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

    Science.gov (United States)

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

    2015-05-08

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

  3. Checkpoint kinase1 (CHK1) is an important biomarker in breast cancer having a role in chemotherapy response

    Science.gov (United States)

    Al-kaabi, M M; Alshareeda, A T; Jerjees, D A; Muftah, A A; Green, A R; Alsubhi, N H; Nolan, C C; Chan, S; Cornford, E; Madhusudan, S; Ellis, I O; Rakha, E A

    2015-01-01

    Background: Checkpoint kinase1 (CHK1), which is a key component of DNA-damage-activated checkpoint signalling response, may have a role in breast cancer (BC) pathogenesis and influence response to chemotherapy. This study investigated the clinicopathological significance of phosphorylated CHK1 (pCHK1) protein in BC. Method: pCHK1 protein expression was assessed using immunohistochemistry in a large, well-characterized annotated series of early-stage primary operable invasive BC prepared as tissue microarray (n=1200). Result: pCHK1 showed nuclear and/or cytoplasmic expression. Tumours with nuclear expression showed positive associations with favourable prognostic features such as lower grade, lower mitotic activity, expression of hormone receptor and lack of expression of KI67 and PI3K (P<0.001). On the other hand, cytoplasmic expression was associated with features of poor prognosis such as higher grade, triple-negative phenotype and expression of KI67, p53, AKT and PI3K. pCHK1 expression showed an association with DNA damage response (ATM, RAD51, BRCA1, KU70/KU80, DNA-PKCα and BARD1) and sumoylation (UBC9 and PIASγ) biomarkers. Subcellular localisation of pCHK1 was associated with the expression of the nuclear transport protein KPNA2. Positive nuclear expression predicted better survival outcome in patients who did not receive chemotherapy in the whole series and in ER-positive tumours. In ER-negative and triple-negative subgroups, nuclear pCHK1 predicted shorter survival in patients who received cyclophosphamide, methotrexate and 5-florouracil chemotherapy. Conclusions: Our data suggest that pCHK1 may have prognostic and predictive significance in BC. Subcellular localisation of pCHK1 protein is related to its function. PMID:25688741

  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. DNA topoisomerase 2 mutant allele mildly delays the mitotic progression and activates the checkpoint protein kinase Chk1 in fission yeast Schizosaccharomyces pombe.

    Science.gov (United States)

    Yadav, Sudhanshu; Verma, Sumit Kumar; Ahmed, Shakil

    2011-08-01

    DNA topoisomerases are specialized nuclear enzymes that perform topological modifications on double-stranded DNA (dsDNA) and hence are essential for DNA metabolism such as replication, transcription, recombination, condensation and segregation. In a genetic screen, we identified a temperature-sensitive mutant allele of topoisomerase 2 that exhibits conditional synthetic lethality with a chk1 knockout strain. The mutant allele of topoisomerase 2 is defective in chromosome segregation at a non-permissive temperature and there was increase in chromosome segregation defects in the double mutant of top2-10 and chk1 delete at a non-permissive temperature. More importantly, topoisomearse 2 mutant cells mildly delay the mitotic progression at non-permissive temperature that is mediated by checkpoint protein kinase Chk1. Additionally, top2-10 mutant cells also activate the Chk1 at a non-permissive temperature and this activation of Chk1 takes place at the time of mitosis. Interestingly, top2-10 mutant cells retain their viability at a non-permissive temperature if the cells are not allowed to enter into mitosis. Taking together our results, we speculate that in the top2-10 mutant, the segregation of entangled chromatids during mitosis could result in delaying the mitotic progression through the activation of Chk1 kinase.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    Science.gov (United States)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  9. The fork and the kinase: a DNA replication tale from a CHK1 perspective.

    Science.gov (United States)

    González Besteiro, Marina A; Gottifredi, Vanesa

    2015-01-01

    Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress.

  10. 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...... by a combined action of Chk1 and Chk2 kinases. Finally, phosphorylation of Chk1 by ATM was required to fully accelerate the IR-induced degradation of Cdc25A. Our results provide evidence that the mammalian S phase checkpoint functions via amplification of physiologically operating, Chk1-dependent mechanisms....

  11. Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase: a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy.

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    Martino-Echarri, Estefania; Henderson, Beric R; Brocardo, Mariana G

    2014-10-30

    5-fluorouracil (5-FU) is the first line component used in colorectal cancer (CRC) therapy however even in combination with other chemotherapeutic drugs recurrence is common. Mutations of the adenomatous polyposis coli (APC) gene are considered as the initiating step of transformation in familial and sporadic CRCs. We have previously shown that APC regulates the cellular response to DNA replication stress and recently hypothesized that APC mutations might therefore influence 5-FU resistance. To test this, we compared CRC cell lines and show that those expressing truncated APC exhibit a limited response to 5-FU and arrest in G1/S-phase without undergoing lethal damage, unlike cells expressing wild-type APC. In SW480 APC-mutant CRC cells, 5-FU-dependent apoptosis was restored after transient expression of full length APC, indicating a direct link between APC and drug response. Furthermore, we could increase sensitivity of APC truncated cells to 5-FU by inactivating the Chk1 kinase using drug treatment or siRNA-mediated knockdown. Our findings identify mutant APC as a potential tumor biomarker of resistance to 5-FU, and importantly we show that APC-mutant CRC cells can be made more sensitive to 5-FU by use of Chk1 inhibitors.

  12. 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...

  13. Design of targeted libraries against the human Chk1 kinase using PGVL Hub.

    Science.gov (United States)

    Peng, Zhengwei; Hu, Qiyue

    2011-01-01

    PGVL Hub is a Pfizer internal desktop tool for chemical library and singleton design. In this chapter, we give a short introduction to PGVL Hub, the core workflow it supports, and the rich design capabilities it provides. By re-creating two legacy targeted libraries against the human checkpoint kinase 1 (Chk1) as a showcase, we illustrate how PGVL Hub could be used to help library designers carry out the steps in library design and realize design objectives such as SAR expansion and improvement in both kinase selectivity and compound aqueous solubility. Finally we share several tips about library design and usage of PGVL Hub.

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

    Directory of Open Access Journals (Sweden)

    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.

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

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    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.

  16. 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

    Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7-ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA...... 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......) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase h. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin-proteasome machinery with the DNA damage bypass pathway to guard against replication collapse...

  17. The stress-activated protein kinases p38α/β and JNK1/2 cooperate with Chk1 to inhibit mitotic entry upon DNA replication arrest.

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    Llopis, Alba; Salvador, Noelia; Ercilla, Amaia; Guaita-Esteruelas, Sandra; Barrantes, Ivan del Barco; Gupta, Jalaj; Gaestel, Matthias; Davis, Roger J; Nebreda, Angel R; Agell, Neus

    2012-10-01

    Accurate DNA replication is crucial for the maintenance of genome integrity. To this aim, cells have evolved complex surveillance mechanisms to prevent mitotic entry in the presence of partially replicated DNA. ATR and Chk1 are key elements in the signal transduction pathways of DNA replication checkpoint; however, other kinases also make significant contributions. We show here that the stress kinases p38 and JNK are activated when DNA replication is blocked, and that their activity allows S/M, but not G 2/M, checkpoint maintenance when Chk1 is inhibited. Activation of both kinases by DNA replication inhibition is not mediated by the caffeine-sensitive kinases ATR or ATM. Phosphorylation of MKK3/6 and MKK4, p38 and JNK upstream kinases was also observed upon DNA replication inhibition. Using a genetic approach, we dissected the p38 pathway and showed that both p38α and p38β isoforms collaborate to inhibit mitotic entry. We further defined MKK3/6 and MK2/3 as the key upstream and downstream elements in the p38 signaling cascade after replication arrest. Accordingly, we found that the stress signaling pathways collaborate with Chk1 to keep cyclin B1/Cdk1 complexes inactive when DNA replication is inhibited, thereby preventing cell cycle progression when DNA replication is stalled. Our results show a complex response to replication stress, where multiple pathways are activated and fulfill overlapping roles to prevent mitotic entry with unreplicated DNA.

  18. 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].

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

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

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

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  2. 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.

  3. CHK1 as a therapeutic target to bypass chemoresistance in AML.

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    David, Laure; Fernandez-Vidal, Anne; Bertoli, Sarah; Grgurevic, Srdana; Lepage, Benoît; Deshaies, Dominique; Prade, Naïs; Cartel, Maëlle; Larrue, Clément; Sarry, Jean-Emmanuel; Delabesse, Eric; Cazaux, Christophe; Didier, Christine; Récher, Christian; Manenti, Stéphane; Hoffmann, Jean-Sébastien

    2016-09-13

    The nucleoside analog cytarabine, an inhibitor of DNA replication fork progression that results in DNA damage, is currently used in the treatment of acute myeloid leukemia (AML). We explored the prognostic value of the expression of 72 genes involved in various aspects of DNA replication in a set of 198 AML patients treated by cytarabine-based chemotherapy. We unveiled that high expression of the DNA replication checkpoint gene CHEK1 is a prognostic marker associated with shorter overall, event-free, and relapse-free survivals and determined that the expression of CHEK1 can predict more frequent and earlier postremission relapse. CHEK1 encodes checkpoint kinase 1 (CHK1), which is activated by the kinase ATR when DNA replication is impaired by DNA damage. High abundance of CHK1 in AML patient cells correlated with higher clonogenic ability and more efficient DNA replication fork progression upon cytarabine treatment. Exposing the patient cells with the high abundance of CHK1 to SCH900776, an inhibitor of the kinase activity of CHK1, reduced clonogenic ability and progression of DNA replication in the presence of cytarabine. These results indicated that some AML cells rely on an efficient CHK1-mediated replication stress response for viability and that therapeutic strategies that inhibit CHK1 could extend current cytarabine-based treatments and overcome drug resistance. Furthermore, monitoring CHEK1 expression could be used both as a predictor of outcome and as a marker to select AML patients for CHK1 inhibitor treatments.

  4. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

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

    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 (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  5. ATR, Claspin and the Rad9-Rad1-Hus1 complex regulate Chk1 and Cdc25A in the absence of DNA damage

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Syljuåsen, Randi G; Lukas, Jiri

    2004-01-01

    The ATR and Chk1 kinases are essential to maintain genomic integrity. ATR, with Claspin and the Rad9-Rad1-Hus1 complex, activates Chk1 after DNA damage. Chk1-mediated phosphorylation of the Cdc25A phosphatase is required for the mammalian S-phase checkpoint. Here, we show that during physiological...... S phase the regulation of the Chk1-Cdc25A pathway depends on ATR, Claspin, Rad9, and Hus1. Human cells with chemically or genetically ablated ATR showed inhibition of Chk1-dependent phosphorylation of Cdc25A, and they accumulated Cdc25A without external DNA damage. Furthermore, si......RNA-mediated depletion of Claspin, Rad9 and Hus1 also stabilized Cdc25A. ATR ablation also inhibited the activatory phosphorylation of Chk1 on serine 345. Thus, the ATR-Chk1-Cdc25A pathway represents an integral part of physiological S-phase progression, and interference with this mechanism undermines viability...

  6. Chk1 inhibition activates p53 through p38 MAPK in tetraploid cancer cells.

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    Vitale, Ilio; Senovilla, Laura; Galluzzi, Lorenzo; Criollo, Alfredo; Vivet, Sonia; Castedo, Maria; Kroemer, Guido

    2008-07-01

    We have previously shown that tetraploid cancer cells succumb through a p53-dependent apoptotic pathway when checkpoint kinase 1 (Chk1) is depleted by small interfering RNAs (siRNAs) or inhibited with 7-hydroxystaurosporine (UCN-01). Here, we demonstrate that Chk1 inhibition results in the activating phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Depletion of p38 MAPK by transfection with a siRNA targeting the alpha isoform of p38 MAPK (p38alpha MAPK) abolishes the phosphorylation of p53 on serines 15 and 46 that is induced by Chk1 knockdown. The siRNA-mediated downregulation and pharmacological inhibition of p38alpha MAPK (with SB 203580) also reduces cell death induced by Chk1 knockdown or UCN-01. These results underscore the role of p38 MAPK as a pro-apoptotic kinase in the p53-dependant pathway for the therapeutic elimination of polyploidy cells.

  7. 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.

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

    Science.gov (United States)

    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

  9. Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation.

    Directory of Open Access Journals (Sweden)

    Wen Deng

    Full Text Available Nasopharyngeal carcinoma (NPC is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1 is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.

  10. 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 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...

  11. 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.

  12. Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation.

    Science.gov (United States)

    Petsalaki, Eleni; Dandoulaki, Maria; Morrice, Nick; Zachos, George

    2014-09-15

    Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Bloom's syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

  13. Targeting checkpoint kinase 1 in cancer therapeutics.

    Science.gov (United States)

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

    2007-04-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Chk1 suppressed cell death

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  17. 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.

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

    Science.gov (United States)

    Gao, Xiaodong; Han, Liping; Ren, Yujie

    2016-05-05

    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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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...

  6. DNA Damage Activates a Spatially Distinct Late Cytoplasmic Cell-Cycle Checkpoint Network Controlled by MK2-Mediated RNA Stabilization

    NARCIS (Netherlands)

    Reinhardt, H. Christian; Hasskamp, Pia; Schmedding, Ingolf; Morandell, Sandra; van Vugt, Marcel A. T. M.; Wang, XiaoZhe; Linding, Rune; Ong, Shao-En; Weaver, David; Carr, Steven A.; Yaffe, Michael B.

    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 pa

  7. MAP kinase meets mitosis: A role for Raf Kinase Inhibitory Protein in spindle checkpoint regulation

    Directory of Open Access Journals (Sweden)

    Rosner Marsha

    2007-01-01

    Full Text Available Abstract Raf Kinase Inhibitory Protein (RKIP is an evolutionarily conserved protein that functions as a modulator of signaling by the MAP kinase cascade. Implicated as a metastasis suppressor, Raf Kinase Inhibitory Protein depletion correlates with poor prognosis for breast, prostate and melanoma tumors but the mechanism is unknown. Recent evidence indicates that Raf Kinase Inhibitory Protein regulates the mitotic spindle assembly checkpoint by controlling Aurora B Kinase activity, and the mechanism involves Raf/MEK/ERK signaling. In contrast to elevated MAP kinase signaling during the G1, S or G2 phases of the cell cycle that activates checkpoints and induces arrest or senescence, loss of RKIP during M phase leads to bypass of the spindle assembly checkpoint and the generation of chromosomal abnormalities. These results reveal a role for Raf Kinase Inhibitory Protein and the MAP kinase cascade in ensuring the fidelity of chromosome segregation prior to cell division. Furthermore, these data highlight the need for precise titration of the MAP kinase signal to ensure the integrity of the spindle assembly process and provide a mechanism for generating genomic instability in tumors. Finally, these results raise the possibility that RKIP status in tumors could influence the efficacy of treatments such as poisons that stimulate the Aurora B-dependent spindle assembly checkpoint.

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

    Science.gov (United States)

    Chan, Leon Y; Amon, Angelika

    2009-07-15

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

  9. 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

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

    OpenAIRE

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

    2011-01-01

    Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extr...

  11. 细胞周期监测点激酶1与DNA损伤应答信号通路在肿瘤中的研究进展%Research progress of checkpoint kinase 1 and DNA damage response pathway in tumors

    Institute of Scientific and Technical Information of China (English)

    张瑶

    2016-01-01

    乳腺癌治疗过程中产生的治疗耐受已经成为其复发和转移的主要原因,耐受机制可能与激活DNA损伤应答反应有关.为了保持细胞基因的完整性,DNA损伤应答通路有较为复杂的信号转导通路系统,其中包括细胞周期监测点、DNA修复、转录和细胞凋亡.在癌症治疗中,各种细胞毒性药物以及放疗导致的基因损伤可以诱发DNA的损伤应答,对损伤反应的应答修复功能限制了放化疗的疗效.因此,为了提高乳腺癌放化疗的敏感性,满足找准靶向治疗药物的迫切需要,有必要将DNA损伤应答通路作为靶点治疗的机制研究,特别是对细胞周期监测点激酶1(CHK1)功能抑制药物的研究.最新的观点认为CHK1是DNA损伤应答激活的主要标志物,表明CHK1不仅激活了细胞周期检查位点的调节,而且直接影响了DNA修复和细胞凋亡.因此CHK1在DNA损伤应答机制中的作用能够促进CHK1抑制剂成为放化疗耐受肿瘤患者的一种新的治疗手段.%The main reason of recurrence and metastasis in breast cancer is the resistance for the radiotherapy and chemotherapy,and the mechanism of radio-resistance and chemo-resistance may be related to the DNA damage response (DDR).There is a complicated system of the DDR pathway,including cell cycle checkpoint,DNA repair,transcription and apoptosis to maintain the integrity of cell genes.In the cancer treatment,DDR occurs in various kinds of cytotoxic drugs and radiation to cause genetic damage,which limits the curative effect of chemotherapy and radiotherapy.This promotes the targeted therapy of DDR pathway,especially checkpoint kinase 1 (CHK1).Recently,the new viewpoint supports that CHK1 is a main marker of the DDR pathway activation,which shows that CHK1 not only activates the check point but also affects the DNA repair and apoptosis directly.Thus,the role of CHK1 in DDR will promote CHK1 inhibitor to be one of the new treatment strategies for the cancer

  12. 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...

  13. 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.

  14. 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

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

    Science.gov (United States)

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

    2011-01-01

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

  16. 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.

  17. 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.

  18. Microcephalin is a DNA damage response protein involved in regulation of CHK1 and BRCA1.

    Science.gov (United States)

    Xu, Xingzhi; Lee, Juhie; Stern, David F

    2004-08-13

    Microcephalin (MCPH1) is the first gene identified among at least six loci that contribute to the autosomal recessive disease, primary microcephaly. MCPH1, like NFBD1/MDC1, 53BP1, and BRCA1, encodes a protein with twin carboxyl-terminal BRCT domains (PTCB). Here, we report that Mcph1 forms ionizing radiation-induced foci. Down-regulation of Mcph1, like other PTCBs, by siRNA, impairs ionizing radiation-induced intra-S-phase and G(2)/M checkpoints. Inhibition of the expression of Mcph1 decreases both protein and transcript levels of endogenous Brca1 but not exogenous Brca1. Mcph1 inhibition also decreases both endogenous and heterologous Chk1 transcripts and protein. We conclude that Mcph1 is involved in DNA damage-induced cellular responses, and we propose that regulation of Brca1 and/or Chk1 by Mcph1 may contribute to these cellular responses.

  19. 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.

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

    Science.gov (United States)

    Khan, Saman; Ahmed, Shakil

    2015-01-01

    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.

  1. 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.

  2. The oxidative stress responsive transcription factor Pap1 confers DNA damage resistance on checkpoint-deficient fission yeast cells.

    Directory of Open Access Journals (Sweden)

    Carrie Belfield

    Full Text Available Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to chk1-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of chk1-deficient cells with mutations in genes encoding DNA ligase (cdc17 or DNA polymerase δ (cdc6, but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to chk1 mutants, as it also improved survival of rad1- and rad9-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active.

  3. 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.

  4. 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).

  5. 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.

  6. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    OpenAIRE

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

    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 (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel...

  7. Chk1防止DNA损伤的S期肿瘤细胞进行异常的有丝分裂%Chk1 prevents abnormal mitosis of S-phase HeLa cells containing DNA damage

    Institute of Scientific and Technical Information of China (English)

    李小方; Tarsha Ward; 姚雪彪; 吴家睿

    2009-01-01

    为探讨在p53失活的肿瘤细胞中DNA甲基化试剂引发的DNA损伤对于细胞周期的影响,我们将同步化在G1,S和G2/M期的HeLa细胞分别进行甲磺酸甲酯(MMS)处理.MMS的处理结果表明,各个时相的细胞周期进程均发生延迟或阻滞,其中S期细胞对药物最为敏感.进一步的分子机理研究表明,3个时相中ATM-Chk2和p38 MAPK通路均被激活,但是Chk1仅在S期中被活化,提示Chk1特异地参与了S期的DNA损伤检查点(DNAdamage checkpoint)或者DNA复制检查点(DNA replication checkpoint)的作用.为了进一步确定Chk1在S期的检查点功能,用专一的小分子抑制剂抑制Chk1的磷酸化.发现被MMS处理的S期细胞能在未完成复制的情况下进行异常的有丝分裂,提示Chk1主要是在HeLa细胞S期的DNA损伤检查点而不是DNA复制检查点发挥其作用.另外,本研究还检查了参与G2/M期进程的cyclin B1的表达变化情况.在MMS处理的S期细胞中,cyclin B1表达量不能上调;而在加入Chk1抑制剂处理后,cyclin B1则有所增加.这一结果进一步支持DNA损伤S期细胞在Chk1失活时进入异常有丝分裂的推论.研究结果表明,Chk1是MMS诱发的HeLa细胞S期DNA损伤检查点的专一性的重要蛋白激酶;当MMS引发DNA损伤后,上游蛋白激酶对Chk1进行磷酸化,从而激活了S期的DNA损伤检查点.阻止细胞进入G2/M期.由于这一过程不依赖于p53的活性,因此Chk1有可能作为p53失活的肿瘤细胞的药物靶标.

  8. 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.

  9. WEE1 inhibition targets cell cycle checkpoints for triple negative breast cancers to overcome cisplatin resistance

    Science.gov (United States)

    Zheng, Hongping; Shao, Fangyuan; Martin, Scots; Xu, Xiaoling; Deng, Chu-Xia

    2017-01-01

    Cisplatin is one of the most commonly used therapeutic drugs for cancer therapy, yet prolonged cisplatin treatment frequently results in drug resistance. To enhance therapeutic effect of cisplatin, we conducted a high throughput screening using a kinase library containing 704 kinases against triple negative breast cancer (TNBC) cells. We demonstrated that cisplatin activates ATR, CHK1 and WEE1, which shut down DNA replication and attenuate cisplatin induced-lethality. WEE1 inhibition sensitizes TNBCs and cisplatin resistant cancer cells to cisplatin-induced lethality, because it not only impairs DNA replication checkpoint more profoundly than inhibition of ATR or CHK1, but also defects G2-M cell cycle checkpoint. Finally, we demonstrated that combined cisplatin treatment and WEE1 inhibition synergistically inhibits xenograft cancer growth accompanied by markedly reduced expression of TNBC signature genes. Thus targeting DNA replication and G2-M cell cycle checkpoint simultaneously by cisplatin and WEE1 inhibition is promising for TNBCs treatment, and for overcoming their cisplatin resistance. PMID:28262781

  10. WEE1 inhibition targets cell cycle checkpoints for triple negative breast cancers to overcome cisplatin resistance.

    Science.gov (United States)

    Zheng, Hongping; Shao, Fangyuan; Martin, Scots; Xu, Xiaoling; Deng, Chu-Xia

    2017-03-06

    Cisplatin is one of the most commonly used therapeutic drugs for cancer therapy, yet prolonged cisplatin treatment frequently results in drug resistance. To enhance therapeutic effect of cisplatin, we conducted a high throughput screening using a kinase library containing 704 kinases against triple negative breast cancer (TNBC) cells. We demonstrated that cisplatin activates ATR, CHK1 and WEE1, which shut down DNA replication and attenuate cisplatin induced-lethality. WEE1 inhibition sensitizes TNBCs and cisplatin resistant cancer cells to cisplatin-induced lethality, because it not only impairs DNA replication checkpoint more profoundly than inhibition of ATR or CHK1, but also defects G2-M cell cycle checkpoint. Finally, we demonstrated that combined cisplatin treatment and WEE1 inhibition synergistically inhibits xenograft cancer growth accompanied by markedly reduced expression of TNBC signature genes. Thus targeting DNA replication and G2-M cell cycle checkpoint simultaneously by cisplatin and WEE1 inhibition is promising for TNBCs treatment, and for overcoming their cisplatin resistance.

  11. 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 direct role...

  12. 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.

  13. 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.

  14. Mutation analysis of the checkpoint kinase 2 gene in colorectal cancer cell lines

    Institute of Scientific and Technical Information of China (English)

    LIU Wei-dong; ZHONG Bai-yun; ZHANG Yang-de; CHOI Gyu-seog

    2007-01-01

    Background Checkpoint kinase 2 (CHK2) is a DNA damage-activated protein kinase which is involved in cell cycle checkpoint control.CHK2 gene could be a candidate gene for colorectal cancer susceptibility.But there are few systematic repots on mutation of CHK2 in colorectal cancer.Methods The mutations of all 14 exons of CHK2 in 56 colorecfal cancer cell lines were screened systematically.using denaturing high-performance liquid chromatography (DHPLC) to screen the mismatches of the CHK2 exons amplified products,and then the suspected mutant cell lines were scanned by nucleotide sequence analysis.Results VACO400 in CHK2 exon 1a was suspected to have mutation by DHPLC and confirmed by sequence,but this was nonsense mutation.C106,CX-1,HT-29,SK01,SW480,SW620 and VACO400 in CHK2 exon 1b were confirmed to have the same nonsense mutation in 11609 A>G.DLD-1 and HCT-15 in CHK2 exon 2 were confirmed to have missense mutation R145W.which was heterozygous C>T missense mutation at nucleotide 433.leading to an Arg>Trp substitution within the FHA domain.Conclusions The CHK2 mutation in colorectal cancer is a low frequency event.There are just 10 cell lines to have sequence variations in all the 14 exons in 56 colorectal cancer cell lines and only DLD-1/HCT-15 had heterozygous missense mutation.These findings may give useful information of susceptibility of colorectal cancer as single nucleotide polymorphysim.

  15. 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.

  16. 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.

  17. 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

  18. Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

    Science.gov (United States)

    Ganapathy, Suthakar; Fagman, Johan B; Shen, Ling; Yu, Tianqi; Zhou, Xiaodong; Dai, Wei; Makriyannis, Alexandros; Chen, Changyan

    2016-12-20

    Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

  19. Discovery of pyrazolo[1,5-a]pyrimidine-based CHK1 inhibitors: A template-based approach-Part 2

    Energy Technology Data Exchange (ETDEWEB)

    Labroli, Marc; Paruch, Kamil; Dwyer, Michael P.; Alvarez, Carmen; Keertikar, Kartik; Poker, Cory; Rossman, Randall; Duca, Jose S.; Fischmann, Thierry O.; Madison, Vincent; Parry, David; Davis, Nicole; Seghezzi, Wolfgang; Wiswell, Derek; Guzi, Timothy J. [Merck

    2013-11-20

    Previous efforts by our group have established pyrazolo[1,5-a]pyrimidine as a viable core for the development of potent and selective CDK inhibitors. As part of an effort to utilize the pyrazolo[1,5-a]pyrimidine core as a template for the design and synthesis of potent and selective kinase inhibitors, we focused on a key regulator in the cell cycle progression, CHK1. Continued SAR development of the pyrazolo[1,5-a]pyrimidine core at the C5 and C6 positions, in conjunction with previously disclosed SAR at the C3 and C7 positions, led to the discovery of potent and selective CHK1 inhibitors.

  20. 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.

  1. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4.

    Science.gov (United States)

    Falk, Jill E; Chan, Leon Y; Amon, Angelika

    2011-08-02

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother-bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.

  2. 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.

  3. 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.

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

    DEFF Research Database (Denmark)

    Tibelius, Alexandra; Marhold, Joachim; Zentgraf, Hanswalter

    2009-01-01

    in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently...

  5. Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

    Science.gov (United States)

    Cornago, M; Garcia-Alberich, C; Blasco-Angulo, N; Vall-Llaura, N; Nager, M; Herreros, J; Comella, J X; Sanchis, D; Llovera, M

    2014-10-02

    Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis.

  6. The depletion of ATM inhibits colon cancer proliferation and migration via B56γ2-mediated Chk1/p53/CD44 cascades.

    Science.gov (United States)

    Liu, Rui; Tang, Jiajia; Ding, Chaodong; Liang, Weicheng; Zhang, Li; Chen, Tianke; Xiong, Yan; Dai, Xiaowei; Li, Wenfeng; Xu, Yunsheng; Hu, Jin; Lu, Liting; Liao, Wanqin; Lu, Xincheng

    2017-04-01

    Ataxia-telangiectasia mutated (ATM) protein kinase is a major guardian of genomic stability, and its well-established function in cancer is tumor suppression. Here, we report an oncogenic role of ATM. Using two isogenic sets of human colon cancer cell lines that differed only in their ATM status, we demonstrated that ATM deficiency significantly inhibits cancer cell proliferation, migration, and invasion. The tumor-suppressive function of ATM depletion is not modulated by the compensatory activation of ATR, but it is associated with B56γ2-mediated Chk1/p53/CD44 signaling pathways. Under normal growth conditions, the depletion of ATM prevents B56γ2 ubiquitination and degradation, which activates PP2A-mediated Chk1/p53/p21 signaling pathways, leading to senescence and cell cycle arrest. CD44 was validated as a novel ATM target based on its ability to rescue cell migration and invasion defects in ATM-depleted cells. The activation of p53 induced by ATM depletion suppresses CD44 transcription, thus resulting in epithelial-mesenchymal transition (EMT) and cell migration suppression. Our study suggests that ATM has tumorigenic potential in post-formed colon neoplasia, and it supports ATM as an appealing target for improving cancer therapy.

  7. 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.

  8. 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.

  9. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  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. 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.

  12. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

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    Laura Merlini

    Full Text Available The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC. This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  13. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

    Science.gov (United States)

    Merlini, Laura; Fraschini, Roberta; Boettcher, Barbara; Barral, Yves; Lucchini, Giovanna; Piatti, Simonetta

    2012-01-01

    The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  14. Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

    Science.gov (United States)

    Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

    2013-07-31

    During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

  15. 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

    as a result of defective chromosome segregation when irradiated cells entered their first mitosis, either prematurely without S and G(2) checkpoint arrest in the presence of CEP-3891 or after a prolonged S and G(2) checkpoint arrest in the absence of CEP-3891. The nuclear fragmentation was clearly...

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

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    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.

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

    Science.gov (United States)

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

    2014-12-04

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

  18. Rad3-Cds1 mediates coupling of initiation of meiotic recombination with DNA replication. Mei4-dependent transcription as a potential target of meiotic checkpoint.

    Science.gov (United States)

    Ogino, Keiko; Masai, Hisao

    2006-01-20

    Premeiotic S-phase and meiotic recombination are known to be strictly coupled in Saccharomyces cerevisiae. However, the checkpoint pathway regulating this coupling has been largely unknown. In fission yeast, Rad3 is known to play an essential role in coordination of DNA replication and cell division during both mitotic growth and meiosis. Here we have examined whether the Rad3 pathway also regulates the coupling of DNA synthesis and recombination. Inhibition of premeiotic S-phase with hydroxyurea completely abrogates the progression of meiosis, including the formation of DNA double-strand breaks (DSBs). DSB formation is restored in rad3 mutant even in the presence of hydroxyurea, although repair of DSBs does not take place or is significantly delayed, indicating that the subsequent recombination steps may be still inhibited. Examination of the roles of downstream checkpoint kinases reveals that Cds1, but not Chk1 or Mek1, is required for suppression of DSB in the presence of hydroxyurea. Transcriptional induction of some rec+ genes essential for DSB occurs at a normal timing and to a normal level in the absence of DNA synthesis in both the wild-type and cds1delta cells. On the other hand, the transcriptional induction of the mei4+ transcription factor and cdc25+ phosphatase, which is significantly suppressed by hydroxyurea in the wild-type cells, occurs almost to a normal level in cds1delta cells even in the presence of hydroxyurea. These results show that the Rad3-Cds1 checkpoint pathway coordinates initiation of meiotic recombination and meiotic cell divisions with premeiotic DNA synthesis. Because mei4+ is known to be required for DSB formation and cdc25+ is required for activation of meiotic cell divisions, we propose an intriguing possibility that the Rad3-Cds1 meiotic checkpoint pathway may target transcription of these factors.

  19. 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.

  20. 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.

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

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    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.

  2. 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

  3. 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

  4. 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.

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

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    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.

  6. 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.

  7. 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

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    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

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

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    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.

  9. Mangiferin induces cell cycle arrest at G2/M phase through ATR-Chk1 pathway in HL-60 leukemia cells.

    Science.gov (United States)

    Peng, Z G; Yao, Y B; Yang, J; Tang, Y L; Huang, X

    2015-05-12

    This study aimed to determine the effect of mangiferin on the cell cycle in HL-60 leukemia cells and expression of the cell cycle-regulatory genes Wee1, Chk1 and CDC25C and to further investigate the molecular mechanisms of the antileukemic action of mangiferin. The inhibitory effect of mangiferin on HL-60 leukemia cell proliferation was determined by the MTT assay. The impact of mangiferin on the HL-60 cell cycle was evaluated by flow cytometry. After the cells were treated with different concentrations of mangiferin, the expression levels of Wee1, Chk1 and CDC25C mRNA were determined by RT-PCR, and Western blot was used to evaluate the expression levels of cdc25c, cyclin B1, and Akt proteins. The inhibition of HL-60 cell growth by mangiferin was dose- and time-dependent. After treatment for 24 h, cells in G2/M phase increased, and G2/M phase arrest appeared with increased mRNA expression of Wee1, Chk1 and CDC25C. Mangiferin inhibited Chk1 and cdc25c mRNA expression at high concentrations and induced Wee1 mRNA expression in a dose-dependent manner. It significantly inhibited ATR, Chk1, Wee1, Akt, and ERK1/2 phosphorylation but increased cdc2 and cyclin B1 phosphorylation. Furthermore, mangiferin reduced cdc25c, cyclin B1, and Akt protein levels while inducing Wee1 protein expression. It also antagonized the phosphorylation effect of vanadate on ATR, and the phosphorylation effect of EGF on Wee1. These findings indicated that mangiferin inhibits cell cycle progression through the ATR-Chk1 stress response DNA damage pathway, leading to cell cycle arrest at G2/M phase in leukemia cells.

  10. The mismatch repair system modulates curcumin sensitivity through induction of DNA strand breaks and activation of G2-M checkpoint.

    Science.gov (United States)

    Jiang, Zhihua; Jin, ShunQian; Yalowich, Jack C; Brown, Kevin D; Rajasekaran, Baskaran

    2010-03-01

    The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of gammaH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G(2)-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.

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

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    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.

  12. ERK1/2 signaling plays an important role in topoisomerase II poison-induced G2/M checkpoint activation.

    Science.gov (United States)

    Kolb, Ryan H; Greer, Patrick M; Cao, Phu T; Cowan, Kenneth H; Yan, Ying

    2012-01-01

    Topo II poisons, which target topoisomerase II (topo II) to generate enzyme mediated DNA damage, have been commonly used for anti-cancer treatment. While clinical evidence demonstrate a capability of topo II poisons in inducing apoptosis in cancer cells, accumulating evidence also show that topo II poison treatment frequently results in cell cycle arrest in cancer cells, which was associated with subsequent resistance to these treatments. Results in this report indicate that treatment of MCF-7 and T47D breast cancer cells with topo II poisons resulted in an increased phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and an subsequent induction of G2/M cell cycle arrest. Furthermore, inhibition of ERK1/2 activation using specific inhibitors markedly attenuated the topo II poison-induced G2/M arrest and diminished the topo II poison-induced activation of ATR and Chk1 kinases. Moreover, decreased expression of ATR by specific shRNA diminished topo II poison-induced G2/M arrest but had no effect on topo II poison-induced ERK1/2 activation. In contrast, inhibition of ERK1/2 signaling had little, if any, effect on topo II poison-induced ATM activation. In addition, ATM inhibition by either incubation of cells with ATM specific inhibitor or transfection of cells with ATM specific siRNA did not block topo II poison-induced G2/M arrest. Ultimately, inhibition of ERK1/2 signaling greatly enhanced topo II poison-induced apoptosis. These results implicate a critical role for ERK1/2 signaling in the activation of G2/M checkpoint response following topo II poison treatment, which protects cells from topo II poison-induced apoptosis.

  13. Induction of DNA damage and G2 cell cycle arrest by diepoxybutane through the activation of the Chk1-dependent pathway in mouse germ cells.

    Science.gov (United States)

    Dong, Jianyun; Wang, Zhi; Zou, Peng; Zhang, Guowei; Dong, Xiaomei; Ling, Xi; Zhang, Xi; Liu, Jinyi; Ye, Dongqing; Cao, Jia; Ao, Lin

    2015-03-16

    1,2:3,4-Diepoxybutane (DEB) is a major carcinogenic metabolite of 1,3-butadiene (BD), which has been shown to cause DNA strand breaks in cells through its potential genotoxicity. The adverse effect of DEB on male reproductive cells in response to DNA damage has not been thoroughly studied, and the related mechanism is yet to be elucidated. Using mouse spermatocyte-derived GC-2 cells, we demonstrated in the present study that DEB caused the proliferation inhibition and marked cell cycle arrest at the G2 phase but not apoptosis. DEB also induced DNA damage as evidenced by γ-H2AX expression, the comet assay, and the cytokinesis-block micronucleus assay. Meanwhile, DEB triggered the Chk1/Cdc25c/Cdc2 signal pathway, which could be abated in the presence of UCN-01 or Chk1 siRNA. GC-2 cells exposed to DEB experienced ROS generation and pretreatment of N-acetyl-l-cysteine, partly attenuated DEB-induced DNA damage, and G2 arrest. Furthermore, measurement of testicular cells showed an increased proportion of tetraploid cells in mice administrated with DEB, alongside the enhanced expression of p-Chk1. Also, the defective reproductive phenotypes, including reduced sperm motility, increased sperm malformation, and histological abnormality of testes, were observed. In conclusion, these results suggest DEB induces DNA damage and G2 cell cycle arrest by activating the Chk1-dependent pathway, while oxidative stress may be associated with eliciting toxicity in male reproductive cells.

  14. Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage

    Directory of Open Access Journals (Sweden)

    Hervé Técher

    2016-02-01

    Full Text Available Mammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates.

  15. 口腔黏膜癌变过程中Chk1和Chk2的表达及意义%Expression of Chk1 and Chk2 proteins in precancerous lesions and squamous cell carcinomas of oral mucosa

    Institute of Scientific and Technical Information of China (English)

    金刚石; 蔡扬; 周瑛

    2014-01-01

    目的 探讨Chk1和Chk2在口腔黏膜癌变过程中的作用及意义.方法 采用免疫组化SABC法检测10例口腔正常黏膜组织、29例口腔黏膜癌前病变组织及40例口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)组织中Chk1和Chk2蛋白的表达,分析二者表达的相关性及二者在口腔黏膜癌变过程中的作用及意义.结果 (1) Chk1和Chk2在口腔黏膜癌前病变组织及OSCC组织中的阳性率明显低于口腔正常黏膜组织,差异均有统计学意义(P<0.05).(2)Ⅰ+Ⅱ期OSCC组织中Chk1的阳性率明显高于Ⅲ+Ⅳ期,差异有统计学意义(P <0.05);Chk2在伴有淋巴结转移的OSCC组织中的阳性率明显低于无淋巴结转移组,差异有统计学意义(P<0.05).(3)Chk1在Chk2阳性的OSCC组织中的阳性率为60% (6/10),在Chk2阴性的OSCC组织中阳性率为20% (6/30),Chk1和Chk2表达呈正相关(r=0.378,P<0.05).结论 细胞周期调控因子Chk1和Chk2表达下调是口腔黏膜癌变过程中的早期事件,可能是口腔黏膜上皮发生癌变的重要因素之一;Chk1和Chk2在OSCC 组织中的失表达有可能作为临床评估OSCC预后的参考指标之一.

  16. 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.

  17. 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.

  18. 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

  19. Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasis and function

    OpenAIRE

    Wang, Yanyan; Huang, Gonghua; Vogel, Peter; Neale, Geoffrey; Reizis, Boris; Chi, Hongbo

    2012-01-01

    Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c+ cells induced markedly elevated apopt...

  20. 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.

  1. 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

  2. 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.

  3. 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...

  4. 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...

  5. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  6. Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control.

    OpenAIRE

    Saka, Y.; Fantes, P; Sutani, T; McInerny, C; Creanor, J; Yanagida, M

    1994-01-01

    Fission yeast temperature-sensitive cut5 (cell untimely torn) mutants are defective in initiation and/or elongation of DNA replication but allow mitosis and cell division at a restrictive temperature. We show that the cut5 protein (identical to rad4) (i) is an essential component of the replication checkpoint system but not the DNA damage checkpoint, and (ii) negatively regulates the activation of M phase kinase at mitotic entry. Even if the replication checkpoint has been activated previousl...

  7. Unprotected Drosophila melanogaster telomeres activate the spindle assembly checkpoint.

    Science.gov (United States)

    Musarò, Mariarosaria; Ciapponi, Laura; Fasulo, Barbara; Gatti, Maurizio; Cenci, Giovanni

    2008-03-01

    In both yeast and mammals, uncapped telomeres activate the DNA damage response (DDR) and undergo end-to-end fusion. Previous work has shown that the Drosophila HOAP protein, encoded by the caravaggio (cav) gene, is required to prevent telomeric fusions. Here we show that HOAP-depleted telomeres activate both the DDR and the spindle assembly checkpoint (SAC). The cell cycle arrest elicited by the DDR was alleviated by mutations in mei-41 (encoding ATR), mus304 (ATRIP), grp (Chk1) and rad50 but not by mutations in tefu (ATM). The SAC was partially overridden by mutations in zw10 (also known as mit(1)15) and bubR1, and also by mutations in mei-41, mus304, rad50, grp and tefu. As expected from SAC activation, the SAC proteins Zw10, Zwilch, BubR1 and Cenp-meta (Cenp-E) accumulated at the kinetochores of cav mutant cells. Notably, BubR1 also accumulated at cav mutant telomeres in a mei-41-, mus304-, rad50-, grp- and tefu-dependent manner. Our results collectively suggest that recruitment of BubR1 by dysfunctional telomeres inhibits Cdc20-APC function, preventing the metaphase-to-anaphase transition.

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

    Science.gov (United States)

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly

    2015-01-01

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA-protein crosslinks (DPC) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. PMID:25817892

  9. 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

  10. The Error-Prone DNA Polymerase κ Promotes Temozolomide Resistance in Glioblastoma through Rad17-Dependent Activation of ATR-Chk1 Signaling.

    Science.gov (United States)

    Peng, Chenghao; Chen, Zhengxin; Wang, Shuai; Wang, Hong-Wei; Qiu, Wenjin; Zhao, Lin; Xu, Ran; Luo, Hui; Chen, Yuanyuan; Chen, Dan; You, Yongping; Liu, Ning; Wang, Huibo

    2016-04-15

    The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol κ, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol κ in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol κ disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression. Further investigation of the relationship between Pol κ and temozolomide revealed that Pol κ inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol κ-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol κ. Finally, we found that Pol κ overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. ©2016 AACR.

  11. Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation

    DEFF Research Database (Denmark)

    Biskup, Edyta; Naym, David Gram; Gniadecki, Robert

    2016-01-01

    and require more aggressive therapies. OBJECTIVE: The aim of this project was to investigate whether inhibition of Ataxia Telangiectasia and Rad3 related kinase (ATR) may enhance efficacy of phototherapy. METHODS: CTCL cell lines (MyLa2000, SeAx and Mac2a) served as in vitro cell models. ATR and Chk1 were...

  12. Regulatory effects of Rock2 on cell cycle checkpoint Cdc25A%Rock2对细胞周期检查点Cdc25A的调节作用

    Institute of Scientific and Technical Information of China (English)

    刘天德; 余新; 袁荣发; 王庆诺; 杨志强; 邵江华

    2012-01-01

    AIM; To invesligale the regulalory effecls of Rho - associated coiled - coil - containing prolein ki-nase 2(Rock2) on the cell cycle checkpoinl cell division cycle 25A(Cdc25A). METHODS; The prolein expression levels of Rock2 and Cdc25A in 51 pairs of hepalocellular carcinoma and the adjacenl lissues were delecled by Weslern blol-ling. shRock2 plasmids were constructed, selecled and slably lransfecled inlo hepalocellular carcinoma Huh -7 and HepG2 cells. The prolein expression of Cdc25A in the cells was determined by Weslern blolling. Based on the Rock2 interfering sequences, we designed the primers and changed the 4 indicated bases via sile - specific mulagenesis. The Rock2 - mulanl plasmid was verified by sequencing and was lransfecled inlo slable Rock2 - knockdown cells. The prolein expression of Cdc25A was delecled by Weslern blolling, and the cell proliferation was measured by MTT assay. The prolein levels of checkpoint kinase( Chk) 1/Chk2 were also delected in slable Rock2 - knockdown cells. The interaction between Rock2 and Cdc25A was measured by co - immunoprecipilalion, and the co - localization of Rock2 and Cdc25A was delected by confo-cal laser scanning microscopy. RESULTS; Rock2 and Cdc25A were apparently up - regulated in hepalocellular carcinoma,with a significantly positive correlation. The protein expression of Cdc25A was significantly down - regulated in slable Rock2 - knockdown cells. The expression of Chkl and Chk2 was not changed following knockdown of Rock2. The co - immunoprecipilation resulls verified thai Rock2 bound to Cdc25A. The resulls of confocal laser scanning microscopy showed thai Rock2 and Cdc25A were co -localized in hepalocellular carcinoma cells. CONCLUSION; Rock2 positively regulates the cell cycle checkpoint Cdc25A, which is independenl of Chkl/Chk2 and this may provide a new larget gene for Ireat-ment of hepalocellular carcinoma.%目的:探讨肝癌细胞中Rho相关含卷曲螺旋蛋白激酶2(Rock2)对细胞周期检

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. Inactivation of ATM/ATR DNA damage checkpoint promotes androgen induced chromosomal instability in prostate epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yung-Tuen Chiu

    Full Text Available The ATM/ATR DNA damage checkpoint functions in the maintenance of genetic stability and some missense variants of the ATM gene have been shown to confer a moderate increased risk of prostate cancer. However, whether inactivation of this checkpoint contributes directly to prostate specific cancer predisposition is still unknown. Here, we show that exposure of non-malignant prostate epithelial cells (HPr-1AR to androgen led to activation of the ATM/ATR DNA damage response and induction of cellular senescence. Notably, knockdown of the ATM gene expression in HPr-1AR cells can promote androgen-induced TMPRSS2: ERG rearrangement, a prostate-specific chromosome translocation frequently found in prostate cancer cells. Intriguingly, unlike the non-malignant prostate epithelial cells, the ATM/ATR DNA damage checkpoint appears to be defective in prostate cancer cells, since androgen treatment only induced a partial activation of the DNA damage response. This mechanism appears to preserve androgen induced autophosphorylation of ATM and phosphorylation of H2AX, lesion processing and repair pathway yet restrain ATM/CHK1/CHK2 and p53 signaling pathway. Our findings demonstrate that ATM/ATR inactivation is a crucial step in promoting androgen-induced genomic instability and prostate carcinogenesis.

  19. Fisetin induces G2/M phase cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2 activation in human endometrial cancer cells

    Directory of Open Access Journals (Sweden)

    Zhan-Ying Wang

    2015-06-01

    Full Text Available Endometrial cancer is one of the most prevalent gynaecological malignancies where, currently available therapeutic options remain limited. Recently phytochemicals are exploited for their efficiency in cancer therapy. The present study investigates the anti-proliferative effect of fisetin, a flavonoid on human endometrial cancer cells (KLE and Hec1 A. Fisetin (20-100 µM effectively reduced the viability of Hec1 A and KLE cells and potentially altered the cell population at G2/M stage. Expression levels of the cell cycle proteins (cyclin B1, p-Cdc2, p-Cdc25C, p-Chk1, Chk2, p-ATM, cyclin B1, H2AX, p21 and p27 were analyzed. Fisetin suppressed cyclin B1 expression and caused inactiva-tion of Cdc25C and Cdc2 by increasing their phosphorylation levels and further activated ATM, Chk1 and Chk2. Increased levels of p21 and p27 were observed as well. These results suggest that fisetin induced G2/M cell cycle arrest via inactivating Cdc25c and Cdc2 through activation of ATM, Chk1 and Chk2.

  20. 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

  1. 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

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

    OpenAIRE

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

    2011-01-01

    Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome–microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the...

  3. The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

    OpenAIRE

    Lancelot, Nathalie; Charier, Gaëlle; Couprie, Joël; Duband-Goulet, Isabelle; Alpha-Bazin, Béatrice; Quémeneur, Eric; Ma, Emilie; Marsolier-Kergoat, Marie-Claude; Ropars, Virginie; Charbonnier, Jean-Baptiste; Miron, Simona; Craescu, Constantin,; Callebaut, Isabelle; Gilquin, Bernard; Zinn-Justin, Sophie

    2007-01-01

    International audience; DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem t...

  4. 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.

  5. Checkpoint Blockade in Cancer Immunotherapy

    Science.gov (United States)

    Korman, Alan J.; Peggs, Karl S.; Allison, James P.

    2007-01-01

    The progression of a productive immune response requires that a number of immunological checkpoints be passed. Passage may require the presence of excitatory costimulatory signals or the avoidance of negative or coinhibitory signals, which act to dampen or terminate immune activity. The immunoglobulin superfamily occupies a central importance in this coordination of immune responses, and the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4):B7.1/B7.2 receptor/ligand grouping represents the archetypal example of these immune regulators. In part the role of these checkpoints is to guard against the possibility of unwanted and harmful self-directed activities. While this is a necessary function, aiding in the prevention of autoimmunity, it may act as a barrier to successful immunotherapies aimed at targeting malignant self-cells that largely display the same array of surface molecules as the cells from which they derive. Therapies aimed at overcoming these mechanisms of peripheral tolerance, in particular by blocking the inhibitory checkpoints, offer the potential to generate antitumor activity, either as monotherapies or in synergism with other therapies that directly or indirectly enhance presentation of tumor epitopes to the immune system. Such immunological molecular adjuvants are showing promise in early clinical trials. This review focuses on the results of the archetypal example of checkpoint blockade, anti-CTLA-4, in preclinical tumor models and clinical trials, while also highlighting other possible targets for immunological checkpoint blockade. PMID:16730267

  6. Constitutive Mad1 targeting to kinetochores uncouples checkpoint signalling from chromosome biorientation.

    Science.gov (United States)

    Maldonado, Maria; Kapoor, Tarun M

    2011-04-01

    Accurate chromosome segregation depends on biorientation, whereby sister chromatids attach to microtubules from opposite spindle poles. The spindle-assembly checkpoint is a surveillance mechanism in eukaryotes that inhibits anaphase until all chromosomes have bioriented. In present models, the recruitment of the spindle-assembly checkpoint protein Mad2, through Mad1, to non-bioriented kinetochores is needed to stop cell-cycle progression. However, it is unknown whether Mad1-Mad2 targeting to kinetochores is sufficient to block anaphase. Furthermore, it is unclear whether regulators of biorientation (for example, Aurora kinases) have checkpoint functions downstream of Mad1-Mad2 recruitment or whether they act upstream to quench the primary error signal. Here, we engineered a Mad1 construct that localizes to bioriented kinetochores. We show that the kinetochore localization of Mad1 is sufficient for a metaphase arrest that depends on Mad1-Mad2 binding. By uncoupling the checkpoint from its primary error signal, we show that Aurora, Mps1 and BubR1 kinases, but not Polo-like kinase, are needed to maintain checkpoint arrest when Mad1 is present on kinetochores. Together, our data suggest a model in which the biorientation errors, which recruit Mad1-Mad2 to kinetochores, may be signalled not only through Mad2 template dynamics, but also through the activity of widely conserved kinases, to ensure the fidelity of cell division.

  7. 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...

  8. 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

  9. 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.

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

    Science.gov (United States)

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

    2011-04-20

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

  11. Differential effect of schisandrin B stereoisomers on ATR-mediated DNA damage checkpoint signaling.

    Science.gov (United States)

    Tatewaki, Naoto; Nishida, Hiroshi; Yoshida, Masaaki; Ando, Hidehiro; Kondo, Seizo; Sakamaki, Toshiyuki; Konishi, Tetsuya

    2013-01-01

    We have previously reported that schisandrin B (SchB) is a specific inhibitor of ATR (ataxia telangiectasia and Rad-3-related) protein kinase. Since SchB consists of a mixture of its diastereomers gomisin N (GN) and γ-schisandrin (γ-Sch), the inhibitory action of SchB might result from a stereospecific interaction between one of the stereoisomers of SchB and ATR. Therefore, we investigated the effect of GN and γ-Sch on UV (UVC at 254 nm)-induced activation of DNA damage checkpoint signaling in A549 cells. UV-induced cell death (25 - 75 J/m(2)) was amplified by the presence of the diastereomers, especially GN. At the same time, GN, but not γ-Sch, inhibited the phosphorylation of checkpoint proteins such as p53, structural maintenance of chromosomes 1, and checkpoint kinase 1 in UV-irradiated cells. Moreover, GN inhibited the G2/M checkpoint during UV-induced DNA damage. The in vitro kinase activity of immunoaffinity-purified ATR was dose-dependently inhibited by GN (IC50: 7.28 μM) but not by γ-Sch. These results indicate that GN is the active component of SchB and suggest that GN inhibits the DNA damage checkpoint signaling by stereospecifically interacting with ATR.

  12. 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.

  13. Phenotypic checkpoints regulate neuronal development.

    Science.gov (United States)

    Ben-Ari, Yehezkel; Spitzer, Nicholas C

    2010-11-01

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

  14. 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

  15. 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.

  16. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    Science.gov (United States)

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  17. 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

  18. 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.

  19. 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.

  20. Contribution of ATM and ATR kinase pathways to p53-mediated response in etoposide and methyl methanesulfonate induced DNA damage.

    Science.gov (United States)

    Sun, Bin; Ross, Susan M; Rowley, Sean; Adeleye, Yeyejide; Clewell, Rebecca A

    2017-03-01

    p53 is a key integrator of cellular response to DNA damage, supporting post-translational repair and driving transcription-mediated responses including cell cycle arrest, apoptosis, and repair. DNA damage sensing kinases recognize different types of DNA damage and initiate specific responses through various post-translational modifications of p53. This study evaluated chemical specificity of the p53 pathway response by manipulating p53 or its upstream kinases and assessing the effect on DNA damage and cellular responses to prototype chemicals: etoposide (ETP, topoisomerase II inhibitor) and methyl methane sulfonate (MMS, alkylating agent). p53-deficient cells demonstrated reduced accumulation of the p53 target proteins MDM2, p21, and Wip1; reduced apoptotic response; and increased DNA damage (p-H2AX and micronuclei) with both chemicals. However, p53 was not essential for cell cycle arrest in HT1080 or HCT116 cells. The two chemicals induced different patterns of kinase activation, particularly in terms of Chk 1, Chk 2, p38, and ERK 1/2. However, inhibition of the ATM pathway showed a greater effect on p53 activtation, apoptosis, and accumulation of DNA damage than ATR-Chk 1 or the MAP kinases regardless of the chemical used. These results indicate that ATM is the predominant upstream kinase responsible for activation of the p53-mediated DNA damage response for both MMS and ETP, though the downstream kinase response is markedly different. Environ. Mol. Mutagen. 58:72-83, 2017. © 2017 Wiley Periodicals, Inc.

  1. Slipping past the spindle assembly checkpoint.

    Science.gov (United States)

    Subramanian, Radhika; Kapoor, Tarun M

    2013-11-01

    Error-free genome segregation depends on the spindle assembly checkpoint (SAC), a signalling network that delays anaphase onset until chromosomes have established proper spindle attachments. Three reports now quantitatively examine the sensitivity and robustness of the SAC response.

  2. 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.

  3. 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

    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...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....... 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...

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

    Science.gov (United States)

    Paek, Andrew L; Weinert, Ted

    2010-11-24

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

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

    Science.gov (United States)

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

    2016-08-17

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

  6. 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.

  7. 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.

  8. A Checkpoint Storage System for Desktop Grid Computing

    CERN Document Server

    Kiswany, Samer Al; Vazhkudai, Sudharshan S

    2007-01-01

    Checkpointing is an indispensable technique to provide fault tolerance for long-running high-throughput applications like those running on desktop grids. In these environments, a checkpoint storage system can offer multiple benefits: reduce the load on a traditional file system, offer high-performance through specialization, and, finally, optimize checkpoint data management by taking into account application semantics. Such a storage system can present a unifying abstraction to checkpoint operations, while hiding the fact that there are no dedicated resources to store the checkpoint data. This paper presents a dedicated checkpoint storage system for desktop grid environments. Our solution uses scavenged disk space from participating desktops to build an inexpensive storage space, offering a traditional file system interface for easy integration with checkpointing applications. This paper presents the architecture of our checkpoint storage system, key write optimizations for high-speed I/O, support for increme...

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

    Science.gov (United States)

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

    2015-10-15

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

  10. 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…

  11. User Process Checkpoint/Restart. Revision 1.1.

    Science.gov (United States)

    2007-11-02

    This document describes the design of the Tera user process checkpoint facility. Checkpoint is a means for saving the state of an executing process...or group of processes and restarting them later on demand. The motivation for providing checkpoint on the Tera is to allow long running computationally

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  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. Targeting KIT on innate immune cells to enhance the antitumor activity of checkpoint inhibitors.

    Science.gov (United States)

    Stahl, Maximilian; Gedrich, Richard; Peck, Ronald; LaVallee, Theresa; Eder, Joseph Paul

    2016-06-01

    Innate immune cells such as mast cells and myeloid-derived suppressor cells are key components of the tumor microenvironment. Recent evidence indicates that levels of myeloid-derived suppressor cells in melanoma patients are associated with poor survival to checkpoint inhibitors. This suggests that targeting both the innate and adaptive suppressive components of the immune system will maximize clinical benefit and elicit more durable responses in cancer patients. Preclinical data suggest that targeting signaling by the receptor tyrosine kinase KIT, particularly on mast cells, may modulate innate immune cell numbers and activity in tumors. Here, we review data highlighting the importance of the KIT signaling in regulating antitumor immune responses and the potential benefit of combining selective KIT inhibitors with immune checkpoint inhibitors.

  18. 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.

  19. The spindle checkpoint and chromosome segregation in meiosis.

    Science.gov (United States)

    Gorbsky, Gary J

    2015-07-01

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

  20. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.

    Science.gov (United States)

    Santaguida, Stefano; Tighe, Anthony; D'Alise, Anna Morena; Taylor, Stephen S; Musacchio, Andrea

    2010-07-12

    The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD-ZWILCH-ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome-microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery.

  1. Targeting immune checkpoints in malignant glioma

    Science.gov (United States)

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

    2017-01-01

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

  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. Renal effects of immune checkpoint inhibitors.

    Science.gov (United States)

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

    2016-12-26

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

  4. 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 for this proc...

  5. 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.

  6. Phospho-Bcl-x(L)(Ser62) plays a key role at DNA damage-induced G(2) checkpoint.

    Science.gov (United States)

    Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

    2012-06-01

    Accumulating evidence suggests that Bcl-xL, an anti-apoptotic member of the Bcl-2 family, also functions in cell cycle progression and cell cycle checkpoints. Analysis of a series of phosphorylation site mutants reveals that cells expressing Bcl-xL(Ser62Ala) mutant are less stable at the G 2 checkpoint and enter mitosis more rapidly than cells expressing wild-type Bcl-xL or Bcl-xL phosphorylation site mutants, including Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala and Thr115Ala. Analysis of the dynamic phosphorylation and location of phospho-Bcl-xL(Ser62) in unperturbed, synchronized cells and during DNA damage-induced G 2 arrest discloses that a pool of phospho-Bcl-xL(Ser62) accumulates into nucleolar structures in etoposide-exposed cells during G 2 arrest. In a series of in vitro kinase assays, pharmacological inhibitors and specific siRNAs experiments, we found that Polo kinase 1 and MAPK9/JNK2 are major protein kinases involved in Bcl-xL(Ser62) phosphorylation and accumulation into nucleolar structures during the G 2 checkpoint. In nucleoli, phospho-Bcl-xL(Ser62) binds to and co-localizes with Cdk1(cdc2), the key cyclin-dependent kinase required for entry into mitosis. These data indicate that during G 2 checkpoint, phospho-Bcl-xL(Ser62) stabilizes G 2 arrest by timely trapping of Cdk1(cdc2) in nucleolar structures to slow mitotic entry. It also highlights that DNA damage affects the dynamic composition of the nucleolus, which now emerges as a piece of the DNA damage response.

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

    Science.gov (United States)

    Archer, Charles Jens; Inglett, Todd Alan

    2009-01-13

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

  8. 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.

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

    Science.gov (United States)

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

    2014-03-01

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

  10. Checkpointing and rollback recovery for network of workstations

    Institute of Scientific and Technical Information of China (English)

    汪东升; 郑纬民; 王鼎兴; 沈美明

    1999-01-01

    Network of workstations (NOW) now becomes one of the main trends of parallel computing. But for long-running scientific programs, it needs effective fault tolerance for its changing property. Checkpointing and rollback recovery is a solution to this problem. First the main problems upon rollback recovery are discussed, the different checkpointing techniques for NOW are analyzed, and then the design and implementation of ChaRM (checkpoint-based rollback recovery and process migration) system are described. The comparison of three coordinated checkpointing systems is given.

  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 topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint.

    Science.gov (United States)

    Warsi, Tariq H; Navarro, Michelle S; Bachant, Jeff

    2008-10-01

    Centromeric (CEN) chromatin is placed under mechanical tension and stretches as kinetochores biorient on the mitotic spindle. This deformation could conceivably provide a readout of biorientation to error correction mechanisms that monitor kinetochore-spindle interactions, but whether CEN chromatin acts in a tensiometer capacity is unresolved. Here, we report observations linking yeast Topoisomerase II (Top2) to both CEN mechanics and assessment of interkinetochore tension. First, in top2-4 and sumoylation-resistant top2-SNM mutants CEN chromatin stretches extensively during biorientation, resulting in increased sister kinetochore separation and preanaphase spindle extension. Our data indicate increased CEN stretching corresponds with alterations to CEN topology induced in response to tension. Second, Top2 potentiates aspects of the tension checkpoint. Mutations affecting the Mtw1 kinetochore protein activate Ipl1 kinase to detach kinetochores and induce spindle checkpoint arrest. In mtw1top2-4 and mtw1top2-SNM mutants, however, kinetochores are resistant to detachment and checkpoint arrest is attenuated. For top2-SNM cells, CEN stretching and checkpoint attenuation occur even in the absence of catenation linking sister chromatids. In sum, Top2 seems to play a novel role in CEN compaction that is distinct from decatenation. Perturbations to this function may allow weakened kinetochores to stretch CENs in a manner that mimics tension or evades Ipl1 surveillance.

  13. Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae

    Science.gov (United States)

    Osés-Ruiz, Míriam; Sakulkoo, Wasin; Littlejohn, George R.; Martin-Urdiroz, Magdalena

    2017-01-01

    To cause rice blast disease, the fungal pathogen Magnaporthe oryzae develops a specialized infection structure called an appressorium. This dome-shaped, melanin-pigmented cell generates enormous turgor and applies physical force to rupture the rice leaf cuticle using a rigid penetration peg. Appressorium-mediated infection requires septin-dependent reorientation of the F-actin cytoskeleton at the base of the infection cell, which organizes polarity determinants necessary for plant cell invasion. Here, we show that plant infection by M. oryzae requires two independent S-phase cell-cycle checkpoints. Initial formation of appressoria on the rice leaf surface requires an S-phase checkpoint that acts through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, appressorium repolarization involves a novel, DDR-independent S-phase checkpoint, triggered by appressorium turgor generation and melanization. This second checkpoint specifically regulates septin-dependent, NADPH oxidase-regulated F-actin dynamics to organize the appressorium pore and facilitate entry of the fungus into host tissue. PMID:28028232

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

    Science.gov (United States)

    2010-11-15

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

  15. 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...

  16. 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.

  17. Heat induction of a novel Rad9 variant from a cryptic translation initiation site reduces mitotic commitment.

    Science.gov (United States)

    Janes, Simon; Schmidt, Ulrike; Ashour Garrido, Karim; Ney, Nadja; Concilio, Susanna; Zekri, Mohamed; Caspari, Thomas

    2012-10-01

    Exposure of human cells to heat switches the activating signal of the DNA damage checkpoint from genotoxic to temperature stress. This change reduces mitotic commitment at the expense of DNA break repair. The thermal alterations behind this switch remain elusive despite the successful use of heat to sensitise cancer cells to DNA breaks. Rad9 is a highly conserved subunit of the Rad9-Rad1-Hus1 (9-1-1) checkpoint-clamp that is loaded by Rad17 onto damaged chromatin. At the DNA, Rad9 activates the checkpoint kinases Rad3(ATR) and Chk1 to arrest cells in G2. Using Schizosaccharomyces pombe as a model eukaryote, we discovered a new variant of Rad9, Rad9-M50, whose expression is specifically induced by heat. High temperatures promote alternative translation from a cryptic initiation codon at methionine-50. This process is restricted to cycling cells and is independent of the temperature-sensing mitogen-activated protein kinase (MAPK) pathway. While full-length Rad9 delays mitosis in the presence of DNA lesions, Rad9-M50 functions in a remodelled checkpoint pathway to reduce mitotic commitment at elevated temperatures. This remodelled pathway still relies on Rad1 and Hus1, but acts independently of Rad17. Heat-induction of Rad9-M50 ensures that the kinase Chk1 remains in a hypo-phosphorylated state. Elevated temperatures specifically reverse the DNA-damage-induced modification of Chk1 in a manner dependent on Rad9-M50. Taken together, heat reprogrammes the DNA damage checkpoint at the level of Chk1 by inducing a Rad9 variant that can act outside of the canonical 9-1-1 complex.

  18. Thymidine kinase 1 regulatory fine-tuning through tetramer formation

    DEFF Research Database (Denmark)

    Mutahir, Zeeshan; Clausen, Anders R.; Andersson, Karl-Magnus;

    2013-01-01

    Abstract: Thymidine kinase 1 (TK1) provides a crucial precursor, deoxythymidine monophosphate, for nucleic acid synthesis, and the activity of TK1 increases by up to 200-fold during the S-phase of cell division in humans. An important part of the regulatory checkpoints is the ATP and enzyme...

  19. Expanded CAG/CTG repeat DNA induces a checkpoint response that impacts cell proliferation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Rangapriya Sundararajan

    2011-03-01

    Full Text Available Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2, a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.

  20. Expanded CAG/CTG repeat DNA induces a checkpoint response that impacts cell proliferation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sundararajan, Rangapriya; Freudenreich, Catherine H

    2011-03-01

    Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases.

  1. 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.

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-01

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

  4. Combination approaches with immune checkpoint blockade in cancer therapy

    Directory of Open Access Journals (Sweden)

    Maarten Swart

    2016-11-01

    Full Text Available In healthy individuals, immune checkpoint molecules prevent autoimmune responses and limit immune cell-mediated tissue damage. Tumors frequently exploit these molecules to evade eradication by the immune system. Over the past years, immune checkpoint blockade of cytotoxic T lymphocyte antigen-4 (CTLA-4 and programmed death-1 (PD-1 emerged as promising strategies to activate anti-tumor cytotoxic T cell responses. Although complete regression and long-term survival is achieved in some patients, not all patients respond. This review describes promising, novel combination approaches involving immune checkpoint blockade, aimed at increasing response-rates to the single treatments.

  5. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    Directory of Open Access Journals (Sweden)

    Pushpavalli Sreerangam NCVL

    2013-01-01

    Full Text Available Abstract Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using

  6. 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.

  7. 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.

  8. 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.

  9. Immune checkpoint receptors in regulating immune reactivity in rheumatic disease

    OpenAIRE

    Ceeraz, Sabrina; Nowak, Elizabeth C.; Burns, Christopher M.; Noelle, Randolph J.

    2014-01-01

    Immune checkpoint regulators are critical modulators of the immune system, allowing the initiation of a productive immune response and preventing the onset of autoimmunity. Co-inhibitory and co-stimulatory immune checkpoint receptors are required for full T-cell activation and effector functions such as the production of cytokines. In autoimmune rheumatic diseases, impaired tolerance leads to the development of diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Sjogren’s...

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins.

    Science.gov (United States)

    Lisby, Michael; Barlow, Jacqueline H; Burgess, Rebecca C; Rothstein, Rodney

    2004-09-17

    DNA repair is an essential process for preserving genome integrity in all organisms. In eukaryotes, recombinational repair is choreographed by multiprotein complexes that are organized into centers (foci). Here, we analyze the cellular response to DNA double-strand breaks (DSBs) and replication stress in Saccharomyces cerevisiae. The Mre11 nuclease and the ATM-related Tel1 kinase are the first proteins detected at DSBs. Next, the Rfa1 single-strand DNA binding protein relocalizes to the break and recruits other key checkpoint proteins. Later and only in S and G2 phase, the homologous recombination machinery assembles at the site. Unlike the response to DSBs, Mre11 and recombination proteins are not recruited to hydroxyurea-stalled replication forks unless the forks collapse. The cellular response to DSBs and DNA replication stress is likely directed by the Mre11 complex detecting and processing DNA ends in conjunction with Sae2 and by RP-A recognizing single-stranded DNA and recruiting additional checkpoint and repair proteins.

  16. Checkpoint modulation--A new way to direct the immune system against renal cell carcinoma.

    Science.gov (United States)

    Bedke, Jens; Kruck, Stephan; Gakis, Georgios; Stenzl, Arnulf; Goebell, Peter J

    2015-01-01

    The introduction of targeted therapies like the tyrosine kinase (TKI) and mammalian target of rapamycin (mTOR) inhibitors has improved patients' survival in general. Nevertheless the prognosis remains limited. Therapies with a new mode of action are urgently warranted, especially those who would provoke long-term responders or long-lasting complete remissions as observed with unspecific immunotherapy with the cytokines interleukin-2 and interferon-α. In the recent years a deeper understanding of the underlying immunology of T cell activation led to the development of checkpoint inhibitors, which are mainly monocloncal antibodies and which enhances the presence of the co-stimulatory signals needed for T cell activation or priming. This review discusses the clinical data and ongoing studies available for the inhibition of the PD-1 (CD279) and CTLA-4 (CD152) axis in mRCC. In addition, potential future immunological targets are discussed. This approach of T-cell activation or re-activation by immunological checkpoint inhibition holds the inherent promise to directly affect the tumor cell and thereby to potentially cure a subset of patients with mRCC.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. The versatile functions of ATM kinase

    Directory of Open Access Journals (Sweden)

    Rebecca J Boohaker

    2014-02-01

    Full Text Available Ataxia-telangiectasia mutated (ATM kinase, the mutation of which causes the autosomal recessive disease ataxia-telangiectasia, plays an essential role in the maintenance of genome stability. Extensive studies have revealed that activated ATM signals to a massive list of proteins to facilitate cell cycle checkpoints, DNA repair, and many other aspects of physiological responses in the event of DNA double-strand breaks. ATM also plays functional roles beyond the well-characterized DNA damage response (DDR. In this review article, we discuss the recent findings on the molecular mechanisms of ATM in DDR, the mitotic spindle checkpoint, as well as hyperactive ATM signaling in cancer invasion and metastasis.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Mechanisms and Components of the DNA Damage Checkpoint

    Science.gov (United States)

    2002-09-01

    Saccharomyces cerevisiae DNA damage checkpoint. Molecular Cell 9: 1055-1065. (reprint included as Appendix 2) "* Schwartz, M.F., Duong, J.K., Sun, Z., Pradhan...phosphorylation sites couple Rad53 to the Saccharomyces cerevisiae DNA damage checkpoint. Molecular Cell 9, 1055-1065. 13 Molecular Cell , Vol. 9,1055-1065...Cambridge, Massachusetts 02139. 1999), and mutation of conserved amino acids in the Molecular Cell 1056 A Rad9 B ,•o 0, 1 sitesN NC -T6 RVTQSA o- 0~ --T240

  6. 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).

  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. CANCER IMMUNOTHERAPY BASED ON THE BLOCKADE OF IMMUNE CHECKPOINTS

    Directory of Open Access Journals (Sweden)

    A. V. Bogolyubova

    2015-01-01

    Full Text Available Immune checkpoints represent the system of inhibitory mechanisms regulating the activation of the immune response, preventing the autoimmune processes and modulating the immune response by decreasing the immune cell-mediated damage of tissues and organs. Tumor cells may utilize these checkpoints to prevent the activation of tumor-specific lymphocytes, thereby acquiring resistance against the immune response. The blockade of inhibitory signal that is transduced in immune checkpoints leading to the reactivation of antitumor immune response is a promising method of tumor immunotherapy. Since the majority of immune checkpoints are based on the ligand-receptor interactions, one of contemporary modalities of anti-tumor therapy is based on the development of ligandor receptor-blocking therapeutic monoclonal antibodies, as well as soluble recombinant receptors capable of competing for a ligand and thereby modulating the signal transduction. In the past few years, this field of tumor immunotherapy experienced an impressive success; however, the potential tradeoff for altering of the natural suppressive mechanisms is the development of the autoimmune reactions.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Gradual implementation of the meiotic recombination program via checkpoint pathways controlled by global DSB levels.

    Science.gov (United States)

    Joshi, Neeraj; Brown, M Scott; Bishop, Douglas K; Börner, G Valentin

    2015-03-05

    During meiosis, Spo11-induced double-strand breaks (DSBs) are processed into crossovers, ensuring segregation of homologous chromosomes (homologs). Meiotic DSB processing entails 5' end resection and preferred strand exchange with the homolog rather than the sister chromatid (homolog bias). In many organisms, DSBs appear gradually along the genome. Here we report unexpected effects of global DSB levels on local recombination events. Early-occurring, low-abundance "scout" DSBs lack homolog bias. Their resection and interhomolog processing are controlled by the conserved checkpoint proteins Tel1(ATM) kinase and Pch2(TRIP13) ATPase. Processing pathways controlled by Mec1(ATR) kinase take over these functions only above a distinct DSB threshold, resulting in progressive strengthening of the homolog bias. We conclude that Tel1(ATM)/Pch2 and Mec1(ATR) DNA damage response pathways are sequentially activated during wild-type meiosis because of their distinct sensitivities to global DSB levels. Moreover, relative DSB order controls the DSB repair pathway choice and, ultimately, recombination outcome.

  14. 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.

  15. 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.

  16. Clinical impact of checkpoint inhibitors as novel cancer therapies.

    Science.gov (United States)

    Shih, Kent; Arkenau, Hendrik-Tobias; Infante, Jeffrey R

    2014-11-01

    Immune responses are tightly regulated via signaling through numerous co-stimulatory and co-inhibitory molecules. Exploitation of these immune checkpoint pathways is one of the mechanisms by which tumors evade and/or escape the immune system. A growing understanding of the biology of immune checkpoints and tumor immunology has led to the development of monoclonal antibodies designed to target co-stimulatory and co-inhibitory molecules in order to re-engage the immune system and restore antitumor immune responses. Anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies were among the first to be tested in the clinic, and ipilimumab was the first immune checkpoint inhibitor approved for an anticancer indication. Agents targeting the programmed death 1 (PD-1) pathway, either PD-1 or one of its ligands, programmed death ligand 1, are in active clinical development for numerous cancers, including advanced melanoma and lung cancer. Understanding the different mechanisms of action, safety profiles, and response patterns associated with inhibition of the CTLA-4 and PD-1 pathways may improve patient management as these therapies are moved in to the clinical practice setting and may also provide a rationale for combination therapy with different inhibitors. Additional immune checkpoint molecules with therapeutic potential, including lymphocyte activation gene-3 and glucocorticoid-induced tumor necrosis factor receptor-related gene, also have inhibitors in early stages of clinical development. Clinical responses and safety data reported to date on immune checkpoint inhibitors suggest these agents may have the potential to markedly improve outcomes for patients with cancer.

  17. 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...

  18. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error.

    Science.gov (United States)

    Vogt, E; Kirsch-Volders, M; Parry, J; Eichenlaub-Ritter, U

    2008-03-12

    The spindle assembly checkpoint (SAC) monitors attachment to microtubules and tension on chromosomes in mitosis and meiosis. It represents a surveillance mechanism that halts cells in M-phase in the presence of unattached chromosomes, associated with accumulation of checkpoint components, in particular, Mad2, at the kinetochores. A complex between the anaphase promoting factor/cylosome (APC/C), its accessory protein Cdc20 and proteins of the SAC renders APC/C inactive, usually until all chromosomes are properly assembled at the spindle equator (chromosome congression) and under tension from spindle fibres. Upon release from the SAC the APC/C can target proteins like cyclin B and securin for degradation by the proteasome. Securin degradation causes activation of separase proteolytic enzyme, and in mitosis cleavage of cohesin proteins at the centromeres and arms of sister chromatids. In meiosis I only the cohesin proteins at the sister chromatid arms are cleaved. This requires meiosis specific components and tight regulation by kinase and phosphatase activities. There is no S-phase between meiotic divisions. Second meiosis resembles mitosis. Mammalian oocytes arrest constitutively at metaphase II in presence of aligned chromosomes, which is due to the activity of the cytostatic factor (CSF). The SAC has been identified in spermatogenesis and oogenesis, but gender-differences may contribute to sex-specific differential responses to aneugens. The age-related reduction in expression of components of the SAC in mammalian oocytes may act synergistically with spindle and other cell organelles' dysfunction, and a partial loss of cohesion between sister chromatids to predispose oocytes to errors in chromosome segregation. This might affect dose-response to aneugens. In view of the tendency to have children at advanced maternal ages it appears relevant to pursue studies on consequences of ageing on the susceptibility of human oocytes to the induction of meiotic error by

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. Sister chromatid tension and the spindle assembly checkpoint.

    Science.gov (United States)

    Nezi, Luigi; Musacchio, Andrea

    2009-12-01

    The spindle assembly checkpoint (SAC) is a feedback control system that monitors the state of kinetochore/microtubule attachment during mitosis and halts cell cycle progression until all chromosomes are properly aligned at the metaphase plate. The state of chromosome-microtubule attachment is implicated as a crucial factor in the checkpoint response. On the contrary, lack of tension in the centromere-kinetochore region of sister chromatids has been shown to regulate a pathway of correction of undesired chromosome-microtubule connections, while the presence of tension is believed to promote the stabilization of attachments. We discuss how tension-sensitive phenomena, such as attachment correction and stabilization, relate to the SAC and we speculate on the existence of a single pathway linking error correction and SAC activation.

  4. Update on immune checkpoint inhibitors in gynecological cancers

    Science.gov (United States)

    2017-01-01

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

  5. Minimum Process Coordinated Checkpointing Scheme for Ad Hoc Networks

    CERN Document Server

    Tuli, Ruchi

    2011-01-01

    The wireless mobile ad hoc network (MANET) architecture is one consisting of a set of mobile hosts capable of communicating with each other without the assistance of base stations. This has made possible creating a mobile distributed computing environment and has also brought several new challenges in distributed protocol design. In this paper, we study a very fundamental problem, the fault tolerance problem, in a MANET environment and propose a minimum process coordinated checkpointing scheme. Since potential problems of this new environment are insufficient power and limited storage capacity, the proposed scheme tries to reduce the amount of information saved for recovery. The MANET structure used in our algorithm is hierarchical based. The scheme is based for Cluster Based Routing Protocol (CBRP) which belongs to a class of Hierarchical Reactive routing protocols. The protocol proposed by us is nonblocking coordinated checkpointing algorithm suitable for ad hoc environments. It produces a consistent set of...

  6. Checkpointing Shared Memory Programs at the Application-level

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-09-08

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

  7. Emerging role of checkpoint blockade therapy in lymphoma

    Science.gov (United States)

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

    2017-01-01

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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Zwint-1 is required for spindle assembly checkpoint function and kinetochore-microtubule attachment during oocyte meiosis.

    Science.gov (United States)

    Woo Seo, Dong; Yeop You, Seung; Chung, Woo-Jae; Cho, Dong-Hyung; Kim, Jae-Sung; Su Oh, Jeong

    2015-10-21

    The key step for faithful chromosome segregation during meiosis is kinetochore assembly. Defects in this process result in aneuploidy, leading to miscarriages, infertility and various birth defects. However, the roles of kinetochores in homologous chromosome segregation during meiosis are ill-defined. Here we found that Zwint-1 is required for homologous chromosome segregation during meiosis. Knockdown of Zwint-1 accelerated the first meiosis by abrogating the kinetochore recruitment of Mad2, leading to chromosome misalignment and a high incidence of aneuploidy. Although Zwint-1 knockdown did not affect Aurora C kinase activity, the meiotic defects following Zwint-1 knockdown were similar to those observed with ZM447439 treatment. Importantly, the chromosome misalignment following Aurora C kinase inhibition was not restored after removing the inhibitor in Zwint-1-knockdown oocytes, whereas the defect was rescued after the inhibitor washout in the control oocytes. These results suggest that Aurora C kinase-mediated correction of erroneous kinetochore-microtubule attachment is primarily regulated by Zwint-1. Our results provide the first evidence that Zwint-1 is required to correct erroneous kinetochore-microtubule attachment and regulate spindle checkpoint function during meiosis.

  15. Subcellular localization of the mitotic checkpoint kinase MPS1: Mechanisms and regulation

    NARCIS (Netherlands)

    Nijenhuis, W.

    2014-01-01

    Faithful segregation of chromosomes during mitosis is required for the maintenance of genomic stability. The missegregation of chromosomes during mitosis causes aneuploidy and may be an initial step in the development of cancer. To ensure that both daughter cells are endowed with a complete set of c

  16. Checkpoint Kinase-Dependent Regulation of DNA Repair and Genome Instability in Breast Cancer

    Science.gov (United States)

    2009-06-01

    Slaaby, A. M. Carr, and O. Nielsen. 2005. Ddb1 controls genome stability and meiosis in fission yeast. Genes Dev 19:853-62. 13. Hook, S. S., J. J. Lin...stability and meiosis in fission yeast. Genes Dev. 19:853–862. 26. Hu, J., C. M. McCall, T. Ohta, and Y. Xiong. 2004. Targeted ubiquitination of CDT1 by

  17. Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies.

    Science.gov (United States)

    Caydasi, Ayse Koca; Pereira, Gislene

    2009-01-01

    In many polarized cells, the accuracy of chromosome segregation depends on the correct positioning of the mitotic spindle. In budding yeast, the spindle positioning checkpoint (SPOC) delays mitotic exit when the anaphase spindle fails to extend toward the mother-daughter axis. However it remains to be established how spindle orientation is translated to SPOC components at the yeast spindle pole bodies (SPB). Here, we used photobleaching techniques to show that the dynamics with which Bub2-Bfa1 turned over at SPBs significantly increased upon SPOC activation. A version of Bfa1 that was stably associated with SPBs rendered the cells SPOC deficient without affecting other Bub2-Bfa1 functions, demonstrating the functional importance of regulating the dynamics of Bfa1 SPB association. In addition, we established that the SPOC kinase Kin4 is the major regulator of Bfa1 residence time at SPBs. We suggest that upon SPOC activation Bfa1-Bub2 spreads throughout the cytoplasm, thereby inhibiting mitotic exit.

  18. 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.

  19. 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.

  20. 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.

  1. Monitoring spindle orientation: Spindle position checkpoint in charge

    Directory of Open Access Journals (Sweden)

    Pereira Gislene

    2010-12-01

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

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

    Science.gov (United States)

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

    2010-12-11

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

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

    Science.gov (United States)

    Day, Daphne; Hansen, Aaron R

    2016-12-01

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

  4. 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...

  5. 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...

  6. 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

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

    Science.gov (United States)

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

    2012-01-01

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

  8. 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.

  9. 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.

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

    Science.gov (United States)

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

    2017-02-17

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

  11. 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.

  12. Spindle assembly checkpoint and its regulators in meiosis.

    Science.gov (United States)

    Sun, Shao-Chen; Kim, Nam-Hyung

    2012-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-09

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

  14. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes......, 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......-subunit affecting: (i) stability, (ii) enzyme specificity and (iii) enzyme activity. The question where CK-2 and its subunits are located throughout the cell cycle has also been addressed, mainly because of the large discrepancies that still exist between results obtained by different investigators. Tissue...

  15. 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

  16. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes.

    Science.gov (United States)

    Gryaznova, Yuliya; Koca Caydasi, Ayse; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-05-09

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.

  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. 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...

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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).

  4. 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.

  5. 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.

  6. 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.

  7. 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.

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

    Science.gov (United States)

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

    2017-01-01

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

  9. Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Hickson, Ian D

    2006-01-01

    Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3......, causes impaired S-phase progression and the persistence of abnormal DNA structures (X-shaped DNA molecules) after exposure to methylmethanesulfonate. The impaired S-phase progression is due to a persistent checkpoint-mediated cell cycle delay and can be overridden by addition of caffeine. Hence......, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α

    Science.gov (United States)

    Wang, Ling; Guo, Qingyuan; Fisher, Laura A; Liu, Dongxu; Peng, Aimin

    2015-01-01

    In addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55α, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55α and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery. PMID:25483054

  15. Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.

    Science.gov (United States)

    Lewis, Cody W; Golsteyn, Roy M

    2016-11-16

    We have examined the relationship between checkpoint adaptation (mitosis with damaged DNA) and micronuclei. Micronuclei in cancer cells are linked to genomic change, and may induce chromothripsis (chromosome shattering). We measured the cytotoxicity of the cancer drug cisplatin in M059K (glioma fibroblasts, IC50 15 μM). Nearly 100% of M059K cells were positive for histone γH2AX staining after 48 h treatment with a cytotoxic concentration of cisplatin. The proportion of micronucleated cells, as confirmed by microscopy using DAPI and lamin A/C staining, increased from 24% to 48%, and the total micronuclei in surviving cells accumulated over time. Promoting entry into mitosis with a checkpoint inhibitor increased the number of micronuclei in cells whereas blocking checkpoint adaptation with a Cdk inhibitor reduced the number of micronuclei. Interestingly, some micronuclei underwent asynchronous DNA replication, relative to the main nuclei, as measured by deoxy-bromo-uracil (BrdU) staining. These micronuclei stained positive for histone γH2AX, which was linked to DNA replication, suggesting that micronuclei arise from checkpoint adaptation and that micronuclei may continue to damage DNA. By contrast the normal cell line WI-38 did not undergo checkpoint adaptation when treated with cisplatin and did not show changes in micronuclei number. These data reveal that the production of micronuclei by checkpoint adaptation is part of a process that contributes to genomic change.

  16. 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.

  17. 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.

  18. bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast

    Directory of Open Access Journals (Sweden)

    Qun eRen

    2012-08-01

    Full Text Available Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H2O2 treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kietochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

  19. 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

  20. 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.

  1. Amino acids and mTOR mediate distinct metabolic checkpoints in mammalian G1 cell cycle.

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    Mahesh Saqcena

    Full Text Available OBJECTIVE: In multicellular organisms, cell division is regulated by growth factors (GFs. In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains - especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase. METHODS: We used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs, the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [(3H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot. RESULTS: We report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR - the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints. CONCLUSION: The data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.

  2. Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells.

    Science.gov (United States)

    Zhou, Xiaodong; Kim, Sung-Hoon; Shen, Ling; Lee, Hyo-Jung; Chen, Changyan

    2014-01-01

    Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.

  3. The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond

    Directory of Open Access Journals (Sweden)

    Maia André F

    2008-05-01

    Full Text Available Abstract During mitosis, correct bipolar chromosome attachment to the mitotic spindle is an essential prerequisite for the equal segregation of chromosomes. The spindle assembly checkpoint can prevent chromosome segregation as long as not all chromosome pairs have obtained bipolar attachment to the spindle. The chromosomal passenger complex plays a crucial role during chromosome alignment by correcting faulty chromosome-spindle interactions (e.g. attachments that do not generate tension. In the process of doing so, the chromosomal passenger complex generates unattached chromosomes, a specific situation that is known to promote checkpoint activity. However, several studies have implicated an additional, more direct role for the chromosomal passenger complex in enforcing the mitotic arrest imposed by the spindle assembly checkpoint. In this review, we discuss the different roles played by the chromosomal passenger complex in ensuring proper mitotic checkpoint function. Additionally, we discuss the possibility that besides monitoring the presence of unattached kinetochores, the spindle assembly checkpoint may also be capable of responding to chromosome-microtubule interactions that do not generate tension and we propose experimental set-ups to study this.

  4. The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond.

    Science.gov (United States)

    Vader, Gerben; Maia, André F; Lens, Susanne Ma

    2008-05-28

    During mitosis, correct bipolar chromosome attachment to the mitotic spindle is an essential prerequisite for the equal segregation of chromosomes. The spindle assembly checkpoint can prevent chromosome segregation as long as not all chromosome pairs have obtained bipolar attachment to the spindle. The chromosomal passenger complex plays a crucial role during chromosome alignment by correcting faulty chromosome-spindle interactions (e.g. attachments that do not generate tension). In the process of doing so, the chromosomal passenger complex generates unattached chromosomes, a specific situation that is known to promote checkpoint activity. However, several studies have implicated an additional, more direct role for the chromosomal passenger complex in enforcing the mitotic arrest imposed by the spindle assembly checkpoint. In this review, we discuss the different roles played by the chromosomal passenger complex in ensuring proper mitotic checkpoint function. Additionally, we discuss the possibility that besides monitoring the presence of unattached kinetochores, the spindle assembly checkpoint may also be capable of responding to chromosome-microtubule interactions that do not generate tension and we propose experimental set-ups to study this.

  5. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    Science.gov (United States)

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

  6. Preserving Yeast Genetic Heritage through DNA Damage Checkpoint Regulation and Telomere Maintenance

    Directory of Open Access Journals (Sweden)

    Huilin Zhou

    2012-10-01

    Full Text Available In order to preserve genome integrity, extrinsic or intrinsic DNA damages must be repaired before they accumulate in cells and trigger other mutations and genome rearrangements. Eukaryotic cells are able to respond to different genotoxic stresses as well as to single DNA double strand breaks (DSBs, suggesting highly sensitive and robust mechanisms to detect lesions that trigger a signal transduction cascade which, in turn, controls the DNA damage response (DDR. Furthermore, cells must be able to distinguish natural chromosomal ends from DNA DSBs in order to prevent inappropriate checkpoint activation, DDR and chromosomal rearrangements. Since the original discovery of RAD9, the first DNA damage checkpoint gene identified in Saccharomyces cerevisiae, many genes that have a role in this pathway have been identified, including MRC1, MEC3, RAD24, RAD53, DUN1, MEC1 and TEL1. Extensive studies have established most of the genetic basis of the DNA damage checkpoint and uncovered its different functions in cell cycle regulation, DNA replication and repair, and telomere maintenance. However, major questions concerning the regulation and functions of the DNA damage checkpoint remain to be answered. First, how is the checkpoint activity coupled to DNA replication and repair? Second, how do cells distinguish natural chromosome ends from deleterious DNA DSBs? In this review we will examine primarily studies performed using Saccharomyces cerevisiae as a model system.

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

    Science.gov (United States)

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

    2017-01-01

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

  8. 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.

  9. 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.

  10. Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

    Science.gov (United States)

    Lima, Michelle; Bouzid, Hana; Soares, Daniele G; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M; Henriques, João A P; Larsen, Annette K; Escargueil, Alexandre E

    2016-05-03

    Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

  11. Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint

    DEFF Research Database (Denmark)

    Zhang, Gang; Lischetti, Tiziana; Hayward, Daniel G;

    2015-01-01

    , we show that a distinct region in Bub1 mediates kinetochore localization of BubR1 through direct binding, but surprisingly removal of this region increases checkpoint strength. Our work thus uncovers how Bub1 coordinates checkpoint signalling by distinct domains for RZZ and BubR1 recruitment...

  12. 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...

  13. 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.

  14. 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

    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...

  15. Functional and Physical Interaction between Rad24 and Rfc5 in the Yeast Checkpoint Pathways

    OpenAIRE

    Shimomura, Toshiyasu; Ando, Seiko; Matsumoto, Kunihiro; Sugimoto, Katsunori

    1998-01-01

    The RFC5 gene encodes a small subunit of replication factor C (RFC) complex in Saccharomyces cerevisiae and has been shown to be required for the checkpoints which respond to replication block and DNA damage. Here we describe the isolation of RAD24, known to play a role in the DNA damage checkpoint, as a dosage-dependent suppressor of rfc5-1. RAD24 overexpression suppresses the sensitivity of rfc5-1 cells to DNA-damaging agents and the defect in DNA damage-induced Rad53 phosphorylation. Rad24...

  16. 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.

  17. 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.

  18. 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.

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

    NARCIS (Netherlands)

    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

  20. 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...

  1. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle

    NARCIS (Netherlands)

    Shaltiel, Indra A.; Krenning, Lenno; Bruinsma, Wytse; Medema, René H.

    2015-01-01

    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cyc

  2. Restarting the cell cycle when the checkpoint comes to a halt

    NARCIS (Netherlands)

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

    2005-01-01

    The DNA damage checkpoint coordinates a block in cell proliferation with the DNA repair process that follows when lesions are inflicted on the genome. However, we do not know exactly how cell division can recommence following a DNA damage-induced arrest. Recent work from our lab has identified Polo-

  3. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    DEFF Research Database (Denmark)

    McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel

    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...

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

    Science.gov (United States)

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

    2015-08-04

    The mechanisms 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 duration ≥ 50 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. We propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

  5. 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.

  6. 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

  7. DISTURBED ANTIGEN PRESENTATION IN CLASSICAL HODGKIN LYMPHOMA; IMPLICATIONS FOR IMMUNE CHECKPOINT INHIBITOR THERAPY?

    NARCIS (Netherlands)

    Nijland, M.; Visser, Lydia; Veenstra, Rianne; Kushekhar, K.; van Imhoff, G.; Berg, van den Anke; Diepstra, A.

    2016-01-01

    Immune checkpoint inhibitors are being tested in clinical trials and show great promise in the treatment of classical Hodgkin lymphoma (cHL). The proposed mechanism of action of these inhibitors consists of reactivating T lymphocytes that have become unresponsive as a consequence of inhibitory mecha

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

    DEFF Research Database (Denmark)

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

    2006-01-01

    We present an approach to the synthesis of fault-tolerant hard real-time systems for safety-critical applications. We use checkpointing with rollback recovery and active replication for tolerating transient faults. Processes are statically scheduled and communications are performed using the time...

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

    Directory of Open Access Journals (Sweden)

    Paula M Checchi

    2011-09-01

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

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

    Science.gov (United States)

    Checchi, Paula M; Engebrecht, JoAnne

    2011-09-01

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

  11. 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

    2016-09-19

    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.

  12. Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

    Science.gov (United States)

    Chatterjee, Ritam; Chattopadhyay, Sukalpa; Law, Sujata

    2016-11-01

    Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

  13. 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.

  14. 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...

  15. 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.

  16. Precision Therapy for Lung Cancer: Tyrosine Kinase Inhibitors and Beyond.

    Science.gov (United States)

    Rajan, Arun; Schrump, David S

    2015-01-01

    For patients with advanced cancers there has been a concerted effort to transition from a generic treatment paradigm to one based on tumor-specific biologic, and patient-specific clinical characteristics. This approach, known as precision therapy has been made possible owing to widespread availability and a reduction in the cost of cutting-edge technologies that are used to study the genomic, proteomic, and metabolic attributes of individual tumors. This review traces the evolution of precision therapy for lung cancer from the identification of molecular subsets of the disease to the development and approval of tyrosine kinase, as well as immune checkpoint inhibitors for lung cancer therapy. Challenges of the precision therapy era including the emergence of acquired resistance, identification of untargetable mutations, and the effect on clinical trial design are discussed. We conclude by highlighting newer applications for the concept of precision therapy.

  17. 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....

  18. 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.

  19. The G2/M DNA damage checkpoint inhibits mitosis through Tyr15 phosphorylation of p34cdc2 in Aspergillus nidulans.

    Science.gov (United States)

    Ye, X S; Fincher, R R; Tang, A; Osmani, S A

    1997-01-02

    It is possible to cause G2 arrest in Aspergillus nidulans by inactivating either p34cdc2 or NIMA. We therefore investigated the negative control of these two mitosis-promoting kinases after DNA damage. DNA damage caused rapid Tyr15 phosphorylation of p34cdc2 and transient cell cycle arrest but had little effect on the activity of NIMA. Dividing cells deficient in Tyr15 phosphorylation of p34cdc2 were sensitive to both MMS and UV irradiation and entered lethal premature mitosis with damaged DNA. However, non-dividing quiescent conidiospores of the Tyr15 mutant strain were not sensitive to DNA damage. The UV and MMS sensitivity of cells unable to tyrosine phosphorylate p34cdc2 is therefore caused by defects in DNA damage checkpoint regulation over mitosis. Both the nimA5 and nimT23 temperature-sensitive mutations cause an arrest in G2 at 42 degrees C. Addition of MMS to nimT23 G2-arrested cells caused a marked delay in their entry into mitosis upon downshift to 32 degrees C and this delay was correlated with a long delay in the dephosphorylation and activation of p34cdc2. Addition of MMS to nimA5 G2-arrested cells caused inactivation of the H1 kinase activity of p34cdc2 due to an increase in its Tyr15 phosphorylation level and delayed entry into mitosis upon return to 32 degrees C. However, if Tyr15 phosphorylation of p34cdc2 was prevented then its H1 kinase activity was not inactivated upon MMS addition to nimA5 G2-arrested cells and they rapidly progressed into a lethal mitosis upon release to 32 degrees C. Thus, Tyr15 phosphorylation of p34cdc2 in G2 arrests initiation of mitosis after DNA damage in A. nidulans.

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

    OpenAIRE

    Checchi, Paula M.; JoAnne Engebrecht

    2011-01-01

    Meiosis is a specialized form of cellular division that results in the precise halving of the genome to produce gametes for sexual reproduction. Checkpoints function during meiosis to detect errors and subsequently to activate a signaling cascade that prevents the formation of aneuploid gametes. Indeed, asynapsis of a homologous chromosome pair elicits a checkpoint response that can in turn trigger germline apoptosis. In a heterogametic germ line, however, sex chromosomes proceed through meio...

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

    OpenAIRE

    Mihailo Mirkovic; Lukas H. Hutter; Béla Novák; Raquel A. Oliveira

    2015-01-01

    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 r...

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

    OpenAIRE

    Mirkovic, Mihailo; Hutter, Lukas H.; Novák, Béla; Oliveira, Raquel A.

    2015-01-01

    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 r...

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

    Energy Technology Data Exchange (ETDEWEB)

    Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

    2012-10-12

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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

    2016-02-02

    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.

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

    Directory of Open Access Journals (Sweden)

    José Henrique Teixeira

    2014-01-01

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

  14. The chromosomal passenger complex and the spindle assembly checkpoint: kinetochore-microtubule error correction and beyond

    OpenAIRE

    Maia André F; Vader Gerben; Lens Susanne MA

    2008-01-01

    Abstract During mitosis, correct bipolar chromosome attachment to the mitotic spindle is an essential prerequisite for the equal segregation of chromosomes. The spindle assembly checkpoint can prevent chromosome segregation as long as not all chromosome pairs have obtained bipolar attachment to the spindle. The chromosomal passenger complex plays a crucial role during chromosome alignment by correcting faulty chromosome-spindle interactions (e.g. attachments that do not generate tension). In ...

  15. Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model.

    Science.gov (United States)

    Reardon, David A; Gokhale, Prafulla C; Klein, Sarah R; Ligon, Keith L; Rodig, Scott J; Ramkissoon, Shakti H; Jones, Kristen L; Conway, Amy Saur; Liao, Xiaoyun; Zhou, Jun; Wen, Patrick Y; Van Den Abbeele, Annick D; Hodi, F Stephen; Qin, Lei; Kohl, Nancy E; Sharpe, Arlene H; Dranoff, Glenn; Freeman, Gordon J

    2016-02-01

    Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti-PD-1, anti-PD-L1, or anti-CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti-CTLA-4 plus anti-PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8(+) and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma.

  16. 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.

  17. A novel ATM-dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma.

    Science.gov (United States)

    Spoerri, Loredana; Brooks, Kelly; Chia, KeeMing; Grossman, Gavriel; Ellis, Jonathan J; Dahmer-Heath, Mareike; Škalamera, Dubravka; Pavey, Sandra; Burmeister, Bryan; Gabrielli, Brian

    2016-05-01

    Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM-dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM-dependent checkpoint arrest, and over-expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM-dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over-express PLK1, and a significant proportion of melanomas have high levels of PLK1 over-expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM-dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.

  18. 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.

  19. 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.

  20. 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)....

  1. 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.

  2. 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.

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

    Science.gov (United States)

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

    2016-01-01

    Despite compelling antitumour activity of antibodies targeting the programmed death 1 (PD-1): programmed death ligand 1 (PD-L1) immune checkpoint in lung cancer, resistance to these therapies has increasingly been observed. In this study, to elucidate mechanisms of adaptive resistance, we analyse the tumour immune microenvironment in the context of anti-PD-1 therapy in two fully immunocompetent mouse models of lung adenocarcinoma. In tumours progressing following response to anti-PD-1 therapy, we observe upregulation of alternative immune checkpoints, notably T-cell immunoglobulin mucin-3 (TIM-3), in PD-1 antibody bound T cells and demonstrate a survival advantage with addition of a TIM-3 blocking antibody following failure of PD-1 blockade. Two patients who developed adaptive resistance to anti-PD-1 treatment also show a similar TIM-3 upregulation in blocking antibody-bound T cells at treatment failure. These data suggest that upregulation of TIM-3 and other immune checkpoints may be targetable biomarkers associated with adaptive resistance to PD-1 blockade. PMID:26883990

  4. 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

  5. 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

  6. Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance.

    Science.gov (United States)

    Khair, Lyne; Chang, Ya-Ting; Subramanian, Lakxmi; Russell, Paul; Nakamura, Toru M

    2010-06-01

    While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

  7. Suppression of the DNA damage response in acute myeloid leukemia versus myelodysplastic syndrome.

    Science.gov (United States)

    Boehrer, S; Adès, L; Tajeddine, N; Hofmann, W K; Kriener, S; Bug, G; Ottmann, O G; Ruthardt, M; Galluzzi, L; Fouassier, C; Tailler, M; Olaussen, K A; Gardin, C; Eclache, V; de Botton, S; Thepot, S; Fenaux, P; Kroemer, G

    2009-06-04

    The molecular mechanisms responsible for the evolution from the preleukemic entities of low-risk myelodysplastic syndrome (MDS) to the less favorable forms of high-risk MDS, as well as those enabling transformation to acute myeloid leukemia (AML), are still incompletely understood. Abundant evidence from solid tumors demonstrates that preneoplastic lesions activate signaling pathways of a DNA damage response (DDR), which functions as an 'anticancer barrier' hindering tumorigenesis. Testing the hypothesis that subgroups of MDS and AML differ with respect to DDR, we first assessed markers of DDR (phosphorylation of ATM, Chk-1, Chk-2 and H2AX) in cell lines representing different entities of MDS (P39, MOLM-13) and AML (MV4-11, KG-1) before and after gamma-irradiation. Although gamma-irradiation induced apoptosis and G(2)/M arrest and a concomitant increase in the phosphorylation of ATM, Chk-1 and H2AX in MDS-derived cell lines, this radiation response was attenuated in the AML-derived cell lines. It is noteworthy that KG-1, but not P39 cells exhibit signs of an endogenous activation of the DDR. Similarly, we found that the frequency of P-ATM(+) cells detectable in bone marrow (BM) biopsies increased in samples from patients with AML as compared with high-risk MDS samples and significantly correlated with the percentage of BM blasts. In contrast, the frequency of gamma-H2AX(+) cells was heterogeneous in all subgroups of AML and MDS. Whereas intermediate-1 MDS samples contained as little P-Chk-1 and P-Chk-2 as healthy controls, staining for both checkpoint kinases increased in intermediate-2 and high-risk MDS, yet declined to near-to-background levels in AML samples. Thus the activation of Chk-1 and Chk-2 behaves in accord with the paradigm established for solid tumors, whereas ATM is activated during and beyond transformation. In conclusion, we demonstrate the heterogeneity of the DDR response in MDS and AML and provide evidence for its selective suppression in AML

  8. 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.

  9. The 14-3-3 protein Bmh1 functions in the spindle position checkpoint by breaking Bfa1 asymmetry at yeast centrosomes.

    Science.gov (United States)

    Caydasi, Ayse Koca; Micoogullari, Yagmur; Kurtulmus, Bahtiyar; Palani, Saravanan; Pereira, Gislene

    2014-07-15

    In addition to their well-known role in microtubule organization, centrosomes function as signaling platforms and regulate cell cycle events. An important example of such a function is the spindle position checkpoint (SPOC) of budding yeast. SPOC is a surveillance mechanism that ensures alignment of the mitotic spindle along the cell polarity axis. Upon spindle misalignment, phosphorylation of the SPOC component Bfa1 by Kin4 kinase engages the SPOC by changing the centrosome localization of Bfa1 from asymmetric (one centrosome) to symmetric (both centrosomes). Here we show that, unexpectedly, Kin4 alone is unable to break Bfa1 asymmetry at yeast centrosomes. Instead, phosphorylation of Bfa1 by Kin4 creates a docking site on Bfa1 for the 14-3-3 family protein Bmh1, which in turn weakens Bfa1-centrosome association and promotes symmetric Bfa1 localization. Consistently, BMH1-null cells are SPOC deficient. Our work thus identifies Bmh1 as a new SPOC component and refines the molecular mechanism that breaks Bfa1 centrosome asymmetry upon SPOC activation.

  10. 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.

  11. 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

  12. Combining molecular dynamics simulation and ligand-receptor contacts analysis as a new approach for pharmacophore modeling: beta-secretase 1 and check point kinase 1 as case studies.

    Science.gov (United States)

    Hatmal, Ma'mon M; Jaber, Shadi; Taha, Mutasem O

    2016-12-01

    Ligand-based pharmacophore modeling require relatively long lists of active compounds, while a pharmacophore based on a single ligand-receptor crystallographic structure is often promiscuous. These problems prompted us to combine molecular dynamics (MD) simulation with ligand-receptor contacts analysis as means to develop valid pharmacophore model(s). The particular ligand-receptor complex is allowed to perturb over a few nano-seconds using MD simulation. Subsequently, ligand-receptor contact points (≤2.5 Å) are identified. Ligand-receptor contacts maintained above certain threshold during molecular dynamics simulation are considered critical and used to guide pharmacophore development. We termed this method as Molecular-Dynamics Based Ligand-Receptor Contact Analysis. We implemented this new methodology to develop valid pharmacophore models for check point kinase 1 (Chk1) and beta-secretase 1 (BACE1) inhibitors as case studies. The resulting pharmacophore models were validated by receiver operating characteristic curved analysis against inhibitors obtained from CHEMBL database.

  13. Combining molecular dynamics simulation and ligand-receptor contacts analysis as a new approach for pharmacophore modeling: beta-secretase 1 and check point kinase 1 as case studies

    Science.gov (United States)

    Hatmal, Ma'mon M.; Jaber, Shadi; Taha, Mutasem O.

    2016-12-01

    Ligand-based pharmacophore modeling require relatively long lists of active compounds, while a pharmacophore based on a single ligand-receptor crystallographic structure is often promiscuous. These problems prompted us to combine molecular dynamics (MD) simulation with ligand-receptor contacts analysis as means to develop valid pharmacophore model(s). The particular ligand-receptor complex is allowed to perturb over a few nano-seconds using MD simulation. Subsequently, ligand-receptor contact points (≤2.5 Å) are identified. Ligand-receptor contacts maintained above certain threshold during molecular dynamics simulation are considered critical and used to guide pharmacophore development. We termed this method as Molecular-Dynamics Based Ligand-Receptor Contact Analysis. We implemented this new methodology to develop valid pharmacophore models for check point kinase 1 (Chk1) and beta-secretase 1 (BACE1) inhibitors as case studies. The resulting pharmacophore models were validated by receiver operating characteristic curved analysis against inhibitors obtained from CHEMBL database.

  14. Structural and Functional Characterization of the Protein Kinase Mps1 in Arabidopsis thaliana

    Science.gov (United States)

    de Oliveira, Eduardo Alves Gamosa; Romeiro, Nelilma Correia; Ribeiro, Elane da Silva; Santa-Catarina, Claudete; Oliveira, Antônia Elenir Amâncio; Silveira, Vanildo; de Souza Filho, Gonçalo Apolinário; Venancio, Thiago Motta; Cruz, Marco Antônio Lopes

    2012-01-01

    In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes. PMID:23049844

  15. 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.

  16. A preliminary study on the radiation-resistance mechanism in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Qi Liao

    2013-01-01

    Full Text Available Aim: The present study was designed to explore the radiation-resistance mechanism by interfering in checkpoints kinase 1 (CHK1 and DNA-activated protein kinase (DNA-PK genes with short hairpin RNA (shRNA transfection into Skov3 cells derived from ovarian cancer and HeLa cells derived from cervical cancer. Materials and Methods: The cultured Skov3 and HeLa cells were transfected with plasmid vectors containing CHK1 shRNA and DNA-PK shRNA, respectively, through Lipofectimine™ 2000 mediation, and cultured for 20 hours before exposure to 2 Gy X-radiation. The cells were harvested 4 and 28 after X-irradiation respectively then washed 3 times with PBS. These cells were stained with Annexin V/PI and applied by flow cytometer to analyze alteration of apoptosis with software CellQuest. Results: The apoptotic response in Skov3 cells to X-radiation was significantly lower than that in HeLa cells at 4 hour (t = 15.22, P < 0.001 and 28 hours (t = 15.78, P < 0.001 of post-irradiation. The shRNA might not affect the apoptosis of Skov3 and HeLa cells, while shRNA-transfection significantly enhanced the apoptotic response in Skov3 cells to X-radiation as compared with that in HeLa cells. Conclusions: The present work suggests that the CHK1 and DNA-PK genes are very likely to play a role in developing a radiation resistance in ovarian cancer.

  17. 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

  18. 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.

  19. BMI1 attenuates etoposide-induced G2/M checkpoints via reducing ATM activation.

    Science.gov (United States)

    Wei, F; Ojo, D; Lin, X; Wong, N; He, L; Yan, J; Xu, S; Major, P; Tang, D

    2015-06-04

    The BMI1 protein contributes to stem cell pluripotency and oncogenesis via multiple functions, including its newly identified role in DNA damage response (DDR). Although evidence clearly demonstrates that BMI1 facilitates the repair of double-stranded breaks via homologous recombination (HR), it remains unclear how BMI1 regulates checkpoint activation during DDR. We report here that BMI1 has a role in G2/M checkpoint activation in response to etoposide (ETOP) treatment. Ectopic expression of BMI1 in MCF7 breast cancer and DU145 prostate cancer cells significantly reduced ETOP-induced G2/M arrest. Conversely, knockdown of BMI1 in both lines enhanced the arrest. Consistent with ETOP-induced activation of the G2/M checkpoints via the ATM pathway, overexpression and knockdown of BMI1, respectively, reduced and enhanced ETOP-induced phosphorylation of ATM at serine 1981 (ATM pS1981). Furthermore, the phosphorylation of ATM targets, including γH2AX, threonine 68 (T68) on CHK2 (CHK2 pT68) and serine 15 (S15) on p53 were decreased in overexpression and increased in knockdown BMI1 cells in response to ETOP. In line with the requirement of NBS1 in ATM activation, we were able to show that BMI1 associates with NBS1 and that this interaction altered the binding of NBS1 with ATM. BMI1 consists of a ring finger (RF), helix-turn-helix-turn-helix-turn (HT), proline/serine (PS) domain and two nuclear localization signals (NLS). Although deletion of either RF or HT did not affect the association of BMI1 with NBS1, the individual deletions of PS and one NLS (KRMK) robustly reduced the interaction. Stable expression of these BMI1 mutants decreased ETOP-induced ATM pS1981 and CHK2 pT68, but not ETOP-elicited γH2AX in MCF7 cells. Furthermore, ectopic expression of BMI1 in non-transformed breast epithelial MCF10A cells also compromised ETOP-initiated ATM pS1981 and γH2AX. Taken together, we provide compelling evidence that BMI1 decreases ETOP-induced G2/M checkpoint activation via

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

    Science.gov (United States)

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

    2007-06-01

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

  1. Error-prone mammalian female meiosis from silencing the spindle assembly checkpoint without normal interkinetochore tension.

    Science.gov (United States)

    Kolano, Agnieszka; Brunet, Stéphane; Silk, Alain D; Cleveland, Don W; Verlhac, Marie-Hélène

    2012-07-03

    It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.

  2. An Adaptive Checkpointing Scheme for Peer-to-Peer Based Volunteer Computing Work Flows

    CERN Document Server

    Ni, Lei

    2007-01-01

    Volunteer Computing, sometimes called Public Resource Computing, is an emerging computational model that is very suitable for work-pooled parallel processing. As more complex grid applications make use of work flows in their design and deployment it is reasonable to consider the impact of work flow deployment over a Volunteer Computing infrastructure. In this case, the inter work flow I/O can lead to a significant increase in I/O demands at the work pool server. A possible solution is the use of a Peer-to- Peer based parallel computing architecture to off-load this I/O demand to the workers; where the workers can fulfill some aspects of work flow coordination and I/O checking, etc. However, achieving robustness in such a large scale system is a challenging hurdle towards the decentralized execution of work flows and general parallel processes. To increase robustness, we propose and show the merits of using an adaptive checkpoint scheme that efficiently checkpoints the status of the parallel processes accordin...

  3. Differences in spindle association of the mitotic checkpoint protein Mad2 in mammalian spermatogenesis and oogenesis.

    Science.gov (United States)

    Kallio, M; Eriksson, J E; Gorbsky, G J

    2000-09-01

    We have investigated expression and subcellular localization of the spindle checkpoint protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.

  4. Estimated Interval-Based Checkpointing (EIC on Spot Instances in Cloud Computing

    Directory of Open Access Journals (Sweden)

    Daeyong Jung

    2014-01-01

    Full Text Available In cloud computing, users can rent computing resources from service providers according to their demand. Spot instances are unreliable resources provided by cloud computing services at low monetary cost. When users perform tasks on spot instances, there is an inevitable risk of failures that causes the delay of task execution time, resulting in a serious deterioration of quality of service (QoS. To deal with the problem on spot instances, we propose an estimated interval-based checkpointing (EIC using weighted moving average. Our scheme sets the thresholds of price and execution time based on history. Whenever the actual price and the execution time cross over the thresholds, the system saves the state of spot instances. The Bollinger Bands is adopted to inform the ranges of estimated cost and execution time for user's discretion. The simulation results reveal that, compared to the HBC and REC, the EIC reduces the number of checkpoints and the rollback time. Consequently, the task execution time is decreased with EIC by HBC and REC. The EIC also provides the benefit of the cost reduction by HBC and REC, on average. We also found that the actual cost and execution time fall within the estimated ranges suggested by the Bollinger Bands.

  5. Role of Clinical Pharmacology in the Development and Approval of Immunotherapies Targeting Immune Checkpoints.

    Science.gov (United States)

    Rahman, A

    2016-12-01

    Immune surveillance plays a critical role in preventing the development and progression of cancer. Immune modulators, such as interferon-gamma or interleukin-2, have been a part of the cancer treatment armament over the past few decades. However, new understandings regarding the role of the costimulatory and coinhibitory molecules associated with T-cells and antigen-presenting cells as well as tumor necrosis factor receptors and ligands have ushered the new era of immunotherapy for cancer treatment. We now know that primary cancer cells evade screening by the innate immune system, proliferate, and form metastases by upregulating immune inhibitory pathways referred to as immune checkpoints. The recent development of therapies that target immune checkpoints, such as cytotoxic T lymphocyte antigen 4, programmed cell death 1, programmed cell death ligand 1, indoleamine 2,3-dioxygenase, T-cell immunoglobulin and mucin domain 3, and lymphocyte activation gene 3 precisely target the immune system and give new hope for treating various types of cancer. In select marker-enriched populations, immunotherapies provide high response rates as well as durable responses in terms of progression-free survival and overall survival. Numerous factors, such as patient's immune system, the expression of targets on both immune and cancer cells, maintenance of an effective drug exposure, and tolerability to these agents may play a role in this unique observation.

  6. Checkpoint for DNA integrity at the first mitosis after oocyte activation.

    Science.gov (United States)

    Liu, Lin; Trimarchi, James R; Smith, Peter J S; Keefe, David L

    2002-06-01

    Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.

  7. 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

    2016-10-01

    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.

  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. Enterococcus faecalis phosphomevalonate kinase.

    Science.gov (United States)

    Doun, Stephanie S; Burgner, John W; Briggs, Scott D; Rodwell, Victor W

    2005-05-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

  12. 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.

  13. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

    Lee, Y.; Munnik, T.; Munnik, T.

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate (PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its

  14. 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

  15. Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint.

    Directory of Open Access Journals (Sweden)

    Martha Klovstad

    2008-02-01

    Full Text Available Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.

  16. 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...

  17. 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

    We present an approach to the synthesis of fault-tolerant hard real-time systems for safety-critical applications. We use checkpointing with rollback recovery and active replication for tolerating transient faults. Processes and communications are statically scheduled. Our synthesis approach deci...

  18. 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.

  19. Cyclin-dependent kinase activity controls the onset of the HCMV lytic cycle.

    Directory of Open Access Journals (Sweden)

    Martin Zydek

    Full Text Available The onset of human cytomegalovirus (HCMV lytic infection is strictly synchronized with the host cell cycle. Infected G0/G1 cells support viral immediate early (IE gene expression and proceed to the G1/S boundary where they finally arrest. In contrast, S/G2 cells can be infected but effectively block IE gene expression and this inhibition is not relieved until host cells have divided and reentered G1. During latent infection IE gene expression is also inhibited, and for reactivation to occur this block to IE gene expression must be overcome. It is only poorly understood which viral and/or cellular activities maintain the block to cell cycle or latency-associated viral IE gene repression and whether the two mechanisms may be linked. Here, we show that the block to IE gene expression during S and G2 phase can be overcome by both genotoxic stress and chemical inhibitors of cellular DNA replication, pointing to the involvement of checkpoint-dependent signaling pathways in controlling IE gene repression. Checkpoint-dependent rescue of IE expression strictly requires p53 and in the absence of checkpoint activation is mimicked by proteasomal inhibition in a p53 dependent manner. Requirement for the cyclin dependent kinase (CDK inhibitor p21 downstream of p53 suggests a pivotal role for CDKs in controlling IE gene repression in S/G2 and treatment of S/G2 cells with the CDK inhibitor roscovitine alleviates IE repression independently of p53. Importantly, CDK inhibiton also overcomes the block to IE expression during quiescent infection of NTera2 (NT2 cells. Thus, a timely block to CDK activity not only secures phase specificity of the cell cycle dependent HCMV IE gene expression program, but in addition plays a hitherto unrecognized role in preventing the establishment of a latent-like state.

  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.

  1. Adapt or die: how eukaryotic cells respond to prolonged activation of the spindle assembly checkpoint.

    Science.gov (United States)

    Rossio, Valentina; Galati, Elena; Piatti, Simonetta

    2010-12-01

    Many cancer-treating compounds used in chemotherapies, the so-called antimitotics, target the mitotic spindle. Spindle defects in turn trigger activation of the SAC (spindle assembly checkpoint), a surveillance mechanism that transiently arrests cells in mitosis to provide the time for error correction. When the SAC is satisfied, it is silenced. However, after a variable amount of time, cells escape from the mitotic arrest, even if the SAC is not satisfied, through a process called adaptation or mitotic slippage. Adaptation weakens the killing properties of antimitotics, ultimately giving rise to resistant cancer cells. We summarize here the mechanisms underlying this process and propose a strategy to identify the factors involved using budding yeast as a model system. Inhibition of factors involved in SAC adaptation could have important therapeutic applications by potentiating the ability of antimitotics to cause cell death.

  2. Joined at the hip: kinetochores, microtubules, and spindle assembly checkpoint signaling.

    Science.gov (United States)

    Sacristan, Carlos; Kops, Geert J P L

    2015-01-01

    Error-free chromosome segregation relies on stable connections between kinetochores and spindle microtubules. The spindle assembly checkpoint (SAC) monitors such connections and relays their absence to the cell cycle machinery to delay cell division. The molecular network at kinetochores that is responsible for microtubule binding is integrated with the core components of the SAC signaling system. Molecular-mechanistic understanding of how the SAC is coupled to the kinetochore-microtubule interface has advanced significantly in recent years. The latest insights not only provide a striking view of the dynamics and regulation of SAC signaling events at the outer kinetochore but also create a framework for understanding how that signaling may be terminated when kinetochores and microtubules connect.

  3. 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.  

  4. Prospect of the use of checkpoint inhibitors in hepatocellular cancer treatments

    Science.gov (United States)

    Raufi, Ali; Tirona, Maria Tria

    2017-01-01

    Hepatocellular cancer (HCC) is a very fatal disease due to limited therapeutic options as well as due to its association with underlying chronic liver disease in the majority of cases. The immune evasion in HCC signifies a major barrier to the delivery of effective immunotherapy. Sorafenib is the only Food and Drug Administration-approved drug available with an overall response rate of 2%–3% and overall survival of 2.8 months. Chemotherapy has not been used routinely because of the relative refractoriness of advanced HCC. The introduction of immune checkpoint inhibitors (cytotoxic T-lymphocyte antigen 4, programmed death 1, and programmed death-ligand 1) has opened a new horizon for cancer immunotherapy. Future direction in immunotherapy for HCC is to rationally combine it with other treatment modalities, including surgery, radiofrequency ablation, and cytotoxic agents, to maximize its therapeutic efficacy.

  5. 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

  6. Mitosis-specific phosphorylation of PML at T409 regulates spindle checkpoint.

    Science.gov (United States)

    Jin, J; Liu, J

    2016-08-31

    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.

  7. 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.

  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. Immune-checkpoint status in penile squamous cell carcinoma: a North American cohort.

    Science.gov (United States)

    Cocks, Margaret; Taheri, Diana; Ball, Mark W; Bezerra, Stephania M; Del Carmen Rodriguez, Maria; Ricardo, Bernardo F P; Bivalacqua, Trinity J; Sharma, Rajni B; Meeker, Alan; Chaux, Alcides; Burnett, Arthur L; Netto, George J

    2017-01-01

    Penile squamous cell carcinoma (SCC) is primarily treated by surgical resection. Locally advanced and metastatic diseases require a multidisciplinary treatment approach. However, mortality and morbidity remain high, and novel molecular and immunotherapeutic targets are actively being sought. We investigated the expression of immune-checkpoint markers in penile cancers. Fifty-three invasive penile SCCs diagnosed between 1985 and 2013 were retrieved from our surgical pathology archives. Representative formalin-fixed, paraffin-embedded archival blocks were used for the construction of 2 high-density tissue microarrays. Tissue microarrays were stained with immunohistochemistry for PD-L1, FOXP3, CD8, and Ki-67. PD-L1 was investigated using rabbit monoclonal anti-PD-L1 antibody (Cell Signaling, Boston, MA; E1L3N, 1:100). Overall, 21 (40%) of 53 penile SCCs had positive PD-L1 expression. PD-L1 was expressed by a significant proportion of advanced penile SCC. Forty-four percent (15/34) of stage pT2 or more SCC and 38% (6/16) of tumors with lymph node metastasis were positive for PD-L1. PD-L1 expression did not correlate with patient age, tumor location, histologic subtype, tumor stage, anatomic depth of invasion, or tumor grade. FOXP3 expression in tumoral immune cells was found in 26 (49%) of 53 cases. FOXP3 expression in stromal immune cells correlated with tumor thickness (P = .0086). The ratio of CD8/FOXP3 was greater than 1 in 62% of cases in tumor-infiltrating immune cells and 34% of cases in stromal immune cells. Our current study is the largest to assess expression of PD-L1 in a clinically well-annotated North American cohort of penile SCC. Our findings support a rationale for targeting immune-checkpoint inhibitor pathways in advanced penile SCC.

  10. Lichenoid Dermatologic Toxicity From Immune Checkpoint Blockade Therapy: A Detailed Examination of the Clinicopathologic Features.

    Science.gov (United States)

    Tetzlaff, Michael T; Nagarajan, Priyadharsini; Chon, Susan; Huen, Auris; Diab, Adi; Omar, Pacha; Aung, Phyu P; Torres-Cabala, Carlos A; Mays, Steven R; Prieto, Victor G; Curry, Jonathan L

    2017-02-01

    Immunotherapy targeting the programmed cell death 1 (PD-1) receptor has demonstrated tremendous promise in the treatment of advanced solid tumors. Dermatologic toxicities, however, are an emerging consequence of this therapy and have been clearly associated with immune checkpoint blockade antibodies. Distinctive clinical and histologic subtypes of dermatologic toxicity secondary to immunotherapy are emerging and include rare autoimmune bullous reactions (eg, bullous pemphigoid) and lichenoid eruptions. We report three patients who developed lichenoid dermatitis while receiving anti-PD-1 antibody therapy. The mean time to onset of lichenoid dermatologic toxicity was 42 days (range: 1-75 days) from initiation of anti-PD-1 antibody therapy. Lesions most frequently presented on the extremities and trunk as pustules, papules, and plaques. The face was not commonly involved. Of the five skin biopsies examined, all demonstrated dense band-like lymphocytic infiltrate, hyperkeratosis, hypergranulosis, saw-tooth rete ridge pattern, and dyskeratosis. Acanthosis was a feature in all of the skin biopsies, and in one, epidermal hyperplasia was prominent. In several skin biopsies, histologic features supporting a lichenoid drug eruption were present, including parakeratosis, spongiosis, periadnexal/perivascular inflammation, and eosinophils. Furthermore, the histologic features varied in skin biopsy specimens taken from the same patient at different sites, supporting a drug reaction. All patients' skin lesions improved with use of steroids: two were treated with topical steroids and one with systemic steroids. Recognition of the histopathologic patterns of dermatologic toxicities resulting from immune checkpoint blockade therapy will become increasingly important for ensuring appropriate management of dermatologic toxicities and optimal patient care.

  11. 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.

  12. The Possible Crosstalk of MOB2 With NDR1/2 Kinases in Cell Cycle and DNA Damage Signaling

    Science.gov (United States)

    Gundogdu, Ramazan; Hergovich, Alexander

    2017-01-01

    This article is the authors’ opinion of the roles of the signal transducer Mps one binder 2 (MOB2) in the control of cell cycle progression and the DNA Damage Response (DDR). We recently found that endogenous MOB2 is required to prevent the accumulation of endogenous DNA damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. In this regard, it is noteworthy that MOB2 has been linked biochemically to the regulation of the NDR1/2 (aka STK38/STK38L) protein kinases, which themselves have functions at different steps of the cell cycle. Therefore, we are speculating in this article about the possible connections of MOB2 with NDR1/2 kinases in cell cycle and DDR Signaling.

  13. 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

  14. Enterococcus faecalis phosphomevalonate kinase

    OpenAIRE

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone D...

  15. 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.

  16. A regimen combining the Wee1 inhibitor AZD1775 with HDAC inhibitors targets human acute myeloid leukemia cells harboring various genetic mutations.

    Science.gov (United States)

    Zhou, L; Zhang, Y; Chen, S; Kmieciak, M; Leng, Y; Lin, H; Rizzo, K A; Dumur, C I; Ferreira-Gonzalez, A; Dai, Y; Grant, S

    2015-04-01

    AZD1775 targets the cell cycle checkpoint kinase Wee1 and potentiates genotoxic agent cytotoxicity through p53-dependent or -independent mechanisms. Here, we report that AZD1775 interacted synergistically with histone deacetylase inhibitors (HDACIs, for example, Vorinostat), which interrupt the DNA damage response, to kill p53-wild type (wt) or -deficient as well as FLT3-ITD leukemia cells in association with pronounced Wee1 inhibition and diminished cdc2/Cdk1 Y15 phosphorylation. Similarly, Wee1 shRNA knockdown significantly sensitized cells to HDACIs. Although AZD1775 induced Chk1 activation, reflected by markedly increased Chk1 S296/S317/S345 phosphorylation leading to inhibitory T14 phosphorylation of cdc2/Cdk1, these compensatory responses were sharply abrogated by HDACIs. This was accompanied by premature mitotic entry, multiple mitotic abnormalities and accumulation of early S-phase cells displaying increased newly replicated DNA, culminating in robust DNA damage and apoptosis. The regimen was active against patient-derived acute myelogenous leukemia (AML) cells harboring either wt or mutant p53 and various next-generation sequencing-defined mutations. Primitive CD34(+)/CD123(+)/CD38(-) populations enriched for leukemia-initiating progenitors, but not normal CD34(+) hematopoietic cells, were highly susceptible to this regimen. Finally, combining AZD1775 with Vorinostat in AML murine xenografts significantly reduced tumor burden and prolonged animal survival. A strategy combining Wee1 with HDACI inhibition warrants further investigation in AML with poor prognostic genetic aberrations.

  17. When genome integrity and cell cycle decisions collide: roles of polo kinases in cellular adaptation to DNA damage.

    Science.gov (United States)

    Serrano, Diego; D'Amours, Damien

    2014-09-01

    The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.

  18. Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

    OpenAIRE

    Naiki, Takahiro; Kondo, Tae; Nakada, Daisuke; Matsumoto, Kunihiro; Sugimoto, Katsunori

    2001-01-01

    RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Δ mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that a...

  19. A comparative study of the aneugenic and polyploidy-inducing effects of fisetin and two model Aurora kinase inhibitors.

    Science.gov (United States)

    Gollapudi, P; Hasegawa, L S; Eastmond, D A

    2014-06-01

    Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. The dawn of Aurora kinase research: from fly genetics to the clinic.

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    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

  5. 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

  6. Using ergonomics checkpoints to support a participatory ergonomics intervention in an industrially developing country (IDC)--a case study.

    Science.gov (United States)

    Helali, Faramarz

    2009-01-01

    To achieve ergonomics awareness in 3 subsidiary companies, an intervention team was formed. The aims of this study were to implement basic ergonomics through a participatory ergonomics intervention process that can support a continuous learning process and lead to an improvement in health and safety as well as in the work systems in the organization. The findings of this study (i.e., method, continuous learning and integration) were key to making the participatory ergonomics intervention successful. Furthermore, 4 issues of the ergonomics checkpoints (i.e., work schedules, work tasks, healthy work organization and learning) for assessing the work system were found suitable for both changing work schedules and for improving the work system. This paper describes the result of this project and also the experiences gained and the conclusions reached from using the International Labour Office's ergonomics checkpoints in the industries of industrially developing country.

  7. Current status and perspectives in translational biomarker research for PD-1/PD-L1 immune checkpoint blockade therapy

    Directory of Open Access Journals (Sweden)

    Weijie Ma

    2016-05-01

    Full Text Available Abstract Modulating immune inhibitory pathways has been a major recent breakthrough in cancer treatment. Checkpoint blockade antibodies targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4 and programed cell-death protein 1 (PD-1 have demonstrated acceptable toxicity, promising clinical responses, durable disease control, and improved survival in some patients with advanced melanoma, non-small cell lung cancer (NSCLC, and other tumor types. About 20 % of advanced NSCLC patients and 30 % of advanced melanoma patients experience tumor responses from checkpoint blockade monotherapy, with better clinical responses seen with the combination of anti-PD-1 and anti-CTLA-4 antibodies. Given the power of these new therapies, it is important to understand the complex and dynamic nature of host immune responses and the regulation of additional molecules in the tumor microenvironment and normal organs in response to the checkpoint blockade therapies. In this era of precision oncology, there remains a largely unmet need to identify the patients who are most likely to benefit from immunotherapy, to optimize the monitoring assays for tumor-specific immune responses, to develop strategies to improve clinical efficacy, and to identify biomarkers so that immune-related adverse events can be avoided. At this time, PD-L1 immunohistochemistry (IHC staining using 22C3 antibody is the only FDA-approved companion diagnostic for patients with NSCLC-treated pembrolizumab, but more are expected to come to market. We here summarize the current knowledge, clinical efficacy, potential immune biomarkers, and associated assays for immune checkpoint blockade therapies in advanced solid tumors.

  8. 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.

  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. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    Science.gov (United States)

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  11. 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

    displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles......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...... 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...

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

    Science.gov (United States)

    Butt, A Q; Mills, K H G

    2014-09-18

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

  13. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    Energy Technology Data Exchange (ETDEWEB)

    Manet, Oliva-Trastoy; Berthonaud, V.; Chevalier, A.; Ducrot, C.; Marsolier-Kergoat, M.C.; Mann, C.; Leteurtre, F. [CEA Saclay, DSV, DBJC, SBGM, Lab. du Controle du Cycle Cellulaire, 91 - Gif-sur-Yvette (France)

    2006-07-01

    The DNA checkpoints are signal transduction pathways that sense DNA damage and coordinate various responses such as cell cycle arrests, DNA repair or cell death. These pathways are particularly well conserved in eukaryotes and the family of the 'Checkpoint Kinases 2' genes (or CHK2) plays a major role in them. This family includes the Rad53 protein of the yeast Saccharomyces cerevisiae and its Chk2 human homologue. Rad53 plays a central part in DNA checkpoint: rad53d mutants (whose RAD53 gene has been deleted) are hypersensitive to all genotoxic stresses. Mice Chk2-1- cells are defective in the G1, the intra-S, and the G2/M checkpoints. Mutations in CHK2 have been associated to many forms o f cancer, either sporadic or hereditary which demonstrates Chk2 tumor suppressor function. Chk2 proteins are characterized by several conserved elements: (i) an N-terminal domain with a series of SQ/TQ motifs, preferential phosphorylation sites for the ATM/ATR kinases, (ii) an FHA domain (ForkHead Associated) that binds specifically to phosphorylated residues within TXXY motifs (with the Y residue depending on the FHA domain and conferring an extra specificity) and (iii) a kinase domain including an activation loop. The Chk2 protein is activated by phosphorylation of its threonine T68, mainly by ATM, upon DNA double-strand breaks. This phosphorylation allows for the homo-dimerization of Chk2 through the binding of phospho-T68 from one molecule to the FHA domain of another molecule. It results in trans auto-phosphorylations, especially at threonines T383 and T387 in the activation T-loop. Fully active Chk2 becomes monomeric and, diffusing through the whole nucleus, phosphorylates its targets (CDC25 A and CDC25C/cell cycle arrest; p53, E2F, PML/apoptosis; BRCA2/DNA repair). Chk2/Rad53 inactivation occurs in two cases: once the DNA lesions have been repaired (it is called recovery) or, under certain conditions, in the presence of unrepaired DNA damage (it is then called

  14. 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

    2015-12-01

    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.

  15. IL-6 contributes to an immune tolerance checkpoint in post germinal center B cells.

    Science.gov (United States)

    Yan, Yi; Wang, Ying-Hua; Diamond, Betty

    2012-02-01

    The generation of a B cell repertoire involves producing and subsequently purging autoreactive B cells. Receptor editing, clonal deletion and anergy are key mechanisms of central B cell tolerance. Somatic mutation of antigen-activated B cells within the germinal center produces a second wave of autoreactivity; but the regulatory mechanisms that operate at this phase of B cell activation are poorly understood. We recently identified a post germinal center tolerance checkpoint, where receptor editing is re-induced to extinguish autoreactivity that is generated by somatic hypermutation. Re-induction of the recombinase genes RAG1 and RAG2 in antigen-activated B cells requires antigen to engage the B cell receptor and IL-7 to signal through the IL-7 receptor. We demonstrate that this process requires IL-6 to upregulate IL-7 receptor expression on post germinal center B cells. Diminishing IL-6 by blocking antibody or haplo-insufficiency leads to reduced expression of the IL-7 receptor and RAG and increased titers of anti-DNA antibodies following immunization with a peptide mimetope of DNA. The dependence on IL-6 to initiate receptor editing is B cell intrinsic. Interestingly, estradiol decreases IL-6 expression thereby increasing the anti-DNA response. Our data reveal a novel regulatory cascade to control post germinal center B cell autoreactivity.

  16. Management of adverse events related to new cancer immunotherapy (immune checkpoint inhibitors).

    Science.gov (United States)

    Bourke, Jack M; O'Sullivan, Michael; Khattak, Muhammad A

    2016-11-07

    New immunotherapies have significantly improved survival in certain advanced cancers in recent years, particularly metastatic melanoma and lung cancer. The most effective of these therapies are the immune checkpoint inhibitors (ICIs) such as ipilimumab, nivolumab and pembrolizumab. The use of ICIs will continue to increase in the coming years as evidence of their benefit in a range of other cancers builds. ICIs are associated with novel immune-related adverse events (irAEs), which can involve a wide range of organs. The most common irAEs involve the skin (rash, pruritus), gastrointestinal tract (diarrhoea, colitis) and endocrine system (thyroid, pituitary). While severity is generally mild, life-threatening complications can occur if not recognised and treated promptly. Due to the diverse manifestations of irAEs, patients may present to doctors who are not familiar with these drugs, which creates the potential for delays in management. Management of irAEs depends on severity and the organ affected. Systemic steroids are often required and ICI therapy may be withheld or discontinued. Additional immunosuppressive medications may be necessary in steroid-refractory cases. This review provides an overview of the potential toxicities and their management for general clinicians. Broader awareness of these issues among medical professionals will hopefully reduce unnecessary delays in diagnosis and treatment. Patient and carer education regarding irAEs is extremely important; patients and carers should be advised to seek urgent medical attention if required.

  17. Neurological adverse events associated with immune checkpoint inhibitors: Review of the literature.

    Science.gov (United States)

    Cuzzubbo, S; Javeri, F; Tissier, M; Roumi, A; Barlog, C; Doridam, J; Lebbe, C; Belin, C; Ursu, R; Carpentier, A F

    2017-03-01

    Immune checkpoint inhibitors (ICIs) targeting CTLA4 and PD1 constitute a promising class of cancer treatment but are associated with several immune-related disorders. We here review the literature reporting neurological adverse events (nAEs) associated with ICIs. A systematic search of literature, up to February 2016, mentioning nAEs in patients treated with ICIs was conducted. Eligible studies included case reports and prospective trials. One case seen in our ward was also added. Within the 59 clinical trials (totalling 9208 patients) analysed, the overall incidence of nAEs was 3.8% with anti-CTLA4 antibodies, 6.1% with anti-PD1 antibodies, and 12.0% with the combination of both. The clinical spectrum of neurological disorders was highly heterogeneous. Most of these nAEs were grade 1-2 and consisted of non-specific symptoms such as headache (55%). The incidence of high grade nAEs was below 1% for all types of treatment. Headaches, encephalopathies and meningitis were the most commonly reported (21%, 19% and 15%, respectively). Among the 27 case reports, the most common nAEs were encephalopathies, meningoradiculoneuritis, Guillain-Barré like syndromes and myasthenic syndromes. The median time of nAEs onset was 6 weeks. In most cases, drug interruption and steroids led to neurological recovery, even in conditions where steroids are not usually recommended such as Guillain-Barré syndrome.

  18. Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

    Science.gov (United States)

    Ganesan, Raja; Hos, Nina Judith; Gutierrez, Saray; Fischer, Julia; Stepek, Joanna Magdalena; Daglidu, Evmorphia; Krönke, Martin

    2017-01-01

    During intracellular infections, autophagy significantly contributes to the elimination of pathogens, regulation of pro-inflammatory signaling, secretion of immune mediators and in coordinating the adaptive immune system. Intracellular pathogens such as S. Typhimurium have evolved mechanisms to circumvent autophagy. However, the regulatory mechanisms targeted by S. Typhimurium to modulate autophagy have not been fully resolved. Here we report that cytosolic energy loss during S. Typhimurium infection triggers transient activation of AMPK, an important checkpoint of mTOR activity and autophagy. The activation of AMPK is regulated by LKB1 in a cytosolic complex containing Sirt1 and LKB1, where Sirt1 is required for deacetylation and subsequent activation of LKB1. S. Typhimurium infection targets Sirt1, LKB1 and AMPK to lysosomes for rapid degradation resulting in the disruption of the AMPK-mediated regulation of mTOR and autophagy. The degradation of cytosolic Sirt1/LKB1/AMPK complex was not observed with two mutant strains of S. Typhimurium, ΔssrB and ΔssaV, both compromising the pathogenicity island 2 (SPI2). The results highlight virulence factor-dependent degradation of host cell proteins as a previously unrecognized strategy of S. Typhimurium to evade autophagy. PMID:28192515

  19. 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...... in response to double strand breaks (DSBs) in G1, S and G2 phase in vivo. These foci are dependent on Mec3 (9-1-1 complex) as well as Rad24 (clamp loader), but independent of the HR protein Rad52. Nevertheless, these Dpb11 foci colocalize with Rad52 in S and G1 phase, and a single defined DSB is sufficient...... for recruitment. Also, the chicken homologue TopBP1 colocalizes with RPA1 as well as Rad51 when DNA damage is induced. Previously, dpb11 mutants have been shown to be sensitive to DNA-damaging agents that cause DSBs, DNA alkylation and stalled replication forks. Interestingly, we found the point mutants dpb11-PF...

  20. Absence of a conventional spindle mitotic checkpoint in the binucleated single-celled parasite Giardia intestinalis.

    Science.gov (United States)

    Markova, Kristyna; Uzlikova, Magdalena; Tumova, Pavla; Jirakova, Klara; Hagen, Guy; Kulda, Jaroslav; Nohynkova, Eva

    2016-10-01

    The spindle assembly checkpoint (SAC) joins the machinery of chromosome-to-spindle microtubule attachment with that of the cell cycle to prevent missegregation of chromosomes during mitosis. Although a functioning SAC has been verified in a limited number of organisms, it is regarded as an evolutionarily conserved safeguard mechanism. In this report, we focus on the existence of the SAC in a single-celled parasitic eukaryote, Giardia intestinalis. Giardia belongs to Excavata, a large and diverse supergroup of unicellular eukaryotes in which SAC control has been nearly unexplored. We show that Giardia cells with absent or defective mitotic spindles due to the inhibitory effects of microtubule poisons do not arrest in mitosis; instead, they divide without any delay, enter the subsequent cell cycle and even reduplicate DNA before dying. We identified a limited repertoire of kinetochore and SAC components in the Giardia genome, indicating that this parasite is ill equipped to halt mitosis before the onset of anaphase via SAC control of chromosome-spindle microtubule attachment. Finally, based on overexpression, we show that Giardia Mad2, a core SAC protein in other eukaryotes, localizes along intracytoplasmic portions of caudal flagellar axonemes, but never within nuclei, even in mitotic cells with blocked spindles, where the SAC should be active. These findings are consistent with the absence of a conventional SAC, known from yeast and metazoans, in the parasitic protist Giardia.

  1. Prussian blue nanoparticle-based photothermal therapy combined with checkpoint inhibition for photothermal immunotherapy of neuroblastoma.

    Science.gov (United States)

    Cano-Mejia, Juliana; Burga, Rachel A; Sweeney, Elizabeth E; Fisher, John P; Bollard, Catherine M; Sandler, Anthony D; Cruz, Conrad Russell Y; Fernandes, Rohan

    2017-02-01

    We describe "photothermal immunotherapy," which combines Prussian blue nanoparticle (PBNP)-based photothermal therapy (PTT) with anti-CTLA-4 checkpoint inhibition for treating neuroblastoma, a common, hard-to-treat pediatric cancer. PBNPs exhibit pH-dependent stability, which makes them suitable for intratumorally-administered PTT. PBNP-based PTT is able to lower tumor burden and prime an immune response, specifically an increased infiltration of lymphocytes and T cells to the tumor area, which is complemented by the antitumor effects of anti-CTLA-4 immunotherapy, providing a more durable treatment against neuroblastoma in an animal model. We observe 55.5% survival in photothermal immunotherapy-treated mice at 100days compared to 12.5%, 0%, 0%, and 0% survival in mice receiving: anti-CTLA-4 alone, PBNPs alone, PTT alone, and no treatment, respectively. Additionally, long-term surviving, photothermal immunotherapy-treated mice exhibit protection against neuroblastoma rechallenge, suggesting the development of immunity against these tumors. Our findings suggest the potential of photothermal immunotherapy in improving treatments for neuroblastoma.

  2. 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.

  3. 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.

  4. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

    Science.gov (United States)

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-03-03

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

  5. cGAS is essential for the antitumor effect of immune checkpoint blockade

    Science.gov (United States)

    Wang, Hua; Hu, Shuiqing; Chen, Xiang; Shi, Heping; Chen, Chuo; Sun, Lijun; Chen, Zhijian J.

    2017-01-01

    cGMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates innate immune responses. cGAS catalyzes the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce type I interferons (IFNs) and other immune modulatory molecules. Here we show that cGAS is indispensable for the antitumor effect of immune checkpoint blockade in mice. Wild-type, but not cGAS-deficient, mice exhibited slower growth of B16 melanomas in response to a PD-L1 antibody treatment. Consistently, intramuscular delivery of cGAMP inhibited melanoma growth and prolonged the survival of the tumor-bearing mice. The combination of cGAMP and PD-L1 antibody exerted stronger antitumor effects than did either treatment alone. cGAMP treatment activated dendritic cells and enhanced cross-presentation of tumor-associated antigens to CD8 T cells. These results indicate that activation of the cGAS pathway is important for intrinsic antitumor immunity and that cGAMP may be used directly for cancer immunotherapy. PMID:28137885

  6. 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

  7. The bacterial cell cycle checkpoint protein Obg and its role in programmed cell death

    Directory of Open Access Journals (Sweden)

    Liselot Dewachter

    2016-03-01

    Full Text Available The phenomenon of programmed cell death (PCD, in which cells initiate their own demise, is not restricted to multicellular organisms. Unicellular organisms, both eukaryotes and prokaryotes, also possess pathways that mediate PCD. We recently identified a PCD mechanism in Escherichia coli that is triggered by a mutant isoform of the essential GTPase ObgE (Obg of E. coli. Importantly, the PCD pathway mediated by mutant Obg (Obg* differs fundamentally from other previously described bacterial PCD pathways and thus constitutes a new mode of PCD. ObgE was previously proposed to act as a cell cycle checkpoint protein able to halt cell division. The implication of ObgE in the regulation of PCD further increases the similarity between this protein and eukaryotic cell cycle regulators that are capable of doing both. Moreover, since Obg is conserved in eukaryotes, the elucidation of this cell death mechanism might contribute to the understanding of PCD in higher organisms. Additionally, if Obg*-mediated PCD is conserved among different bacterial species, it will be a prime target for the development of innovative antibacterials that artificially induce this pathway.

  8. 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.

  9. 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.

  10. Spindle assembly checkpoint signalling is uncoupled from chromosomal position in mouse oocytes.

    Science.gov (United States)

    Gui, Liming; Homer, Hayden

    2012-06-01

    The spindle assembly checkpoint (SAC) averts aneuploidy by coordinating proper bipolar chromosomal attachment with anaphase-promoting complex/cyclosome (APC/C)-mediated securin and cyclin B1 destruction required for anaphase onset. The generation of a Mad2-based signal at kinetochores is central to current models of SAC-based APC/C inhibition. During mitosis, kinetochores of polar-displaced chromosomes, which are at greatest risk of mis-segregating, recruit the highest levels of Mad2, thereby ensuring that SAC activation is proportionate to aneuploidy risk. Paradoxically, although an SAC operates in mammalian oocytes, meiosis I (MI) is notoriously error prone and polar-displaced chromosomes do not prevent anaphase onset. Here we find that Mad2 is not preferentially recruited to the kinetochores of polar chromosomes of wild-type mouse oocytes, in which polar chromosomes are rare, or of oocytes depleted of the kinesin-7 motor CENP-E, in which polar chromosomes are more abundant. Furthermore, in CENP-E-depleted oocytes, although polar chromosomal displacement intensified during MI and the capacity to form stable end-on attachments was severely compromised, all kinetochores nevertheless became devoid of Mad2. Thus, it is possible that the ability of the SAC to robustly discriminate chromosomal position might be compromised by the propensity of oocyte kinetochores to become saturated with unproductive attachments, thereby predisposing to aneuploidy. Our data also reveal novel functions for CENP-E in oocytes: first, CENP-E stabilises BubR1, thereby impacting MI progression; and second, CENP-E mediates bi-orientation by promoting kinetochore reorientation and preventing chromosomal drift towards the poles.

  11. MYSM1-dependent checkpoints in B cell lineage differentiation and B cell-mediated immune response.

    Science.gov (United States)

    Förster, Michael; Farrington, Kyo; Petrov, Jessica C; Belle, Jad I; Mindt, Barbara C; Witalis, Mariko; Duerr, Claudia U; Fritz, Jörg H; Nijnik, Anastasia

    2017-03-01

    MYSM1 is a chromatin-binding histone deubiquitinase. MYSM1 mutations in humans result in lymphopenia whereas loss of Mysm1 in mice causes severe hematopoietic abnormalities, including an early arrest in B cell development. However, it remains unknown whether MYSM1 is required at later checkpoints in B cell development or for B cell-mediated immune responses. We analyzed conditional mouse models Mysm1(fl/fl)Tg.mb1-cre, Mysm1(fl/fl)Tg.CD19-cre, and Mysm1(fl/fl)Tg.CD21-cre with inactivation of Mysm1 at prepro-B, pre-B, and follicular B cell stages of development. We show that loss of Mysm1 at the prepro-B cell stage in Mysm1(fl/fl)Tg.mb1-cre mice results in impaired B cell differentiation, with an ∼2-fold reduction in B cell numbers in the lymphoid organs. Mysm1(fl/fl)Tg.mb1-cre B cells also showed increased expression of activation markers and impaired survival and proliferation. In contrast, Mysm1 was largely dispensable from the pre-B cell stage onward, with Mysm1(fl/fl)Tg.CD19-cre and Mysm1(fl/fl)Tg.CD21-cre mice showing no alterations in B cell numbers and largely normal responses to stimulation. MYSM1, therefore, has an essential role in B cell lineage specification but is dispensable at later stages of development. Importantly, MYSM1 activity at the prepro-B cell stage of development is important for the normal programming of B cell responses to stimulation once they complete their maturation process.

  12. How oocytes try to get it right: spindle checkpoint control in meiosis.

    Science.gov (United States)

    Touati, Sandra A; Wassmann, Katja

    2016-06-01

    The generation of a viable, diploid organism depends on the formation of haploid gametes, oocytes, and spermatocytes, with the correct number of chromosomes. Halving the genome requires the execution of two consecutive specialized cell divisions named meiosis I and II. Unfortunately, and in contrast to male meiosis, chromosome segregation in oocytes is error prone, with human oocytes being extraordinarily "meiotically challenged". Aneuploid oocytes, that are with the wrong number of chromosomes, give rise to aneuploid embryos when fertilized. In humans, most aneuploidies are lethal and result in spontaneous abortions. However, some trisomies survive to birth or even adulthood, such as the well-known trisomy 21, which gives rise to Down syndrome (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012). A staggering 20-25 % of oocytes ready to be fertilized are aneuploid in humans. If this were not bad enough, there is an additional increase in meiotic missegregations as women get closer to menopause. A woman above 40 has a risk of more than 30 % of getting pregnant with a trisomic child. Worse still, in industrialized western societies, child birth is delayed, with women getting their first child later in life than ever. This trend has led to an increase of trisomic pregnancies by 70 % in the last 30 years (Nagaoka et al. in Nat Rev Genet 13:493-504, 2012; Schmidt et al. in Hum Reprod Update 18:29-43, 2012). To understand why errors occur so frequently during the meiotic divisions in oocytes, we review here the molecular mechanisms at works to control chromosome segregation during meiosis. An important mitotic control mechanism, namely the spindle assembly checkpoint or SAC, has been adapted to the special requirements of the meiotic divisions, and this review will focus on our current knowledge of SAC control in mammalian oocytes. Knowledge on how chromosome segregation is controlled in mammalian oocytes may help to identify risk factors important for questions

  13. Checkpoint modulation - A new way to direct the immune system against renal cell carcinoma

    OpenAIRE

    Bedke, Jens; Kruck, Stephan; Gakis, Georgios; Stenzl, Arnulf; Goebell, Peter J.

    2015-01-01

    The introduction of targeted therapies like the tyrosine kinase (TKI) and mammalian target of rapamycin (mTOR) inhibitors has improved patients´ survival in general. Nevertheless the prognosis remains limited. Therapies with a new mode of action are urgently warranted, especially those who would provoke long-term responders or long-lasting complete remissions as observed with unspecific immunotherapy with the cytokines interleukin-2 and interferon-α. In the recent years a deeper understanding...

  14. Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency.

    Science.gov (United States)

    Sanvisens, Nerea; Romero, Antonia M; Zhang, Caiguo; Wu, Xiaorong; An, Xiuxiang; Huang, Mingxia; Puig, Sergi

    2016-04-29

    Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function.

  15. 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.

  16. Cellular and biomolecular responses of human ovarian cancer cells to cytostatic dinuclear platinum(II) complexes.

    Science.gov (United States)

    Lin, Miaoxin; Wang, Xiaoyong; Zhu, Jianhui; Fan, Damin; Zhang, Yangmiao; Zhang, Junfeng; Guo, Zijian

    2011-03-01

    Polynuclear platinum(II) complexes represent a class of potential anticancer agents that have shown promising pharmacological properties in preclinical studies. The nature of cellular responses induced by these complexes, however, is poorly understood. In this research, the cellular responses of human ovarian cancer COC1 cells to dinuclear platinum(II) complexes {[cis-Pt(NH₃)₂Cl]₂L¹}(NO₃)₂ (1) and {[cis-Pt(NH₃)₂Cl]₂L²}(NO₃)₂ (2) (L¹ = α,α'-diamino-p-xylene, L² = 4,4'-methylenedianiline) has been studied using cisplatin as a reference. The effect of platinum complexes on the proliferation, death mode, mitochondrial membrane potential, and cell cycle progression has been examined by MTT assay and flow cytometry. The activation of cell cycle checkpoint kinases (CHK1/2), extracellular signal-regulated kinases (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK) of the cells by the complexes has also been analyzed using phospho-specific flow cytometry. Complex 1 is more cytotoxic than complex 2 and cisplatin at most concentrations; complex 2 and cisplatin are comparably cytotoxic. These complexes kill the cells through an apoptotic or apoptosis-like pathway characterized by exposure of phosphatidylserine and dissipation of mitochondrial membrane potential. Complex 1 shows the strongest inductive effect on the morphological changes of the cells, followed by cisplatin and complex 2. Complexes 1 and 2 arrest the cell cycle in G2 or M phase, while cisplatin arrests the cell cycle in S phase. The influence of these complexes on CHK1/2, ERK1/2, and p38 MAPK varies with the dose of the drugs or reaction time. Activation of phospho-ERK1/2 and phospho-p38 MAPK by these complexes is closely related to the cytostatic activity. The results demonstrate that dinuclear platinum(II) complexes can induce some cellular responses different from those caused by cisplatin.

  17. Immunotherapy for the treatment of breast cancer: checkpoint blockade, cancer vaccines, and future directions in combination immunotherapy.

    Science.gov (United States)

    McArthur, Heather L; Page, David B

    2016-11-01

    Immunotherapy encompasses both vaccines that direct immune responses to tumor-associated antigens, and checkpoint blocking antibodies that inhibit immune system suppression by targeting key pathways mediated by cytotoxic T-lymphocyte-associated antigen 4, programmed death 1 (PD-1), and programmed death ligand 1 (PD-L1). Both of these approaches currently are being explored as potential strategies for the treatment of breast cancer. Recent studies suggest that immunotherapy is poised to change the therapeutic landscape for some breast cancers. Specifically, overall response rates of 19% with PD-1/PD-L1-directed antibodies have been reported in 2 small studies of women with PD-L1-positive, heavily pretreated advanced triple-negative breast cancer. In combination with nab-paclitaxel, confirmed response rates were 46% in a PD-L1-unselected population in the first-line metastatic triple-negative breast cancer setting. Checkpoint-blocking antibodies also have been evaluated in small studies of women with hormone receptor-positive metastatic breast cancer, and in women whose breast cancers lack PD-L1 expression, with more modest response rates. It has been hypothesized that some breast cancers are not inherently recognized by the immune system; however, preclinical and preliminary clinical data suggest that inherently modest immunogenicity may be overcome with novel vaccination strategies, as well as strategies that combine immune checkpoint blockade with methods of optimizing antigen presentation, such as tumor ablation, radiation, chemotherapy, or other approaches. If ongoing registrational trials support the use of immunotherapy, it could revolutionize the care of early-stage and metastatic breast cancer, and ideally improve cure rates.

  18. Yeast Dam1p is required to maintain spindle integrity during mitosis and interacts with the Mps1p kinase.

    Science.gov (United States)

    Jones, M H; Bachant, J B; Castillo, A R; Giddings, T H; Winey, M

    1999-07-01

    We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.

  19. 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.

  20. ZTF-8 interacts with the 9-1-1 complex and is required for DNA damage response and double-strand break repair in the C. elegans germline.

    Directory of Open Access Journals (Sweden)

    Hyun-Min Kim

    2014-10-01

    Full Text Available Germline mutations in DNA repair genes are linked to tumor progression. Furthermore, failure in either activating a DNA damage checkpoint or repairing programmed meiotic double-strand breaks (DSBs can impair chromosome segregation. Therefore, understanding the molecular basis for DNA damage response (DDR and DSB repair (DSBR within the germline is highly important. Here we define ZTF-8, a previously uncharacterized protein conserved from worms to humans, as a novel factor involved in the repair of both mitotic and meiotic DSBs as well as in meiotic DNA damage checkpoint activation in the C. elegans germline. ztf-8 mutants exhibit specific sensitivity to γ-irradiation and hydroxyurea, mitotic nuclear arrest at S-phase accompanied by activation of the ATL-1 and CHK-1 DNA damage checkpoint kinases, as well as accumulation of both mitotic and meiotic recombination intermediates, indicating that ZTF-8 functions in DSBR. However, impaired meiotic DSBR progression partially fails to trigger the CEP-1/p53-dependent DNA damage checkpoint in late pachytene, also supporting a role for ZTF-8 in meiotic DDR. ZTF-8 partially co-localizes with the 9-1-1 DDR complex and interacts with MRT-2/Rad1, a component of this complex. The human RHINO protein rescues the phenotypes observed in ztf-8 mutants, suggesting functional conservation across species. We propose that ZTF-8 is involved in promoting repair at stalled replication forks and meiotic DSBs by transducing DNA damage checkpoint signaling via the 9-1-1 pathway. Our findings define a conserved function for ZTF-8/RHINO in promoting genomic stability in the germline.

  1. An RNA Polymerase II-coupled function for histone H3K36 methylation in checkpoint activation and DSB repair

    OpenAIRE

    Jha, Deepak Kumar; Brian D Strahl

    2014-01-01

    Histone modifications are major determinants of DNA double-strand break (DSB) response and repair. Here we elucidate a DSB repair function for transcription-coupled Set2 methylation at H3 lysine 36 (H3K36me). Cells devoid of Set2/H3K36me are hypersensitive to DNA-damaging agents and site-specific DSBs, fail to properly activate the DNA-damage checkpoint, and show genetic interactions with DSB-sensing and repair machinery. Set2/H3K36me3 is enriched at DSBs, and loss of Set2 results in altered ...

  2. 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.

  3. Mode of ATM-dependent suppression of chromosome translocation.

    Science.gov (United States)

    Yamauchi, Motohiro; Suzuki, Keiji; Oka, Yasuyoshi; Suzuki, Masatoshi; Kondo, Hisayoshi; Yamashita, Shunichi

    2011-12-09

    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.

  4. 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......Blockade of checkpoint inhibitors has recently shown very convincing results in the treatment of cancer. One key target is CTLA-4, which has been demonstrated to be a potent negative regulator of lymphocyte activation. The treatment with the FDA-approved fully human CTLA-4 monoclonal antibody...... Ipilimumab increases anticancer T-cell reactivity and overall survival of metastatic cancer patients. Indole-amine 2,3-dioxygenase (IDO) is another checkpoint inhibitor which suppresses T-cell immunity by the depletion of tryptophan in the T-cell microenvironment, and also inhibition of IDO by L-1...

  5. 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.

  6. 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.

  7. DNA injury induced by 5-aminouracil and caffeine in G2 checkpoints path of higher plant cells.

    Science.gov (United States)

    Del Campo, A; Bracho, M; Marcano, L; Guíñez, J; De la Torre, C

    2005-08-01

    This work evaluated the qualitative and quantitative cellular changes induced by treatment with 5-aminouracil (5-AU) and a combination of 5-AU and caffeine in plant cells in relation to DNA damage, repaired damage, and residual damage. As biological material, Allium cepa L. root tips were used, grown in filtered water, in darkness, with aeration at constant temperature of 25 degrees C +/- 0.5. Cell populations were synchronized using 5 mM caffeine in order to study the effects of 5-AU and caffeine/5-AU combined treatment on the DNA content and their incidence in the entrance to mitosis. The results showed a delay in the G2 period due to induced DNA damage by the 5-AU and caffeine/5-AU combined treatment, shown by aberrant metaphases, anaphases and telophases. The effect of caffeine in the combined treatment was heightened in spite of lengthening the checkpoints route that retains the cells in G2. The existence of G2 checkpoints was shown in the cell population studied, inducing lesions in the DNA, chromosomic aberrations and cellular instability.

  8. 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

  9. Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage, Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

    Directory of Open Access Journals (Sweden)

    Lucy H. Swift

    2014-02-01

    Full Text Available When a human cell detects damaged DNA, it initiates the DNA damage response (DDR that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.

  10. Rheumatic immune-related adverse events of checkpoint therapy for cancer: case series of a new nosological entity

    Science.gov (United States)

    Calabrese, C; Kirchner, E; Kontzias, K; Velcheti, V; Calabrese, L H

    2017-01-01

    Immunotherapy of cancer with checkpoint inhibitors has been associated with a spectrum of autoimmune and systemic inflammatory reactions known as immune-related adverse events (irAEs). Rheumatic irAEs are infrequently reported and extensively described. Here, we report our experience over an 18-month period with 15 patients evaluated in the rheumatology department for rheumatic irAEs. We identified 13 patients without pre-existing autoimmune disease (AID) who subsequently developed rheumatic irAEs, and two with established AID referred pre-emptively. irAEs encountered included: inflammatory arthritis, sicca syndrome, polymyalgia rheumatica-like symptoms and myositis. All cases required glucocorticoids, and three required a biological agent. Rheumatic irAEs led to temporary or permanent cessation of immunotherapy in all but five patients. One patient with pre-existing AID experienced a flare after starting immunotherapy. Our findings underscore that rheumatic irAEs are complex, at times require additional immunosuppressive therapy, and may influence ongoing immunotherapy regimens for the primary disease. Similar irAEs will be increasingly seen as checkpoint inhibitors adopted as standard of care in the community.

  11. 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

  12. Enhanced NF-κB activation and cellular function in macrophages lacking IκB kinase 1 (IKK1)

    Science.gov (United States)

    Li, Qiutang; Lu, Qingxian; Bottero, Virginie; Estepa, Gabriela; Morrison, Lisa; Mercurio, Frank; Verma, Inder M.

    2005-01-01

    IκB kinase (IKK) complex plays a key regulatory role in macrophages for NF-κB activation during both innate and adaptive immune responses. Because IKK1–/– mice died at birth, we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria, more efficient antigen-presenting capacity, elevated secretion of several key proinflammatory cytokines and chemokines, and known NFκB target genes. Increased NFκB activity in IKK1 mutant ELDM was the result of prolonged degradation of IκBα in response to infectious pathogens. The delayed restoration of IκBα in pathogen-activated IKK1–/– ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IκBα kinase activity in innate and adaptive immunity. PMID:16116086

  13. A kinome-wide RNAi screen in Drosophila Glia reveals that the RIO kinases mediate cell proliferation and survival through TORC2-Akt signaling in glioblastoma.

    Directory of Open Access Journals (Sweden)

    Renee D Read

    Full Text Available Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK and Pi-3 kinase (PI3K signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

  14. Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals.

    Science.gov (United States)

    Gao, Xueliang; Wang, Haizhen; Yang, Jenny J; Chen, Jing; Jie, Jiang; Li, Liangwei; Zhang, Yinwei; Liu, Zhi-Ren

    2013-05-31

    Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

  15. Sertraline, an antidepressant, induces apoptosis in hepatic cells through the mitogen-activated protein kinase pathway.

    Science.gov (United States)

    Chen, Si; Xuan, Jiekun; Wan, Liqing; Lin, Haixia; Couch, Letha; Mei, Nan; Dobrovolsky, Vasily N; Guo, Lei

    2014-02-01

    Sertraline is generally used for the treatment of depression and is also approved for the treatment of panic, obsessive-compulsive, and posttraumatic stress disorders. Previously, using rat primary hepatocytes and isolated mitochondria, we demonstrated that sertraline caused hepatic cytotoxicity and mitochondrial impairment. In the current study, we investigated and characterized molecular mechanisms of sertraline toxicity in human hepatoma HepG2 cells. Sertraline decreased cell viability and induced apoptosis in a dose- and time-dependent manner. Sertraline activated the intrinsic checkpoint protein caspase-9 and caused the release of cytochrome c from mitochondria to cytosol; this process was Bcl-2 family dependent because antiapoptotic Bcl-2 family proteins were decreased. Pretreatment of the HepG2 cells with caspase-3, caspase-8, and caspase-9 inhibitors partially but significantly reduced the release of lactate dehydrogenase, indicating that sertraline-induced apoptosis is mediated by both intrinsic and extrinsic apoptotic pathways. Moreover, sertraline markedly increased the expression of tumor necrosis factor (TNF) and the phosphorylation of JNK, extracellular signal-regulated kinase (ERK1/2), and p38. In sertraline-treated cells, the induction of apoptosis and cell death was shown to be the result of activation of JNK, but not ERK1/2 or p38 in the mitogen-activated protein kinase (MAPK) pathway. Furthermore, silencing MAP4K4, the upstream kinase of JNK, attenuated both apoptosis and cell death caused by sertraline. Taken together, our findings suggest that sertraline induced apoptosis in HepG2 cells at least partially via activation of the TNF-MAP4K4-JNK cascade signaling pathway.

  16. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  17. 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.

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

    Science.gov (United States)

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

    2010-12-01

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

  19. 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

  20. 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.

  1. Deferasirox in pyruvate kinase deficiency

    OpenAIRE

    Deeren, Dries

    2008-01-01

    Deferasirox in pyruvate kinase deficiency phone: +32-51-237437 (Deeren, Dries) (Deeren, Dries) Department of Haematology, Heilig-Hartziekenhuis Roeselare-Menen vzw - Wilgenstraat 2 - B-8800 - Roeselare - BELGIUM (Deeren, Dries) BELGIUM Registration: 2008-09-10 Received: 2008-09-05 Accepted: 2008-09-10 ePublished: 2008-09-23

  2. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  3. 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

  4. Identification of potential Plk1 targets in a cell-cycle specific proteome through structural dynamics of kinase and Polo box-mediated interactions.

    Directory of Open Access Journals (Sweden)

    Nousheen Bibi

    Full Text Available Polo like kinase 1 (Plk1 is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD. To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets.

  5. Identification of Potential Plk1 Targets in a Cell-Cycle Specific Proteome through Structural Dynamics of Kinase and Polo Box-Mediated Interactions

    Science.gov (United States)

    Bibi, Nousheen; Parveen, Zahida; Rashid, Sajid

    2013-01-01

    Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets. PMID:23967120

  6. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    OpenAIRE

    WANG Ling; Fisher, Laura A.; Wahl, James K.; Peng, Aimin

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that pla...

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

    Science.gov (United States)

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

    2017-03-28

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

  8. Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice.

    Science.gov (United States)

    Yun, Yan; Holt, Janet E; Lane, Simon I R; McLaughlin, Eileen A; Merriman, Julie A; Jones, Keith T

    2014-01-01

    Currently, maternal aging in women, based on mouse models, is thought to raise oocyte aneuploidy rates, because chromosome cohesion deteriorates during prophase arrest, and Sgo2, a protector of centromeric cohesion, is lost. Here we show that the most common mouse strain, C57Bl6/J, is resistant to maternal aging, showing little increase in aneuploidy or Sgo2 loss. Instead it demonstrates significant kinetochore-associated loss in the spindle assembly checkpoint