing and avoid another. They allow replication of chromosome ends, an issue mostly about telomerase, which we seem to understand (though details of its regulation are works in progress). Telomeres must also avoid being detected as DNA breaks. This is important for two reasons: DNA breaks activate checkpoints that cause arrest of cell division, and DNA breaks engage repair machinery. Clearly, normal telomeres neither activate cell cycle arrest nor allow themselves to be repaired; arrest blocks cell division, and repair fuses chromosomes.

  16. Immune-Related Adverse Events Associated with Immune Checkpoint Inhibitors.

    PubMed

    Day, Daphne; Hansen, Aaron R

    2016-12-01

    Immune checkpoint inhibitors (ICIs), including antibodies targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1), have shown durable treatment responses in multiple tumor types by enhancing antitumor immunity. However, removal of self-tolerance can induce autoimmunity and produce a unique immune-driven toxicity profile, termed immune-related adverse events (irAEs). As ICIs gain approval for a growing number of indications, it is imperative clinicians increase their knowledge of and ability to manage irAEs. This review examines the etiology, presentation, kinetics, and treatment of irAEs and aims to provide practical guidance for clinicians.

  17. Cdh1 is an antagonist of the spindle assembly checkpoint.

    PubMed

    Nagai, Masayoshi; Ushimaru, Takashi

    2014-10-01

    The spindle assembly checkpoint (SAC) monitors unsatisfied connections of microtubules to kinetochores and prevents anaphase onset by inhibition of the ubiquitin ligase E3 anaphase-promoting complex or cyclosome (APC/C) in association with the activator Cdc20. Another APC/C activator, Cdh1, exists permanently throughout the cell cycle but it becomes active from telophase to G1. Here, we show that Cdh1 is partially active and mediates securin degradation even in SAC-active metaphase cells. Additionally, Cdh1 mediates Cdc20 degradation in metaphase, promoting formation of the APC/C-Cdh1. These results indicate that Cdh1 opposes the SAC and promotes anaphase transition.

  18. Polycomb Group-Dependent, Heterochromatin Protein 1-Independent, Chromatin Structures Silence Retrotransposons in Somatic Tissues Outside Ovaries

    PubMed Central

    Dufourt, J.; Brasset, E.; Desset, S.; Pouchin, P.; Vaury, C.

    2011-01-01

    Somatic cells are equipped with different silencing mechanisms that protect the genome against retrotransposons. In Drosophila melanogaster, a silencing pathway implicating the argonaute protein PIWI represses retrotransposons in cells surrounding the oocyte, whereas a PIWI-independent pathway is involved in other somatic tissues. Here, we show that these two silencing mechanisms result in distinct chromatin structures. Using sensor transgenes, we found that, in somatic tissues outside of the ovaries, these transgenes adopt a heterochromatic configuration implicating hypermethylation of H3K9 and K27. We identified the Polycomb repressive complexes (PRC1 and 2), but not heterochromatin protein 1 to be necessary factors for silencing. Once established, the compact structure is stably maintained through cell divisions. By contrast, in cells where the silencing is PIWI-dependent, the transgenes display an open and labile chromatin structure. Our data suggest that a post-transcriptional gene silencing (PTGS) mechanism is responsible for the repression in the ovarian somatic cells, whereas a mechanism that couples PTGS to transcriptional gene silencing operates to silence retrotransposons in the other somatic tissues. PMID:21908513

  19. A petunia ocs element binding factor, PhOBF1, plays an important role in antiviral RNA silencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus-induced gene silencing (VIGS) is a common strategy of reverse genetics for characterizing function of genes in plant. The detailed mechanism governing RNA silencing efficiency triggered by virus is largely unclear. Here, we revealed that a petunia (Petunia hybrida) ocs element binding factor, ...

  20. viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence.

    PubMed

    Csorba, Tibor; Kontra, Levente; Burgyán, József

    2015-05-01

    RNA silencing is a homology-dependent gene inactivation mechanism that regulates a wide range of biological processes including antiviral defense. To deal with host antiviral responses viruses evolved mechanisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Besides working as silencing suppressors, these proteins may also fulfill other functions during infection. In many cases the interplay between the suppressor function and other "unrelated" functions remains elusive. We will present host factors implicated in antiviral pathways and summarize the current status of knowledge about the diverse viral suppressors' strategies acting at various steps of antiviral silencing in plants. Besides, we will consider the multi-functionality of these versatile proteins and related biochemical processes in which they may be involved in fine-tuning the plant-virus interaction. Finally, we will present the current applications and discuss perspectives of the use of these proteins in molecular biology and biotechnology.

  1. Communication to Enhance Silence: The Trappist Experience

    ERIC Educational Resources Information Center

    Jaksa, James A.; Stech, Ernest L.

    1978-01-01

    Investigates perceptions of the amount of interpersonal communication and attitudes towards communication frequency after the Trappist monk's rule of enforced silence and solitude was lifted in 1969. Concludes that increased interpersonal communication resulted in increased self-awareness and therefore more meaningful and effective silence. (MH)

  2. Silence as the voice of trauma.

    PubMed

    Ritter, Maria

    2014-06-01

    Silence is a key to the unspoken world of the patient. Rather than interpreting silence as a defensive maneuver, the analyst may understand this disruption as a royal road to the patient's traumatic experiences. The author proposes to recognize traumatic silences in the analytic process and the transference as a re-experiencing of past, unpredictable traumatic affective states and memories. Silences in this context are both a repeat of a disconnecting experience as well as a manifestation of a silencing identification with the original silencer. The clinical material illustrates effects of a German mother's World War II (WWII) personal traumata and collective shame-based silence on her daughter's self and good object development. In the daughter's analysis, the patient and the analyst, who herself experienced similar WWII traumata, face the pain of trauma recovery and un-silencing. The author suggests that the deadening effect of past traumata may be reversed by an analytic process of re-membering and re-speaking for both the patient and analyst. This allows for a more transparent, subjective experience in the transference and a verbal integration of ego functions.

  3. Silence amenity engineering: Past and present

    NASA Astrophysics Data System (ADS)

    Fujita, Hajime; Yokono, Yasuyuki

    1993-09-01

    Recent historical development of the noise control engineering, from mere noise reduction to silence amenity engineering, is reviewed, with social and psychological backgrounds behind it. Philosophical view points for fundamental approach to the silence amenity engineering and examples of noise source control in vibration and aerodynamic noises are described.

  4. Classroom Silence: Voices from Japanese EFL Learners

    ERIC Educational Resources Information Center

    Harumi, Seiko

    2011-01-01

    This article explores Japanese EFL learners' classroom silence in a Japanese EFL context. The existence of silence in second language learning contexts can be a source of conflict between students and teachers and even among students themselves. It can also be an obstacle to acquiring the target language. In order to tackle this problem and to…

  5. Oomycete pathogens encode RNA silencing suppressors.

    PubMed

    Qiao, Yongli; Liu, Lin; Xiong, Qin; Flores, Cristina; Wong, James; Shi, Jinxia; Wang, Xianbing; Liu, Xigang; Xiang, Qijun; Jiang, Shushu; Zhang, Fuchun; Wang, Yuanchao; Judelson, Howard S; Chen, Xuemei; Ma, Wenbo

    2013-03-01

    Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

  6. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

    PubMed Central

    Ji, Zhejian; Gao, Haishan; Jia, Luying; Li, Bing; Yu, Hongtao

    2017-01-01

    The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment. DOI: http://dx.doi.org/10.7554/eLife.22513.001 PMID:28072388

  7. Twitter as a Tool to Warn Others about Sobriety Checkpoints: A Pilot Observational Study

    ERIC Educational Resources Information Center

    Seitz, Christopher M.; Orsini, Muhsin Michael; Fearnow-Kenney, Melodie; Hatzudis, Kiki; Wyrick, David L.

    2012-01-01

    Anecdotal evidence suggests that young people use the website Twitter as a tool to warn drivers about the locations of sobriety checkpoints. Researchers investigated this claim by independently analyzing the website's content regarding a sample of 10 sobriety checkpoints that were conducted in cities throughout the United States during the weekend…

  8. Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei.

    PubMed

    Alsford, Sam; Horn, David

    2012-11-01

    Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G2/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G2/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.

  9. Molecular Pathways: Immune Checkpoint Antibodies and their Toxicities.

    PubMed

    Cousin, Sophie; Italiano, Antoine

    2016-09-15

    The emergence of immune checkpoint inhibitors for solid tumor treatments represents a major oncologic advance. Since the approval of ipilimumab, a cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibody, for the treatment of metastatic melanoma, many drugs, especially those targeting PD-1/PD-L1, have demonstrated promising antitumor effects in many types of cancer. By reactivating the immune system, these immunotherapies have led to the development of new toxicity profiles, also called immune-related adverse events (irAE). IrAEs can involve many organ systems, and their management is radically different from that of cytotoxic drugs; irAEs require immunosuppressive treatments, such as corticoids or TNFα antibody. In addition, the occurrence of irAEs has raised significant questions. Here, we summarize progress that has been made toward answering these questions, focusing on (i) the impact of immunotherapy dose on irAE occurrence, (ii) the correlation between irAE and patient outcome, (iii) the safety of immune checkpoint inhibitors in patients already treated for autoimmune disease, and (iv) the suspected effect on tumor growth of steroids used for the management of irAEs. Clin Cancer Res; 22(18); 4550-5. ©2016 AACR.

  10. A Splicing-Dependent Transcriptional Checkpoint Associated with Prespliceosome Formation

    PubMed Central

    Chathoth, Keerthi T.; Barrass, J. David; Webb, Shaun; Beggs, Jean D.

    2014-01-01

    Summary There is good evidence for functional interactions between splicing and transcription in eukaryotes, but how and why these processes are coupled remain unknown. Prp5 protein (Prp5p) is an RNA-stimulated adenosine triphosphatase (ATPase) required for prespliceosome formation in yeast. We demonstrate through in vivo RNA labeling that, in addition to a splicing defect, the prp5-1 mutation causes a defect in the transcription of intron-containing genes. We present chromatin immunoprecipitation evidence for a transcriptional elongation defect in which RNA polymerase that is phosphorylated at Ser5 of the largest subunit’s heptad repeat accumulates over introns and that this defect requires Cus2 protein. A similar accumulation of polymerase was observed when prespliceosome formation was blocked by a mutation in U2 snRNA. These results indicate the existence of a transcriptional elongation checkpoint that is associated with prespliceosome formation during cotranscriptional spliceosome assembly. We propose a role for Cus2p as a potential checkpoint factor in transcription. PMID:24560925

  11. The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes.

    PubMed

    Valli, Adrian; Busnadiego, Idoia; Maliogka, Varvara; Ferrero, Diego; Castón, José R; Rodríguez, José Francisco; García, Juan Antonio

    2012-01-01

    RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

  12. Identifying security checkpoints locations to protect the major U.S. urban areas

    SciTech Connect

    Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; Rodriguez, Erick; Stroud, Phillip D.

    2015-09-01

    Transit networks are integral to the economy and to society, but at the same time they could allow terrorists to transport weapons of mass destruction into any city. Road networks are especially vulnerable, because they lack natural checkpoints unlike air networks that have security measures in place at all major airports. One approach to mitigate this risk is ensuring that every road route passes through at least one security checkpoint. Using the Ford-Fulkerson maximum-flow algorithm, we generate a minimum set of checkpoint locations within a ring-shaped buffer area surrounding the 50 largest US urban areas. We study how the number of checkpoints changes as we increase the buffer width to perform a cost-benefit analysis and to identify groups of cities that behave similarly. The set of required checkpoints is surprisingly small (10-124) despite the hundreds of thousands of road arcs in those areas, making it feasible to protect all major cities.

  13. Transcription factors of M-phase cyclin CLB2 in the yeast cell wall integrity checkpoint.

    PubMed

    Sekiya, Mizuho; Nogami, Satoru; Ohya, Yoshikazu

    2009-08-01

    The cell wall integrity checkpoint coordinates cell wall synthesis and mitosis in the budding yeast, Saccharomyces cerevisiae. It has been reported that this checkpoint arrests the cell cycle at G2/M phase with repression of the M phase cyclin Clb2p at the transcriptional level, under perturbation of cell wall synthesis. We demonstrate that an override of this checkpoint with accumulation of CLB2 mRNA is induced when negative CLB2 transcription factors are deleted or when positive CLB2 transcription factors are overproduced in cell wall-defective cells. Our data imply that transcription factors for CLB2 are involved in the cell wall integrity checkpoint system and suggest that there are multiple regulation pathways of the checkpoint.

  14. Fhit loss in lung preneoplasia: relation to DNA damage response checkpoint activation

    PubMed Central

    Cirombella, Roberto; Montrone, Giuseppe; Stoppacciaro, Antonella; Giglio, Simona; Volinia, Stefano; Graziano, Paolo; Huebner, Kay; Vecchione, Andrea

    2009-01-01

    Loss of heterozygosity at the FHIT locus is coincident with activation of DNA damage response checkpoint proteins; thus damage at fragile loci may trigger checkpoint activation. We examined preneoplastic lesions adjacent to non-small cell lung carcinomas for alterations to expression of Fhit and activated checkpoint proteins. Expression scores were analyzed for pair-wise associations and correlations among proteins and type of lesion. Hyperplastic and dysplastic lesions were positive for nuclear H2AX expression; 12/20 dysplastic lesions were negative for Fhit expression. Fhit positive lesions showed expression of most checkpoint proteins examined, while Fhit negative lesions showed absence of expression of Chk1 and phosphoChk1. The results show that loss of expression of Fhit is significantly directly correlated with absence of activated Chk1 in dysplasia, and suggest a connection between loss of Fhit and modulation of checkpoint activity. PMID:19931269

  15. Blocking immune checkpoints in prostate, kidney, and urothelial cancer: An overview.

    PubMed

    Alme, Angela K B; Karir, Beerinder S; Faltas, Bishoy M; Drake, Charles G

    2016-04-01

    Despite a long history of immunotherapeutic approaches to treatment, most genitourinary malignancies are not cured by existing immunotherapy regimens. More recently, cell surface molecules known as immune checkpoints have become the focus of efforts to develop more effective immunotherapies. Interactions between these molecules and their ligands inhibit the proliferation and function of tumor-specific lymphocytes. A monoclonal antibody blocking 1 of these checkpoints was approved for the treatment of metastatic melanoma and is now being tested in other malignancies. The objective responses seen in these early trials of checkpoint blockade are driving renewed enthusiasm for cancer immunotherapy. There are several ongoing and planned trials in genitourinary malignancies of single-agent inhibitors, as well as combinations targeting multiple checkpoints or adding other types of therapies to checkpoint blockade.

  16. Blocking Immune Checkpoints in Prostate, Kidney and Urothelial cancer: An Overview

    PubMed Central

    Alme, Angela K.B.; Karir, Beerinder S.; Faltas, Bishoy M.; Drake, Charles G.

    2016-01-01

    Despite a long history of immunotherapeutic approaches to treatment, most genitourinary malignancies are not cured by existing immunotherapy regimens. More recently, cell-surface molecules known as immune checkpoints have become the focus of efforts to develop more effective immunotherapies. Interactions between these molecules and their ligands inhibit the proliferation and function of tumor-specific lymphocytes. A monoclonal antibody blocking one of these checkpoints was approved for the treatment of metastatic melanoma and is now being tested in other malignancies. The objective responses seen in these early trials of checkpoint blockade are driving renewed enthusiasm for cancer immunotherapy. There are several ongoing and planned trials in genitourinary malignancies of single-agent inhibitors, as well as combinations targeting multiple checkpoints or adding other types of therapies to checkpoint blockade. PMID:26923782

  17. Microarray Analysis of LTR Retrotransposon Silencing Identifies Hdac1 as a Regulator of Retrotransposon Expression in Mouse Embryonic Stem Cells

    PubMed Central

    Madej, Monika J.; Taggart, Mary; Gautier, Philippe; Garcia-Perez, Jose Luis; Meehan, Richard R.; Adams, Ian R.

    2012-01-01

    Retrotransposons are highly prevalent in mammalian genomes due to their ability to amplify in pluripotent cells or developing germ cells. Host mechanisms that silence retrotransposons in germ cells and pluripotent cells are important for limiting the accumulation of the repetitive elements in the genome during evolution. However, although silencing of selected individual retrotransposons can be relatively well-studied, many mammalian retrotransposons are seldom analysed and their silencing in germ cells, pluripotent cells or somatic cells remains poorly understood. Here we show, and experimentally verify, that cryptic repetitive element probes present in Illumina and Affymetrix gene expression microarray platforms can accurately and sensitively monitor repetitive element expression data. This computational approach to genome-wide retrotransposon expression has allowed us to identify the histone deacetylase Hdac1 as a component of the retrotransposon silencing machinery in mouse embryonic stem cells, and to determine the retrotransposon targets of Hdac1 in these cells. We also identify retrotransposons that are targets of other retrotransposon silencing mechanisms such as DNA methylation, Eset-mediated histone modification, and Ring1B/Eed-containing polycomb repressive complexes in mouse embryonic stem cells. Furthermore, our computational analysis of retrotransposon silencing suggests that multiple silencing mechanisms are independently targeted to retrotransposons in embryonic stem cells, that different genomic copies of the same retrotransposon can be differentially sensitive to these silencing mechanisms, and helps define retrotransposon sequence elements that are targeted by silencing machineries. Thus repeat annotation of gene expression microarray data suggests that a complex interplay between silencing mechanisms represses retrotransposon loci in germ cells and embryonic stem cells. PMID:22570599

  18. A High Incidence of Meiotic Silencing of Unsynapsed Chromatin Is Not Associated with Substantial Pachytene Loss in Heterozygous Male Mice Carrying Multiple Simple Robertsonian Translocations

    PubMed Central

    Vasco, Chiara; Berríos, Soledad; Parra, María Teresa; Viera, Alberto; Rufas, Julio S.; Zuccotti, Maurizio; Garagna, Silvia; Fernández-Donoso, Raúl

    2009-01-01

    Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian

  19. Strategies to modulate the immune system in breast cancer: checkpoint inhibitors and beyond

    PubMed Central

    Migali, Cristina; Milano, Monica; Trapani, Dario; Criscitiello, Carmen; Esposito, Angela; Locatelli, Marzia; Minchella, Ida; Curigliano, Giuseppe

    2016-01-01

    Is breast cancer (BC) immunogenic? Many data suggest that it is. Many observations demonstrated the prognostic role of tumor-infiltrating lymphocytes (TILs) in triple negative (TN) and human epidermal growth factor receptor 2 (HER-2)-positive BC. TNBCs are poorly differentiated tumors with high genetic instability and very high heterogeneity. This heterogeneity enhances the ‘danger signals’ and select clone variants that could be more antigenic or, in other words, that could more strongly stimulate a host immune antitumor response. The response to chemotherapy is at least partly dependent on an immunological reaction against those tumor cells that are dying during the chemotherapy. One of the mechanisms whereby chemotherapy can stimulate the immune system to recognize and destroy malignant cells is commonly known as immunogenic cell death (ICD). ICD elicits an adaptive immune response. Which are the clinical implications of all ‘immunome’ data produced in the last years? First, validate prognostic or predictive role of TILs. Second, validate immune genomic signatures that may be predictive and prognostic in patients with TN disease. Third, incorporate an ‘immunoscore’ into traditional classification of BC, thus providing an essential prognostic and potentially predictive tool in the pathology report. Fourth, implement clinical trials for BC in the metastatic setting with drugs that target immune-cell–intrinsic checkpoints. Blockade of one of these checkpoints, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) or the programmed cell death 1 (PD-1) receptor may provide proof of concepts for the activity of an immune-modulation approach in the treatment of a BC. PMID:27583028

  20. Overcoming Barriers in Oncolytic Virotherapy with HDAC Inhibitors and Immune Checkpoint Blockade.

    PubMed

    Marchini, Antonio; Scott, Eleanor M; Rommelaere, Jean

    2016-01-06

    Oncolytic viruses (OVs) target and destroy cancer cells while sparing their normal counterparts. These viruses have been evaluated in numerous studies at both pre-clinical and clinical levels and the recent Food and Drug Administration (FDA) approval of an oncolytic herpesvirus-based treatment raises optimism that OVs will become a therapeutic option for cancer patients. However, to improve clinical outcome, there is a need to increase OV efficacy. In addition to killing cancer cells directly through lysis, OVs can stimulate the induction of anti-tumour immune responses. The host immune system thus represents a "double-edged sword" for oncolytic virotherapy: on the one hand, a robust anti-viral response will limit OV replication and spread; on the other hand, the immune-mediated component of OV therapy may be its most important anti-cancer mechanism. Although the relative contribution of direct viral oncolysis and indirect, immune-mediated oncosuppression to overall OV efficacy is unclear, it is likely that an initial period of vigorous OV multiplication and lytic activity will most optimally set the stage for subsequent adaptive anti-tumour immunity. In this review, we consider the use of histone deacetylase (HDAC) inhibitors as a means of boosting virus replication and lessening the negative impact of innate immunity on the direct oncolytic effect. We also discuss an alternative approach, aimed at potentiating OV-elicited anti-tumour immunity through the blockade of immune checkpoints. We conclude by proposing a two-phase combinatorial strategy in which initial OV replication and spread is maximised through transient HDAC inhibition, with anti-tumour immune responses subsequently enhanced by immune checkpoint blockade.

  1. Overcoming Barriers in Oncolytic Virotherapy with HDAC Inhibitors and Immune Checkpoint Blockade

    PubMed Central

    Marchini, Antonio; Scott, Eleanor M.; Rommelaere, Jean

    2016-01-01

    Oncolytic viruses (OVs) target and destroy cancer cells while sparing their normal counterparts. These viruses have been evaluated in numerous studies at both pre-clinical and clinical levels and the recent Food and Drug Administration (FDA) approval of an oncolytic herpesvirus-based treatment raises optimism that OVs will become a therapeutic option for cancer patients. However, to improve clinical outcome, there is a need to increase OV efficacy. In addition to killing cancer cells directly through lysis, OVs can stimulate the induction of anti-tumour immune responses. The host immune system thus represents a “double-edged sword” for oncolytic virotherapy: on the one hand, a robust anti-viral response will limit OV replication and spread; on the other hand, the immune-mediated component of OV therapy may be its most important anti-cancer mechanism. Although the relative contribution of direct viral oncolysis and indirect, immune-mediated oncosuppression to overall OV efficacy is unclear, it is likely that an initial period of vigorous OV multiplication and lytic activity will most optimally set the stage for subsequent adaptive anti-tumour immunity. In this review, we consider the use of histone deacetylase (HDAC) inhibitors as a means of boosting virus replication and lessening the negative impact of innate immunity on the direct oncolytic effect. We also discuss an alternative approach, aimed at potentiating OV-elicited anti-tumour immunity through the blockade of immune checkpoints. We conclude by proposing a two-phase combinatorial strategy in which initial OV replication and spread is maximised through transient HDAC inhibition, with anti-tumour immune responses subsequently enhanced by immune checkpoint blockade. PMID:26751469

  2. Detailed Modeling, Design, and Evaluation of a Scalable Multi-level Checkpointing System

    SciTech Connect

    Moody, A T; Bronevetsky, G; Mohror, K M; de Supinski, B R

    2010-04-09

    High-performance computing (HPC) systems are growing more powerful by utilizing more hardware components. As the system mean-time-before-failure correspondingly drops, applications must checkpoint more frequently to make progress. However, as the system memory sizes grow faster than the bandwidth to the parallel file system, the cost of checkpointing begins to dominate application run times. A potential solution to this problem is to use multi-level checkpointing, which employs multiple types of checkpoints with different costs and different levels of resiliency in a single run. The goal is to design light-weight checkpoints to handle the most common failure modes and rely on more expensive checkpoints for less common, but more severe failures. While this approach is theoretically promising, it has not been fully evaluated in a large-scale, production system context. To this end we have designed a system, called the Scalable Checkpoint/Restart (SCR) library, that writes checkpoints to storage on the compute nodes utilizing RAM, Flash, or disk, in addition to the parallel file system. We present the performance and reliability properties of SCR as well as a probabilistic Markov model that predicts its performance on current and future systems. We show that multi-level checkpointing improves efficiency on existing large-scale systems and that this benefit increases as the system size grows. In particular, we developed low-cost checkpoint schemes that are 100x-1000x faster than the parallel file system and effective against 85% of our system failures. This leads to a gain in machine efficiency of up to 35%, and it reduces the the load on the parallel file system by a factor of two on current and future systems.

  3. Personalized gene silencing therapeutics for Huntington disease.

    PubMed

    Kay, C; Skotte, N H; Southwell, A L; Hayden, M R

    2014-07-01

    Gene silencing offers a novel therapeutic strategy for dominant genetic disorders. In specific diseases, selective silencing of only one copy of a gene may be advantageous over non-selective silencing of both copies. Huntington disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin gene (HTT). Silencing both expanded and normal copies of HTT may be therapeutically beneficial, but preservation of normal HTT expression is preferred. Allele-specific methods can selectively silence the mutant HTT transcript by targeting either the expanded CAG repeat or single nucleotide polymorphisms (SNPs) in linkage disequilibrium with the expansion. Both approaches require personalized treatment strategies based on patient genotypes. We compare the prospect of safe treatment of HD by CAG- and SNP-specific silencing approaches and review HD population genetics used to guide target identification in the patient population. Clinical implementation of allele-specific HTT silencing faces challenges common to personalized genetic medicine, requiring novel solutions from clinical scientists and regulatory authorities.

  4. Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins

    PubMed Central

    LV, TING-ZHUO; WANG, GUANG-SHUN

    2015-01-01

    Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins. PMID:26170956

  5. Epigenetic silencing of tumor suppressor genes: Paradigms, puzzles, and potential.

    PubMed

    Kazanets, Anna; Shorstova, Tatiana; Hilmi, Khalid; Marques, Maud; Witcher, Michael

    2016-04-01

    Cancer constitutes a set of diseases with heterogeneous molecular pathologies. However, there are a number of universal aberrations common to all cancers, one of these being the epigenetic silencing of tumor suppressor genes (TSGs). The silencing of TSGs is thought to be an early, driving event in the oncogenic process. With this in consideration, great efforts have been made to develop small molecules aimed at the restoration of TSGs in order to limit tumor cell proliferation and survival. However, the molecular forces that drive the broad epigenetic reprogramming and transcriptional repression of these genes remain ill-defined. Undoubtedly, understanding the molecular underpinnings of transcriptionally silenced TSGs will aid us in our ability to reactivate these key anti-cancer targets. Here, we describe what we consider to be the five most logical molecular mechanisms that may account for this widely observed phenomenon: 1) ablation of transcription factor binding, 2) overexpression of DNA methyltransferases, 3) disruption of CTCF binding, 4) elevation of EZH2 activity, 5) aberrant expression of long non-coding RNAs. The strengths and weaknesses of each proposed mechanism is highlighted, followed by an overview of clinical efforts to target these processes.

  6. Polycomb-Mediated Gene Silencing in Arabidopsis thaliana

    PubMed Central

    Kim, Dong-Hwan; Sung, Sibum

    2014-01-01

    Polycomb group (PcG) proteins are conserved chromatin regulators involved in the control of key developmental programs in eukaryotes. They collectively provide the transcriptional memory unique to each cell identity by maintaining transcriptional states of developmental genes. PcG proteins form multi-protein complexes, known as Polycomb repressive complex 1 (PRC1) and Polycomb repressive complex 2 (PRC2). PRC1 and PRC2 contribute to the stable gene silencing in part through catalyzing covalent histone modifications. Components of PRC1 and PRC2 are well conserved from plants to animals. PcG-mediated gene silencing has been extensively investigated in efforts to understand molecular mechanisms underlying developmental programs in eukaryotes. Here, we describe our current knowledge on PcG-mediated gene repression which dictates developmental programs by dynamic layers of regulatory activities, with an emphasis given to the model plant Arabidopsis thaliana. PMID:25410906

  7. Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Stueland, C S; Lew, D J; Cismowski, M J; Reed, S I

    1993-01-01

    In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation. Images PMID:8388545

  8. Epigenetic silencing of TIMP4 in heart failure.

    PubMed

    Chaturvedi, Pankaj; Tyagi, Suresh C

    2016-11-01

    Tissue inhibitor of matrix metalloprotease 4 (TIMP4) is endogenously one of the key modulators of matrix metalloprotease 9 (MMP9) and we have reported earlier that cardiac specific TIMP4 instigates contractility and helps in differentiation of cardiac progenitor cells. Although studies show that the expression of TIMP4 goes down in heart failure but the mechanism is unknown. This study aims to determine the mechanism of silencing of TIMP4 in heart failure progression created by aorta-vena cava (AV) fistula. We hypothesize that there is epigenetic silencing of TIMP4 in heart failure. To validate this hypothesis, we created heart failure model by creating AV fistula in C57BL/6 mice and looked into the promoter methylation (methylation specific PCR, high resolution melting, methylation sensitive restriction enzyme and Na bisulphite treatment followed by sequencing), histone modification (ChIP assay) and microRNAs that regulate TIMP4 (mir122a) and MMP9 (mir29b and mir455-5p). The physiological parameters in terms of cardiac function after AV fistula were assessed by echocardiography. We observed that there are 7 CpG islands in the TIMP4 promoter which get methylated during the progression of heart failure which leads to its epigenetic silencing. In addition, the up-regulated levels of mir122a in part, contribute to regulation of TIMP4. Consequently, MMP9 gets up-regulated and leads to cardiac remodeling. This is a novel report to explain the epigenetic silencing of TIMP4 in heart failure.

  9. ATM Dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition.

    PubMed

    Harding, Shane M; Boiarsky, Jonathan A; Greenberg, Roger A

    2015-10-13

    Resolution of DNA double-strand breaks (DSBs) is essential for the suppression of genome instability. DSB repair in transcriptionally active genomic regions represents a unique challenge that is associated with ataxia telangiectasia mutated (ATM) kinase-mediated transcriptional silencing. Despite emerging insights into the underlying mechanisms, how DSB silencing connects to DNA repair remains undefined. We observe that silencing within the rDNA depends on persistent DSBs. Non-homologous end-joining was the predominant mode of DSB repair allowing transcription to resume. ATM-dependent rDNA silencing in the presence of persistent DSBs led to the large-scale reorganization of nucleolar architecture, with movement of damaged chromatin to nucleolar cap regions. These findings identify ATM-dependent temporal and spatial control of DNA repair and provide insights into how communication between DSB signaling and ongoing transcription promotes genome integrity.

  10. Parallelization and checkpointing of GPU applications through program transformation

    SciTech Connect

    Solano-Quinde, Lizandro Damian

    2012-01-01

    GPUs have emerged as a powerful tool for accelerating general-purpose applications. The availability of programming languages that makes writing general-purpose applications for running on GPUs tractable have consolidated GPUs as an alternative for accelerating general purpose applications. Among the areas that have benefited from GPU acceleration are: signal and image processing, computational fluid dynamics, quantum chemistry, and, in general, the High Performance Computing (HPC) Industry. In order to continue to exploit higher levels of parallelism with GPUs, multi-GPU systems are gaining popularity. In this context, single-GPU applications are parallelized for running in multi-GPU systems. Furthermore, multi-GPU systems help to solve the GPU memory limitation for applications with large application memory footprint. Parallelizing single-GPU applications has been approached by libraries that distribute the workload at runtime, however, they impose execution overhead and are not portable. On the other hand, on traditional CPU systems, parallelization has been approached through application transformation at pre-compile time, which enhances the application to distribute the workload at application level and does not have the issues of library-based approaches. Hence, a parallelization scheme for GPU systems based on application transformation is needed. Like any computing engine of today, reliability is also a concern in GPUs. GPUs are vulnerable to transient and permanent failures. Current checkpoint/restart techniques are not suitable for systems with GPUs. Checkpointing for GPU systems present new and interesting challenges, primarily due to the natural differences imposed by the hardware design, the memory subsystem architecture, the massive number of threads, and the limited amount of synchronization among threads. Therefore, a checkpoint/restart technique suitable for GPU systems is needed. The goal of this work is to exploit higher levels of parallelism and

  11. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    PubMed Central

    Decker, Logan M.; Xiao, Hua; Boone, Erin C.; Vierling, Michael M.; Shanker, Benjamin S.; Kingston, Shanika L.; Boone, Shannon F.; Haynes, Jackson B.; Shiu, Patrick K.T.

    2017-01-01

    In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD), which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi) proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC). Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute) of the perinuclear meiotic silencing complex (MSC), directly linking the two cellular factors. PMID:28179391

  12. RNA silencing is required for Arabidopsis defence against Verticillium wilt disease

    PubMed Central

    Ellendorff, Ursula; Fradin, Emilie F.; de Jonge, Ronnie; Thomma, Bart P. H. J.

    2009-01-01

    RNA silencing is a conserved mechanism in eukaryotes that plays an important role in various biological processes including regulation of gene expression. RNA silencing also plays a role in genome stability and protects plants against invading nucleic acids such as transgenes and viruses. Recently, RNA silencing has been found to play a role in defence against bacterial plant pathogens in Arabidopsis through modulating host defence responses. In this study, it is shown that gene silencing plays a role in plant defence against multicellular microbial pathogens; vascular fungi belonging to the Verticillium genus. Several components of RNA silencing pathways were tested, of which many were found to affect Verticillium defence. Remarkably, no altered defence towards other fungal pathogens that include Alternaria brassicicola, Botrytis cinerea, and Plectosphaerella cucumerina, but also the vascular pathogen Fusarium oxysporum, was recorded. Since the observed differences in Verticillium susceptibility cannot be explained by notable differences in root architecture, it is speculated that the gene silencing mechanisms affect regulation of Verticillium-specific defence responses. PMID:19098131

  13. RNA silencing is required for Arabidopsis defence against Verticillium wilt disease.

    PubMed

    Ellendorff, Ursula; Fradin, Emilie F; de Jonge, Ronnie; Thomma, Bart P H J

    2009-01-01

    RNA silencing is a conserved mechanism in eukaryotes that plays an important role in various biological processes including regulation of gene expression. RNA silencing also plays a role in genome stability and protects plants against invading nucleic acids such as transgenes and viruses. Recently, RNA silencing has been found to play a role in defence against bacterial plant pathogens in Arabidopsis through modulating host defence responses. In this study, it is shown that gene silencing plays a role in plant defence against multicellular microbial pathogens; vascular fungi belonging to the Verticillium genus. Several components of RNA silencing pathways were tested, of which many were found to affect Verticillium defence. Remarkably, no altered defence towards other fungal pathogens that include Alternaria brassicicola, Botrytis cinerea, and Plectosphaerella cucumerina, but also the vascular pathogen Fusarium oxysporum, was recorded. Since the observed differences in Verticillium susceptibility cannot be explained by notable differences in root architecture, it is speculated that the gene silencing mechanisms affect regulation of Verticillium-specific defence responses.

  14. The capacity of target silencing by Drosophila PIWI and piRNAs

    PubMed Central

    Post, Christina; Clark, Josef P.; Sytnikova, Yuliya A.; Chirn, Gung-Wei

    2014-01-01

    Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because protein-coding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism. PMID:25336588

  15. Silences: Irish women and abortion.

    PubMed

    Fletcher, R

    1995-01-01

    Notably absent from the public debate on abortion in Ireland have been the voices of women who have experienced induced abortion. Interviews with six acquaintances of the author who underwent abortion identified four themes underlying women's post-abortion silence. First, women fear public condemnation and personal rejection. Second, women are concerned that any emotional ambivalence they express about the abortion experience will be misconstrued as anti-abortion sentiment. Third, women worry that speaking out about their experience would be upsetting to friends and family. Fourth, women report frustration about the lack of a suitable public forum for voicing the complexities inherent in the abortion issue. The women's perception that their experience did not fit neatly with the rhetoric of either pro- or anti-abortion groups caused them to feel alienated from a political discourse that tends to depersonalize abortion. Although none of the women regretted the abortion decision, they continued to struggle with unresolved conflicts over taking responsibility for ending some form of life. A cycle has been created in which women do not feel safe to discuss their personal experiences until a more favorable political climate exists, yet the public perception of abortion is unlikely to change until more women's voices are heard. Feminist leaders are urged to address this dilemma.

  16. Selective killing of cancer cells by β-lapachone: Direct checkpoint activation as a strategy against cancer

    PubMed Central

    Li, Youzhi; Sun, Xiangao; LaMont, J. Thomas; Pardee, Arthur B.; Li, Chiang J.

    2003-01-01

    Most chemotherapeutic drugs kill cancer cells by indirectly activating checkpoint-mediated apoptosis after creating nonselective damage to DNA or microtubules, which accounts for their toxicity toward normal cells. We seek to target cancer cells by directly activating checkpoint regulators without creating such damage. Here, we show that β-lapachone selectively induces apoptosis in cancer cells without causing the death of nontransformed cells in culture. This unusual selectivity against cancer cells is preceded by activation of S-phase checkpoint and selective induction of E2F1, a regulator of checkpoint-mediated apoptosis. This study suggests direct checkpoint activation as a strategy against cancer. PMID:12598645

  17. Immune checkpoint inhibitors renal side effects and management.

    PubMed

    Rassy, Elie El; Kourie, Hampig R; Rizkallah, Jamale; Karak, Fadi El; Hanna, Colette; Chelala, Dania N; Ghosn, Marwan

    2016-12-01

    The choice of immunotherapy in the treatment of cancer has improved the prognosis of many patients affected by various malignancies. The high expectations foreseen with immunotherapy have led to fast approvals despite the incomplete understanding of the toxicity profiles in the different organs, including the kidneys. The high prevalence of chronic kidney disease in cancer patients complicates the issue further and requires a better knowledge of the renal safety profile to ensure an optimal safe treatment. This review summarizes the present knowledge of renal adverse events secondary to immune checkpoint inhibitors and discusses their pathophysiology, clinical presentation and adequate management. We also advocate the need for a multidisciplinary approach in patients with immune-related toxic adverse events.

  18. Genetic instability in cancer cells by impaired cell cycle checkpoints.

    PubMed

    Nakanishi, Makoto; Shimada, Midori; Niida, Hiroyuki

    2006-10-01

    Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy.

  19. VISTA is an immune checkpoint molecule for human T cells

    PubMed Central

    Lines, J. Louise; Sempere, Lorenzo F.; Wang, Li; Pantazi, Eirini; Mak, Justin; O’Connell, Samuel; Ceeraz, Sabrina; Suriawinata, Arief A.; Yan, Shaofeng; Ernstoff, Marc S.; Noelle, Randolph

    2014-01-01

    VISTA is a potent negative regulator of T cell function that is expressed on hematopoietic cells and leukocytes. VISTA levels are heightened within the tumor microenvironment where its blockade can enhance anti-tumor immune responses in mice. In humans, blockade of the related PD-1 pathway has shown great potential in clinical immunotherapy trials. Here we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells, blunted production of T cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T cell activation, induces Foxp3 expression and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer. PMID:24691993

  20. EMODnet MedSea Checkpoint for sustainable Blue Growth

    NASA Astrophysics Data System (ADS)

    Moussat, Eric; Pinardi, Nadia; Manzella, Giuseppe; Blanc, Frederique

    2016-04-01

    The EMODNET checkpoint is a wide monitoring system assessment activity aiming to support the sustainable Blue Growth at the scale of the European Sea Basins by: 1) Clarifying the observation landscape of all compartments of the marine environment including Air, Water, Seabed, Biota and Human activities, pointing out to the existing programs, national, European and international 2) Evaluating fitness for use indicators that will show the accessibility and usability of observation and modeling data sets and their roles and synergies based upon selected applications by the European Marine Environment Strategy 3) Prioritizing the needs to optimize the overall monitoring Infrastructure (in situ and satellite data collection and assembling, data management and networking, modeling and forecasting, geo-infrastructure) and release recommendations for evolutions to better meet the application requirements in view of sustainable Blue Growth The assessment is designed for : - Institutional stakeholders for decision making on observation and monitoring systems - Data providers and producers to know how their data collected once for a given purpose could fit other user needs - End-users interested in a regional status and possible uses of existing monitoring data Selected end-user applications are of paramount importance for: (i) the blue economy sector (offshore industries, fisheries); (ii) marine environment variability and change (eutrophication, river inputs and ocean climate change impacts); (iii) emergency management (oil spills); and (iv) preservation of natural resources and biodiversity (Marine Protected Areas). End-user applications generate innovative products based on the existing observation landscape. The fitness for use assessment is made thanks to the comparison of the expected product specifications with the quality of the product derived from the selected data. This involves the development of checkpoint information and indicators based on Data quality and

  1. Small RNA-Mediated Epigenetic Myostatin Silencing.

    PubMed

    Roberts, Thomas C; Andaloussi, Samir El; Morris, Kevin V; McClorey, Graham; Wood, Matthew Ja

    2012-05-15

    Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

  2. Role of CCT chaperonin in the disassembly of mitotic checkpoint complexes.

    PubMed

    Kaisari, Sharon; Sitry-Shevah, Danielle; Miniowitz-Shemtov, Shirly; Teichner, Adar; Hershko, Avram

    2017-01-31

    The mitotic checkpoint system prevents premature separation of sister chromatids in mitosis and thus ensures the fidelity of chromosome segregation. When this checkpoint is active, a mitotic checkpoint complex (MCC), composed of the checkpoint proteins Mad2, BubR1, Bub3, and Cdc20, is assembled. MCC inhibits the ubiquitin ligase anaphase promoting complex/cyclosome (APC/C), whose action is necessary for anaphase initiation. When the checkpoint signal is turned off, MCC is disassembled, a process required for exit from checkpoint-arrested state. Different moieties of MCC are disassembled by different ATP-requiring processes. Previous work showed that Mad2 is released from MCC by the joint action of the TRIP13 AAA-ATPase and the Mad2-binding protein p31(comet) Now we have isolated from extracts of HeLa cells an ATP-dependent factor that releases Cdc20 from MCC and identified it as chaperonin containing TCP1 or TCP1-Ring complex (CCT/TRiC chaperonin), a complex known to function in protein folding. Bacterially expressed CCT5 chaperonin subunits, which form biologically active homooligomers [Sergeeva, et al. (2013) J Biol Chem 288(24):17734-17744], also promote the disassembly of MCC. CCT chaperonin further binds and disassembles subcomplexes of MCC that lack Mad2. Thus, the combined action of CCT chaperonin with that of TRIP13 ATPase promotes the complete disassembly of MCC, necessary for the inactivation of the mitotic checkpoint.

  3. Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

    PubMed

    Santaguida, Stefano; Vernieri, Claudio; Villa, Fabrizio; Ciliberto, Andrea; Musacchio, Andrea

    2011-04-20

    Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

  4. KSRP: A Checkpoint for Inflammatory Cytokine Production in Astrocytes

    PubMed Central

    LI, XUELIN; LIN, WEI-JYE; CHEN, CHING-YI; SI, YING; ZHANG, XIAOWEN; LU, LIANG; SUSWAM, ESTHER; ZHENG, LEI; KING, PETER H.

    2013-01-01

    Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate-rich elements (ARE) in the 3′ untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH-type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF-α and IL-1β, whose expression increased 2 to 4-fold at the RNA level in astrocytes isolated from KSRP−/− mice compared to littermate controls. Upon activation, KSRP−/− astrocytes produced TNF-α and IL-1β at levels that exceeded control cells by 15-fold or more. Conditioned media from KSRP−/− astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half-life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post-transcriptional levels. Our results outline a critical role for KSRP in regulating pro-inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases. PMID:22847996

  5. TSLC1 gene silencing in cutaneous melanoma.

    PubMed

    You, Yan; Ma, Liangjuan; You, Min; Li, Xiaomei; Wang, Shuhai; Li, Hongli; Wu, Debin; Yang, Huimin; Li, Zhen Yu

    2010-06-01

    Tumor suppressor in lung cancer 1 (TSLC1) is a tumor suppressor gene that encodes a member of the immunoglobulin superfamily, which is involved in the progression of some types of cancer. Several studies have shown that loss of TSLC1 expression is strongly correlated to methylation of the gene promoter, thus leading to poor prognosis in these cancers. However, the role of TSLC1 in cutaneous melanoma (CM) has not been examined. The purpose of this study was to understand the molecular mechanisms and clinical significance of TSLC1 inactivation in CM. The expression and promoter methylation of TSLC1 were analyzed in 120 CMs. TSLC1 expression was examined by immunohistochemistry, whereas its methylation status was determined by methylation-specific PCR. TSLC1 expression was lost in 84 of 120 (70%) CMs; 36 (30%) CMs were scored as positive for TSLC1 protein expression. The TSLC1 promoter was methylated in 58 (48.33%) of 120 CMs. The incidence of the loss of expression and methylation of TSLC1 significantly increased as the tumor stage advanced (P=0.032 and 0.0021, respectively). Furthermore, in CM, disease-related survival was significantly shorter in patients with tumors losing TSLC1 or harboring methylated TSLC1 (P=0.0003 and 0.0329, respectively). The epigenetic silencing of TSLC1 through methylation is an important event in the pathogenesis of CM, and TSLC1 provides an indicator for poor prognosis.

  6. Engineering nanoparticles to silence bacterial communication

    PubMed Central

    Miller, Kristen P.; Wang, Lei; Chen, Yung-Pin; Pellechia, Perry J.; Benicewicz, Brian C.; Decho, Alan W.

    2015-01-01

    The alarming spread of bacterial resistance to traditional antibiotics has warranted the study of alternative antimicrobial agents. Quorum sensing (QS) is a chemical cell-to-cell communication mechanism utilized by bacteria to coordinate group behaviors and establish infections. QS is integral to bacterial survival, and therefore provides a unique target for antimicrobial therapy. In this study, silicon dioxide nanoparticles (Si-NP) were engineered to target the signaling molecules [i.e., acylhomoserine lactones (HSLs)] used for QS in order to halt bacterial communication. Specifically, when Si-NP were surface functionalized with β-cyclodextrin (β-CD), then added to cultures of bacteria (Vibrio fischeri), whose luminous output depends upon HSL-mediated QS, the cell-to-cell communication was dramatically reduced. Reductions in luminescence were further verified by quantitative polymerase chain reaction (qPCR) analyses of luminescence genes. Binding of HSLs to Si-NPs was examined using nuclear magnetic resonance (NMR) spectroscopy. The results indicated that by delivering high concentrations of engineered NPs with associated quenching compounds, the chemical signals were removed from the immediate bacterial environment. In actively-metabolizing cultures, this treatment blocked the ability of bacteria to communicate and regulate QS, effectively silencing and isolating the cells. Si-NPs provide a scaffold and critical stepping-stone for more pointed developments in antimicrobial therapy, especially with regard to QS—a target that will reduce resistance pressures imposed by traditional antibiotics. PMID:25806030

  7. Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1

    PubMed Central

    Balmus, Gabriel; Zhu, Min; Mukherjee, Sucheta; Lyndaker, Amy M.; Hume, Kelly R.; Lee, Jaesung; Riccio, Mark L.; Reeves, Anthony P.; Sutter, Nathan B.; Noden, Drew M.; Peters, Rachel M.; Weiss, Robert S.

    2012-01-01

    The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients. PMID:22575700

  8. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    PubMed

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  9. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis.

    PubMed

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-06-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

  10. A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex.

    PubMed

    Sutton, A; Heller, R C; Landry, J; Choy, J S; Sirko, A; Sternglanz, R

    2001-05-01

    In the yeast Saccharomyces cerevisiae, a and alpha mating-type information is stored in transcriptionally silenced cassettes called HML and HMR. Silencing of these loci, maintained by the formation of a specialized type of heterochromatin, requires trans-acting proteins and cis-acting elements. Proteins required for silencing include the Sir2 NAD(+)-dependent deacetylase, Sir3, and Sir4. Factors that bind to the cis elements at HMR and HML and that are important for silencing include the origin recognition complex (ORC). Mutations of any of these Sir proteins or combinations of cis elements result in loss of silencing. SUM1-1 was previously identified as a dominant mutation that restores silencing to HMR in the absence of either the Sir proteins or some of the cis elements. We have investigated the novel mechanism whereby Sum1-1 causes Sir-independent silencing at HMR and present the following findings: Sum1-1 requires the Sir2 homolog, Hst1, for silencing and most probably requires the NAD(+)-dependent deacetylase activity of this protein. Sum1-1 interacts strongly with ORC, and this strong interaction is dependent on HMR DNA. Furthermore, ORC is required for Sum1-1-mediated silencing at HMR. These observations lead to a model for Sum1-1 silencing of HMR in which Sum1-1 is recruited to HMR by binding to ORC. Sum1-1, in turn, recruits Hst1. Hst1 then deacetylates histones or other chromatin-associated proteins to cause chromatin condensation and transcriptional silencing.

  11. Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

    PubMed

    Yoon, Ju-Yeon; Han, Kyoung-Sik; Park, Han-Yong; Choi, Seung-Kook

    2012-06-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and β-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

  12. TAK1 regulates caspase 8 activation and necroptotic signaling via multiple cell death checkpoints

    PubMed Central

    Guo, Xiaoyun; Yin, Haifeng; Chen, Yi; Li, Lei; Li, Jing; Liu, Qinghang

    2016-01-01

    Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGFβ-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, although the underlying molecular regulatory mechanisms are not well defined. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8-mediated apoptotic signaling through both NFκB-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNFα-induced cell death through the induction of RIP1 phosphorylation/activation and necrosome formation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role for the adaptor protein TNF receptor-associated protein with death domain (TRADD) in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key pro-survival signaling proteins and necrosome formation. Thus, we identified new regulatory mechanisms underlying the critical role of TAK1 in cell survival through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis. PMID:27685625

  13. Polycomb group protein gene silencing, non-coding RNA, stem cells, and cancer.

    PubMed

    Gieni, Randall S; Hendzel, Michael J

    2009-10-01

    Epigenetic programming is an important facet of biology, controlling gene expression patterns and the choice between developmental pathways. The Polycomb group proteins (PcGs) silence gene expression, allowing cells to both acquire and maintain identity. PcG silencing is important for stemness, X chromosome inactivation (XCI), genomic imprinting, and the abnormally silenced genes in cancers. Stem and cancer cells commonly share gene expression patterns, regulatory mechanisms, and signalling pathways. Many microRNA species have oncogenic or tumor suppressor activity, and disruptions in these networks are common in cancer; however, long non-coding (nc)RNA species are also important. Many of these directly guide PcG deposition and gene silencing at the HOX locus, during XCI, and in examples of genomic imprinting. Since inappropriate HOX expression and loss of genomic imprinting are hallmarks of cancer, disruption of long ncRNA-mediated PcG silencing likely has a role in oncogenesis. Aberrant silencing of coding and non-coding loci is critical for both the genesis and progression of cancers. In addition, PcGs are commonly abnormally overexpressed years prior to cancer pathology, making early PcG targeted therapy an option to reverse tumor formation, someday replacing the blunt instrument of eradication in the cancer therapy arsenal.

  14. Strategies for silencing and escape: the ancient struggle between transposable elements and their hosts.

    PubMed

    Lisch, Damon; Slotkin, R Keith

    2011-01-01

    Over the past several years, there has been an explosion in our understanding of the mechanisms by which plant transposable elements (TEs) are epigenetically silenced and maintained in an inactive state over long periods of time. This highly efficient process results in vast numbers of inactive TEs; indeed, the majority of many plant genomes are composed of these quiescent elements. This observation has led to the rather static view that TEs represent an essentially inert portion of plant genomes. However, recent work has demonstrated that TE silencing is a highly dynamic process that often involves transcription of TEs at particular times and places during plant development. Plants appear to use transcripts from silenced TEs as an ongoing source of information concerning the mobile portion of the genome. In contrast to our understanding of silencing pathways, we know relatively little about the ways in which TEs evade silencing. However, vast differences in TE content between even closely related plant species suggest that they are often wildly successful at doing so. Here, we discuss TE activity in plants as the result of a constantly shifting balance between host strategies for TE silencing and TE strategies for escape and amplification.

  15. Light intensity and temperature affect systemic spread of silencing signal in transient agroinfiltration studies.

    PubMed

    Patil, Basavaprabhu L; Fauquet, Claude M

    2015-06-01

    RNA silencing is a sequence-specific post-transcriptional gene inactivation mechanism that operates in diverse organisms and that can extend beyond its site of initiation, owing to the movement of the silencing signal, called non-autonomous gene silencing. Previous studies have shown that several factors manifest the movement of the silencing signal, such as the size (21 or 24 nucleotides) of the secondary small interfering RNA (siRNA) produced, the steady-state concentration of siRNAs and their cognate messenger RNA (mRNA) or a change in the sink-source status of plant parts affecting phloem translocation. Our study shows that both light intensity and temperature have a significant impact on the systemic movement of the silencing signal in transient agroinfiltration studies in Nicotiana benthamiana. At higher light intensities (≥ 450 μE/m(2)/s) and higher temperatures (≥ 30 °C), gene silencing was localized to leaf tissue that was infiltrated, without any systemic spread. Interestingly, in these light and temperature conditions (≥ 450 μE/m(2) /s and ≥ 30 °C), the N. benthamiana plants showed recovery from the viral symptoms. However, the reduced systemic silencing and reduced viral symptom severity at higher light intensities were caused by a change in the sink-source status of the plant, ultimately affecting the phloem translocation of small RNAs or the viral genome. In contrast, at lower light intensities (<300 μE/m(2)/s) with a constant temperature of 25 °C, there was strong systemic movement of the silencing signal in the N. benthamiana plants and reduced recovery from virus infections. The accumulation of gene-specific siRNAs was reduced at higher temperature as a result of a reduction in the accumulation of transcript on transient agroinfiltration of RNA interference (RNAi) constructs, mostly because of poor T-DNA transfer activity of Agrobacterium, possibly also accompanied by reduced phloem translocation.

  16. The Paf1 complex represses small RNA-mediated epigenetic gene silencing

    PubMed Central

    Flury, Valentin; Stadler, Michael Beda; Batki, Julia; Bühler, Marc

    2015-01-01

    RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically post-transcriptional gene silencing (PTGS) and chromatin-dependent gene silencing1. Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymerase-associated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of bi-stable heterochromatin by trans-acting siRNAs, impairment of both transcription termination and nascent transcript release is imperative to confer stability to the repressed state. Our work uncovers a novel mechanism for small RNA- mediated epigenome regulation and highlights fundamental roles for Paf1C and the RNAi machinery in building

  17. ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair.

    PubMed

    Somyajit, Kumar; Basavaraju, Shivakumar; Scully, Ralph; Nagaraju, Ganesh

    2013-05-01

    The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G2 phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G2/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.

  18. Expression of immune checkpoints in T cells of esophageal cancer patients

    PubMed Central

    Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

    2016-01-01

    Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer. PMID:27577071

  19. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  20. Lymphocyte-activation gene-3, an important immune checkpoint in cancer.

    PubMed

    He, Yayi; Rivard, Christopher J; Rozeboom, Leslie; Yu, Hui; Ellison, Kim; Kowalewski, Ashley; Zhou, Caicun; Hirsch, Fred R

    2016-09-01

    Immunotherapy has recently become widely used in lung cancer. Many oncologists are focused on cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed cell death ligand-1 (PD-L1) and programmed cell death-1 (PD-1). Immunotherapy targeting the PD-1/PD-L1 checkpoints has shown promising efficacy in non-small cell lung cancer (NSCLC), but questions remain to be answered. Among them is whether the simultaneous inhibition of other checkpoints could improve outcomes. Lymphocyte-activation gene-3 (LAG-3) is another vital checkpoint that may have a synergistic interaction with PD-1/PD-L1. Here we review the LAG-3 function in cancer, clinical trials with agents targeting LAG-3 and the correlation of LAG-3 with other checkpoints.

  1. RNA Quality Control as a Key to Suppressing RNA Silencing of Endogenous Genes in Plants.

    PubMed

    Liu, Lin; Chen, Xuemei

    2016-06-06

    RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASEs (RDRs) and proceeds through small interfering (si) RNA-directed, ARGONAUTE (AGO)-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate posttranscriptional gene silencing of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome profiles. The tug of war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways.

  2. The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death

    PubMed Central

    Dewachter, Liselot; Verstraeten, Natalie; Fauvart, Maarten; Michiels, Jan

    2016-01-01

    The phenomenon of programmed cell death (PCD), in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli). Importantly, the PCD pathway mediated by mutant Obg (Obg*) differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

  3. Repair versus Checkpoint Functions of BRCA1 Are Differentially Regulated by Site of Chromatin Binding.

    PubMed

    Goldstein, Michael; Kastan, Michael B

    2015-07-01

    The product of the Brca1 tumor-suppressor gene is involved in multiple aspects of the cellular DNA damage response (DDR), including activation of cell-cycle arrests and DNA double-stranded break (DSB) repair by homologous recombination. Prior reports demonstrated that BRCA1 recruitment to areas of DNA breakage depended on RAP80 and the RNF8/RNF168 E3 ubiquitin ligases. Here, we extend these findings by showing that RAP80 is only required for the binding of BRCA1 to regions flanking the DSB, whereas BRCA1 binding directly to DNA breaks requires Nijmegen breakage syndrome 1 (NBS1). These differential recruitment mechanisms differentially affect BRCA1 functions: (i) RAP80-dependent recruitment of BRCA1 to chromatin flanking DNA breaks is required for BRCA1 phosphorylation at serine 1387 and 1423 by ATM and, consequently, for the activation of S and G(2) checkpoints; and (ii) BRCA1 interaction with NBS1 upon DSB induction results in an NBS1-dependent recruitment of BRCA1 directly to the DNA break and is required for nonhomologous end-joining repair. Together, these findings illustrate that spatially distinct fractions of BRCA1 exist at the DSB site, which are recruited by different mechanisms and execute different functions in the DDR.

  4. IL-6 contributes to an immune tolerance checkpoint in post germinal center B cells.

    PubMed

    Yan, Yi; Wang, Ying-Hua; Diamond, Betty

    2012-02-01

    The generation of a B cell repertoire involves producing and subsequently purging autoreactive B cells. Receptor editing, clonal deletion and anergy are key mechanisms of central B cell tolerance. Somatic mutation of antigen-activated B cells within the germinal center produces a second wave of autoreactivity; but the regulatory mechanisms that operate at this phase of B cell activation are poorly understood. We recently identified a post germinal center tolerance checkpoint, where receptor editing is re-induced to extinguish autoreactivity that is generated by somatic hypermutation. Re-induction of the recombinase genes RAG1 and RAG2 in antigen-activated B cells requires antigen to engage the B cell receptor and IL-7 to signal through the IL-7 receptor. We demonstrate that this process requires IL-6 to upregulate IL-7 receptor expression on post germinal center B cells. Diminishing IL-6 by blocking antibody or haplo-insufficiency leads to reduced expression of the IL-7 receptor and RAG and increased titers of anti-DNA antibodies following immunization with a peptide mimetope of DNA. The dependence on IL-6 to initiate receptor editing is B cell intrinsic. Interestingly, estradiol decreases IL-6 expression thereby increasing the anti-DNA response. Our data reveal a novel regulatory cascade to control post germinal center B cell autoreactivity.

  5. The Spindle Assembly Checkpoint Safeguards Genomic Integrity of Skeletal Muscle Satellite Cells.

    PubMed

    Kollu, Swapna; Abou-Khalil, Rana; Shen, Carl; Brack, Andrew S

    2015-06-09

    To ensure accurate genomic segregation, cells evolved the spindle assembly checkpoint (SAC), whose role in adult stem cells remains unknown. Inducible perturbation of a SAC kinase, Mps1, and its downstream effector, Mad2, in skeletal muscle stem cells shows the SAC to be critical for normal muscle growth, repair, and self-renewal of the stem cell pool. SAC-deficient muscle stem cells arrest in G1 phase of the cell cycle with elevated aneuploidy, resisting differentiation even under inductive conditions. p21(CIP1) is responsible for these SAC-deficient phenotypes. Despite aneuploidy's correlation with aging, we find that aged proliferating muscle stem cells display robust SAC activity without elevated aneuploidy. Thus, muscle stem cells have a two-step mechanism to safeguard their genomic integrity. The SAC prevents chromosome missegregation and, if it fails, p21(CIP1)-dependent G1 arrest limits cellular propagation and tissue integration. These mechanisms ensure that muscle stem cells with compromised genomes do not contribute to tissue homeostasis.

  6. Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    PubMed Central

    Etemad, Banafsheh; Kuijt, Timo E. F.; Kops, Geert J. P. L.

    2015-01-01

    The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome–spindle interactions is monitored by the SAC: kinetochore–microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore–microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces. PMID:26621779

  7. Stepwise cytoskeletal polarization as a series of checkpoints in innate but not adaptive cytolytic killing

    NASA Astrophysics Data System (ADS)

    Wülfing, Christoph; Purtic, Bozidar; Klem, Jennifer; Schatzle, John D.

    2003-06-01

    Cytolytic killing is a major effector mechanism in the elimination of virally infected and tumor cells. The innate cytolytic effectors, natural killer (NK) cells, and the adaptive effectors, cytotoxic T cells (CTL), despite differential immune recognition, both use the same lytic mechanism, cytolytic granule release. Using live cell video fluorescence microscopy in various primary cell models of NK cell and CTL killing, we show here that on tight target cell contact, a majority of the NK cells established cytoskeletal polarity required for effective lytic function slowly or incompletely. In contrast, CTLs established cytoskeletal polarity rapidly. In addition, NK cell killing was uniquely sensitive to minor interference with cytoskeletal dynamics. We propose that the stepwise NK cell cytoskeletal polarization constitutes a series of checkpoints in NK cell killing. In addition, the use of more deliberate progression to effector function to compensate for inferior immune recognition specificity provides a mechanistic explanation for how the same effector function can be used in the different functional contexts of the innate and adaptive immune response.

  8. Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

    PubMed Central

    Ganesan, Raja; Hos, Nina Judith; Gutierrez, Saray; Fischer, Julia; Stepek, Joanna Magdalena; Daglidu, Evmorphia; Krönke, Martin

    2017-01-01

    During intracellular infections, autophagy significantly contributes to the elimination of pathogens, regulation of pro-inflammatory signaling, secretion of immune mediators and in coordinating the adaptive immune system. Intracellular pathogens such as S. Typhimurium have evolved mechanisms to circumvent autophagy. However, the regulatory mechanisms targeted by S. Typhimurium to modulate autophagy have not been fully resolved. Here we report that cytosolic energy loss during S. Typhimurium infection triggers transient activation of AMPK, an important checkpoint of mTOR activity and autophagy. The activation of AMPK is regulated by LKB1 in a cytosolic complex containing Sirt1 and LKB1, where Sirt1 is required for deacetylation and subsequent activation of LKB1. S. Typhimurium infection targets Sirt1, LKB1 and AMPK to lysosomes for rapid degradation resulting in the disruption of the AMPK-mediated regulation of mTOR and autophagy. The degradation of cytosolic Sirt1/LKB1/AMPK complex was not observed with two mutant strains of S. Typhimurium, ΔssrB and ΔssaV, both compromising the pathogenicity island 2 (SPI2). The results highlight virulence factor-dependent degradation of host cell proteins as a previously unrecognized strategy of S. Typhimurium to evade autophagy. PMID:28192515

  9. PD-1 and its ligands are important immune checkpoints in cancer.

    PubMed

    Dong, Yinan; Sun, Qian; Zhang, Xinwei

    2017-01-10

    Checkpoint programmed death-1 (PD-1)/programmed cell death ligands (PD-Ls) have been identified as negative immunoregulatory molecules that promote immune evasion of tumor cells. The interaction of PD-1 and PD-Ls inhibits the function of T cells and tumor-infiltrating lymphocytes (TIL) while increasing the function of immunosuppressive regulatory T cells (Tregs). This condition causes the tumor cells to evade immune response. Thus, the blockade of PD-1/PD-L1 enhances anti-tumor immunity by reducing the number and/or the suppressive activity of Tregs and by restoring the activity of effector T cells. Furthermore, some monoclonal antibodies blockading PD-1/PD-Ls axis have achieved good effect and received Food and Drug Administration approval. The role of PD-1/PD-Ls in tumors has been well studied, but little is known on the mechanism by which PD-1 blocks T-cell activation. In this study, we provide a brief overview on the discovery and regulatory mechanism of PD-1 and PD-L1 dysregulation in tumors, as well as the function and signaling pathway of PD-1 and its ligands; their roles in tumor evasion and clinical treatment were also studied.

  10. Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition

    PubMed Central

    Wang, Z; Zeng, Q; Chen, T; Liao, K; Bu, Y; Hong, S; Hu, G

    2015-01-01

    NFBD1 functions in cell cycle checkpoint activation and DNA repair following ionizing radiation (IR). In this study, we defined the NFBD1 as a tractable molecular target to radiosensitize nasopharyngeal carcinoma (NPC) cells. Silencing NFBD1 using lentivirus-mediated shRNA-sensitized NPC cells to radiation in a dose-dependent manner, increasing apoptotic cell death, decreasing clonogenic survival and delaying DNA damage repair. Furthermore, downregulation of NFBD1 inhibited the amplification of the IR-induced DNA damage signal, and failed to accumulate and retain DNA damage-response proteins at the DNA damage sites, which leaded to defective checkpoint activation following DNA damage. We also implicated the involvement of NFBD1 in IR-induced Rad51 and DNA-dependent protein kinase catalytic subunit foci formation. Xenografts models in nude mice showed that silencing NFBD1 significantly enhanced the antitumor activity of IR, leading to tumor growth inhibition of the combination therapy. Our studies suggested that a combination of gene therapy and radiation therapy may be an effective strategy for human NPC treatment. PMID:26247734

  11. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    SciTech Connect

    Park, Jeong-Min; Choi, Ji Ye; Yi, Joo Mi; Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok; Kang, Tae-Hong

    2015-06-05

    Nucleotide excision repair (NER) is the sole mechanism of UV-induced DNA lesion repair in mammals. A single round of NER requires multiple components including seven core NER factors, xeroderma pigmentosum A–G (XPA–XPG), and many auxiliary effector proteins including ATR serine/threonine kinase. The XPA protein helps to verify DNA damage and thus plays a rate-limiting role in NER. Hence, the regulation of XPA is important for the entire NER kinetic. We found that NDR1, a novel XPA-interacting protein, modulates NER by modulating the UV-induced DNA-damage checkpoint. In quiescent cells, NDR1 localized mainly in the cytoplasm. After UV irradiation, NDR1 accumulated in the nucleus. The siRNA knockdown of NDR1 delayed the repair of UV-induced cyclobutane pyrimidine dimers in both normal cells and cancer cells. It did not, however, alter the expression levels or the chromatin association levels of the core NER factors following UV irradiation. Instead, the NDR1-depleted cells displayed reduced activity of ATR for some set of its substrates including CHK1 and p53, suggesting that NDR1 modulates NER indirectly via the ATR pathway. - Highlights: • NDR1 is a novel XPA-interacting protein. • NDR1 accumulates in the nucleus in response to UV irradiation. • NDR1 modulates NER (nucleotide excision repair) by modulating the UV-induced DNA-damage checkpoint response.

  12. The mismatch repair system modulates curcumin sensitivity through induction of DNA strand breaks and activation of G2-M checkpoint.

    PubMed

    Jiang, Zhihua; Jin, ShunQian; Yalowich, Jack C; Brown, Kevin D; Rajasekaran, Baskaran

    2010-03-01

    The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of gammaH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G(2)-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.

  13. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4

    PubMed Central

    Falk, Jill E.; Chan, Leon Y.; Amon, Angelika

    2011-01-01

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother–bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell. PMID:21709215

  14. Moving towards a customized approach for drug development: lessons from clinical trials with immune checkpoint inhibitors in lung cancer

    PubMed Central

    Pilotto, Sara; Carbognin, Luisa; Karachaliou, Niki; Garassino, Marina; Cuppone, Federica; Petraglia, Sandra; Rosell, Rafael; Tortora, Giampaolo

    2015-01-01

    Lung cancer has recently been discovered to be an immunological targetable disease, on the basis of the exciting results of the randomized trials with immune checkpoint inhibitors. Nevertheless, the survival benefit appears to not be entirely captured by the usual outcome measures, thus requiring a deep reflection about the appropriateness of the traditional statistical methodologies in this context. The intrinsic biological differences existing both in terms of mechanism of action and kinetic between immunotherapy and chemotherapy or targeted therapy, impact on patients’ outcome, requiring a global revolution in the way to design clinical studies with the ideal aim to evolve towards trials carefully ‘customized’ on the basis of the investigational drug, the specific disease and the biological background. The exciting data recently obtained with immune checkpoint inhibitors, offer an ideal context and background to explore the major questions and future perspectives about the development of immunotherapeutic agents. In this regard, the choice of adequate endpoints, the use of modified statistical methods and the potential introduction of predictive biomarkers for immunotherapy clinical trials, will be discuss in this review in order to provide practical and rationale suggestions aimed to improve the existing model for cancer immunotherapy investigation. PMID:26798579

  15. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4.

    PubMed

    Falk, Jill E; Chan, Leon Y; Amon, Angelika

    2011-08-02

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother-bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.

  16. Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

    PubMed Central

    Nair, Jayasree S.; Ho, Alan L.; Tse, Archie N.; Coward, Jesse; Cheema, Haider; Ambrosini, Grazia; Keen, Nicholas

    2009-01-01

    The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein. PMID:19225156

  17. Loss of PTEN Is Associated with Resistance to Anti-PD-1 Checkpoint Blockade Therapy in Metastatic Uterine Leiomyosarcoma.

    PubMed

    George, Suzanne; Miao, Diana; Demetri, George D; Adeegbe, Dennis; Rodig, Scott J; Shukla, Sachet; Lipschitz, Mikel; Amin-Mansour, Ali; Raut, Chandrajit P; Carter, Scott L; Hammerman, Peter; Freeman, Gordon J; Wu, Catherine J; Ott, Patrick A; Wong, Kwok-Kin; Van Allen, Eliezer M

    2017-02-21

    Response to immune checkpoint blockade in mesenchymal tumors is poorly characterized, but immunogenomic dissection of these cancers could inform immunotherapy mediators. We identified a treatment-naive patient who has metastatic uterine leiomyosarcoma and has experienced complete tumor remission for >2 years on anti-PD-1 (pembrolizumab) monotherapy. We analyzed the primary tumor, the sole treatment-resistant metastasis, and germline tissue to explore mechanisms of immunotherapy sensitivity and resistance. Both tumors stained diffusely for PD-L2 and showed sparse PD-L1 staining. PD-1(+) cell infiltration significantly decreased in the resistant tumor (p = 0.039). Genomically, the treatment-resistant tumor uniquely harbored biallelic PTEN loss and had reduced expression of two neoantigens that demonstrated strong immunoreactivity with patient T cells in vitro, suggesting long-lasting immunological memory. In this near-complete response to PD-1 blockade in a mesenchymal tumor, we identified PTEN mutations and reduced expression of genes encoding neoantigens as potential mediators of resistance to immune checkpoint therapy.

  18. Epigenetic chromatin silencing: bistability and front propagation

    NASA Astrophysics Data System (ADS)

    Sedighi, Mohammad; Sengupta, Anirvan M.

    2007-12-01

    The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.

  19. Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.

    PubMed

    Lewis, Cody W; Golsteyn, Roy M

    2016-11-16

    We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

  20. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    NASA Astrophysics Data System (ADS)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  1. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  2. Characterization of silencing suppressor p24 of Grapevine leafroll-associated virus 2.

    PubMed

    Li, Mingjun; Zhang, Jiao; Feng, Ming; Wang, Xianyou; Luo, Chen; Wang, Qi; Cheng, Yuqin

    2016-12-20

    Grapevine leafroll-associated virus 2 (GLRaV-2) p24 has been reported to be an RNA silencing suppressor (RSS). However, the mechanisms underlying p24's suppression of RNA silencing are unknown. Using Agrobacterium infiltration-mediated RNA silencing assays, we showed that GLRaV-2 p24 is a strong RSS triggered by positive-sense green fluorescent protein (GFP) RNA, and that silencing suppression by p24 effectively blocks the accumulation of small interfering RNAs. Deletion analyses showed that the region of amino acids 1-188, which contains all predicted α-helices and β-strands, is required for the RSS activity of p24. Hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, previously shown to be critical for p24 self-interaction, are also crucial for silencing suppression, and western blotting results suggested that a lack of self-interaction ability results in decreased p24 accumulation in plants. The mutants showed greatly weakened or a lack of RSS activity. Substitution with two basic residues at positions 2 or 86, putatively involved in RNA binding, totally abolished the RSS activity of p24, suggesting that p24 uses an RNA-binding strategy to suppress RNA silencing. Our results also showed that W54 in the WG/GW-like motif (W54/G55) is crucial for the RSS activity of p24, whereas p24 does not physically interact with AGO1 of Nicotiana benthamiana. Furthermore, p24 did not promote AGO1 degradation, but significantly up-regulated AGO1 mRNA expression, and this effect was correlated with the RSS activity of p24, indicating that p24 may interfere with microRNA-directed processes. The presented results contribute to our understanding of viral suppression of RNA silencing and the molecular mechanisms underlying GLRaV-2 infection.

  3. The P0 protein encoded by cotton leafroll dwarf virus (CLRDV) inhibits local but not systemic RNA silencing.

    PubMed

    Delfosse, Verónica C; Agrofoglio, Yamila C; Casse, María F; Kresic, Iván Bonacic; Hopp, H Esteban; Ziegler-Graff, Véronique; Distéfano, Ana J

    2014-02-13

    Plants employ RNA silencing as a natural defense mechanism against viruses. As a counter-defense, viruses encode silencing suppressor proteins (SSPs) that suppress RNA silencing. Most, but not all, the P0 proteins encoded by poleroviruses have been identified as SSP. In this study, we demonstrated that cotton leafroll dwarf virus (CLRDV, genus Polerovirus) P0 protein suppressed local silencing that was induced by sense or inverted repeat transgenes in Agrobacterium co-infiltration assay in Nicotiana benthamiana plants. A CLRDV full-length infectious cDNA clone that is able to infect N. benthamiana through Agrobacterium-mediated inoculation also inhibited local silencing in co-infiltration assays, suggesting that the P0 protein exhibits similar RNA silencing suppression activity when expressed from the full-length viral genome. On the other hand, the P0 protein did not efficiently inhibit the spread of systemic silencing signals. Moreover, Northern blotting indicated that the P0 protein inhibits the generation of secondary but not primary small interfering RNAs. The study of CLRDV P0 suppression activity may contribute to understanding the molecular mechanisms involved in the induction of cotton blue disease by CLRDV infection.

  4. An essential role for the Saccharomyces cerevisiae DEAD-box helicase DHH1 in G1/S DNA-damage checkpoint recovery.

    PubMed Central

    Bergkessel, Megan; Reese, Joseph C

    2004-01-01

    The eukaryotic cell cycle displays a degree of plasticity in its regulation; cell cycle progression can be transiently arrested in response to environmental stresses. While the signaling pathways leading to cell cycle arrest are beginning to be well understood, the regulation of the release from arrest has not been well characterized. Here we show that DHH1, encoding a DEAD-box RNA helicase orthologous to the human putative proto-oncogene p54/RCK, is important in release from DNA-damage-induced cell cycle arrest at the G1/S checkpoint. DHH1 mutants are not defective for DNA repair and recover normally from the G2/M and replication checkpoints, suggesting a specific function for Dhh1p in recovery from G1/S checkpoint arrest. Dhh1p has been suggested to play a role in partitioning mRNAs between translatable and nontranslatable pools, and our results implicate this modulation of mRNA metabolism in the recovery from G1/S cell cycle arrest following DNA damage. Furthermore, the high degree of conservation between DHH1 and its human ortholog suggests that this mechanism is conserved among all eukaryotes and potentially important in human disease. PMID:15166134

  5. SOCS: potential immune checkpoint molecules for cancer immunotherapy.

    PubMed

    Chikuma, Shunsuke; Kanamori, Mitsuhiro; Mise-Omata, Setsuko; Yoshimura, Akihiko

    2017-02-11

    Inhibition of immune checkpoint molecules, PD-1 and CTLA4, has been shown to be a promising cancer treatment. PD-1 and CTLA4 inhibit TCR and co-stimulatory signals, respectively. The third T cell activation signal represents the signals from the cytokine receptors. The cytokine Interferon-γ (IFNγ) plays an important role in anti-tumor immunity by activating cytotoxic T cells (CTLs). Most cytokines use the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and the suppressors of cytokine signaling (SOCS) family of proteins are major negative regulators of the JAK/STAT pathway. Among SOCS proteins, CIS, SOCS1, and SOCS3 proteins can be considered the third immunocheckpoint molecules since they regulate cytokine signals that control the polarization of CD4(+) T cells and the maturation of CD8(+) T cells. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in terms of their anti-tumor immunity and potential applications. This article is protected by copyright. All rights reserved.

  6. Compensatory upregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agent checkpoint blockade in metastatic ovarian cancer.

    PubMed

    Huang, Ruea-Yea; Francois, Ariel; McGray, Aj Robert; Miliotto, Anthony; Odunsi, Kunle

    2017-01-01

    Tumor-associated or -infiltrating lymphocytes (TALs or TILs) co-express multiple immune inhibitory receptors that contribute to immune suppression in the ovarian tumor microenvironment (TME). Dual blockade of PD-1 along with LAG-3 or CTLA-4 has been shown to synergistically enhance T-cell effector function, resulting in a delay in murine ovarian tumor growth. However, the mechanisms underlying this synergy and the relative contribution of other inhibitory receptors to immune suppression in the ovarian TME are unknown. Here, we report that multiple immune checkpoints are expressed in TALs and TILs isolated from ovarian tumor-bearing mice. Importantly, blockade of PD-1, LAG-3, or CTLA-4 alone using genetic ablation or blocking antibodies conferred a compensatory upregulation of the other checkpoint pathways, potentiating their capacity for local T-cell suppression that, in turn, could be overcome through combinatorial blockade strategies. Whereas single-agent blockade led to tumor outgrowth in all animals, dual antibody blockade against PD-1/CTLA-4 or triple blockade against PD-1/LAG-3/CTLA-4 resulted in tumor-free survival in 20% of treated mice. In contrast, dual blockade of LAG-3 and CTLA-4 pathways using PD-1 knockout mice led to tumor-free survival in 40% of treated mice, suggesting a hierarchical ordering of checkpoint function. Durable antitumor immunity was most strongly associated with increased numbers of CD8(+) T cells, the frequency of cytokine-producing effector T cells, reduced frequency of Tregs and arginine-expressing monocytic myeloid-derived suppressor cells in the peritoneal TME. These data provide a basis for combinatorial checkpoint blockade in clinical intervention for ovarian cancer.

  7. Compensatory upregulation of PD-1, LAG-3, and CTLA-4 limits the efficacy of single-agent checkpoint blockade in metastatic ovarian cancer

    PubMed Central

    Francois, Ariel; McGray, AJ Robert; Miliotto, Anthony; Odunsi, Kunle

    2017-01-01

    ABSTRACT Tumor-associated or -infiltrating lymphocytes (TALs or TILs) co-express multiple immune inhibitory receptors that contribute to immune suppression in the ovarian tumor microenvironment (TME). Dual blockade of PD-1 along with LAG-3 or CTLA-4 has been shown to synergistically enhance T-cell effector function, resulting in a delay in murine ovarian tumor growth. However, the mechanisms underlying this synergy and the relative contribution of other inhibitory receptors to immune suppression in the ovarian TME are unknown. Here, we report that multiple immune checkpoints are expressed in TALs and TILs isolated from ovarian tumor-bearing mice. Importantly, blockade of PD-1, LAG-3, or CTLA-4 alone using genetic ablation or blocking antibodies conferred a compensatory upregulation of the other checkpoint pathways, potentiating their capacity for local T-cell suppression that, in turn, could be overcome through combinatorial blockade strategies. Whereas single-agent blockade led to tumor outgrowth in all animals, dual antibody blockade against PD-1/CTLA-4 or triple blockade against PD-1/LAG-3/CTLA-4 resulted in tumor-free survival in 20% of treated mice. In contrast, dual blockade of LAG-3 and CTLA-4 pathways using PD-1 knockout mice led to tumor-free survival in 40% of treated mice, suggesting a hierarchical ordering of checkpoint function. Durable antitumor immunity was most strongly associated with increased numbers of CD8+ T cells, the frequency of cytokine-producing effector T cells, reduced frequency of Tregs and arginine-expressing monocytic myeloid-derived suppressor cells in the peritoneal TME. These data provide a basis for combinatorial checkpoint blockade in clinical intervention for ovarian cancer. PMID:28197366

  8. Transcriptionally Silenced Transgenes in Maize Are Activated by Three Mutations Defective in Paramutation

    PubMed Central

    McGinnis, Karen M.; Springer, Catherine; Lin, Yan; Carey, Charles C.; Chandler, Vicki

    2006-01-01

    Plants with mutations in one of three maize genes, mop1, rmr1, and rmr2, are defective in paramutation, an allele-specific interaction that leads to meiotically heritable chromatin changes. Experiments reported here demonstrate that these genes are required to maintain the transcriptional silencing of two different transgenes, suggesting that paramutation and transcriptional silencing of transgenes share mechanisms. We hypothesize that the transgenes are silenced through an RNA-directed chromatin mechanism, because mop1 encodes an RNA-dependent RNA polymerase. In all the mutants, DNA methylation was reduced in the active transgenes relative to the silent transgenes at all of the CNG sites monitored within the transgene promoter. However, asymmetrical methylation persisted at one site within the reactivated transgene in the rmr1-1 mutant. With that one mutant, rmr1-1, the transgene was efficiently resilenced upon outcrossing to reintroduce the wild-type protein. In contrast, with the mop1-1 and rmr2-1 mutants, the transgene remained active in a subset of progeny even after the wild-type proteins were reintroduced by outcrossing. Interestingly, this immunity to silencing increased as the generations progressed, consistent with a heritable chromatin state being formed at the transgene in plants carrying the mop1-1 and rmr2-1 mutations that becomes more resistant to silencing in subsequent generations. PMID:16702420

  9. A PTEN-regulated checkpoint regulates surface delivery of delta opioid receptors.

    PubMed

    Shiwarski, Daniel J; Tipton, Alycia; Giraldo, Melissa D; Schmidt, Brigitte F; Gold, Michael S; Pradhan, Amynah A; Puthenveedu, Manojkumar A

    2017-03-06

    The delta opioid receptor (δR) is a promising alternate target for pain management, because δR agonists show decreased abuse potential compared to current opioid analgesics that target the mu opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface, and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENTOpioid analgesics like morphine, which target the mu opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. While the delta opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular

  10. Evolution and Functional Trajectory of Sir1 in Gene Silencing

    PubMed Central

    Ellahi, Aisha

    2016-01-01

    We used the budding yeasts Saccharomyces cerevisiae and Torulaspora delbrueckii to examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles of SIR and RNA interference (RNAi) genes in T. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins in T. delbrueckii revealed a different topography of chromatin at the HML and HMR loci than was observed in S. cerevisiae. S. cerevisiae Sir1, enriched at the silencers of HMLα and HMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast to S. cerevisiae SIR1's partially dispensable role in silencing, the T. delbrueckii SIR1 paralog KOS3 was essential for silencing. KOS3 was also found at telomeres with T. delbrueckii Sir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping in T. delbrueckii revealed single silencers at HML and HMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. The KOS3 gene mapped near HMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing, T. delbrueckii RNAi genes AGO1 and DCR1 did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin. PMID:26811328

  11. High Dub3 expression in mouse ESCs couples the G1/S checkpoint to pluripotency.

    PubMed

    van der Laan, Siem; Tsanov, Nikolay; Crozet, Carole; Maiorano, Domenico

    2013-11-07

    The molecular mechanism underlying G1/S checkpoint bypass in mouse embryonic stem cells (ESCs) remains unknown. DNA damage blocks S phase entry by inhibiting the CDK2 kinase through destruction of its activator, the Cdc25A phosphatase. We observed high Cdc25A levels in G1 that persist even after DNA damage in mouse ESCs. We also found higher expression of Dub3, a deubiquitylase that controls Cdc25A protein abundance. Moreover, we demonstrate that the Dub3 gene is a direct target of Esrrb, a key transcription factor of the self-renewal machinery. We show that Dub3 expression is strongly downregulated during neural conversion and precedes Cdc25A destabilization, while forced Dub3 expression in ESCs becomes lethal upon differentiation, concomitant to cell-cycle remodeling and lineage commitment. Finally, knockdown of either Dub3 or Cdc25A induced spontaneous differentiation of ESCs. Altogether, these findings couple the self-renewal machinery to cell-cycle control through a deubiquitylase in ESCs.

  12. Decreased somatic hypermutation induces an impaired peripheral B cell tolerance checkpoint

    PubMed Central

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M.; Ng, Yen-Shing; Massad, Christopher; Delmotte, Fabien R.; Yamakawa, Natsuko; Glauzy, Salome; Chamberlain, Nicolas; Kinnunen, Tuure; Menard, Laurence; Lavoie, Aubert; Walter, Jolan E.; Notarangelo, Luigi D.; Bruneau, Julie; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D.; Cunningham-Rundles, Charlotte; Kuijpers, Taco W.; Kracker, Sven; Kaneko, Hideo; Nonoyama, Shigeaki; Durandy, Anne

    2016-01-01

    Patients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation of autoreactive B cells in blood is unclear. Here, we analyzed B cell tolerance in AID-deficient patients, patients with autosomal dominant AID mutations (AD-AID), asymptomatic AICDA heterozygotes (AID+/–), and patients with uracil N-glycosylase (UNG) deficiency, which impairs CSR but not SHM. The low frequency of autoreactive mature naive B cells in UNG-deficient patients resembled that of healthy subjects, revealing that impaired CSR does not interfere with the peripheral B cell tolerance checkpoint. In contrast, we observed decreased frequencies of SHM in memory B cells from AD-AID patients and AID+/– subjects, who were unable to prevent the accumulation of autoreactive mature naive B cells. In addition, the individuals with AICDA mutations, but not UNG-deficient patients, displayed Tregs with defective suppressive capacity that correlated with increases in circulating T follicular helper cells and enhanced cytokine production. We conclude that SHM, but not CSR, regulates peripheral B cell tolerance through the production of mutated antibodies that clear antigens and prevent sustained interleukin secretions that interfere with Treg function. PMID:27701145

  13. Decreased somatic hypermutation induces an impaired peripheral B cell tolerance checkpoint.

    PubMed

    Cantaert, Tineke; Schickel, Jean-Nicolas; Bannock, Jason M; Ng, Yen-Shing; Massad, Christopher; Delmotte, Fabien R; Yamakawa, Natsuko; Glauzy, Salome; Chamberlain, Nicolas; Kinnunen, Tuure; Menard, Laurence; Lavoie, Aubert; Walter, Jolan E; Notarangelo, Luigi D; Bruneau, Julie; Al-Herz, Waleed; Kilic, Sara Sebnem; Ochs, Hans D; Cunningham-Rundles, Charlotte; van der Burg, Mirjam; Kuijpers, Taco W; Kracker, Sven; Kaneko, Hideo; Sekinaka, Yujin; Nonoyama, Shigeaki; Durandy, Anne; Meffre, Eric

    2016-11-01

    Patients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation of autoreactive B cells in blood is unclear. Here, we analyzed B cell tolerance in AID-deficient patients, patients with autosomal dominant AID mutations (AD-AID), asymptomatic AICDA heterozygotes (AID+/-), and patients with uracil N-glycosylase (UNG) deficiency, which impairs CSR but not SHM. The low frequency of autoreactive mature naive B cells in UNG-deficient patients resembled that of healthy subjects, revealing that impaired CSR does not interfere with the peripheral B cell tolerance checkpoint. In contrast, we observed decreased frequencies of SHM in memory B cells from AD-AID patients and AID+/- subjects, who were unable to prevent the accumulation of autoreactive mature naive B cells. In addition, the individuals with AICDA mutations, but not UNG-deficient patients, displayed Tregs with defective suppressive capacity that correlated with increases in circulating T follicular helper cells and enhanced cytokine production. We conclude that SHM, but not CSR, regulates peripheral B cell tolerance through the production of mutated antibodies that clear antigens and prevent sustained interleukin secretions that interfere with Treg function.

  14. Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

    PubMed Central

    Salmela, Anna-Leena; Pouwels, Jeroen; Varis, Asta; Kukkonen, Anu M.; Toivonen, Pauliina; Halonen, Pasi K.; Perälä, Merja; Kallioniemi, Olli; Gorbsky, Gary J.; Kallio, Marko J.

    2009-01-01

    Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 μg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound. PMID:19395653

  15. The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

    SciTech Connect

    Salmela, Anna-Leena; Pouwels, Jeroen; Kukkonen-Macchi, Anu; Waris, Sinikka; Toivonen, Pauliina; Jaakkola, Kimmo; Maeki-Jouppila, Jenni; Kallio, Lila; Kallio, Marko J.

    2012-03-10

    The spindle assembly checkpoint (SAC) is a conserved mechanism that ensures the fidelity of chromosome distribution in mitosis by preventing anaphase onset until the correct bipolar microtubule-kinetochore attachments are formed. Errors in SAC function may contribute to tumorigenesis by inducing numerical chromosome anomalies (aneuploidy). On the other hand, total disruption of SAC can lead to massive genomic imbalance followed by cell death, a phenomena that has therapeutic potency. We performed a cell-based high-throughput screen with a compound library of 2000 bioactives for novel SAC inhibitors and discovered a plant-derived phenolic compound eupatorin (3 Prime ,5-dihydroxy-4 Prime ,6,7-trimethoxyflavone) as an anti-mitotic flavonoid. The premature override of the microtubule drug-imposed mitotic arrest by eupatorin is dependent on microtubule-kinetochore attachments but not interkinetochore tension. Aurora B kinase activity, which is essential for maintenance of normal SAC signaling, is diminished by eupatorin in cells and in vitro providing a mechanistic explanation for the observed forced mitotic exit. Eupatorin likely has additional targets since eupatorin treatment of pre-mitotic cells causes spindle anomalies triggering a transient M phase delay followed by impaired cytokinesis and polyploidy. Finally, eupatorin potently induces apoptosis in multiple cancer cell lines and suppresses cancer cell proliferation in organotypic 3D cell culture model.

  16. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.

    PubMed

    Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

    2016-06-01

    Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.

  17. Immunotherapy Combinations With Checkpoint Inhibitors in Metastatic Melanoma: Current Approaches and Future Directions.

    PubMed

    Atkins, Michael

    2015-12-01

    Based on the complexity of the immune response to cancer and the mechanisms of tumor evasion, it is likely that therapeutic modulation of multiple immune-mediated pathways will be needed to maximally induce tumor regression in patients with advanced melanoma. The rationale of using combination checkpoint inhibitor-based regimens may include the concomitant effects on re-activation of T cells, increased trafficking of tumor reactive lymphocytes into the tumor tissue, and enhanced killing of cancer cells. The administration of nivolumab in combination with ipilimumab demonstrated increased response rates, tumor shrinkage, and median progression-free survival using combined therapy compared with either treatment alone. Although toxicity was also increased, this trial established proof of principle that combination immunotherapy could enhance the efficacy seen with single-agent programmed cell death protein-1 (PD-1) pathway blockade for the unselected patient. Current and future trials are evaluating alternative schedules and other combinations of immunotherapies to determine if they could provide similar efficacy with less toxicity. In addition, efforts are underway to determine how best to integrate combination immunotherapy with other treatment approaches for patients with advanced melanoma.

  18. Spindle assembly checkpoint inactivation fails to suppress neuroblast tumour formation in aurA mutant Drosophila

    PubMed Central

    Caous, Renaud; Pascal, Aude; Romé, Pierre; Richard-Parpaillon, Laurent; Karess, Roger; Giet, Régis

    2015-01-01

    Tissue homeostasis requires accurate control of cell proliferation, differentiation and chromosome segregation. Drosophila sas-4 and aurA mutants present brain tumours with extra neuroblasts (NBs), defective mitotic spindle assembly and delayed mitosis due to activation of the spindle assembly checkpoint (SAC). Here we inactivate the SAC in aurA and sas-4 mutants to determine whether the generation of aneuploidy compromises NB proliferation. Inactivation of the SAC in the sas-4 mutant impairs NB proliferation and disrupts euploidy. By contrast, disrupting the SAC in the aurA mutant does not prevent NB amplification, tumour formation or chromosome segregation. The monitoring of Mad2 and cyclin B dynamics in live aurA NBs reveals that SAC satisfaction is not coupled to cyclin B degradation. Thus, the NBs of aurA mutants present delayed mitosis, with accurate chromosome segregation occurring in a SAC-independent manner. We report here the existence of an Aurora A-dependent mechanism promoting efficient, timed cyclin B degradation. PMID:26568519

  19. Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis

    PubMed Central

    Schweizer, Nina; Ferrás, Cristina; Kern, David M.; Logarinho, Elsa; Cheeseman, Iain M.

    2013-01-01

    Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response. PMID:24344181

  20. Absence of a conventional spindle mitotic checkpoint in the binucleated single-celled parasite Giardia intestinalis.

    PubMed

    Markova, Kristyna; Uzlikova, Magdalena; Tumova, Pavla; Jirakova, Klara; Hagen, Guy; Kulda, Jaroslav; Nohynkova, Eva

    2016-10-01

    The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

  1. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints

    PubMed Central

    Zhao, Hui; Watkins, Janis L.; Piwnica-Worms, Helen

    2002-01-01

    Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G2 checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G2 checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks. PMID:12399544

  2. TMV induces RNA decay pathways to modulate gene silencing and disease symptoms.

    PubMed

    Conti, Gabriela; Zavallo, Diego; Venturuzzi, Andrea L; Rodriguez, Maria C; Crespi, Martin; Asurmendi, Sebastian

    2017-01-01

    RNA decay pathways comprise a combination of RNA degradation mechanisms that are implicated in gene expression, development and defense responses in eukaryotes. These mechanisms are known as the RNA Quality Control or RQC pathways. In plants, another important RNA degradation mechanism is the post-transcriptional gene silencing (PTGS) mediated by small RNAs (siRNAs). Notably, the RQC pathway antagonizes PTGS by preventing the entry of dysfunctional mRNAs into the silencing pathway to avoid global degradation of mRNA by siRNAs. Viral transcripts must evade RNA degrading mechanisms, thus viruses encode PTGS suppressor proteins to counteract viral RNA silencing. Here, we demonstrate that tobacco plants infected with TMV and transgenic lines expressing TMV MP and CP (coat protein) proteins (which are not linked to the suppression of silencing) display increased transcriptional levels of RNA decay genes. These plants also showed accumulation of cytoplasmic RNA granules with altered structure, increased rates of RNA decay for transgenes and defective transgene PTGS amplification. Furthermore, knockdown of RRP41 or RRP43 RNA exosome components led to lower levels of TMV accumulation with milder symptoms after infection, several developmental defects and miRNA deregulation. Thus, we propose that TMV proteins induce RNA decay pathways (in particular exosome components) to impair antiviral PTGS and this defensive mechanism would constitute an additional counter-defense strategy that lead to disease symptoms.

  3. High-performance genetically targetable optical neural silencing by light-driven proton pumps.

    PubMed

    Chow, Brian Y; Han, Xue; Dobry, Allison S; Qian, Xiaofeng; Chuong, Amy S; Li, Mingjie; Henninger, Michael A; Belfort, Gabriel M; Lin, Yingxi; Monahan, Patrick E; Boyden, Edward S

    2010-01-07

    The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of 'optogenetic' voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.

  4. Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast.

    PubMed

    Ren, Qun; Liou, Liang-Chun; Gao, Qiuqiang; Bao, Xiaoming; Zhang, Zhaojie

    2012-01-01

    Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

  5. Concordance of immune checkpoints within tumor immune contexture and their prognostic significance in gastric cancer.

    PubMed

    Dai, Congqi; Geng, Ruixuan; Wang, Chenchen; Wong, Angela; Qing, Min; Hu, Jianjun; Sun, Yu; Lo, A W I; Li, Jin

    2016-12-01

    Checkpoint blockade therapy has emerged as a novel approach for cancer immunotherapy in several malignancies. However, patient prognosis and disease progression relevant to immune checkpoints in gastric tumor microenvironment are not defined. This study aims to investigate the expression and prognostic significance of immune checkpoints within gastric cancer. In the study, a cohort of 398 cancer tissues from stage I to IV gastric cancer patients were assessed for programmed cell death 1 ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) infiltration using immunohistochemistry to ascertain their survival correlation. The data revealed that higher TIL density correlated with less risk of disease progression, and exhibited survival benefits in gastric cancer patients, and PD-L1 positivity showed a significant association with the presence of high TIL infiltration. Furthermore, real-time quantitative polymerase chain reaction was performed to detect expression of multiple immune checkpoints with the relation to clinical outcome in 139 samples randomly selected from the same cohort, and higher messenger RNA levels of most immune checkpoints were associated with favorable outcome, while consistently showing a positive correlation with interferon gamma levels. In situ hybridization was used to determine the localization of Epstein-Barr virus (EBV) in 97 specimens, and showed EBV-positive gastric cancer samples correlated with PD-L1 expression and increased TIL density. These results suggest that induction of immune checkpoint within gastric cancer patients reflects a high immune infiltration density, especially in those with EBV-associated gastric cancer, which may direct patient selection for checkpoint blockade therapy.

  6. Embracing Silence and the Emptiness between Unspoken Words

    ERIC Educational Resources Information Center

    VanSlyke-Briggs, Kjersti

    2014-01-01

    This article examines the use of silence as a constructive teaching tool in the classroom rather than as a punitive measure. The author offers suggestions for the inclusion of silence to benefit students specifically in a literature high school classroom.

  7. Surprised by Bird, Bard, and Bach: Language, Silence, and Transcendence.

    ERIC Educational Resources Information Center

    Suhor, Charles

    1991-01-01

    Argues the importance of the relationships among silence and literature, the arts, and other experiences that point toward transcendence. Suggests that English teachers can expand the repertoire of classroom activities and teaching techniques that make use of silence. (KEH)

  8. The Arabidopsis acetylated histone-binding protein BRAT1 forms a complex with BRP1 and prevents transcriptional silencing

    PubMed Central

    Zhang, Cui-Jun; Hou, Xiao-Mei; Tan, Lian-Mei; Shao, Chang-Rong; Huang, Huan-Wei; Li, Yong-Qiang; Li, Lin; Cai, Tao; Chen, She; He, Xin-Jian

    2016-01-01

    Transposable elements and other repetitive DNA sequences are usually subject to DNA methylation and transcriptional silencing. However, anti-silencing mechanisms that promote transcription in these regions are not well understood. Here, we describe an anti-silencing factor, Bromodomain and ATPase domain-containing protein 1 (BRAT1), which we identified by a genetic screen in Arabidopsis thaliana. BRAT1 interacts with an ATPase domain-containing protein, BRP1 (BRAT1 Partner 1), and both prevent transcriptional silencing at methylated genomic regions. Although BRAT1 mediates DNA demethylation at a small set of loci targeted by the 5-methylcytosine DNA glycosylase ROS1, the involvement of BRAT1 in anti-silencing is largely independent of DNA demethylation. We also demonstrate that the bromodomain of BRAT1 binds to acetylated histone, which may facilitate the prevention of transcriptional silencing. Thus, BRAT1 represents a potential link between histone acetylation and transcriptional anti-silencing at methylated genomic regions, which may be conserved in eukaryotes. PMID:27273316

  9. Media Credibility and the Spiral of Silence.

    ERIC Educational Resources Information Center

    Lee, Hye-ryeon

    The Spiral of Silence theory (Elisabeth Noelle-Neumann, l973) suggests that highly consonant media content has a strong impact upon individuals' perception of the opinion climate as well as upon their opinion expression. Noting that the theory lacks empirical investigation, a study took advantage of a controlled media system in Cheongju, South…

  10. Deconstructing the silences: gay social memory.

    PubMed

    Loong, Lionel Loh Han

    2012-01-01

    Adopting a Foucaultian perceptive, this article deconstructs the silences in the Singaporean gay community. The collective absences in homosexuals' social memory is not simply reflective of a fragmented community, but must be comprehended in relation to the role of the state and media in shaping particular discourses.

  11. Silence in the Context of "Child Voice"

    ERIC Educational Resources Information Center

    Lewis, Ann

    2010-01-01

    Recent decades have seen growing enthusiasm internationally for the concept and practice of "child voice". This was encapsulated in, and stimulated, by Article 12 of the 1989 United Nations Convention on the Rights of the Child. This article presents the case for incorporating the equally important concept of "child silence" in both research and…

  12. Muted Colors: Gender and Classroom Silence.

    ERIC Educational Resources Information Center

    Fredericksen, Elaine

    2000-01-01

    Highlights some causes for silence in schoolgirls and other marginalized students. Suggests ways teachers can help these students participate more fully as speakers and writers in language arts classes. Shows how language arts instruction can change students' attitudes about themselves as gendered subjects, agents, and communicators. (SR)

  13. Behold: Silence and Attention in Education

    ERIC Educational Resources Information Center

    Lewin, David

    2014-01-01

    Educators continually ask about the best means to engage students and how best to capture attention. These concerns often make the problematic assumption that students can directly govern their own attention. In order to address the role and limits of attention in education, some theorists have sought to recover the significance of silence or…

  14. Ideas for Creating and Overcoming Student Silences

    ERIC Educational Resources Information Center

    Woods, Donald R.; Sheardown, Heather

    2009-01-01

    The key idea is that 50 minutes of teacher talk with passive student listening is relatively ineffective in developing student learning. Teachers can create silences for productive active student learning. Students can also change from passive listeners to active talker-discussers of their learning. Ideas are given about how to overcome silences…

  15. Parenting a Precocious Preschooler: Breaking the Silence

    ERIC Educational Resources Information Center

    Fish, Leigh Ann

    2016-01-01

    Precocity in the very young should be a valid topic of discussion in parental and educational circles, yet too frequently those conversations are slow to occur or are absent altogether. Many parents and educators remain silent about raising and nurturing precocious preschoolers, and author Leigh Ann Fish believe that the silence is due to a lack…

  16. Cancer-associated TERT promoter mutations abrogate telomerase silencing

    PubMed Central

    Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

    2015-01-01

    Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells. DOI: http://dx.doi.org/10.7554/eLife.07918.001 PMID:26194807

  17. Histone Methylation and Epigenetic Silencing in Breast Cancer

    DTIC Science & Technology

    2010-07-01

    by asterisks in Figure 1A. Similar results implicating YY1 in Polycomb silencing of a Hox target gene in differentiating human ES cells have...demonstrate reporter silencing by a Hox gene DNA fragment targeted by PRC2 and PRC1 in mesenchymal stem cells (Woo et al. 2010). We transfected SKBR3...of histone H3, an epigenetic mark linked to gene silencing and implicated in tumor suppressor silencing during breast cancer progression. Progress

  18. The Sound of Silence: The Case of Virtual Team Organising

    ERIC Educational Resources Information Center

    Panteli, N.; Fineman, S.

    2005-01-01

    In this paper we discuss the role of silence within a virtual organising context. The paper raises issues related to the construction of silence in the virtual team context and the implications it has on team interactions. By drawing upon existing studies on virtual teams, we argue that members' silence may not always have negative effects on team…

  19. Silenced Voices and Extraordinary Conversations... Re-Imagining Schools.

    ERIC Educational Resources Information Center

    Fine, Michelle; Weis, Lois

    This collection of papers examines the crisis in public education, focusing on poor and minority children. There are seven chapters in two parts. After "Introduction: Silenced Voices and Extraordinary Conversations" (Michelle Fine and Lois Weis), Part 1, "Scenes of Silencing," includes: (1) "Silencing and Nurturing Voice…

  20. Mutuality, Self-Silencing, and Disordered Eating in College Women

    ERIC Educational Resources Information Center

    Wechsler, Lisa S.; Riggs, Shelley A.; Stabb, Sally D.; Marshall, David M.

    2006-01-01

    The current study examined patterns of association among mutuality, self-silencing, and disordered eating in an ethnically diverse sample of college women (N = 149). Partner mutuality and overall self-silencing were negatively correlated and together were associated with six disordered eating indices. All four self-silencing subscales were…

  1. Choosing Silence for Equality in and through Schooling

    ERIC Educational Resources Information Center

    Lees, Helen E.

    2016-01-01

    This article considers silences and equality as combined from a theoretical perspective. Equality in and through chosen, deliberate and regular silence experience is seen as an equaliser: if no one is speaking no one can dominate. The article uses a bifurcated concept of silence: weak, negative forms and strong, positive forms. Only the strong…

  2. Unpacking the Unspoken: Silence in Collective Memory and Forgetting

    ERIC Educational Resources Information Center

    Vinitzky-Seroussi, Vered; Teeger, Chana

    2010-01-01

    Collective memory quite naturally brings to mind notions of mnemonic speech and representation. In this article, however, we propose that collective silences be thought of as a rich and promising arena through which to understand how groups deal with their collective pasts. In so doing, we explore two types of silence: overt silence and covert…

  3. After the Blackbird Whistles: Listening to Silence in Classrooms

    ERIC Educational Resources Information Center

    Schultz, Katherine

    2010-01-01

    Background/Context: Students spend a large part of their time in schools in silence. However, teachers tend to spend most of their time attending to student talk. Anthropological and linguistic research has contributed to an understanding of silence in particular communities, offering explanations for students' silence in school. This research…

  4. Raising rural women's voices: From self-silencing to self-expression.

    PubMed

    Bogar, Sandra; Ganos, Emmy; Hoormann, Kelly; Bub-Standal, Caryn; Beyer, Kirsten M M

    2016-12-29

    Within the context of a community-academic partnership, we undertook a mixed-methods study to identify and explore health status, priorities, and management strategies among aging Wisconsin rural women. A questionnaire measuring diverse wellness needs was administered to women participating in personal development programming offered by a rural nonprofit organization. A subgroup participated in qualitative interviews to deepen the understanding of identified health priorities and methods of coping and healing. Findings provide insight into the prevalence of self-silencing among rural women and highlight mechanisms that help to facilitate the dismantling of self-silencing.

  5. Checks and balances between cohesin and polycomb in gene silencing and transcription.

    PubMed

    Dorsett, Dale; Kassis, Judith A

    2014-06-02

    The cohesin protein complex was discovered for its roles in sister chromatid cohesion and segregation, and the Polycomb group (PcG) proteins for their roles in epigenetic gene silencing during development. Cohesin also controls gene transcription via multiple mechanisms. Genetic and molecular evidence from Drosophila argue that cohesin and the PRC1 PcG complex interact to control transcription of many active genes that are critical for development, and that via these interactions cohesin also controls the availability of PRC1 for gene silencing.

  6. Lactate dehydrogenase A silencing in IDH mutant gliomas

    PubMed Central

    Chesnelong, Charles; Chaumeil, Myriam M.; Blough, Michael D.; Al-Najjar, Mohammad; Stechishin, Owen D.; Chan, Jennifer A.; Pieper, Russell O.; Ronen, Sabrina M.; Weiss, Samuel; Luchman, H. Artee; Cairncross, J. Gregory

    2014-01-01

    Background Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. Methods We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (mt) and IDH wild-type (wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. Results We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDHmt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDHmt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDHwt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDHmt glioblastomas. Conclusion To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDHmt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis. PMID:24366912

  7. Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

    PubMed Central

    Szittya, György; Silhavy, Dániel; Molnár, Attila; Havelda, Zoltán; Lovas, Ágnes; Lakatos, Lóránt; Bánfalvi, Zsófia; Burgyán, József

    2003-01-01

    Temperature dramatically affects plant–virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21–26 nucleotide) interfering (si) RNAs—the central molecules of RNA silencing-mediated defence pathways—are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes. PMID:12554663

  8. Identifying security checkpoints locations to protect the major U.S. urban areas

    DOE PAGES

    Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; ...

    2015-09-01

    Transit networks are integral to the economy and to society, but at the same time they could allow terrorists to transport weapons of mass destruction into any city. Road networks are especially vulnerable, because they lack natural checkpoints unlike air networks that have security measures in place at all major airports. One approach to mitigate this risk is ensuring that every road route passes through at least one security checkpoint. Using the Ford-Fulkerson maximum-flow algorithm, we generate a minimum set of checkpoint locations within a ring-shaped buffer area surrounding the 50 largest US urban areas. We study how the numbermore » of checkpoints changes as we increase the buffer width to perform a cost-benefit analysis and to identify groups of cities that behave similarly. The set of required checkpoints is surprisingly small (10-124) despite the hundreds of thousands of road arcs in those areas, making it feasible to protect all major cities.« less

  9. Caffeine stabilizes Cdc25 independently of Rad3 in Schizosaccharomyces pombe contributing to checkpoint override.

    PubMed

    Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

    2014-05-01

    Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both Schizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S. pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S. pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.

  10. The nucleoporin Nup153 affects spindle checkpoint activity due to an association with Mad1

    PubMed Central

    Shimi, Takeshi

    2010-01-01

    The nucleoporin Nup153 is known to play pivotal roles in nuclear import and export in interphase cells and as the cell transitions into mitosis, Nup153 is involved in nuclear envelope breakdown. In this study, we demonstrate that the interaction of Nup153 with the spindle assembly checkpoint protein Mad1 is important in the regulation of the spindle checkpoint. Overexpression of human Nup153 in HeLa cells leads to the appearance of multinucleated cells and induces the formation of multipolar spindles. Importantly, it causes inactivation of the spindle checkpoint due to hypophosphorylation of Mad1. Depletion of Nup153 using RNA interference results in the decline of Mad1 at nuclear pores during interphase and more significantly causes a delayed dissociation of Mad1 from kinetochores in metaphase and an increase in the number of unresolved midbodies. In the absence of Nup153 the spindle checkpoint remains active. In vitro studies indicate direct binding of Mad1 to the N-terminal domain of Nup153. Importantly, Nup153 binding to Mad1 affects Mad1's phosphorylation status, but not its ability to interact with Mad2. Our data suggest that Nup153 levels regulate the localization of Mad1 during the metaphase/anaphase transition thereby affecting its phoshorylation status and in turn spindle checkpoint activity and mitotic exit. PMID:21327106

  11. Closed MAD2 (C-MAD2) is selectively incorporated into the mitotic checkpoint complex (MCC)

    PubMed Central

    Tipton, Aaron R; Tipton, Michael; Yen, Tim

    2011-01-01

    The mitotic checkpoint is a specialized signal transduction pathway that monitors kinetochore-microtubule attachment to achieve faithful chromosome segregation. MAD2 is an evolutionarily conserved mitotic checkpoint protein that exists in open (O) and closed (C) conformations. The increase of intracellular C-MAD2 level during mitosis, through O→C-MAD2 conversion as catalyzed by unattached kinetochores, is a critical signaling event for the mitotic checkpoint. However, it remains controversial whether MAD2 is an integral component of the effector of the mitotic checkpoint—the mitotic checkpoint complex (MCC). We show here that endogenous human MCC is assembled by first forming a BUBR1:BUB3:CDC20 complex in G2 and then selectively incorporating C-MAD2 during mitosis. Nevertheless, MCC can be induced to form in G1/S cells by expressing a C-conformation locked MAD2 mutant, indicating intracellular level of C-MAD2 as a major limiting factor for MCC assembly. In addition, a recombinant MCC containing C-MAD2 exhibits effective inhibitory activity toward APC/C isolated from mitotic HeLa cells, while a recombinant BUBR1:BUB3:CDC20 ternary complex is ineffective at comparable concentrations despite association with APC/C. These results help establish a direct connection between a major signal transducer (C-MAD2) and the potent effector (MCC) of the mitotic checkpoint, and provide novel insights into protein-protein interactions during assembly of a functional MCC. PMID:22037211

  12. TIGIT and CD96: new checkpoint receptor targets for cancer immunotherapy.

    PubMed

    Dougall, William C; Kurtulus, Sema; Smyth, Mark J; Anderson, Ana C

    2017-03-01

    While therapies targeting the co-inhibitory or immune checkpoint receptors PD-1 and CTLA-4 have shown remarkable success in many cancers, not all patients benefit from these therapies. This has catalyzed enormous interest in the targeting of other immune checkpoint receptors. In this regard, TIGIT and CD96 have recently entered the limelight as novel immune checkpoint receptor targets. TIGIT and CD96 together with the co-stimulatory receptor CD226 form a pathway that is analogous to the CD28/CTLA-4 pathway, in which shared ligands and differential receptor:ligand affinities fine-tune the immune response. Although the roles of TIGIT and CD96 as immune checkpoint receptors in T cell and natural killer cell biology are just beginning to be uncovered, accumulating data support the targeting of these receptors for improving anti-tumor immune responses. A clear understanding of the immune cell populations regulated by TIGIT and CD96 is key to the design of immunotherapies that target these receptors in combination with other existing immune checkpoint blockade therapies.

  13. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    PubMed

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

  14. FAP positive fibroblasts induce immune checkpoint blockade resistance in colorectal cancer via promoting immunosuppression.

    PubMed

    Chen, Lingling; Qiu, Xiangting; Wang, Xinhua; He, Jian

    2017-03-13

    Immune checkpoint blockades that significantly prolonged survival of melanoma patients have been less effective on colorectal cancer (CRC) patients. Growing evidence suggested that fibroblast activation protein-alpha (FAP) on cancer associate fibroblasts (CAFs) has critical roles in regulating antitumor immune response by inducing tumor-promoting inflammation. In this study, we explored the roles of FAP in regulating the tumor immunity and immune checkpoint blockades resistance in CRC experimental systems. We found that CAFs with high FAP expression could induce immune checkpoint blockade resistance in CRC mouse model. Mechanistically, CAFs with high FAP expression promoted immunosuppression in the CRC tumor immune microenvironment by up-regulating CCL2 secretion, recruiting myeloid cells, and decreasing T-cell activity. In human CRC samples, FAP expression was proportional to myeloid cells number, but inversely related to T-cell number. High FAP expression also predicted poor survival of CRC patients. Taken together, our study suggested that high FAP expression in CAFs is one reason leading to immune checkpoint blockades resistance in CRC patients and FAP is an optional target for reversing immune checkpoint blockades resistance.

  15. RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei.

    PubMed

    Yang, Xiaofeng; Figueiredo, Luisa M; Espinal, Amin; Okubo, Eiji; Li, Bibo

    2009-04-03

    Trypanosoma brucei expresses variant surface glycoprotein (VSG) genes in a strictly monoallelic fashion in its mammalian hosts, but it is unclear how this important virulence mechanism is enforced. Telomere position effect, an epigenetic phenomenon, has been proposed to play a critical role in VSG regulation, yet no telomeric protein has been identified whose disruption led to VSG derepression. We now identify tbRAP1 as an intrinsic component of the T. brucei telomere complex and a major regulator for silencing VSG expression sites (ESs). Knockdown of tbRAP1 led to derepression of all VSGs in silent ESs, but not VSGs located elsewhere, and resulted in stronger derepression of genes located within 10 kb from telomeres than genes located further upstream. This graduated silencing pattern suggests that telomere integrity plays a key role in tbRAP1-dependent silencing and VSG regulation.

  16. RNAi mediated gene silencing against betasatellite associated with Croton yellow vein mosaic begomovirus.

    PubMed

    Sahu, Anurag Kumar; Marwal, Avinash; Nehra, Chitra; Choudhary, Devendra Kumar; Sharma, Pradeep; Gaur, Rajarshi Kumar

    2014-11-01

    Plant viruses encode suppressors of posttranscriptional gene silencing, an adaptive antiviral defense responses that confines virus infection. Previously, we identified single-stranded DNA satellite (also known as DNA-β) of ~1,350 nucleotides in length associated with Croton yellow vein mosaic begomovirus (CYVMV) in croton plants. The expression of genes from DNA-β requires the begomovirus for packaged, replication, insect transmission and movement in plants. The present study demonstrates the effect of the βC1 gene on the silencing pathway as analysed by using both transgenic systems and transient Agrobacterium tumefaciens based delivery. Plants that carry an intron-hairpin construct covering the βC1 gene accumulated cognate small-interfering RNAs and remained symptom-free after exposure to CYVMV and its satellite. These results suggest that βC1 interferes with silencing mechanism.

  17. The Nuclear Matrix Protein Megator Regulates Stem Cell Asymmetric Division through the Mitotic Checkpoint Complex in Drosophila Testes.

    PubMed

    Liu, Ying; Singh, Shree Ram; Zeng, Xiankun; Zhao, Jiangsha; Hou, Steven X

    2015-12-01

    In adult Drosophila testis, asymmetric division of germline stem cells (GSCs) is specified by an oriented spindle and cortically localized adenomatous coli tumor suppressor homolog 2 (Apc2). However, the molecular mechanism underlying these events remains unclear. Here we identified Megator (Mtor), a nuclear matrix protein, which regulates GSC maintenance and asymmetric division through the spindle assembly checkpoint (SAC) complex. Loss of Mtor function results in Apc2 mis-localization, incorrect centrosome orientation, defective mitotic spindle formation, and abnormal chromosome segregation that lead to the eventual GSC loss. Expression of mitotic arrest-deficient-2 (Mad2) and monopolar spindle 1 (Mps1) of the SAC complex effectively rescued the GSC loss phenotype associated with loss of Mtor function. Collectively our results define a new role of the nuclear matrix-SAC axis in regulating stem cell maintenance and asymmetric division.

  18. DRG1 is a potential oncogene in lung adenocarcinoma and promotes tumor progression via spindle checkpoint signaling regulation

    PubMed Central

    Lu, Li; Lv, Yanrong; Dong, Ji; Hu, Shaohua; Peng, Ruiyun

    2016-01-01

    Developmentally regulated GTP binding protein 1 (DRG1), a member of the DRG family, plays important roles in regulating cell growth. However, the molecular basis of DRG1 in cell proliferation regulation and the relationship between DRG1 and tumor progression remain poorly understood. Here, we demonstrate that DRG1 is elevated in lung adenocarcinomas while weakly expressed in adjacent lung tissues. DRG1 knockdown causes growth inhibition of tumor cells by significantly increasing the proportion of cells in M phase. Overexpression of DRG1 leads to chromosome missegregation which is an important index for tumorigenesis. Interestingly, ectopic of DRG1 reduces taxol induced apoptosis of lung adenocarcinoma cells. Mechanistic analyses confirm that DRG1 localizes at mitotic spindles in dividing cells and binds to spindle checkpoint signaling proteins in vivo. These studies highlight the expanding role of DRG1 in tumorigenesis and reveal a mechanism of DRG1 in taxol resistance. PMID:27626498

  19. How oocytes try to get it right: spindle checkpoint control in meiosis.

    PubMed

    Touati, Sandra A; Wassmann, Katja

    2016-06-01

    The generation of a viable, diploid organism depends on the formation of haploid gametes, oocytes, and spermatocytes, with the correct number of chromosomes. Halving the genome requires the execution of two consecutive specialized cell divisions named meiosis I and II. Unfortunately, and in contrast to male meiosis, chromosome segregation in oocytes is error prone, with human oocytes being extraordinarily "meiotically challenged". Aneuploid oocytes, that are with the wrong number of chromosomes, give rise to aneuploid embryos when fertilized. In humans, most aneuploidies are lethal and result in spontaneous abortions. However, some trisomies survive to birth or even adulthood, such as the well-known trisomy 21, which gives rise to Down syndrome (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012). A staggering 20-25 % of oocytes ready to be fertilized are aneuploid in humans. If this were not bad enough, there is an additional increase in meiotic missegregations as women get closer to menopause. A woman above 40 has a risk of more than 30 % of getting pregnant with a trisomic child. Worse still, in industrialized western societies, child birth is delayed, with women getting their first child later in life than ever. This trend has led to an increase of trisomic pregnancies by 70 % in the last 30 years (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012; Schmidt et al. in Hum Reprod Update 18:29-43, 2012). To understand why errors occur so frequently during the meiotic divisions in oocytes, we review here the molecular mechanisms at works to control chromosome segregation during meiosis. An important mitotic control mechanism, namely the spindle assembly checkpoint or SAC, has been adapted to the special requirements of the meiotic divisions, and this review will focus on our current knowledge of SAC control in mammalian oocytes. Knowledge on how chromosome segregation is controlled in mammalian oocytes may help to identify risk factors important for questions

  20. Patterning of Virus-Infected Glycine max Seed Coat Is Associated with Suppression of Endogenous Silencing of Chalcone Synthase Genes

    PubMed Central

    Senda, Mineo; Masuta, Chikara; Ohnishi, Shizen; Goto, Kazunori; Kasai, Atsushi; Sano, Teruo; Hong, Jin-Sung; MacFarlane, Stuart

    2004-01-01

    Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction. PMID:15037735

  1. Patterning of virus-infected Glycine max seed coat is associated with suppression of endogenous silencing of chalcone synthase genes.

    PubMed

    Senda, Mineo; Masuta, Chikara; Ohnishi, Shizen; Goto, Kazunori; Kasai, Atsushi; Sano, Teruo; Hong, Jin-Sung; MacFarlane, Stuart

    2004-04-01

    Most commercial Glycine max (soybean) varieties have yellow seeds because of loss of pigmentation in the seed coat. It has been suggested that inhibition of seed coat pigmentation in yellow G. max may be controlled by homology-dependent silencing of chalcone synthase (CHS) genes. Our analysis of CHS mRNA and short-interfering RNAs provide clear evidence that the inhibition of seed coat pigmentation in yellow G. max results from posttranscriptional rather than transcriptional silencing of the CHS genes. Furthermore, we show that mottling symptoms present on the seed coat of G. max plants infected with some viruses can be caused by suppression of CHS posttranscriptional gene silencing (PTGS) by a viral silencing suppressor protein. These results demonstrate that naturally occurring PTGS plays a key role in expression of a distinctive phenotype in plants and present a simple clear example of the elucidation of the molecular mechanism for viral symptom induction.

  2. Dynactin is involved in a checkpoint to monitor cell wall synthesis in Saccharomyces cerevisiae.

    PubMed

    Suzuki, Masaya; Igarashi, Ryoji; Sekiya, Mizuho; Utsugi, Takahiko; Morishita, Shinichi; Yukawa, Masashi; Ohya, Yoshikazu

    2004-09-01

    Checkpoint controls ensure the completion of cell cycle events with high fidelity in the correct order. Here we show the existence of a novel checkpoint that ensures coupling of cell wall synthesis and mitosis. In response to a defect in cell wall synthesis, S. cerevisiae cells arrest the cell-cycle before spindle pole body separation. This arrest results from the regulation of the M-phase cyclin Clb2p at the transcriptional level through the transcription factor Fkh2p. Components of the dynactin complex are required to achieve the G2 arrest whilst keeping cells highly viable. Thus, the dynactin complex has a function in a checkpoint that monitors cell wall synthesis.

  3. Challenges and opportunities for checkpoint blockade in T-cell lymphoproliferative disorders.

    PubMed

    Phillips, Tycel; Devata, Sumana; Wilcox, Ryan A

    2016-01-01

    The T-cell lymphoproliferative disorders are a heterogeneous group of non-Hodgkin's lymphomas (NHL) for which current therapeutic strategies are inadequate, as most patients afflicted with these NHL will succumb to disease progression within 2 years of diagnosis. Appreciation of the genetic and immunologic landscape of these aggressive NHL, including PD-L1 (B7-H1, CD274) expression by malignant T cells and within the tumor microenvironment, provides a strong rationale for therapeutic targeting this immune checkpoint. While further studies are needed, the available data suggests that responses with PD-1 checkpoint blockade alone will unlikely approach those achieved in other lymphoproliferative disorders. Herein, we review the unique challenges posed by the T-cell lymphoproliferative disorders and discuss potential strategies to optimize checkpoint blockade in these T-cell derived malignancies.

  4. DGK-α: A Checkpoint in Cancer-Mediated Immuno-Inhibition and Target for Immunotherapy

    PubMed Central

    Noessner, Elfriede

    2017-01-01

    Immunotherapy is moving to the forefront of cancer treatments owing to impressive durable responses achieved with checkpoint blockade antibodies and adoptive T-cell therapy. Still, improvements are necessary since, overall, only a small percentage of patients benefit from current therapies. Here, I summarize evidence that DGK-α may represent an immunological checkpoint suppressing the activity of cytotoxic immunocytes in the tumor microenvironment. DGK-inhibitors can restore the antitumor function of tumor-suppressed adaptive and innate cytotoxic immunocytes. The activity of DGK-inhibitors lays downstream of current checkpoint blockade antibodies. Thus, synergistic effects are expected from combination strategies. Moreover, DGK-inhibitors may permit a double-strike attack on tumor cells as DGK-inhibition may not only re-instate immunological tumor attack but also may harm tumor cells directly by interfering with oncogenic survival pathways. Together, DGK-inhibitors have very promising characteristics and may be beneficially included into the armamentarium of cancer immunotherapeutics. PMID:28316970

  5. Is there a role for therapeutic cancer vaccines in the age of checkpoint inhibitors?

    PubMed Central

    Dillman, Robert O.

    2017-01-01

    ABSTRACT Because of the recent success of monoclonal antibody checkpoint inhibitors, and the disappointing results of most therapeutic cancer vaccine trials, it has been questioned whether there is any potential role for such products going forward. In my opinion the answer is “yes” based on the following: [1] there is a persistent unmet clinical need because the majority of patients do not benefit from anti-checkpoint therapy, [2] there is evidence that not all patients make immune responses to their tumors, [3] there is evidence that immune responses to autologous tumor antigens can be induced by patient-specific vaccines, [4] there is clinical evidence from the pre-checkpoint era that suggests survival can be positively impacted by such patient-specific vaccines, and [5] the 2 available therapeutic vaccines that have received regulatory approval are quite limited in terms of their therapeutic benefit. PMID:27808593

  6. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    PubMed

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  7. Interplays between Soil-Borne Plant Viruses and RNA Silencing-Mediated Antiviral Defense in Roots

    PubMed Central

    Andika, Ida Bagus; Kondo, Hideki; Sun, Liying

    2016-01-01

    Although the majority of plant viruses are transmitted by arthropod vectors and invade the host plants through the aerial parts, there is a considerable number of plant viruses that infect roots via soil-inhabiting vectors such as plasmodiophorids, chytrids, and nematodes. These soil-borne viruses belong to diverse families, and many of them cause serious diseases in major crop plants. Thus, roots are important organs for the life cycle of many viruses. Compared to shoots, roots have a distinct metabolism and particular physiological characteristics due to the differences in development, cell composition, gene expression patterns, and surrounding environmental conditions. RNA silencing is an important innate defense mechanism to combat virus infection in plants, but the specific information on the activities and molecular mechanism of RNA silencing-mediated viral defense in root tissue is still limited. In this review, we summarize and discuss the current knowledge regarding RNA silencing aspects of the interactions between soil-borne viruses and host plants. Overall, research evidence suggests that soil-borne viruses have evolved to adapt to the distinct mechanism of antiviral RNA silencing in roots. PMID:27695446

  8. Less Efficient G2-M Checkpoint Is Associated with an Increased Risk of Lung Cancer in African Americans

    PubMed Central

    Zheng, Yun-Ling; Loffredo, Christopher A.; Alberg, Anthony J.; Yu, Zhipeng; Jones, Raymond T.; Perlmutter, Donna; Enewold, Lindsey; Krasna, Mark J.; Yung, Rex; Shields, Peter G.; Harris, Curtis C.

    2006-01-01

    Cell cycle checkpoints play critical roles in the maintenance of genomic integrity. The inactivation of checkpoint genes by genetic and epigenetic mechanisms is frequent in all cancer types, as a less-efficient cell cycle control can lead to genetic instability and tumorigenesis. In an on-going case-control study consisting of 216 patients with non–small cell lung cancer, 226 population-based controls, and 114 hospital-based controls, we investigated the relationship of γ-radiation-induced G2-M arrest and lung cancer risk. Peripheral blood lymphocytes were cultured for 90 hours, exposed to 1.0 Gy γ-radiation, and harvested at 3 hours after γ-radiation treatment. γ-Radiation-induced G2-M arrest was measured as the percentage of mitotic cells in untreated cultures minus the percentage of mitotic cells in γ-radiation-treated cultures from the same subject. The mean percentage of γ-radiation-induced G2-M arrest was significantly lower in cases than in population controls (1.18 versus 1.44, P < 0.01) and hospital controls (1.18 versus 1.40, P = 0.01). When dichotomized at the 50th percentile value in combined controls (population and hospital controls), a lower level of γ-radiation-induced G2-M arrest was associated with an increased risk of lung cancer among African Americans after adjusting for baseline mitotic index, age, gender, and pack-years of smoking [adjusted odd ratio (OR), 2.25; 95% confidence interval (95% CI), 0.97–5.20]. A significant trend of an increased risk of lung cancer with a decreased level of G2-M arrest was observed (Ptrend = 0.02) among African Americans, with a lowest-versus-highest quartile adjusted OR of 3.74 (95% CI, 0.98–14.3). This trend was most apparent among African American females (Ptrend < 0.01), with a lowest-versus-highest quartile adjusted OR of 11.75 (95% CI, 1.47–94.04). The results suggest that a less-efficient DNA damage–induced G2-M checkpoint is associated with an increased risk of lung cancer among African

  9. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing.

    PubMed

    Golden, Ryan J; Chen, Beibei; Li, Tuo; Braun, Juliane; Manjunath, Hema; Chen, Xiang; Wu, Jiaxi; Schmid, Vanessa; Chang, Tsung-Cheng; Kopp, Florian; Ramirez-Martinez, Andres; Tagliabracci, Vincent S; Chen, Zhijian J; Xie, Yang; Mendell, Joshua T

    2017-02-09

    MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.

  10. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  11. CAR T-cell intrinsic PD-1 checkpoint blockade: A two-in-one approach for solid tumor immunotherapy.

    PubMed

    Chen, Nan; Morello, Aurore; Tano, Zachary; Adusumilli, Prasad S

    2017-01-01

    PD-L1/2 expression in solid tumors inhibits chimeric antigen receptor (CAR) T-cell efficacy. A PD-1 dominant negative receptor expressed in CAR T cells provides cell-intrinsic checkpoint blockade and augments antitumor efficacy. A combinatorial immunotherapeutic strategy of combining CAR T cells with checkpoint blockade is a promising treatment approach for solid tumors.

  12. Homology-dependent Gene Silencing in Paramecium

    PubMed Central

    Ruiz, Françoise; Vayssié, Laurence; Klotz, Catherine; Sperling, Linda; Madeddu, Luisa

    1998-01-01

    Microinjection at high copy number of plasmids containing only the coding region of a gene into the Paramecium somatic macronucleus led to a marked reduction in the expression of the corresponding endogenous gene(s). The silencing effect, which is stably maintained throughout vegetative growth, has been observed for all Paramecium genes examined so far: a single-copy gene (ND7), as well as members of multigene families (centrin genes and trichocyst matrix protein genes) in which all closely related paralogous genes appeared to be affected. This phenomenon may be related to posttranscriptional gene silencing in transgenic plants and quelling in Neurospora and allows the efficient creation of specific mutant phenotypes thus providing a potentially powerful tool to study gene function in Paramecium. For the two multigene families that encode proteins that coassemble to build up complex subcellular structures the analysis presented herein provides the first experimental evidence that the members of these gene families are not functionally redundant. PMID:9529389

  13. Silencing nociceptor neurons reduces allergic airway inflammation

    PubMed Central

    Talbot, Sébastien; Abdulnour, Raja-Elie E.; Burkett, Patrick R.; Lee, Seungkyu; Cronin, Shane J.F.; Pascal, Maud A.; Laedermann, Cedric; Foster, Simmie L.; Tran, Johnathan V.; Lai, Nicole; Chiu, Isaac M.; Ghasemlou, Nader; DiBiase, Matthew; Roberson, David; Von Hehn, Christian; Agac, Busranour; Haworth, Oliver; Seki, Hiroyuki; Penninger, Josef M.; Kuchroo, Vijay K.; Bean, Bruce P.; Levy, Bruce D.; Woolf, Clifford J.

    2015-01-01

    Summary Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8+ sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large pore ion channels to specifically block nociceptors, substantially reduced ovalbumin or house dust mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4+ and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma. PMID:26119026

  14. Transcriptional Silencing by Polycomb-Group Proteins

    PubMed Central

    Grossniklaus, Ueli; Paro, Renato

    2014-01-01

    Polycomb-group (PcG) genes encode chromatin proteins involved in stable and heritable transcriptional silencing. PcG proteins participate in distinct multimeric complexes that deposit, or bind to, specific histone modifications (e.g., H3K27me3 and H2AK119ub1) to prevent gene activation and maintain repressed chromatin domains. PcG proteins are evolutionary conserved and play a role in processes ranging from vernalization and seed development in plants, over X-chromosome inactivation in mammals, to the maintenance of stem cell identity. PcG silencing is medically relevant as it is often observed in human disorders, including cancer, and tissue regeneration, which involve the reprogramming of PcG-controlled target genes. PMID:25367972

  15. Vaccines combined with immune checkpoint antibodies promote cytotoxic T cell activity and tumor eradication

    PubMed Central

    Ali, Omar A.; Lewin, Sarah A.; Dranoff, Glenn; Mooney, David J.

    2015-01-01

    We demonstrate that a poly(lactide-co-glycolide) (PLG) cancer vaccine can be used in combination with immune checkpoint antibodies, anti–CTLA-4 or anti–PD-1, to enhance cytotoxic T cell (CTL) activity and induce the regression of solid B16 tumors in mice. Combination therapy obviated the need for vaccine boosting and significantly skewed intratumoral reactions toward CTL activity, resulting in the regression of B16 tumors up to 50mm2 in size and 75% survival rates. These data suggests that combining material-based cancer vaccines with checkpoint antibodies has the potential to mediate tumor regression in humans. PMID:26669718

  16. FAK-inhibition opens the door to checkpoint immunotherapy in Pancreatic Cancer.

    PubMed

    Symeonides, Stefan N; Anderton, Stephen M; Serrels, Alan

    2017-01-01

    Immunotherapy has had remarkable success in the treatment of some cancer types. However, pancreatic cancer has remained largely refractory to immunotherapy, including immune checkpoint inhibitors. Recently, Jiang and colleagues identified a key role for FAK in regulating the composition of the fibrotic and immuno-suppressive pancreatic tumour niche, and showed that FAK inhibitors can be used in combination with immune checkpoint blockade and gemcitabine chemotherapy to significantly delay pancreatic tumour progression. This study further supports the use of FAK inhibitors in combination with immunotherapy.

  17. PD-1 and PD-L1 Immune Checkpoint Blockade to Treat Breast Cancer.

    PubMed

    Hartkopf, Andreas D; Taran, Florin-Andrei; Wallwiener, Markus; Walter, Christina B; Krämer, Bernhard; Grischke, Eva-Maria; Brucker, Sara Y

    2016-12-01

    Immune checkpoint inhibition represents a major recent breakthrough in the treatment of malignant diseases including breast cancer. Blocking the programmed death receptor-1 (PD-1) and its ligand, PD-L1, has shown impressive antitumor activity and may lead to durable long-term disease control, especially in the triple-negative subtypes of breast cancer (TNBC). Although immune checkpoint blockade is generally well tolerated, specific immune-related adverse events (irAEs) may occur. This review summarizes the clinical efficacy, perspectives, and future challenges of using PD-1/PD-L1-directed antibodies in the treatment of breast cancer.

  18. Use of common time base for checkpointing and rollback recovery in a distributed system

    NASA Technical Reports Server (NTRS)

    Ramanathan, Parameswaran; Shin, Kang G.

    1993-01-01

    An approach to checkpointing and rollback recovery in a distributed computing system using a common time base is proposed. A common time base is established in the system using a hardware clock synchronization algorithm. This common time base is coupled with the idea of pseudo-recovery points to develop a checkpointing algorithm that has the following advantages: reduced wait for commitment for establishing recovery lines, fewer messages to be exchanged, and less memory requirement. These advantages are assessed quantitatively by developing a probabilistic model.

  19. A Single dicer Gene Is Required for Efficient Gene Silencing Associated with Two Classes of Small Antisense RNAs in Mucor circinelloides▿ †

    PubMed Central

    de Haro, Juan P.; Calo, Silvia; Cervantes, María; Nicolás, Francisco E.; Torres-Martínez, Santiago; Ruiz-Vázquez, Rosa M.

    2009-01-01

    RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2− transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores. PMID:19666782

  20. Targeted Gene Silencing to Induce Permanent Sterility

    PubMed Central

    Dissen, Gregory A.; Lomniczi, Alejandro; Boudreau, Ryan L.; Chen, Yong Hong; Davidson, Beverly L.; Ojeda, Sergio R.

    2012-01-01

    Contents A nonsurgical method to induce sterility would be a useful tool to control feral populations of animals. Our laboratories have experience with approaches aimed at targeting brain cells in vivo with vehicles that deliver a payload of either inhibitory RNAs or genes intended to correct cellular dysfunction. A combination/modification of these methods may provide a useful framework for the design of approaches that can be used to sterilize cats and dogs. For this approach to succeed it has to meet several conditions: It needs to target a gene essential for fertility. It must involve a method that can selectively silence the gene of interest. It also needs to deliver the silencing agent via a minimally invasive method. Finally, the silencing effect needs to be sustained for many years, so that expansion of the targeted population can be effectively prevented. In this article we discuss this subject and provide a succinct account of our previous experience with: a) molecular reagents able to disrupt reproductive cyclicity when delivered to regions of the brain involved in the control of reproduction, and b) molecular reagents able to ameliorate neuronal disease when delivered systemically using a novel approach of gene therapy. PMID:22827375

  1. Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model.

    PubMed

    Chakrabarti, Lina; Morgan, Clifford; Sandler, Anthony D

    2015-01-01

    Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.

  2. Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation

    PubMed Central

    Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H. F. M.; Stadler, Michael B.; Turner, James M. A.

    2015-01-01

    During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions. PMID:26509798

  3. Silencing of X-Linked MicroRNAs by Meiotic Sex Chromosome Inactivation.

    PubMed

    Royo, Hélène; Seitz, Hervé; ElInati, Elias; Peters, Antoine H F M; Stadler, Michael B; Turner, James M A

    2015-10-01

    During the pachytene stage of meiosis in male mammals, the X and Y chromosomes are transcriptionally silenced by Meiotic Sex Chromosome Inactivation (MSCI). MSCI is conserved in therian mammals and is essential for normal male fertility. Transcriptomics approaches have demonstrated that in mice, most or all protein-coding genes on the X chromosome are subject to MSCI. However, it is unclear whether X-linked non-coding RNAs behave in a similar manner. The X chromosome is enriched in microRNA (miRNA) genes, with many exhibiting testis-biased expression. Importantly, high expression levels of X-linked miRNAs (X-miRNAs) have been reported in pachytene spermatocytes, indicating that these genes may escape MSCI, and perhaps play a role in the XY-silencing process. Here we use RNA FISH to examine X-miRNA expression in the male germ line. We find that, like protein-coding X-genes, X-miRNAs are expressed prior to prophase I and are thereafter silenced during pachynema. X-miRNA silencing does not occur in mouse models with defective MSCI. Furthermore, X-miRNAs are expressed at pachynema when present as autosomally integrated transgenes. Thus, we conclude that silencing of X-miRNAs during pachynema in wild type males is MSCI-dependent. Importantly, misexpression of X-miRNAs during pachynema causes spermatogenic defects. We propose that MSCI represents a chromosomal mechanism by which X-miRNAs, and other potential X-encoded repressors, can be silenced, thereby regulating genes with critical late spermatogenic functions.

  4. Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

    PubMed Central

    Kuser-Abali, Gamze; Alptekin, Ahmet; Cinar, Bekir

    2014-01-01

    Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival. PMID:24499724

  5. Endogenous Small RNA Mediates Meiotic Silencing of a Novel DNA Transposon.

    PubMed

    Wang, Yizhou; Smith, Kristina M; Taylor, John W; Freitag, Michael; Stajich, Jason E

    2015-06-23

    Genome defense likely evolved to curtail the spread of transposable elements and invading viruses. A combination of effective defense mechanisms has been shown to limit colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used laboratory "wild-type" strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA, also simply called meiotic silencing, prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and is proposed to involve the production of small RNA, so-called masiRNAs, by proteins homologous to those involved in RNA interference-silencing pathways in animals, fungi, and plants. Here, we demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild-type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. We present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element.

  6. MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

    PubMed Central

    Dahlmans, Vivian E. H.; Spaapen, Frank; Salvaing, Juliette; Vanhove, Jolien; Geijselaers, Claudia; Bartels, Stefanie J. J.; Partouns, Iris; Neumann, Dietbert; Speel, Ernst-Jan; Takihara, Yoshihiro; Wouters, Bradly G.; Voncken, Jan Willem

    2015-01-01

    Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent. PMID:25853770

  7. Myeloid PTEN deficiency impairs tumor-immune surveillance via immune-checkpoint inhibition

    PubMed Central

    Kuttke, M.; Sahin, E.; Pisoni, J.; Percig, S.; Vogel, A.; Kraemmer, D.; Hanzl, L.; Brunner, J. S.; Paar, H.; Soukup, K.; Halfmann, A.; Dohnal, A. M.; Steiner, C. W.; Blüml, S.; Basilio, J.; Hochreiter, B.; Salzmann, M.; Hoesel, B.; Lametschwandtner, G.; Eferl, R.; Schmid, J. A.; Schabbauer, G.

    2016-01-01

    ABSTRACT Tumor–host interaction is determined by constant immune surveillance, characterized by tumor infiltration of myeloid and lymphoid cells. A malfunctioning or diverted immune response promotes tumor growth and metastasis. Recent advances had been made, by treating of certain tumor types, such as melanoma, with T-cell checkpoint inhibitors. This highlights the importance of understanding the molecular mechanisms underlying the crosstalk between tumors and their environment, in particular myeloid and lymphoid cells. Our aim was to study the contribution of the myeloid PI3K/PTEN-signaling pathway in the regulation of tumor-immune surveillance in murine models of cancer. We made use of conditional PTEN-deficient mice, which exhibit sustained activation of the PI3K-signaling axis in a variety of myeloid cell subsets such as macrophages and dendritic cells (DCs). In colitis-associated colon cancer (CAC), mice deficient in myeloid PTEN showed a markedly higher tumor burden and decreased survival. We attributed this observation to the increased presence of immune-modulatory conventional CD8α+ DCs in the spleen, whereas other relevant myeloid cell subsets were largely unaffected. Notably, we detected enhanced surface expression of PD-L1 and PD-L2 on these DCs. As a consequence, tumoricidal T-cell responses were hampered or redirected. Taken together, our findings indicated an unanticipated role for the PI3K/PTEN-signaling axis in the functional regulation of splenic antigen-presenting cells (APCs). Our data pointed at potential, indirect, tumoricidal effects of subclass-specific PI3K inhibitors, which are currently under clinical investigation for treatment of tumors, via myeloid cell activation. PMID:27622019

  8. Cohabitation of insulators and silencing elements in yeast subtelomeric regions.

    PubMed Central

    Fourel, G; Revardel, E; Koering, C E; Gilson, E

    1999-01-01

    In budding yeast, the telomeric DNA is flanked by a combination of two subtelomeric repetitive sequences, the X and Y' elements. We have investigated the influence of these sequences on telomeric silencing. The telomere-proximal portion of either X or Y' dampened silencing when located between the telomere and the reporter gene. These elements were named STARs, for subtelomeric anti-silencing regions. STARs can also counteract silencer-driven repression at the mating-type HML locus. When two STARs bracket a reporter gene, its expression is no longer influenced by surrounding silencing elements, although these are still active on a second reporter gene. In addition, an intervening STAR uncouples the silencing of neighboring genes. STARs thus display the hallmarks of insulators. Protection from silencing is recapitulated by multimerized oligonucleotides representing Tbf1p- and Reb1p-binding sites, as found in STARs. In contrast, sequences located more centromere proximal in X and Y' elements reinforce silencing. They can promote silencing downstream of an insulated expressed domain. Overall, our results suggest that the silencing emanating from telomeres can be propagated in a discontinuous manner via a series of subtelomeric relay elements. PMID:10228166

  9. Homology-Dependent Silencing by an Exogenous Sequence in the Drosophila Germline

    PubMed Central

    Pöyhönen, Maria; de Vanssay, Augustin; Delmarre, Valérie; Hermant, Catherine; Todeschini, Anne Laure; Teysset, Laure; Ronsseray, Stéphane

    2012-01-01

    The study of P transposable element repression in Drosophila melanogaster led to the discovery of the trans-silencing effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences) represses in trans, in the female germline, a homologous P-lacZ transgene inserted in euchromatin. TSE shows variegation in ovaries and displays a maternal effect as well as epigenetic transmission through meiosis. In addition, TSE is highly sensitive to mutations affecting heterochromatin components (including HP1) and the Piwi-interacting RNA silencing pathway (piRNA), a homology-dependent silencing mechanism that functions in the germline. TSE appears thus to involve the piRNA-based silencing proposed to play a major role in P repression. Under this hypothesis, TSE may also be established when homology between the telomeric and target loci involves sequences other than P elements, including sequences exogenous to the D. melanogaster genome. We have tested whether TSE can be induced via lacZ sequence homology. We generated a piggyBac-otu-lacZ transgene in which lacZ is under the control of the germline ovarian tumor promoter, resulting in strong expression in nurse cells and the oocyte. We show that all piggyBac-otu-lacZ transgene insertions are strongly repressed by maternally inherited telomeric P-lacZ transgenes. This repression shows variegation between egg chambers when it is incomplete and presents a maternal effect, two of the signatures of TSE. Finally, this repression is sensitive to mutations affecting aubergine, a key player of the piRNA pathway. These data show that TSE can occur when silencer and target loci share solely a sequence exogenous to the D. melanogaster genome. This functionally supports the hypothesis that TSE represents a general repression mechanism which can be co-opted by new transposable elements to regulate their activity after a transfer to the D. melanogaster

  10. Transcriptome analysis of antigenic variation in Plasmodium falciparum - var silencing is not dependent on antisense RNA

    PubMed Central

    Ralph, Stuart A; Bischoff, Emmanuel; Mattei, Denise; Sismeiro, Odile; Dillies, Marie-Agnès; Guigon, Ghislaine; Coppee, Jean-Yves; David, Peter H; Scherf, Artur

    2005-01-01

    Background Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36. Results In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript. Conclusion Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci. PMID:16277748

  11. Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo.

    PubMed

    Riu, Efren; Chen, Zhi-Ying; Xu, Hui; He, Chen-Yi; Kay, Mark A

    2007-07-01

    One of the major obstacles to success in non-viral gene therapy is transcriptional silencing of the DNA vector. The mechanisms underlying gene silencing/repression in mammalian cells are complex and remain unclear. Because changes in chromatin structure and, in particular, histone modifications are involved in transcriptional regulation of endogenous genes, we hypothesized that changes in the pattern of histone modifications were related to the observed transcriptional silencing of exogenous DNA vectors. We used antibodies against specific modified histones to perform chromatin immunoprecipitation (ChIP) analyses on liver lysates from mice transfected with two types of plasmids: (i) DNA minicircles (MCs) devoid of bacterial plasmid backbone DNA, which showed marked persistence of transgene expression, and (ii) their parental plasmids, which were silenced over time. Silencing of the transgene from the parental vectors was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the MC DNA was consistent with euchromatin. Our data indicates that (i) episomal vectors undergo chromatinization in vivo, and (ii) both persistence and silencing of transgene expression are associated with specific histone modifications.

  12. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  13. A leukemia fusion protein attenuates the spindle checkpoint and promotes aneuploidy

    PubMed Central

    Boyapati, Anita; Yan, Ming; Peterson, Luke F.; Biggs, Joseph R.; Le Beau, Michelle M.

    2007-01-01

    The 8;21 chromosomal translocation occurs in 15% to 40% of patients with the FAB M2 subtype of acute myeloid leukemia (AML). This chromosomal abnormality fuses part of the AML1/RUNX1 gene to the ETO/MTG8 gene and generates the AML1-ETO protein. We previously identified a C-terminal truncated AML1-ETO protein (AEtr) in a mouse leukemia model. AEtr is almost identical to the AML1-ETO exon 9a isoform expressed in leukemia patients. Here, we describe a novel function of AEtr in the development of aneuploidy through spindle checkpoint attenuation. AEtr cells had a reduced mitotic index following nocodazole treatment, suggesting a failure in a subset of cells to arrest in mitosis with a functional spindle checkpoint. Additionally, primary leukemia cells and cell lines expressing AEtr were aneuploid. Moreover, AEtr cells had reduced levels of several spindle checkpoint proteins including BubR1 and securin following treatment with the spindle poison nocodazole. These results suggest that inactivation of the spindle checkpoint may contribute to the development of aneuploidy described in t(8;21) leukemia patients. PMID:17197431

  14. An assessment of the checkpoint bioassay concept for full scale wastewater UV reactor validation.

    PubMed

    Maka, P P; Lawryshyn, Y A

    2011-01-01

    In an effort to help policy makers and manufacturers understand the impact of parameter uncertainties on UV reactor performance, a numerical bioassay model was developed by integrating a UV reactor model based on computational fluid dynamics with a Monte Carlo model developed to account for parameter uncertainty. For the model implemented, it was determined that reactor performance uncertainty was less than 6%. The integrated model was used to evaluate several checkpoint bioassay criteria including one currently used by the California Department of Public Health. The model showed that these criteria failed to take into account the fact that in an ideal case, a full scale reactor will pass a single checkpoint test 50% of the time. In reality, differences in equipment measurement errors between the system validation and checkpoint bioassay, and limitations of the power law form of the dose monitoring equation in accurately representing system validation data will result in poorer than expected performance. It was suggested that such checkpoint criteria be modified by crediting the inherent over-sizing of full scale reactors.

  15. Myoelectric silence following unopposed passive stretch in normal man.

    PubMed

    Angel, R W; Waxman, S G; Kocsis, J D

    1980-08-01

    The response to unopposed passive muscle stretch applied during sustained contraction was studied in normal man. When the subject did not resist the stretching force, the initial response was a brief cessation of EMG activity in the elongated muscle. The myoelectric silence was observed repeatedly in muscles of the upper and lower limbs. The response to passive stretch is discussed in relation to the lengthening reaction and the inverse myotatic reflex. The silent period observed under these experimental conditions is unlikely to be caused by Renshaw inhibition, a pause in spindle afferent discharge, or activity of the group II afferent reflex pathway. Possible mechanisms include autogenetic inhibition and a stretch-evoked decrease of fusimotor activity.

  16. Arabidopsis HDA6 Regulates Locus-Directed Heterochromatin Silencing in Cooperation with MET1

    PubMed Central

    Matsui, Akihiro; Kurihara, Yukio; Morosawa, Taeko; Ishida, Junko; Tanaka, Maho; Endo, Takaho; Kakutani, Tetsuji; Toyoda, Tetsuro; Kimura, Hiroshi; Yokoyama, Shigeyuki; Shinozaki, Kazuo; Seki, Motoaki

    2011-01-01

    Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA–methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure

  17. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

    PubMed Central

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. PMID:24955955

  18. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

    PubMed

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

  19. Evolution of Neoantigen Landscape during Immune Checkpoint Blockade in Non-Small Cell Lung Cancer.

    PubMed

    Anagnostou, Valsamo; Smith, Kellie N; Forde, Patrick M; Niknafs, Noushin; Bhattacharya, Rohit; White, James; Zhang, Theresa; Adleff, Vilmos; Phallen, Jillian; Wali, Neha; Hruban, Carolyn; Guthrie, Violeta B; Rodgers, Kristen; Naidoo, Jarushka; Kang, Hyunseok; Sharfman, William; Georgiades, Christos; Verde, Franco; Illei, Peter; Li, Qing Kay; Gabrielson, Edward; Brock, Malcolm V; Zahnow, Cynthia A; Baylin, Stephen B; Scharpf, Robert B; Brahmer, Julie R; Karchin, Rachel; Pardoll, Drew M; Velculescu, Victor E

    2017-03-01

    Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.

  20. Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing

    PubMed Central

    Casas-Mollano, J. Armando; Rohr, Jennifer; Kim, Eun-Jeong; Balassa, Eniko; van Dijk, Karin; Cerutti, Heriberto

    2008-01-01

    Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER-LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga. PMID:18493041

  1. Functional validation of a constitutive autonomous silencer element.

    PubMed

    Qi, Heyuan; Liu, Mingdong; Emery, David W; Stamatoyannopoulos, George

    2015-01-01

    Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.

  2. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  3. Small silencing RNAs: an expanding universe

    PubMed Central

    Ghildiyal, Megha; Zamore, Phillip D.

    2009-01-01

    Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats. PMID:19148191

  4. Small silencing RNAs: an expanding universe.

    PubMed

    Ghildiyal, Megha; Zamore, Phillip D

    2009-02-01

    Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats.

  5. Root-Knot Nematode Parasitism Suppresses Host RNA Silencing.

    PubMed

    Walsh, E; Elmore, J M; Taylor, C G

    2017-04-12

    Root-knot nematodes damage crops around the world by developing complex feeding sites from normal root cells of their hosts. The ability to initiate and maintain this feeding site (composed of individual "giant cells") is essential to their parasitism process. RNA silencing pathways in plants serve a diverse set of functions, from directing growth and development to defending against invading pathogens. Influencing a host's RNA silencing pathways as a pathogenicity strategy has been well-documented for viral plant pathogens, but recently, it has become clear that silencing pathways also play an important role in other plant pathosystems. To determine if RNA silencing pathways play a role in nematode parasitism, we tested the susceptibility of plants that express a viral suppressor of RNA silencing. We observed an increase in susceptibility to nematode parasitism in plants expressing viral suppressors of RNA silencing. Results from studies utilizing a silenced reporter gene suggest that active suppression of RNA silencing pathways may be occurring during nematode parasitism. With these studies, we provide further evidence to the growing body of plant-biotic interaction research that suppression of RNA silencing is important in the successful interaction between a plant-parasitic animal and its host.

  6. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

    PubMed

    Finnigan, Gregory C; Sterling, Sarah M; Duvalyan, Angela; Liao, Elizabeth N; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-07-15

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.

  7. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Sterling, Sarah M.; Duvalyan, Angela; Liao, Elizabeth N.; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-01-01

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611–950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611–950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379–1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. PMID:27193302

  8. Robustness of CDK2 in Triggering Cellular Senescence based on Probability of DNA-damaged Cells Passing G1/S Checkpoint

    NASA Astrophysics Data System (ADS)

    Ling, Hong; Samarasinghe, Sandhya; Kulasiri, Don

    2011-06-01

    Recent experiments have shown that cellular senescence, a mechanism employed by cells for thwarting cell proliferation, plays an important role in protecting cells against cancer; therefore, a deeper understanding of cellular senescence can lead to effective cancer treatment. Inhibition of CDK2 is thought to be the critical trigger for cellular senescence. In this study, we first implement a mathematical model of G1/S transition involving the DNA-damage pathway and show that cellular senescence can be achieved by lowering CDK2. The robustness of CDK2 in triggering cellular senescence is determined from the probability (β) of DNA-damaged cells passing G1/S checkpoint for normal CDK2 and CDK2-deficient situations based on different thresholds of the peak time of two important biomarkers, CycE and E2F. The comparison of the values of β under the normal CDK2 and lower CDK2 levels reveals that reducing CDK2 levels can decrease the percentage of damaged cells passing G1/S checkpoint; more importantly, 50% reduction of CDK2 achieves 65% reduction in the percentage of damaged cells passing the G1/S checkpoint. These results point out that the developed model can highlight the possibility of lowering the bar for cellular senescence by reducing CDK2 levels. The results of investigation of β for the different thresholds of the peak times of other biomarkers show that β is insensitive to these perturbations of the peak time indicating that CDK2 activity is robust in lowering the senescence bar for low and high levels of DNA-damage. Furthermore, a mathematical formulation of robustness indicates that the robustness of CDK2-triggered senescence increases with decreasing levels of CDK2, and is slightly greater for low-level DNA damage condition.

  9. Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex

    PubMed Central

    Egan, Emily D.; Braun, Craig R.; Gygi, Steven P.; Moazed, Danesh

    2014-01-01

    RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing. PMID:24713849

  10. The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

    SciTech Connect

    Fusaro, Adriana F.; Correa, Regis L.; Nakasugi, Kenlee; Jackson, Craig; Kawchuk, Lawrence; Vaslin, Maite F.S.; Waterhouse, Peter M.

    2012-05-10

    The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0{sup PE}, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0{sup PE} has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0{sup PE} destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

  11. Virus Counterdefense: Diverse Strategies for Evading the RNA-Silencing Immunity

    PubMed Central

    Li, Feng; Ding, Shou-Wei

    2009-01-01

    Viruses are obligate, intracellular pathogens that must manipulate and exploit host molecular mechanisms to prosper in the hostile cellular environment. Here we review the strategies used by viruses to evade the immunity controlled by 21- to 26-nt small RNAs. Viral suppressors of RNA silencing (VSRs) are encoded by genetically diverse viruses infecting plants, invertebrates, and vertebrates. VSRs target key steps in the small RNA pathways by inhibiting small RNA production,sequestering small RNAs,orpreventing short- and long-distance spread of RNA silencing. However, although VSRs are required for infection, explicit data demonstrating a role of silencing suppression in virus infection are available only for a few VSRs. A subset of VSRs bind double-stranded RNA, but a distinct protein fold is revealed for each of the four VSRs examined. We propose that VSR families are evolved independently as a viral adaptation to immunity. Unresolved issues on the role of RNA silencing in virus-host interactions are highlighted. PMID:16768647

  12. TRB3 Gene Silencing Alleviates Diabetic Cardiomyopathy in a Type 2 Diabetic Rat Model

    PubMed Central

    Ti, Yun; Xie, Guo-lu; Wang, Zhi-hao; Bi, Xiao-lei; Ding, Wen-yuan; Wang, Jia; Jiang, Gui-hua; Bu, Pei-li; Zhang, Yun; Zhong, Ming; Zhang, Wei

    2011-01-01

    OBJECTIVE Tribbles 3 (TRB3) is associated with insulin resistance, an important trigger in the development of diabetic cardiomyopathy (DCM). We sought to determine whether TRB3 plays a major role in modulating DCM and the mechanisms involved. RESEARCH DESIGN AND METHODS The type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin. We evaluated the characteristics of type 2 DCM by serial echocardiography and metabolite tests, Western blot analysis for TRB3 expression, and histopathologic analyses of cardiomyocyte density, lipids accumulation, cardiac inflammation, and fibrosis area. We then used gene silencing to investigate the role of TRB3 in the pathophysiologic features of DCM. RESULTS Rats with DCM showed severe insulin resistance, left ventricular dysfunction, aberrant lipids deposition, cardiac inflammation, fibrosis, and TRB3 overexpression. We found that the silencing of TRB3 ameliorated metabolic disturbance and insulin resistance; myocardial hypertrophy, lipids accumulation, inflammation, fibrosis, and elevated collagen I-to-III content ratio in DCM rats were significantly decreased. These anatomic findings were accompanied by significant improvements in cardiac function. Furthermore, with TRB3 gene silencing, the inhibited phosphorylation of Akt was restored and the increased phosphorylation of extracellular signal–regulated kinase 1/2 and Jun NH2-terminal kinase in DCM was significantly decreased. Conclusions. TRB3 gene silencing may exert a protective effect on DCM by improving selective insulin resistance, implicating its potential role for treatment of human DCM. PMID:21933987

  13. Compromised virus-induced gene silencing in RDR6-deficient plants.

    PubMed

    Vaistij, Fabián E; Jones, Louise

    2009-03-01

    RNA silencing in plants serves as a potent antiviral defense mechanism through the action of small interfering RNAs (siRNAs), which direct RNA degradation. siRNAs can be derived directly from the viral genome or via the action of host-encoded RNA-dependent RNA polymerases (RDRs). Plant genomes encode multiple RDRs, and it has been demonstrated that plants defective for RDR6 hyperaccumulate several classes of virus. In this study, we compared the effectiveness of virus-induced gene silencing (VIGS) and RNA-directed DNA methylation (RdDM) in wild-type and RDR6-deficient Nicotiana benthamiana plants. For the potexvirus Potato virus X (PVX) and the potyvirus Plum pox virus (PPV), the efficiency of both VIGS and RdDM were compromised in RDR6-defective plants despite accumulating high levels of viral siRNAs similar to infection of wild-type plants. The reduced efficiency of VIGS and RdDM was unrelated to the size class of siRNA produced and, at least for PVX, was not dependent on the presence of the virus-encoded silencing suppressor protein, 25K. We suggest that primary siRNAs produced from PVX and PPV in the absence of RDR6 may not be good effectors of silencing and that RDR6 is required to produce secondary siRNAs that drive a more effective antiviral response.

  14. Identification of BMP2 as an epigenetically silenced growth inhibitor in rhabdomyosarcoma.

    PubMed

    Wolf, Sebastian; Hagl, Beate; Kappler, Roland

    2014-05-01

    Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of infancy and although therapy has improved over the years, mortality is still fairly high. The establishment of new treatments has been hampered by the limited knowledge of the molecular mechanisms driving development of RMS. One characteristic of cancer cells is aberrant DNA methylation, which could lead to silencing of tumor suppressor genes. However, only a few epigenetically silenced genes have been described in RMS so far. We performed an expression profiling analysis of three RMS cell lines that were treated with the demethylating agent 5'-aza-2'-deoxycytidine (5-Aza‑dC) facilitating re-expression of epigenetically silenced genes. This treatment induced the gene BMP2 (bone morphogenetic protein 2) throughout all cell lines. Detailed methylation analysis of CpG sites in the BMP2 promoter region by bisulfite sequencing and methylation-specific PCR revealed that a high degree of DNA methylation is causatively associated with the suppression of BMP2 in RMS cells. Consequently, treatment of the RMS cell lines with 5-Aza-dC resulted in DNA demethylation of the BMP2 promoter, most prominently in alveolar RMS. Supplementation of recombinant human BMP2 (rhBMP2) led to a reduced viability of RMS cells. Altogether, these findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS, thereby providing a rationale for the development of a new treatment strategy for RMS.

  15. Clustering heterochromatin: Sir3 promotes telomere clustering independently of silencing in yeast

    PubMed Central

    Ruault, Myriam; De Meyer, Arnaud; Loïodice, Isabelle

    2011-01-01

    A general feature of the nucleus is the organization of repetitive deoxyribonucleic acid sequences in clusters concentrating silencing factors. In budding yeast, we investigated how telomeres cluster in perinuclear foci associated with the silencing complex Sir2–Sir3–Sir4 and found that Sir3 is limiting for telomere clustering. Sir3 overexpression triggers the grouping of telomeric foci into larger foci that relocalize to the nuclear interior and correlate with more stable silencing in subtelomeric regions. Furthermore, we show that Sir3′s ability to mediate telomere clustering can be separated from its role in silencing. Indeed, nonacetylable Sir3, which is unable to spread into subtelomeric regions, can mediate telomere clustering independently of Sir2–Sir4 as long as it is targeted to telomeres by the Rap1 protein. Thus, arrays of Sir3 binding sites at telomeres appeared as the sole requirement to promote trans-interactions between telomeres. We propose that similar mechanisms involving proteins able to oligomerize account for long-range interactions that impact genomic functions in many organisms. PMID:21300849

  16. Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA

    PubMed Central

    Rothé, Benjamin; Leal-Esteban, Lucia; Bernet, Florian; Urfer, Séverine; Doerr, Nicholas; Weimbs, Thomas; Iwaszkiewicz, Justyna

    2015-01-01

    Loss of the RNA-binding protein Bicaudal-C (Bicc1) provokes renal and pancreatic cysts as well as ectopic Wnt/β-catenin signaling during visceral left-right patterning. Renal cysts are linked to defective silencing of Bicc1 target mRNAs, including adenylate cyclase 6 (AC6). RNA binding of Bicc1 is mediated by N-terminal KH domains, whereas a C-terminal sterile alpha motif (SAM) self-polymerizes in vitro and localizes Bicc1 in cytoplasmic foci in vivo. To assess a role for multimerization in silencing, we conducted structure modeling and then mutated the SAM domain residues which in this model were predicted to polymerize Bicc1 in a left-handed helix. We show that a SAM-SAM interface concentrates Bicc1 in cytoplasmic clusters to specifically localize and silence bound mRNA. In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway. Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects. We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms. PMID:26217012

  17. Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers.