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Sample records for checkpoint kinase chk1

  1. A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1

    DEFF Research Database (Denmark)

    Blasius, Melanie; Forment, Josep V; Thakkar, Neha;

    2011-01-01

    BACKGROUND: The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1...

  2. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw;

    2005-01-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination......, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51...

  3. The regulatory beta-subunit of protein kinase CK2 accelerates the degradation of CDC25A phosphatase through the checkpoint kinase Chk1

    DEFF Research Database (Denmark)

    Kreutzer, Jan Nicolas; Guerra, Barbara

    2007-01-01

    Human CDC25 phosphatases play an important role in cell cycle regulation by removing inhibitory phosphate groups on cyclin-CDKs. Chk1 has been shown to phosphorylate CDC25 family members down-regulating their phosphatase activity through distinct mechanisms. The kinase activity of Chk1 is evident...... cell cycle progression is shown to enhance CDC25A degradation, and this occurs in a manner similar to that by which CDC25A is down-regulated upon activation of cellular checkpoint responses. By using RNA interference to specifically deplete cells of Chk1, we demonstrate that Chk1 mediates the down-regulation...... cell cycle regulation and indicate the mechanism by which CDC25A turnover might be regulated by Chk1 in the absence of DNA damage....

  4. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  5. A kinome screen identifies checkpoint kinase 1 (CHK1 as a sensitizer for RRM1-dependent gemcitabine efficacy.

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    Jun Zhou

    Full Text Available Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1 and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003 and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.

  6. Phosphorylation-dependent interactions between Crb2 and Chk1 are essential for DNA damage checkpoint.

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    Meng Qu

    2012-07-01

    Full Text Available In response to DNA damage, the eukaryotic genome surveillance system activates a checkpoint kinase cascade. In the fission yeast Schizosaccharomyces pombe, checkpoint protein Crb2 is essential for DNA damage-induced activation of downstream effector kinase Chk1. The mechanism by which Crb2 mediates Chk1 activation is unknown. Here, we show that Crb2 recruits Chk1 to double-strand breaks (DSBs through a direct physical interaction. A pair of conserved SQ/TQ motifs in Crb2, which are consensus phosphorylation sites of upstream kinase Rad3, is required for Chk1 recruitment and activation. Mutating both of these motifs renders Crb2 defective in activating Chk1. Tethering Crb2 and Chk1 together can rescue the SQ/TQ mutations, suggesting that the main function of these phosphorylation sites is promoting interactions between Crb2 and Chk1. A 19-amino-acid peptide containing these SQ/TQ motifs is sufficient for Chk1 binding in vitro when one of the motifs is phosphorylated. Remarkably, the same peptide, when tethered to DSBs by fusing with either recombination protein Rad22/Rad52 or multi-functional scaffolding protein Rad4/Cut5, can rescue the checkpoint defect of crb2Δ. The Rad22 fusion can even bypass the need for Rad9-Rad1-Hus1 (9-1-1 complex in checkpoint activation. These results suggest that the main role of Crb2 and 9-1-1 in DNA damage checkpoint signaling is recruiting Chk1 to sites of DNA lesions.

  7. NEK11: linking CHK1 and CDC25A in DNA damage checkpoint signaling

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Melixetian, Marina; Klein, Ditte Kjaersgaard;

    2010-01-01

    The DNA damage induced G(2)/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events...... is required for beta-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1...

  8. The Transcription Factor E4F1 Coordinates CHK1-Dependent Checkpoint and Mitochondrial Functions

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    Geneviève Rodier

    2015-04-01

    Full Text Available Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

  9. The transcription factor E4F1 coordinates CHK1-dependent checkpoint and mitochondrial functions.

    Science.gov (United States)

    Rodier, Geneviève; Kirsh, Olivier; Baraibar, Martín; Houlès, Thibault; Lacroix, Matthieu; Delpech, Hélène; Hatchi, Elodie; Arnould, Stéphanie; Severac, Dany; Dubois, Emeric; Caramel, Julie; Julien, Eric; Friguet, Bertrand; Le Cam, Laurent; Sardet, Claude

    2015-04-14

    Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

  10. Inhibition of Chk1 by CEP-3891 accelerates mitotic nuclear fragmentation in response to ionizing Radiation

    DEFF Research Database (Denmark)

    Syljuåsen, Randi G; Sørensen, Claus Storgaard; Nylandsted, Jesper;

    2004-01-01

    The human checkpoint kinase Chk1 has been suggested as a target for cancer treatment. Here, we show that a new inhibitor of Chk1 kinase, CEP-3891, efficiently abrogates both the ionizing radiation (IR)-induced S and G(2) checkpoints. When the checkpoints were abrogated by CEP-3891, the majority (64...

  11. ATR-Chk1-APC/C-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

    DEFF Research Database (Denmark)

    Yamada, M.; Watanabe, K.; Mistrik, M.;

    2013-01-01

    replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosomeCdh1 (APC/C) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C through...... degradation of Cdh1 upon replication block, thereby stabilizing APC/C substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4...

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

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    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

  13. Blocking CHK1 Expression Induces Apoptosis and Abrogates the G2 Checkpoint Mechanism

    Directory of Open Access Journals (Sweden)

    Yan Luo

    2001-01-01

    Full Text Available Checkpoint kinase 1 (Chki is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chki expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chki inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chki is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.

  14. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Syljuåsen, Randi G; Falck, Jacob;

    2003-01-01

    Chk1 kinase coordinates cell cycle progression and preserves genome integrity. Here, we show that chemical or genetic ablation of human Chk1 triggered supraphysiological accumulation of the S phase-promoting Cdc25A phosphatase, prevented ionizing radiation (IR)-induced degradation of Cdc25A...

  15. Polo-like kinase-1 controls proteasome-dependent degradation of claspin during checkpoint recovery

    NARCIS (Netherlands)

    Mamely, Ivan; van Vugt, Marcel A. T. M.; Smits, Veronique A. J.; Semple, Jennifer I.; Lemmens, Bennie; Perrakis, Anastassis; Medema, Rene H.; Freire, Raimundo

    2006-01-01

    DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin [1].

  16. Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint.

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    Junchao Duan

    Full Text Available Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH release were observed in human umbilical vein endothelial cells (HUVECs as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS generation caused oxidative damage followed by the production of malondialdehyde (MDA as well as the inhibition of superoxide dismutase (SOD and glutathione peroxidase (GSH-Px. Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.

  17. Targeting lung cancer through inhibition of checkpoint kinases

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    Randi Gussgard Syljuåsen

    2015-02-01

    Full Text Available 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.

  18. Identification of inhibitors of checkpoint kinase 1 through template screening.

    Science.gov (United States)

    Matthews, Thomas P; Klair, Suki; Burns, Samantha; Boxall, Kathy; Cherry, Michael; Fisher, Martin; Westwood, Isaac M; Walton, Michael I; McHardy, Tatiana; Cheung, Kwai-Ming J; Van Montfort, Rob; Williams, David; Aherne, G Wynne; Garrett, Michelle D; Reader, John; Collins, Ian

    2009-08-13

    Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells. PMID:19572549

  19. Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.

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    Carla Manuela Abreu

    2013-04-01

    Full Text Available The mediators of the DNA damage response (DDR are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR-specific protein kinases.

  20. Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

    Institute of Scientific and Technical Information of China (English)

    LI XiaoFang; WARD Tarsha; YAO XueBiao; WU JiaRui

    2009-01-01

    To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells,synchronized HeLa cells at G1,S and G2/M phases were treated with methyl methanesulfnate (MMS).The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases,while the S-phase cells were the most sensitive to MMS.Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS;whereas Chk1 was activated only in S phase under the drug treatment,indicating that Chk1 specifically participated in S-phase checkpoints.To analyze the role of Chk1 in S-phase checkpoints,we administered a specific Chk1 inhibitor,UCN-01,to the S-phase cells.The results showed that the S-phase cells treated with MMS+UCN-01 could enter aberrant mitosis without finishing DNA replication,indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint.In addition,MMS treatment alone inhibited the accumulation of cyclin B1,a key component of M-phase CDK-cyclin complex,in the S-phase cells,whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells.This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited.Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint.As p53 is not involved in this process,Chk1 may be a potential target for p53-deficient tumor therapy.

  1. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Institute of Scientific and Technical Information of China (English)

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    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-kB 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 Cdkl 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 Chkl 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.

  2. Structure-based design, discovery and development of checkpoint kinase inhibitors as potential anti-cancer therapies

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    Matthews, Thomas P; Jones, Alan M; Collins, Ian

    2014-01-01

    Introduction Checkpoint kinase inhibitors offer the promise of enhancing the effectiveness of widely prescribed cancer chemotherapies and radiotherapy by inhibiting the DNA damage response, as well as the potential for single agent efficacy. Areas covered This article surveys structural insights into the checkpoint kinases CHK1 and CHK2 that have been exploited to enhance the selectivity and potency of small molecule inhibitors. The use of mechanistic cellular assays to guide the optimisation of inhibitors is reviewed. The status of the current clinical candidates and emerging new clinical contexts for CHK1 and CHK2 inhibitors are discussed, including the prospects for single agent efficacy. Expert opinion Protein bound water molecules play key roles in structural features that can be targeted to gain high selectivity for either enzyme. The results of early phase clinical trials of checkpoint inhibitors have been mixed, but significant progress has been made in testing the combination of CHK1 inhibitors with genotoxic chemotherapy. Second generation CHK1 inhibitors are likely to benefit from increased selectivity and oral bioavailability. While the optimum therapeutic context for CHK2 inhibition remains unclear, the emergence of single agent preclinical efficacy for CHK1 inhibitors in specific tumour types exhibiting constitutive replication stress represents exciting progress in exploring the therapeutic potential of these agents. PMID:23594139

  3. Abrogation of Chk1-mediated S/G2 checkpoint by UCN-01 enhances ara-C-induced cytotoxicity in human colon cancer cells

    Institute of Scientific and Technical Information of China (English)

    Rong-guang SHAO; Chun-Xia CAO; Yves POMMIER

    2004-01-01

    AIM: To investigate whether 7-hydroxystaurosporine (UCN-01) affects cell cycle progression in arabinosylcytosine (ara-C) treated human colon carcinoma HT-29 cells. METHODS: Cytotoxicity, DNA synthesis, cell cycle distribution,protein level, and kinase activity were determined by clonogenic assay, flow cytometry, DNA synthesis assay,immunoblotting, and kinase assays, respectively. RESULTS: UCN-01 abrogated an S/G2-phase checkpoint in HT29 cells treated with ara-C. When UCN-01 was added after treatment with ara-C, the rate of recovery of DNA synthesis was enhanced and colony-forming ability diminished. Thus, premature recovery of DNA synthesis was associated with increased cytotoxicity. Measurements of cyclin A and B protein levels, Cdk2 and Cdc2 kinase activities, Cdc25C phosphorylation, and Chkl kinase activity were consistent with UCN-01-induced abrogation of the S/G2-phase checkpoint in ara-C treated cells. CONCLUSION: The abrogation of the S/G2 checkpoint may be due to inhibition of Chkl kinase by UCN-01. The enhanced cytotoxicity produced when UCN-01 was combined with ara-C suggested a rationale for the use of this drug combination for tumors that might be susceptible to cell cycle checkpoint abrogation.

  4. Checkpoint Kinases Regulate a Global Network of Transcription Factors in Response to DNA Damage

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    Eric J. Jaehnig

    2013-07-01

    Full Text Available DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1 in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.

  5. Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

    DEFF Research Database (Denmark)

    Gatei, Magtouf; Sloper, Katie; Sørensen, Claus Storgaard;

    2003-01-01

    . In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have......In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2....... Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced...

  6. Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

    NARCIS (Netherlands)

    de Vries, HI; Uyetake, L; Lemstra, W; Brunsting, JF; Su, TT; Kampinga, HH; Sibon, OCM

    2005-01-01

    Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-

  7. Kinase signaling in the spindle checkpoint.

    Science.gov (United States)

    Kang, Jungseog; Yu, Hongtao

    2009-06-01

    The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects. PMID:19228686

  8. Chk1 suppressed cell death

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    Meuth Mark

    2010-09-01

    Full Text Available Abstract The role of Chk1 in the cellular response to DNA replication stress is well established. However recent work indicates a novel role for Chk1 in the suppression of apoptosis following the disruption of DNA replication or DNA damage. This review will consider these findings in the context of known pathways of Chk1 signalling and potential applications of therapies that target Chk1.

  9. Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition

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    Guertin Amy D

    2012-11-01

    Full Text Available Abstract Background Inhibition of kinases involved in the DNA damage response sensitizes cells to genotoxic agents by abrogating checkpoint-induced cell cycle arrest. CHK1 and WEE1 act in a pathway upstream of CDK1 to inhibit cell cycle progression in response to damaged DNA. Therapeutic targeting of either CHK1 or WEE1, in combination with chemotherapy, is under clinical evaluation. These studies examine the overlap and potential for synergy when CHK1 and WEE1 are inhibited in cancer cell models. Methods Small molecules MK-8776 and MK-1775 were used to selectively and potently inhibit CHK1 and WEE1, respectively. Results In vitro, the combination of MK-8776 and MK-1775 induces up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone at a fixed concentration. This requires aberrant cyclin-dependent kinase activity but does not appear to be dependent on p53 status alone. Furthermore, DNA damage takes place primarily in S-phase cells, implying disrupted DNA replication. When dosed together, the combination of MK-8776 and MK-1775 induced more intense and more durable DNA damage as well as anti-tumor efficacy than either MK-8776 or MK-1775 dosed alone. DNA damage induced by the combination was detected in up to 40% of cells in a treated xenograft tumor model. Conclusions These results highlight the roles of WEE1 and CHK1 in maintaining genomic integrity. Importantly, the strong synergy observed upon inhibition of both kinases suggests unique yet complimentary anti-tumor effects of WEE1 and CHK1 inhibition. This demonstration of DNA double strand breaks in the absence of a DNA damaging chemotherapeutic provides preclinical rationale for combining WEE1 and CHK1 inhibitors as a cancer treatment regimen.

  10. The radioresistance to killing of A1-5 cells derives from activation of the Chk1 pathway

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    Hu, B.; Zhou, X. Y.; Wang, X.; Zeng, Z. C.; Iliakis, G.; Wang, Y.

    2001-01-01

    Checkpoints respond to DNA damage by arresting the cell cycle to provide time for facilitating repair. In mammalian cells, the G(2) checkpoint prevents the Cdc25C phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. Both Chk1 and Chk2, the checkpoint kinases, can phosphorylate Cdc25C and inactivate its in vitro phosphatase activity. Therefore, both Chk1 and Chk2 are thought to regulate the activation of the G(2) checkpoint. Here we report that A1-5, a transformed rat embryo fibroblast cell line, shows much more radioresistance associated with a much stronger G(2) arrest response when compared with its counterpart, B4, although A1-5 and B4 cells have a similar capacity for nonhomologous end-joining DNA repair. These phenotypes of A1-5 cells are accompanied by a higher Chk1 expression and a higher phosphorylation of Cdc2. On the other hand, Chk2 expression increases slightly following radiation; however, it has no difference between A1-5 and B4 cells. Caffeine or UCN-01 abolishes the extreme radioresistance with the strong G(2) arrest and at the same time reduces the phosphorylation of Cdc2 in A1-5 cells. In addition, Chk1 but not Chk2 antisense oligonucleotide sensitizes A1-5 cells to radiation-induced killing and reduces the G(2) arrest of the cells. Taken together these results suggest that the Chk1/Cdc25C/Cdc2 pathway is the major player for the radioresistance with G(2) arrest in A1-5 cells.

  11. Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

    Directory of Open Access Journals (Sweden)

    Neel M Fofaria

    Full Text Available In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR and checkpoint kinase 1 (Chk1. Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb. Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis.

  12. A subset of cancer cell lines is acutely sensitive to the Chk1 inhibitor MK-8776 as monotherapy due to CDK2 activation in S phase.

    Science.gov (United States)

    Sakurikar, Nandini; Thompson, Ruth; Montano, Ryan; Eastman, Alan

    2016-01-12

    DNA damage activates Checkpoint kinase 1 (Chk1) to halt cell cycle progression thereby preventing further DNA replication and mitosis until the damage has been repaired. Consequently, Chk1 inhibitors have emerged as promising anticancer therapeutics in combination with DNA damaging drugs, but their single agent activity also provides a novel approach that may be particularly effective in a subset of patients. From analysis of a large panel of cell lines, we demonstrate that 15% are very sensitive to the Chk1 inhibitor MK-8776. Upon inhibition of Chk1, sensitive cells rapidly accumulate DNA double-strand breaks in S phase in a CDK2- and cyclin A-dependent manner. In contrast, resistant cells can continue to grow for at least 7 days despite continued inhibition of Chk1. Resistance can be circumvented by inhibiting Wee1 kinase and thereby directly activating CDK2. Hence, sensitivity to Chk1 inhibition is regulated upstream of CDK2 and correlates with accumulation of CDC25A. We conclude that cells poorly tolerate CDK2 activity in S phase and that a major function of Chk1 is to ensure it remains inactive. Indeed, inhibitors of CDK1 and CDK2 arrest cells in G1 or G2, respectively, but do not prevent progression through S phase demonstrating that neither kinase is required for S phase progression. Inappropriate activation of CDK2 in S phase underlies the sensitivity of a subset of cell lines to Chk1 inhibitors, and this may provide a novel therapeutic opportunity for appropriately stratified patients. PMID:26595527

  13. In Silico Exploration of 1,7-Diazacarbazole Analogs as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Xiaodong Gao

    2016-05-01

    Full Text Available Checkpoint kinase 1 (Chk1 is an important serine/threonine kinase with a self-protection function. The combination of Chk1 inhibitors and anti-cancer drugs can enhance the selectivity of tumor therapy. In this work, a set of 1,7-diazacarbazole analogs were identified as potent Chk1 inhibitors through a series of computer-aided drug design processes, including three-dimensional quantitative structure–activity relationship (3D-QSAR modeling, molecular docking, and molecular dynamics simulations. The optimal QSAR models showed significant cross-validated correlation q2 values (0.531, 0.726, fitted correlation r2 coefficients (higher than 0.90, and standard error of prediction (less than 0.250. These results suggested that the developed models possess good predictive ability. Moreover, molecular docking and molecular dynamics simulations were applied to highlight the important interactions between the ligand and the Chk1 receptor protein. This study shows that hydrogen bonding and electrostatic forces are key interactions that confer bioactivity.

  14. Discovery of checkpoint kinase inhibitor (S)-5-(3-fluorophenyl)-N-(piperidin-3-yl)-3-ureidothiophene-2-carboxamide (AZD7762) by structure-based design and optimization of thiophenecarboxamide ureas.

    Science.gov (United States)

    Oza, Vibha; Ashwell, Susan; Almeida, Lynsie; Brassil, Patrick; Breed, Jason; Deng, Chun; Gero, Thomas; Grondine, Michael; Horn, Candice; Ioannidis, Stephanos; Liu, Dongfang; Lyne, Paul; Newcombe, Nicholas; Pass, Martin; Read, Jon; Ready, Shannon; Rowsell, Siân; Su, Mei; Toader, Dorin; Vasbinder, Melissa; Yu, Dingwei; Yu, Yan; Xue, Yafeng; Zabludoff, Sonya; Janetka, James

    2012-06-14

    Checkpoint kinases CHK1 and CHK2 are activated in response to DNA damage that results in cell cycle arrest, allowing sufficient time for DNA repair. Agents that lead to abrogation of such checkpoints have potential to increase the efficacy of such compounds as chemo- and radiotherapies. Thiophenecarboxamide ureas (TCUs) were identified as inhibitors of CHK1 by high throughput screening. A structure-based approach is described using crystal structures of JNK1 and CHK1 in complex with 1 and 2 and of the CHK1-3b complex. The ribose binding pocket of CHK1 was targeted to generate inhibitors with excellent cellular potency and selectivity over CDK1and IKKβ, key features lacking from the initial compounds. Optimization of 3b resulted in the identification of a regioisomeric 3-TCU lead 12a. Optimization of 12a led to the discovery of the clinical candidate 4 (AZD7762), which strongly potentiates the efficacy of a variety of DNA-damaging agents in preclinical models. PMID:22551018

  15. Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

    Science.gov (United States)

    Sproul, Christopher D; Rao, Shangbang; Ibrahim, Joseph G; Kaufmann, William K; Cordeiro-Stone, Marila

    2013-11-15

    The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.

  16. Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

    International Nuclear Information System (INIS)

    Research highlights: → The most important therapeutic tool in brain metastasis is radiation therapy. → Radiosensitivity of cancer cells was enhanced with treatment of Chk1 inhibitor. → Depletion of Chk1 in cancer cells showed an enhancement of sensitivity to radiation. → Chk1 can be a good target for enhancement of radiosensitivity. -- Abstract: The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.

  17. Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Heekyoung [Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Cancer Stem Cell Research Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Yoon, Su Jin [Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Jin, Juyoun [Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Cancer Stem Cell Research Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Choi, Seung Ho [Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); Seol, Ho Jun; Lee, Jung-Il [Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Gangnam-Gu, Seoul 135-710 (Korea, Republic of); and others

    2011-03-04

    Research highlights: {yields} The most important therapeutic tool in brain metastasis is radiation therapy. {yields} Radiosensitivity of cancer cells was enhanced with treatment of Chk1 inhibitor. {yields} Depletion of Chk1 in cancer cells showed an enhancement of sensitivity to radiation. {yields} Chk1 can be a good target for enhancement of radiosensitivity. -- Abstract: The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.

  18. Suppressed miR-424 expression via upregulation of target gene Chk1 contributes to the progression of cervical cancer.

    Science.gov (United States)

    Xu, J; Li, Y; Wang, F; Wang, X; Cheng, B; Ye, F; Xie, X; Zhou, C; Lu, W

    2013-02-21

    MicroRNAs (miRNAs) act as important gene regulators in human genomes and their aberrant expression links to many malignancies. We previously identified a different characteristic miRNA expression profile in cervical cancer from that in cervical normal tissues, including the downregulated miR-424. However, the role and mechanism of miR-424 in cervical cancer still remain unknown. Here, we focused on identifying the tumor-suppressive function and clinical significance of miR-424 and exploring the mechanistic relevance by characterizing its target. We showed a significantly decreased expression of miR-424 in 147 cervical cancer tissues versus 74 cervical normal tissues by performing quantitative RT-PCR. In 147 cervical cancer tissue samples, low-level expression of miR-424 was positively correlated with poor tumor differentiation, advanced clinical stage, lymph node metastasis and other poor prognostic clinicopathological parameters. Further in vitro observations showed that enforced expression of miR-424 inhibited cell growth by both enhancing apoptosis and blocking G1/S transition, and suppressed cell migration and invasion in two human cervical cancer cell lines, SiHa and CaSki, implying that miR-424 functions as a tumor suppressor in the progression of cervical cancer. Interestingly, overexpression of miR-424 inhibited the expression of protein checkpoint kinase 1 (Chk1) and phosphorylated Chk1 (p-Chk1) at residues Ser345 and decreased the activity of luciferase-reporter containing the 3'-untranslated region (UTR) of Chk1 with predicted miR-424-binding site. Moreover, miR-424 expression levels were inversely correlated with Chk1 and p-Chk1 protein levels in both cervical cancer and normal tissues. Furthermore, RNAi-mediated knockdown of Chk1 decreased matrix metalloproteinase 9 expression and phenocopied the tumor suppressive effects of miR-424 in cell models. Taken together, our results identify a crucial tumor suppressive role of miR-424 in the progression of

  19. The nucleolus stress response is coupled to an ATR-Chk1-mediated G2 arrest.

    Science.gov (United States)

    Ma, Hanhui; Pederson, Thoru

    2013-05-01

    We report experiments on the connection between nucleolar stress and cell cycle progression, using HeLa cells engineered with the fluorescent ubiquitinylation-based cell cycle indicator. Nucleolar stress elicited by brief exposure of cells to a low concentration of actinomycin D that selectively inhibits rRNA synthesis had no effect on traverse of G1 or S, but stalled cells in very late interphase. Additional experiments revealed that a switch occurs during a specific temporal window during nucleolar stress and that the subsequent cell cycle arrest is not triggered simply by the stress-induced decline in the synthesis of rRNA or by a ribosome starvation phenomenon. Further experiments revealed that this nucleolus stress-induced cell cycle arrest involves the action of a G2 checkpoint mediated by the ataxia telangiectasia and Rad3-related protein (ATR)-checkpoint kinase 1 (Chk1) pathway. Based on analysis of the cell cycle stages at which this nucleolar stress effect is put into action, to become manifest later, our results demonstrate a feedforward mechanism that leads to G2 arrest and identify ATR and Chk1 as molecular agents of the requisite checkpoint.

  20. Protein kinase C controls activation of the DNA integrity checkpoint

    Science.gov (United States)

    Soriano-Carot, María; Quilis, Inma; Bañó, M. Carmen; Igual, J. Carlos

    2014-01-01

    The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans. PMID:24792164

  1. Nuclear Chk1 prevents premature mitotic entry.

    Science.gov (United States)

    Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

    2011-07-01

    Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry.

  2. Nuclear Chk1 prevents premature mitotic entry.

    Science.gov (United States)

    Matsuyama, Makoto; Goto, Hidemasa; Kasahara, Kousuke; Kawakami, Yoshitaka; Nakanishi, Makoto; Kiyono, Tohru; Goshima, Naoki; Inagaki, Masaki

    2011-07-01

    Chk1 inhibits the premature activation of the cyclin-B1-Cdk1. However, it remains controversial whether Chk1 inhibits Cdk1 in the centrosome or in the nucleus before the G2-M transition. In this study, we examined the specificity of the mouse monoclonal anti-Chk1 antibody DCS-310, with which the centrosome was stained. Conditional Chk1 knockout in mouse embryonic fibroblasts reduced nuclear but not centrosomal staining with DCS-310. In Chk1(+/myc) human colon adenocarcinoma (DLD-1) cells, Chk1 was detected in the nucleus but not in the centrosome using an anti-Myc antibody. Through the combination of protein array and RNAi technologies, we identified Ccdc-151 as a protein that crossreacted with DCS-310 on the centrosome. Mitotic entry was delayed by expression of the Chk1 mutant that localized in the nucleus, although forced immobilization of Chk1 to the centrosome had little impact on the timing of mitotic entry. These results suggest that nuclear but not centrosomal Chk1 contributes to correct timing of mitotic entry. PMID:21628425

  3. Tumour growth environment modulates Chk1 signalling pathways and Chk1 inhibitor sensitivity

    Science.gov (United States)

    Massey, Andrew J.

    2016-01-01

    Clinical development of Chk1 inhibitors is currently focussed on evaluating activity as monotherapy and as potentiators of chemotherapy. To aid translation of pre-clinical studies, we sought to understand the effects of the tumour growth environment on Chk1 signalling and sensitivity to small molecule Chk1 inhibition. Spheroid culture altered Chk1 signalling to a more xenograft like state but decreased sensitivity to Chk1 inhibition. Growth in low serum did not alter DDR signalling but increased the sensitivity of A2058 and U2OS tumour cells to Chk1 inhibition. An analysis of the expression levels of replication associated proteins identified a correlation between Cdc6 and pChk1 (S296) as well as total Chk1 in xenograft derived samples and between Cdc6 and total Chk1 in anchorage-dependent growth derived protein samples. No apparent correlation between Chk1 or Cdc6 expression and sensitivity to Chk1 inhibition in vitro was observed. A database analysis revealed upregulation of CDC6 mRNA expression in tumour compared to normal tissue and a correlation between CDC6 and CHEK1 mRNA expression in human cancers. We suggest that Cdc6 overexpression in human tumours requires a concomitant increase in Chk1 to counterbalance the deleterious effects of origin hyperactivation-induced DNA damage. PMID:27775084

  4. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization

    DEFF Research Database (Denmark)

    Reinhardt, H Christian; Hasskamp, Pia; Schmedding, Ingolf;

    2010-01-01

    Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1...... expression as part of the DNA damage response in cancer cells....

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

    Science.gov (United States)

    Diril, M Kasim; Bisteau, Xavier; Kitagawa, Mayumi; Caldez, Matias J; Wee, Sheena; Gunaratne, Jayantha; Lee, Sang Hyun; Kaldis, Philipp

    2016-09-01

    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

  6. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    Science.gov (United States)

    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. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

    DEFF Research Database (Denmark)

    Syljuåsen, Randi G; Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg;

    2005-01-01

    -nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by...... increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose...... that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in...

  8. Suppression of allelic recombination and aneuploidy by cohesin is independent of Chk1 in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shay Covo

    Full Text Available Sister chromatid cohesion (SCC, which is established during DNA replication, ensures genome stability. Establishment of SCC is inhibited in G2. However, this inhibition is relived and SCC is established as a response to DNA damage, a process known as Damage Induced Cohesion (DIC. In yeast, Chk1, which is a kinase that functions in DNA damage signal transduction, is considered an activator of SCC through DIC. Nonetheless, here we show that, unlike SCC mutations, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. We suggest that Chk1 has a redundant role in the control of DIC or that DIC is redundant for maintaining genome stability.

  9. Suppression of Allelic Recombination and Aneuploidy by Cohesin Is Independent of Chk1 in Saccharomyces cerevisiae

    Science.gov (United States)

    Gordenin, Dmitry A.; Resnick, Michael A.

    2014-01-01

    Sister chromatid cohesion (SCC), which is established during DNA replication, ensures genome stability. Establishment of SCC is inhibited in G2. However, this inhibition is relived and SCC is established as a response to DNA damage, a process known as Damage Induced Cohesion (DIC). In yeast, Chk1, which is a kinase that functions in DNA damage signal transduction, is considered an activator of SCC through DIC. Nonetheless, here we show that, unlike SCC mutations, loss of CHK1 did not increase spontaneous or damage-induced allelic recombination or aneuploidy. We suggest that Chk1 has a redundant role in the control of DIC or that DIC is redundant for maintaining genome stability. PMID:25551702

  10. Phosphorylation of pRB at Ser612 by Chk1/2 leads to a complex between pRB and E2F-1 after DNA damage.

    Science.gov (United States)

    Inoue, Yasumichi; Kitagawa, Masatoshi; Taya, Yoichi

    2007-04-18

    The retinoblastoma tumor suppressor protein (pRB) plays a critical role in the control of cell proliferation and in the DNA damage checkpoints. pRB inhibits cell cycle progression through interactions with the E2F family of transcription factors. Here, we report that DNA damage induced not only the dephosphorylation of pRB at Cdk phosphorylation sites and the binding of pRB to E2F-1, but also the phosphorylation of pRB at Ser612. Phosphorylation of pRB at Ser612 enhanced the formation of a complex between pRB and E2F-1. Substitution of Ser612 with Ala decreased pRB-E2F-1 binding and the transcriptional repression activity. Until now, Ser612 of pRB has been thought to be phosphorylated by Cdk2. However, the phosphorylation of pRB at Ser612 was conducted by Chk1/2 after DNA damage, and inhibition of ATM-Chk1/2 activity suppressed the phosphorylation of Ser612 and the binding of pRB to E2F-1. These results suggest that Ser612 is phosphorylated by Chk1/2 after DNA damage, leading to the formation of pRB-E2F-1. This is the first report that pRB is phosphorylated in vivo by a kinase other than Cdk.

  11. A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition

    Science.gov (United States)

    Gill, Martin R.; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A.; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A.

    2016-08-01

    Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)]2+ before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing.

  12. A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition.

    Science.gov (United States)

    Gill, Martin R; Harun, Siti Norain; Halder, Swagata; Boghozian, Ramon A; Ramadan, Kristijan; Ahmad, Haslina; Vallis, Katherine A

    2016-01-01

    Ruthenium(II) polypyridyl complexes can intercalate DNA with high affinity and prevent cell proliferation; however, the direct impact of ruthenium-based intercalation on cellular DNA replication remains unknown. Here we show the multi-intercalator [Ru(dppz)2(PIP)](2+) (dppz = dipyridophenazine, PIP = 2-(phenyl)imidazo[4,5-f][1,10]phenanthroline) immediately stalls replication fork progression in HeLa human cervical cancer cells. In response to this replication blockade, the DNA damage response (DDR) cell signalling network is activated, with checkpoint kinase 1 (Chk1) activation indicating prolonged replication-associated DNA damage, and cell proliferation is inhibited by G1-S cell-cycle arrest. Co-incubation with a Chk1 inhibitor achieves synergistic apoptosis in cancer cells, with a significant increase in phospho(Ser139) histone H2AX (γ-H2AX) levels and foci indicating increased conversion of stalled replication forks to double-strand breaks (DSBs). Normal human epithelial cells remain unaffected by this concurrent treatment. Furthermore, pre-treatment of HeLa cells with [Ru(dppz)2(PIP)](2+) before external beam ionising radiation results in a supra-additive decrease in cell survival accompanied by increased γ-H2AX expression, indicating the compound functions as a radiosensitizer. Together, these results indicate ruthenium-based intercalation can block replication fork progression and demonstrate how these DNA-binding agents may be combined with DDR inhibitors or ionising radiation to achieve more efficient cancer cell killing. PMID:27558808

  13. Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM–Chk1/2–Cdc25C pathway

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Yong-Cheng [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Su, Nan [Department of Quality Detection and Management, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan (China); Shi, Xiao-Jing; Zhao, Wen; Ke, Yu [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China); Zi, Xiaolin [Department of Urology, University of California, Irvine, Orange, CA (United States); Department of Pharmacology, University of California, Irvine, Orange, CA (United States); Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA (United States); Zhao, Ning-Min; Qin, Yu-Hua; Zhao, Hong-Wei [Clinical Pharmacology Laboratory, Henan Province People' s Hospital, No. 7, Wei Wu Road, Zhengzhou, Henan (China); Liu, Hong-Min, E-mail: liuhm@zzu.edu.cn [School of Pharmaceutical Sciences, Zhengzhou University, No. 100, Science Avenue, Zhengzhou, Henan (China)

    2015-01-15

    Jaridonin, a novel diterpenoid from Isodon rubescens, has been shown previously to inhibit proliferation of esophageal squamous cancer cells (ESCC) through G2/M phase cell cycle arrest. However, the involved mechanism is not fully understood. In this study, we found that the cell cycle arrest by Jaridonin was associated with the increased expression of phosphorylation of ATM at Ser1981 and Cdc2 at Tyr15. Jaridonin also resulted in enhanced phosphorylation of Cdc25C via the activation of checkpoint kinases Chk1 and Chk2, as well as in increased phospho-H2A.X (Ser139), which is known to be phosphorylated by ATM in response to DNA damage. Furthermore, Jaridonin-mediated alterations in cell cycle arrest were significantly attenuated in the presence of NAC, implicating the involvement of ROS in Jaridonin's effects. On the other hand, addition of ATM inhibitors reversed Jaridonin-related activation of ATM and Chk1/2 as well as phosphorylation of Cdc25C, Cdc2 and H2A.X and G2/M phase arrest. In conclusion, these findings identified that Jaridonin-induced cell cycle arrest in human esophageal cancer cells is associated with ROS-mediated activation of ATM–Chk1/2–Cdc25C pathway. - Highlights: • Jaridonin induced G2/M phase arrest through induction of redox imbalance. • Jaridonin increased the level of ROS through depleting glutathione in cell. • ATM–Chk1/2–Cdc25C were involved in Jaridonin-induced cell cycle arrest. • Jaridonin selectively inhibited cancer cell viability and cell cycle progression.

  14. Role of swi7H4 mutant allele of DNA polymerase α in the DNA damage checkpoint response.

    Directory of Open Access Journals (Sweden)

    Saman Khan

    Full Text Available Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.

  15. Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.

    Science.gov (United States)

    Stojic, Lovorka; Mojas, Nina; Cejka, Petr; Di Pietro, Massimiliano; Ferrari, Stefano; Marra, Giancarlo; Jiricny, Josef

    2004-06-01

    S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage. PMID:15175264

  16. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition.

    Science.gov (United States)

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W; Ideker, Trey

    2015-11-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  17. Hyperactive Cdc2 kinase interferes with the response to broken replication forks by trapping S.pombe Crb2 in its mitotic T215 phosphorylated state.

    Science.gov (United States)

    Mahyous Saeyd, Salah Adam; Ewert-Krzemieniewska, Katarzyna; Liu, Boyin; Caspari, Thomas

    2014-07-01

    Although it is well established that Cdc2 kinase phosphorylates the DNA damage checkpoint protein Crb2(53BP1) in mitosis, the full impact of this modification is still unclear. The Tudor-BRCT domain protein Crb2 binds to modified histones at DNA lesions to mediate the activation of Chk1 by Rad3ATR kinase. We demonstrate here that fission yeast cells harbouring a hyperactive Cdc2CDK1 mutation (cdc2.1w) are specifically sensitive to the topoisomerase 1 inhibitor camptothecin (CPT) which breaks DNA replication forks. Unlike wild-type cells, which delay only briefly in CPT medium by activating Chk1 kinase, cdc2.1w cells bypass Chk1 to enter an extended cell-cycle arrest which depends on Cds1 kinase. Intriguingly, the ability to bypass Chk1 requires the mitotic Cdc2 phosphorylation site Crb2-T215. This implies that the presence of the mitotic phosphorylation at Crb2-T215 channels Rad3 activity towards Cds1 instead of Chk1 when forks break in S phase. We also provide evidence that hyperactive Cdc2.1w locks cells in a G1-like DNA repair mode which favours non-homologous end joining over interchromosomal recombination. Taken together, our data support a model such that elevated Cdc2 activity delays the transition of Crb2 from its G1 to its G2 mode by blocking Srs2 DNA helicase and Casein Kinase 1 (Hhp1).

  18. A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation.

    Science.gov (United States)

    Gemble, Simon; Buhagiar-Labarchède, Géraldine; Onclercq-Delic, Rosine; Biard, Denis; Lambert, Sarah; Amor-Guéret, Mounira

    2016-08-15

    Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.

  19. STAT3 interrupts ATR-Chk1 signaling to allow oncovirus-mediated cell proliferation.

    Science.gov (United States)

    Koganti, Siva; Hui-Yuen, Joyce; McAllister, Shane; Gardner, Benjamin; Grasser, Friedrich; Palendira, Umaimainthan; Tangye, Stuart G; Freeman, Alexandra F; Bhaduri-McIntosh, Sumita

    2014-04-01

    DNA damage response (DDR) is a signaling network that senses DNA damage and activates response pathways to coordinate cell-cycle progression and DNA repair. Thus, DDR is critical for maintenance of genome stability, and presents a powerful defense against tumorigenesis. Therefore, to drive cell-proliferation and transformation, viral and cellular oncogenes need to circumvent DDR-induced cell-cycle checkpoints. Unlike in hereditary cancers, mechanisms that attenuate DDR and disrupt cell-cycle checkpoints in sporadic cancers are not well understood. Using Epstein-Barr virus (EBV) as a source of oncogenes, we have previously shown that EBV-driven cell proliferation requires the cellular transcription factor STAT3. EBV infection is rapidly followed by activation and increased expression of STAT3, which mediates relaxation of the intra-S phase cell-cycle checkpoint; this facilitates viral oncogene-driven cell proliferation. We now show that replication stress-associated DNA damage, which results from EBV infection, is detected by DDR. However, signaling downstream of ATR is impaired by STAT3, leading to relaxation of the intra-S phase checkpoint. We find that STAT3 interrupts ATR-to-Chk1 signaling by promoting loss of Claspin, a protein that assists ATR to phosphorylate Chk1. This loss of Claspin which ultimately facilitates cell proliferation is mediated by caspase 7, a protein that typically promotes cell death. Our findings demonstrate how STAT3, which is constitutively active in many human cancers, suppresses DDR, fundamental to tumorigenesis. This newly recognized role for STAT3 in attenuation of DDR, discovered in the context of EBV infection, is of broad interest as the biology of cell proliferation is central to both health and disease.

  20. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1.

    Directory of Open Access Journals (Sweden)

    Claudia Breit

    Full Text Available The spindle assembly checkpoint (SAC monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.

  1. HPV-positive HNSCC cell lines but not primary human fibroblasts are radiosensitized by the inhibition of Chk1

    International Nuclear Information System (INIS)

    Purpose: Despite the comparably high cure rates observed for HPV-positive HNSCC, there is still a great need for specific tumor radiosensitization due to the often severe side effects resulting from intense radiochemotherapy. We recently demonstrated that HPV-positive HNSCC cell lines are characterized by a defect in DNA double-strand break repair associated with a pronounced G2-arrest. Here we tested whether abrogation of this radiation-induced G2-arrest by the inhibition of Chk1 results in specific radiosensitization of HPV-positive HNSCC cells. Materials and methods: Experiments were performed with five HPV and p16-positive (93-VU-147T, UM-SCC-47, UT-SCC-45, UD-SCC-2, UPCI-SCC-154) and two HPV and p16-negative HNSCC cell lines, as well as two normal human fibroblast strains. Chk1 was inhibited by the selective inhibitor PF-00477736. Cell cycle distribution was determined by flow cytometry, Chk1-activity via Western blot and cell survival by colony formation assay. Results: With the exception of UPCI-SCC-154, the inhibition of Chk1 was found to abolish the pronounced radiation-induced G2-arrest in all HPV-positive cells utilized. All tumor cell lines that demonstrated the abrogation of G2-arrest also demonstrated radiosensitization. Notably, in G1-arrest-proficient normal human fibroblasts no radiosensitization was induced. Conclusion: Abrogation of the G2 checkpoint through the inhibition of Chk1 may be used to selectively increase the cellular radiosensitivity of HPV-positive HNSCC without affecting the surrounding normal tissue

  2. In vivo role of checkpoint kinase 2 in signaling telomere dysfunction

    OpenAIRE

    García-Beccaria, María; Martínez, Paula; Flores, Juana M.; Blasco, Maria A.

    2014-01-01

    Checkpoint kinase 2 (CHK2) is a downstream effector of the DNA damage response (DDR). Dysfunctional telomeres, either owing to critical shortening or disruption of the shelterin complex, activate a DDR, which eventually results in cell cycle arrest, senescence and/or apoptosis. Successive generations of telomerase-deficient (Terc) mice show accelerated aging and shorter lifespan due to tissue atrophy and impaired organ regeneration associated to progressive telomere shortening. In contrast, m...

  3. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D

    2016-01-29

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  4. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-09-26

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

  6. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia;

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a...

  7. The Aurora B kinase in chromosome biorientation and spindle checkpoint signalling

    Directory of Open Access Journals (Sweden)

    Veronica eKrenn

    2015-10-01

    Full Text Available 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.

  8. Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106

    Energy Technology Data Exchange (ETDEWEB)

    Borst, Gerben R., E-mail: g.borst@nki.nl [The Institute of Cancer Research, London (United Kingdom); Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam (Netherlands); McLaughlin, Martin; Kyula, Joan N.; Neijenhuis, Sari; Khan, Aadil; Good, James; Zaidi, Shane [The Institute of Cancer Research, London (United Kingdom); Powell, Ned G. [HPV Research Group, School of Medicine, Cardiff University, Cardiff (United Kingdom); Meier, Pascal; Collins, Ian; Garrett, Michelle D. [The Institute of Cancer Research, London (United Kingdom); Verheij, Marcel [Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Amsterdam (Netherlands); Harrington, Kevin J. [The Institute of Cancer Research, London (United Kingdom)

    2013-03-15

    Purpose: To explore the activity of a potent Chk1 inhibitor (SAR-020106) in combination with radiation. Methods and Materials: Colony and mechanistic in vitro assays and a xenograft in vivo model. Results: SAR-020106 suppressed-radiation-induced G{sub 2}/M arrest and reduced clonogenic survival only in p53-deficient tumor cells. SAR-020106 promoted mitotic entry following irradiation in all cell lines, but p53-deficient cells were likely to undergo apoptosis or become aneuploid, while p53 wild-type cells underwent a postmitotic G{sub 1} arrest followed by subsequent normal cell cycle re-entry. Following combined treatment with SAR-020106 and radiation, homologous-recombination-mediated DNA damage repair was inhibited in all cell lines. A significant increase in the number of pan-γH2AX-staining apoptotic cells was observed only in p53-deficient cell lines. Efficacy was confirmed in vivo in a clinically relevant human head-and-neck cell carcinoma xenograft model. Conclusion: The Chk1 inhibitor SAR-020106 is a potent radiosensitizer in tumor cell lines defective in p53 signaling.

  9. Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression.

    Science.gov (United States)

    Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian; Kohlwein, Sepp D

    2015-03-10

    Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle-dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle-dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A(Cdc55)) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle. PMID:25713391

  10. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production. PMID:27503884

  11. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  12. Targeting radioresistant breast cancer cells by single agent CHK1 inhibitor via enhancing replication stress

    Science.gov (United States)

    Du, Zhanwen; Gao, Jinnan; Yang, Shuming; Gorityala, Shashank; Xiong, Xiahui; Deng, Ou; Ma, Zhefu; Yan, Chunhong; Susana, Gonzalo; Xu, Yan; Zhang, Junran

    2016-01-01

    Radiotherapy (RT) remains a standard therapeutic modality for breast cancer patients. However, intrinsic or acquired resistance limits the efficacy of RT. Here, we demonstrate that CHK1 inhibitor AZD7762 alone significantly inhibited the growth of radioresistant breast cancer cells (RBCC). Given the critical role of ATR/CHK1 signaling in suppressing oncogene-induced replication stress (RS), we hypothesize that CHK1 inhibition leads to the specific killing for RBCC due to its abrogation in the suppression of RS induced by oncogenes. In agreement, the expression of oncogenes c-Myc/CDC25A/c-Src/H-ras/E2F1 and DNA damage response (DDR) proteins ATR/CHK1/BRCA1/CtIP were elevated in RBCC. AZD7762 exposure led to significantly higher levels of RS in RBCC, compared to the parental cells. The mechanisms by which CHK1 inhibition led to specific increase of RS in RBCC were related to the interruptions in the replication fork dynamics and the homologous recombination (HR). In summary, RBCC activate oncogenic pathways and thus depend upon mechanisms controlled by CHK1 signaling to maintain RS under control for survival. Our study provided the first example where upregulating RS by CHK1 inhibitor contributes to the specific killing of RBCC, and highlight the importance of the CHK1 as a potential target for treatment of radioresistant cancer cells. PMID:27167194

  13. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736.

    Science.gov (United States)

    Restelli, Valentina; Chilà, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-11-10

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL. PMID:26439697

  14. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    Science.gov (United States)

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)–dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  15. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

    Science.gov (United States)

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-11-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  16. Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

    Science.gov (United States)

    Man, Wing Yu; Mak, Joyce P Y; Poon, Randy Y C

    2014-01-01

    Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.

  17. Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

    Science.gov (United States)

    Man, Wing Yu; Mak, Joyce P Y; Poon, Randy Y C

    2014-01-01

    Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint. PMID:24238094

  18. CHK1 Inhibition Radiosensitizes Head and Neck Cancers to Paclitaxel-Based Chemoradiotherapy.

    Science.gov (United States)

    Barker, Holly E; Patel, Radhika; McLaughlin, Martin; Schick, Ulrike; Zaidi, Shane; Nutting, Christopher M; Newbold, Katie L; Bhide, Shreerang; Harrington, Kevin J

    2016-09-01

    Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer-related deaths, with increasingly more cases arising due to high-risk human papillomavirus (HPV) infection. Cisplatin-based chemoradiotherapy is a standard-of-care for locally advanced head and neck cancer but is frequently ineffective. Research into enhancing radiation responses as a means of improving treatment outcomes represents a high priority. Here, we evaluated a CHK1 inhibitor (CCT244747) as a radiosensitiser and investigated whether a mechanistically rational triple combination of radiation/paclitaxel/CHK1 inhibitor delivered according to an optimized schedule would provide added benefit. CCT244747 abrogated radiation-induced G2 arrest in the p53-deficient HNSCC cell lines, HN4 and HN5, causing cells to enter mitosis with unrepaired DNA damage. The addition of paclitaxel further increased cell kill and significantly reduced tumor growth in an HN5 xenograft model. Importantly, a lower dose of paclitaxel could be used when CCT244747 was included, therefore potentially limiting toxicity. Triple therapy reduced the expression of several markers of radioresistance. Moreover, the more radioresistant HN5 cell line exhibited greater radiation-mediated CHK1 activation and was more sensitive to triple therapy than HN4 cells. We analyzed CHK1 expression in a panel of head and neck tumors and observed that primary tumors from HPV(+) patients, who went on to recur postradiotherapy, exhibited significantly stronger expression of total, and activated CHK1. CHK1 may serve as a biomarker for identifying tumors likely to recur and, therefore, patients who may benefit from concomitant treatment with a CHK1 inhibitor and paclitaxel during radiotherapy. Clinical translation of this strategy is under development. Mol Cancer Ther; 15(9); 2042-54. ©2016 AACR.

  19. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete;

    2013-01-01

    knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation......DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed....... Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA...

  20. Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase.

    Science.gov (United States)

    Colombo, Riccardo; Caldarelli, Marina; Mennecozzi, Milena; Giorgini, Maria Laura; Sola, Francesco; Cappella, Paolo; Perrera, Claudia; Depaolini, Stefania Re; Rusconi, Luisa; Cucchi, Ulisse; Avanzi, Nilla; Bertrand, Jay Aaron; Bossi, Roberto Tiberio; Pesenti, Enrico; Galvani, Arturo; Isacchi, Antonella; Colotta, Francesco; Donati, Daniele; Moll, Jürgen

    2010-12-15

    MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.

  1. Cdk2 is required for p53-independent G2/M checkpoint control.

    Directory of Open Access Journals (Sweden)

    Jon H Chung

    2010-02-01

    Full Text Available The activation of phase-specific cyclin-dependent kinases (Cdks is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G(2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G(2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G(2/M checkpoint activation.

  2. Checkpoint kinase inhibitors: SAR and radioprotective properties of a series of 2-arylbenzimidazoles.

    Science.gov (United States)

    Arienti, Kristen L; Brunmark, Anders; Axe, Frank U; McClure, Kelly; Lee, Alice; Blevitt, Jon; Neff, Danielle K; Huang, Liming; Crawford, Shelby; Pandit, Chennagiri R; Karlsson, Lars; Breitenbucher, J Guy

    2005-03-24

    The discovery of a series of novel, potent, and highly selective inhibitors of the DNA damage control kinase chk2 is disclosed. Here we report the first SAR study around inhibitors of this kinase. High-throughput screening of purified human chk2 led to the identification of a novel series of 2-arylbenzimidazole inhibitors of the kinase. Optimization was facilitated using homology models of chk2 and docking of inhibitors, leading to the highly potent 2-arylbenzimidazole 2h (IC(50) 15 nM). Compound 2h is an ATP-competitive inhibitor of chk2 that dose dependently protects human CD4(+) and CD8(+) T-cells from apoptosis due to ionizing radiation. This work suggests that a selective small molecule inhibitor of chk2 could be a useful adjuvant to radiotherapy, increasing the therapeutic window of such treatment. PMID:15771432

  3. [Preparation and identification of monoclonal antibodies against chicken cell cycle checkpoint kinase 2 (cChk2)].

    Science.gov (United States)

    Gao, Junna; Lian, Xue; Sun, Haiwei; Lu, Zejun; Zhang, Xunhai; Jung, Yong-Sam; Chen, Hongjun; Qian, Yingjuan

    2016-09-01

    Objective To prepare monoclonal antibodies (mAbs) against chicken cell cycle checkpoint kinase 2 (cChk2). Methods The cChk2 gene was amplified by reverse transcription PCR (RT-PCR) and subcloned into the prokaryotic expression vector pGEX-4T-3. After induced by IPTG, cChk2 was expressed in BL21 (DE3) E.coli cells and analyzed by SDS-PAGE to determine its soluability. BALB/c mice were immunized with cChk2 protein peritoneally. Indirect immunofluorescence assay (IFA) and Western blotting were used to detect anti-serum; if the detection result was positive, IFA and limited dilution was performed to screen hybridoma clones that produced antibodies against cChk2. Results cChk2 was mainly expressed in inclusion bodies. The anti-sera were able to recognize Chk2. Nine positive hybridoma clones were obtained and identified as 1F4, 2D9, 2G1, 3D9, 3E3, 4B5, 4E2, 5C9 and 5F7. Conclusion The study has prepared mAbs against cChk2 with a good specificity and a high titer. PMID:27609583

  4. Centromere-tethered Mps1 pombe homolog (Mph1) kinase is a sufficient marker for recruitment of the spindle checkpoint protein Bub1, but not Mad1.

    Science.gov (United States)

    Ito, Daisuke; Saito, Yu; Matsumoto, Tomohiro

    2012-01-01

    The spindle checkpoint delays the onset of anaphase until all of the chromosomes properly achieve bipolar attachment to the spindle. It has been shown that unattached kinetochores are the site that emits a signal for activation of the checkpoint. Although the components of the checkpoint such as Bub1, Mad1 and Mad2 selectively accumulate at unattached kinetochores, the answer to how they recognize unattached kinetochores has remained elusive. Mps1 pombe homolog (Mph1) kinase has been shown to function upstream of most of the components of the checkpoint and thus it is thought to recognize unattached kinetochores by itself and recruit other components. In this study we have expressed a fusion protein of Mph1 and Ndc80 (a kinetochore protein of the outer plate) and shown that the fusion protein arrests cell cycle progression in a spindle-checkpoint\\x{2013}dependent manner in fission yeast. When expression of Mad2 is turned off, the cells grow normally with Mph1 constitutively localized at centromeres/kinetochores. Under this condition, Bub1 can be found with Mph1 throughout the cell cycle, indicating that localization of Mph1 at centromeres/kinetochores is sufficient to recruit Bub1. In contrast, Mad1 is found to transiently localize at kinetochores, which are presumably unattached to the spindle, but soon it dissociates from kinetochores. We propose that Mph1 is a sufficient marker for recruitment of Bub1. Mad1, in contrast, requires an additional condition/component for stable association with kinetochores. PMID:22184248

  5. MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis.

    Science.gov (United States)

    Tenorio-Gómez, María; de Sena-Tomás, Carmen; Pérez-Martín, Jose

    2015-01-01

    DNA damage response (DDR) leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-01

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

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

    International Nuclear Information System (INIS)

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

  8. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong-Min; Choi, Ji Ye [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Yi, Joo Mi [Research Center, Dongnam Institute of Radiological & Medical Sciences, Busan (Korea, Republic of); Chung, Jin Woong; Leem, Sun-Hee; Koh, Sang Seok [Department of Biological Science, Dong-A University, Busan (Korea, Republic of); Kang, Tae-Hong, E-mail: thkang@dau.ac.kr [Department of Biological Science, Dong-A University, Busan (Korea, Republic of)

    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.

  9. NDR1 modulates the UV-induced DNA-damage checkpoint and nucleotide excision repair

    International Nuclear Information System (INIS)

    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

  10. Clonning and expression analysis of Chk1 gene in Daphnia pulex%蚤状溞(Daphnia pulex)Chk1基因的克隆与表达分析

    Institute of Scientific and Technical Information of China (English)

    方增冰; 戴新宇; 邹秀; 郭晓鸽; 徐善良; 王丹丽; 赵云龙

    2015-01-01

    The full-length cDNA of a Chk1 (DpChk1) was cloned from cladoceran Daphnia pulex using rapid amplification of comple-mentary DNA ends ( RACE) method.The DpChk1 cDNA is 1767 bp in length;and it has a 1497-bp open reading frame that encodes a 498-amino-acid polypeptide containing three conserved Ser-Gln (SQ), and Thr-Gln (TQ) sequence.In addition, DpChk1 shared homology of 51%-55% with gene in Rhipicephalus pulchellus, Megachile rotundata, Metaseiulus occidentalis, Acyrthosiphon pisum and Drosophila melanogaster.Phylogenetic analysis revealed that DpChk 1 protein has a close genetic relationship with Phylum arthropo-da such as Acyrthosiphon pisum, Megachile rotundata, Drosophila melanogaster, Rhipicephalus pulchellus, Metaseiulus occidenat lis and so on.qPCR results (real-time quantitative PCR) showed that the DpChk1 expression was significantly higher (P<0.05) in ephippial female than in parthenogenetic female and was the lowest in the resting egg .Therefore, Chk1 was closely related to the reproduction conversion of Daphnia pulex.%用RACE技术从蚤状溞( Daphnia pulex)中克隆到Chk1基因cDNA全长为1767 bp,开放阅读框为1497 bp,编码了498个氨基酸,其结构中存在3个保守的Ser-Gln ( SQ)和Thr-Gln ( TQ)序列。同源性比对结果显示,蚤状溞Chk1基因与丽色扇头蜱、切叶蜂、转基因捕食螨、豌豆长管蚜和黑腹果蝇等的同源性均为51%~55%。进化分析发现,蚤状溞Chk1基因与豌豆长管蚜、切叶蜂、黑腹果蝇、丽色扇头蜱和转基因捕食螨等节肢动物亲缘关系最近。Real Time PCR实验结果表明,Chk1 mRNA在两性溞的表达量显著高于孤雌溞( P<0.05),且在休眠卵中表达量最低。推测Chk1基因可能在蚤状溞的生殖转化调控中发挥重要作用。

  11. MRN- and 9-1-1-Independent Activation of the ATR-Chk1 Pathway during the Induction of the Virulence Program in the Phytopathogen Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    María Tenorio-Gómez

    Full Text Available DNA damage response (DDR leads to DNA repair, and depending on the extent of the damage, to further events, including cell death. Evidence suggests that cell differentiation may also be a consequence of the DDR. During the formation of the infective hypha in the phytopathogenic fungus Ustilago maydis, two DDR kinases, Atr1 and Chk1, are required to induce a G2 cell cycle arrest, which in turn is essential to display the virulence program. However, the triggering factor of DDR in this process has remained elusive. In this report we provide data suggesting that no DNA damage is associated with the activation of the DDR during the formation of the infective filament in U. maydis. We have analyzed bulk DNA replication during the formation of the infective filament, and we found no signs of impaired DNA replication. Furthermore, using RPA-GFP fusion as a surrogate marker of the presence of DNA damage, we were unable to detect any sign of DNA damage at the cellular level. In addition, neither MRN nor 9-1-1 complexes, both instrumental to transmit the DNA damage signal, are required for the induction of the above mentioned cell cycle arrest, as well as for virulence. In contrast, we have found that the claspin-like protein Mrc1, which in other systems serves as scaffold for Atr1 and Chk1, was required for both processes. We discuss possible alternative ways to trigger the DDR, independent of DNA damage, in U. maydis during virulence program activation.

  12. Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability

    Directory of Open Access Journals (Sweden)

    Doerfler Lillian

    2011-10-01

    Full Text Available Abstract Background Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. Results We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Conclusions Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.

  13. In silico analysis of deleterious single nucleotide polymorphisms in human BUB1 mitotic checkpoint serine/threonine kinase B gene.

    Science.gov (United States)

    Akhoundi, Fatemeh; Parvaneh, Nikpour; Modjtaba, Emadi-Baygi

    2016-09-01

    One of the major challenges in the analysis of human genetic variation is to distinguish mutations that are functionally neutral from those that contribute to disease. BubR1 is a key protein mediating spindle-checkpoint activation that plays a role in the inhibition of the anaphase-promoting complex/cyclosome (APC/C), delaying the onset of anaphase and ensuring proper chromosome segregation. Owing to the importance of BUB1B gene in mitotic checkpoint a functional analysis using different in silico approaches was undertaken to explore the possible associations between genetic mutations and phenotypic variation. In this work we found that 3 nsSNPs I82N, P334L and R814H have a functional effect on protein function and stability. A literature search revealed that R814H was already implicated in human diseases. Additionally, 2 SNPs in the 5' UTR region was predicted to exhibit a pattern change in the internal ribosome entry site (IRES), and eight MicroRNA binding sites were found to be highly affected due to 3' UTR SNPs. These in silico predictions will provide useful information in selecting the target SNPs that are likely to have functional impact on the BUB1B gene. PMID:27331020

  14. Berberine, a genotoxic alkaloid, induces ATM-Chk1 mediated G2 arrest in prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang Yu; Liu Qiao; Liu Zhaojian; Li Boxuan; Sun Zhaoliang; Zhou Haibin; Zhang Xiyu; Gong Yaoqin [Ministry of Education Key Laboratory of Experimental Teratology and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan (China); Shao Changshun, E-mail: changshun.shao@gmail.com [Ministry of Education Key Laboratory of Experimental Teratology and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan (China)

    2012-06-01

    Berberine has been shown to possess anti-tumor activity against a wide spectrum of cancer cells. It inhibits cancer cell proliferation by inducing cell cycle arrest, at G1 and/or G2/M, and apoptosis. While it has been documented that berberine induces G1 arrest by activating the p53-p21 cascade, it remains unclear what mechanism underlies the berberine-induced G2/M arrest, which is p53-independent. In this study, we tested the anti-proliferative effect of berberine on murine prostate cancer cell line RM-1 and characterized the underlying mechanisms. Berberine dose-dependently induced DNA double-strand breaks and apoptosis. At low concentrations, berberine was observed to induce G1 arrest, concomitant with the activation of p53-p21 cascade. Upon exposure to berberine at a higher concentration (50 {mu}M) for 24 h, cells exhibited G2/M arrest. Pharmacological inhibition of ATM by KU55933, or Chk1 by UCN-01, could efficiently abrogate the G2/M arrest in berberine-treated cells. Downregulation of Chk1 by RNA interference also abolished the G2/M arrest caused by berberine, confirming the role of Chk1 in the pathway leading to G2/M arrest. Abrogation of G2/M arrest by ATM inhibition forced more cells to undergo apoptosis in response to berberine treatment. Chk1 inhibition by UCN-01, on the other hand, rendered cells more sensitive to berberine only when p53 was inhibited. Our results suggest that combined administration of berberine and caffeine, or other ATM inhibitor, may accelerate the killing of cancer cells.

  15. Staurosporine is chemoprotective by inducing G1 arrest in a Chk1- and pRb-dependent manner.

    Science.gov (United States)

    Murray, Mollianne McGahren; Bui, Tuyen; Smith, Michelle; Bagheri-Yarmand, Rozita; Wingate, Hannah; Hunt, Kelly K; Keyomarsi, Khandan

    2013-10-01

    Chemotherapeutic agents have been the mainstay of cancer therapy for years. However, their effectiveness has been limited by toxicities they impart on normal cells. Staurosporine (ST) has been shown to arrest normal, but not breast cancer, cells in G1. Therefore, ST may become a chemoprotective agent, arresting normal cells while allowing tumor cells to enter cell cycle phases where they are sensitive to chemotherapeutic agents. Understanding the mechanism of ST-mediated G1 arrest may allow for a beneficial chemoprotective treatment strategy for patients. We utilized 76NE6 (pRb+/p53-), 76NF2V (pRb+/p53+) and 76NE7 (pRb-/P53+) non-tumorigenic human mammary epithelial cell lines to understand the role of the Rb and p53 pathways in ST-directed G1 arrest. CDK4 was downregulated by ST in Rb+ cells, but its presence could not reverse the arrest, neither did its stable downregulation alter ST-mediated cellular response. ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4. Further assessment of this pathway revealed that Chk1 expression and activity were required for the Rb-dependent arrest. For example, pRb+ cells with small interfering RNA to Chk1 had approximately 60% less cells in G1 phase compared with controls and pRb- cells do not arrest upon ST. Furthermore, Chk1 expression facilitates the release of the Rb+ cells from G1 arrest. Collectively, our data suggest that pRb cooperates with Chk1 to mediate a G1 arrest only in pRb+ cells. The elucidation of this pathway can help identify novel agents to protect cancer patients against the debilitating effects of chemotherapy.

  16. Inactivating the spindle checkpoint kinase Bub1 during embryonic development results in a global shutdown of proliferation

    Directory of Open Access Journals (Sweden)

    Taylor Stephen S

    2009-09-01

    Full Text Available Abstract Background Bub1 is a component of the spindle assembly checkpoint, a surveillance mechanism that maintains chromosome stability during M-phase. Bub1 is essential during the early stages of embryogenesis, with homozygous BUB1-null mice dying shortly after day E3.5. Bub1 is also required later during embryogenesis; inactivation of BUB1 on day E10.5 appears to rapidly block all further development. However, the mechanism(s responsible for this phenotype remain unclear. Findings Here we show that inactivating BUB1 on day E10.5 stalls embryogenesis within 48 hours. This is accompanied by a global shutdown of proliferation, widespread apoptosis and haemorrhaging. Conclusion Our results suggest that Bub1 is required throughout the developing embryo for cellular proliferation. Therefore, Bub1 has been shown to be essential in all scenarios analyzed thus far in mice: proliferation of cultured fibroblasts, spermatogenesis, oogenesis and both early and late embryonic development. This likely reflects the fact that Bub1 has dual functions during mitosis, being required for both SAC function and chromosome alignment.

  17. Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

    NARCIS (Netherlands)

    Weerdt, B.C.M. van de; Vugt, M.A.T.M. van; Lindon, C.; Kauw, J.J.W.; Rozendaal, M.J.; Klompmaker, R.; Wolthuis, R.M.F.; Medema, R.H.

    2005-01-01

    Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plk1 through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210

  18. Uncoupling anaphase-promoting complex/cyclosome activity from spindle assembly checkpoint control by deregulating polo-like kinase 1

    NARCIS (Netherlands)

    van de Weerdt, BCM; van Vugt, MATM; Lindon, C; Kauw, JJW; Rozendaal, MJ; Klompmaker, R; Wolthuis, RMF; Medema, RH

    2005-01-01

    Polo-like kinase 1 (Plk1) plays a role in numerous events in mitosis, but how the multiple functions of Plk1 are separated is poorly understood. We studied regulation of Plkl through two putative phosphorylation residues, Ser-137 and Thr-210. Using phospho-specific antibodies, we found that Thr-210

  19. Expression of Chk1 in Primary Liver Cancer and Liver Non-tumor Tissue%Chk1在原发性肝癌和肝非肿瘤组织中的表达

    Institute of Scientific and Technical Information of China (English)

    马瑞波; 杨大刚; 孙诚谊; 王众

    2012-01-01

    目的:探讨细胞周期检测点启动DNA损伤修复的主要激酶Chk1蛋白在肝非肿瘤组织和原发性肝癌组织中的表达差异、以及与临床病理特征之间的关系.方法:应用免疫组织化学法检测15例肝非肿瘤组织(其中正常肝组织7例、肝硬化组织8例)和41例原发性肝癌组织Chk1蛋白的表达.结果:Chk1蛋白在原发性肝癌组织中表达阳性率为73.2%,在肝非肿瘤组织中表达阳性率为26.7%差异有统计学意义(P<0.05) ;Chk1蛋白在不同分化程度的原发性肝癌患者中的表达有差异,有统计学意义(P<0.05).结论:Chk1在原发性肝癌中高表达可能与原发性肝癌的发生、发展有关.%To investigate the expression difference of Chkl protien in primary liver cancer and non-tumor liver tissue, and to explore the probable relationship between its expression and clini-copathologic features of liver cancer. Methods: Immunohistochemistry was applied to detect the expressions of Chkl in 15 cases with liver non-tumor tissue (7 cases with normal liver tissue, 8 cases with cirrhosis tissue) and 41 cases with primary liver cancer with various differentiation degrees. Results : The positive expression rate of Chkl in primary liver cancer (73. 2% ) was higher than that of non-tumor liver tissue (26.1% ) {P <0. 05}. Significant differences were found in Chkl expressions among primary liver cancer with various differentiation degrees ( P < 0. 05 ). Conclusions: The high expression level of Chkl in primary liver cancer indicates that Chkl may be related to genesis and progression of primary liver cancer.

  20. Targeting the Checkpoint to Kill Cancer Cells

    Directory of Open Access Journals (Sweden)

    Jan Benada

    2015-08-01

    Full Text Available Cancer treatments such as radiotherapy and most of the chemotherapies act by damaging DNA of cancer cells. Upon DNA damage, cells stop proliferation at cell cycle checkpoints, which provides them time for DNA repair. Inhibiting the checkpoint allows entry to mitosis despite the presence of DNA damage and can lead to cell death. Importantly, as cancer cells exhibit increased levels of endogenous DNA damage due to an excessive replication stress, inhibiting the checkpoint kinases alone could act as a directed anti-cancer therapy. Here, we review the current status of inhibitors targeted towards the checkpoint effectors and discuss mechanisms of their actions in killing of cancer cells.

  1. Phosphorylation of microtubule-binding protein Hec1 by mitotic kinase Aurora B specifies spindle checkpoint kinase Mps1 signaling at the kinetochore.

    Science.gov (United States)

    Zhu, Tongge; Dou, Zhen; Qin, Bo; Jin, Changjiang; Wang, Xinghui; Xu, Leilei; Wang, Zhaoyang; Zhu, Lijuan; Liu, Fusheng; Gao, Xinjiao; Ke, Yuwen; Wang, Zhiyong; Aikhionbare, Felix; Fu, Chuanhai; Ding, Xia; Yao, Xuebiao

    2013-12-13

    The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.

  2. The effect of ataxia-telangiectasia mutated kinase-dependent hyperphosphorylation of checkpoint kinase-2 on oligodeoxynucleotide 7909 containing CpG motifs-enhanced sensitivity to X-rays in human lung adenocarcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Liu XQ

    2015-06-01

    Full Text Available Xiaoqun Liu,1,* Xiangdong Liu,2,* Tiankui Qiao,1 Wei Chen,1 Sujuan Yuan1 1Department of Oncology, 2Department of Ophthalmology, Affiliated Jinshan Hospital, Fudan University, Shanghai, People’s Republic of China *These authors contributed equally to this work Objective: The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2 on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909 in human lung adenocarcinoma A549 cells. Methods: In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+X-ray, ATM kinase-small interfering RNA (siRNA+CpG+X-ray (ATM-siRNA, and Chk2-siRNA+CpG+X-ray (Chk2-siRNA groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry. Results: Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively, though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01 when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis

  3. Determinants of mitotic catastrophe on abrogation of the G2 DNA damage checkpoint by UCN-01.

    Science.gov (United States)

    On, Kin Fan; Chen, Yue; Ma, Hoi Tang; Chow, Jeremy P H; Poon, Randy Y C

    2011-05-01

    Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G(2) DNA damage checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01-treated cells were immediately eliminated during the first mitosis after checkpoint abrogation, about half remained viable and progressed into G(1). Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly checkpoint, by either depleting MAD2 or overexpressing the MAD2-binding protein p31(comet), suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31(comet) or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA damage and the effectiveness of the spindle-assembly checkpoint in determining whether checkpoint-abrogated cells are eliminated during mitosis.

  4. miR-107 activates ATR/Chk1 pathway and suppress cervical cancer invasion by targeting MCL1.

    Directory of Open Access Journals (Sweden)

    Chengyan Zhou

    Full Text Available MicroRNAs (miRNAs are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRNAs may function as oncogenes or tumor suppressors. Here we showed that miR-107 directly targeted MCL1 and activated ATR/Chk1 pathway to inhibit proliferation, migration and invasiveness of cervical cancer cells. Moreover, we found that MCL1 was frequently up-regulated in cervical cancer, and knockdown of MCL1 markedly inhibited cancer cell proliferation, migration and invasion, whereas ectopic expression of MCL1 significantly enhances these properties. The restoration of MCL1 expression can counteract the effect of miR-107 on the cancer cells. Together, miR-107 is a new regulator of MCL1, and both miR-107 and MCL1 play important roles in the pathogenesis of cervical cancer. We have therefore identified a mechanism for ATR/Chk1 pathway which involves an increase in miR-107 leading to a decrease in MCL1. Correspondingly, our results revealed that miR-107 affected ATR/Chk1 signalling and gene expression, and implicated miR-107 as a therapeutic target in human cervical cancer. We also demonstrated that taxol attenuated migration and invasion in cervical cancer cells by activating the miR-107, in which miR-107 play an important role in regulating the expression of MCL1. Elucidation of this discovered MCL1 was directly regulated by miR-107 will greatly enhance our understanding of the mechanisms responsible for cervical cancer and will provide an additional arm for the development of anticancer therapies.

  5. ‘The octet’: eight protein kinases that control mammalian DNA replication

    Directory of Open Access Journals (Sweden)

    Melvin L. Depamphilis

    2012-09-01

    Full Text Available Development of a fertilized human egg into an average sized adult requires about 29 trillion cell divisions, thereby producing enough DNA to stretch to the Sun and back 200 times (DePamphilis and Bell, 2011! Even more amazing is the fact that throughout these mitotic cell cycles, the human genome is duplicated once and only once each time a cell divides. If a cell accidentally begins to re-replicate its nuclear DNA prior to cell division, checkpoint pathways trigger apoptosis. And yet, some cells are developmentally programmed to respond to environmental cues by switching from mitotic cell cycles to endocycles, a process in which multiple S phases occur in the absence of either mitosis or cytokinesis. Endocycles allow production of viable, differentiated, polyploid cells that no longer proliferate. What is surprising is that among the 516 (Manning et al., 2002 to 557 (BioMart web site protein kinases encoded by the human genome, only eight regulate nuclear DNA replication directly. These are Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, Cdc7, Chk1 and Chk2. Even more remarkable is the fact that only four of these enzymes (Cdk1, Cdk7, Cdc7 and Chk1 are essential for mammalian development. Here we describe how these protein kinases determine when DNA replication occurs during mitotic cell cycles, how mammalian cells switch from mitotic cell cycles to endocycles, and how cancer cells can be selectively targeted for destruction by inducing them to begin a second S phase before mitosis is complete.

  6. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

    Science.gov (United States)

    Deegan, Tom D; Yeeles, Joseph Tp; Diffley, John Fx

    2016-05-01

    The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner. Sld3 binds specifically to DDK-phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho-MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK-independent replication. Thus, Sld3 is an essential "reader" of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase. PMID:26912723

  7. Replication factor C3 of Schizosaccharomyces pombe, a small subunit of replication factor C complex, plays a role in both replication and damage checkpoints.

    Science.gov (United States)

    Shimada, M; Okuzaki, D; Tanaka, S; Tougan, T; Tamai, K K; Shimoda, C; Nojima, H

    1999-12-01

    We report here the isolation and functional analysis of the rfc3(+) gene of Schizosaccharomyces pombe, which encodes the third subunit of replication factor C (RFC3). Because the rfc3(+) gene was essential for growth, we isolated temperature-sensitive mutants. One of the mutants, rfc3-1, showed aberrant mitosis with fragmented or unevenly separated chromosomes at the restrictive temperature. In this mutant protein, arginine 216 was replaced by tryptophan. Pulsed-field gel electrophoresis suggested that rfc3-1 cells had defects in DNA replication. rfc3-1 cells were sensitive to hydroxyurea, methanesulfonate (MMS), and gamma and UV irradiation even at the permissive temperature, and the viabilities after these treatments were decreased. Using cells synchronized in early G2 by centrifugal elutriation, we found that the replication checkpoint triggered by hydroxyurea and the DNA damage checkpoint caused by MMS and gamma irradiation were impaired in rfc3-1 cells. Association of Rfc3 and Rad17 in vivo and a significant reduction of the phosphorylated form of Chk1 in rfc3-1 cells after treatments with MMS and gamma or UV irradiation suggested that the checkpoint signal emitted by Rfc3 is linked to the downstream checkpoint machinery via Rad17 and Chk1. From these results, we conclude that rfc3(+) is required not only for DNA replication but also for replication and damage checkpoint controls, probably functioning as a checkpoint sensor. PMID:10588638

  8. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  9. A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

    OpenAIRE

    Theesfeld, Chandra L.; Zyla, Trevin R.; Bardes, Elaine G.S.; Lew, Daniel J.

    2003-01-01

    Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon...

  10. Melanoma therapy: Check the checkpoints.

    Science.gov (United States)

    Furue, Masutaka; Kadono, Takafumi

    2016-02-01

    Recent mutational and translational studies have revealed that the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a key role in melanomagenesis. Mutations in NRAS and BRAF are found in the majority of melanomas resulting in the formation of constitutively active NRAS and BRAF molecules, which leads to the proliferation and survival of melanoma cells through the activation of MEK/ERK signals. Inhibitors of BRAF or MEK significantly extend the progression-free survival and overall survival of melanoma patients compared with conventional chemotherapies. Combining BRAF and MEK inhibitors further enhances the clinical effectiveness. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is an immune checkpoint molecule that downregulates T-cell activation by binding to B7 (CD80/CD86) molecules on antigen-presenting cells. Programmed death receptor ligand 1 on melanoma cells negatively regulates T-cell function by binding to the programmed death-1 (PD-1) receptor on T cells. Antibodies against CTLA-4 and PD-1 also enhance the survival of melanoma patients. In this review, we summarize the clinical effectiveness and adverse events of the BRAF inhibitors, MEK inhibitors and anti-immune checkpoint antibodies in melanoma treatment.

  11. Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

    International Nuclear Information System (INIS)

    Chk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence. Growth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models. MK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval. There are two reasons why delayed

  12. Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

    Institute of Scientific and Technical Information of China (English)

    Yan REN; Jia Rui WU

    2004-01-01

    DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern Ⅱ was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.

  13. Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.

    OpenAIRE

    Osmani, A H; O'Donnell, K; Pu, R T; Osmani, S A

    1991-01-01

    Mutation of nimA reversibly arrests cells in late G2 and nimA overexpression promotes premature mitosis. Here we demonstrate that the product of nimA (designated NIMA) has protein kinase activity that can phosphorylate beta-casein but not histone proteins. NIMA kinase activity is cell cycle regulated being 20-fold higher at mitosis when compared to S-phase arrested cells. NIMA activation is normally required in G2 to initiate chromosome condensation, to nucleate spindle pole body microtubules...

  14. Checkpoint Kinase ATR Promotes Nucleotide Excision Repair of UV-induced DNA Damage via Physical Interaction with Xeroderma Pigmentosum Group A*

    Science.gov (United States)

    Shell, Steven M.; Li, Zhengke; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka; Brosey, Chris; Serrano, Moises A.; Chazin, Walter J.; Musich, Phillip R.; Zou, Yue

    2009-01-01

    In response to DNA damage, eukaryotic cells activate a series of DNA damage-dependent pathways that serve to arrest cell cycle progression and remove DNA damage. Coordination of cell cycle arrest and damage repair is critical for maintenance of genomic stability. However, this process is still poorly understood. Nucleotide excision repair (NER) and the ATR-dependent cell cycle checkpoint are the major pathways responsible for repair of UV-induced DNA damage. Here we show that ATR physically interacts with the NER factor Xeroderma pigmentosum group A (XPA). Using a mass spectrometry-based protein footprinting method, we found that ATR interacts with a helix-turn-helix motif in the minimal DNA-binding domain of XPA where an ATR phosphorylation site (serine 196) is located. XPA-deficient cells complemented with XPA containing a point mutation of S196A displayed a reduced repair efficiency of cyclobutane pyrimidine dimers as compared with cells complemented with wild-type XPA, although no effect was observed for repair of (6-4) photoproducts. This suggests that the ATR-dependent phosphorylation of XPA may promote NER repair of persistent DNA damage. In addition, a K188A point mutation of XPA that disrupts the ATR-XPA interaction inhibits the nuclear import of XPA after UV irradiation and, thus, significantly reduced DNA repair efficiency. By contrast, the S196A mutation has no effect on XPA nuclear translocation. Taken together, our results suggest that the ATR-XPA interaction mediated by the helix-turn-helix motif of XPA plays an important role in DNA-damage responses to promote cell survival and genomic stability after UV irradiation. PMID:19586908

  15. Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide

    Energy Technology Data Exchange (ETDEWEB)

    Jobson, Andrew G.; Lountos, George T.; Lorenzi, Philip L.; Llamas, Jenny; Connelly, John; Cerna, David; Tropea, Joseph E.; Onda, Akikazu; Zoppoli, Gabriele; Kondapaka, Sudhir; Zhang, Guangtao; Caplen, Natasha J.; Cardellina, II, John H.; Yoo, Stephen S.; Monks, Anne; Self, Christopher; Waugh, David S.; Shoemaker, Robert H.; Pommier, Yves; (NIH)

    2010-04-05

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {l_brace}4-[1-(guanidinohydrazone)-ethyl]-phenyl{r_brace}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

  16. Distinct and combined roles of the MAP kinases of Cochliobolus heterostrophus in virulence and stress responses.

    Science.gov (United States)

    Igbaria, Aeid; Lev, Sophie; Rose, Mark S; Lee, Bee Na; Hadar, Ruthi; Degani, Ofir; Horwitz, Benjamin A

    2008-06-01

    Pathogenicity mitogen-activated protein kinases (MAPKs), related to yeast FUS3/KSS1, are essential for virulence in fungi, including Cochliobolus heterostrophus, a necrotrophic pathogen causing Southern corn leaf blight. We compared the phenotypes of mutants in three MAPK genes: HOG1, MPS1, and CHK1. The chk1 and mps1 mutants show autolytic appearance, light pigmentation, and dramatic reduction in virulence and conidiation. Similarity of mps1 and chk1 mutants is reflected by coregulation by these two MAPKs of several genes. Unlike chk1, mps1 mutants are female-fertile and form normal-looking appressoria. HOG1 mediates resistance to hyperosmotic and, to a lesser extent, oxidative stress, and is required for stress upregulation of glycerol-3-phosphate phosphatase, transaldolase, and a monosaccharide transporter. Hog1, but not Mps1 or Chk1, was rapidly phosphorylated in response to increased osmolarity. The hog1 mutants have smaller appressoria and cause decreased disease symptoms on maize leaves. Surprisingly, loss of MPS1 in a wild-type or hog1 background improved resistance to some stresses. All three MAPKs contribute to the regulation of central developmental functions under normal and stress conditions, and full virulence cannot be achieved without appropriate input from all three pathways. PMID:18473669

  17. HCLK2 is required for activity of the DNA damage response kinase ATR

    DEFF Research Database (Denmark)

    Rendtlew Danielsen, Jannie M; Larsen, Dorthe Helena; Schou, Kenneth Bødtker;

    2008-01-01

    ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activati...

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

    DEFF Research Database (Denmark)

    van Vugt, Marcel A T M; Gardino, Alexandra K; Linding, Rune;

    2010-01-01

    the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation......DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular...... of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity...

  19. Circumvention of Mcl-1-Dependent Drug Resistance by Simultaneous Chk1 and MEK1/2 Inhibition in Human Multiple Myeloma Cells

    Science.gov (United States)

    Pei, Xin-Yan; Dai, Yun; Felthousen, Jessica; Chen, Shuang; Takabatake, Yukie; Zhou, Liang; Youssefian, Leena E.; Sanderson, Michael W.; Bodie, Wesley W.; Kramer, Lora B.; Orlowski, Robert Z.; Grant, Steven

    2014-01-01

    The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM) cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+ MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM. PMID:24594907

  20. Circumvention of Mcl-1-dependent drug resistance by simultaneous Chk1 and MEK1/2 inhibition in human multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Xin-Yan Pei

    Full Text Available The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+ MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM.

  1. Development of cell-cycle checkpoint therapy for solid tumors.

    Science.gov (United States)

    Tamura, Kenji

    2015-12-01

    Cellular proliferation is tightly controlled by several cell-cycle checkpoint proteins. In cancer, the genes encoding these proteins are often disrupted and cause unrestrained cancer growth. The proteins are over-expressed in many malignancies; thus, they are potential targets for anti-cancer therapies. These proteins include cyclin-dependent kinase, checkpoint kinase, WEE1 kinase, aurora kinase and polo-like kinase. Cyclin-dependent kinase inhibitors are the most advanced cell-cycle checkpoint therapeutics available. For instance, palbociclib (PD0332991) is a first-in-class, oral, highly selective inhibitor of CDK4/6 and, in combination with letrozole (Phase II; PALOMA-1) or with fulvestrant (Phase III; PALOMA-3), it has significantly prolonged progression-free survival, in patients with metastatic estrogen receptor-positive, HER2-negative breast cancer, in comparison with that observed in patients using letrozole, or fulvestrant alone, respectively. In this review, we provide an overview of the current compounds available for cell-cycle checkpoint protein-directed therapy for solid tumors. PMID:26486823

  2. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    Energy Technology Data Exchange (ETDEWEB)

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  3. Checkpoint adaptation and recovery: back with Polo after the break

    NARCIS (Netherlands)

    Vugt, M.A.T.M. van; Medema, R.H.

    2004-01-01

    S. cerevisiae cells that are unable to repair a double strand break ultimately escape the DNA damage checkpoint arrest and enter mitosis. This process called 'adaptation' depends on functional Cdc5, a Polo-like kinase, and was long thought to be limited to single-cell organisms. However, the recent

  4. Sustaining the spindle assembly checkpoint to improve cancer therapy.

    Science.gov (United States)

    Visconti, Roberta; Della Monica, Rosa; Grieco, Domenico

    2016-01-01

    To prevent chromosome segregation errors, the spindle assembly checkpoint (SAC) delays mitosis exit until proper spindle assembly. We found that the FCP1 phosphatase and its downstream target WEE1 kinase oppose the SAC, promoting mitosis exit despite malformed spindles. We further showed that targeting this pathway might be useful for cancer therapy. PMID:27308561

  5. Checkpoint adaptation and recovery : back with Polo after the break

    NARCIS (Netherlands)

    van Vugt, Marcel A T M; Medema, René H

    2004-01-01

    S. cerevisiae cells that are unable to repair a double strand break ultimately escape the DNA damage checkpoint arrest and enter mitosis. This process called 'adaptation' depends on functional Cdc5, a Polo-like kinase, and was long thought to be limited to single-cell organisms. However, the recent

  6. Checkpoint responses to replication stalling: inducing tolerance and preventing mutagenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kai, Mihoko; Wang, Teresa S.-F

    2003-11-27

    Replication mutants often exhibit a mutator phenotype characterized by point mutations, single base frameshifts, and the deletion or duplication of sequences flanked by homologous repeats. Mutation in genes encoding checkpoint proteins can significantly affect the mutator phenotype. Here, we use fission yeast (Schizosaccharomyces pombe) as a model system to discuss the checkpoint responses to replication perturbations induced by replication mutants. Checkpoint activation induced by a DNA polymerase mutant, aside from delay of mitotic entry, up-regulates the translesion polymerase DinB (Pol{kappa}). Checkpoint Rad9-Rad1-Hus1 (9-1-1) complex, which is loaded onto chromatin by the Rad17-Rfc2-5 checkpoint complex in response to replication perturbation, recruits DinB onto chromatin to generate the point mutations and single nucleotide frameshifts in the replication mutator. This chain of events reveals a novel checkpoint-induced tolerance mechanism that allows cells to cope with replication perturbation, presumably to make possible restarting stalled replication forks. Fission yeast Cds1 kinase plays an essential role in maintaining DNA replication fork stability in the face of DNA damage and replication fork stalling. Cds1 kinase is known to regulate three proteins that are implicated in maintaining replication fork stability: Mus81-Eme1, a hetero-dimeric structure-specific endonuclease complex; Rqh1, a RecQ-family helicase involved in suppressing inappropriate recombination during replication; and Rad60, a protein required for recombinational repair during replication. These Cds1-regulated proteins are thought to cooperatively prevent mutagenesis and maintain replication fork stability in cells under replication stress. These checkpoint-regulated processes allow cells to survive replication perturbation by preventing stalled replication forks from degenerating into deleterious DNA structures resulting in genomic instability and cancer development.

  7. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    Energy Technology Data Exchange (ETDEWEB)

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  8. Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis.

    Directory of Open Access Journals (Sweden)

    Nataliya Kotelevets

    Full Text Available Sphingosine kinases (SK catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P, thereby promoting oncogenic processes. Breast (MDA-MB-231, lung (NCI-H358, and colon (HCT 116 carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

  9. Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Sible Jill C

    2007-10-01

    Full Text Available Abstract Background The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT of early development. Cell divisions 2–12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk activity. Results In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, Δ34-Xic1, was injected in embryos and also shown to induce apoptosis. Conclusion Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and Δ34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in X. laevis embryos.

  10. Mangrove dolabrane-type of diterpenes tagalsins suppresses tumor growth via ROS-mediated apoptosis and ATM/ATR-Chk1/Chk2-regulated cell cycle arrest.

    Science.gov (United States)

    Neumann, Jennifer; Yang, Yi; Köhler, Rebecca; Giaisi, Marco; Witzens-Harig, Mathias; Liu, Dong; Krammer, Peter H; Lin, Wenhan; Li-Weber, Min

    2015-12-01

    Natural compounds are an important source for drug development. With an increasing cancer rate worldwide there is an urgent quest for new anti-cancer drugs. In this study, we show that a group of dolabrane-type of diterpenes, collectively named tagalsins, isolated from the Chinese mangrove genus Ceriops has potent cytotoxicity on a panel of hematologic cancer cells. Investigation of the molecular mechanisms by which tagalsins kill malignant cells revealed that it induces a ROS-mediated damage of DNA. This event leads to apoptosis induction and blockage of cell cycle progression at S-G2 phase via activation of the ATM/ATR-Chk1/Chk2 check point pathway. We further show that tagalsins suppress growth of human T-cell leukemia xenografts in vivo. Tagalsins show only minor toxicity on healthy cells and are well tolerated by mice. Our study shows a therapeutic potential of tagalsins for the treatment of hematologic malignancies and a new source of anticancer drugs. PMID:26061604

  11. Checkpointing in speculative versioning caches

    Science.gov (United States)

    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.

  12. Cambridge checkpoint English workbook 2

    CERN Document Server

    Reynolds, John

    2014-01-01

    Build confidence and understanding throughout the year with hundreds of additional practice questions. This Workbook supports our bestselling Checkpoint series, with exercises specifically matched to the Cambridge Progression tests and the Checkpoint tests. - Develops understanding and builds confidence ahead of assessment with exercises matched to the tests - Ensures a thorough understanding of all aspects of the course by following the structure of the relevant textbook - Saves planning time with exercises that are suitable for use in class or as homework This Workbook is

  13. Cambridge checkpoint English workbook 3

    CERN Document Server

    Reynolds, John

    2014-01-01

    Build confidence and understanding throughout the year with hundreds of additional practice questions. This Workbook supports our bestselling Checkpoint series, with exercises specifically matched to the Cambridge Progression tests and the Checkpoint tests. - Develops understanding and builds confidence ahead of assessment with exercises matched to the tests - Ensures a thorough understanding of all aspects of the course by following the structure of the relevant textbook - Saves planning time with exercises that are suitable for use in class or as homework This Workbook is

  14. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    OpenAIRE

    Ivanova, Tsvetomira Georgieva, 1978-; Alves-Rodrigues, Isabel; G??mez Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo Hernando, Elena; Ayt?? del Olmo, Jos??

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint....

  15. Compiler-assisted static checkpoint insertion

    Science.gov (United States)

    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.

  16. The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

    Science.gov (United States)

    Blaikley, Elizabeth J; Tinline-Purvis, Helen; Kasparek, Torben R; Marguerat, Samuel; Sarkar, Sovan; Hulme, Lydia; Hussey, Sharon; Wee, Boon-Yu; Deegan, Rachel S; Walker, Carol A; Pai, Chen-Chun; Bähler, Jürg; Nakagawa, Takuro; Humphrey, Timothy C

    2014-05-01

    DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability. PMID:24623809

  17. Efficient Incremental Checkpointing of Java Programs

    DEFF Research Database (Denmark)

    Lawall, Julia Laetitia; Muller, Gilles

    2000-01-01

    This paper investigates the optimization of language-level checkpointing of Java programs. First, we describe how to systematically associate incremental checkpoints with Java classes. While being safe, the genericness of this solution induces substantial execution overhead. Second, to solve...

  18. Cambridge checkpoint English workbook 1

    CERN Document Server

    Reynolds, John

    2013-01-01

    This Workbook supports our bestselling Checkpoint English series, with exercises specifically matched to the Cambridge Progression tests and the Checkpoint English tests. - Offers plenty of additional questions for use in class or as homework. - Includes clearly identified questions on grammar and punctuation, comprehension, use of language and essay planning. - Follows the structure of the relevant textbook to ensure a thorough understanding of all aspects of the course. - Provides a space for Students to write their answers. This Workbook is matched to the Cambridge Secondary 1 Curriculum Fr

  19. Msc1 Acts Through Histone H2A.Z to Promote Chromosome Stability in Schizosaccharomyces pombe

    OpenAIRE

    Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C.

    2007-01-01

    As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expres...

  20. Network support for system initiated checkpoints

    Science.gov (United States)

    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.

  1. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    Science.gov (United States)

    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

  2. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions.

    Science.gov (United States)

    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

  3. Checkpointing for a hybrid computing node

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. DNA damage checkpoint recovery and cancer development

    International Nuclear Information System (INIS)

    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

  5. DNA damage checkpoint recovery and cancer development

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Haiyong [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Zhang, Xiaoshan [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States); Teng, Lisong, E-mail: lsteng@zju.edu.cn [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Legerski, Randy J., E-mail: rlegersk@mdanderson.org [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States)

    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

  6. ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling.

    Directory of Open Access Journals (Sweden)

    Erhong Meng

    Full Text Available OBJECTIVE: Aldehyde dehydrogenase (ALDH expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. METHODS: Isogenic ovarian cancer cell lines for platinum sensitivity (A2780 and platinum resistant (A2780/CP70 as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. RESULTS: ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01. ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ and replication checkpoint (pS317 Chk1 were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. CONCLUSION: This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling.

  7. A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

    Science.gov (United States)

    Theesfeld, Chandra L.; Zyla, Trevin R.; Bardes, Elaine G.S.; Lew, Daniel J.

    2003-01-01

    Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation. PMID:12925763

  8. Simulated Microgravity Promotes Cell Apoptosis Through Suppressing Uev1A/TICAM/TRAF/NF-κB-Regulated Anti-Apoptosis and p53/PCNA- and ATM/ATR-Chk1/2-Controlled DNA-Damage Response Pathways.

    Science.gov (United States)

    Zhao, Tuo; Tang, Xin; Umeshappa, Channakeshava Sokke; Ma, Hong; Gao, Haijun; Deng, Yulin; Freywald, Andrew; Xiang, Jim

    2016-09-01

    Microgravity has been known to induce cell death. However, its underlying mechanism is less studied. In this study, BL6-10 melanoma cells were cultured in flasks under simulated microgravity (SMG). We examined cell apoptosis, and assessed expression of genes associated with apoptosis and genes regulating apoptosis in cells under SMG. We demonstrate that SMG induces cell morphological changes and microtubule alterations by confocal microscopy, and enhances apoptosis by flow cytometry, which was associated with up- and down-regulation of pro-apoptotic and anti-apoptotic genes, respectively. Moreover, up- and down-regulation of pro-apoptotic (Caspases 3, 7, 8) and anti-apoptotic (Bcl2 and Bnip3) molecules was confirmed by Western blotting analysis. Western blot analysis also indicates that SMG causes inhibition of an apoptosis suppressor, pNF-κB-p65, which is complemented by the predominant localization of NF-κB-p65 in the cytoplasm. SMG also reduces expression of molecules regulating the NF-κB pathway including Uev1A, TICAM, TRAF2, and TRAF6. Interestingly, 10 DNA repair genes are down-regulated in cells exposed to SMG, among which down-regulation of Parp, Ercc8, Rad23, Rad51, and Ku70 was confirmed by Western blotting analysis. In addition, we demonstrate a significant inhibition of molecules involved in the DNA-damage response, such as p53, PCNA, ATM/ATR, and Chk1/2. Taken together, our work reveals that SMG promotes the apoptotic response through a combined modulation of the Uev1A/TICAM/TRAF/NF-κB-regulated apoptosis and the p53/PCNA- and ATM/ATR-Chk1/2-controlled DNA-damage response pathways. Thus, our investigation provides novel information, which may help us to determine the cause of negative alterations in human physiology occurring at spaceflight environment. J. Cell. Biochem. 117: 2138-2148, 2016. © 2016 Wiley Periodicals, Inc. PMID:26887372

  9. Visualizing the spindle checkpoint in Drosophila spermatocytes

    OpenAIRE

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint ma...

  10. A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens.

    Science.gov (United States)

    Lev, S; Sharon, A; Hadar, R; Ma, H; Horwitz, B A

    1999-11-01

    Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted. The ability of Deltachk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets. PMID:10557357

  11. GRID COMPUTING AND CHECKPOINT APPROACH

    Directory of Open Access Journals (Sweden)

    Pankaj gupta

    2011-05-01

    Full Text Available Grid computing is a means of allocating the computational power of alarge number of computers to complex difficult computation or problem. Grid computing is a distributed computing paradigm thatdiffers from traditional distributed computing in that it is aimed toward large scale systems that even span organizational boundaries. In this paper we investigate the different techniques of fault tolerance which are used in many real time distributed systems. The main focus is on types of fault occurring in the system, fault detection techniques and the recovery techniques used. A fault can occur due to link failure, resource failure or by any other reason is to be tolerated for working the system smoothly and accurately. These faults can be detected and recovered by many techniques used accordingly. An appropriate fault detector can avoid loss due to system crash and reliable fault tolerance technique can save from system failure. This paper provides how these methods are applied to detect and tolerate faults from various Real Time Distributed Systems. The advantages of utilizing the check pointing functionality are obvious; however so far the Grid community has notdeveloped a widely accepted standard that would allow the Gridenvironment to consciously utilize low level check pointing packages.Therefore, such a standard named Grid Check pointing Architecture isbeing designed. The fault tolerance mechanism used here sets the jobcheckpoints based on the resource failure rate. If resource failureoccurs, the job is restarted from its last successful state using acheckpoint file from another grid resource. A critical aspect for anautomatic recovery is the availability of checkpoint files. A strategy to increase the availability of checkpoints is replication. Grid is a form distributed computing mainly to virtualizes and utilize geographically distributed idle resources. A grid is a distributed computational and storage environment often composed of

  12. The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

    OpenAIRE

    Marquitz, Aron R.; Harrison, Jacob C.; Bose, Indrani; Zyla, Trevin R.; McMillan, John N.; Lew, Daniel J.

    2002-01-01

    The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (G...

  13. Intellectual property issues of immune checkpoint inhibitors.

    Science.gov (United States)

    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

  14. Overlapped checkpointing with hardware assist

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, Christopher J [Los Alamos National Laboratory; Nunez, James A [Los Alamos National Laboratory; Wang, Jun [U. OF CENTRAL FLORIDA (UCF)

    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.

  15. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    Science.gov (United States)

    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.

  16. Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula.

    Science.gov (United States)

    Boivin, Stéphane; Kazmierczak, Théophile; Brault, Mathias; Wen, Jiangqi; Gamas, Pascal; Mysore, Kirankumar S; Frugier, Florian

    2016-10-01

    Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE-domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock-down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume-specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation. PMID:27341695

  17. Clinical Development of Immune Checkpoint Inhibitors

    Directory of Open Access Journals (Sweden)

    Ayumu Ito

    2015-01-01

    Full Text Available Recent progress in cancer immunotherapy has been remarkable. Most striking are the clinical development and approval of immunomodulators, also known as immune checkpoint inhibitors. These monoclonal antibodies (mAb are directed to immune checkpoint molecules, which are expressed on immune cells and mediate signals to attenuate excessive immune reactions. Although mAbs targeting tumor associated antigens, such as anti-CD20 mAb and anti-Her2 mAb, directly recognize tumor cells and induce cell death, immune checkpoint inhibitors restore and augment the antitumor immune activities of cytotoxic T cells by blocking immune checkpoint molecules on T cells or their ligands on antigen presenting and tumor cells. Based on preclinical data, many clinical trials have demonstrated the acceptable safety profiles and efficacies of immune checkpoint inhibitors in a variety of cancers. The first in class approved immune checkpoint inhibitor is ipilimumab, an anti-CTLA-4 (cytotoxic T lymphocyte antigen-4 mAb. Two pivotal phase III randomized controlled trials demonstrated a survival benefit in patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA approved ipilimumab for metastatic melanoma. Several clinical trials have since investigated new agents, alone and in combination, for various cancers. In this review, we discuss the current development status of and future challenges in utilizing immune checkpoint inhibitors.

  18. Visualizing the spindle checkpoint in Drosophila spermatocytes

    Science.gov (United States)

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila. PMID:11256627

  19. REVIEW OF CHECKPOINTING ALGORITHMS IN DISTRIBUTED SYSTEMS

    Directory of Open Access Journals (Sweden)

    Poonam Gahlan

    2010-06-01

    Full Text Available Checkpointing is the process of saving the status information. Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at a later time. Mobile computing raises many new issues such as lack of stablestorage, low bandwidth of wireless channel, high mobility, and limited battery life. Coordinated checkpointing is an attractive approach for transparently adding fault tolerance to distributed applications since it avoids domino effects and minimizes the stable storage requirement. This paper presents the review of the algorithms,which have been reported in the literature for checkpointing. This paper also covers backward error recovery techniques for distributed systems specially the distributed mobile systems.

  20. Ndd1 turnover by SCF(Grr1 is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Ellen R Edenberg

    2015-04-01

    Full Text Available In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1, phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1. In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

  1. A Nonblocking Coordinated Checkpointing Algorithm for Mobile Computing Systems

    Directory of Open Access Journals (Sweden)

    Rachit Garg

    2010-05-01

    Full Text Available A checkpoint algorithm for mobile computing systems needs to handle many new issues like: mobility, low bandwidth of wireless channels, lack of stable storage on mobile nodes, disconnections, limited battery power and high failure rate of mobile nodes. These issues make traditional checkpointing techniques unsuitable for such environments. Minimum-process coordinated checkpointing is an attractive approach to introduce fault tolerance in mobile distributed systems transparently. This approach is domino-free, requires at most two checkpoints of a process on stable storage, and forces only a minimum number of processes to checkpoint. But, it requires extra synchronization messages, blocking of the underlying computation or taking some useless checkpoints. In this paper, we propose a nonblocking coordinated checkpointing algorithm for mobile computing systems, which requires only a minimum number of processes to take permanent checkpoints. We reduce the message complexity as compared to the Cao-Singhal algorithm [4], while keeping the number of useless checkpoints unchanged. We also address the related issues like: failures during checkpointing, disconnections, concurrent initiations of the algorithm and maintaining exact dependencies among processes. Finally, the paper presents an optimization technique, which significantly reduces the number of useless checkpoints at the cost of minor increase in the message complexity. In coordinated checkpointing, if a single process fails to take its tentative checkpoint; all the checkpoint effort is aborted. We try to reduce this effort by taking soft checkpoints in the first phase at Mobile Hosts.

  2. The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

    Science.gov (United States)

    Marquitz, Aron R.; Harrison, Jacob C.; Bose, Indrani; Zyla, Trevin R.; McMillan, John N.; Lew, Daniel J.

    2002-01-01

    The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint. PMID:12145202

  3. Targeting WEE1 Kinase in Cancer.

    Science.gov (United States)

    Matheson, Christopher J; Backos, Donald S; Reigan, Philip

    2016-10-01

    WEE1 kinase plays a crucial role in the G2-M cell-cycle checkpoint arrest for DNA repair before mitotic entry. Normal cells repair damaged DNA during G1 arrest; however, cancer cells often have a deficient G1-S checkpoint and depend on a functional G2-M checkpoint for DNA repair. WEE1 is expressed at high levels in various cancer types including breast cancers, leukemia, melanoma, and adult and pediatric brain tumors. Many of these cancers are treated with DNA-damaging agents; therefore, targeting WEE1 for inhibition and compromising the G2-M checkpoint presents an opportunity to potentiate therapy. In this review we summarize the current WEE1 inhibitors, the potential for further inhibitor development, and the challenges in the clinic for the WEE1 inhibitor strategy. PMID:27427153

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Dpb11/TopBP1 contributes to genomicstability via homologous recombinationand checkpoint signaling

    DEFF Research Database (Denmark)

    Germann, Susanne Manuela

    Homologous recombination (HR) is essential for maintaining genome integrity and is a major pathway for repairing (DSBs). DPB11 is an essential gene conserved from yeast to human (TopBP1), which is involved in initiation of DNA replication and DNA checkpoint signaling. We found that Dpb11 forms foci...... signaling. Importantly, Dpb11 foci are independent of checkpoint kinases Mec1 and Tel1, as well as Rad9, further strengthening the upstream position of Dpb11 in the DNA damage checkpoint response. Moreover, dpb11-PF has a defect in S-phase checkpoint function, albeit to a lesser extent than dpb11-1. Altered...... rates of heteroallelic and direct repeat recombination implicate a role for Dpb11 in homologous recombination. Physical monitoring of mating-type switching as a model for DSB repair revealed that the repair kinetics of dpb11-PF are delayed. Finally, we found Dpb11 in budding yeast as well as TopBP1...

  6. Robust Cell Size Checkpoint from Spatiotemporal Positive Feedback Loop in Fission Yeast

    Directory of Open Access Journals (Sweden)

    Jie Yan

    2013-01-01

    Full Text Available Cells must maintain appropriate cell size during proliferation. Size control may be regulated by a size checkpoint that couples cell size to cell division. Biological experimental data suggests that the cell size is coupled to the cell cycle in two ways: the rates of protein synthesis and the cell polarity protein kinase Pom1 provide spatial information that is used to regulate mitosis inhibitor Wee1. Here a mathematical model involving these spatiotemporal regulations was developed and used to explore the mechanisms underlying the size checkpoint in fission yeast. Bifurcation analysis shows that when the spatiotemporal regulation is coupled to the positive feedback loops (active Cdc2 promotes its activator, Cdc25, and suppress its inhibitor, Wee1, the mitosis-promoting factor (MPF exhibits a bistable steady-state relationship with the cell size. The switch-like response from the positive feedback loops naturally generates the cell size checkpoint. Further analysis indicated that the spatial regulation provided by Pom1 enhances the robustness of the size checkpoint in fission yeast. This was consistent with experimental data.

  7. Molecular Mechanisms of DNA Replication Checkpoint Activation

    Directory of Open Access Journals (Sweden)

    Bénédicte Recolin

    2014-03-01

    Full Text Available 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.

  8. Checkpoint triggering in a computer system

    Science.gov (United States)

    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.

  9. Garbage Collection in Uncoordinated Checkpointing Algorithms

    Institute of Scientific and Technical Information of China (English)

    LIU Yunlong; CHEN Junliang

    1999-01-01

    In this paper, the hard problem of thethorough garbage collection in uncoordinated checkpointing algorithms isstudied. After introduction of the traditional garbage collectingscheme, with which only obsolete checkpoints can be discarded, it isshown that this kind of traditional method may fail to discard anycheckpoint in some special cases, and it is necessary and urgent to finda thorough garbage collecting method, with which all the checkpointsuseless for any future rollback-recovery including the obsolete ones canbe discarded. Then, the Thorough Garbage Collection Theorem is proposedand proved, which ensures the feasibility of the thorough garbagecollection, and gives the method to calculate the set of the usefulcheckpoints as well.

  10. Analysis of the tolerance to DNA alkylating damage in MEC1 and RAD53 checkpoint mutants of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Alfonso Gallego-Sánchez

    Full Text Available Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.

  11. Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint.

    Science.gov (United States)

    Keaton, Mignon A; Lew, Daniel J

    2006-12-01

    The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial. PMID:17055334

  12. Checkpointing and Recovery in Distributed and Database Systems

    Science.gov (United States)

    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…

  13. Selection of checkpoints provided by the ergonomic checkpoints in agriculture tool for mechanized sugarcane harvesting

    Directory of Open Access Journals (Sweden)

    Ana Lucy Rodrigues Ferreira

    2014-11-01

    Full Text Available The changing work dynamics of sugarcane harvesting owing to increasing mechanization has submitted workers to new working conditions, including interaction with machinery and equipment, thereby changing the profile of work-related diseases and injuries. One of the ways to solve problems resulting from the impact of mechanization on working conditions is the use of instruments that allow risk identification from man-labor ratio. This study aimed at selecting checkpoints applicable to mechanized sugarcane harvesting provided by the Ergonomic Checkpoints in Agriculture tool. A literature review of the mechanical sugarcane harvesting stages was conducted and, in light of its particularities, checkpoints provided by the aforementioned tool were analyzed. As a result, there were identified thirty-four checkpoints with potential application to mechanical sugarcane harvesting.

  14. Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection

    Institute of Scientific and Technical Information of China (English)

    Fan Zhao; Qing-Jun Ma; Hui Zhong; Ning-Bo Hou; Xiao-Li Yang; Xiang He; Yu Liu; Yan-Hong Zhang; Cong-Wen Wei; Ting Song; Li Li

    2008-01-01

    AIM: To explore whether acute cellular DNA damage response is induced upon hepatitis B virus (HBV) infection and the effects of the HBV infection. METHODS: We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chk1 phosphorylation loci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet (UV) radiation and ionizing radiation (IR)-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells.RESULTS: We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chkl, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h after HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increased apoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection. CONCLUSION: HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

  15. A chemical tool box defines mitotic and interphase roles for Mps1 kinase

    OpenAIRE

    Lan, Weijie; Don W Cleveland

    2010-01-01

    In this issue, three groups (Hewitt et al. 2010. J. Cell Biol. doi:10.1083/jcb.201002133; Maciejowski et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001050; Santaguida et al. 2010. J. Cell Biol. doi:10.1083/jcb.201001036) use chemical inhibitors to analyze the function of the mitotic checkpoint kinase Mps1. These studies demonstrate that Mps1 kinase activity ensures accurate chromosome segregation through its recruitment to kinetochores of mitotic checkpoint proteins, formation of interphase a...

  16. Multiple Defects of Cell Cycle Checkpoints in U937-ASPI3K, an U937 Cell Mutant Stably Expressing Anti-Sense ATM Gene cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    (Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and nable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Synchronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S, G2/M cell cycle checkpoint profiles were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.

  17. The effect of the intra-S-phase checkpoint on origins of replication in human cells.

    Science.gov (United States)

    Karnani, Neerja; Dutta, Anindya

    2011-03-15

    Although many chemotherapy drugs activate the intra-S-phase checkpoint pathway to block S-phase progression, not much is known about how and where the intra-S-phase checkpoint regulates origins of replication in human chromosomes. A genomic analysis of replication in human cells in the presence of hydroxyurea (HU) revealed that only the earliest origins fire, but the forks stall within 2 kb and neighboring clusters of dormant origins are activated. The initiation events are located near expressed genes with a preference for transcription start and end sites, and when they are located in intergenic regions they are located near regulatory factor-binding regions (RFBR). The activation of clustered neo-origins by HU suggests that there are many potential replication initiation sites in permissive parts of the genome, most of which are not used in a normal S phase. Consistent with this redundancy, we see multiple sites bound to MCM3 (representative of the helicase) in the region flanking three out of three origins studied in detail. Bypass of the intra-S-phase checkpoint by caffeine activates many new origins in mid- and late-replicating parts of the genome. The intra-S-phase checkpoint suppresses origin firing after the loading of Mcm10, but before the recruitment of Cdc45 and AND-1/CTF4; i.e., after helicase loading but before helicase activation and polymerase loading. Interestingly, Cdc45 recruitment upon checkpoint bypass was accompanied by the restoration of global Cdk2 kinase activity and decrease in both global and origin-bound histone H3 Lys 4 trimethylation (H3K4me3), consistent with the suggestion that both of these factors are important for Cdc45 recruitment.

  18. Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination

    DEFF Research Database (Denmark)

    Germann, Susanne Manuela; Østergaard, Vibe Hallundbæk; Haas, Caroline;

    2011-01-01

    DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its...... homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1...... and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant...

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

    Energy Technology Data Exchange (ETDEWEB)

    Mohror, Kathryn [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Moody, Adam [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Bronevetsky, Greg [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); de Supinski, Bronis R. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    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.

  20. Comparison of the Radiosensitizing Effect of ATR, ATM and DNA-PK Kinase Inhibitors on Cervical Carcinoma Cells.

    Science.gov (United States)

    Vávrová, J; Zárybnická, L; Jošt, P; Tichý, A; Řezáčová, M; Šinkorová, Z; Pejchal, J

    2016-01-01

    Here, we compared the effects of inhibitors of three phosphatidylinositol-3-kinase-related kinases, ATM, ATR a DNA-PK, on radiosensitization of cervical carcinoma cells. We demonstrated that DNA-PK inhibitor NU7441 enhanced phosphorylation of Chk1 and Chk2 kinases 2 h after irradiation of HeLa cells at a dose of 8 Gy in contrast to ATM kinase inhibitor KU55933, which completely blocked the Chk2 kinase phosphorylation on threonine 68, and ATR kinase inhibitor VE-821, which blocked the Chk1 kinase phosphorylation on serine 345. Most HeLa cells were accumulated in G2 phase of the cell cycle 24 h after irradiation at a high dose of 15 Gy, which was even potentiated after adding the inhibitors NU7441 and KU55933. Compared to all other irradiated groups, inhibitor VE-821 increased the number of cells in S phase and reduced the number of cells in G2 phase 24 h after irradiation at the high dose of 15 Gy. HeLa cells entered the mitotic cycle with unrepaired DNA, which resulted in cell death and the radiosensitizing effect of VE-821. Short-term application of the inhibitors (2 h before and 30 min after the irradiation by the dose of 8 Gy) significantly decreased the colony-forming ability of HeLa cells. Using real-time monitoring of cell proliferation by the xCELLigence system we demonstrated that while the radiosensitizing effect of VE-821 (ATR inhibitor) is manifested early after the irradiation, the radiosensitizing effect of KU55933 (ATM inhibitor) and NU7441 (DNA-PK inhibitor) is only observed as late as 72 h after the irradiation. PMID:27643582

  1. Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via an unanticipated binding Site

    DEFF Research Database (Denmark)

    Bolanos-Garcia, Victor M; Lischetti, Tiziana; Matak-Vinković, Dijana;

    2011-01-01

    The maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central...

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

    Science.gov (United States)

    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. Replication licensing and the DNA damage checkpoint

    OpenAIRE

    Cook, Jeanette Gowen

    2009-01-01

    Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings sug...

  4. Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides.

    Science.gov (United States)

    Wanrooij, Paulina H; Tannous, Elias; Kumar, Sandeep; Navadgi-Patil, Vasundhara M; Burgers, Peter M

    2016-01-01

    Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.

  5. Immune Checkpoint Inhibitors in Older Adults.

    Science.gov (United States)

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: 75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient. PMID:27287329

  6. Immune Checkpoint Inhibitors in Older Adults.

    Science.gov (United States)

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: 75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient.

  7. DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

    2013-06-01

    Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

  8. Biological significance of the focus on DNA damage checkpoint factors remained after irradiation of ionizing radiation

    International Nuclear Information System (INIS)

    This paper reviews recent reports on the focus formation and participation to checkpoint of (such phosphorylated (P-d) as below) ATM and H2AX, MDC1, 53BP1 and NBS1, and discusses their role in DNA damage checkpoint induction mainly around authors' studies. When the cell is irradiated by ionizing radiation, the subtype histone like H2AX is P-d and the formed focus', seen in the nucleus on immuno-fluorographic observation, represents the P-d H2AX at the damaged site of DNA. The role of P-d ATM (the product of causative gene of ataxia-telangiectasia mutation, a protein kinase) has been first shown by laser beam irradiation. Described are discussions on the roles and functions after irradiation in focus formation and DNA damage checkpoint of P-d H2AX (a specific histone product by the radiation like γ-ray as above), P-d ATM, MDC1 (a mediator of DNA damage check point protein 1), 53BP1, (a p53 binding protein) and NBS1 (the product of the causative gene of Nijmegen Breakage Syndrome). Authors have come to point out the remained focal size increase as implications of the efficient repair of damaged DNA, and the second cycled p53 accumulation, of tumor suppression. Thus evaluation of biological significance of these aspects, scarcely noted hitherto, is concluded important. (S.I.)

  9. Efficient Checkpointing of Virtual Machines using Virtual Machine Introspection

    Energy Technology Data Exchange (ETDEWEB)

    Aderholdt, Ferrol [Tennessee Technological University; Han, Fang [Tennessee Technological University; Scott, Stephen L [ORNL; Naughton, III, Thomas J [ORNL

    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.

  10. The meiotic recombination checkpoint is regulated by checkpoint rad+ genes in fission yeast.

    Science.gov (United States)

    Shimada, Midori; Nabeshima, Kentaro; Tougan, Takahiro; Nojima, Hiroshi

    2002-06-01

    During the course of meiotic prophase, intrinsic double-strand breaks (DSBs) must be repaired before the cell can engage in meiotic nuclear division. Here we investigate the mechanism that controls the meiotic progression in Schizosaccharomyces pombe that have accumulated excess meiotic DSBs. A meiotic recombination-defective mutant, meu13Delta, shows a delay in meiotic progression. This delay is dependent on rec12+, namely on DSB formation. Pulsed-field gel electrophoresis analysis revealed that meiotic DSB repair in meu13Delta was retarded. We also found that the delay in entering nuclear division was dependent on the checkpoint rad+, cds1+ and mek1+ (the meiotic paralog of Cds1/Chk2). This implies that these genes are involved in a checkpoint that provides time to repair DSBs. Consistently, the induction of an excess of extrinsic DSBs by ionizing radiation delayed meiotic progression in a rad17(+)-dependent manner. dmc1Delta also shows meiotic delay, however, this delay is independent of rec12+ and checkpoint rad+. We propose that checkpoint monitoring of the status of meiotic DSB repair exists in fission yeast and that defects other than DSB accumulation can cause delays in meiotic progression. PMID:12032093

  11. DNA Damage and Repair Biomarkers in Cervical Cancer Patients Treated with Neoadjuvant Chemotherapy: An Exploratory Analysis.

    Directory of Open Access Journals (Sweden)

    Patrizia Vici

    Full Text Available Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1, a key G2/M checkpoint kinase, and γ-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1, a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, γ-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. γ-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and γ-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and γ-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings.

  12. DNA Damage and Repair Biomarkers in Cervical Cancer Patients Treated with Neoadjuvant Chemotherapy: An Exploratory Analysis.

    Science.gov (United States)

    Vici, Patrizia; Buglioni, Simonetta; Sergi, Domenico; Pizzuti, Laura; Di Lauro, Luigi; Antoniani, Barbara; Sperati, Francesca; Terrenato, Irene; Carosi, Mariantonia; Gamucci, Teresa; Dattilo, Rosanna; Bartucci, Monica; Vincenzoni, Cristina; Mariani, Luciano; Vizza, Enrico; Sanguineti, Giuseppe; Gadducci, Angiolo; Vitale, Ilio; Barba, Maddalena; De Maria, Ruggero; Mottolese, Marcella; Maugeri-Saccà, Marcello

    2016-01-01

    Cervical cancer cells commonly harbour a defective G1/S checkpoint owing to the interaction of viral oncoproteins with p53 and retinoblastoma protein. The activation of the G2/M checkpoint may thus become essential for protecting cancer cells from genotoxic insults, such as chemotherapy. In 52 cervical cancer patients treated with neoadjuvant chemotherapy, we investigated whether the levels of phosphorylated Wee1 (pWee1), a key G2/M checkpoint kinase, and γ-H2AX, a marker of DNA double-strand breaks, discriminated between patients with a pathological complete response (pCR) and those with residual disease. We also tested the association between pWee1 and phosphorylated Chk1 (pChk1), a kinase acting upstream Wee1 in the G2/M checkpoint pathway. pWee1, γ-H2AX and pChk1 were retrospectively assessed in diagnostic biopsies by immunohistochemistry. The degrees of pWee1 and pChk1 expression were defined using three different classification methods, i.e., staining intensity, Allred score, and a multiplicative score. γ-H2AX was analyzed both as continuous and categorical variable. Irrespective of the classification used, elevated levels of pWee1 and γ-H2AX were significantly associated with a lower rate of pCR. In univariate and multivariate analyses, pWee1 and γ-H2AX were both associated with reduced pCR. Internal validation conducted through a re-sampling without replacement procedure confirmed the robustness of the multivariate model. Finally, we found a significant association between pWee1 and pChk1. The message conveyed by the present analysis is that biomarkers of DNA damage and repair may predict the efficacy of neoadjuvant chemotherapy in cervical cancer. Further studies are warranted to prospectively validate these encouraging findings. PMID:26930412

  13. Immune checkpoints in cancer clinical trials

    Institute of Scientific and Technical Information of China (English)

    Elad Sharon; Howard Streicher; Priscila Goncalves; Helen XChen

    2014-01-01

    Immunology-based therapy is rapidly developing into an effective treatment option for a surprising range of cancers. We have learned over the last decade that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called“immune checkpoints.” These checkpoints serve to control or turn off the immune response when it is no longer needed to prevent tissue injury and autoimmunity. Cancer cells have learned or evolved to use these mechanisms to evade immune control and elimination. The development of a new therapeutic class of drugs that inhibit these inhibitory pathways has recently emerged as a potent strategy in oncology. Three sets of agents have emerged in clinical trials exploiting this strategy. These agents are antibody-based therapies targeting cytotoxic T-lymphocyte antigen4 (CTLA4), programmed cell death1 (PD-1), and programmed cell death ligand 1 (PD-L1). These inhibitors of immune inhibition have demonstrated extensive activity as single agents and in combinations. Clinical responses have been seen in melanoma, renal cellcarcinoma, non-smal celllung cancer, and several other tumor types. Despite the autoimmune or inflammatory immune-mediated adverse effects which have been seen, the responses and overall survival benefits exhibited thus far warrant further clinical development.

  14. DMTCP: Scalable User-Level Transparent Checkpointing for Cluster Computations

    CERN Document Server

    Ansel, Jason; Arya, Kapil

    2008-01-01

    As the size of clusters increases, failures are becoming increasingly frequent. Applications must become fault tolerant if they are to run for extended periods of time. We present DMTCP (Distributed MultiThreaded CheckPointing), the first user-level distributed checkpointing package not dependent on a specific message passing library. This contrasts with existing approaches either specific to libraries such as MPI or requiring kernel modification. DMTCP provides fault tolerance through checkpointing. DMTCP transparently checkpoints general cluster computations consisting of many nodes, processes, and threads. DMTCP automatically accounts for TCP/IP sockets, UNIX domain sockets, pipes, ptys (pseudo-terminals), signal handlers, ordinary file descriptors, shared file descriptors, and other operating system artifacts. We demonstrate checkpointing and restart of applications communicating through MPICH2, OpenMPI, and sockets directly. These applications were written with a variety of languages including Fortran, C...

  15. Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair.

    Directory of Open Access Journals (Sweden)

    Qi Wu

    Full Text Available DNA interstrand crosslinks (ICLs are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSalpha and MutSbeta mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.

  16. Premature Sister Chromatid Separation Is Poorly Detected by the Spindle Assembly Checkpoint as a Result of System-Level Feedback

    Directory of Open Access Journals (Sweden)

    Mihailo Mirkovic

    2015-10-01

    Full Text Available Sister chromatid cohesion, mediated by the cohesin complex, is essential for faithful mitosis. Nevertheless, evidence suggests that the surveillance mechanism that governs mitotic fidelity, the spindle assembly checkpoint (SAC, is not robust enough to halt cell division when cohesion loss occurs prematurely. The mechanism behind this poor response is not properly understood. Using developing Drosophila brains, we show that full sister chromatid separation elicits a weak checkpoint response resulting in abnormal mitotic exit after a short delay. Quantitative live-cell imaging approaches combined with mathematical modeling indicate that weak SAC activation upon cohesion loss is caused by weak signal generation. This is further attenuated by several feedback loops in the mitotic signaling network. We propose that multiple feedback loops involving cyclin-dependent kinase 1 (Cdk1 gradually impair error-correction efficiency and accelerate mitotic exit upon premature loss of cohesion. Our findings explain how cohesion defects may escape SAC surveillance.

  17. A Previously Unknown Unique Challenge for Inhibitors of SYK ATP-Binding Site: Role of SYK as A Cell Cycle Checkpoint Regulator

    Directory of Open Access Journals (Sweden)

    Fatih M. Uckun

    2014-11-01

    Full Text Available The identification of SYK as a molecular target in B-lineage leukemia/lymphoma cells prompted the development of SYK inhibitors as a new class of anti-cancer drug candidates. Here we report that induction of the SYK gene expression in human cells causes a significant down-regulation of evolutionarily conserved genes associated with mitosis and cell cycle progression providing unprecedented evidence that SYK is a master regulator of cell cycle regulatory checkpoint genes in human cells. We further show that SYK regulates the G2 checkpoint by physically associating with and inhibiting the dual-specificity phosphatase CDC25C via phosphorylation of its S216 residue. SYK depletion by RNA interference or treatment with the chemical SYK inhibitor prevented nocodazole-treated human cell lines from activating the G2 checkpoint via CDC25C S216-phosphorylation and resulted in polyploidy. Our study provides genetic and biochemical evidence that spleen tyrosine kinase (SYK has a unique role in the activation of the G2 checkpoint in both non-lymphohematopoietic and B-lineage lymphoid cells. This previously unknown role of SYK as a cell cycle checkpoint regulator represents an unforeseen and significant challenge for inhibitors of SYK ATP binding site.

  18. Protein phosphatases acting on the replication checkpoint

    OpenAIRE

    Conde, Rui Miguel Esteves Antunes Seabra

    2010-01-01

    A génese de um cancro está dependente da acumulação de mutações genéticas que dão origem a instabilidade genómica, que por sua vez resulta na proliferação descontrolada. Para prevenir a acumulação destas mutações, as células têm mecanismos de controlo (checkpoints) que suspendem o ciclo celular e accionam as vias de reparação do ADN. Estes eventos são muitas vezes regulados por dinâmicas de (des)fosforilação de proteínas. As proteínas fosfatases (PPs), enzimas responsáveis p...

  19. Managing Adverse Events With Immune Checkpoint Agents.

    Science.gov (United States)

    Dadu, Ramona; Zobniw, Chrystia; Diab, Adi

    2016-01-01

    Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4 and anti programmed cell death 1/programmed cell death 1 ligand antibodies) have shown impressive clinical activity in multiple cancer types. Despite achieving great clinical success, challenges and limitations of these drugs as monotherapy or various combinational strategies include the development of a unique set of immune-related adverse events (irAEs) that can be severe and even fatal. Therefore, identification of patients at risk, prevention, consistent communication between patients and medical team, rapid recognition, and treatment of irAEs are critical in optimizing treatment outcomes. This review focuses on the description of more common irAEs and provides a suggested approach for management of specific irAEs. PMID:27111908

  20. FORMAL VERIFICATION OF DISTRIBUTED CHECKPOINTING USING EVENT-B

    Directory of Open Access Journals (Sweden)

    Girish Chandra

    2015-10-01

    Full Text Available The development of complex system makes challenging task for correct software development. Due to faulty specification, software may involve errors. The traditional testing methods are not sufficient to verify the correctness of such complex system. In order to capture correct system requirements and rigorous reasoning about the problems, formal methods are required. Formal methods are mathematical techniques that provide precise specification of problems with their solutions and proof of correctness. In this paper, we have done formal verification of check pointing process in a distributed database system using Event B. Event-B is an event driven formal method which is used to develop formal models of distributed database systems. In a distributed database system, the database is stored at different sites that are connected together through the network. Checkpoint is a recovery point which contains the state information about the site. In order to do recovery of a distributed transaction a global checkpoint number (GCPN is required. A global checkpoint number decides which transaction will be included for recovery purpose. All transactions whose timestamp are less than global checkpoint number will be marked as before checkpoint transaction (BCPT and will be considered for recovery purpose. The transactions whose timestamp are greater than GCPN will be marked as after checkpoint transaction (ACPT and will be part of next global checkpoint number.

  1. Keeping checkpoint/restart viable for exascale systems.

    Energy Technology Data Exchange (ETDEWEB)

    Riesen, Rolf E.; Bridges, Patrick G. (IBM Research, Ireland, Mulhuddart, Dublin); Stearley, Jon R.; Laros, James H., III; Oldfield, Ron A.; Arnold, Dorian (University of New Mexico, Albuquerque, NM); 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.

  2. Anaphase onset before complete DNA replication with intact checkpoint responses

    DEFF Research Database (Denmark)

    Torres-Rosell, Jordi; De Piccoli, Giacomo; Cordon-Preciado, Violeta;

    2007-01-01

    Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most...... significantly at natural replication-impeding loci like the ribosomal DNA gene cluster. In the absence of Smc5-Smc6, chromosome nondisjunction occurs as a consequence of mitotic entry with unfinished replication despite intact checkpoint responses. Eliminating processes that obstruct replication fork...

  3. A New Adaptive Checkpointing Strategy for Mobile Computing

    Institute of Scientific and Technical Information of China (English)

    MENChaoguang; ZUODecheng; YANGXiaozong

    2005-01-01

    Adaptive checkpointing strategy is an efficient recovery scheme, which is suitable for mobile computing system. However, all existing adaptive checkpointing schemes are not correct to recover system when failure occurs in some special period. In this paper, the issues that will lead to system inconsistency are first discussed and then a new adaptive strategy that can recover system to correct consistent state is proposed. Our algorithm improves system recovery performance because only failure process needs rollback through logging.

  4. The DNA damage response signaling cascade regulates proliferation of the phytopathogenic fungus Ustilago maydis in planta.

    Science.gov (United States)

    de Sena-Tomás, Carmen; Fernández-Álvarez, Alfonso; Holloman, William K; Pérez-Martín, José

    2011-04-01

    In the phytopathogenic fungus Ustilago maydis, the dikaryotic state dominates the period of growth occurring during the infectious phase. Dikaryons are cells in which two nuclei, one from each parent cell, share a single cytoplasm for a period of time without undergoing nuclear fusion. In fungal cells, maintenance of the dikaryotic state requires an intricate cell division process that often involves the formation of a structure known as the clamp connection as well as the sorting of one of the nuclei to this structure to ensure that each daughter dikaryon inherits a balance of each parental genome. Here, we describe an atypical role of the DNA damage checkpoint kinases Chk1 and Atr1 during pathogenic growth of U. maydis. We found that Chk1 and Atr1 collaborate to control cell cycle arrest during the induction of the virulence program in U. maydis and that Chk1 and Atr1 work together to control the dikaryon formation. These findings uncover a link between a widely conserved signaling cascade and the virulence program in a phytopathogen. We propose a model in which adjustment of the cell cycle by the Atr1-Chk1 axis controls fidelity in dikaryon formation. Therefore, Chk1 and Atr1 emerge as critical cell type regulators in addition to their roles in the DNA damage response.

  5. Discovery of a Small-Molecule BMP Sensitizer for Human Embryonic Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Lingling Feng

    2016-05-01

    Full Text Available Sorely missing from the “toolkit” for directed differentiation of stem/progenitor cells are agonists of the BMP-signaling pathway. Using a high-throughput chemical screen, we discovered that PD407824, a checkpoint kinase 1 (CHK1 inhibitor, increases the sensitivity of cells to sub-threshold amounts of BMP4. We show utility of the compound in the directed differentiation of human embryonic stem cells toward mesoderm or cytotrophoblast stem cells. Blocking CHK1 activity using pharmacological compounds or CHK1 knockout using single guide RNA (sgRNA confirmed that CHK1 inhibition increases the sensitivity to BMP4 treatment. Additional mechanistic studies indicate that CHK1 inhibition depletes p21 levels, thereby activating CDK8/9, which then phosphorylates the SMAD2/3 linker region, leading to decreased levels of SMAD2/3 protein and enhanced levels of nuclear SMAD1. This study provides insight into mechanisms controlling the BMP/transforming growth factor beta (TGF-β signaling pathways and a useful pharmacological reagent for directed differentiation of stem cells.

  6. DMTCP: bringing interactive checkpoint-restart to Python

    Science.gov (United States)

    Arya, Kapil; Cooperman, Gene

    2015-01-01

    DMTCP (Distributed MultiThreaded CheckPointing) is a mature checkpoint-restart package. It operates in user space without kernel privilege, and adapts to application-specific requirements through plugins. While DMTCP has been able to checkpoint Python and IPython ‘from the outside’ for many years, a Python module has recently been created to support DMTCP. IPython support is included through a new DMTCP plugin. A checkpoint can be requested interactively within a Python session or under the control of a specific Python program. Further, the Python program can execute specific Python code prior to checkpoint, upon resuming (within the original process) and upon restarting (from a checkpoint image). Applications of DMTCP are demonstrated for: (i) Python-based graphics using virtual network client, (ii) a fast/slow technique to use multiple hosts or cores to check one (Cython Behnel S et al 2011 Comput. Sci. Eng. 13 31-39) computation in parallel, and (iii) a reversible debugger, FReD, with a novel reverse-expression watchpoint feature for locating the cause of a bug.

  7. An Analysis of Checkpointing Algorithms for Distributed Mobile Systems

    Directory of Open Access Journals (Sweden)

    Ajay Khunteta

    2010-07-01

    Full Text Available Distributed snapshots are an important building block for distributed systems, and are useful for constructing efficient checkpointing protocols, among other uses. Direct application of these algorithms to mobile systems is not easible, however, due to differences in the environment in which mobile systems operate, relative to general distributed systems. The mobile computing environment introduces newchallenges in the area of fault-tolerant computing. Compared to traditional distributed environments, wireless networks are typically slower, providing lower throughput and latency, comparing to wireline networks. In addition, the mobile hosts have limited computation esources, are often exposed to harsh operating environment that makes them more likely to fail, and can roam while operating. Over the past two decades, intensive research work has been carried out on providing efficient checkpointing protocols in traditional distributed computing. Recently, more attention has been paid to providing checkpointing protocols for mobile systems. Some of these protocols have been adapted from the traditional distributed environment; others have been created from scratch for mobile systems. Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at alater time. Checkpointing is the process of saving the status information. This paper surveys the algorithms which have been reported in the literature for checkpointing in Mobile Distributed systems.

  8. A survey of checkpointing algorithms for parallel and distributed computers

    Indian Academy of Sciences (India)

    S Kalaiselvi; V Rajaraman

    2000-10-01

    Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at a later time. Checkpointing is the process of saving the status information. This paper surveysthe algorithms which have been reported in the literature for checkpointing parallel/distributed systems. It has been observed that most of the algorithms published for checkpointing in message passing systems are based on the seminal article by Chandy and Lamport. A large number of articles have been published in this area by relaxing the assumptions made in this paper and by extending it to minimise the overheads of coordination and context saving. Checkpointing for sharedmemory systems primarily extend cache coherence protocolstomaintain a consistent memory. All of them assume that the main memory is safe for storing the context. Recently algorithms have been published for distributed shared memory systems, which extend the cache coherence protocols used in shared memory systems. They however also include methods for storing the status of distributed memory in stable storage. Most of the algorithms assume that there is no knowledge about the programs being executed.It is howeverfelt that in development of parallel programs the user has to do a fair amount of work in distributing tasks and this information can be effectively used to simplify checkpointing and rollback recovery.

  9. A Tunable Checkpointing Algorithm for Distributed Mobile Applications

    Directory of Open Access Journals (Sweden)

    Sungchae Lim

    2011-11-01

    Full Text Available The aim of a distributed checkpointing algorithm is to efficiently restore the execution state of distributed applications in face of hardware or software failures. Originally, such algorithms were devised for fixed networking systems, of which computing components communicate with each other via wired networks. Therefore, those algorithms usually suffer from heavy networking costs coming from frequent data transits over wireless networks, if they are used in the wireless computing environment. In this paper, to reduce usage of wireless communications, our checkpointing algorithm allows the distributed mobile application to tune the level of its checkpointing strictness. The strictness is defined by the maximum rollback distance (MRD that says how many recent local checkpoints can be rolled back in the worst case. Since our algorithm have more flexibility in checkpointing schedule due to the use of MRD, it is possible to reduce the number of enforced local checkpointing. In particular, the amount of data transited on wirelesses networks becomes smaller than in earlier methods; thus, our algorithm provides less communication cost and shortened blocking time.

  10. LAMMER kinase contributes to genome stability in Ustilago maydis.

    Science.gov (United States)

    de Sena-Tomás, Carmen; Sutherland, Jeanette H; Milisavljevic, Mira; Nikolic, Dragana B; Pérez-Martín, José; Kojic, Milorad; Holloman, William K

    2015-09-01

    Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.

  11. A genetic screen for replication initiation defective (rid mutants in Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Locovei Alexandra M

    2010-08-01

    Full Text Available Abstract In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase or DNA damage repair (in G2. Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20 are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular

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

    Science.gov (United States)

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

    2015-01-01

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

  13. Targeting of Carbon Ion-Induced G2 Checkpoint Activation in Lung Cancer Cells Using Wee-1 Inhibitor MK-1775.

    Science.gov (United States)

    Ma, Hongyu; Takahashi, Akihisa; Sejimo, Yukihiko; Adachi, Akiko; Kubo, Nobuteru; Isono, Mayu; Yoshida, Yukari; Kanai, Tatsuaki; Ohno, Tatsuya; Nakano, Takashi

    2015-12-01

    The potent inhibitor of the cell cycle checkpoint regulatory factor Wee-1, MK-1775, has been reported to enhance non-small cell lung cancer (NSCLC) cell sensitivity to photon radiation by abrogating radiation-induced G2 arrest. However, little is known about the effects of this sensitizer after exposure to carbon (C)-ion radiation. The purpose of this study was therefore to investigate the effects of C ions in combination with MK-1775 on the killing of NSCLC cells. Human NSCLC H1299 cells were exposed to X rays or C ions (290 MeV/n, 50 keV/μm at the center of a 6 cm spread-out Bragg peak) in the presence of MK-1775. The cell cycle was analyzed using flow cytometry and Western blotting. Radiosensitivity was determined using clonogenic survival assays. The mechanisms underlying MK-1775 radiosensitization were studied by observing H2AX phosphorylation and mitotic catastrophe. G2 checkpoint arrest was enhanced 2.3-fold by C-ion exposure compared with X-ray exposure. Radiation-induced G2 checkpoint arrest was abrogated by MK-1775. Exposure to radiation resulted in a significant reduction in the mitotic ratio and increased phosphorylation of cyclin-dependent kinase 1 (Cdk1), the primary downstream mediator of Wee-1-induced G2 arrest. The Wee-1 inhibitor, MK-1775 restored the mitotic ratio and suppressed Cdk1 phosphorylation. In addition, MK-1775 increased H1299 cell sensitivity to C ions and X rays independent of TP53 status. MK-1775 also significantly increased H2AX phosphorylation and mitotic catastrophe in irradiated cells. These results suggest that the G2 checkpoint inhibitor MK-1775 can enhance the sensitivity of human NSCLC cells to C ions as well as X rays. PMID:26645158

  14. Immune checkpoint inhibitors and prostate cancer: a new frontier?

    Directory of Open Access Journals (Sweden)

    Alessandra Modena

    2016-04-01

    Full Text Available Despite recent advances in the treatment of metastatic castrationresistant 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-04-15

    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.

  17. Berkeley lab checkpoint/restart (BLCR) for Linux clusters

    Science.gov (United States)

    Hargrove, Paul H.; Duell, Jason C.

    2006-09-01

    This article describes the motivation, design and implementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-level checkpoint/restart implementation for Linux clusters that targets the space of typical High Performance Computing applications, including MPI. Application-level solutions, including both checkpointing and fault-tolerant algorithms, are recognized as more time and space efficient than system-level checkpoints, which cannot make use of any application-specific knowledge. However, system-level checkpointing allows for preemption, making it suitable for responding to ''fault precursors'' (for instance, elevated error rates from ECC memory or network CRCs, or elevated temperature from sensors). Preemption can also increase the efficiency of batch scheduling; for instance reducing idle cycles (by allowing for shutdown without any queue draining period or reallocation of resources to eliminate idle nodes when better fitting jobs are queued), and reducing the average queued time (by limiting large jobs to running during off-peak hours, without the need to limit the length of such jobs). Each of these potential uses makes BLCR a valuable tool for efficient resource management in Linux clusters.

  18. Berkeley lab checkpoint/restart (BLCR) for Linux clusters

    International Nuclear Information System (INIS)

    This article describes the motivation, design and implementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-level checkpoint/restart implementation for Linux clusters that targets the space of typical High Performance Computing applications, including MPI. Application-level solutions, including both checkpointing and fault-tolerant algorithms, are recognized as more time and space efficient than system-level checkpoints, which cannot make use of any application-specific knowledge. However, system-level checkpointing allows for preemption, making it suitable for responding to ''fault precursors'' (for instance, elevated error rates from ECC memory or network CRCs, or elevated temperature from sensors). Preemption can also increase the efficiency of batch scheduling; for instance reducing idle cycles (by allowing for shutdown without any queue draining period or reallocation of resources to eliminate idle nodes when better fitting jobs are queued), and reducing the average queued time (by limiting large jobs to running during off-peak hours, without the need to limit the length of such jobs). Each of these potential uses makes BLCR a valuable tool for efficient resource management in Linux clusters

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

    Science.gov (United States)

    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.

  20. Keeping it together in times of stress: checkpoint function at stalled replication forks

    OpenAIRE

    Berens, Theresa J.; David P Toczyski

    2012-01-01

    In this issue, De Piccoli et al. (2012) show that, contrary to current models of DNA replication checkpoint function, replication proteins remain associated with each other and with replicating DNA when replication is stressed in checkpoint-deficient cells.

  1. Low-Overhead Non-Blocking Checkpointing Scheme for Mobile Computing Systems

    Institute of Scientific and Technical Information of China (English)

    MEN Chaoguang; CAO Liujuan; WANG Liwen; XU Zhenpeng

    2007-01-01

    When applied to mobile computing systems, checkpoint protocols for distributed computing systems would face many new challenges, such as low wireless bandwidth, frequent disconnections, and lack of stable storage at mobile hosts. This paper proposes a novel checkpoint protocol to effectively reduce the coordinating overhead. By using a communication vector, only a few processes participate in the checkpointing event. During checkpointing, the scheme can save the time used to trace the dependency tree by sending checkpoint requests to dependent processes at once. In addition, processes are non-blocking in this scheme, since the inconsistency is resolved by the piggyback technique. Hence the unnecessary and orphan messages can be avoided. Compared with the traditional coordinated checkpoint approach, the proposed non-blocking algorithm obtains a minimal number of processes to take checkpoints. It also reduces the checkpoint latency, which brings less overhead to mobile host with limited resources.

  2. American Chemical Society - 240th national meeting - chemistry for preventing and combating disease: part 2.

    Science.gov (United States)

    Kibble, Alexandra

    2010-10-01

    The 240th National Meeting of the American Chemical Society, held in Boston, included topics covering new therapeutic research. This conference report highlights selected presentations on (S)-adenosylhomocysteine (AHCY) inhibitors for the treatment of Alzheimer's disease, 2,4-diphenyl-1H-imidazole analogs as cannabinoid CB2 agonists for the treatment of pain, checkpoint kinase 1 (Chk1) and Aurora kinase B as therapeutic targets for cancer treatment, pyridylmethylthio derivatives as VEGFR2 inhibitors, and Janus kinase 2 (JAK2) for the treatment of myeloproliferative disorders. Investigational drugs discussed include L-002259713 (Merck & Co), AZD-1480 (AstraZeneca), CYT-387 (YM Biosciences) and ruxolitinib (Incyte).

  3. Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

    Science.gov (United States)

    Thebault, Philippe; Chirgadze, Dimitri Y; Dou, Zhen; Blundell, Tom L; Elowe, Sabine; Bolanos-Garcia, Victor M

    2012-12-15

    The SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1 nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.

  4. Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1.

    Science.gov (United States)

    Althoff, Friederike; Karess, Roger E; Lehner, Christian F

    2012-06-01

    Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.

  5. BRCA1 and its phosphorylation involved in caffeine-inhibitable event upstream of G2 checkpoint

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Caffeine,which specifically inhibits ATM/ATR kinases,efficiently abrogates the ionizing radiation(IR)-induced G2 arrest and increases the sensitivity of various tumor cells to IR.Mechanisms for the effect of caffeine remain to be elucidated.As a target of ATM/ATR kinases,BRCA1 becomes activated and phosphorylated in response to IR.Thus,in this work,we investigated the possible role of BRCA1 in the effect of caffeine on G2 checkpoint and observed how BRCA1 phosphorylation was regulated in this process.For these purposes,the BRCA1 protein level and the phosphorylation states were analyzed by Western blotting by using an antibody against BRCA1 and phospho-specific antibodies against Ser-1423 and Ser-1524 residues in cells exposed to a combination of IR and caffeine.The results showed that caffeine down-regulated IR-induced BRCA1 expression and specifically abolished BRCA1 phosphorylation of Ser-1524,which was followed by an override of G2 arrest by caffeine.In addition,the ability of BRCA1 to transactivate p21 may be required for MCF-7 but not necessary for Hela response to caffeine.These data suggest that BRCA1 may be a potential target of caffeine.BRCA1 and its phosphorylation are most likely to be involved in the caffeine-inhibitable event upstream of G2 arrest.

  6. Harnessing the Power of Onco-Immunotherapy with Checkpoint Inhibitors

    Directory of Open Access Journals (Sweden)

    Karishma R. Rajani

    2015-11-01

    Full Text Available Oncolytic viruses represent a diverse class of replication competent viruses that curtail tumor growth. These viruses, through their natural ability or through genetic modifications, can selectively replicate within tumor cells and induce cell death while leaving normal cells intact. Apart from the direct oncolytic activity, these viruses mediate tumor cell death via the induction of innate and adaptive immune responses. The field of oncolytic viruses has seen substantial advancement with the progression of numerous oncolytic viruses in various phases of clinical trials. Tumors employ a plethora of mechanisms to establish growth and subsequently metastasize. These include evasion of immune surveillance by inducing up-regulation of checkpoint proteins which function to abrogate T cell effector functions. Currently, antibodies blocking checkpoint proteins such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4 and anti-programmed cell death-1 (PD-1 have been approved to treat cancer and shown to impart durable clinical responses. These antibodies typically need pre-existing active immune tumor microenvironment to establish durable clinical outcomes and not every patient responds to these therapies. This review provides an overview of published pre-clinical studies demonstrating superior therapeutic efficacy of combining oncolytic viruses with checkpoint blockade compared to monotherapies. These studies provide compelling evidence that oncolytic therapy can be potentiated by coupling it with checkpoint therapies.

  7. Localization of checkpoint and repair proteins in eukaryotes

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2005-01-01

    In eukaryotes, the cellular response to DNA damage depends on the type of DNA structure being recognized by the checkpoint and repair machinery. DNA ends and single-stranded DNA are hallmarks of double-strand breaks and replication stress. These two structures are recognized by distinct sets of p...

  8. Attachment issues : kinetochore transformations and spindle checkpoint silencing

    NARCIS (Netherlands)

    Etemad, Banafsheh; Kops, Geert Jpl

    2016-01-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetoc

  9. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    Directory of Open Access Journals (Sweden)

    Brian J. Ahn

    2013-11-01

    Full Text Available Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas.

  10. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Brian J. [Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Pollack, Ian F. [Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Okada, Hideho, E-mail: okadah@upmc.edu [Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States)

    2013-11-01

    Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas.

  11. Checkpointing for graceful degradation in distributed embedded systems

    Science.gov (United States)

    Sababha, Belal Hussein

    Graceful degradation is an approach to developing dependable safety-critical embedded applications, where redundant active or standby resources are used to cope with faults through a system reconfiguration at run-time. Compared to traditional hardware and software redundancy, it is a promising technique that may achieve dependability with a significant reduction in cost, size, weight, and power requirements. Reconfiguration at run-time necessitates using proper checkpointing protocols to support state reservation to ensure correct task restarts after a system reconfiguration. One of the most common checkpointing protocols are communication induced checkpointing (CIC) protocols, which are well developed and understood for large parallel and information systems, but not much has been done for resource limited embedded systems. This work implements and evaluates some of the most common CIC protocols in a periodic resource constrained distributed embedded system for graceful degradation purposes. A test-bed has been developed and used for the evaluation of the various protocols. The implemented protocols are thoroughly studied and performances are contrasted. Specifically the periodicity property and how it benefits checkpointing in embedded systems is investigated. This work introduces a unique effort of CIC protocol implementation and evaluation in the field of distributed embedded systems. Other than providing a test-bed for graceful degradation support, this work shows that some checkpointing protocols that are not efficient in large information systems and supercomputers perform well in embedded systems. We show that a simple index-based CIC protocol, such as the BCS protocol, is more appropriate in embedded system applications compared to other protocols that piggyback a significant amount of information to reduce the number of forced checkpoints. Finally, this work proposes a whole graceful degradation approach to achieve fault tolerance in resource constrained

  12. Loss of ATM kinase activity leads to embryonic lethality in mice

    DEFF Research Database (Denmark)

    Daniel, J.A.; Pellegrini, M.; Filsuf, D.;

    2012-01-01

    Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine wh...

  13. WEE1 kinase targeting combined with DNA-damaging cancer therapy catalyzes mitotic catastrophe

    NARCIS (Netherlands)

    P.C. De Witt Hamer; S.E. Mir; D. Noske; C.J.F. van Noorden; T. Würdinger

    2011-01-01

    WEE1 kinase is a key molecule in maintaining G₂-cell-cycle checkpoint arrest for premitotic DNA repair. Whereas normal cells repair damaged DNA during G₁-arrest, cancer cells often have a deficient G₁-arrest and largely depend on G₂-arrest. The molecular switch for the G₂-M transition is held by WEE

  14. Cell size checkpoint control by the retinoblastoma tumor suppressor pathway.

    Directory of Open Access Journals (Sweden)

    Su-Chiung Fang

    2006-10-01

    Full Text Available Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription.

  15. Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange.

    Science.gov (United States)

    Oliver, Antony W; Paul, Angela; Boxall, Katherine J; Barrie, S Elaine; Aherne, G Wynne; Garrett, Michelle D; Mittnacht, Sibylle; Pearl, Laurence H

    2006-07-12

    The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.

  16. The DNA damage checkpoint response to replication stress: A Game of Forks.

    Directory of Open Access Journals (Sweden)

    Rachel eJossen

    2013-03-01

    Full Text Available Conditions challenging replication fork progression, collectively referred to as replication stress, represent a major source of genomic instability and are associated to cancer onset. The replication checkpoint, a specialized branch of the DNA damage checkpoint, monitors fork problems and triggers a cellular response aimed at preserving genome integrity. Here, we review the mechanisms by which the replication checkpoint monitors and responds to replication stress, focusing on the checkpoint-mediated pathways contributing to protect replication fork integrity. We discuss how cells achieve checkpoint signaling inactivation once replication stress is overcome and how a failure to timely revert checkpoint-mediated changes in cellular physiology might impact on replication dynamics and genome integrity. We also highlight the checkpoint function as an anti-cancer barrier preventing cells malignant transformation following oncogene-induced replication stress.

  17. DNA-damage response network at the crossroads of cell-cycle checkpoints,cellular senescence and apoptosis

    Institute of Scientific and Technical Information of China (English)

    SCHMITT Estelle; PAQUET Claudie; BEAUCHEMIN Myriam; BERTRAND Richard

    2007-01-01

    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.

  18. Pch2 acts through Xrs2 and Tel1/ATM to modulate interhomolog bias and checkpoint function during meiosis.

    Directory of Open Access Journals (Sweden)

    Hsuan-Chung Ho

    2011-11-01

    Full Text Available Proper segregation of chromosomes during meiosis requires the formation and repair of double-strand breaks (DSBs to form crossovers. Repair is biased toward using the homolog as a substrate rather than the sister chromatid. Pch2 is a conserved member of the AAA(+-ATPase family of proteins and is implicated in a wide range of meiosis-specific processes including the recombination checkpoint, maturation of the chromosome axis, crossover control, and synapsis. We demonstrate a role for Pch2 in promoting and regulating interhomolog bias and the meiotic recombination checkpoint in response to unprocessed DSBs through the activation of axial proteins Hop1 and Mek1 in budding yeast. We show that Pch2 physically interacts with the putative BRCT repeats in the N-terminal region of Xrs2, a member of the MRX complex that acts at sites of unprocessed DSBs. Pch2, Xrs2, and the ATM ortholog Tel1 function in the same pathway leading to the phosphorylation of Hop1, independent of Rad17 and the ATR ortholog Mec1, which respond to the presence of single-stranded DNA. An N-terminal deletion of Xrs2 recapitulates the pch2Δ phenotypes for signaling unresected breaks. We propose that interaction with Xrs2 may enable Pch2 to remodel chromosome structure adjacent to the site of a DSB and thereby promote accessibility of Hop1 to the Tel1 kinase. In addition, Xrs2, like Pch2, is required for checkpoint-mediated delay conferred by the failure to synapse chromosomes.

  19. Checkpoint signaling from a single DNA interstrand crosslink

    OpenAIRE

    Ben-Yehoyada, Merav; Wang, Lily C; Kozekov, Ivan D.; Rizzo, Carmelo J.; Gottesman, Max E.; Gautier, Jean

    2009-01-01

    DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as Mitomycin C and Cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from and repairing of a single, site-specific ICL in cell-free extract derived from Xenopus...

  20. Learning common lessons and checkpoints form human error incidents. Maintenance

    International Nuclear Information System (INIS)

    CRIEPI has been conducting detailed and structured analyses of all human error incidents collected from Japanese Licensee Event Reports using J-HPES (Japanese version of HPES) as an analysis method. Results obtained by the analyses have been stored in J-HPES database. This paper described the process to analyze J-HPES data concerning maintenance systematically to extract problems identified in the process of error action and checkpoints for preventing errors. Human error actions of these J-HPES data are classified by viewpoints of error mode and work type. As to each of these error categories, problems are extracted based on J-HPES causal relation charts in following three viewpoints: acts at workplace, activities, and preconditions of job. Moreover, checkpoints for preventing errors are developed referring proposed countermeasures in J-HPES database. In order to share these results, we started to issue 4-pages booklets 'Catch the Point' periodically. In future, based on these results, we will publish a teaching material. We also have a plan to store the contents of Catch the Point in a database, which facilitate users to find necessary checkpoints and hazards before they start their activities. (author)

  1. Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair.

    Science.gov (United States)

    Wang, Zheng; Lai, Song-Tao; Ma, Ning-Yi; Deng, Yun; Liu, Yong; Wei, Dong-Ping; Zhao, Jian-Dong; Jiang, Guo-Liang

    2015-12-01

    Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study. PMID:26304716

  2. Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair.

    Science.gov (United States)

    Wang, Zheng; Lai, Song-Tao; Ma, Ning-Yi; Deng, Yun; Liu, Yong; Wei, Dong-Ping; Zhao, Jian-Dong; Jiang, Guo-Liang

    2015-12-01

    Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study.

  3. In Vitro Characterization of Derrone as an Aurora Kinase Inhibitor.

    Science.gov (United States)

    Hoang, Nhung Thi My; Phuong, Thuong Thien; Nguyen, Trang Thi Nhu; Tran, Yen Thi Hai; Nguyen, Anh Thi Ngoc; Nguyen, Thanh Lai; Bui, Khanh Thi Van

    2016-06-01

    Among mitotic kinases, Aurora kinases are the most widely studied, since their expression is restricted to mitosis. They play a key role in chromosome segregation and cell polyploidy. Aurora kinases are important therapeutic targets, and several research groups have directed their efforts toward the identification of kinase inhibitors. The aim of this study is to screen and characterize Aurora kinase inhibitors from natural substances extracted from plants that are used in the Vietnamese pharmacopoeia. We have characterized in vitro Derrone, extracted from Erythrina orientalis L. MURR, as a novel Aurora kinase inhibitor. This compound exhibited an ability to inhibit the phosphorylation of histone H3 at ser10 both in kinase assay and at the cellular level. The compound was more effective against Aurora kinase B, with a lower IC50 value as compared to Aurora A. Moreover, it impaired the mitotic spindle checkpoint and led to endoreduplication in cancer cells, a phenomenon caused by an Aurora B inhibitor. Interestingly, using the xCelligence system and real-time cell analysis (RTCA) software, we set up a comparison of cell proliferation profiles between cancer cells treated with Derrone and VX680-a well-known Aurora kinase inhibitor-and we found that these profiles exhibited considerable similarity in cell morphology, growth, and death. Additionally, Derrone significantly inhibited the formation and growth of MCF7 tumor spheroids. PMID:26983907

  4. Protein kinase TTK interacts and co-localizes with CENP-E to the kinetochore of human cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Spindle checkpoint is an important biochemical signaling cascade during mitosis which monitors the fidelity of chromosome segregation, and is mediated by protein kinases Mps1 and Bub1/BubR1. Our recent studies show that kinesin-related motor protein CENP-E interacts with BubR1 and participates in spindle checkpoint signaling. To elucidate the molecular mechanisms underlying spindle checkpoint signaling, we carried out proteomic dissection of human cell kinetochore and revealed protein kinase TTK, human homologue of yeast Mps1. Our studies show that TTK is localized to the kinetochore of human cells, and interacts with CENP-E, suggesting that TTK may play an important role in chromosome segregation during mitosis.

  5. Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis.

    OpenAIRE

    Ye, X S; Fincher, R R; Tang, A.; O'Donnell, K; Osmani, S A

    1996-01-01

    We demonstrate that there are at least two S-phase checkpoint mechanisms controlling mitosis in Aspergillus. The first responds to the rate of DNA replication and inhibits mitosis via tyrosine phosphorylation of p34cdc2. Cells unable to tyrosine phosphorylate p34cdc2 are therefore viable but are unable to tolerate low levels of hydroxyurea and prematurely enter lethal mitosis when S-phase is slowed. However, if the NIMA mitosis-promoting kinase is inactivated then non-tyrosine-phosphorylated ...

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

    OpenAIRE

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

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint...

  7. Cisplatin-Induced DNA Damage Activates Replication Checkpoint Signaling Components that Differentially Affect Tumor Cell SurvivalS⃞

    OpenAIRE

    Wagner, Jill M.; Karnitz, Larry M.

    2009-01-01

    Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical r...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  10. Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells.

    Science.gov (United States)

    Takubo, Keiyo; Nagamatsu, Go; Kobayashi, Chiharu I; Nakamura-Ishizu, Ayako; Kobayashi, Hiroshi; Ikeda, Eiji; Goda, Nobuhito; Rahimi, Yasmeen; Johnson, Randall S; Soga, Tomoyoshi; Hirao, Atsushi; Suematsu, Makoto; Suda, Toshio

    2013-01-01

    Defining the metabolic programs that underlie stem cell maintenance will be essential for developing strategies to manipulate stem cell capacity. Mammalian hematopoietic stem cells (HSCs) maintain cell cycle quiescence in a hypoxic microenvironment. It has been proposed that HSCs exhibit a distinct metabolic phenotype under these conditions. Here we directly investigated this idea using metabolomic analysis and found that HSCs generate adenosine-5'-triphosphate by anaerobic glycolysis through a pyruvate dehydrogenase kinase (Pdk)-dependent mechanism. Elevated Pdk expression leads to active suppression of the influx of glycolytic metabolites into mitochondria. Pdk overexpression in glycolysis-defective HSCs restored glycolysis, cell cycle quiescence, and stem cell capacity, while loss of both Pdk2 and Pdk4 attenuated HSC quiescence, glycolysis, and transplantation capacity. Moreover, treatment of HSCs with a Pdk mimetic promoted their survival and transplantation capacity. Thus, glycolytic metabolic status governed by Pdk acts as a cell cycle checkpoint that modulates HSC quiescence and function. PMID:23290136

  11. Assays Used to Study the DNA Replication Checkpoint in Fission Yeast

    OpenAIRE

    Noguchi, Eishi; Ansbach, Alison B.; Noguchi, Chiaki; Russell, Paul

    2009-01-01

    The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components a...

  12. Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

    OpenAIRE

    De Souza, Colin P. C.; Ye, Xiang S.; Osmani, Stephen A.

    1999-01-01

    The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode ...

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

    Directory of Open Access Journals (Sweden)

    Tanzima Zerin Islam

    2013-01-01

    Full Text Available 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 of 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.

  14. Action-oriented use of ergonomic checkpoints for healthy work design in different settings.

    Science.gov (United States)

    Kogi, Kazutaka

    2007-12-01

    Recent experiences in the action-oriented use of ergonomic checkpoints in different work settings are reviewed. The purpose is to know what features are useful for healthy work design adjusted to each local situation. Based on the review results, common features of ergonomic checkpoints used in participatory training programs for improving workplace conditions in small enterprises, construction sites, home work and agriculture in industrially developing countries in Asia are discussed. These checkpoints generally compile practical improvement options in a broad range of technical areas, such as materials handling, workstation design, physical environment and work organization. Usually, "action checklists" comprising the tiles of the checkpoints are used together. A clear focus is placed on readily applicable low-cost options. Three common features of these various checkpoints appear to be important. First, the checkpoints represent typical good practices in multiple areas. Second, each how-to section of these checkpoints presents simple improvements reflecting basic ergonomic principles. Examples of these principles include easy reach, fewer and faster transport, elbow-level work, coded displays, isolated or screened hazards and shared teamwork. Third, the illustrated checkpoints accompanied by corresponding checklists are used as group work tools in short-term training courses. Many practical improvements achieved are displayed in websites for inter-country work improvement networks. It is suggested to promote the use of locally adjusted checkpoints in various forms of participatory action-oriented training in small-scale workplaces and in agriculture particularly in industrially developing countries.

  15. Action-oriented use of ergonomic checkpoints for healthy work design in different settings.

    Science.gov (United States)

    Kogi, Kazutaka

    2007-12-01

    Recent experiences in the action-oriented use of ergonomic checkpoints in different work settings are reviewed. The purpose is to know what features are useful for healthy work design adjusted to each local situation. Based on the review results, common features of ergonomic checkpoints used in participatory training programs for improving workplace conditions in small enterprises, construction sites, home work and agriculture in industrially developing countries in Asia are discussed. These checkpoints generally compile practical improvement options in a broad range of technical areas, such as materials handling, workstation design, physical environment and work organization. Usually, "action checklists" comprising the tiles of the checkpoints are used together. A clear focus is placed on readily applicable low-cost options. Three common features of these various checkpoints appear to be important. First, the checkpoints represent typical good practices in multiple areas. Second, each how-to section of these checkpoints presents simple improvements reflecting basic ergonomic principles. Examples of these principles include easy reach, fewer and faster transport, elbow-level work, coded displays, isolated or screened hazards and shared teamwork. Third, the illustrated checkpoints accompanied by corresponding checklists are used as group work tools in short-term training courses. Many practical improvements achieved are displayed in websites for inter-country work improvement networks. It is suggested to promote the use of locally adjusted checkpoints in various forms of participatory action-oriented training in small-scale workplaces and in agriculture particularly in industrially developing countries. PMID:18572793

  16. The Spindle Assembly Checkpoint Safeguards Genomic Integrity of Skeletal Muscle Satellite Cells

    Directory of Open Access Journals (Sweden)

    Swapna Kollu

    2015-06-01

    Full Text Available 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. p21CIP1 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, p21CIP1-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.

  17. The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis.

    Science.gov (United States)

    Salmela, Anna-Leena; Pouwels, Jeroen; Kukkonen-Macchi, Anu; Waris, Sinikka; Toivonen, Pauliina; Jaakkola, Kimmo; Mäki-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',5-dihydroxy-4',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.

  18. The flavonoid eupatorin inactivates the mitotic checkpoint leading to polyploidy and apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Salmela, Anna-Leena [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Turku Graduate School of Biomedical Sciences, Turku (Finland); Turku Centre for Biotechnology, P.O. Box 123, University of Turku (Finland); Pouwels, Jeroen; Kukkonen-Macchi, Anu [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Waris, Sinikka; Toivonen, Pauliina [Turku Centre for Biotechnology, P.O. Box 123, University of Turku (Finland); Jaakkola, Kimmo [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Maeki-Jouppila, Jenni [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Turku Centre for Biotechnology, P.O. Box 123, University of Turku (Finland); Drug Discovery Graduate School, University of Turku (Finland); Kallio, Lila, E-mail: lila.kallio@vtt.fi [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Kallio, Marko J. [VTT Technical Research Centre of Finland, Medical Biotechnology, P.O. Box 106, Turku (Finland); Turku Centre for Biotechnology, P.O. Box 123, University of Turku (Finland); Centre of Excellence for Translational Genome-Scale Biology, P.O. Box 106, Academy of Finland (Finland)

    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.

  19. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    , no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-specific expression of CK-2 at the mRNA and at the protein level has also been given attention. The fact that the enzyme activity is surprisingly high in brain and low in heart and lung may be indicative of involvement of CK-2 in processes other than proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)...

  20. Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

    DEFF Research Database (Denmark)

    Sedgwick, Garry G; Larsen, Marie Sofie Yoo; Lischetti, Tiziana;

    2016-01-01

    The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly t...

  1. Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A

    NARCIS (Netherlands)

    Wolthuis, Rob; Clay-Farrace, Lori; van Zon, Wouter; Yekezare, Mona; Koop, Lars; Ogink, Janneke; Medema, Rene; Pines, Jonathon

    2008-01-01

    Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin 131 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase

  2. Centrosome-associated regulators of the G2/M checkpoint as targets for cancer therapy

    Directory of Open Access Journals (Sweden)

    Broaddus Russell R

    2009-02-01

    Full Text Available Abstract In eukaryotic cells, control mechanisms have developed that restrain cell-cycle transitions in response to stress. These regulatory pathways are termed cell-cycle checkpoints. The G2/M checkpoint prevents cells from entering mitosis when DNA is damaged in order to afford these cells an opportunity to repair the damaged DNA before propagating genetic defects to the daughter cells. If the damage is irreparable, checkpoint signaling might activate pathways that lead to apoptosis. Since alteration of cell-cycle control is a hallmark of tumorigenesis, cell-cycle regulators represent potential targets for therapy. The centrosome has recently come into focus as a critical cellular organelle that integrates G2/M checkpoint control and repairs signals in response to DNA damage. A growing number of G2/M checkpoint regulators have been found in the centrosome, suggesting that centrosome has an important role in G2/M checkpoint function. In this review, we discuss centrosome-associated regulators of the G2/M checkpoint, the dysregulation of this checkpoint in cancer, and potential candidate targets for cancer therapy.

  3. Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

    DEFF Research Database (Denmark)

    Hein, Jamin B; Nilsson, Jakob

    2014-01-01

    The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co-activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind...

  4. Priming the pancreatic cancer tumor microenvironment for checkpoint-inhibitor immunotherapy

    OpenAIRE

    Lutz, Eric R.; Kinkead, Heather; Jaffee, Elizabeth M.; Zheng, Lei

    2014-01-01

    Single agent immunotherapy is effective against several cancers, but has failed against poorly immunogenic cancers, including pancreatic cancer. Evaluation of pancreatic tumors following treatment with an experimental vaccine (Lutz et al. Cancer Immunology Research 2014) suggests that vaccination primes the tumor microenvironment (TME) for checkpoint-inhibitor immunotherapy, and supports a new platform for evaluating checkpoint-inhibitors in poorly immunogenic cancers.

  5. Immunotherapy of Ovarian Cancer: The Role of Checkpoint Inhibitors

    Directory of Open Access Journals (Sweden)

    Francesca De Felice

    2015-01-01

    Full Text Available Ovarian cancer is the most important cause of gynecological cancer-related mortality, with the majority of women presenting with advanced disease. Although surgery and chemotherapy can improve survival rates, it is necessary to integrate alternative strategies to improve the outcomes. Advances in understanding the role of immune system in the pathogenesis of cancer have led to the rapid evolvement of immunotherapy, which might establish a sustained immune system response against recurring cancer cells. Recently, it has emerged that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called “immune checkpoints,” which turn off the immune system. Similarly, cancer cells are able to use these checkpoints to avoid immune control and rejection. Inhibition of these inhibitory pathways represents a potent strategy in the fight against cancer and is currently under investigation with encouraging results in some cancers, such as melanoma. In ovarian cancer researches are still in an early phase, but with promising results. In this review we will explore the rationale of immunotherapy in ovarian cancer with a special focus on these emerging molecules.

  6. Cenp-meta is required for sustained spindle checkpoint

    Directory of Open Access Journals (Sweden)

    Thomas Rubin

    2014-05-01

    Full Text Available Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC. Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.

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

    Science.gov (United States)

    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.

  8. Parallelization and checkpointing of GPU applications through program transformation

    Energy Technology Data Exchange (ETDEWEB)

    Solano-Quinde, Lizandro Damian [Iowa State Univ., Ames, IA (United States)

    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 beneffited 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

  9. Parallelization and checkpointing of GPU applications through program transformation

    Energy Technology Data Exchange (ETDEWEB)

    Solano-Quinde, Lizandro Damian [Iowa State Univ., Ames, IA (United States)

    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

  10. Activation of a DNA damage checkpoint response in a TAF1-defective cell line.

    Science.gov (United States)

    Buchmann, Ann M; Skaar, Jeffrey R; DeCaprio, James A

    2004-06-01

    Although the link between transcription and DNA repair is well established, defects in the core transcriptional complex itself have not been shown to elicit a DNA damage response. Here we show that a cell line with a temperature-sensitive defect in TBP-associated factor 1 (TAF1), a component of the TFIID general transcription complex, exhibits hallmarks of an ATR-mediated DNA damage response. Upon inactivation of TAF1, ATR rapidly localized to subnuclear foci and contributed to the phosphorylation of several downstream targets, including p53 and Chk1, resulting in cell cycle arrest. The increase in p53 expression and the G(1) phase arrest could be blocked by caffeine, an inhibitor of ATR. In addition, dominant negative forms of ATR but not ATM were able to override the arrest in G(1). These results suggest that a defect in TAF1 can elicit a DNA damage response. PMID:15169897

  11. Protein Kinase D family kinases

    OpenAIRE

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lo...

  12. Probabilistic Checkpointing Protocol to Sensor Network Fault-Tolerant

    Directory of Open Access Journals (Sweden)

    Titouna Faiza

    2012-09-01

    Full Text Available A wireless sensor network WSN is a collection of autonomous sensors nodes organized into a cooperative network. A sensor node transmits the data quantity to the sink. Indeed, a failed sink may abort the overall mission of the network. Due to their crucial functions, sinks must be designed and maintained to be robust enough in order to face trouble coming from the harsh environment. Thus, as a keystone of a WSN, a sink has to be provided with ability to recover from failures. In this paper, we propose a new protocol avoiding to the sink to be a central point of failure. First, we model a sensor node failure estimation problem through a causal network. Then, we show how the checkpointing process ensures the recovery of the network. This approach reduces both energy consumption and communication bandwidth requirements, and prolongs the lifetime of WSN. Interesting results are given by simulation

  13. Attachment issues: kinetochore transformations and spindle checkpoint silencing.

    Science.gov (United States)

    Etemad, Banafsheh; Kops, Geert J P L

    2016-04-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. PMID:26947988

  14. Checkpoint Blockade in Cancer Immunotherapy: Squaring the Circle

    Directory of Open Access Journals (Sweden)

    Maria A.V. Marzolini

    2015-03-01

    Full Text Available Manipulating the complex interaction between the immune system and tumour cells has been the focus of cancer research for many years, but it is only in the past decade that significant progress has been made in the field of cancer immunotherapy resulting in clinically effective treatments. The blockade of co-inhibitory immune checkpoints, essential for maintaining lymphocyte homeostasis and self-tolerance, by immunomodulatory monoclonal antibodies has resulted in the augmentation of anti-tumour responses. The greatest successes so far have been seen with the blockade of cytotoxic T lymphocyte associated antigen-4, which has resulted in the first Phase III clinical trial showing an overall survival benefit in metastatic melanoma, and in the blockade of the programmed cell death protein-1 axis. This concise review will focus on the clinical advances made by the blockade of these two pathways and their role in current cancer treatment strategies.

  15. EMODnet MedSea Checkpoint for sustainable Blue Growth

    Science.gov (United States)

    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

  16. A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

    DEFF Research Database (Denmark)

    Menzel, Tobias; Nähse-Kumpf, Viola; Kousholt, Arne Nedergaard;

    2011-01-01

    To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2......, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main...

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

    Institute of Scientific and Technical Information of China (English)

    Jennifer Kleponis; Richard Skelton; Lei Zheng

    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-inifltrating 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.

  18. Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

    Directory of Open Access Journals (Sweden)

    Simon R Stockwell

    Full Text Available Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical

  19. A novel role for the GTPase-activating protein Bud2 in the spindle position checkpoint.

    Directory of Open Access Journals (Sweden)

    Scott A Nelson

    Full Text Available The spindle position checkpoint (SPC ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP, and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A, was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN. Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.

  20. Performance comparison of hierarchical checkpoint protocols grid computing

    Directory of Open Access Journals (Sweden)

    Ndeye Massata NDIAYE

    2012-06-01

    Full Text Available Grid infrastructure is a large set of nodes geographically distributed and connected by a communication. In this context, fault tolerance is a necessity imposed by the distribution that poses a number of problems related to the heterogeneity of hardware, operating systems, networks, middleware, applications, the dynamic resource, the scalability, the lack of common memory, the lack of a common clock, the asynchronous communication between processes. To improve the robustness of supercomputing applications in the presence of failures, many techniques have been developed to provide resistance to these faults of the system. Fault tolerance is intended to allow the system to provide service as specified in spite of occurrences of faults. It appears as an indispensable element in distributed systems. To meet this need, several techniques have been proposed in the literature. We will study the protocols based on rollback recovery. These protocols are classified into two categories: coordinated checkpointing and rollback protocols and log-based independent checkpointing protocols or message logging protocols. However, the performance of a protocol depends on the characteristics of the system, network and applications running. Faced with the constraints of large-scale environments, many of algorithms of the literature showed inadequate. Given an application environment and a system, it is not easy to identify the recovery protocol that is most appropriate for a cluster or hierarchical environment, like grid computing. While some protocols have been used successfully in small scale, they are not suitable for use in large scale. Hence there is a need to implement these protocols in a hierarchical fashion to compare their performance in grid computing. In this paper, we propose hierarchical version of four well-known protocols. We have implemented and compare the performance of these protocols in clusters and grid computing using the Omnet++ simulator

  1. Spindle assembly checkpoint protein expression correlates with cellular proliferation and shorter time to recurrence in ovarian cancer.

    LENUS (Irish Health Repository)

    McGrogan, Barbara

    2014-07-01

    Ovarian carcinoma (OC) is the most lethal of the gynecological malignancies, often presenting at an advanced stage. Treatment is hampered by high levels of drug resistance. The taxanes are microtubule stabilizing agents, used as first-line agents in the treatment of OC that exert their apoptotic effects through the spindle assembly checkpoint. BUB1-related protein kinase (BUBR1) and mitotic arrest deficient 2 (MAD2), essential spindle assembly checkpoint components, play a key role in response to taxanes. BUBR1, MAD2, and Ki-67 were assessed on an OC tissue microarray platform representing 72 OC tumors of varying histologic subtypes. Sixty-one of these patients received paclitaxel and platinum agents combined; 11 received platinum alone. Overall survival was available for all 72 patients, whereas recurrence-free survival (RFS) was available for 66 patients. Increased BUBR1 expression was seen in serous carcinomas, compared with other histologies (P = .03). Increased BUBR1 was significantly associated with tumors of advanced stage (P = .05). Increased MAD2 and BUBR1 expression also correlated with increased cellular proliferation (P < .0002 and P = .02, respectively). Reduced MAD2 nuclear intensity was associated with a shorter RFS (P = .03), in ovarian tumors of differing histologic subtype (n = 66). In this subgroup, for those women who received paclitaxel and platinum agents combined (n = 57), reduced MAD2 intensity also identified women with a shorter RFS (P < .007). For the entire cohort of patients, irrespective of histologic subtype or treatment, MAD2 nuclear intensity retained independent significance in a multivariate model, with tumors showing reduced nuclear MAD2 intensity identifying patients with a poorer RFS (P = .05).

  2. The pachytene checkpoint and its relationship to evolutionary patterns of polyploidization and hybrid sterility.

    Science.gov (United States)

    Li, X C; Barringer, B C; Barbash, D A

    2009-01-01

    Sterility is a commonly observed phenotype in interspecific hybrids. Sterility may result from chromosomal or genic incompatibilities, and much progress has been made toward understanding the genetic basis of hybrid sterility in various taxa. The underlying mechanisms causing hybrid sterility, however, are less well known. The pachytene checkpoint is a meiotic surveillance system that many organisms use to detect aberrant meiotic products, in order to prevent the production of defective gametes. We suggest that activation of the pachytene checkpoint may be an important mechanism contributing to two types of hybrid sterility. First, the pachytene checkpoint may form the mechanistic basis of some gene-based hybrid sterility phenotypes. Second, the pachytene checkpoint may be an important mechanism that mediates chromosomal-based hybrid sterility phenotypes involving gametes with non-haploid (either non-reduced or aneuploid) chromosome sets. Studies in several species suggest that the strength of the pachytene checkpoint is sexually dimorphic, observations that warrant future investigation into whether such variation may contribute to differences in patterns of sterility between male and female interspecific hybrids. In addition, plants seem to lack the pachytene checkpoint, which correlates with increased production of unreduced gametes and a higher incidence of polyploid species in plants versus animals. Although the pachytene checkpoint occurs in many animals and in fungi, at least some of the genes that execute the pachytene checkpoint are different among organisms. This finding suggests that the penetrance of the pachytene checkpoint, and even its presence or absence can evolve rapidly. The surprising degree of evolutionary flexibility in this meiotic surveillance system may contribute to the observed variation in patterns of hybrid sterility and in rates of polyploidization.

  3. Thyroid hormone receptor interacting protein 13 (TRIP13) AAA-ATPase is a novel mitotic checkpoint-silencing protein.

    Science.gov (United States)

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

    2014-08-22

    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 p31(comet). 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 p31(comet)-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.

  4. The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds.

    Science.gov (United States)

    Sasi, Nanda Kumar; Tiwari, Kanchan; Soon, Fen-Fen; Bonte, Dorine; Wang, Tong; Melcher, Karsten; Xu, H Eric; Weinreich, Michael

    2014-01-01

    Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.

  5. The potent Cdc7-Dbf4 (DDK kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds.

    Directory of Open Access Journals (Sweden)

    Nanda Kumar Sasi

    Full Text Available Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA. We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.

  6. Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies.

    Science.gov (United States)

    Sanmamed, M F; Chester, C; Melero, I; Kohrt, H

    2016-07-01

    The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy. PMID:26912558

  7. Coupling end resection with the checkpoint response at DNA double-strand breaks.

    Science.gov (United States)

    Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

    2016-10-01

    DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast. PMID:27141941

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

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K;

    2015-01-01

    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...

  9. Cambridge checkpoint English revision guide for the Cambridge secondary 1 test

    CERN Document Server

    Reynolds, John

    2013-01-01

    With Checkpoint English Revision Guide for the Cambridge Secondary 1 test you can aim for the best grade with the help of relevant and accessible notes, examiner advice plus questions and answers on each key topic. - Clear explanations of every topic covered in the Cambridge Secondary 1 Checkpoint English syllabus. - Builds revision skills you need for success in the test. - Exam tips wirtten by test setters and examiners giving you their expert advice. This text has not been through the Cambridge endorsement process.

  10. A conserved checkpoint monitors meiotic chromosome synapsis inCaenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Cambridge checkpoint maths revision guide for the Cambridge secondary 1 test

    CERN Document Server

    Smith, Alan

    2013-01-01

    With Checkpoint Maths Revision Guide for the Cambridge Secondary 1 test you can aim for the best grade with the help of relevant and accessible notes, examiner advice plus questions and answers on each key topic. - Clear explanations of every topic covered in the Cambridge Secondary 1 Checkpoint Maths syllabus. - Builds revision skills you need for success in the test. - Exam tips wirtten by test setters and examiners giving you their expert advice. This text has not been through the Cambridge endorsement process.

  12. Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation.

    Directory of Open Access Journals (Sweden)

    Katherine L Furniss

    Full Text Available By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.

  13. The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80

    OpenAIRE

    Tung, Kuei-Shu; Hong, Eun-Jin Erica; Roeder, G. Shirleen

    2000-01-01

    In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase. We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I. Herein, we show that the Ndt80 protein accumulates a...

  14. A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint

    OpenAIRE

    Hong, Eun-Jin Erica; Roeder, G. Shirleen

    2002-01-01

    The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates. We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae. During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs). Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair. The Rad24 che...

  15. A Minimum-Process Coordinated Checkpointing Protocol For Mobile Distributed System

    Directory of Open Access Journals (Sweden)

    Praveen Kumar

    2010-05-01

    Full Text Available While dealing with Mobile Distributed systems, we come across some issues like: mobility, low bandwidth of wireless channels and lack of stable storage on mobile nodes, disconnections, limited battery power and high failure rate of mobile nodes. These issues make traditional checkpointing techniques designed for Distributed systems unsuitable for Mobile environments. In this paper, we design a minimum process algorithm for Mobile Distributed systems, where no useless checkpoints are taken and an effort has been made to optimize the blocking of processes. We propose to delay the processing of selective messages at the receiver end only during the checkpointing period. A Process is allowed to perform its normal computations and send messages during its blocking period. In this way, we try to keep blocking of processes to bare minimum. We captured the transitive dependencies during the normal execution by piggybacking dependency vectors onto computational messages. In this way, we try to reduce the Checkpointing time by avoiding formation of Checkpointing tree. The Z-dependencies are well taken care of. The proposed scheme forces zero useless checkpoints at the cost of very small blocking.

  16. RAG-mediated DNA double-strand breaks activate a cell type-specific checkpoint to inhibit pre-B cell receptor signals.

    Science.gov (United States)

    Bednarski, Jeffrey J; Pandey, Ruchi; Schulte, Emily; White, Lynn S; Chen, Bo-Ruei; Sandoval, Gabriel J; Kohyama, Masako; Haldar, Malay; Nickless, Andrew; Trott, Amanda; Cheng, Genhong; Murphy, Kenneth M; Bassing, Craig H; Payton, Jacqueline E; Sleckman, Barry P

    2016-02-01

    DNA double-strand breaks (DSBs) activate a canonical DNA damage response, including highly conserved cell cycle checkpoint pathways that prevent cells with DSBs from progressing through the cell cycle. In developing B cells, pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly, leading to RAG-mediated DNA DSBs. The pre-BCR also promotes cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here, we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor, resulting in suppression of pre-BCR signaling. This regulatory circuit prevents the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and a RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes. PMID:26834154

  17. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  18. Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

    Directory of Open Access Journals (Sweden)

    Wen-Bin Ou

    2011-01-01

    Full Text Available The receptor tyrosine kinases (RTKs epidermal growth factor receptor (EGFR and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3 in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90. Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17demethoxygeldanamycin (17-AAG had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of down-stream signaling intermediates (AKT, mitogen-activated protein kinase, and S6; upregulated the p53, p21, and p27 cell cycle checkpoints; induced G2 phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G1 apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.

  19. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

    Directory of Open Access Journals (Sweden)

    Smith Charlotte M

    2013-01-01

    Full Text Available Abstract Background During metaphase clathrin stabilises the mitotic spindle kinetochore(K-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

  20. The complement of protein kinases of the microsporidium Encephalitozoon cuniculi in relation to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Vivares Christian P

    2007-09-01

    . The difficulty in assigning clear homology relationships for nine out of the twenty-nine microsporidian conventional protein kinases despite its compact genome reflects the phylogenetic distance between microsporidia and other eukaryotes. Indeed, the E. cuniculi genome presents a high proportion of genes in which evolution has been accelerated by up to four-fold. There are no orthologues of the protein kinases that constitute MAP kinase pathways and many other protein kinases with roles in nutrient signalling are absent from the E. cuniculi kinome. However, orthologous kinases can nonetheless be identified that correspond to members of the yeast kinomes with roles in some of the most fundamental cellular processes. For example, E. cuniculi has clear orthologues of virtually all the major conserved protein kinases that regulate the core cell cycle machinery (Aurora, Polo, DDK, CDK and Chk1. A comprehensive comparison of the homology relationships between the budding and fission yeast kinomes indicates that, despite an estimated 800 million years of independent evolution, the two model fungi share ~85% of their protein kinases. This will facilitate the annotation of many of the as yet uncharacterised fission yeast kinases, and also those of novel fungal genomes.

  1. Smurf2 as a novel mitotic regulator: From the spindle assembly checkpoint to tumorigenesis

    Directory of Open Access Journals (Sweden)

    Moore Finola E

    2009-07-01

    Full Text Available Abstract The execution of the mitotic program with high fidelity is dependent upon precise spatiotemporal regulation of posttranslational protein modifications. For example, the timely polyubiquitination of critical mitotic regulators by Anaphase Promoting Complex/Cyclosome (APC/C is essential for the metaphase to anaphase transition and mitotic exit. The spindle assembly checkpoint prevents unscheduled activity of APC/C-Cdc20 in early mitosis, allowing bipolar attachment of kinetochores to mitotic spindle and facilitating equal segregation of sister chromatids. The critical effector of the spindle checkpoint, Mitotic arrest deficient 2 (Mad2, is recruited to unattached kinetochores forming a complex with other regulatory proteins to efficiently and cooperatively inhibit APC/C-Cdc20. A weakened and/or dysfunctional spindle checkpoint has been linked to the development of genomic instability in both cell culture and animal models, and evidence suggests that aberrant regulation of the spindle checkpoint plays a critical role in human carcinogenesis. Recent studies have illuminated a network of both degradative and non-degradative ubiquitination events that regulate the metaphase to anaphase transition and mitotic exit. Within this context, our recent work showed that the HECT (Homologous to E6-AP C-terminus-family E3 ligase Smurf2 (Smad specific ubiquitin regulatory factor 2, known as a negative regulator of transforming growth factor-beta (TGF-β signaling, is required for a functional spindle checkpoint by promoting the functional localization and stability of Mad2. Here we discuss putative models explaining the role of Smurf2 as a new regulator in the spindle checkpoint. The dynamic mitotic localization of Smurf2 to the centrosome and other critical mitotic structures provides implications about mitotic checkpoint control dependent on various ubiquitination events. Finally, deregulated Smurf2 activity may contribute to carcinogenesis by

  2. In-silico modeling of the mitotic spindle assembly checkpoint.

    Directory of Open Access Journals (Sweden)

    Bashar Ibrahim

    Full Text Available BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((MSAC is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.

  3. Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

    Science.gov (United States)

    Ahmed, Shakil; Dul, Barbara; Qiu, Xinxing; Walworth, Nancy C

    2007-11-01

    As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage. PMID:17947424

  4. Combining targeted therapy and immune checkpoint inhibitors in the treatment of metastatic melanoma

    Institute of Scientific and Technical Information of China (English)

    Teresa Kim; Rodabe N Amaria; Christine Spencer; Alexandre Reuben; Zachary A Cooper; Jennifer A Wargo

    2014-01-01

    Melanoma is the deadliest form of skin cancer and has an incidence that is rising faster than any other solid tumor. Metastatic melanoma treatment has considerably progressed in the past ifve years with the introduction of targeted therapy (BARF and MEK inhibitors) and immune checkpoint blockade (anti-CTLA4, anti-PD-1, and anti-PD-L1). However, each treatment modality has limitations. Treatment with targeted therapy has been associated with a high response rate, but with short-term responses. Conversely, treatment with immune checkpoint blockade has a lower response rate, but with long-term responses. Targeted therapy affects antitumor immunity, and synergy may exist when targeted therapy is combined with immunotherapy. hTis article presents a brief review of the rationale and evidence for the potential synergy between targeted therapy and immune checkpoint blockade. Challenges and directions for future studies are also proposed.

  5. Immune checkpoints aberrations and gastric cancer; assessment of prognostic value and evaluation of therapeutic potentials.

    Science.gov (United States)

    Abdel-Rahman, Omar

    2016-01-01

    Till now, the prognosis of advanced gastric cancer looked dreadful; thus the search for newer better approaches for this lethal disease has been a strategic target for cancer researchers. In recent years, important immunobiological aspects of the tumor have been revealed with the subsequent proposal of immune check point inhibitors to target these pathways. Clinically, unselected use of immune checkpoint inhibitors in gastric cancer has been deemed with failure; in contrast to the clear success of more recent studies reporting on the use of pembrolizumab in molecularly selected patients. This may illustrate that any future use of immune checkpoint inhibitors in gastric cancer has to be molecularly supported. This review provides a delicate dissection of the clinical and immunobiological considerations underlying the use of these agents in addition to a thorough review of the published clinical data of immune checkpoint inhibitors in gastric cancer. PMID:26321371

  6. Hierarchical Non-blocking Coordinated Checkpointing Algorithms for Mobile Distributed Computing

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    2010-01-01

    Full Text Available Mobile system typically uses wireless communication which is based on electromagnetic waves and utilizes a shared broadcast medium. This has made possible creating a mobile distributed computing environment and has brought us several new challenges in distributed protocol design. So many issues such as range of transmission, limited power supply due to battery capacity and mobility of processes. These new issue makes traditional recovery algorithm unsuitable. In this paper, we propose hierarchical non blocking coordinated checkpointing algorithms suitable for mobile distributed computing. The algorithm is non-blocking, requires minimum message logging, has minimum stable storage requirement and produce a consistent set of checkpoints. This algorithm requires minimum number of processes to take checkpoint.

  7. Sustained spindle-assembly checkpoint response requires de novo transcription and translation of cyclin B1.

    Directory of Open Access Journals (Sweden)

    Ana Lúcia Mena

    Full Text Available BACKGROUND: Microtubule-targeting drugs induce mitotic delay at pro-metaphase by preventing the spindle assembly checkpoint to be satisfied. However, especially after prolonged treatments, cells can escape this arrest in a process called mitotic slippage. The mechanisms underlying the spindle assembly checkpoint and slippage are not fully understood. It has been generally accepted that during mitosis there is a temporary shutdown of high-energy-consuming processes, such as transcription and translation. However, the synthesis of specific proteins is maintained or up-regulated since protein synthesis is necessary for entry into and progression through mitosis. METHODOLOGY/PRINCIPAL FINDINGS: In this work we investigated whether the mitotic arrest caused by the mitotic checkpoint is independent of transcription and translation. By using immunofluorescent microscopy and western blotting, we demonstrate that inhibition of either of these processes induces a shortening of the mitotic arrest caused by the nocodazole treatment, and ultimately leads to mitotic slippage. Our western blotting and RTQ-PCR results show that inhibition of transcription during mitotic arrest does not affect the expression of the spindle checkpoint proteins, whereas it induces a significant decrease in the mRNA and protein levels of Cyclin B1. The exogenous expression of Cyclin B1 substantially rescued the mitotic phenotype in nocodazole cells treated with the inhibitors of transcription and translation. CONCLUSIONS/SIGNIFICANCE: This work emphasizes the importance of transcription and translation for the maintenance of the spindle assembly checkpoint, suggesting the existence of a mechanism dependent on cyclin B1 gene regulation during mitosis. We propose that continuous transcription of mitotic regulators is required to sustain the activation of the spindle assembly checkpoint.

  8. CK (Creatine Kinase) Test

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Creatine Kinase Share this page: Was this page helpful? Also known as: CK; Total CK; Creatine Phosphokinase; CPK Formal name: Creatine Kinase Related tests: ...

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

    International Nuclear Information System (INIS)

    Highlights: ► RNAi is linked to the cell cycle checkpoint in fission yeast. ► Ptr1 co-purifies with Ago1. ► The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. ► ago1+ and ptr1+ regulate the cell cycle checkpoint via the same pathway. ► Mutations in ago1+ and ptr1+ lead to the nuclear accumulation of poly(A)+ 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+, the overexpression of ago1+ alleviated the cell cycle defect in dcr1Δ. 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+ is dependent on ptr1+. Nuclear accumulation of poly(A)+ RNAs was detected in mutants of ago1+ and ptr1+, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  10. Functions of spindle check-point and its relationship to chromosome instability

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    It is generally believed that the equal distribution of genetic materials to two daughter cells during mitosis is the key to cell health and development. During the dynamic process, spindle checkpoint plays a very important role in chromosome movements and final sister chromatid separation. The equal and precise segregation of chromosomes contributes to the genomic stability while aberrant separations result in chromosome instability that causes pathogenesis of certain diseases such as Down's syndrome and cancers. Kinetochore and its regulatory proteins consist of the spindle checkpoint and determine the spatial and temporal orders of chromosome segregation.

  11. A direct role of Mad1 in the spindle assembly checkpoint beyond Mad2 kinetochore recruitment

    DEFF Research Database (Denmark)

    Kruse, Thomas; Larsen, Marie Sofie Yoo; Sedgwick, Garry G;

    2014-01-01

    The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi-oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O-Mad2) or active closed (C-Mad2) conformation...... in the SAC beyond recruitment of C-Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C-Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C-terminal globular...

  12. Timeless links replication termination to mitotic kinase activation.

    Science.gov (United States)

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-05-06

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  13. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu

    Full Text Available The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1. Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  14. Cross talk of tyrosine kinases with the DNA damage signaling pathways.

    Science.gov (United States)

    Mahajan, Kiran; Mahajan, Nupam P

    2015-12-15

    Tyrosine kinases respond to extracellular and intracellular cues by activating specific cellular signaling cascades to regulate cell cycle, growth, proliferation, differentiation and survival. Likewise, DNA damage response proteins (DDR) activated by DNA lesions or chromatin alterations recruit the DNA repair and cell cycle checkpoint machinery to restore genome integrity and cellular homeostasis. Several new examples have been uncovered in recent studies which reveal novel epigenetic and non-epigenetic mechanisms by which tyrosine kinases interact with DDR proteins to dictate cell fate, i.e. survival or apoptosis, following DNA damage. These studies reveal the ability of tyrosine kinases to directly regulate the activity of DNA repair and cell cycle check point proteins by tyrosine phosphorylation. In addition, tyrosine kinases epigenetically regulate DNA damage signaling pathways by modifying the core histones as well as chromatin modifiers at critical tyrosine residues. Thus, deregulated tyrosine kinase driven epigenomic alterations have profound implications in cancer, aging and genetic disorders. Consequently, targeting oncogenic tyrosine kinase induced epigenetic alterations has gained significant traction in overcoming cancer cell resistance to various therapies. This review discusses mechanisms by which tyrosine kinases interact with DDR pathways to regulate processes critical for maintaining genome integrity as well as clinical strategies for targeted cancer therapies. PMID:26546517

  15. Naphthalimides Induce G2 Arrest Through the ATM-Activated Chk2-Executed Pathway in HCT116 Cells

    Directory of Open Access Journals (Sweden)

    Hong Zhu

    2009-11-01

    Full Text Available Naphthalimides, particularly amonafide and 2-(2-dimethylamino-6-thia-2-aza-benzo[def]chrysene-1,3-diones (R16, have been identified to possess anticancer activities and to induce G2-M arrest through inhibiting topoisomerase II accompanied by Chk1 degradation. The current study was designed to precisely dissect the signaling pathway(s responsible for the naphthalimide-induced cell cycle arrest in human colon carcinoma HCT116 cells. Using phosphorylated histone H3 and mitotic protein monoclonal 2 as mitosis markers, we first specified the G2 arrest elicited by the R16 and amonafide. Then, R16 and amonafide were revealed to induce phosphorylation of the DNA damage sensor ataxia telangiectasia-mutated (ATM responding to DNA double-strand breaks (DSBs. Inhibition of ATM by both the pharmacological inhibitor caffeine and the specific small interference RNA (siRNA rescued the G2 arrest elicited by R16, indicating its ATM-dependent characteristic. Furthermore, depletion of Chk2, but not Chk1 with their corresponding siRNA, statistically significantly reversed the R16- and amonafide-triggered G2 arrest. Moreover, the naphthalimides phosphorylated Chk2 in an ATM-dependent manner but induced Chk1 degradation. These data indicate that R16 and amonafide preferentially used Chk2 as evidenced by the differential ATM-executed phosphorylation of Chk1 and Chk2. Thus, a clear signaling pathway can be established, in which ATM relays the DNA DSBs signaling triggered by the naphthalimides to the checkpoint kinases, predominantly to Chk2,which finally elicits G2 arrest. The mechanistic elucidation not only favors the development of the naphthalimides as anticancer agents but also provides an alternative strategy of Chk2 inhibition to potentiate the anticancer activities of these agents.

  16. Spindle checkpoint deficiency is tolerated by murine epidermal cells but not hair follicle stem cells

    NARCIS (Netherlands)

    Foijer, Floris; DiTommaso, Tia; Donati, Giacomo; Hautaviita, Katta; Xie, Stephanie Z.; Heath, Emma; Smyth, Ian; Watt, Fiona M.; Sorger, Peter K.; Bradley, Allan

    2013-01-01

    The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-me

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

    Science.gov (United States)

    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.

  18. Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    NARCIS (Netherlands)

    Etemad, B.; Kuijt, T.E.F.; Kops, G.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

  19. Constitutive Cdk2 activity promotes aneuploidy while altering the spindle assembly and tetraploidy checkpoints

    DEFF Research Database (Denmark)

    Jahn, Stephan C; Corsino, Patrick E; Davis, Bradley J;

    2013-01-01

    instability. Expression of these complexes in the MCF10A cell line leads to retinoblastoma protein (Rb) hyperphosphorylation, a subsequent increase in proliferation rate, and increased expression of the spindle assembly checkpoint protein Mad2. This results in a strengthening of the spindle assembly...

  20. Synthesis of Fault-Tolerant Embedded Systems with Checkpointing and Replication

    DEFF Research Database (Denmark)

    Izosimov, Viacheslav; Pop, Paul; Eles, Petru;

    2006-01-01

    -triggered protocol. Our synthesis approach decides the assignment of fault-tolerance policies to processes, the optimal placement of checkpoints and the mapping of processes to processors such that transient faults are tolerated and the timing constraints of the application are satisfied. We present several...

  1. Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Bhatt, Shweta; Gupta, Manoj; Khamaisi, Mogher; Martinez, Rachael; Gritsenko, Marina A.; Wagner, Bridget; Guye, Patrick; Busskamp, Volker; Shirakawa, Jun; Wu, Gongxiong; Liew, Chong Wee; Clauss, Therese RW; Valdez, Ivan; EL Ouaaman, Abdelfattah; Dirice, Ercument; Takatani, Tomozumi; Keenan, Hillary; Smith, Richard D.; Church, George; Weiss, Ron; Wagers, Amy J.; Qian, Weijun; King, George L.; Kulkami, Rohit N.

    2015-08-04

    Themechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D(disease durationR50 years) with severe (Medalist +C) or absent to mild complications (Medalist *C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist *C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage.WeproposemiR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

  2. Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

    International Nuclear Information System (INIS)

    Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of γ-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

  3. Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, Pilar [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Barquinero, Joan Francesc [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Duran, Assumpta [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Caballin, Maria Rosa [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Ribas, Montserrat [Servei de Radiofisica i Radioproteccio de l' Hospital de la Santa Creu i Sant Pau, 08025 Barcelona (Spain); Barrios, Leonardo, E-mail: Lleonard.Barrios@uab.cat [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain)

    2009-11-02

    Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of {gamma}-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

  4. Studying S-phase DNA Damage Checkpoints using the Fission Yeast Schizosaccharomyces pombe

    Science.gov (United States)

    Willis, Nicholas; Rhind, Nicholas

    2016-01-01

    Slowing of replication in response to DNA damage is a universal response to DNA damage during S-phase. Originally discovered to be defective in checkpoint mutant cells in metazoans, this S-phase DNA damage checkpoint response has been extensively studied in yeast. Unlike other checkpoints that completely arrest cell cycle, the S-phase DNA damage checkpoint slows but does not completely halt replication in response to DNA damage. An analysis of mutants defective in the slowing response requires a sensitive assay to measure this quantitative effect. The use of centrifugal elutriation to synchronize cells and improved techniques in preparing cells for flow cytometry allow for more sensitive and accurate measurement of cells’ ability to slow replication in the presence of DNA damage. This chapter describes the use of transient cdc10-M17 temperature sensitive allele arrest and release combined with centrifugal elutriation to synchronize cells in G1. The S-phase progression of these cells is then assayed by flow cytometry of isolated nuclei, which allows sensitive determination of replication kinetics. PMID:21870281

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

    Science.gov (United States)

    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

  6. Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

    OpenAIRE

    Harris, Peter S; Venkataraman, Sujatha; Alimova, Irina; Birks, Diane K; Balakrishnan, Ilango; Cristiano, Brian; Donson, Andrew M; Dubuc, Adrian M.; Taylor, Michael D.; Foreman, Nicholas K.; Reigan, Philip; Vibhakar, Rajeev

    2014-01-01

    Background Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. Methods To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle...

  7. Selective disruption of aurora C kinase reveals distinct functions from aurora B kinase during meiosis in mouse oocytes.

    Directory of Open Access Journals (Sweden)

    Ahmed Z Balboula

    2014-02-01

    Full Text Available Aurora B kinase (AURKB is the catalytic subunit of the chromosomal passenger complex (CPC, an essential regulator of chromosome segregation. In mitosis, the CPC is required to regulate kinetochore microtubule (K-MT attachments, the spindle assembly checkpoint, and cytokinesis. Germ cells express an AURKB homolog, AURKC, which can also function in the CPC. Separation of AURKB and AURKC function during meiosis in oocytes by conventional approaches has not been successful. Therefore, the meiotic function of AURKC is still not fully understood. Here, we describe an ATP-binding-pocket-AURKC mutant, that when expressed in mouse oocytes specifically perturbs AURKC-CPC and not AURKB-CPC function. Using this mutant we show for the first time that AURKC has functions that do not overlap with AURKB. These functions include regulating localized CPC activity and regulating chromosome alignment and K-MT attachments at metaphase of meiosis I (Met I. We find that AURKC-CPC is not the sole CPC complex that regulates the spindle assembly checkpoint in meiosis, and as a result most AURKC-perturbed oocytes arrest at Met I. A small subset of oocytes do proceed through cytokinesis normally, suggesting that AURKC-CPC is not the sole CPC complex during telophase I. But, the resulting eggs are aneuploid, indicating that AURKC is a critical regulator of meiotic chromosome segregation in female gametes. Taken together, these data suggest that mammalian oocytes contain AURKC to efficiently execute meiosis I and ensure high-quality eggs necessary for sexual reproduction.

  8. Complex Haploinsufficiency-Based Genetic Analysis of the NDR/Lats Kinase Cbk1 Provides Insight into Its Multiple Functions in Candida albicans.

    Science.gov (United States)

    Saputo, Sarah; Norman, Kaitlyn L; Murante, Thomas; Horton, Brooke N; Diaz, Jacinto De La Cruz; DiDone, Louis; Colquhoun, Jennifer; Schroeder, Jeremy W; Simmons, Lyle A; Kumar, Anuj; Krysan, Damian J

    2016-07-01

    Although the analysis of genetic interactions and networks is a powerful approach to understanding biology, it has not been applied widely to the pathogenic yeast Candida albicans Here, we describe the use of both screening and directed genetic interaction studies based on complex haploinsufficiency to probe the function of the R: egulation of A: ce2 and M: orphogenesis (RAM) pathway in C. albicans A library of 5200 Tn7-mutagenized derivatives of a parental strain heterozygous at CBK1, the key kinase in the RAM pathway, was screened for alterations in serum-induced filamentation. Following confirmation of phenotypes and identification of insertion sites by sequencing, a set of 36 unique double heterozygous strains showing complex haploinsufficiency was obtained. In addition to a large set of genes regulated by the RAM transcription factor Ace2, genes related to cell wall biosynthesis, cell cycle, polarity, oxidative stress, and nitrogen utilization were identified. Follow-up analysis led to the first demonstration that the RAM pathway is required for oxidative stress tolerance in a manner related to the two-component-regulated kinase Chk1 and revealed a potential direct connection between the RAM pathway and the essential Mps1 spindle pole-related kinase. In addition, genetic interactions with CDC42-related genes MSB1, a putative scaffold protein, and RGD3, a putative Rho GTPase-activating protein (GAP) were identified. We also provide evidence that Rgd3 is a GAP for Cdc42 and show that its localization and phosphorylation are dependent on Cbk1. PMID:27206715

  9. The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

    DEFF Research Database (Denmark)

    Lischetti, Tiziana; Zhang, Gang; Sedgwick, Garry G;

    2014-01-01

    Improperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyse the binding of two checkpoint proteins, Mad2 and BubR1, to Cdc20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of Cdc20 kinetochore localization...... on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second Cdc20 binding site in BubR1 in promoting both efficient SAC signalling and SAC...

  10. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael;

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea...... that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell....

  11. Collective synchronization of divisions in Drosophila development

    Science.gov (United States)

    Vergassola, Massimo

    Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

  12. Cdc7p-Dbf4p regulates mitotic exit by inhibiting Polo kinase.

    Directory of Open Access Journals (Sweden)

    Charles T Miller

    2009-05-01

    Full Text Available Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN, presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.

  13. Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

    Directory of Open Access Journals (Sweden)

    Jessica W Luzwick

    Full Text Available Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

  14. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    Science.gov (United States)

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  15. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  16. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age

    OpenAIRE

    Marangos, P; Stevense, M.; Niaka, K.; Lagoudaki, M.; Nabti, I.; Jessberger, R.; Carroll, J.

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes fro...

  17. Overview and management of toxicities of immune checkpoint-blocking drugs

    Directory of Open Access Journals (Sweden)

    Economopoulou Panagiota

    2016-03-01

    Full Text Available Immunotherapy is considered to be the most important breakthrough in cancer management in the past few years. This success was based on the scientific understanding of immune mechanisms due to improvement in preclinical science and the introduction of new methods of investigation. Immune checkpoint inhibitors (ICIs are among the most promising drugs in the field of immune-oncology; they represent monoclonal antibodies that modulate the effects of immune checkpoints, such as cytotoxic T lymphocyte Antigen 4 (CTLA-4 and Programmed Cell Death protein 1 (PD-1, which are co-inhibitory signals responsible for immune suppression. Despite clinical benefits, ICIs are immune activating agents that are associated with a number of important side effects (immune-related adverse events-irAEs, attributed to organ-specific inflammation. Herein, we review the toxicities of ICIs, highlighting the importance of early identification and management.

  18. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis.

    Science.gov (United States)

    Dubin, Krista; Callahan, Margaret K; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G; Wolchok, Jedd D

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy. PMID:26837003

  19. Cloud object store for checkpoints of high performance computing applications using decoupling middleware

    Energy Technology Data Exchange (ETDEWEB)

    Bent, John M.; Faibish, Sorin; Grider, Gary

    2016-04-19

    Cloud object storage is enabled for checkpoints of high performance computing applications using a middleware process. A plurality of files, such as checkpoint files, generated by a plurality of processes in a parallel computing system are stored by obtaining said plurality of files from said parallel computing system; converting said plurality of files to objects using a log structured file system middleware process; and providing said objects for storage in a cloud object storage system. The plurality of processes may run, for example, on a plurality of compute nodes. The log structured file system middleware process may be embodied, for example, as a Parallel Log-Structured File System (PLFS). The log structured file system middleware process optionally executes on a burst buffer node.

  20. Immune Checkpoint Inhibitors: A New Opportunity in the Treatment of Ovarian Cancer?

    Directory of Open Access Journals (Sweden)

    Gloria Mittica

    2016-07-01

    Full Text Available Epithelial ovarian cancer (EOC is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70% and prognosis is globally poor. New therapeutic agents are needed to improve survival. Since EOC is strongly immunogenic, immune checkpoint inhibitors are under evaluation for their capacity to contrast the “turn off” signals expressed by the tumor to escape the immune system and usually responsible for self-tolerance maintenance. This article reviews the literature on anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies in EOC and highlights their possible lines of development. Further studies are needed to better define the prognostic role of the immune checkpoint inhibitors, to identify predictors of response and the optimal clinical setting in EOC.

  1. Social and ethical checkpoints for bottom-up synthetic biology, or protocells.

    Science.gov (United States)

    Bedau, Mark A; Parke, Emily C; Tangen, Uwe; Hantsche-Tangen, Brigitte

    2009-12-01

    An alternative to creating novel organisms through the traditional "top-down" approach to synthetic biology involves creating them from the "bottom up" by assembling them from non-living components; the products of this approach are called "protocells." In this paper we describe how bottom-up and top-down synthetic biology differ, review the current state of protocell research and development, and examine the unique ethical, social, and regulatory issues raised by bottom-up synthetic biology. Protocells have not yet been developed, but many expect this to happen within the next five to ten years. Accordingly, we identify six key checkpoints in protocell development at which particular attention should be given to specific ethical, social and regulatory issues concerning bottom-up synthetic biology, and make ten recommendations for responsible protocell science that are tied to the achievement of these checkpoints. PMID:19816801

  2. Immune Checkpoint Inhibitors: A New Opportunity in the Treatment of Ovarian Cancer?

    Science.gov (United States)

    Mittica, Gloria; Genta, Sofia; Aglietta, Massimo; Valabrega, Giorgio

    2016-01-01

    Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70%) and prognosis is globally poor. New therapeutic agents are needed to improve survival. Since EOC is strongly immunogenic, immune checkpoint inhibitors are under evaluation for their capacity to contrast the "turn off" signals expressed by the tumor to escape the immune system and usually responsible for self-tolerance maintenance. This article reviews the literature on anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies in EOC and highlights their possible lines of development. Further studies are needed to better define the prognostic role of the immune checkpoint inhibitors, to identify predictors of response and the optimal clinical setting in EOC. PMID:27447625

  3. Negative immune checkpoints on T lymphocytes and their relevance to cancer immunotherapy.

    Science.gov (United States)

    Śledzińska, Anna; Menger, Laurie; Bergerhoff, Katharina; Peggs, Karl S; Quezada, Sergio A

    2015-12-01

    The term 'inhibitory checkpoint' refers to the broad spectrum of co-receptors expressed by T cells that negatively regulate T cell activation thus playing a crucial role in maintaining peripheral self-tolerance. Co-inhibitory receptor ligands are highly expressed by a variety of malignancies allowing evasion of anti-tumour immunity. Recent studies demonstrate that manipulation of these co-inhibitory pathways can remove the immunological brakes that impede endogenous immune responses against tumours. Antibodies that block the interactions between co-inhibitory receptors and their ligands have delivered very promising clinical responses, as has been shown by recent successful trials targeting the CTLA-4 and PD-1 pathways. In this review, we discuss the mechanisms of action and expression pattern of co-inhibitory receptors on different T cells subsets, emphasising differences between CD4(+) and CD8(+) T cells. We also summarise recent clinical findings utilising immune checkpoint blockade.

  4. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis

    OpenAIRE

    Dubin, Krista; Callahan, Margaret K.; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G.; Wolchok, Jedd D.

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-med...

  5. Sudden Telomere Lengthening Triggers a Rad53-dependent Checkpoint in Saccharomyces cerevisiae

    OpenAIRE

    Viscardi, Valeria; Baroni, Enrico; Romano, Michele; Lucchini, Giovanna; Longhese, Maria Pia

    2003-01-01

    Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent...

  6. Handoff Based Secure Checkpointing and Log Based Rollback Recovery for Mobile Hosts

    Directory of Open Access Journals (Sweden)

    Priyanka Dey and Suparna Biswas

    2012-10-01

    Full Text Available An efficient fault tolerant algorithm based on movement-based secure checkpointing and logging formobile computing system is proposed here. The recovery scheme proposed here combines independentcheckpointing and message logging. Here we consider mobility rate of the user in checkpointing so thatmobile host can manage recovery information such as checkpoints and logs properly so that a mobilehost takes less recovery time after failure. Mobile hosts save checkpoints when number of hand-offexceeds a predefined hand-off threshold value. Current approaches save logs in base station. But thisapproach maximizes recovery time if message passing frequency is large. If a mobile host saves log in itsown memory, recovery cost will be less because log retrieval time will be small after failure. But there isa probability of memory crash of a mobile host. In that case logs can not be retrieved if it is saved only inmobile node. If the failure is transient then logs can be retrieved from the memory of mobile node.Hence in this algorithm mobile hosts also save log in own memory and base station. In case of crashrecovery, log will be retrieved from base station and in case of transient failure recovery logs will beretrieved from mobile host. In this algorithm recovery probability is optimized and total recovery time isreduced in comparison to existing works. Logs are very small in size. Hence saving logs in mobile hostsdoes not cause much memory overhead. Hand-off threshold is a function of mobility rate, messagepassing frequency and failure rate of mobile hosts. This algorithm describes a secure check pointingtechnique as a method for providing fault tolerance while preventing information leakage through thecheckpoint data.

  7. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    Sagar P Mahale

    Full Text Available The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC. However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division.

  8. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

    Science.gov (United States)

    Mahale, Sagar P.; Sharma, Amit; Mylavarapu, Sivaram V. S.

    2016-01-01

    The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division. PMID:27441562

  9. DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint.

    Science.gov (United States)

    Sansam, Christopher L; Shepard, Jennifer L; Lai, Kevin; Ianari, Alessandra; Danielian, Paul S; Amsterdam, Adam; Hopkins, Nancy; Lees, Jacqueline A

    2006-11-15

    Checkpoint genes maintain genomic stability by arresting cells after DNA damage. Many of these genes also control cell cycle events in unperturbed cells. By conducting a screen for checkpoint genes in zebrafish, we found that dtl/cdt2 is an essential component of the early, radiation-induced G2/M checkpoint. We subsequently found that dtl/cdt2 is required for normal cell cycle control, primarily to prevent rereplication. Both the checkpoint and replication roles are conserved in human DTL. Our data indicate that the rereplication reflects a requirement for DTL in regulating CDT1, a protein required for prereplication complex formation. CDT1 is degraded in S phase to prevent rereplication, and following DNA damage to prevent origin firing. We show that DTL associates with the CUL4-DDB1 E3 ubiquitin ligase and is required for CDT1 down-regulation in unperturbed cells and following DNA damage. The cell cycle defects of Dtl-deficient zebrafish are suppressed by reducing Cdt1 levels. In contrast, the early G2/M checkpoint defect appears to be Cdt1-independent. Thus, DTL promotes genomic stability through two distinct mechanisms. First, it is an essential component of the CUL4-DDB1 complex that controls CDT1 levels, thereby preventing rereplication. Second, it is required for the early G2/M checkpoint.

  10. Efficient checkpointing schemes for depletion perturbation solutions on memory-limited architectures

    International Nuclear Information System (INIS)

    We describe a methodology for decreasing the memory footprint and machine I/O load associated with the need to access a forward solution during an adjoint solve. Specifically, we are interested in the depletion perturbation equations, where terms in the adjoint Bateman and transport equations depend on the forward flux solution. Checkpointing is the procedure of storing snapshots of the forward solution to disk and using these snapshots to recompute the parts of the forward solution that are necessary for the adjoint solve. For large problems, however, the storage cost of just a few copies of an angular flux vector can exceed the available RAM on the host machine. We propose a methodology that does not checkpoint the angular flux vector; instead, we write and store converged source moments, which are typically of a much lower dimension than the angular flux solution. This reduces the memory footprint and I/O load of the problem, but requires that we perform single sweeps to reconstruct flux vectors on demand. We argue that this trade-off is exactly the kind of algorithm that will scale on advanced, memory-limited architectures. We analyze the cost, in terms of FLOPS and memory footprint, of five checkpointing schemes. We also provide computational results that support the analysis and show that the memory-for-work trade off does improve time to solution. (authors)

  11. Efficient checkpointing schemes for depletion perturbation solutions on memory-limited architectures

    Energy Technology Data Exchange (ETDEWEB)

    Stripling, H. F.; Adams, M. L.; Hawkins, W. D. [Texas A and M University, Department of Nuclear Engineering, 3133 TAMU, College Station, TX 77843-3133 (United States)

    2013-07-01

    We describe a methodology for decreasing the memory footprint and machine I/O load associated with the need to access a forward solution during an adjoint solve. Specifically, we are interested in the depletion perturbation equations, where terms in the adjoint Bateman and transport equations depend on the forward flux solution. Checkpointing is the procedure of storing snapshots of the forward solution to disk and using these snapshots to recompute the parts of the forward solution that are necessary for the adjoint solve. For large problems, however, the storage cost of just a few copies of an angular flux vector can exceed the available RAM on the host machine. We propose a methodology that does not checkpoint the angular flux vector; instead, we write and store converged source moments, which are typically of a much lower dimension than the angular flux solution. This reduces the memory footprint and I/O load of the problem, but requires that we perform single sweeps to reconstruct flux vectors on demand. We argue that this trade-off is exactly the kind of algorithm that will scale on advanced, memory-limited architectures. We analyze the cost, in terms of FLOPS and memory footprint, of five checkpointing schemes. We also provide computational results that support the analysis and show that the memory-for-work trade off does improve time to solution. (authors)

  12. Immunologic checkpoints in cancer therapy: focus on the programmed death-1 (PD-1 receptor pathway

    Directory of Open Access Journals (Sweden)

    Momtaz P

    2014-11-01

    Full Text Available Parisa Momtaz,1,2 Michael A Postow1,2 1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Weill Cornell Medical College, New York, NY, USA Abstract: T-lymphocytes have the potential to recognize cancer antigens as foreign and therefore eliminate them. However, immune checkpoints such as cytotoxic T-lymphocyte-associated antigen (CTLA-4 and programmed cell death (PD-1 receptor and its ligands (PD-L1, PD-L2 suppress the activity of T-lymphocytes. Advances in the understanding of immunology and its role in cancer have led to the development of immune checkpoint inhibitors that block CTLA-4 and PD-1 and result in durable responses in patients with a wide range of cancers. PD-1 and PD-L1 inhibitors are currently in many stages of clinical investigation, and the anti-PD-1 antibody, pembrolizumab, was recently approved by the US Food and Drug Administration. Many questions remain to be answered, such as the optimal administration schedule, biomarkers that associate with benefit, and potential for use of PD-1 agents in combination approaches. Nonetheless, immunotherapy with PD-1 blocking antibodies is now becoming an integral part in the management of cancer. Keyword: immune checkpoints, immunotherapy, programmed cell death protein-1, cytotoxic T-lymphocyte antigen 4

  13. Immune checkpoint inhibitors: the new frontier in non–small cell lung cancer treatment

    Directory of Open Access Journals (Sweden)

    El-Osta HE

    2016-08-01

    Full Text Available Hazem El-Osta, Kamran Shahid, Glenn M Mills, Prakash Peddi Department of Medicine, Division of Hematology-Oncology, Louisiana State University Health Sciences Center, Shreveport, LA, USA Abstract: Lung cancer is the major cause for cancer-related death in the US. Although advances in chemotherapy and targeted therapy have improved the outcome of metastatic non-small-cell lung cancer, its prognosis remains dismal. A deeper understanding of the complex interaction between the immune system and tumor microenvironment has identified immune checkpoint inhibitors as new avenue of immunotherapy. Rather than acting directly on the tumor, these therapies work by removing the inhibition exerted by tumor cell or other immune cells on the immune system, promoting antitumoral immune response. To date, two programmed death-1 inhibitors, namely nivolumab and pembrolizumab, have received the US Food and Drug Administration approval for the treatment of advanced non-small-cell lung cancer that failed platinum-based chemotherapy. This manuscript provides a brief overview of the pathophysiology of cancer immune evasion, summarizes pertinent data on completed and ongoing clinical trials involving checkpoint inhibitors, discusses the different strategies to optimize their function, and outlines various challenges that are faced in this promising yet evolving field. Keywords: checkpoint inhibitors, immunotherapy, nivolumab, non-small-cell lung cancer, pembrolizumab, programmed death-1, programmed death ligand-1

  14. p53 activates G₁ checkpoint following DNA damage by doxorubicin during transient mitotic arrest.

    Science.gov (United States)

    Hyun, Sun-Yi; Jang, Young-Joo

    2015-03-10

    Recovery from DNA damage is critical for cell survival. The serious damage is not able to be repaired during checkpoint and finally induces cell death to prevent abnormal cell growth. In this study, we demonstrated that 8N-DNA contents are accumulated via re-replication during prolonged recovery period containing serious DNA damage in mitotic cells. During the incubation for recovery, a mitotic delay and initiation of an abnormal interphase without cytokinesis were detected. Whereas a failure of cytokinesis occurred in cells with no relation with p53/p21, re-replication is an anomalous phenomenon in the mitotic DNA damage response in p53/p21 negative cells. Cells with wild-type p53 are accumulated just prior to the initiation of DNA replication through a G₁ checkpoint after mitotic DNA damage, even though p53 does not interrupt pre-RC assembly. Finally, these cells undergo cell death by apoptosis. These data suggest that p53 activates G₁ checkpoint in response to mitotic DNA damage. Without p53, cells with mitotic DNA damage undergo re-replication leading to accumulation of damage.

  15. Immune checkpoint blockade therapy: The 2014 Tang prize in biopharmaceutical science

    Directory of Open Access Journals (Sweden)

    Ya-Shan Chen

    2015-02-01

    Full Text Available The first Tang Prize for Biopharmaceutical Science has been awarded to Prof. James P. Allison and Prof. Tasuku Honjo for their contributions leading to an entirely new way to treat cancer by blocking the molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 and programmed cell death protein 1 (PD-1 that turn off immune response. The treatment, called "immune checkpoint blockade therapy," has opened a new therapeutic era. Here the discoveries of the immune checkpoints and how they contribute to the maintenance of self-tolerance, as well as how to protect tissues from the excess immune responses causing damage are reviewed. The efforts made by Prof. Allison and Prof. Honjo for developing the most promising approaches to activate therapeutic antitumor immunity are also summarized. Since these certain immune checkpoint pathways appear to be one of the major mechanisms resulting in immune escape of tumors, the presence of anti-CTLA-4 and/or anti-PD-1 should contribute to removal of the inhibition signals for T cell activation. Subsequently, it will enhance specific T cell activation and, therefore, strengthen antitumor immunity.

  16. Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1.

    Directory of Open Access Journals (Sweden)

    Zhen Dou

    Full Text Available BACKGROUND: Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC, a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1 is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. METHODOLOGY/PRINCIPAL FINDINGS: Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. CONCLUSIONS/SIGNIFICANCE: hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase.

  17. p53 deficiency enhances mitotic arrest and slippage induced by pharmacological inhibition of Aurora kinases.

    Science.gov (United States)

    Marxer, M; Ma, H T; Man, W Y; Poon, R Y C

    2014-07-01

    A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.

  18. Tumor suppressor protein DAB2IP participates in chromosomal stability maintenance through activating spindle assembly checkpoint and stabilizing kinetochore-microtubule attachments

    Science.gov (United States)

    Yu, Lan; Shang, Zeng-Fu; Abdisalaam, Salim; Lee, Kyung-Jong; Gupta, Arun; Hsieh, Jer-Tsong; Asaithamby, Aroumougame; Chen, Benjamin P.C.; Saha, Debabrata

    2016-01-01

    Defects in kinetochore-microtubule (KT-MT) attachment and the spindle assembly checkpoint (SAC) during cell division are strongly associated with chromosomal instability (CIN). CIN has been linked to carcinogenesis, metastasis, poor prognosis and resistance to cancer therapy. We previously reported that the DAB2IP is a tumor suppressor, and that loss of DAB2IP is often detected in advanced prostate cancer (PCa) and is indicative of poor prognosis. Here, we report that the loss of DAB2IP results in impaired KT-MT attachment, compromised SAC and aberrant chromosomal segregation. We discovered that DAB2IP directly interacts with Plk1 and its loss inhibits Plk1 kinase activity, thereby impairing Plk1-mediated BubR1 phosphorylation. Loss of DAB2IP decreases the localization of BubR1 at the kinetochore during mitosis progression. In addition, the reconstitution of DAB2IP enhances the sensitivity of PCa cells to microtubule stabilizing drugs (paclitaxel, docetaxel) and Plk1 inhibitor (BI2536). Our findings demonstrate a novel function of DAB2IP in the maintenance of KT-MT structure and SAC regulation during mitosis which is essential for chromosomal stability. PMID:27568005

  19. Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation.

    Directory of Open Access Journals (Sweden)

    Gary M R Deyter

    2010-11-01

    Full Text Available The master regulators of the cell cycle are cyclin-dependent kinases (Cdks, which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3-depleted embryos is dependent on an intact spindle assembly checkpoint (SAC and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3-dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC-dependent block in anaphase chromosome segregation.

  20. The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast.

    Science.gov (United States)

    Craig, R; Norbury, C

    1998-12-18

    Entry into mitosis is normally blocked in eukaryotic cells that have not completed replicative DNA synthesis; this 'S-M' checkpoint control is fundamental to the maintenance of genomic integrity. Mutants of the fission yeast Schizosaccharomyces pombe defective in the S-M checkpoint fail to arrest the cell cycle when DNA replication is inhibited and hence attempt mitosis and cell division with unreplicated chromosomes, resulting in the 'cut' phenotype. In an attempt to identify conserved molecules involved in the S-M checkpoint we have screened a regulatable murine cDNA library in S. pombe and have identified cDNAs that induce the cut phenotype in cells arrested in S phase by hydroxyurea. One such cDNA encodes a novel protein with multiple calmodulin-binding motifs that, in addition to its effects on the S-M checkpoint, perturbed mitotic spindle functions, although spindle pole duplication was apparently normal. Both aspects of the phenotype induced by this cDNA product, which we term Sha1 (for spindle and hydroxyurea checkpoint abnormal), were suppressed by simultaneous overexpression of calmodulin. Sha1 is structurally related to the product of the Drosophila gene abnormal spindle (asp). These data suggest that calmodulin-binding protein(s) are important in the co-ordination of mitotic spindle functions with mitotic entry in fission yeast, and probably also in multicellular eukaryotes. PMID:9819352

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

  2. Bub3 is a spindle assembly checkpoint protein regulating chromosome segregation during mouse oocyte meiosis.

    Directory of Open Access Journals (Sweden)

    Mo Li

    Full Text Available In mitosis, the spindle assembly checkpoint (SAC prevents anaphase onset until all chromosomes have been attached to the spindle microtubules and aligned correctly at the equatorial metaphase plate. The major checkpoint proteins in mitosis consist of mitotic arrest-deficient (Mad1-3, budding uninhibited by benzimidazole (Bub1, Bub3, and monopolar spindle 1(Mps1. During meiosis, for the formation of a haploid gamete, two consecutive rounds of chromosome segregation occur with only one round of DNA replication. To pull homologous chromosomes to opposite spindle poles during meiosis I, both sister kinetochores of a homologue must face toward the same pole which is very different from mitosis and meiosis II. As a core member of checkpoint proteins, the individual role of Bub3 in mammalian oocyte meiosis is unclear. In this study, using overexpression and RNA interference (RNAi approaches, we analyzed the role of Bub3 in mouse oocyte meiosis. Our data showed that overexpressed Bub3 inhibited meiotic metaphase-anaphase transition by preventing homologous chromosome and sister chromatid segregations in meiosis I and II, respectively. Misaligned chromosomes, abnormal polar body and double polar bodies were observed in Bub3 knock-down oocytes, causing aneuploidy. Furthermore, through cold treatment combined with Bub3 overexpression, we found that overexpressed Bub3 affected the attachments of microtubules and kinetochores during metaphase-anaphase transition. We propose that as a member of SAC, Bub3 is required for regulation of both meiosis I and II, and is potentially involved in kinetochore-microtubule attachment in mammalian oocytes.

  3. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.

    Science.gov (United States)

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  4. From Phosphosites to Kinases

    DEFF Research Database (Denmark)

    Munk, Stephanie; Refsgaard, Jan C; Olsen, Jesper V;

    2016-01-01

    Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations...... sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available...

  5. Phosphatidylinositol kinase from rabbit reticulocytes

    International Nuclear Information System (INIS)

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of [32P]ATP and Mg2+. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements

  6. Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1

    DEFF Research Database (Denmark)

    Tibelius, Alexandra; Marhold, Joachim; Zentgraf, Hanswalter;

    2009-01-01

    Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations...

  7. Anti-TNF-refractory colitis after checkpoint inhibitor therapy: Possible role of CMV-mediated immunopathogenesis.

    Science.gov (United States)

    Lankes, Katharina; Hundorfean, Gheorghe; Harrer, Thomas; Pommer, Ansgar J; Agaimy, Abbas; Angelovska, Irena; Tajmir-Riahi, Azadeh; Göhl, Jonas; Schuler, Gerold; Neurath, Markus F; Hohenberger, Werner; Heinzerling, Lucie

    2016-06-01

    Immune-related adverse events (irAEs) induced by checkpoint inhibitors are well known. Since fatal outcomes have been reported early detection and adequate management are crucial. In particular, colitis is frequently observed and can result in intestinal perforation. This is the first report of an autoimmune colitis that was treated according to algorithms but became resistant due to a CMV reactivation. The 32-y-old male patient with metastatic melanoma treated within an anti-PD-1/ipilimumab combination study developed severe immune-mediated colitis (CTCAE grade 3) with up to 18 watery stools per day starting 2 weeks after treatment initiation. After improving upon therapy with immunosuppressive treatment (high dose steroids and infliximab) combined with parenteral nutrition diarrhea again exacerbated. Additionally, the patient had asymptomatic grade 3 CTCAE amylase and lipase elevation. Colitis was monitored by weekly endoscopies and colon biopsies were analyzed histologically with CMV staining, multi-epitope ligand cartography (MELC) and qRT-PCR for inflammatory genes. In the course, CMV reactivation was detected in the colon and treated with antiviral medication in parallel to a reduction of corticosteroids. Subsequently, symptoms improved. The patient showed a complete response for 2 y now including regression of bone metastases. CMV reactivation under checkpoint inhibitor therapy in combination with immunosuppressive treatment for autoimmune side effects has to be considered in these patients and if present treated. Potentially, CMV reactivation is underdiagnosed. Treatment algorithms should include CMV diagnostics. PMID:27471608

  8. Tumor-suppressor genes, cell cycle regulatory checkpoints, and the skin

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2015-01-01

    Full Text Available The cell cycle (or cell-division cycle is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH terms "tumor suppressor′s genes," "skin," and "cell cycle regulatory checkpoints." We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses.

  9. DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.

    Science.gov (United States)

    Magiera, Maria M; Gueydon, Elisabeth; Schwob, Etienne

    2014-01-20

    Deoxyribonucleic acid (DNA) replication and chromosome segregation must occur in ordered sequence to maintain genome integrity during cell proliferation. Checkpoint mechanisms delay mitosis when DNA is damaged or upon replication stress, but little is known on the coupling of S and M phases in unperturbed conditions. To address this issue, we postponed replication onset in budding yeast so that DNA synthesis is still underway when cells should enter mitosis. This delayed mitotic entry and progression by transient activation of the S phase, G2/M, and spindle assembly checkpoints. Disabling both Mec1/ATR- and Mad2-dependent controls caused lethality in cells with deferred S phase, accompanied by Rad52 foci and chromosome missegregation. Thus, in contrast to acute replication stress that triggers a sustained Mec1/ATR response, multiple pathways cooperate to restrain mitosis transiently when replication forks progress unhindered. We suggest that these surveillance mechanisms arose when both S and M phases were coincidently set into motion by a unique ancestral cyclin-Cdk1 complex.

  10. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy

    Science.gov (United States)

    Chen, Qian; Xu, Ligeng; Liang, Chao; Wang, Chao; Peng, Rui; Liu, Zhuang

    2016-01-01

    A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours. PMID:27767031

  11. 有丝分裂关键激酶抑制剂的研究进展%Progress on the research of key kinase inhibitors in mitosis

    Institute of Scientific and Technical Information of China (English)

    陈葆峰; 解鸿波; 沙宇; 程卯生

    2012-01-01

    目的 综述有丝分裂关键激酶抑制剂近年来的研究进展.方法 根据已报道的有丝分裂中关键激酶抑制剂的文献,将对有丝分裂过程中与肿瘤发生联系密切的激酶抑制剂,如Aurora激酶抑制剂、CDK(cyclin-dependent kinase)激酶抑制剂、PLK(Polo-like kinase)激酶抑制剂、CHK(check-point kinase)激酶抑制剂等目前的研究进展进行综述.结果 有丝分裂关键激酶抑制剂已在临床前研究及临床研究中显示出很好的抗肿瘤活性.结论 随着研究的不断深入,有丝分裂关键激酶抑制剂将在肿瘤治疗中发挥更大的作用.%Objective To review the recent progress on the research of key kinase inhibitors in mitosis. Methods According to the references to the key kinase inhibitors in mitosis reported, the kinase inhibitors were introduced closely related to the occurrence of the cancer in the progress of mitosis, such as Aurora kinase in- . hibitors, CDK (cyclin-dependent kinase) inhibitors, PLK (Polo-like kinase) inhibitors and CHK (checkpoint kinase)inhibitors. Results The potent anti-tumor activity of key kinase inhibitors in mitosis had been revealed in the preclinical and clinical research. Conclusions With the further research,key kinase inhibitors in mitosis shows more significant roles in the therapy of cancer.

  12. Kinase Inhibitors from Marine Sponges

    Directory of Open Access Journals (Sweden)

    Ana Zivanovic

    2011-10-01

    Full Text Available Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included.

  13. RNA-dependent protein kinase (PKR) depletes nutrients, inducing phosphorylation of AMP-activated kinase in lung cancer.

    Science.gov (United States)

    Guo, Chengcheng; Hao, Chuncheng; Shao, RuPing; Fang, Bingliang; Correa, Arlene M; Hofstetter, Wayne L; Roth, Jack A; Behrens, Carmen; Kalhor, Neda; Wistuba, Ignacio I; Swisher, Stephen G; Pataer, Apar

    2015-05-10

    We have demonstrated that RNA-dependent protein kinase (PKR) and its downstream protein p-eIF2α are independent prognostic markers for overall survival in lung cancer. In the current study, we further investigate the interaction between PKR and AMPK in lung tumor tissue and cancer cell lines. We examined PKR protein expression in 55 frozen primary lung tumor tissues by Western blotting and analyzed the association between PKR expression and expression of 139 proteins on tissue samples examined previously by Reverse Phase Protein Array (RPPA) from the same 55 patients. We observed that biomarkers were either positively (phosphorylated AMP-activated kinase(T172) [p-AMPK]) or negatively (insulin receptor substrate 1, meiotic recombination 11, ATR interacting protein, telomerase, checkpoint kinase 1, and cyclin E1) correlated with PKR. We further confirmed that induction of PKR with expression vectors in lung cancer cells causes activation of the AMPK protein independent of the LKB1, TAK1, and CaMKKβ pathway. We found that PKR causes nutrient depletion, which increases AMP levels and decreases ATP levels, causing AMPK phosphorylation. We further demonstrated that inhibiting AMPK expression with compound C or siRNA enhanced PKR-mediated cell death. We next explored the combination of PKR and p-AMPK expression in NSCLC patients and observed that expression of p-AMPK predicted a poor outcome for adenocarcinoma patients with high PKR expression and a better prognosis for those with low PKR expression. These findings were consistent with our in vitro results. AMPK might rescue cells facing metabolic stresses, such as ATP depletion caused by PKR. Our data indicate that PKR causes nutrient depletion, which induces the phosphorylation of AMPK. AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation. PMID:25798539

  14. Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

    DEFF Research Database (Denmark)

    Sironi, L; Melixetian, M; Faretta, M;

    2001-01-01

    Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms...

  15. Design Optimization of Time- and Cost-Constrained Fault-Tolerant Embedded Systems with Checkpointing and Replication

    DEFF Research Database (Denmark)

    Pop, Paul; Izosimov, Viacheslav; Eles, Petru;

    2009-01-01

    decides the assignment of fault-tolerance policies to processes, the optimal placement of checkpoints and the mapping of processes to processors such that multiple transient faults are tolerated and the timing constraints of the application are satisfied. We present several design optimization approaches...

  16. Cell cycle re-entry mechanisms after DNA damage checkpoints Giving it some gas to shut off the breaks!

    NARCIS (Netherlands)

    van Vugt, Marcel A. T. M.; Yaffe, Michael B.

    2010-01-01

    In order to maintain genetic integrity, cells are equipped with cell cycle checkpoints that detect DNA damage, orchestrate repair, and if necessary, eliminate severely damaged cells by inducing apoptotic cell death. The mitotic machinery is now emerging as an important determinant of the cellular re

  17. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  18. Chromosome 17p deletion in human medulloblastoma: a missing checkpoint in the Hedgehog pathway.

    Science.gov (United States)

    De Smaele, Enrico; Di Marcotullio, Lucia; Ferretti, Elisabetta; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2004-10-01

    Although deregulation of Hedgehog signalling is considered to play a crucial oncogenic role and commonly occurrs in medulloblastoma, genetic lesions in components of this pathway are observed in a minority of cases. The recent identification of a novel putative tumor suppressor (REN(KCTD11)) on chromosome 17p13.2, a region most frequently lost in human medulloblastoma, highlights the role of allelic deletion of the gene in this brain malignancy, leading to the loss of growth inhibitory activity via suppression of Gli-dependent activation of Hedgehog target genes. The presence on 17p13 of another tumor suppressor gene (p53) whose inactivation cooperates with Hedgehog pathway for medulloblastoma formation, suggests that 17p deletion unveils haploinsufficiency conditions leading to abrogation of either direct and indirect checkpoints of Hedgehog signalling in cancer. PMID:15467454

  19. Checkpoint and Replication Oriented Fault Tolerant Mechanism for MapReduce Framework

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2013-09-01

    Full Text Available MapReduce is an emerging programming paradigm and an associated implementation for processing and generating big data which has been widely applied in data-intensive systems. In cloud environment, node and task failure is no longer accidental but a common feature of large-scale systems. In MapReduce framework, although the rescheduling based fault-tolerant method is simple to implement, it failed to fully consider the location of distributed data, the computation and storage overhead. Thus, a single node failure will increase the completion time dramatically. In this paper, a Checkpoint and Replication Oriented Fault Tolerant scheduling algorithm (CROFT is proposed, which takes both task and node failure into consideration. Preliminary experiments show that with less storage and network overhead. CROFT will significantly reduce the completion time at failure time, and the overall performance of MapReduce can be improved at least over 30% than original mechanism in Hadoop.  

  20. Meiosis I in Xenopus oocytes is not error-prone despite lacking spindle assembly checkpoint.

    Science.gov (United States)

    Liu, Dandan; Shao, Hua; Wang, Hongmei; Liu, X Johné

    2014-01-01

    The spindle assembly checkpoint, SAC, is a surveillance mechanism to control the onset of anaphase during cell division. SAC prevents anaphase initiation until all chromosome pairs have achieved bipolar attachment and aligned at the metaphase plate of the spindle. In doing so, SAC is thought to be the key mechanism to prevent chromosome nondisjunction in mitosis and meiosis. We have recently demonstrated that Xenopus oocyte meiosis lacks SAC control. This prompted the question of whether Xenopus oocyte meiosis is particularly error-prone. In this study, we have karyotyped a total of 313 Xenopus eggs following in vitro oocyte maturation. We found no hyperploid egg, out of 204 metaphase II eggs with countable chromosome spreads. Therefore, chromosome nondisjunction is very rare during Xenopus oocyte meiosis I, despite the lack of SAC. PMID:24646611

  1. Mitosis-specific phosphorylation of PML at T409 regulates spindle checkpoint.

    Science.gov (United States)

    Jin, J; Liu, J

    2016-01-01

    During mitosis, Promyelocytic leukemia nuclear bodies (PML NBs) change dramatically in morphology and composition, but little is known about function of PML in mitosis. Here, we show that PML is phosphorylated at T409 (PML p409) in a mitosis-specific manner. More importantly, PML p409 contributes to maintain the duration of pro-metaphase and regulates spindle checkpoint. Deficient PML p409 caused a shortening of pro-metaphase and challenged the nocodazole-triggered mitotic arrest. T409A mutation led to a higher frequency of misaligned chromosomes on metaphase plate, and subsequently death in late mitosis. In addition, inhibition of PML p409 repressed growth of tumor cells, suggesting that PML p409 is a potential target for cancer therapy. Collectively, our study demonstrated an important phosphorylated site of PML, which contributed to explore the role of PML in mitosis. PMID:27609478

  2. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies.

    Science.gov (United States)

    Naidoo, J; Page, D B; Li, B T; Connell, L C; Schindler, K; Lacouture, M E; Postow, M A; Wolchok, J D

    2015-12-01

    Immune checkpoint antibodies that augment the programmed cell death protein 1 (PD-1)/PD-L1 pathway have demonstrated antitumor activity across multiple malignancies, and gained recent regulatory approval as single-agent therapy for the treatment of metastatic malignant melanoma and nonsmall-cell lung cancer. Knowledge of toxicities associated with PD-1/PD-L1 blockade, as well as effective management algorithms for these toxicities, is pivotal in order to optimize clinical efficacy and safety. In this article, we review selected published and presented clinical studies investigating single-agent anti-PD-1/PD-L1 therapy and trials of combination approaches with other standard anticancer therapies, in multiple tumor types. We summarize the key adverse events reported in these studies and their management algorithms. PMID:26371282

  3. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  4. Pyruvate Dehydrogenase Kinase 4

    OpenAIRE

    Cadoudal, Thomas; Distel, Emilie; Durant, Sylvie; Fouque, Françoise; Blouin, Jean-Marc; Collinet, Martine; Bortoli, Sylvie; Forest, Claude; Benelli, Chantal

    2008-01-01

    OBJECTIVE—Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled ...

  5. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  6. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, Ian W.H., E-mail: Ian.Jarvis@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Bergvall, Christoffer, E-mail: Christoffer.Bergvall@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Bottai, Matteo, E-mail: Matteo.Bottai@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Westerholm, Roger, E-mail: Roger.Westerholm@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Stenius, Ulla, E-mail: Ulla.Stenius@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Dreij, Kristian, E-mail: Kristian.Dreij@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden)

    2013-02-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  7. Cyclin-dependent kinases.

    Science.gov (United States)

    Malumbres, Marcos

    2014-01-01

    Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  8. Response to BRAF inhibition in melanoma is enhanced when combined with immune checkpoint blockade.

    Science.gov (United States)

    Cooper, Zachary A; Juneja, Vikram R; Sage, Peter T; Frederick, Dennie T; Piris, Adriano; Mitra, Devarati; Lo, Jennifer A; Hodi, F Stephen; Freeman, Gordon J; Bosenberg, Marcus W; McMahon, Martin; Flaherty, Keith T; Fisher, David E; Sharpe, Arlene H; Wargo, Jennifer A

    2014-07-01

    BRAF-targeted therapy results in objective responses in the majority of patients; however, the responses are short lived (∼6 months). In contrast, treatment with immune checkpoint inhibitors results in a lower response rate, but the responses tend to be more durable. BRAF inhibition results in a more favorable tumor microenvironment in patients, with an increase in CD8(+) T-cell infiltrate and a decrease in immunosuppressive cytokines. There is also increased expression of the immunomodulatory molecule PDL1, which may contribute to the resistance. On the basis of these findings, we hypothesized that BRAF-targeted therapy may synergize with the PD1 pathway blockade to enhance antitumor immunity. To test this hypothesis, we developed a BRAF(V600E)/Pten(-/-) syngeneic tumor graft immunocompetent mouse model in which BRAF inhibition leads to a significant increase in the intratumoral CD8(+) T-cell density and cytokine production, similar to the effects of BRAF inhibition in patients. In this model, CD8(+) T cells were found to play a critical role in the therapeutic effect of BRAF inhibition. Administration of anti-PD1 or anti-PDL1 together with a BRAF inhibitor led to an enhanced response, significantly prolonging survival and slowing tumor growth, as well as significantly increasing the number and activity of tumor-infiltrating lymphocytes. These results demonstrate synergy between combined BRAF-targeted therapy and immune checkpoint blockade. Although clinical trials combining these two strategies are ongoing, important questions still remain unanswered. Further studies using this new melanoma mouse model may provide therapeutic insights, including optimal timing and sequence of therapy.

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

    Directory of Open Access Journals (Sweden)

    Antonio Marchini

    2016-01-01

    Full Text Available 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.

  10. The role of vitamin D in asthma.

    Science.gov (United States)

    Luong, Khanh vinh quoc; Nguyen, Lan Thi Hoàng

    2012-04-01

    Vitamin D metabolites are important immune-modulatory hormones and are able to suppress Th2-mediated allergic airway disease. Some genetic factors that may contribute to asthma are regulated by vitamin D, such as vitamin D receptor (VDR), human leukocyte antigen genes (HLA), human Toll-like receptors (TLR), matrix metalloproteinases (MMPs), a disintegrin and metalloprotein-33 (ADAM-33), and poly(ADP-ribosyl) polymerase- 1 (PARP-1). Vitamin D has also been implicated in asthma through its effects on the obesity, bacillus Calmettee Guérin (BCG) vaccination and high vitamin D level, vitamin D supplement, checkpoint protein kinase 1 (Chk1), plasminogen activator inhibitor-1 (PAI-1) and gamma delta T cells (gdT). Vitamin D plays a role in asthma and exerts its action through either genomic and/or non-genomic ways.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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

  12. Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

    Directory of Open Access Journals (Sweden)

    Colin P De Souza

    Full Text Available Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs, the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN specifically at SPBs in the basal region of G1 cells and that

  13. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  14. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten;

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  15. Map kinases in fungal pathogens.

    Science.gov (United States)

    Xu, J R

    2000-12-01

    MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens. PMID:11273677

  16. Expression of constitutively active CDK1 stabilizes APC-Cdh1 substrates and potentiates premature spindle assembly and checkpoint function in G1 cells.

    Directory of Open Access Journals (Sweden)

    Yan Ma

    Full Text Available Mitotic progression in eukaryotic cells depends upon the activation of cyclin-dependent kinase 1 (CDK1, followed by its inactivation through the anaphase-promoting complex (APC/cyclosome-mediated degradation of M-phase cyclins. Previous work revealed that expression of a constitutively active CDK1 (CDK1AF in HeLa cells permitted their division, but yielded G1 daughter cells that underwent premature S-phase and early mitotic events. While CDK1AF was found to impede the sustained activity of APC-Cdh1, it was unknown if this defect improperly stabilized mitotic substrates and contributed to the occurrence of these premature M phases. Here, we show that CDK1AF expression in HeLa cells improperly stabilized APC-Cdh1 substrates in G1-phase daughter cells, including mitotic kinases and the APC adaptor, Cdc20. Division of CDK1AF-expressing cells produced G1 daughters with an accelerated S-phase onset, interrupted by the formation of premature bipolar spindles capable of spindle assembly checkpoint function. Further characterization of these phenotypes induced by CDK1AF expression revealed that this early spindle formation depended upon premature CDK1 and Aurora B activities, and their inhibition induced rapid spindle disassembly. Following its normal M-phase degradation, we found that the absence of Wee1 in these prematurely cycling daughter cells permitted the endogenous CDK1 to contribute to these premature mitotic events, since expression of a non-degradable Wee1 reduced the number of cells that exhibited premature cyclin B1oscillations. Lastly, we discovered that Cdh1-ablated cells could not be forced into a premature M phase, despite cyclin B1 overexpression and proteasome inhibition. Together, these results demonstrate that expression of constitutively active CDK1AF hampers the destruction of critical APC-Cdh1 targets, and that this type of condition could prevent newly divided cells from properly maintaining a prolonged interphase state. We

  17. Mode of ATM-dependent suppression of chromosome translocation

    Energy Technology Data Exchange (ETDEWEB)

    Yamauchi, Motohiro, E-mail: motoyama@nagasaki-u.ac.jp [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi [Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer We addressed how ATM suppresses frequency of chromosome translocation. Black-Right-Pointing-Pointer We found ATM/p53-dependent G1 checkpoint suppresses translocation frequency. Black-Right-Pointing-Pointer We found ATM and DNA-PKcs function in a common pathway to suppress translocation. -- Abstract: It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs.

  18. Therapeutic targeting of Janus kinases

    OpenAIRE

    Pesu, Marko; Laurence, Arian; Kishore, Nandini; Zwillich, Sam; Chan, Gary; O’Shea, John J.

    2008-01-01

    Cytokines play pivotal roles in immunity and inflammation, and targeting cytokines and their receptors is an effective means of treating such disorders. Type I and II cytokine receptors associate with Janus family kinases (JAKs) to effect intracellular signaling. These structurally unique protein kinases play essential and specific roles in immune cell development and function. One JAK, JAK3, has particularly selective functions. Mutations of this kinase underlie severe combined immunodeficie...

  19. Visualizing autophosphorylation in histidine kinases

    OpenAIRE

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two dir...

  20. Polo-like kinase 1 as target for cancer therapy

    Directory of Open Access Journals (Sweden)

    Weiß Lily

    2012-12-01

    Full Text Available Abstract Polo-like kinase 1 (Plk1 is an interesting molecule both as a biomarker and as a target for highly specific cancer therapy for several reasons. Firstly, it is over-expressed in many cancers and can serve as a biomarker to monitor treatment efficacy of Plk1 inhibitors. Furthermore, the Plk1 enzyme is expressed only in dividing cells and is a major regulator of the cell cycle. It controls entry into mitosis and regulates the spindle checkpoint. The expression of Plk1 in normal cells is not nearly as strong as that in cancer cells, which makes Plk1 a discriminating tartget for the development of cancer-specific small molecule drugs. RNA interference experiments in vitro and in vivo have indicated that downregulation of Plk1 expression represents an attractive concept for cancer therapy. Over the years, a number of Plk1 inhibitors have been discovered. Many of these inhibitors are substances that compete with ATP for the substrate binding site. The ATP-competitive inhibitor BI 6727 is currently being clinically tested in cancer patients. Another drug in development, poloxin, is the first Polo-box domain inhibitor of Plk1. This compound is a derivative of the natural product, thymoquinone, derived from Nigella sativa. A novel and promising strategy is to synthesize bifunctional inhibitors that combine the high binding affinity of ATP inhibitors with the specificity of competitive inhibitors.

  1. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc. PMID:21235885

  2. The dawn of Aurora kinase research: from fly genetics to the clinic.

    Directory of Open Access Journals (Sweden)

    Mar eCarmena

    2015-11-01

    Full Text Available Aurora kinases comprise a family of highly conserved serine-threonine protein kinases that play a pivotal role in the regulation of cell cycle. Aurora kinases are not only involved in the control of multiple processes during cell division but also coordinate chromosomal and cytoskeletal events, contributing to the regulation of checkpoints and ensuring the smooth progression of the cell cycle.Because of their fundamental contribution to cell cycle regulation, Aurora kinases were originally identified in independent genetic screens designed to find genes involved in the regulation of cell division. The first aurora mutant was part of a collection of mutants isolated in C. Nusslein-Volhard’s laboratory. This collection was screened in D. M. Glover’s laboratory in search for mutations disrupting the centrosome cycle in embryos derived from homozygous mutant mothers. The mutants identified were given names related to the polar regions, and included not only aurora but also the equally famous polo. Ipl1, the only Aurora in yeast, was identified in a genetic screen looking for mutations that caused chromosome segregation defects. The discovery of a second Aurora-like kinase in mammals opened a new chapter in the research of Aurora kinases. The rat kinase AIM was found to be highly homologous to the fly and yeast proteins, but localised at the midzone and midbody and was proposed to have a role in cytokinesis. Homologs of the equatorial Aurora (Aurora B were identified in metazoans ranging from flies to humans. Xenopus Aurora B was found to be in a complex with the chromosomal passenger INCENP, and both proteins were shown to be essential in flies for chromosome structure, segregation, central spindle formation and cytokinesis. Fifteen years on, Aurora kinase research is an active field of research. After the successful introduction of the first anti-mitotic agents in cancer therapy, both Auroras have become the focus of attention as targets for

  3. In vitro expression levels of cell-cycle checkpoint proteins are associated with cellular DNA repair capacity in peripheral blood lymphocytes: a multivariate analysis

    OpenAIRE

    Fan, You-Hong; Hu, Zhibin; Li, Chunying; Wang, Li-E; Guo, Zhaozheng; Qiao, Yawei; Zhang, Li; Zhang, Wei; Mao, Li; Wei, Qingyi

    2007-01-01

    DNA repair should occur after cells sense DNA damage signals and undergo cell-cycle arrest to provide sufficient time for DNA repair, and suboptimal DNA repair capacity (DRC) in peripheral lymphocytes has been suggested as a cancer susceptibility marker. Numerous studies showed a functional link between DNA damage sensing, cell-cycle checkpoint and DNA repair. We hypothesized that in vitro cell-cycle checkpoint-related protein expression levels in stimulated lymphocytes predict DRC levels. To...

  4. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    Energy Technology Data Exchange (ETDEWEB)

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  5. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    International Nuclear Information System (INIS)

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated β-galactosidase (SA-β-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that inhibits NHEJ

  6. The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress

    Directory of Open Access Journals (Sweden)

    Soriano-Carot María

    2012-02-01

    Full Text Available Abstract Background The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism. Results This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU, methylmetanosulfonate (MMS, phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses. Conclusions Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt

  7. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

    Directory of Open Access Journals (Sweden)

    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  8. Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

    OpenAIRE

    Yamamoto, Ayumu; Kitamura, Kenji; Hihara, Daisuke; Hirose, Yukinobu; Katsuyama, Satoshi; Hiraoka, Yasushi

    2008-01-01

    During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/CCdc20), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to th...

  9. p38γ regulates UV-induced checkpoint signaling and repair of UV-induced DNA damage.

    Science.gov (United States)

    Wu, Chia-Cheng; Wu, Xiaohua; Han, Jiahuai; Sun, Peiqing

    2010-06-01

    In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.

  10. Immune checkpoint blockade in cancer treatment: a double-edged sword cross-targeting the host as an "innocent bystander".

    Science.gov (United States)

    Gelao, Lucia; Criscitiello, Carmen; Esposito, Angela; Goldhirsch, Aron; Curigliano, Giuseppe

    2014-03-01

    Targeted immune checkpoint blockade augments anti-tumor immunity and induces durable responses in patients with melanoma and other solid tumors. It also induces specific "immune-related adverse events" (irAEs). IrAEs mainly include gastrointestinal, dermatological, hepatic and endocrinological toxicities. Off-target effects that arise appear to account for much of the toxicity of the immune checkpoint blockade. These unique "innocent bystander" effects are likely a direct result of breaking immune tolerance upon immune check point blockade and require specific treatment guidelines that include symptomatic therapies or systemic corticosteroids. What do we need going forward to limit immune checkpoint blockade-induced toxicity? Most importantly, we need a better understanding of the roles played by these agents in normal tissues, so that we can begin to predict potentially problematic side effects on the basis of their selectivity profile. Second, we need to focus on the predictive factors of the response and toxicity of the host rather than serially focusing on individual agents. Third, rigorous biomarker-driven clinical trials are needed to further elucidate the mechanisms of both the benefit and toxicity. We will summarize the double-edged sword effect of immunotherapeutics in cancer treatment. PMID:24594636

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

    Energy Technology Data Exchange (ETDEWEB)

    Nakadate, Yusuke [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Kodera, Yasuo; Kitamura, Yuka [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Tachibana, Taro [Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Tamura, Tomohide [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Koizumi, Fumiaki, E-mail: fkoizumi@ncc.go.jp [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    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.

  12. Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation.

    Science.gov (United States)

    Yamaguchi, Masaya; VanderLinden, Ryan; Weissmann, Florian; Qiao, Renping; Dube, Prakash; Brown, Nicholas G; Haselbach, David; Zhang, Wei; Sidhu, Sachdev S; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-08-18

    The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation. PMID:27522463

  13. Dictyostelium nucleomorphin is a member of the BRCT-domain family of cell cycle checkpoint proteins.

    Science.gov (United States)

    Myre, Michael A; O'Day, Danton H

    2004-11-18

    A search of the Dictyostelium genome project database (http://dictybase.org/db/cgi-bin/blast.pl) with nucleomorphin, a protein that regulates the nuclear number, predicted it to be encoded by a larger gene containing a putative breast cancer carboxy-terminus domain (BRCT). Using RT-PCR, Northern and Western blotting we have identified a differentially expressed, 2318 bp cDNA encoding a protein isoform of Dictyostelium NumA with an apparent molecular weight of 70 kDa that we have called NumB. It contains a single amino-terminal BRCT-domain spanning residues 125-201. Starvation of shaking cultures reduces NumA expression by approximately 88+/-5.6%, whereas NumB expression increases approximately 35+/-3.5% from vegetative levels. NumC, a third isoform that is also expressed during development but not growth, remains to be characterized. These findings suggest NumB may be a member of the BRCT-domain containing cell cycle checkpoint proteins. PMID:15535983

  14. Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity

    Science.gov (United States)

    Prestwood, Tyler R.; Spitzer, Matthew H.; Linde, Ian L.; Chabon, Jonathan; Reticker-Flynn, Nathan E.; Bhattacharya, Nupur; Zhang, Hong; Zhang, Xiangyue; Basto, Pamela A.; Burt, Bryan M.; Alonso, Michael N.; Engleman, Edgar G.

    2016-01-01

    BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity. PMID:27812544

  15. Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis.

    Science.gov (United States)

    Schweizer, Nina; Ferrás, Cristina; Kern, David M; Logarinho, Elsa; Cheeseman, Iain M; Maiato, Helder

    2013-12-23

    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.

  16. Checkpoint-Inhibitoren in der Immuntherapie: Ein Meilenstein in der Behandlung des malignen Melanoms.

    Science.gov (United States)

    Wilden, Sophia M; Lang, Berenice M; Mohr, Peter; Grabbe, Stephan

    2016-07-01

    Seit Jahrzehnten ist bekannt, dass Tumoren vom Immunsystem erkannt und zerstört werden können. Diese, vor allem in Tierversuchen gewonnene Erkenntnis konnte jedoch in der Vergangenheit nicht zum Nutzen unserer Patienten umgesetzt werden, da immunonkologische Therapieansätze in den letzten Jahrzehnten in der Anwendung beim Menschen stets versagt haben. Daher hat, mit Ausnahme der adjuvanten Interferontherapie, keines dieser Verfahren den Einzug in die klinische Versorgung gefunden. Langzeitüberleben unter guter Lebensqualität war dabei sehr wenigen Patienten vorbehalten. Mit den neuen immunologischen Therapieansätzen wird jedoch sowohl das Langzeitüberleben als auch die Lebensqualität onkologischer Patienten neu definiert. Auf die neuen "Immun-Checkpoint-Inhibitoren" spricht erstmals ein relevanter Teil der behandelten Patienten an und diese zeigen in der Regel langandauernde Remissionen bis hin zur Heilung. Schon jetzt ist klar, dass die Immuntherapie in Zukunft eine der wesentlichen Therapiesäulen bei der Behandlung des metastasierten Melanoms und auch vieler anderer fortgeschrittener Tumoren bilden wird. In dieser Übersicht werden die wichtigsten neuen Therapiemodalitäten besprochen und sowohl deren Wirkprinzip als auch klinische Daten zum Therapieansprechen und zu erwartenden Nebenwirkungen der Therapie referiert. PMID:27373243

  17. Kinetochore-microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint.

    Science.gov (United States)

    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

  18. Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    Science.gov (United States)

    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

  19. Compact modeling of allosteric multisite proteins: application to a cell size checkpoint.

    Directory of Open Access Journals (Sweden)

    Germán Enciso

    2014-02-01

    Full Text Available We explore a framework to model the dose response of allosteric multisite phosphorylation proteins using a single auxiliary variable. This reduction can closely replicate the steady state behavior of detailed multisite systems such as the Monod-Wyman-Changeux allosteric model or rule-based models. Optimal ultrasensitivity is obtained when the activation of an allosteric protein by its individual sites is concerted and redundant. The reduction makes this framework useful for modeling and analyzing biochemical systems in practical applications, where several multisite proteins may interact simultaneously. As an application we analyze a newly discovered checkpoint signaling pathway in budding yeast, which has been proposed to measure cell growth by monitoring signals generated at sites of plasma membrane growth. We show that the known components of this pathway can form a robust hysteretic switch. In particular, this system incorporates a signal proportional to bud growth or size, a mechanism to read the signal, and an all-or-none response triggered only when the signal reaches a threshold indicating that sufficient growth has occurred.

  20. Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint.

    Science.gov (United States)

    Chen, Jing; Liu, Jian

    2016-09-01

    Chromosome segregation during mitosis hinges on proper assembly of the microtubule spindle that establishes bipolar attachment to each chromosome. Experiments demonstrate allometry of mitotic spindles and a universal scaling relationship between spindle size and cell size across metazoans, which indicates a conserved principle of spindle assembly at play during evolution. However, the nature of this principle is currently unknown. Researchers have focused on deriving the mechanistic underpinning of the size scaling from the mechanical aspects of the spindle assembly process. In this work we take a different standpoint and ask: What is the size scaling for? We address this question from the functional perspectives of spindle assembly checkpoint (SAC). SAC is the critical surveillance mechanism that prevents premature chromosome segregation in the presence of unattached or misattached chromosomes. The SAC signal gets silenced after and only after the last chromosome-spindle attachment in mitosis. We previously established a model that explains the robustness of SAC silencing based on spindle-mediated spatiotemporal regulation of SAC proteins. Here, we refine the previous model, and find that robust and timely SAC silencing entails proper size scaling of mitotic spindle. This finding provides, to our knowledge, a novel, function-oriented angle toward understanding the observed spindle allometry, and the universal scaling relationship between spindle size and cell size in metazoans. In a broad sense, the functional requirement of robust SAC silencing could have helped shape the spindle assembly mechanism in evolution. PMID:27602734

  1. Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

    Science.gov (United States)

    Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

    2015-01-01

    During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470

  2. Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

    Science.gov (United States)

    Fayadat, Laurence; Kopito, Ron R.

    2003-01-01

    To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane. PMID:12631739

  3. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  4. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  5. An association between low-dose hyper-radiosensitivity and the early G2-phase checkpoint in normal fibroblasts of cancer patients.

    Science.gov (United States)

    Słonina, Dorota; Gasińska, Anna; Biesaga, Beata; Janecka, Anna; Kabat, Damian

    2016-03-01

    In our previous study, low-dose hyper-radiosensitivity (HRS) effect was demonstrated for normal fibroblasts (asynchronous and G2-phase enriched) of 4 of the 25 cancer patients investigated. For the rest of patients, HRS was not defined in either of the 2 fibroblast populations. Thus, the study indicated that G2-phase enrichment had no influence on HRS identification. The conclusion contradicts that reported for human tumor cells, and suggests different mechanism of HRS in normal human cells. In the present paper we report, for the first time, the activity of early G2-phase checkpoint after low-dose irradiation in normal fibroblasts of these 4 HRS-positive patients and 4 HRS-negative patients and answer the question regarding the role of this checkpoint in normal human cells. The response of the early G2-phase checkpoint was determined by assessment of the progression of irradiated cells into mitosis using the mitotic marker, phosphorylated histone H3. We found evident differences in the activity of the early G2-phase checkpoint between HRS-positive and HRS-negative fibroblasts. In HRS-positive fibroblasts the checkpoint was not triggered and DNA damage was not recognized after doses lower than 0.2Gy resulting in HRS response. On the contrary, in HRS-negative fibroblasts the early G2-phase checkpoint was activated regardless of the dose in the range 0.1-2Gy. In conclusion, although cell cycle phase has no effect on the presence of HRS effect in normal human fibroblasts, the data reported here indicate that HRS response in these cells is associated with the functioning of early G2-phase checkpoint in a threshold-dose dependent manner, similarly as it takes place in most of human tumor and other cells. PMID:26725161

  6. Spatial collisions and discordant temporalities: everyday life between camp and checkpoint.

    Science.gov (United States)

    Abourahme, Nasser

    2011-01-01

    How do we make sense of the colonial subject that is neither in revolt nor in open crisis? How do people reproduce their lives, fashion routines, etch out some meaning when the political is evacuated, when time is on hold? These questions loom over a contemporary disjuncture in Palestine, marked in part by the splintering and opening up of the field of subjective bonds, attachments and associations to new modalities of production, less circumscribed by previous normative parameters and engendering a host of complexities and ambivalences in politico-social relationalities. Yet most scholarship on Palestine remains caught up in reductive binaries of violence versus resistance and heavily reliant on rigid and aggregated categories, the bulk of it unable to capture entire assemblages of action, subjective dissonance, productive ambiguities and contingent vitalities that inflect so much of contemporary quotidian life. The refugee in particular has emerged as a destabilizing figure, capable of subversively using the spatio-temporality of the camp as the very resource through which to disturb ascribed categorizations. Reading the paradoxical multiplicity of actions that refugees — women, children and the elderly — perform in the space between Qalandia camp and its checkpoint provides an insight into some of what defines contemporary refugee subjectivities — flexibility, a readiness to take risks, an ability to maneuver through different temporal orders and instrumentalize the spatial fragmentation. These subjects, traversing and negotiating liminality in everyday life, point to lived and bodied affirmations of presence and visibility that cannot be understood through frameworks of recognition and rights. PMID:21542208

  7. Immune checkpoint blockade with concurrent electrochemotherapy in advanced melanoma: a retrospective multicenter analysis.

    Science.gov (United States)

    Heppt, Markus V; Eigentler, Thomas K; Kähler, Katharina C; Herbst, Rudolf A; Göppner, Daniela; Gambichler, Thilo; Ulrich, Jens; Dippel, Edgar; Loquai, Carmen; Schell, Beatrice; Schilling, Bastian; Schäd, Susanne G; Schultz, Erwin S; Matheis, Fanny; Tietze, Julia K; Berking, Carola

    2016-08-01

    Growing evidence suggests that concurrent loco-regional and systemic treatment modalities may lead to synergistic anti-tumor effects in advanced melanoma. In this retrospective multicenter study, we evaluate the use of electrochemotherapy (ECT) combined with ipilimumab or PD-1 inhibition. We investigated patients with unresectable or metastatic melanoma who received the combination of ECT and immune checkpoint blockade for distant or cutaneous metastases within 4 weeks. Clinical and laboratory data were collected and analyzed with respect to safety and efficacy. A total of 33 patients from 13 centers were identified with a median follow-up time of 9 months. Twenty-eight patients received ipilimumab, while five patients were treated with a PD-1 inhibitor (pembrolizumab n = 3, nivolumab n = 2). The local overall response rate (ORR) was 66.7 %. The systemic ORR was 19.2 and 40.0 % in the ipilimumab and PD-1 cohort, respectively. The median duration of response was not reached in either group. The median time to disease progression was 2.5 months for the entire population with 2 months for ipilimumab and 5 months for PD-1 blockade. The median overall survival was not reached in patients with ipilimumab and 15 months in the PD-1 group. Severe systemic adverse events were detected in 25.0 % in the ipilimumab group. No treatment-related deaths were observed. This is the first reported evaluation of ECT and simultaneous PD-1 inhibition and the largest published dataset on ECT with concurrent ipilimumab. The local response was lower than reported for ECT only. Ipilimumab combined with ECT was feasible, tolerable and showed a high systemic response rate. PMID:27294607

  8. Protein Crystals of Raf Kinase

    Science.gov (United States)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  9. Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

    OpenAIRE

    Man, Wing Yu; Mak, Joyce PY; Poon, Randy YC

    2013-01-01

    Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 D...

  10. MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells.

    Science.gov (United States)

    Beke, Lijs; Kig, Cenk; Linders, Joannes T M; Boens, Shannah; Boeckx, An; van Heerde, Erika; Parade, Marc; De Bondt, An; Van den Wyngaert, Ilse; Bashir, Tarig; Ogata, Souichi; Meerpoel, Lieven; Van Eynde, Aleyde; Johnson, Christopher N; Beullens, Monique; Brehmer, Dirk; Bollen, Mathieu

    2015-01-01

    Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold. PMID:26431963

  11. Cache-style Parallel Checkpointing for Large-scale Computing System%面向大规模计算系统的Cache式并行检查点

    Institute of Scientific and Technical Information of China (English)

    刘勇燕; 刘勇鹏; 冯华; 迟万庆

    2011-01-01

    Checkpointing is a typical technique for fault tolerance, whereas its scalability is limited by the overhead of file access. According to the multi-level file system architecture, the cache-style parallel checkpointing was introduced,which translates global coordinated checkpointing into local file operation by out-of-order pipelining of checkpoint flushing opportunity. The overhead of write-back is hidden effectively to increase the performance and the scalability of parallel checkpointing.%检查点机制是高性能并行计算系统中重要的容错手段,随着系统规模的增大,并行检查点的可扩展性受文件访问的制约.针对大规模并行计算系统的多级文件系统结构,提出了cache式并行检查点技术.它将全局同步并行检查点转化为局部文件操作,并利用多处理器结构进行乱序流水线式写回调度,将检查点的写回时机合理分布,从而有效地隐藏了检查点的写回开销,保证了并行检查点文件访问的高性能和高可扩展性.

  12. Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

    Energy Technology Data Exchange (ETDEWEB)

    Jacques, David A.; Streamer, Margaret [School of Molecular and Microbial Biosciences, University of Sydney (Australia); Rowland, Susan L.; King, Glenn F. [Institute of Molecular Biology, University of Queensland (Australia); Guss, J. Mitchell; Trewhella, Jill; Langley, David B., E-mail: d.langley@usyd.edu.au [School of Molecular and Microbial Biosciences, University of Sydney (Australia)

    2009-06-01

    The crystal structure of Sda, a DNA-replication/damage checkpoint inhibitor of sporulation in B. subtilis, has been solved via the MAD method. The subunit arrangement in the crystal has enabled a reappraisal of previous biophysical data, resulting in a new model for the behaviour of the protein in solution. The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.

  13. Information-theoretic analysis of x-ray photoabsorption based threat detection system for check-point

    Science.gov (United States)

    Lin, Yuzhang; Allouche, Genevieve G.; Huang, James; Ashok, Amit; Gong, Qian; Coccarelli, David; Stoian, Razvan-Ionut; Gehm, Michael E.

    2016-05-01

    In this work we present an information-theoretic framework for a systematic study of checkpoint x-ray systems using photoabsorption measurements. Conventional system performance analysis of threat detection systems confounds the effect of the system architecture choice with the performance of a threat detection algorithm. However, our system analysis approach enables a direct comparison of the fundamental performance limits of disparate hardware architectures, independent of the choice of a specific detection algorithm. We compare photoabsorptive measurements from different system architectures to understand the affect of system geometry (angular views) and spectral resolution on the fundamental limits of the system performance.

  14. Effect of Spindle Checkpoint on Akt2-mediated Paclitaxel-resistance in A2780 Ovarian Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    周婷; 鲍引娣; 叶双梅; 翁丹卉; 陈刚; 卢运萍; 马丁; 王世宣

    2010-01-01

    Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel(PTX)-induced apoptosis and control of the cell cycle.In addition,some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint.In the present study,we investigated the role of the Akt2/Bub1 cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel(PTX).Recombinant expression plasmid WT-Akt2 was transfected into A2780 ...

  15. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    Directory of Open Access Journals (Sweden)

    Naoto Tatewaki

    Full Text Available Ataxia telangiectasia mutated (ATM kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR. The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15 and Chk1 (Ser317 was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

  16. Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

    Science.gov (United States)

    Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

    2016-01-01

    Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression. PMID:26824362

  17. Tetrandrine: A Potent Abrogator of G2 Checkpoint Function in Tumor Cells and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    (M phase). Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.

  18. Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

    Energy Technology Data Exchange (ETDEWEB)

    Jacques, David A.; Streamer, Margaret; Rowland, Susan L.; King, Glenn F.; Guss, J. Mitchell; Trewhella, J.; Langley, David B.; (Sydney); (Queensland)

    2009-09-02

    The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.

  19. Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer

    International Nuclear Information System (INIS)

    Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA

  20. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    Science.gov (United States)

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

  1. The architecture of the BubR1 tetratricopeptide tandem repeat defines a protein motif underlying mitotic checkpoint-kinetochore communication

    DEFF Research Database (Denmark)

    Bolanos-Garcia, Victor M; Nilsson, Jakob; Blundell, Tom L

    2012-01-01

    The accurate and timely transmission of the genetic material to progeny during successive rounds of cell division is sine qua non for the maintenance of genome stability. Eukaryotic cells have evolved a surveillance mechanism, the mitotic spindle assembly checkpoint (SAC), to prevent premature...... as substitution of BubR1 residues engaged in KNL1 binding impaired the SAC and BubR1 recruitment into checkpoint complexes in stable cell lines. Here we discuss the implications of the disorder-to-order transition of KNL1 upon BubR1 binding for SAC signaling and propose a mechanistic model of how BUBs binding may...

  2. Moving towards a customized approach for drug development: lessons from clinical trials with immune checkpoint inhibitors in lung cancer

    Science.gov (United States)

    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

  3. Stranglehold on the spindle assembly checkpoint: the human papillomavirus E2 protein provokes BUBR1-dependent aneuploidy.

    Science.gov (United States)

    Tan, Chye Ling; Teissier, Sébastien; Gunaratne, Jayantha; Quek, Ling Shih; Bellanger, Sophie

    2015-01-01

    The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event. PMID:25789401

  4. Ermenilerin Kontrol Noktası: İskenderun Limanı Alexandretta Port - Checkpoint For Armenians

    Directory of Open Access Journals (Sweden)

    Naim ÜRKMEZ

    2013-07-01

    Full Text Available The period of Yavuz Sultan Selim and the Ottoman Empire’s entering Alexandretta until the first half of the 19th century had identity of a very small village. Right to the end of the century, the industry revolution’s impact and trading goods’ large amount of transport means gained importance starting especially in the city of Aleppo and then in one of South Anatolia’s natural ports as of the day the preliminary plan came out. After the 1890’s the increase of Armenian activities in Anatolia and the outbreak of revolts in many other places parallel the Ottoman government’s large forces’ involvement to prevent and be able to keep Armenian rebels under control, taking various measures. Abdul Hamid II started under this negative initiative; the Sultan tried to resist in various ways. From these measures in any of the provinces of Aleppo, Mamuretülaziz, Adana, Kayseri, Bitlis, Van, Diyarbekir, or Erzurum, Armenians living in the diaspora and connecting supplier port, as in Alexandretta, have a checkpoint facility. Sultan Abdul Hamid II’s direction of the facility is seen at the checkpoint from the dock outside rising or Anatolia geography’s various residential areas incoming Armenian travelers often reserved monthly lists. In addition to this, England in the grip of Cyprus was able to check Armenians’ entries into Anatolia set by a weapons and bombs training field. It is understood that this matter is elaborated on further in books prepared from lists. These books will come to light with the acceleration of the classification of records. Yavuz Sultan Selim döneminde Osmanlı hâkimiyetine giren İskenderun, 19. yüzyılın ilk yarısına kadar çok küçük bir köy hüviyetinde kalmıştır. Yüzyılın sonuna doğru sanayi devriminin etkisi ve ticari emtianın çokça taşınması vesilesi ile önem kazanmaya başlayan şehir özellikle Halep ve ardı ile Güney Anadolu’nun doğal bir limanı olarak gün geçtikçe ön plana

  5. A multisubstrate deoxyribonucleoside kinase from plants

    DEFF Research Database (Denmark)

    Clausen, Anders R.; Girandon, Lenart; Knecht, Wolfgang;

    2008-01-01

    Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship...... in anti-cancer therapy....

  6. Protein kinase CK2 in human diseases

    DEFF Research Database (Denmark)

    Guerra, Barbara; Issinger, Olaf-Georg

    2008-01-01

    Protein kinase CK2 (formerly referred to as casein kinase II) is an evolutionary conserved, ubiquitous protein kinase. There are two paralog catalytic subunits, i.e. alpha (A1) and alpha' (A2). The alpha and alpha' subunits are linked to two beta subunits to produce a heterotetrameric structure....... The catalytic alpha subunits are distantly related to the CMGC subfamily of kinases, such as the Cdk kinases. There are some peculiarities associated with protein kinase CK2, which are not found with most other protein kinases: (i) the enzyme is constitutively active, (ii) it can use ATP and GTP and...... specifically target this protein kinase [10]. Since not all the aspects of what has been published on CK2 can be covered in this review, we would like to recommend the following reviews; (i) for general information on CK2 [11-18] and (ii) with a focus on aberrant CK2 [19-22]....

  7. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tub

  8. Inhibitors of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    LIU Shiying; JIANG Yuyang; CAO Jian; LIU Feng; MA Li; ZHAO Yufen

    2005-01-01

    Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family. They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer, inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.

  9. Degradation of Activated Protein Kinases by Ubiquitination

    OpenAIRE

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases.

  10. Determinants of homodimerization specificity in histidine kinases

    OpenAIRE

    Ashenberg, Orr; Rozen-Gagnon, Kathryn; Laub, Michael T; Keating, Amy E.

    2011-01-01

    Two-component signal transduction pathways consisting of a histidine kinase and a response regulator are used by prokaryotes to respond to diverse environmental and intracellular stimuli. Most species encode numerous paralogous histidine kinases that exhibit significant structural similarity. Yet in almost all known examples, histidine kinases are thought to function as homodimers. We investigated the molecular basis of dimerization specificity, focusing on the model histidine kinase EnvZ and...

  11. The Level of Europium-154 Contaminating Samarium-153-EDTMP Activates the Radiation Alarm System at the US Homeland Security Checkpoints

    Directory of Open Access Journals (Sweden)

    Mohammed Najeeb Al Hallak

    2009-08-01

    Full Text Available 153Sm-EDTMP is a radiopharmaceutical composed of EDTMP (ethylenediamine-tetramethylenephosphonate and Samarium-153 [1]. 153Sm-EDTMP has an affinity for skeletal tissue and concentrates in areas with increased bone turnover; thus, it is successfully used in relieving pain related to diffuse bone metastases [1]. The manufacturing process of 153Sm-EDTMP leads to contamination with 154Eu (Europium-154 [2]. A previous study only alluded to the retention of 154Eu in the bones after receiving treatment with 153Sm-EDTMP [2]. Activation of the alarm at security checkpoints after 153Sm-EDTMP therapy has not been previously reported. Two out of 15 patients who received 153Sm-EDTMP at Roger Maris Cancer Center (Fargo, N. Dak., USA activated the radiation activity sensors while passing through checkpoints; one at a US airport and the other while crossing theAmerican-Canadian border. We assume that the 154Eu which remained in the patients’ bones activated the sensors. Methods: In order to investigate this hypothesis, we obtained the consent from 3 of our 15 patients who received 153Sm-EDTMP within the previous 4 months to 2 years, including the patient who had activated the radiation alarm at the airport. The patients were scanned with a handheld detector and a gamma camera for energies from 511 keV to 1.3 MeV. Results: All three patients exhibited identical spectral images, and further analysis showed that the observed spectra are the result of 154Eu emissions. Conclusion: Depending on the detection thresholds and windows used by local and federal authorities, the remaining activity of 154Eu retained in patients who received 153Sm-EDTMP could be sufficient enough to increase the count rates above background levels and activate the sensors. At Roger Maris Cancer Center, patients are now informed of the potential consequences of 153Sm-EDTMP therapy prior to initiating treatment. In addition, patients treated with 153Sm-EDTMP at Roger Maris Cancer Center

  12. Checkpoint inhibitors in cancer immunotherapy: Cross reactivity of a CTLA-4 antibody and IDO-inhibitor L-1MT in pigs

    DEFF Research Database (Denmark)

    Al-Shatrawi, Zina Adil; Frøsig, Thomas Mørch; Jungersen, Gregers

    a non-specific activation of porcine T cells. This will be further investigated to provide the basis for in vivo studies investigating checkpoint inhibitor blockade in combination with other cancer immunotherapies. Eventually our goal is to establish pigs as an alternative large animal model...

  13. SCIB2, an antibody DNA vaccine encoding NY-ESO-1 epitopes, induces potent antitumor immunity which is further enhanced by checkpoint blockade.

    Science.gov (United States)

    Xue, Wei; Metheringham, Rachael L; Brentville, Victoria A; Gunn, Barbara; Symonds, Peter; Yagita, Hideo; Ramage, Judith M; Durrant, Lindy G

    2016-06-01

    Checkpoint blockade has demonstrated promising antitumor responses in approximately 10-40% of patients. However, the majority of patients do not make a productive immune response to their tumors and do not respond to checkpoint blockade. These patients may benefit from an effective vaccine that stimulates high-avidity T cell responses in combination with checkpoint blockade. We have previously shown that incorporating TRP-2 and gp100 epitopes into the CDR regions of a human IgG1 DNA (ImmunoBody®: IB) results in significant tumor regression both in animal models and patients. This vaccination strategy is superior to others as it targets antigen to antigen-presenting cells and stimulates high-avidity T cell responses. To broaden the application of this vaccination strategy, 16 NY-ESO-1 epitopes, covering over 80% of HLA phenotypes, were incorporated into the IB (SCIB2). They produced higher frequency and avidity T cell responses than peptide vaccination. These T cells were of sufficient avidity to kill NY-ESO-1-expressing tumor cells, and in vivo controlled the growth of established B16-NY-ESO-1 tumors, resulting in long-term survival (35%). When SCIB2 was given in combination with Treg depletion, CTLA-4 blockade or PD-1 blockade, long-term survival from established tumors was significantly enhanced to 56, 67 and 100%, respectively. Translating these responses into the clinic by using a combination of SCIB2 vaccination and checkpoint blockade can only further improve clinical responses.

  14. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

    Science.gov (United States)

    Kikuchi, Ken; Hettmer, Simone; Aslam, M Imran; Michalek, Joel E; Laub, Wolfram; Wilky, Breelyn A; Loeb, David M; Rubin, Brian P; Wagers, Amy J; Keller, Charles

    2014-01-01

    Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells. PMID:24453992

  15. Cell-cycle dependent expression of a translocation-mediated fusion oncogene mediates checkpoint adaptation in rhabdomyosarcoma.

    Directory of Open Access Journals (Sweden)

    Ken Kikuchi

    2014-01-01

    Full Text Available Rhabdomyosarcoma is the most commonly occurring soft-tissue sarcoma in childhood. Most rhabdomyosarcoma falls into one of two biologically distinct subgroups represented by alveolar or embryonal histology. The alveolar subtype harbors a translocation-mediated PAX3:FOXO1A fusion gene and has an extremely poor prognosis. However, tumor cells have heterogeneous expression for the fusion gene. Using a conditional genetic mouse model as well as human tumor cell lines, we show that that Pax3:Foxo1a expression is enriched in G2 and triggers a transcriptional program conducive to checkpoint adaptation under stress conditions such as irradiation in vitro and in vivo. Pax3:Foxo1a also tolerizes tumor cells to clinically-established chemotherapy agents and emerging molecularly-targeted agents. Thus, the surprisingly dynamic regulation of the Pax3:Foxo1a locus is a paradigm that has important implications for the way in which oncogenes are modeled in cancer cells.

  16. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yueyang [Division of Infectious Diseases, Brigham and Women' s Hospital and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115 (United States); Munger, Karl, E-mail: kmunger@rics.bwh.harvard.edu [Division of Infectious Diseases, Brigham and Women' s Hospital and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115 (United States)

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

  17. Fault-Tolerance through Message-logging and Check-pointing: Disaster Recovery for CORBA-based Distributed Bank Servers

    CERN Document Server

    Vassev, Emil; Kuang, Heng

    2009-01-01

    This report presents results of our endeavor towards developing a failure-recovery variant of a CORBA-based bank server that provides fault tolerance features through message logging and checkpoint logging. In this group of projects, three components were developed to satisfy the requirements: 1) a message-logging protocol for the branch servers of the distributed banking system to log required information; 2) a recovery module that restarts the bank server using the message log to help the restarted bank server process subsequent requests for various operations; 3) a monitor module that periodically checks whether the bank server is down and helps the recovery module restart the bank server if the latter has crashed.

  18. A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint.

    Science.gov (United States)

    Loureiro, Cláudia A; Matos, Ana Margarida; Dias-Alves, Ângela; Pereira, Joana F; Uliyakina, Inna; Barros, Patrícia; Amaral, Margarida D; Matos, Paulo

    2015-05-19

    The peripheral protein quality control (PPQC) checkpoint removes improperly folded proteins from the plasma membrane through a mechanism involving the E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70 interacting protein). PPQC limits the efficacy of some cystic fibrosis (CF) drugs, such as VX-809, that improve trafficking to the plasma membrane of misfolded mutants of the CF transmembrane conductance regulator (CFTR), including F508del-CFTR, which retains partial functionality. We investigated the PPQC checkpoint in lung epithelial cells with F508del-CFTR that were exposed to VX-809. The conformation of the scaffold protein NHERF1 (Na(+)/H(+) exchange regulatory factor 1) determined whether the PPQC recognized "rescued" F508del-CFTR (the portion that reached the cell surface in VX-809-treated cells). Activation of the cytoskeletal regulator Rac1 promoted an interaction between the actin-binding adaptor protein ezrin and NHERF1, triggering exposure of the second PDZ domain of NHERF1, which interacted with rescued F508del-CFTR. Because binding of F508del-CFTR to the second PDZ of NHERF1 precluded the recruitment of CHIP, the coexposure of airway cells to Rac1 activator nearly tripled the efficacy of VX-809. Interference with the NHERF1-ezrin interaction prevented the increase of efficacy of VX-809 by Rac1 activation, but the actin-binding domain of ezrin was not required for the increase in efficacy. Thus, rather than mainly directing anchoring of F508del-CFTR to the actin cytoskeleton, induction of ezrin activation by Rac1 signaling triggered a conformational change in NHERF1, which was then able to bind and stabilize misfolded CFTR at the plasma membrane. These insights into the cell surface stabilization of CFTR provide new targets to improve treatment of CF.

  19. Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer.

    Directory of Open Access Journals (Sweden)

    Maojun Yang

    2008-03-01

    Full Text Available In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2 and N2-Mad2/closed Mad2 (C-Mad2, with C-Mad2 being more active in APC/C(Cdc20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C or asymmetric O-Mad2-C-Mad2 (O-C dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

  20. Phenotypic characterization of autoreactive B cells--checkpoints of B cell tolerance in patients with systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Annett M Jacobi

    Full Text Available DNA-reactive B cells play a central role in systemic lupus erythematosus (SLE; DNA antibodies precede clinical disease and in established disease correlate with renal inflammation and contribute to dendritic cell activation and high levels of type 1 interferon. A number of central and peripheral B cell tolerance mechanisms designed to control the survival, differentiation and activation of autoreactive B cells are thought to be disturbed in patients with SLE. The characterization of DNA-reactive B cells has, however, been limited by their low frequency in peripheral blood. Using a tetrameric configuration of a peptide mimetope of DNA bound by pathogenic anti-DNA antibodies, we can identify B cells producing potentially pathogenic DNA-reactive antibodies. We, therefore, characterized the maturation and differentiation states of peptide, (ds double stranded DNA cross-reactive B cells in the peripheral blood of lupus patients and correlated these with clinical disease activity. Flow cytometric analysis demonstrated a significantly higher frequency of tetramer-binding B cells in SLE patients compared to healthy controls. We demonstrated the existence of a novel tolerance checkpoint at the transition of antigen-naïve to antigen-experienced. We further demonstrate that patients with moderately active disease have more autoreactive B cells in both the antigen-naïve and antigen-experienced compartments consistent with greater impairment in B cell tolerance in both early and late checkpoints in these patients than in patients with quiescent disease. This methodology enables us to gain insight into the development and fate of DNA-reactive B cells in individual patients with SLE and paves the way ultimately to permit better and more customized therapies.

  1. DMPD: Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15081522 Bruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signall...ruton's tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? PubmedID 15081522 Title Bruton...'s tyrosine kinase (Btk)-the critical tyrosine kinase in LPS signalling? Authors

  2. Endocytosis of Receptor Tyrosine Kinases

    Science.gov (United States)

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  3. RIP Kinases Initiate Programmed Necrosis

    Institute of Scientific and Technical Information of China (English)

    Lorenzo Galluzzi; Oliver Kepp; Guido Kroemer

    2009-01-01

    Some lethal stimuli can induce either apoptosis or necrosis, depending on the cell type and/or experimental setting. Until recently,the molecular bases of this phenomenon were largely unknown. Now, two members of the receptor-interacting serine-threonine kinase (RIP) family, RIP1 and RIP3, have been demonstrated to control the switch between apoptotic and necrotic cell death.Some mechanistic details, however, remain controversial.

  4. Thymidine kinase diversity in bacteria

    DEFF Research Database (Denmark)

    Sandrini, Michael; Clausen, A.R.; Munch-Petersen, B.;

    2006-01-01

    Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The....... The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria....

  5. Pantothenate Kinase-Associated Neurodegeneration

    OpenAIRE

    Meitinger, Thomas; Prokisch, Holger; Hartig, Monika B.; Klopstock, Thomas

    2012-01-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is a hereditary progressive disorder and the most frequent form of neurodegeneration with brain iron accumulation (NBIA). PKAN patients present with a progressive movement disorder, dysarthria, cognitive impairment and retinitis pigmentosa. In magnetic resonance imaging, PKAN patients exhibit the pathognonomic "eye of the tiger" sign in the globus pallidus which corresponds to iron accumulation and gliosis as shown in neuropathological e...

  6. Regulation and function of TPL-2,an IκB kinase-regulated MAP kinase kinase kinase

    Institute of Scientific and Technical Information of China (English)

    Thorsten Gantke; Srividya Sriskantharajah; Steven C Ley

    2011-01-01

    The IκB kinase(IKK)complex plays a well-documented role in innate and adaptive immunity.This function has been widely attributed to its role as the central activator of the NF-κB family of transcription factors.However,another important consequence of IKK activation is the regulation of TPL-2,a MEK kinase that is required for activation of ERK-1/2 MAP kinases in myeioid cells following Toll-like receptor and TNF receptor stimulation.In unstimulated cells,TPL-2 is stoichiometrically complexed with the NF-κB inhibitory protein NF-κB1 p105,which blocks TPL-2 access to its substrate MEK,and the ubiquitin-binding protein ABIN-2(A20-binding inhibitor of NF-κB 2),both of which are required to maintain TPL-2 protein stability.Following agonist stimulation,the IKK complex phosphorylates p105,triggering its K48-1inked ubiquitination and degradation by the proteasome.This releases TPL-2 from p105-mediated inhibition,facilitating activation of MEK,in addition to modulating NF-κB activation by liberating associated Rel subunits for translocation into the nucleus.IKK-induced proteolysis of 0105,therefore,can directly regulate both NF-κB and ERK MAP kinase activation via NF-κB1 p105.TPL-2 is critical for production of the proinflammatory cytokine TNF during inflammatory responses.Consequently,there has been considerable interest in the pharmaceutical industry to develop selective TPL-2 inhibitors as drugs for the treatment of TNF-dependent inflammatory,diseases,such as rheumatoid arthritis and inflammatory bowel disease.This review summarizes our current understanding of the regulation of TPL-2 signaling function,and also the complex positive and negative roles of TPL-2 in immune and inflammatory responses.

  7. NPM phosphorylation stimulates Cdk1, overrides G2/M checkpoint and increases leukemic blasts in mice.

    Science.gov (United States)

    Du, Wei; Zhou, Yun; Pike, Suzette; Pang, Qishen

    2010-02-01

    An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CDK) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Simultaneous inactivation of two CDK phosphorylation sites at Ser10 and Ser70 (NPM-AA) induced G(2)/M cell cycle arrest, phosphorylation of Cdk1 at Tyr15 (Cdc2(Tyr15)) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1(Tyr15) and Cdc25C sequestration was suppressed by expression of a phosphomimetic NPM mutant created on the same CDK sites (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 was required for proper interaction between Cdk1 and Cdc25C. Moreover, NPM-EE directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G(2)/M arrest and increased leukemia blasts in a NOD/SCID xenograft model. Thus, these findings reveal a novel function of NPM on regulation of cell cycle progression, in which phosphorylation of NPM controls cell cycle progression at G(2)/M transition through modulation of Cdk1 and Cdc25C activities.

  8. A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

    Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

  9. Immunochemical characterization of rat brain protein kinase

    International Nuclear Information System (INIS)

    Polyclonal antibodies against rat brain protein kinase C (the Ca2+/phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca2+, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca2+/phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of [3H]phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca2+-independent/phospholipid-dependent phorbol ester-binding protein

  10. Molecular Pathways: Targeting ATR in Cancer Therapy.

    Science.gov (United States)

    Karnitz, Larry M; Zou, Lee

    2015-11-01

    The human ATR gene encodes a kinase that is activated by DNA damage and replication stress as a central transducer of a checkpoint signaling pathway. Once activated, ATR phosphorylates multiple substrates, including the kinase Chk1, to regulate cell-cycle progression, replication fork stability, and DNA repair. These events promote cell survival during replication stress and in cells with DNA damage. Accordingly, there has been the tantalizing possibility that ATR inhibitors would be therapeutically useful, especially if they were more effective in tumor versus normal cells. Indeed, multiple studies have demonstrated that alterations that promote tumorigenesis, such as defects in the ATM-p53 pathway, constitutive oncogene activation, and acquisition of the alternative lengthening of telomeres pathway, render tumor cells sensitive to ATR inhibitor monotherapy and/or increase the synergy between ATR inhibitors and genotoxic chemotherapies. Now, nearly two decades after the discovery of ATR, two highly selective and potent ATR inhibitors, AZD6738 and VX-970, are in early-phase clinical trials either as monotherapies or paired with a variety of genotoxic chemotherapies. These trials will generate important insights into the effects of ATR inhibition in humans and the potential role of inhibiting this kinase in the treatment of human malignancies. PMID:26362996

  11. Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

    DEFF Research Database (Denmark)

    Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas;

    2009-01-01

    The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of...... numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites, the...... the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2...

  12. Protein Kinase D family kinases: roads start to segregate.

    Science.gov (United States)

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. PMID:24847910

  13. Diacylglycerol Kinase Inhibition and Vascular Function

    OpenAIRE

    Choi, Hyehun; Allahdadi, Kyan J.; Tostes, Rita C A; Webb, R. Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structu...

  14. MAP kinases and histone modification

    Institute of Scientific and Technical Information of China (English)

    Tamaki Suganuma; Jerry L. Workman

    2012-01-01

    Signal transduction pathways alter the gene expression program in response to extracellular or intracellular cues.Mitogen-activated protein kinases (MAPKs) govern numerous cellular processes including cell growth,stress response,apoptosis,and differentiation.In the past decade,MAPKs have been shown to regulate the transcription machinery and associate with chromatin-modifying complexes.Moreover,recent studies demonstrate that several MAPKs bind directly to chromatin at target genes.This review highlights the recent discoveries of MAPK signaling in regard to histone modifications and chromatin regulation.Evidence suggesting that further unknown mechanisms integrate signal transduction with chromatin biology is discussed.

  15. Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome.

    Science.gov (United States)

    Grosstessner-Hain, Karin; Hegemann, Björn; Novatchkova, Maria; Rameseder, Jonathan; Joughin, Brian A; Hudecz, Otto; Roitinger, Elisabeth; Pichler, Peter; Kraut, Norbert; Yaffe, Michael B; Peters, Jan-Michael; Mechtler, Karl

    2011-11-01

    Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif. PMID:21857030

  16. P21-activated kinase 4 (PAK4) is required for metaphase spindle positioning and anchoring.

    Science.gov (United States)

    Bompard, G; Rabeharivelo, G; Cau, J; Abrieu, A; Delsert, C; Morin, N

    2013-02-14

    The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins. PMID:22450748

  17. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases.

    Science.gov (United States)

    Chen, Ying-Nan P; LaMarche, Matthew J; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G; Dobson, Jason R; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J; Sellers, William R; Stams, Travis; Fortin, Pascal D

    2016-07-01

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers. PMID:27362227

  18. MST kinases in development and disease

    OpenAIRE

    Thompson, Barry J.; Sahai, Erik

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cance...

  19. Protein kinase profiling assays: a technology review.

    Science.gov (United States)

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  20. Suppression of the p53- or pRB-mediated G1 checkpoint is required for E2F-induced S-phase entry

    DEFF Research Database (Denmark)

    Lomazzi, Marina; Moroni, M Cristina; Jensen, Michael R;

    2002-01-01

    Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer. In the absence of other genetic alterations, this deregulation results in lack of differentiation, hyperproliferation and apoptosis. The pRB protein acts as a transcriptional repressor by targeting the E2F...... fibroblasts, increased E2F1 activity can result in S-phase entry in diploid fibroblasts in which the p53-mediated G1 checkpoint is suppressed. In addition, we show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results indicate that, in addition to acting as an E2F......-dependent transcriptional repressor, pRB is also required for the cells to retain the G1 checkpoint in response to unprogrammed proliferative signals....

  1. Inhibition of c-Abl kinase activity renders cancer cells highly sensitive to mitoxantrone.

    Directory of Open Access Journals (Sweden)

    Kemal Alpay

    Full Text Available Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in this context is far from clear. We studied the effect of inhibition of c-Abl kinase activity by imatinib with chemotherapy drugs and found a striking difference in cell survival after combined mitoxantrone (MX and imatinib treatment compared to a panel of other chemotherapy drugs. The combinatory treatment induced apoptosis in HeLa cells and other cancer cell lines but not in primary fibroblasts. The difference in MX and doxorubicin was related to significant augmentation of DNA damage. Transcriptionally active p53 accumulated in cells in which human papillomavirus E6 normally degrades p53. The combination treatment resulted in caspase activation and apoptosis, but this effect did not depend on either p53 or p73 activity. Despite increased p53 activity, the cells arrested in G2 phase became defective in this checkpoint, allowing cell cycle progression. The effect after MX treatment depended partially on c-Abl: Short interfering RNA knockdown of c-Abl rendered HeLa cells less sensitive to MX. The effect of imatinib was decreased by c-Abl siRNA suggesting a role for catalytically inactive c-Abl in the death cascade. These findings indicate that MX has a unique cytotoxic effect when the kinase activity of c-Abl is inhibited. The treatment results in increased DNA damage and c-Abl-dependent apoptosis, which may offer new possibilities for potentiation of cancer chemotherapy.

  2. Mps1 kinase regulates tumor cell viability via its novel role in mitochondria.

    Science.gov (United States)

    Zhang, X; Ling, Y; Guo, Y; Bai, Y; Shi, X; Gong, F; Tan, P; Zhang, Y; Wei, C; He, X; Ramirez, A; Liu, X; Cao, C; Zhong, H; Xu, Q; Ma, R Z

    2016-01-01

    Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells. PMID:27383047

  3. Ataxia telangiectasia-mutated kinase deficiency exacerbates left ventricular dysfunction and remodeling late after myocardial infarction.

    Science.gov (United States)

    Daniel, Laura L; Scofield, Stephanie L C; Thrasher, Patsy; Dalal, Suman; Daniels, Christopher R; Foster, Cerrone R; Singh, Mahipal; Singh, Krishna

    2016-08-01

    Ataxia telangiectasia-mutated kinase (ATM), a cell cycle checkpoint protein, is activated in response to DNA damage and oxidative stress. We have previously shown that ATM deficiency is associated with increased apoptosis and fibrosis and attenuation of cardiac dysfunction early (1-7 days) following myocardial infarction (MI). Here, we tested the hypothesis that enhanced fibrosis and apoptosis, as observed early post-MI during ATM deficiency, exacerbate cardiac dysfunction and remodeling in ATM-deficient mice late post-MI. MIs were induced in wild-type (WT) and ATM heterozygous knockout (hKO) mice by ligation of the left anterior descending artery. Left ventricular (LV) structural and functional parameters were assessed by echocardiography 14 and 28 days post-MI, whereas biochemical parameters were measured 28 days post-MI. hKO-MI mice exhibited exacerbated LV dysfunction as observed by increased LV end-systolic volume and decreased percent fractional shortening and ejection fraction. Infarct size and thickness were not different between the two genotypes. Myocyte cross-sectional area was greater in hKO-MI group. The hKO-MI group exhibited increased fibrosis in the noninfarct and higher expression of α-smooth muscle actin (myofibroblast marker) in the infarct region. Apoptosis and activation of GSK-3β (proapoptotic kinase) were significantly lower in the infarct region of hKO-MI group. Matrix metalloproteinase 2 (MMP-2) expression was not different between the two genotypes. However, MMP-9 expression was significantly lower in the noninfarct region of hKO-MI group. Thus ATM deficiency exacerbates cardiac remodeling late post-MI with effects on cardiac function, fibrosis, apoptosis, and myocyte hypertrophy. PMID:27288435

  4. Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

    Directory of Open Access Journals (Sweden)

    Peter Dinér

    Full Text Available The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG pathway is a conserved mitogen-activated protein kinase (MAPK signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

  5. Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

    Science.gov (United States)

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Grollman, Arthur P; Rosenquist, Thomas

    2015-01-01

    Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells. PMID:24792323

  6. Celecoxib Enhances the Radiosensitizing Effect of 7-Hydroxystaurosporine (UCN-01) in Human Lung Cancer Cell Lines

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Young-Mee; Jeong, In-Hye [Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Pyo, Hongryull, E-mail: Quasar93@yahoo.co.kr [Department of Radiation Oncology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2012-07-01

    Purpose: 7-Hydroxystaurosporine (UCN-01), a Chk1-specific inhibitor, showed promising in vitro and in vivo chemo- or radiosensitizing activity. However, there have been concerns about its limited therapeutic efficacy and risk of side effects. A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. Celecoxib is a cyclooxygenase-2 (COX-2)-specific inhibitor that downregulates ataxia telangiectasia and rad3-related (ATR) protein, an upstream kinase of Chk1. In this study, we investigated whether the addition of celecoxib can potentiate the radiosensitizing effect of UCN-01. Methods and Materials: The cooperative radiosensitizing effects and the underlying molecular mechanisms of UCN-01 plus celecoxib were determined by clonogenic assay, tumor growth delay assay, flow cytometry, and Western blotting. Synergism of the three agents combined (UCN-01 plus celecoxib plus radiation) were evaluated using median drug effect analysis and drug-independent action model analysis. Results: The combination of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The combination of both drugs also cooperatively inhibited IR-induced G{sub 2}/M arrest, and increased the G{sub 2} to mitotic transition. Conclusions: Combined treatment with UCN-01 and celecoxib can exert synergistically enhanced radiosensitizing effects via cooperative inhibition of the ionizing radiation-activated G{sub 2} checkpoint. We propose that this combination strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients.

  7. Immune Reactivation by Cell-Free Fetal DNA in Healthy Pregnancies Re-Purposed to Target Tumors: Novel Checkpoint Inhibition in Cancer Therapeutics

    OpenAIRE

    Enninga, Elizabeth Ann L.; Nevala, Wendy K.; Holtan, Shernan G.; Svetomir N. Markovic

    2015-01-01

    The role of the immune system in cancer progression has become increasingly evident over the past decade. Chronic inflammation in the promotion of tumorigenesis is well established, and cancer-associated tolerance/immune evasion has long been appreciated. Recent developments of immunotherapies targeting cancer-associated inflammation and immune tolerance, such as cancer vaccines, cell therapies, neutralizing antibodies, and immune checkpoint inhibitors, have shown promising clinical results. ...

  8. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    Science.gov (United States)

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  9. Automatic Fault-Tolerance Support in Resource Management System Based on Job Checkpoint/Restart%资源管理系统中基于作业检查点的自动容错

    Institute of Scientific and Technical Information of China (English)

    曹宏嘉; 卢宇彤; 谢旻

    2009-01-01

    An automatic fault-tolerance method based on job checkpoint/restart in resource management systems is pro-posed The key technologies are presented, including the separation of job checkpoint and task checkpoint, management of checkpoint image files, and automatic job restart.Automatic job checkpoint/restart with BLCR is implemented in SLURM and the challenges are discussed. Analysis and experiments show that the checkpoint and restart works correctly, and the time to complete large-scale jobs is reduced effectively.%本文提出了在资源管理系统中基于作业检查点实现自动容错支持,深入分析了作业与任务检查点分离、映像文件管理、自动恢复执行等关键技术.基于BLCR在SLURM中实现了作业的自动检查点/恢复,详细介绍了实现中的关键技术难题.分析与测试表明,检查点与恢复执行功能正确,并能有效缩短大规模作业成功运行所需的时间.

  10. Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

    NARCIS (Netherlands)

    Parikh, Kaushal; Diks, Sander H.; Tuynman, Jurriaan H. B.; Verhaar, Auke; Lowenberg, Mark; Hommes, Daan W.; Joore, Jos; Pandey, Akhilesh; Peppelenbosch, Maikel P.

    2009-01-01

    Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to pred

  11. Identification of a mutation causing a defective spindle assembly checkpoint in high ethyl caproate-producing sake yeast strain K1801.

    Science.gov (United States)

    Goshima, Tetsuya; Nakamura, Ryo; Kume, Kazunori; Okada, Hiroki; Ichikawa, Eri; Tamura, Hiroyasu; Hasuda, Hirokazu; Inahashi, Masaaki; Okazaki, Naoto; Akao, Takeshi; Shimoi, Hitoshi; Mizunuma, Masaki; Ohya, Yoshikazu; Hirata, Dai

    2016-08-01

    In high-quality sake brewing, the cerulenin-resistant sake yeast K1801 with high ethyl caproate-producing ability has been used widely; however, K1801 has a defective spindle assembly checkpoint (SAC). To identify the mutation causing this defect, we first searched for sake yeasts with a SAC-defect like K1801 and found that K13 had such a defect. Then, we searched for a common SNP in only K1801 and K13 by examining 15 checkpoint-related genes in 23 sake yeasts, and found 1 mutation, R48P of Cdc55, the PP2A regulatory B subunit that is important for the SAC. Furthermore, we confirmed that the Cdc55-R48P mutation was responsible for the SAC-defect in K1801 by molecular genetic analyses. Morphological analysis indicated that this mutation caused a high cell morphological variation. But this mutation did not affect the excellent brewing properties of K1801. Thus, this mutation is a target for breeding of a new risk-free K1801 with normal checkpoint integrity. PMID:27191586

  12. Immune Checkpoint Blockade in Cancer Treatment: A Double-Edged Sword Cross-Targeting the Host as an “Innocent Bystander”

    Directory of Open Access Journals (Sweden)

    Lucia Gelao

    2014-03-01

    Full Text Available Targeted immune checkpoint blockade augments anti-tumor immunity and induces durable responses in patients with melanoma and other solid tumors. It also induces specific “immune-related adverse events” (irAEs. IrAEs mainly include gastrointestinal, dermatological, hepatic and endocrinological toxicities. Off-target effects that arise appear to account for much of the toxicity of the immune checkpoint blockade. These unique “innocent bystander” effects are likely a direct result of breaking immune tolerance upon immune check point blockade and require specific treatment guidelines that include symptomatic therapies or systemic corticosteroids. What do we need going forward to limit immune checkpoint blockade-induced toxicity? Most importantly, we need a better understanding of the roles played by these agents in normal tissues, so that we can begin to predict potentially problematic side effects on the basis of their selectivity profile. Second, we need to focus on the predictive factors of the response and toxicity of the host rather than serially focusing on individual agents. Third, rigorous biomarker-driven clinical trials are needed to further elucidate the mechanisms of both the benefit and toxicity. We will summarize the double-edged sword effect of immunotherapeutics in cancer treatment.

  13. Loss of yeast peroxiredoxin Tsa1p induces genome instability through activation of the DNA damage checkpoint and elevation of dNTP levels.

    Directory of Open Access Journals (Sweden)

    Hei-Man Vincent Tang

    2009-10-01

    Full Text Available Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in tsa1Delta cells. Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Delta cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Delta cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.

  14. Novel Library of Selenocompounds as Kinase Modulators

    Directory of Open Access Journals (Sweden)

    Carmen Sanmartín

    2011-07-01

    Full Text Available Although the causes of cancer lie in mutations or epigenic changes at the genetic level, their molecular manifestation is the dysfunction of biochemical pathways at the protein level. The 518 protein kinases encoded by the human genome play a central role in various diseases, a fact that has encouraged extensive investigations on their biological function and three dimensional structures. Selenium (Se is an important nutritional trace element involved in different physiological functions with antioxidative, antitumoral and chemopreventive properties. The mechanisms of action for selenocompounds as anticancer agents are not fully understood, but kinase modulation seems to be a possible pathway. Various organosulfur compounds have shown antitumoral and kinase inhibition effects but, in many cases, the replacement of sulfur by selenium improves the antitumoral effect of compounds. Although Se atom possesses a larger atomic volume and nucleophilic character than sulfur, Se can also formed interactions with aminoacids of the catalytic centers of proteins. So, we propose a novel chemical library that includes organoselenium compounds as kinase modulators. In this study thirteen selenocompounds have been evaluated at a concentration of 3 or 10 µM in a 24 kinase panel using a Caliper LabChip 3000 Drug Discover Platform. Several receptor (EGFR, IGFR1, FGFR1… and non-receptor (Abl kinases have been selected, as well as serine/threonine/lipid kinases (AurA, Akt, CDKs, MAPKs… implicated in main cancer pathways: cell cycle regulation, signal transduction, angiogenesis regulation among them. The obtained results showed that two compounds presented inhibition values higher than 50% in at least four kinases and seven derivatives selectively inhibited one or two kinases. Furthermore, three compounds selectively activated IGF-1R kinase with values ranging from −98% to −211%. In conclusion, we propose that the replacement of sulfur by selenium seems to be

  15. A novel viral thymidylate kinase with dual kinase activity.

    Science.gov (United States)

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  16. Opportunistic autoimmune disorders potentiated by immune-checkpoint inhibitors anti-CTLA-4 and anti-PD-1

    Directory of Open Access Journals (Sweden)

    Yi-Chi eKong

    2014-05-01

    Full Text Available To improve the efficacy of immunotherapy for cancer and autoimmune diseases, recent ongoing and completed clinical trials have focused on specific targets to redirect the immune network toward eradicating a variety of tumors and ameliorating the self-destructive process. In a previous review (Kong et al., Ann. N.Y. Acad. Sci. 1183:222-236, 2010, both systemic immunomodulators and monoclonal antibodies, anti-CTLA-4 and anti-CD52, were discussed regarding therapeutics and autoimmune sequelae, as well as predisposing factors known to exacerbate immune-related adverse events. This review will focus on immune-checkpoint inhibitors, and the data from most clinical trials involve blockade with anti-CTLA-4 such as ipilimumab. However, despite the mild to severe immune-related adverse events observed with ipilimumab in ~60% of patients, overall survival averaged ~22-25% at 3-5 years. To boost overall survival, other monoclonal antibodies targeting programmed death-1 and its ligand are undergoing clinical trials as monotherapy or dual therapy with anti-CTLA-4. Therapeutic combinations may generate different spectrum of opportunistic autoimmune disorders. To simulate clinical scenarios, we have applied regulatory T cell perturbation to murine models combined to examine the balance between thyroid autoimmunity and tumor-specific immunity.

  17. Human Zwint-1 Specifies Localization of Zeste White 10 to Kinetochores and Is Essential for Mitotic Checkpoint Signaling

    Institute of Scientific and Technical Information of China (English)

    HongmeiWang; XiaoyuHu; XiaDing; ZhenDou; ZhihongYank; AndrewW.Shaw; MaikunTcng; DonW.Cleveland; MichaelL.Goldberg; LiwenNiu; XucbiaoYao

    2005-01-01

    Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore, a multiprotein complex assembled onto centromeric DNA of the chromosome. Here we show that Zwint-1 is required and is sufficient for kinetochore localization of Zestc White 10 (ZW10) in HeLa cells. Zwint-1 specifies the kinetochore association of ZW10 by interacting with its N-terminal domain. Suppression of synthesis of Zwint-1 by small interfering RNA abolishes the localization of ZW10 to the kinetochore, demonstrating the requirement of Zwint-1 for ZWl0 kinetochore localization. In addition, dcplction of Zwint-1 affects no mitotic arrest but causes aberrant premature chromo. some segregation. These Zwint-l-suppressed cells dis. play chromosome bridge phenotype with sister chromatids inter-connected. Moreover, Zwint-1 is required for stable association of CENP.F and dynamitin but not BUB1 with the kinetochore. Finally, our studies showthat Zwint-1 is a new component of the mitotic check. point, as cells lacking Zwint-1 fail to arrest in mitosis when exposed to microtubule inhibitors, yielding inter. phase cells with multinuclei. As ZWl0 and Zwint.1 are absent from yeast, we reasoned that metazoans evolved an elaborate spindle checkpoint machinery to ensure faithful chromosome segregation in mitosis.

  18. A DNA damage checkpoint in Caulobacter crescentus inhibits cell division through a direct interaction with FtsW.

    Science.gov (United States)

    Modell, Joshua W; Hopkins, Alexander C; Laub, Michael T

    2011-06-15

    Following DNA damage, cells typically delay cell cycle progression and inhibit cell division until their chromosomes have been repaired. The bacterial checkpoint systems responsible for these DNA damage responses are incompletely understood. Here, we show that Caulobacter crescentus responds to DNA damage by coordinately inducing an SOS regulon and inhibiting the master regulator CtrA. Included in the SOS regulon is sidA (SOS-induced inhibitor of cell division A), a membrane protein of only 29 amino acids that helps to delay cell division following DNA damage, but is dispensable in undamaged cells. SidA is sufficient, when overproduced, to block cell division. However, unlike many other regulators of bacterial cell division, SidA does not directly disrupt the assembly or stability of the cytokinetic ring protein FtsZ, nor does it affect the recruitment of other components of the cell division machinery. Instead, we provide evidence that SidA inhibits division by binding directly to FtsW to prevent the final constriction of the cytokinetic ring.

  19. miR-125b promotes cell death by targeting spindle assembly checkpoint gene MAD1 and modulating mitotic progression.

    Science.gov (United States)

    Bhattacharjya, S; Nath, S; Ghose, J; Maiti, G P; Biswas, N; Bandyopadhyay, S; Panda, C K; Bhattacharyya, N P; Roychoudhury, S

    2013-03-01

    The spindle assembly checkpoint (SAC) is a 'wait-anaphase' mechanism that has evolved in eukaryotic cells in response to the stochastic nature of chromosome-spindle attachments. In the recent past, different aspects of the SAC regulation have been described. However, the role of microRNAs in the SAC is vaguely understood. We report here that Mad1, a core SAC protein, is repressed by human miR-125b. Mad1 serves as an adaptor protein for Mad2 - which functions to inhibit anaphase entry till the chromosomal defects in metaphase are corrected. We show that exogenous expression of miR-125b, through downregulation of Mad1, delays cells at metaphase. As a result of this delay, cells proceed towards apoptotic death, which follows from elevated chromosomal abnormalities upon ectopic expression of miR-125b. Moreover, expressions of Mad1 and miR-125b are inversely correlated in a variety of cancer cell lines, as well as in primary head and neck tumour tissues. We conclude that increased expression of miR-125b inhibits cell proliferation by suppressing Mad1 and activating the SAC transiently. We hypothesize an optimum Mad1 level and thus, a properly scheduled SAC is maintained partly by miR-125b.

  20. Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy

    Science.gov (United States)

    He, Chunbai; Duan, Xiaopin; Guo, Nining; Chan, Christina; Poon, Christopher; Weichselbaum, Ralph R.; Lin, Wenbin

    2016-08-01

    Advanced colorectal cancer is one of the deadliest cancers, with a 5-year survival rate of only 12% for patients with the metastatic disease. Checkpoint inhibitors, such as the antibodies inhibiting the PD-1/PD-L1 axis, are among the most promising immunotherapies for patients with advanced colon cancer, but their durable response rate remains low. We herein report the use of immunogenic nanoparticles to augment the antitumour efficacy of PD-L1 antibody-mediated cancer immunotherapy. Nanoscale coordination polymer (NCP) core-shell nanoparticles carry oxaliplatin in the core and the photosensitizer pyropheophorbide-lipid conjugate (pyrolipid) in the shell (NCP@pyrolipid) for effective chemotherapy and photodynamic therapy (PDT). Synergy between oxaliplatin and pyrolipid-induced PDT kills tumour cells and provokes an immune response, resulting in calreticulin exposure on the cell surface, antitumour vaccination and an abscopal effect. When combined with anti-PD-L1 therapy, NCP@pyrolipid mediates regression of both light-irradiated primary tumours and non-irradiated distant tumours by inducing a strong tumour-specific immune response.