"Isogaba Maware": quality control of genome DNA by checkpoints.

PubMed

Kitazono, A; Matsumoto, T

1998-05-01

Checkpoints maintain the interdependency of cell cycle events by permitting the onset of an event only after the completion of the preceding event. The DNA replication checkpoint induces a cell cycle arrest until the completion of the DNA replication. Similarly, the DNA damage checkpoint arrests cell cycle progression if DNA repair is incomplete. A number of genes that play a role in the two checkpoints have been identified through genetic studies in yeasts, and their homologues have been found in fly, mouse, and human. They form signaling cascades activated by a DNA replication block or DNA damage and subsequently generate the negative constraints on cell cycle regulators. The failure of these signaling cascades results in producing offspring that carry mutations or that lack a portion of the genome. In humans, defects in the checkpoints are often associated with cancer-prone diseases. Focusing mainly on the studies in budding and fission yeasts, we summarize the recent progress.

The human intra-S checkpoint response to UVC-induced DNA damage.

PubMed

Kaufmann, William K

2010-05-01

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

DNA damage checkpoint recovery and cancer development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong, E-mail: lsteng@zju.edu.cn

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 observedmore » 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 recovery pathway plays a role in overriding tumor barrier in tumorigenesis. • Recovery protein dysregulation and human cancer development is correlated.« less

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

NASA Technical Reports Server (NTRS)

Wang, Yi-Min

1993-01-01

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

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.

PubMed

van Brabant, A J; Buchanan, C D; Charboneau, E; Fangman, W L; Brewer, B J

2001-04-01

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.

A TPR domain–containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

PubMed Central

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H.J.; Snel, Berend

2013-01-01

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B–dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. PMID:23569217

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

PubMed

Nijenhuis, Wilco; von Castelmur, Eleonore; Littler, Dene; De Marco, Valeria; Tromer, Eelco; Vleugel, Mathijs; van Osch, Maria H J; Snel, Berend; Perrakis, Anastassis; Kops, Geert J P L

2013-04-15

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Roles of nibrin and AtM/ATR kinases on the G2 checkpoint under endogenous or radio-induced DNA damage.

PubMed

Marcelain, Katherine; De La Torre, Consuelo; González, Patricio; Pincheira, Juana

2005-01-01

Checkpoint response to DNA damage involves the activation of DNA repair and G2 lengthening subpathways. The roles of nibrin (NBS1) and the ATM/ATR kinases in the G2 DNA damage checkpoint, evoked by endogenous and radio-induced DNA damage, were analyzed in control, A-T and NBS lymphoblast cell lines. Short-term responses to G2 treatments were evaluated by recording changes in the yield of chromosomal aberrations in the ensuing mitosis, due to G2 checkpoint adaptation, and also in the duration of G2 itself. The role of ATM/ATR in the G2 checkpoint pathway repairing chromosomal aberrations was unveiled by caffeine inhibition of both kinases in G2. In the control cell lines, nibrin and ATM cooperated to provide optimum G2 repair for endogenous DNA damage. In the A-T cells, ATR kinase substituted successfully for ATM, even though no G2 lengthening occurred. X-ray irradiation (0.4 Gy) in G2 increased chromosomal aberrations and lengthened G2, in both mutant and control cells. However, the repair of radio-induced DNA damage took place only in the controls. It was associated with nibrin-ATM interaction, and ATR did not substitute for ATM. The absence of nibrin prevented the repair of both endogenous and radio-induced DNA damage in the NBS cells and partially affected the induction of G2 lengthening.

Eukaryotic Initiation Factor 4E Binding Protien Family of Protiens: Sentinels at a Translational Control Checkpoint in Lung Tumor Defense

PubMed Central

Kim, Yong Y; Von Weymarn, Linda; Larsson, Ola; Fan, Danhua; Underwood, Jon M; Hecht, Stephen S; Polunovsky, Vitaly A; Bitterman, Peter B

2009-01-01

The usurping of translational control by sustained activation of translation initiation factors is oncogenic. Here we show that the primary negative regulators of these oncogenic initiation factors - the 4E-BP protein family - operate as guardians of a translational control checkpoint in lung tumor defense. When challenged with the tobacco carcinogen NNK, 4ebp1−/−/4ebp2−/− mice showed increased sensitivity to tumorigenesis compared to their wild type counterparts. The 4E-BP deficient state per se creates pro-oncogenic, genome-wide skewing of the molecular landscape - with translational activation of genes governing angiogenesis, growth and proliferation; and translational activation of the precise cytochrome p450 enzyme isoform (CYP2A5) that bioactivates NNK into mutagenic metabolites. Our study provides in vivo proof for a translational control checkpoint in lung tumor defense. PMID:19843855

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

PubMed Central

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

1999-01-01

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling. Images Figure 3 PMID:10229703

Accuracy Investigation of Creating Orthophotomaps Based on Images Obtained by Applying Trimble-UX5 UAV

NASA Astrophysics Data System (ADS)

Hlotov, Volodymyr; Hunina, Alla; Siejka, Zbigniew

2017-06-01

The main purpose of this work is to confirm the possibility of making largescale orthophotomaps applying unmanned aerial vehicle (UAV) Trimble- UX5. A planned altitude reference of the studying territory was carried out before to the aerial surveying. The studying territory has been marked with distinctive checkpoints in the form of triangles (0.5 × 0.5 × 0.2 m). The checkpoints used to precise the accuracy of orthophotomap have been marked with similar triangles. To determine marked reference point coordinates and check-points method of GNSS in real-time kinematics (RTK) measuring has been applied. Projecting of aerial surveying has been done with the help of installed Trimble Access Aerial Imaging, having been used to run out the UX5. Aerial survey out of the Trimble UX5 UAV has been done with the help of the digital camera SONY NEX-5R from 200m and 300 m altitude. These aerial surveying data have been calculated applying special photogrammetric software Pix 4D. The orthophotomap of the surveying objects has been made with its help. To determine the precise accuracy of the got results of aerial surveying the checkpoint coordinates according to the orthophotomap have been set. The average square error has been calculated according to the set coordinates applying GNSS measurements. A-priori accuracy estimation of spatial coordinates of the studying territory using the aerial surveying data have been calculated: mx=0.11 m, my=0.15 m, mz=0.23 m in the village of Remeniv and mx=0.26 m, my=0.38 m, mz=0.43 m in the town of Vynnyky. The accuracy of determining checkpoint coordinates has been investigated using images obtained out of UAV and the average square error of the reference points. Based on comparative analysis of the got results of the accuracy estimation of the made orthophotomap it can be concluded that the value the average square error does not exceed a-priori accuracy estimation. The possibility of applying Trimble UX5 UAV for making large-scale orthophotomaps has been investigated. The aerial surveying output data using UAV can be applied for monitoring potentially dangerous for people objects, the state border controlling, checking out the plots of settlements. Thus, it is important to control the accuracy the got results. Having based on the done analysis and experimental researches it can be concluded that applying UAV gives the possibility to find data more efficiently in comparison with the land surveying methods. As the result, the Trimble UX5 UAV gives the possibility to survey built-up territories with the required accuracy for making orthophotomaps with the following scales 1: 2000, 1: 1000, 1: 500.

Epigenetic inactivation of CHFR in human tumors

PubMed Central

Toyota, Minoru; Sasaki, Yasushi; Satoh, Ayumi; Ogi, Kazuhiro; Kikuchi, Takefumi; Suzuki, Hiromu; Mita, Hiroaki; Tanaka, Nobuyuki; Itoh, Fumio; Issa, Jean-Pierre J.; Jair, Kam-Wing; Schuebel, Kornel E.; Imai, Kohzoh; Tokino, Takashi

2003-01-01

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected. PMID:12810945

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

PubMed Central

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

1999-01-01

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsBrad3 and uvsDrad26 have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIMEcyclinB, but ectopic expression of active nondegradable NIMEcyclinB does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMXcdc2 and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells. PMID:10564263

Cid1, a Fission Yeast Protein Required for S-M Checkpoint Control when DNA Polymerase δ or ɛ Is Inactivated

PubMed Central

Wang, Shao-Win; Toda, Takashi; MacCallum, Robert; Harris, Adrian L.; Norbury, Chris

2000-01-01

The S-M checkpoint is an intracellular signaling pathway that ensures that mitosis is not initiated in cells undergoing DNA replication. We identified cid1, a novel fission yeast gene, through its ability when overexpressed to confer specific resistance to a combination of hydroxyurea, which inhibits DNA replication, and caffeine, which overrides the S-M checkpoint. Cid1 overexpression also partially suppressed the hydroxyurea sensitivity characteristic of DNA polymerase δ mutants and mutants defective in the “checkpoint Rad” pathway. Cid1 is a member of a family of putative nucleotidyltransferases including budding yeast Trf4 and Trf5, and mutation of amino acid residues predicted to be essential for this activity resulted in loss of Cid1 function in vivo. Two additional Cid1-like proteins play similar but nonredundant checkpoint-signaling roles in fission yeast. Cells lacking Cid1 were found to be viable but specifically sensitive to the combination of hydroxyurea and caffeine and to be S-M checkpoint defective in the absence of Cds1. Genetic data suggest that Cid1 acts in association with Crb2/Rhp9 and through the checkpoint-signaling kinase Chk1 to inhibit unscheduled mitosis specifically when DNA polymerase δ or ɛ is inhibited. PMID:10757807

Clinical trial aims to study immunotherapy for central nervous system tumors | Center for Cancer Research

Cancer.gov

A new clinical trial aims to determine whether nivolumab, an immune checkpoint inhibitor, can improve control of cancer for patients with several types of tumors of the central nervous system (CNS). The CNS is composed of the brain and spinal cord and the cause of most CNS tumors in adults is unknown. Learn more...

The problem of suspended and revoked drivers who avoid detection at checkpoints.

PubMed

Parrish, Kelly E; Masten, Scott V

2015-01-01

Although driver license suspension and revocation have been shown to improve traffic safety, suspended or revoked (SR) drivers who continue to drive-which appears to be the majority-are about 3 times more likely to be involved in crashes and to cause a fatal crash. In California and many other U.S. states, drivers are typically mailed notices requesting that they surrender their licenses when they are SR for reasons other than driving under the influence of alcohol or drugs (DUI), yet they frequently do not comply. Typical procedures at DUI checkpoints in California and other U.S. states include inspecting driver licenses and checking for signs of intoxication during brief contacts with law enforcement officers. Hence, these checkpoints are in fact DUI/license checkpoints in California and many other states. The purpose of this study was to estimate the extent to which SR drivers avoid being detected at DUI/license checkpoints for SR driving, because they illegally retained possession of their license cards. Law enforcement officers used electronic license card readers at DUI/license checkpoints in Sacramento, California, to record data for 13,705 drivers. The SR status of all contacted drivers was determined after the checkpoints and compared to law enforcement citation records from the checkpoints. Although only 3% of the drivers contacted at the checkpoints were SR, about 41% of SR drivers were able to pass through undetected because they presented license cards that they illegally retained. Drivers SR for DUI-related reasons were more likely to be detected, whereas those SR for failure to provide proof of financial responsibility (insurance) were less likely to be detected. The fact that many SR drivers are able to pass through DUI/license checkpoints undetected weakens both the specific and general impacts of checkpoints for deterring SR driving and may diminish the effectiveness of suspension and revocation actions for reducing the crash risk posed by problem drivers. Using license card readers that can quickly identify SR drivers in real time during routine traffic stops and at DUI/license checkpoints warrants further consideration.

Topoisomerase II Inhibitors and Poisons, and the Influence of Cell Cycle Checkpoints.

PubMed

D Arcy, Nicholas; Gabrielli, Brian

2017-01-01

Interactions between the decatenation checkpoint and Topoisomerase II (TopoII) are vital for maintaining integrity of the genome. Agents that target this enzyme have been in clinical use in cancer therapy for over 30 years with great success. The types of compounds that have been developed to target TopoII are broadly divided into poisons and catalytic inhibitors. The TopoII poisons are in clinical use as anti-cancer therapies, although in common to most chemotherapeutic agents, they display considerable normal tissue toxicity. Inhibition of the TopoIIb isoform has been implicated in this cytotoxicity. Response to TopoII active agents is determined by several factors, but cell cycle checkpoints play a large role in sensitivity and resistance. The G2/M phase checkpoints are of particular importance in considering the effectiveness of these drugs and are reviewed in this article. Functionality of the ATM dependent decatenation checkpoint may represent a new avenue for selective cancer therapy. Here we review the function of TopoII, the anti-cancer mechanisms and limitations of current catalytic inhibitors and poisons, and their influence on cell cycle checkpoints. We will also assess potential new mechanisms for targeting this enzyme to limit normal tissue toxicity, and how the cell cycle checkpoint triggered by these drugs may provide an alternative and possibly better target for novel therapies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The point of no return: The poly(A)-associated elongation checkpoint.

PubMed

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3' end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved.

Role for the Silencing Protein Dot1 in Meiotic Checkpoint Control

PubMed Central

San-Segundo, Pedro A.; Roeder, G. Shirleen

2000-01-01

During the meiotic cell cycle, a surveillance mechanism called the “pachytene checkpoint” ensures proper chromosome segregation by preventing meiotic progression when recombination and chromosome synapsis are defective. The silencing protein Dot1 (also known as Pch1) is required for checkpoint-mediated pachytene arrest of the zip1 and dmc1 mutants of Saccharomyces cerevisiae. In the absence of DOT1, the zip1 and dmc1 mutants inappropriately progress through meiosis, generating inviable meiotic products. Other components of the pachytene checkpoint include the nucleolar protein Pch2 and the heterochromatin component Sir2. In dot1, disruption of the checkpoint correlates with the loss of concentration of Pch2 and Sir2 in the nucleolus. In addition to its checkpoint function, Dot1 blocks the repair of meiotic double-strand breaks by a Rad54-dependent pathway of recombination between sister chromatids. In vegetative cells, mutation of DOT1 results in delocalization of Sir3 from telomeres, accounting for the impaired telomeric silencing in dot1. PMID:11029058

Replication licensing and the DNA damage checkpoint

PubMed Central

Cook, Jeanette Gowen

2011-01-01

Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings suggest that checkpoints and replication are more intimately associated than previously appreciated, even in the absence of exogenous DNA damage. This review summarizes some of these developments. PMID:19482602

Control of the yeast telomeric senescence survival pathways of recombination by the Mec1 and Mec3 DNA damage sensors and RPA

PubMed Central

Grandin, Nathalie; Charbonneau, Michel

2007-01-01

Saccharomyces cerevisiae telomerase-negative cells undergo homologous recombination on subtelomeric or TG1–3 telomeric sequences, thus allowing Type I or Type II post-senescence survival, respectively. Here, we find that the DNA damage sensors, Mec1, Mec3 and Rad24 control Type II recombination, while the Rad9 adaptor protein and the Rad53 and Chk1 effector kinases have no effect on survivor type selection. Therefore, the Mec1 and Mec3 checkpoint complexes control telomeric recombination independently of their roles in generating and amplifying the Mec1-Rad53-Chk1 kinase cascade. rfa1-t11 mutant cells, bearing a mutation in Replication Protein A (RPA) conferring a defect in recruiting Mec1-Ddc2, were also deficient in both types of telomeric recombination. Importantly, expression of an Rfa1-t11-Ddc2 hybrid fusion protein restored checkpoint-dependent arrest, but did not rescue defective telomeric recombination. Therefore, the Rfa1-t11-associated defect in telomeric recombination is not solely due to its failure to recruit Mec1. We have also isolated novel alleles of RFA1 that were deficient in Type I but not in Type II recombination and proficient in checkpoint control. Therefore, the checkpoint and recombination functions of RPA can be genetically separated, as can the RPA-mediated control of the two types of telomeric recombination. PMID:17202155

Concordance of immune checkpoints within tumor immune contexture and their prognostic significance in gastric cancer.

PubMed

Dai, Congqi; Geng, Ruixuan; Wang, Chenchen; Wong, Angela; Qing, Min; Hu, Jianjun; Sun, Yu; Lo, A W I; Li, Jin

2016-12-01

Checkpoint blockade therapy has emerged as a novel approach for cancer immunotherapy in several malignancies. However, patient prognosis and disease progression relevant to immune checkpoints in gastric tumor microenvironment are not defined. This study aims to investigate the expression and prognostic significance of immune checkpoints within gastric cancer. In the study, a cohort of 398 cancer tissues from stage I to IV gastric cancer patients were assessed for programmed cell death 1 ligand 1 (PD-L1) expression and tumor-infiltrating lymphocyte (TIL) infiltration using immunohistochemistry to ascertain their survival correlation. The data revealed that higher TIL density correlated with less risk of disease progression, and exhibited survival benefits in gastric cancer patients, and PD-L1 positivity showed a significant association with the presence of high TIL infiltration. Furthermore, real-time quantitative polymerase chain reaction was performed to detect expression of multiple immune checkpoints with the relation to clinical outcome in 139 samples randomly selected from the same cohort, and higher messenger RNA levels of most immune checkpoints were associated with favorable outcome, while consistently showing a positive correlation with interferon gamma levels. In situ hybridization was used to determine the localization of Epstein-Barr virus (EBV) in 97 specimens, and showed EBV-positive gastric cancer samples correlated with PD-L1 expression and increased TIL density. These results suggest that induction of immune checkpoint within gastric cancer patients reflects a high immune infiltration density, especially in those with EBV-associated gastric cancer, which may direct patient selection for checkpoint blockade therapy. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Understanding and Managing Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitors in Patients With Advanced Melanoma

PubMed Central

Weinstein, Alyona; Gordon, Ruth-Ann; Kasler, Mary Kate; Burke, Matthew; Ranjan, Smita; Hodgetts, Jackie; Reed, Vanessa; Shames, Yelena; Prempeh-Keteku, Nana; Lingard, Karla

2017-01-01

The immune checkpoint inhibitors ipilimumab, nivolumab, and pembrolizumab represent a substantial improvement in treating advanced melanoma but are associated with adverse events (AEs) likely related to general immunologic enhancement. To ensure that patients receive optimal benefit from these agents, prompt assessment and treatment of AEs are essential. We review the efficacy and safety profiles of these immune checkpoint inhibitors and describe guidelines for managing immune-related AEs. We also present case studies describing the management of toxicities in patients receiving immune checkpoint inhibitor therapy. These cases illustrate the importance of collecting a detailed medical history when administering immunotherapy, as this information is necessary to establish baseline, inform monitoring, and determine the etiology of symptoms. Advanced practice nurses and physician assistants are uniquely positioned to educate patients on the early recognition of AEs and have an important role in establishing appropriate monitoring and open dialogue among services. PMID:29900017

The Scaffold Attachment Factor SAFB1: A New Player in G2/M Checkpoint Control

DTIC Science & Technology

2007-04-01

RNA was obtained from locally advanced breast tumors in 24 patients before they underwent four cycles of neoadjuvant docetaxel treatment . Gene...expression analysis was performed and correlated to the treatment response to determine genes that are differentially expressed in docetaxel-sensitive...decreased sensitivity to drugs, depending on the chemotherapeutic agent used 2) Association of SAFB1 loss with resistance to docetaxel treatment , both

The point of no return: The poly(A)-associated elongation checkpoint

PubMed Central

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

abstract Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3′ end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved. PMID:26853452

Development of cell-cycle checkpoint therapy for solid tumors.

PubMed

Tamura, Kenji

2015-12-01

Cellular proliferation is tightly controlled by several cell-cycle checkpoint proteins. In cancer, the genes encoding these proteins are often disrupted and cause unrestrained cancer growth. The proteins are over-expressed in many malignancies; thus, they are potential targets for anti-cancer therapies. These proteins include cyclin-dependent kinase, checkpoint kinase, WEE1 kinase, aurora kinase and polo-like kinase. Cyclin-dependent kinase inhibitors are the most advanced cell-cycle checkpoint therapeutics available. For instance, palbociclib (PD0332991) is a first-in-class, oral, highly selective inhibitor of CDK4/6 and, in combination with letrozole (Phase II; PALOMA-1) or with fulvestrant (Phase III; PALOMA-3), it has significantly prolonged progression-free survival, in patients with metastatic estrogen receptor-positive, HER2-negative breast cancer, in comparison with that observed in patients using letrozole, or fulvestrant alone, respectively. In this review, we provide an overview of the current compounds available for cell-cycle checkpoint protein-directed therapy for solid tumors. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A cytokinesis checkpoint requiring the yeast homologue of an APC-binding protein

PubMed Central

Muhua, Li; Adames, Neil R.; Murphy, Michael D.; Shields, Colleen R.; Cooper, John A.

2008-01-01

Checkpoint controls ensure that events of the cell-division cycle are completed with fidelity and in the correct order. In budding yeast with a mutation in the motor protein dynein, the mitotic spindle is often misaligned and therefore slow to enter the neck between mother cell and budding daughter cell. When this occurs, cytokinesis (division of the cytoplasm into two) is delayed until the spindle is properly positioned1. Here we describe mutations that abolish this delay, indicating the existence of a new checkpoint mechanism. One mutation lies in the gene encoding the yeast homologue of EB1, a human protein that binds the adenomatous polyposis coli (APC) protein, a tumour suppressor. EB1 is located on microtubules of the mitotic spindle and is important in spindle assembly. EB1 may therefore, by associating with microtubules, contribute to the sensor mechanism that activates the checkpoint. Another mutation affects Stt4, a phosphatidylinositol-4-OH kinase. Cold temperature is an environmental stimulus that causes misalignment of the mitotic spindle in yeast and appears to activate this checkpoint mechanism. PMID:9624007

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

PubMed

Nakada, Daisuke; Hirano, Yukinori; Sugimoto, Katsunori

2004-11-01

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Checkpointing filesystem

DOEpatents

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

2005-05-17

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

Induction of a G1-S checkpoint in fission yeast.

PubMed

Bøe, Cathrine A; Krohn, Marit; Rødland, Gro Elise; Capiaghi, Christoph; Maillard, Olivier; Thoma, Fritz; Boye, Erik; Grallert, Beáta

2012-06-19

Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

PP2ARts1 is a master regulator of pathways that control cell size

PubMed Central

Zapata, Jessica; Dephoure, Noah; MacDonough, Tracy; Yu, Yaxin; Parnell, Emily J.; Mooring, Meghan; Gygi, Steven P.; Stillman, David J.

2014-01-01

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth. PMID:24493588

PD-1 /PD-L1 checkpoint in hematological malignancies.

PubMed

Annibali, O; Crescenzi, A; Tomarchio, V; Pagano, A; Bianchi, A; Grifoni, A; Avvisati, G

2018-04-01

Programmed cell death protein 1 (PD-1), is a cell surface receptor with an important role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1/PDL1 axis represents a checkpoint to control immune responses and it is often used as a mechanism of immune escaping by cancers and infectious diseases. Many data demonstrate its important role in solid tumors and report emerging evidences in lymphoproliferative disorders. In this review, we summarized the available data on the role of PD-1/PD-L1 checkpoint in lymphoproliferative diseases and the therapeutics use of monoclonal blocking antibodies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Cellular miR-2909 RNomics governs the genes that ensure immune checkpoint regulation.

PubMed

Kaul, Deepak; Malik, Deepti; Wani, Sameena

2018-06-20

Cross-talk between coding RNAs and regulatory non-coding microRNAs, within human genome, has provided compelling evidence for the existence of flexible checkpoint control of T-Cell activation. The present study attempts to demonstrate that the interplay between miR-2909 and its effector KLF4 gene has the inherent capacity to regulate genes coding for CTLA4, CD28, CD40, CD134, PDL1, CD80, CD86, IL-6 and IL-10 within normal human peripheral blood mononuclear cells (PBMCs). Based upon these findings, we propose a pathway that links miR-2909 RNomics with the genes coding for immune checkpoint regulators required for the maintenance of immune homeostasis.

Identification of Late Larval Stage Developmental Checkpoints in Caenorhabditis elegans Regulated by Insulin/IGF and Steroid Hormone Signaling Pathways

PubMed Central

Schindler, Adam J.; Baugh, L. Ryan; Sherwood, David R.

2014-01-01

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint. PMID:24945623

Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

PubMed

Thai, Vu; Dephoure, Noah; Weiss, Amit; Ferguson, Jacqueline; Leitao, Ricardo; Gygi, Steven P; Kellogg, Douglas R

2017-03-24

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2A Cdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2A Cdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2A Cdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2A Cdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2A Cdc55 by multiple overlapping mechanisms. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Gyneco-oncological genomics and emerging biomarkers for cancer treatment with immune-checkpoint inhibitors.

PubMed

Curigliano, Giuseppe

2018-05-15

In gynecological cancers tumor infiltrating lymphocytes and upregulation of immune-related gene signatures have been associated with a better prognosis. Knowledge of tumor immunogenicity and associated gene signatures suggests that the tumor immune landscape is a key determinant to define patient prognosis and potentially to predict response to immune-checkpoint inhibitors. The aim of this review is to give an overview of immune gene signatures across gynecology histological cancer types, defining their prognostic and potential predictive role. In the current review we will present data on these gene signatures, on immunohistochemical features and their potential importance to select patients potentially eligible to trials with immune-checkpoint inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

PubMed

Pinto-Fernandez, Adan; Kessler, Benedikt M

2016-01-01

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets

PubMed Central

Pinto-Fernandez, Adan; Kessler, Benedikt M.

2016-01-01

Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors. PMID:27516771

Recovery from the DNA Replication Checkpoint

PubMed Central

Chaudhury, Indrajit; Koepp, Deanna M.

2016-01-01

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

Immune checkpoint inhibitors for nonsmall cell lung cancer treatment.

PubMed

Chen, Yuh-Min

2017-01-01

Immune checkpoint inhibition with blocking antibodies that target cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed cell death protein 1 (PD-1) pathway [PD-1/programmed death-ligand 1 (PD-L1)] have demonstrated promise in a variety of malignancies. While ipilimumab has been approved as a CTLA-4 blocking antibody by the US Food and Drug Administration for the treatment of advanced melanoma, it is still not approved for lung cancer treatment. In contrast, nivolumab and pembrolizumab, both PD-1 blocking antibodies, have been approved for second-line treatment of nonsmall cell lung cancer in 2015 because of their high potency and long-lasting effects in some patient subgroups. Other PD-1 and PD-L1 monoclonal antibodies are also in active development phase. Treatment with such immune checkpoint inhibitors is associated with a unique pattern of immune-related adverse events or side effects. Combination approaches involving CTLA-4 and PD-1/PD-L1 blockade or checkpoint inhibitors with chemotherapy or radiotherapy are being investigated to determine whether they may enhance the efficacy of treatment. Despite many challenges ahead, immunotherapy with checkpoint inhibitors has already become a new and important treatment modality for lung cancer in the last decade following the discovery of targeted therapy. Copyright © 2016. Published by Elsevier Taiwan LLC.

Nanosecond pulsed electric fields and the cell cycle

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.

Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when compared to sham treated cells. CHO cells undergoing mitosis after exposure also exhibit improper separation of chromatids which could indicate loss of function of the mitotic spindle checkpoint. Activation and loss of function of checkpoints in CHO but not Jurkat cells after nsPEF exposure suggests that activation of cell cycle checkpoints could be important in defining the character of cell line specific recovery after nsPEF exposure. Moreover, the increased sensitivity in G2/M phase exhibited by both cell lines indicates that cell cycle phase is an important consideration during nsPEF exposure, particularly when aiming to induce apoptosis.

Patterns of disease control and survival in patients with melanoma brain metastases undergoing immune-checkpoint blockade.

PubMed

Milsch, Laura; Gesierich, Anja; Kreft, Sophia; Livingstone, Elisabeth; Zimmer, Lisa; Goebeler, Matthias; Schadendorf, Dirk; Schilling, Bastian

2018-06-12

Immune-checkpoint blockers (ICBs) significantly prolong overall survival (OS) in patients with advanced melanoma. Limited data are available on the efficacy and clinical benefit in patients with melanoma brain metastases (MBMs). The aim of this study was to determine whether ICB is active in an unselected cohort treated of patients with known brain metastases and if disease control correlates with the survival. A total of 385 patients with metastatic malignant melanoma treated with ICB as monotherapy between 2005 and 2017 in two tertiary referral centres were included. Patient records were searched for the development of brain metastases. Demographic and clinical data of all patients were collected retrospectively. We identified 177 patients with MBM who received ICBs (ipilimumab, nivolumab, pembrolizumab). Patients with and without brain metastases received similar ICB regimens. Prognosis was inferior in patients with brain metastases; patients with >1 brain metastasis showed even poorer survival. For extracranial (ec) metastases, disease control was associated with improved survival. However, when comparing patients with intracranial (ic) disease control during immunotherapy to patients with ic disease progression, no difference in OS could be observed. In our study, ec disease control was the dominant predictive factor for OS in both patients with or without melanoma brain metastases. These data indicate that clinical trials in melanoma patients with brain metastases should address end-points such as symptom control, quality of life or OS in addition to ic response rates. Copyright © 2018 Elsevier Ltd. All rights reserved.

Development of Novel PD1/PD-L1 Antagonists Using Circular Cys-Knotted Micro Proteins

DTIC Science & Technology

2017-06-01

positive bacte- ria.[67] Similar antibacterial activities have been found in cyclo- tides isolated from Hedyota biflora (Rubiaceae family)[68] and C...immune control. Hence, reversing the inhibition of the adaptive immunity can lead to the activation of a patient’s immunity. For example, inhibition of...receptor and PD-L1, to block immune checkpoints, and facilitate antitumor activity . These checkpoint-blocking antibodies have demonstrated clinical

Compiler-assisted static checkpoint insertion

NASA Technical Reports Server (NTRS)

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

1992-01-01

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

DNA damage and polyploidization.

PubMed

Chow, Jeremy; Poon, Randy Y C

2010-01-01

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Unrepaired clustered DNA lesions induce chromosome breakage in human cells

PubMed Central

Asaithamby, Aroumougame; Hu, Burong; Chen, David J.

2011-01-01

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis. PMID:21527720

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

PubMed Central

Recolin, Bénédicte; Van Der Laan, Siem; Maiorano, Domenico

2012-01-01

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation. PMID:22187152

The Role of Replication in Activation of the DNA Damage Checkpoint

DTIC Science & Technology

2006-03-01

St AD Award Number : W81XWH-04-1-0311 TITLE: The Role of Replication in Activation of the DNA Damage Checkpoint PRINCIPAL INVESTIGATOR: Christopher...Van CONTRACTING ORGANIZATION: Stanford University Stanford, CA 94305 REPORT DATE: March 2006 TYPE OF REPORT: Annual Summary PREPARED FOR: U.S. Army...to comply with a collection of information if it does not display a currently valid OMB control number . PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE

Immune Checkpoints in Leprosy: Immunotherapy As a Feasible Approach to Control Disease Progression.

PubMed

Lima, Hayana Ramos; Gasparoto, Thaís Helena; de Souza Malaspina, Tatiana Salles; Marques, Vinícius Rizzo; Vicente, Marina Jurado; Marcos, Elaine Camarinha; Souza, Fabiana Corvolo; Nogueira, Maria Renata Sales; Barreto, Jaison Antônio; Garlet, Gustavo Pompermaier; da Silva, João Santana; Brito-de-Souza, Vânia Nieto; Campanelli, Ana Paula

2017-01-01

Leprosy remains a health problem in several countries. Current management of patients with leprosy is complex and requires multidrug therapy. Nonetheless, antibiotic treatment is insufficient to prevent nerve disabilities and control Mycobacterium leprae . Successful infectious disease treatment demands an understanding of the host immune response against a pathogen. Immune-based therapy is an effective treatment option for malignancies and infectious diseases. A promising therapeutic approach to improve the clinical outcome of malignancies is the blockade of immune checkpoints. Immune checkpoints refer to a wide range of inhibitory or regulatory pathways that are critical for maintaining self-tolerance and modulating the immune response. Programmed cell-death protein-1 (PD-1), programmed cell death ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4, and lymphocyte-activation gene-3 are the most important immune checkpoint molecules. Several pathogens, including M. leprae , are supposed to utilize these mechanisms to evade the host immune response. Regulatory T cells and expression of co-inhibitory molecules on lymphocytes induce specific T-cell anergy/exhaustion, leading to disseminated and progressive disease. From this perspective, we outline how the co-inhibitory molecules PD-1, PD-L1, and Th1/Th17 versus Th2/Treg cells are balanced, how antigen-presenting cell maturation acts at different levels to inhibit T cells and modulate the development of leprosy, and how new interventions interfere with leprosy development.

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

DOE PAGES

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

2013-01-01

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

Multiple Duties for Spindle Assembly Checkpoint Kinases in Meiosis

PubMed Central

Marston, Adele L.; Wassmann, Katja

2017-01-01

Cell division in mitosis and meiosis is governed by evolutionary highly conserved protein kinases and phosphatases, controlling the timely execution of key events such as nuclear envelope breakdown, spindle assembly, chromosome attachment to the spindle and chromosome segregation, and cell cycle exit. In mitosis, the spindle assembly checkpoint (SAC) controls the proper attachment to and alignment of chromosomes on the spindle. The SAC detects errors and induces a cell cycle arrest in metaphase, preventing chromatid separation. Once all chromosomes are properly attached, the SAC-dependent arrest is relieved and chromatids separate evenly into daughter cells. The signaling cascade leading to checkpoint arrest depends on several protein kinases that are conserved from yeast to man. In meiosis, haploid cells containing new genetic combinations are generated from a diploid cell through two specialized cell divisions. Though apparently less robust, SAC control also exists in meiosis. Recently, it has emerged that SAC kinases have additional roles in executing accurate chromosome segregation during the meiotic divisions. Here, we summarize the main differences between mitotic and meiotic cell divisions, and explain why meiotic divisions pose special challenges for correct chromosome segregation. The less-known meiotic roles of the SAC kinases are described, with a focus on two model systems: yeast and mouse oocytes. The meiotic roles of the canonical checkpoint kinases Bub1, Mps1, the pseudokinase BubR1 (Mad3), and Aurora B and C (Ipl1) will be discussed. Insights into the molecular signaling pathways that bring about the special chromosome segregation pattern during meiosis will help us understand why human oocytes are so frequently aneuploid. PMID:29322045

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

PubMed

Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Chronic exposure to particulate chromate induces spindle assembly checkpoint bypass in human lung cells.

PubMed

Wise, Sandra S; Holmes, Amie L; Xie, Hong; Thompson, W Douglas; Wise, John Pierce

2006-11-01

One of the hallmarks of lung cancer is chromosome instability (CIN), particularly a tetraploid phenotype, which is normally prevented by the spindle assembly checkpoint. Hexavalent chromium Cr(VI) is an established human lung carcinogen, and Cr(VI) induces tumors at lung bifurcation sites where Cr(VI) particles impact and persist. However, the effects of Cr(VI) on the spindle assembly checkpoint are unknown and little is known about prolonged exposure to particulate Cr(VI). Accordingly, we investigated particulate Cr(VI)-induced bypass of the spindle assembly checkpoint after several days of exposure in WHTBF-6 cells. We found that lead chromate indeed induces spindle assembly checkpoint bypass in human lung cells, as 72, 96, and 120 h treatments with 0.5 or 1 microg/cm2 lead chromate induced significant increases in the percentage of cells with aberrant mitotic figures. For example, treatment with 1 microg/cm2 lead chromate for 96 h induced 11, 12.3, and 14% of cells with premature anaphase, centromere spreading and premature centromere division, respectively. In addition, we found a disruption of mitosis with more cells accumulating in anaphase; cells treated for 96 h increased from 18% in controls to 31% in cells treated with lead chromate. To confirm involvement of the spindle assembly checkpoint, Mad2 expression was used as a marker. Mad2 expression was decreased in cells exposed to chronic treatments of lead chromate, consistent with disruption of the checkpoint. We also found concentration- and time-dependent increases in tetraploid cells, which continued to grow and form colonies. When cells were treated with chronic lead alone there was no increase in aberrant mitotic cells or polyploidy; however, chronic exposure to a soluble Cr(VI) showed an increase in aberrant mitotic cells and polyploidy. These data suggest that lead chromate does induce CIN and may be one mechanism in the development of Cr(VI)-induced lung cancer.

Use of programmed cell death protein ligand 1 assay to predict the outcomes of non-small cell lung cancer patients treated with immune checkpoint inhibitors

PubMed Central

Tibaldi, Carmelo; Lunghi, Alice; Baldini, Editta

2017-01-01

The recent discovery of immune checkpoints inhibitors, especially anti-programmed cell death protein 1 (PD-1) and anti-programmed cell death protein ligand 1 (PD-L1) monoclonal antibodies, has opened new scenarios in the management of non-small cell lung cancer (NSCLC) and this new class of drugs has achieved a rapid development in the treatment of this disease. However, considering the costs of these drugs and the fact that only a subset of patients experience long-term disease control, the identification of predictive biomarkers for the selection of candidates suitable for treatment has become a priority. The research focused mainly on the expression of the PD-L1 receptor on both tumor cells and/or immune infiltrates determined by immunohistochemistry (IHC). However, different checkpoint inhibitors were tested, different IHC assays were used, different targets were considered (tumor cells, immune infiltrates or both) and different expression thresholds were employed in clinical trials. In some trials the assay was used prospectively to select the patients, while in other trials it was evaluated retrospectively. Some confusion emerges, which makes it difficult to easily compare the literature data and to translate them in practice management. This mini-review shows the possibilities and pitfalls of the PD-L1 expression to predict the activity and efficacy of anti PD1/PD-L1 monoclonal antibodies in the treatment of NSCLC. PMID:28848698

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

PubMed Central

Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

2015-01-01

When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

Immune Checkpoint Inhibitors in the Treatment of Patients with Neuroendocrine Neoplasia.

PubMed

Weber, Matthias M; Fottner, Christian

2018-01-01

Well-differentiated neuroendocrine neoplasms (NENs) are usually controlled by antiproliferative, local ablative and/or radionuclide therapies, whereas poorly differentiated NENs generally require cytotoxic chemotherapy. However, treatment options for patients with advanced/metastatic high-grade NENs remain limited. Review of the literature and international congress abstracts on the efficacy and safety of immunotherapy by checkpoint inhibition in advanced/metastatic NENs. Evidence points to an important role of immune phenomena in the pathogenesis and treatment of neuroendocrine tumors (NETs). Programmed cell death 1 (PD-1) protein and its ligand are mainly expressed in poorly differentiated NENs. Microsatellite instability and high mutational load are more pronounced in high-grade NENs and may predict response to immunotherapy. Clinical experience of immune checkpoint blockade mainly exists for Merkel cell carcinoma, a high-grade cutaneous neuroendocrine carcinoma (NEC), which has led to approval of the anti-PD-1 antibody avelumab. In addition, there is anecdotal evidence for the efficacy of checkpoint inhibitors in large-cell lung NECs, ovarian NECs and others, including gastroenteropancreatic NENs. Currently, phase II studies investigate PDR001, pembrolizumab, combined durvalumab and tremelimumab, and avelumab treatment in patients with advanced/metastatic NENs. Immune checkpoint inhibitors are a promising therapeutic option, especially in progressive NECs or high-grade NETs with high tumor burden, microsatellite instability, and/or mutational load. © 2018 S. Karger GmbH, Freiburg.

Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.

2007-11-15

Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eightmore » cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.« less

Methotrexate increases expression of cell cycle checkpoint genes via Jun-N-terminal kinase activation

PubMed Central

Spurlock, Charles F.; Tossberg, John T.; Fuchs, Howard A.; Olsen, Nancy J.; Aune, Thomas M.

2011-01-01

Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. PMID:22183962

Lazy checkpoint coordination for bounding rollback propagation

NASA Technical Reports Server (NTRS)

Wang, Yi-Min; Fuchs, W. Kent

1992-01-01

Independent checkpointing allows maximum process autonomy but suffers from potential domino effects. Coordinated checkpointing eliminates the domino effect by sacrificing a certain degree of process autonomy. In this paper, we propose the technique of lazy checkpoint coordination which preserves process autonomy while employing communication-induced checkpoint coordination for bounding rollback propagation. The introduction of the notion of laziness allows a flexible trade-off between the cost for checkpoint coordination and the average rollback distance. Worst-case overhead analysis provides a means for estimating the extra checkpoint overhead. Communication trace-driven simulation for several parallel programs is used to evaluate the benefits of the proposed scheme for real applications.

Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae

PubMed Central

Finnigan, Gregory C.; Sterling, Sarah M.; Duvalyan, Angela; Liao, Elizabeth N.; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

2016-01-01

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611–950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611–950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379–1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. PMID:27193302

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.

PubMed

Roque, Telma; Haton, Céline; Etienne, Olivier; Chicheportiche, Alexandra; Rousseau, Laure; Martin, Ludovic; Mouthon, Marc-André; Boussin, François D

2012-03-01

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage. Copyright © 2011 AlphaMed Press.

Characterization of tumor-associated T-lymphocyte subsets and immune checkpoint molecules in head and neck squamous cell carcinoma

PubMed Central

Thelen, Martin; Reuter, Sabrina; Zentis, Peter; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; Wennhold, Kerstin; Garcia-Marquez, Maria; Tharun, Lars; Quaas, Alexander; Schauss, Astrid; Isensee, Jörg; Hucho, Tim; Huebbers, Christian

2017-01-01

The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA−/CCR7−). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the ‘Immunoscore’ (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance. PMID:28574843

The DNA Replication Checkpoint Directly Regulates MBF-Dependent G1/S Transcription▿

PubMed Central

Dutta, Chaitali; Patel, Prasanta K.; Rosebrock, Adam; Oliva, Anna; Leatherwood, Janet; Rhind, Nicholas

2008-01-01

The DNA replication checkpoint transcriptionally upregulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF, the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitro phosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Replacement of two potential phosphorylation sites with phosphomimetic amino acids suffices to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggests that checkpoint regulation of the MBF transcription factor is a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. PMID:18662996

Asynchronous Two-Level Checkpointing Scheme for Large-Scale Adjoints in the Spectral-Element Solver Nek5000

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schanen, Michel; Marin, Oana; Zhang, Hong

Adjoints are an important computational tool for large-scale sensitivity evaluation, uncertainty quantification, and derivative-based optimization. An essential component of their performance is the storage/recomputation balance in which efficient checkpointing methods play a key role. We introduce a novel asynchronous two-level adjoint checkpointing scheme for multistep numerical time discretizations targeted at large-scale numerical simulations. The checkpointing scheme combines bandwidth-limited disk checkpointing and binomial memory checkpointing. Based on assumptions about the target petascale systems, which we later demonstrate to be realistic on the IBM Blue Gene/Q system Mira, we create a model of the expected performance of our checkpointing approach and validatemore » it using the highly scalable Navier-Stokes spectralelement solver Nek5000 on small to moderate subsystems of the Mira supercomputer. In turn, this allows us to predict optimal algorithmic choices when using all of Mira. We also demonstrate that two-level checkpointing is significantly superior to single-level checkpointing when adjoining a large number of time integration steps. To our knowledge, this is the first time two-level checkpointing had been designed, implemented, tuned, and demonstrated on fluid dynamics codes at large scale of 50k+ cores.« less

T cells in chronic lymphocytic leukemia display dysregulated expression of immune checkpoints and activation markers.

PubMed

Palma, Marzia; Gentilcore, Giusy; Heimersson, Kia; Mozaffari, Fariba; Näsman-Glaser, Barbro; Young, Emma; Rosenquist, Richard; Hansson, Lotta; Österborg, Anders; Mellstedt, Håkan

2017-03-01

Chronic lymphocytic leukemia is characterized by impaired immune functions largely due to profound T-cell defects. T-cell functions also depend on co-signaling receptors, inhibitory or stimulatory, known as immune checkpoints, including cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed death-1 (PD-1). Here we analyzed the T-cell phenotype focusing on immune checkpoints and activation markers in chronic lymphocytic leukemia patients (n=80) with different clinical characteristics and compared them to healthy controls. In general, patients had higher absolute numbers of CD3 + cells and the CD8 + subset was particularly expanded in previously treated patients. Progressive patients had higher numbers of CD4 + and CD8 + cells expressing PD-1 compared to healthy controls, which was more pronounced in previously treated patients ( P =0.0003 and P =0.001, respectively). A significant increase in antigen-experienced T cells was observed in patients within both the CD4 + and CD8 + subsets, with a significantly higher PD-1 expression. Higher numbers of CD4 + and CD8 + cells with intracellular CTLA-4 were observed in patients, as well as high numbers of proliferating (Ki67 + ) and activated (CD69 + ) CD4 + and CD8 + cells, more pronounced in patients with active disease. The numbers of Th1, Th2, Th17 and regulatory T cells were substantially increased in patients compared to controls ( P <0.05), albeit decreasing to low levels in pre-treated patients. In conclusion, chronic lymphocytic leukemia T cells display increased expression of immune checkpoints, abnormal subset distribution, and a higher proportion of proliferating cells compared to healthy T cells. Disease activity and previous treatment shape the T-cell profile of chronic lymphocytic leukemia patients in different ways. Copyright© Ferrata Storti Foundation.

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

PubMed

Stockwell, Simon R; Platt, Georgina; Barrie, S Elaine; Zoumpoulidou, Georgia; Te Poele, Robert H; Aherne, G Wynne; Wilson, Stuart C; Sheldrake, Peter; McDonald, Edward; Venet, Mathilde; Soudy, Christelle; Elustondo, Frédéric; Rigoreau, Laurent; Blagg, Julian; Workman, Paul; Garrett, Michelle D; Mittnacht, Sibylle

2012-01-01

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

Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

PubMed Central

Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

2009-01-01

Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

Synaptonemal Complex Components Are Required for Meiotic Checkpoint Function in Caenorhabditis elegans

PubMed Central

Bohr, Tisha; Ashley, Guinevere; Eggleston, Evan; Firestone, Kyra; Bhalla, Needhi

2016-01-01

Synapsis involves the assembly of a proteinaceous structure, the synaptonemal complex (SC), between paired homologous chromosomes, and is essential for proper meiotic chromosome segregation. In Caenorhabditis elegans, the synapsis checkpoint selectively removes nuclei with unsynapsed chromosomes by inducing apoptosis. This checkpoint depends on pairing centers (PCs), cis-acting sites that promote pairing and synapsis. We have hypothesized that the stability of homolog pairing at PCs is monitored by this checkpoint. Here, we report that SC components SYP-3, HTP-3, HIM-3, and HTP-1 are required for a functional synapsis checkpoint. Mutation of these components does not abolish PC function, demonstrating they are bona fide checkpoint components. Further, we identify mutant backgrounds in which the instability of homolog pairing at PCs does not correlate with the synapsis checkpoint response. Altogether, these data suggest that, in addition to homolog pairing, SC assembly may be monitored by the synapsis checkpoint. PMID:27605049

Cell-cycle control in the face of damage--a matter of life or death.

PubMed

Clarke, Paul R; Allan, Lindsey A

2009-03-01

Cells respond to DNA damage or defects in the mitotic spindle by activating checkpoints that arrest the cell cycle. Alternatively, damaged cells can undergo cell death by the process of apoptosis. The correct balance between these pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death. Although the molecular mechanisms of the cell cycle and apoptosis have been elucidated, the links between them have not been clear. Recent work, however, indicates that common components directly link the regulation of apoptosis with cell-cycle checkpoints operating during interphase, whereas in mitosis, the control of apoptosis is directly coupled to the cell-cycle machinery. These findings shed new light on how the balance between cell-cycle progression and cell death is controlled.

Asymmetric cell division during T cell development controls downstream fate

PubMed Central

Pham, Kim; Shimoni, Raz; Charnley, Mirren; Ludford-Menting, Mandy J.; Hawkins, Edwin D.; Ramsbottom, Kelly; Oliaro, Jane; Izon, David; Ting, Stephen B.; Reynolds, Joseph; Lythe, Grant; Molina-Paris, Carmen; Melichar, Heather; Robey, Ellen; Humbert, Patrick O.; Gu, Min

2015-01-01

During mammalian T cell development, the requirement for expansion of many individual T cell clones, rather than merely expansion of the entire T cell population, suggests a possible role for asymmetric cell division (ACD). We show that ACD of developing T cells controls cell fate through differential inheritance of cell fate determinants Numb and α-Adaptin. ACD occurs specifically during the β-selection stage of T cell development, and subsequent divisions are predominantly symmetric. ACD is controlled by interaction with stromal cells and chemokine receptor signaling and uses a conserved network of polarity regulators. The disruption of polarity by deletion of the polarity regulator, Scribble, or the altered inheritance of fate determinants impacts subsequent fate decisions to influence the numbers of DN4 cells arising after the β-selection checkpoint. These findings indicate that ACD enables the thymic microenvironment to orchestrate fate decisions related to differentiation and self-renewal. PMID:26370500

Co-inhibitory immune checkpoints in head and neck squamous cell carcinoma.

PubMed

Deng, W-W; Wu, L; Sun, Z-J

2018-03-01

The upregulation of co-inhibitory immune checkpoints hampers the immune response toward tumor cells and facilitates the tumor cells ability to evade immunosurveillance. Specific inhibitory immune checkpoint delivers inhibitory signals to T cells using multiple mechanisms. More in-depth understanding of the co-inhibitory immune checkpoints could be exploited for head and neck squamous cell carcinoma (HNSCC) treatment. In this review, we summarize the expression and the mechanism of partial co-inhibitory immune checkpoint signals and discuss targeting co-inhibitory immune checkpoints as an immunotherapeutic target for cancer therapy. This review may provide a better understanding of the co-inhibitory immune checkpoints and could promote applications of immunotherapy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

The deterrent capability of sobriety checkpoints : summary of the American literature

DOT National Transportation Integrated Search

1992-03-01

This report reviews and evaluates the scientific literature on sobriety checkpoints in the United States. Concerns about the constitutionality of checkpoint procedures initially limited the number of checkpoint programs in this country as well as con...

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

PubMed

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

2017-01-10

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

The Use of Sobriety Checkpoints for Impaired Driving Enforcement

DOT National Transportation Integrated Search

1990-11-01

Sobriety checkpoints have been a valuable tool for law enforcement's continuing fight to remove impaired drivers from the road. The purpose of the checkpoint is twofold; to apprehend impaired drivers at the physical location of the checkpoint; and se...

Checkpointing for a hybrid computing node

DOE Office of Scientific and Technical Information (OSTI.GOV)

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.

Cycle Checkpoint Abnormalities during Dementia: A Plausible Association with the Loss of Protection against Oxidative Stress in Alzheimer’s Disease

PubMed Central

Katsel, Pavel; Tan, Weilun; Fam, Peter; Purohit, Dushyant P.; Haroutunian, Vahram

2013-01-01

Background Increasing evidence suggests an association between neuronal cell cycle (CCL) events and the processes that underlie neurodegeneration in Alzheimer’s disease (AD). Elevated levels of oxidative stress markers and mitochondrial dysfunction are also among early events in AD. Recent studies have reported the role of CCL checkpoint proteins and tumor suppressors, such as ATM and p53 in the control of glycolysis and oxidative metabolism in cancer, but their involvement in AD remains uncertain. Methods and Findings In this postmortem study, we measured gene expression levels of eight CCL checkpoint proteins in the superior temporal cortex (STC) of persons with varying severities of AD dementia and compare them to those of cognitively normal controls. To assess whether the CCL changes associated with cognitive impairment in AD are specific to dementia, gene expression of the same proteins was also measured in STC of persons with schizophrenia (SZ), which is also characterized by mitochondrial dysfunction. The expression of CCL-checkpoint and DNA damage response genes: MDM4, ATM and ATR was strongly upregulated and associated with progression of dementia (cognitive dementia rating, CDR), appearing as early as questionable or mild dementia (CDRs 0.5–1). In addition to gene expression changes, the downstream target of ATM-p53 signaling - TIGAR, a p53-inducible protein, the activation of which can regulate energy metabolism and protect against oxidative stress was progressively decreased as severity of dementia evolved, but it was unaffected in subjects with SZ. In contrast to AD, different CCL checkpoint proteins, which include p53, CHEK1 and BRCA1 were significantly downregulated in SZ. Conclusions These results support the activation of an ATM signaling and DNA damage response network during the progression of AD dementia, while the progressive decrease in the levels of TIGAR suggests loss of protection initiated by ATM-p53 signaling against intensifying oxidative stress in AD. PMID:23861893

Multiple functions of the S-phase checkpoint mediator.

PubMed

Tanaka, Katsunori

2010-01-01

There is mounting evidence that replication defects are the major source of spontaneous genomic instability in cells, and that S-phase checkpoints are the principal defense against such instability. The S-phase checkpoint mediator protein Mrc1/Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of effector kinase by a sensor kinase. In this review, the multiple functions and the regulation of the S-phase checkpoint mediator are discussed.

Aurora B potentiates Mps1 activation to ensure rapid checkpoint establishment at the onset of mitosis.

PubMed

Saurin, Adrian T; van der Waal, Maike S; Medema, René H; Lens, Susanne M A; Kops, Geert J P L

2011-01-01

The mitotic checkpoint prevents mitotic exit until all chromosomes are attached to spindle microtubules. Aurora B kinase indirectly invokes this checkpoint by destabilizing incorrect attachments; however, a more direct role remains controversial. In contrast, activity of the kinase Mps1 is indispensible for the mitotic checkpoint. Here we show that Aurora B and Hec1 are needed for efficient Mps1 recruitment to unattached kinetochores, allowing rapid Mps1 activation at the onset of mitosis. Live monitoring of cyclin B degradation reveals that this is essential to establish the mitotic checkpoint quickly at the start of mitosis. Delayed Mps1 activation and checkpoint establishment upon Aurora B inhibition or Hec1 depletion are rescued by tethering Mps1 to kinetochores, demonstrating that Mps1 recruitment is the primary role of Aurora B and Hec1 in mitotic checkpoint signalling. These data demonstrate a direct role for Aurora B in initiating the mitotic checkpoint rapidly at the onset of mitosis.

Low-manpower checkpoints: can they provide effective DUI enforcement in small communities?

PubMed

Lacey, John H; Ferguson, Susan A; Kelley-Baker, Tara; Rider, Raamses P

2006-09-01

Sobriety checkpoints can be effective in reducing alcohol-impaired driving. Checkpoints are underutilized, however, partially because police believe a large number of officers are required. This study evaluated the feasibility and impact of conducting small-scale checkpoints in rural communities. Law enforcement agencies in two counties agreed to conduct weekly checkpoints for one year. Two nonadjacent counties did not undertake additional checkpoints. Evaluation included public-awareness surveys and roadside surveys (including blood alcohol concentration [BAC] measurements) of weekend nighttime drivers. Relative to drivers in the comparison counties, the proportion of drivers in the experimental counties with BACs >0.05% was 70% lower. Drivers surveyed at driver's license offices in the experimental counties after program implementation were more likely to report seeing or passing through a checkpoint and were more aware of publicity on driving under the influence (DUI) enforcement. Small rural communities can safely and effectively conduct low-staff sobriety checkpoints on a weekly basis. Such programs can be expected to result in large reductions in drivers operating at higher BACs.

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

PubMed Central

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

2017-01-01

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

S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe

PubMed Central

Lindsay, Howard D.; Griffiths, Dominic J.F.; Edwards, Rhian J.; Christensen, Per U.; Murray, Johanne M.; Osman, Fekret; Walworth, Nancy; Carr, Antony M.

1998-01-01

Checkpoints that respond to DNA structure changes were originally defined by the inability of yeast mutants to prevent mitosis following DNA damage or S-phase arrest. Genetic analysis has subsequently identified subpathways of the DNA structure checkpoints, including the reversible arrest of DNA synthesis. Here, we show that the Cds1 kinase is required to slow S phase in the presence of DNA-damaging agents. Cds1 is phosphorylated and activated by S-phase arrest and activated by DNA damage during S phase, but not during G1 or G2. Activation of Cds1 during S phase is dependent on all six checkpoint Rad proteins, and Cds1 interacts both genetically and physically with Rad26. Unlike its Saccharomyces cerevisiae counterpart Rad53, Cds1 is not required for the mitotic arrest checkpoints and, thus, defines an S-phase specific subpathway of the checkpoint response. We propose a model for the DNA structure checkpoints that offers a new perspective on the function of the DNA structure checkpoint proteins. This model suggests that an intrinsic mechanism linking S phase and mitosis may function independently of the known checkpoint proteins. PMID:9450932

Reducing space overhead for independendent checkpointing

NASA Technical Reports Server (NTRS)

Wang, Yi-Min; Chung, Pi-Yu; Lin, In-Jen; Fuchs, W. Kent

1992-01-01

The main disadvantages of independent checkpointing are the possible domino effect and the associated storage space overhead for maintaining multiple checkpoints. In most previous work, it has been assumed that only the checkpoints older than the current global recovery line can be discarded. Here, we generalize a notion of recovery line to potential recovery line. Only the checkpoints belonging to at least one of the potential recovery lines cannot be discarded. By using the model of maximum-sized antichains on a partially ordered set, an efficient algorithm is developed for finding all non-discardable checkpoints, and we show that the number of non-discardable checkpoints cannot exceed N(N+1)/2, where N is the number of processors. Communication trace driven simulation for several hypercube programs is performed to show the benefit of the proposed algorithm for real applications.

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

PubMed Central

Gubin, Matthew M.; Zhang, Xiuli; Schuster, Heiko; Caron, Etienne; Ward, Jeffrey P.; Noguchi, Takuro; Ivanova, Yulia; Hundal, Jasreet; Arthur, Cora D.; Krebber, Willem-Jan; Mulder, Gwenn E.; Toebes, Mireille; Vesely, Matthew D.; Lam, Samuel S.K.; Korman, Alan J.; Allison, James P.; Freeman, Gordon J.; Sharpe, Arlene H.; Pearce, Erika L.; Schumacher, Ton N.; Aebersold, Ruedi; Rammensee, Hans-Georg; Melief, Cornelis J. M.; Mardis, Elaine R.; Gillanders, William E.; Artyomov, Maxim N.; Schreiber, Robert D.

2014-01-01

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments. PMID:25428507

Template based parallel checkpointing in a massively parallel computer system

DOEpatents

Archer, Charles Jens [Rochester, MN; Inglett, Todd Alan [Rochester, MN

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.

Advances of Immune Checkpoint Inhibitors in Tumor Immunotherapy

NASA Astrophysics Data System (ADS)

Guo, Qiao

2018-01-01

Immune checkpoints are cell surface molecules that can fine-tune the immune responses, they are crucial for modulating the duration and amplitude of immune reactions while maintaining self-tolerance in order to minimize autoimmune responses. Numerous studies have demonstrated that tumors cells can directly express immune-checkpoint molecules, or induce many inhibitory molecules expression in the tumor microenvironment to inhibit the anti-tumor immunity. Releasing these brakes has emerged as an exciting strategy to cure cancer. In the past few years, clinical trials with therapeutic antibodies targeting to the checkpoint molecules CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. In contrast to the conventional treatment, checkpoint inhibitors induce broad and durable antitumor responses. In the future, treatment may involve combination therapy to target different checkpoint molecules and stages of the adaptive immune responses. In this review, we summarized the recent advances of the study and development of other checkpoint molecules in tumor immunotherapy.

Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways

PubMed Central

Rhind, Nicholas; Russell, Paul

2010-01-01

SUMMARY Recent work on the mechanisms of DNA damage and replication cell cycle checkpoints has revealed great similarity between the checkpoint pathways of organisms as diverse as yeasts, flies and humans. However, there are differences in the ways these organisms regulate their cell cycles. To connect the conserved checkpoint pathways with various cell cycle targets requires an adaptable link that can target different cell cycle components in different organisms. The Chk1 and Cds1 protein kinases, downstream effectors in the checkpoint pathways, seem to play just such roles. Perhaps more surprisingly, the two kinases not only have different targets in different organisms but also seem to respond to different signals in different organisms. So, whereas in fission yeast Chk1 is required for the DNA damage checkpoint and Cds1 is specifically involved in the replication checkpoint, their roles seem to be shuffled in metazoans. PMID:11058076

Understanding checkpointing overheads on massive-scale systems : analysis of the IBM Blue Gene/P system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gupta, R.; Naik, H.; Beckman, P.

Providing fault tolerance in high-end petascale systems, consisting of millions of hardware components and complex software stacks, is becoming an increasingly challenging task. Checkpointing continues to be the most prevalent technique for providing fault tolerance in such high-end systems. Considerable research has focussed on optimizing checkpointing; however, in practice, checkpointing still involves a high-cost overhead for users. In this paper, we study the checkpointing overhead seen by various applications running on leadership-class machines like the IBM Blue Gene/P at Argonne National Laboratory. In addition to studying popular applications, we design a methodology to help users understand and intelligently choose anmore » optimal checkpointing frequency to reduce the overall checkpointing overhead incurred. In particular, we study the Grid-Based Projector-Augmented Wave application, the Carr-Parrinello Molecular Dynamics application, the Nek5000 computational fluid dynamics application and the Parallel Ocean Program application-and analyze their memory usage and possible checkpointing trends on 65,536 processors of the Blue Gene/P system.« less

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

PubMed Central

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

2016-01-01

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

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

PubMed

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.

Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. Conclusion The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks. PMID:18093330

SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba.

PubMed

Winnicki, Konrad; Maszewski, Janusz

2012-11-01

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Checkpointing and Recovery in Distributed and Database Systems

ERIC Educational Resources Information Center

Wu, Jiang

2011-01-01

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

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma

PubMed Central

Sekar, Divya; Govene, Luisa; del Río, María-Luisa; Sirait-Fischer, Evelyn; Fink, Annika F.

2018-01-01

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity. PMID:29518903

Downregulation of BTLA on NKT Cells Promotes Tumor Immune Control in a Mouse Model of Mammary Carcinoma.

PubMed

Sekar, Divya; Govene, Luisa; Del Río, María-Luisa; Sirait-Fischer, Evelyn; Fink, Annika F; Brüne, Bernhard; Rodriguez-Barbosa, José I; Weigert, Andreas

2018-03-07

Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.

p53 functions as a cell cycle control protein in osteosarcomas.

PubMed

Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-11-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

PubMed

Finnigan, Gregory C; Sterling, Sarah M; Duvalyan, Angela; Liao, Elizabeth N; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

2016-07-15

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. © 2016 Finnigan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Diverse microRNAs with convergent functions regulate tumorigenesis.

PubMed

Zhu, Min-Yan; Zhang, Wei; Yang, Tao

2016-02-01

MicroRNAs (miRNAs) regulate several biological processes, including tumorigenesis. In order to comprehend the roles of miRNAs in cancer, various screens were performed to investigate the changes in the expression levels of miRNAs that occur in different types of cancer. The present review focuses on the results of five recent screens, whereby a number of overlapping miRNAs were identified to be downregulated or differentially regulated, whereas no miRNAs were observed to be frequently upregulated. Furthermore, the majority of the miRNAs that were common to >1 screen were involved in signaling networks, including wingless-related integration site, receptor tyrosine kinase and transforming growth factor-β, or in cell cycle checkpoint control. The present review will discuss the aforementioned miRNAs implicated in cell cycle checkpoint control and signaling networks.

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed Central

Paulovich, A G; Armour, C D; Hartwell, L H

1998-01-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed

Paulovich, A G; Armour, C D; Hartwell, L H

1998-09-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

Immune Checkpoint Inhibitors: An Innovation in Immunotherapy for the Treatment and Management of Patients with Cancer.

PubMed

Dine, Jennifer; Gordon, RuthAnn; Shames, Yelena; Kasler, Mary Kate; Barton-Burke, Margaret

2017-01-01

Cancer survival rates are generally increasing in the United States. These trends have been partially attributed to improvement in therapeutic strategies. Cancer immunotherapy is an example of one of the newer strategies used to fight cancer, which primes or activates the immune system to produce antitumor effects. The first half of this review paper concisely describes the cell mechanisms that control antitumor immunity and the major immunotherapeutic strategies developed to target these mechanisms. The second half of the review discusses in greater depth immune checkpoint inhibitors that have recently demonstrated tremendous promise for the treatment of diverse solid tumor types, including melanoma, non-small cell lung cancer, and others. More specifically, the mechanisms of action, side effects, and patient and family management and education concerns are discussed to provide oncology nurses up-to-date information relevant to caring for cancer-affected patients treated with immune checkpoint inhibitors. Future directions for cancer immunotherapy are considered.

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-03-14

To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17's RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. © 2016 Isokane et al.

Influenza vaccination of cancer patients during PD-1 blockade induces serological protection but may raise the risk for immune-related adverse events.

PubMed

Läubli, Heinz; Balmelli, Catharina; Kaufmann, Lukas; Stanczak, Michal; Syedbasha, Mohammedyaseen; Vogt, Dominik; Hertig, Astrid; Müller, Beat; Gautschi, Oliver; Stenner, Frank; Zippelius, Alfred; Egli, Adrian; Rothschild, Sacha I

2018-05-22

Immune checkpoint inhibiting antibodies were introduced into routine clinical practice for cancer patients. Checkpoint blockade has led to durable remissions in some patients, but may also induce immune-related adverse events (irAEs). Lung cancer patients show an increased risk for complications, when infected with influenza viruses. Therefore, vaccination is recommended. However, the efficacy and safety of influenza vaccination during checkpoint blockade and its influence on irAEs is unclear. Similarly, the influence of vaccinations on T cell-mediated immune reactions in patients during PD-1 blockade remains poorly defined. We vaccinated 23 lung cancer patients and 11 age-matched healthy controls using a trivalent inactivated influenza vaccine to investigate vaccine-induced immunity and safety during checkpoint blockade. We did not observe significant differences between patients and healthy controls in vaccine-induced antibody titers against all three viral antigens. Influenza vaccination resulted in protective titers in more than 60% of patients/participants. In cancer patients, the post-vaccine frequency of irAEs was 52.2% with a median time to occurrence of 3.2 months after vaccination. Six of 23 patients (26.1%) showed severe grade 3/4 irAEs. This frequency of irAEs might be higher than the rate previously published in the literature and the rate observed in a non-study population at our institution (all grades 25.5%, grade 3/4 9.8%). Although this is a non-randomized trial with a limited number of patients, the increased rate of immunological toxicity is concerning. This finding should be studied in a larger patient population.

The kinetochore proteins CENP-E and CENP-F directly and specifically interact with distinct BUB mitotic checkpoint Ser/Thr kinases.

PubMed

Ciossani, Giuseppe; Overlack, Katharina; Petrovic, Arsen; Huis In 't Veld, Pim J; Koerner, Carolin; Wohlgemuth, Sabine; Maffini, Stefano; Musacchio, Andrea

2018-05-10

The segregation of chromosomes during cell division relies on the function of the kinetochores, protein complexes that physically connect chromosomes with microtubules of the spindle. The metazoan proteins, centromere protein E (CENP-E) and CENP-F, are components of a fibrous layer of mitotic kinetochores named the corona. Several of their features suggest that CENP-E and CENP-F are paralogs: they are very large (comprising approximately 2700 and 3200 residues, respectively), contain abundant predicted coiled-coil structures, are C-terminally prenylated, and are endowed with microtubule-binding sites at their termini. Moreover, CENP-E contains an ATP-hydrolyzing motor domain that promotes microtubule plus end-directed motion. Here, we show that both CENP-E and CENP-F are recruited to mitotic kinetochores independently of the main corona constituent, the Rod-Zwilch-ZW10 (RZZ) complex. We identified specific interactions of CENP-F and CENP-E with budding uninhibited by benzimidazole 1 (BUB1) and BUB1-related (BUBR1) mitotic checkpoint Ser/Thr kinases, respectively, paralogous proteins involved in mitotic checkpoint control and chromosome alignment. Whereas BUBR1 was dispensable for kinetochore localization of CENP-E, BUB1 was stringently required for CENP-F localization. Through biochemical reconstitution, we demonstrated that the CENP-E-BUBR1 and CENP-F-BUB1 interactions are direct and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F and a kinase domain in BUBR1 or BUB1. Our findings are consistent with the existence of structurally similar BUB1-CENP-F and BUBR1-CENP-E complexes, supporting the notion that CENP-E and CENP-F are evolutionarily related. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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.

Experimental evaluation of sobriety checkpoint programs

DOT National Transportation Integrated Search

1995-06-01

Six California communities were selected to participate in the study on the basis of comparability and isolation from each other. Four of the communities' police departments implemented programs of sobriety checkpoints; the checkpoint configurations ...

ATM-dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

PubMed Central

Zhou, Tong; Chou, Jeff; Zhou, Yingchun; Simpson, Dennis A.; Cao, Feng; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

2013-01-01

The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. PMID:17699107

Sobriety checkpoints reduce crash deaths on Tennessee roads

DOT National Transportation Integrated Search

1999-06-19

Sobriety checkpoints are known to be effective in getting alcohol-impaired drivers off the roads. The National Highway Traffic Safety Administration funded equipment and conducted an evaluation of Tennessee's two-year statewide checkpoint demonstrati...

Evaluation of Charlottesville checkpoint operations

DOT National Transportation Integrated Search

1985-05-01

Under a grant from the Virginia Office of Highway Safety, the Charlottesville Police Department implemented a Driver's License and Sobriety Checkpoint Program from December 30, 1983 to December 31, 1984. During the period, 94 checkpoint operations we...

Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

PubMed

Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

2017-11-22

Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

NASA Astrophysics Data System (ADS)

Gaspar, Miguel; Shenk, Thomas

2006-02-01

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

Regulation of kinetochore recruitment of two essential mitotic spindle checkpoint proteins by Mps1 phosphorylation.

PubMed

Xu, Quanbin; Zhu, Songcheng; Wang, Wei; Zhang, Xiaojuan; Old, William; Ahn, Natalie; Liu, Xuedong

2009-01-01

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.

SNM1B/Apollo interacts with astrin and is required for the prophase cell cycle checkpoint.

PubMed

Liu, Lingling; Akhter, Shamima; Bae, Jae-Bum; Mukhopadhyay, Sudit S; Richie, Christopher T; Liu, Xiaojun; Legerski, Randy

2009-02-15

Previously, we have shown that SNM1A is a multifunctional gene involved in both the DNA damage response and in an early mitotic checkpoint in response to spindle stress. Another member of the SNM1 gene family, SNM1B/Apollo, has been shown to have roles in both the response to DNA interstrand cross-linking agents and in telomere protection during S phase. Here, we demonstrate a novel role for SNM1B/Apollo in mitosis in response to spindle stress. SNM1B-deficient cells exhibit a defect in the prophase checkpoint. Loss of the prophase checkpoint induces an extended mitotic delay, which is due to prolonged activation of the spindle checkpoint. In addition, we show that SNM1B/Apollo interacts with the essential microtubule binding protein Astrin. SNM1B/Apollo interacts with Astrin through its conserved metallo-beta-lactamase domain, and disruption of this interaction by point mutations results in a deficient prophase checkpoint. These findings suggest that SNM1B/Apollo and Astrin function together to enforce the prophase checkpoint in response to spindle stress.

Less Efficient G2-M Checkpoint Is Associated with an Increased Risk of Lung Cancer in African Americans

PubMed Central

Zheng, Yun-Ling; Loffredo, Christopher A.; Alberg, Anthony J.; Yu, Zhipeng; Jones, Raymond T.; Perlmutter, Donna; Enewold, Lindsey; Krasna, Mark J.; Yung, Rex; Shields, Peter G.; Harris, Curtis C.

2006-01-01

Cell cycle checkpoints play critical roles in the maintenance of genomic integrity. The inactivation of checkpoint genes by genetic and epigenetic mechanisms is frequent in all cancer types, as a less-efficient cell cycle control can lead to genetic instability and tumorigenesis. In an on-going case-control study consisting of 216 patients with non–small cell lung cancer, 226 population-based controls, and 114 hospital-based controls, we investigated the relationship of γ-radiation-induced G2-M arrest and lung cancer risk. Peripheral blood lymphocytes were cultured for 90 hours, exposed to 1.0 Gy γ-radiation, and harvested at 3 hours after γ-radiation treatment. γ-Radiation-induced G2-M arrest was measured as the percentage of mitotic cells in untreated cultures minus the percentage of mitotic cells in γ-radiation-treated cultures from the same subject. The mean percentage of γ-radiation-induced G2-M arrest was significantly lower in cases than in population controls (1.18 versus 1.44, P < 0.01) and hospital controls (1.18 versus 1.40, P = 0.01). When dichotomized at the 50th percentile value in combined controls (population and hospital controls), a lower level of γ-radiation-induced G2-M arrest was associated with an increased risk of lung cancer among African Americans after adjusting for baseline mitotic index, age, gender, and pack-years of smoking [adjusted odd ratio (OR), 2.25; 95% confidence interval (95% CI), 0.97–5.20]. A significant trend of an increased risk of lung cancer with a decreased level of G2-M arrest was observed (Ptrend = 0.02) among African Americans, with a lowest-versus-highest quartile adjusted OR of 3.74 (95% CI, 0.98–14.3). This trend was most apparent among African American females (Ptrend < 0.01), with a lowest-versus-highest quartile adjusted OR of 11.75 (95% CI, 1.47–94.04). The results suggest that a less-efficient DNA damage–induced G2-M checkpoint is associated with an increased risk of lung cancer among African Americans. Interestingly, we observed a stronger association of DNA damage–induced G2-M arrest and lung cancer among African Americans when compared with Caucasians. If replicated, these results may provide clues to the exceedingly high lung cancer incidence experienced by African Americans. PMID:16230422

Checkpoints couple transcription network oscillator dynamics to cell-cycle progression.

PubMed

Bristow, Sara L; Leman, Adam R; Simmons Kovacs, Laura A; Deckard, Anastasia; Harer, John; Haase, Steven B

2014-09-05

The coupling of cyclin dependent kinases (CDKs) to an intrinsically oscillating network of transcription factors has been proposed to control progression through the cell cycle in budding yeast, Saccharomyces cerevisiae. The transcription network regulates the temporal expression of many genes, including cyclins, and drives cell-cycle progression, in part, by generating successive waves of distinct CDK activities that trigger the ordered program of cell-cycle events. Network oscillations continue autonomously in mutant cells arrested by depletion of CDK activities, suggesting the oscillator can be uncoupled from cell-cycle progression. It is not clear what mechanisms, if any, ensure that the network oscillator is restrained when progression in normal cells is delayed or arrested. A recent proposal suggests CDK acts as a master regulator of cell-cycle processes that have the potential for autonomous oscillatory behavior. Here we find that mitotic CDK is not sufficient for fully inhibiting transcript oscillations in arrested cells. We do find that activation of the DNA replication and spindle assembly checkpoints can fully arrest the network oscillator via overlapping but distinct mechanisms. Further, we demonstrate that the DNA replication checkpoint effector protein, Rad53, acts to arrest a portion of transcript oscillations in addition to its role in halting cell-cycle progression. Our findings indicate that checkpoint mechanisms, likely via phosphorylation of network transcription factors, maintain coupling of the network oscillator to progression during cell-cycle arrest.

Checkpoints: it takes more than time to heal some wounds.

PubMed

Rhind, N; Russell, P

The S-phase DNA damage checkpoint seems to provide a twist on the checkpoint theme. Instead of delaying replication and allowing repair as a consequence, it may activate repair and delay replication as a consequence.

Enhancing Immune Checkpoint Inhibitor Therapy In Kidney Cancer

DTIC Science & Technology

2016-10-01

AWARD NUMBER: W81XWH-15-1-0141 TITLE: Enhancing Immune Checkpoint Inhibitor therapy in Kidney Cancer PRINCIPAL INVESTIGATOR: Hans-Joerg Hammers...Immune Checkpoint Inhibitor therapy in Kidney Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH- 15-1-0141 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...to develop strategies to enhance immune checkpoint inhibition in kidney cancer. The work is designed to test different strategies to induce or

Enhancing Immune Checkpoint Inhibitor Therapy in Kidney Cancer

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-15-1-0141 TITLE: Enhancing Immune Checkpoint Inhibitor therapy in Kidney Cancer PRINCIPAL INVESTIGATOR: Hans-Joerg Hammers...SUBTITLE Enhancing Immune Checkpoint Inhibitor therapy in Kidney Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH- 15-1-0141 5c. PROGRAM ELEMENT NUMBER...immune checkpoint inhibition in kidney cancer . The work is designed to test different strategies to induce or enhance the abscopal in a kidney cancer

A Survey of Rollback-Recovery Protocols in Message-Passing Systems

DTIC Science & Technology

1999-06-01

and M.A. Castillo. "Checkpointing through garbage collection." Technical report. Departamento de Ciencia de la Computation, Escuela de Ingenieria ...between consecutive checkpoints. It can be implemented by using the dirty-bit of the memory protection hardware or by emulating a dirty-bit in software [4...compare the program’s state with the previous checkpoint in software , and writing the difference in a new checkpoint [46]. The required storage and

mus304 encodes a novel DNA damage checkpoint protein required during Drosophila development

PubMed Central

Brodsky, Michael H.; Sekelsky, Jeff J.; Tsang, Garson; Hawley, R. Scott; Rubin, Gerald M.

2000-01-01

Checkpoints block cell cycle progression in eukaryotic cells exposed to DNA damaging agents. We show that several Drosophila homologs of checkpoint genes, mei-41, grapes, and 14-3-3ε, regulate a DNA damage checkpoint in the developing eye. We have used this assay to show that the mutagen-sensitive gene mus304 is also required for this checkpoint. mus304 encodes a novel coiled-coil domain protein, which is targeted to the cytoplasm. Similar to mei-41, mus304 is required for chromosome break repair and for genomic stability. mus304 animals also exhibit three developmental defects, abnormal bristle morphology, decreased meiotic recombination, and arrested embryonic development. We suggest that these phenotypes reflect distinct developmental consequences of a single underlying checkpoint defect. Similar mechanisms may account for the puzzling array of symptoms observed in humans with mutations in the ATM tumor suppressor gene. PMID:10733527

[Genetic Mutation Accumulation and Clinical Outcome of Immune Checkpoint Blockade Therapy].

PubMed

Takahashi, Masanobu

2016-06-01

Immune checkpoint blockade therapy has recently attracted great attention in the area of oncology. In Japan, since 2014, an anti-PD-1 antibody nivolumab and anti-CTLA-4 antibody ipilimumab have been available for the treatment of patients with malignant melanoma, and nivolumab has been available for patients with non-small cell lung cancer. Clinical trials using these drugs and other immune checkpoint inhibitors are currently in progress worldwide. The immune checkpoint blockade therapy is a promising new cancer therapy; however, not all patients with cancer can benefit from this therapy. Recent evidence shows that markers reflecting the extent of genetic mutation accumulation, including mutation burden, non-synonymous mutation that produces neoantigen, and microsatellite instability, possibly serve as promising marker to predict who can benefit from the immune checkpoint blockade therapy. Here, I introduce the recent evidence and discuss the correlation between genetic mutation accumulation and clinical outcome of immune checkpoint blockade therapy.

Linking TGF-beta-mediated Cdc25A inhibition and cytoskeletal regulation through RhoA/p160(ROCK) signaling.

PubMed

Brown, Kimberly; Bhowmick, Neil A

2004-04-01

Transforming growth factor-beta (TGF-beta) can mediate G(1)/S cell-cycle inhibition and changes in the cytoskeletal organization through multiple parallel downstream signaling pathways. Recent findings regarding TGF-beta-mediated cell-cycle checkpoint control and epithelial to mesenchymal transition have converged to the RhoA/p160(ROCK) signaling pathway. The activation of TGF-beta-mediated p160(ROCK)rapidly inhibits the Cdc25A phosphatase as a component of the G(1)/S checkpoint control at the time cytoskeletal re-organization occurs. This can be likened to the ability to preserve genomic integrity in circumstances of genotoxic stress. The inactivation of the RhoA/p160(ROCK) pathway may be a mechanism by which cancer cells bypass growth inhibition even in the presence of TGF-beta.

Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint-restricted Cdc20.

PubMed

Boekhout, Michiel; Wolthuis, Rob

2015-04-15

Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity. © 2015. Published by The Company of Biologists Ltd.

Checkpoints: It takes more than time to heal some wounds

PubMed Central

Rhind, Nicholas; Russell, Paul

2010-01-01

The S-phase DNA damage checkpoint seems to provide a twist on the checkpoint theme. Instead of delaying replication and allowing repair as a consequence, it may activate repair and delay replication as a consequence. PMID:11137027

p53 functions as a cell cycle control protein in osteosarcomas.

PubMed Central

Diller, L; Kassel, J; Nelson, C E; Gryka, M A; Litwak, G; Gebhardt, M; Bressac, B; Ozturk, M; Baker, S J; Vogelstein, B

1990-01-01

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae. Images PMID:2233717

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

PubMed Central

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

2016-01-01

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

Viral-induced Modulation of Multiple Checkpoint Proteins in Cancers.

PubMed

Nuovo, Gerard J; Folcik, Virginia A; Magro, Cynthia

2017-07-01

Therapy with checkpoint inhibitors represents a major advance in cancer treatment. The purpose of this study was to examine the expression patterns of the checkpoint proteins programmed death ligand 1 (PD L1), PD L2, indoleamine 2,3-dioxygenase 1 (IDO1), and cytotoxic T-lymphocyte antigen 4 (CTLA4) in cancers including those associated with viral infections. Normal, noninflamed tissues rarely express checkpoint proteins with exceptions including the placenta and stomach. Expression of PD L1 was noted in 30%, PD L2 in 18%, IDO1 in 13%, and CTLA4 in 14% of 333 nonviral malignancies including endometrial, ovarian, lung, and breast cancers. The expression of each checkpoint protein was significantly higher among 166 cases of viral-related (mostly human papillomavirus) cancers where expression of PD L1 was noted in 84%, PD L2 in 67%, IDO1 in 61%, and CTLA4 in 37% (each P value <0.001); 97% of the viral-related cancers showed expression of at least 1 checkpoint protein. In addition, over 90% of the CD8 cells in the viral-associated cancers were quiescent based on low coexpression of Ki-67 as well as pSTAT1. It is concluded that viral infection in cancers is associated with the increased expression of key checkpoint proteins. This indicates that cancers with productive viral infection may be better targets for checkpoint inhibitor therapy.

SU-E-I-56: Scan Angle Reduction for a Limited-Angle Intrafraction Verification (LIVE) System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, L; Zhang, Y; Yin, F

Purpose: To develop a novel adaptive reconstruction strategy to further reduce the scanning angle required by the limited-angle intrafraction verification (LIVE) system for intrafraction verification. Methods: LIVE acquires limited angle MV projections from the exit fluence of the arc treatment beam or during gantry rotation between static beams. Orthogonal limited-angle kV projections are also acquired simultaneously to provide additional information. LIVE considers the on-board 4D-CBCT images as a deformation of the prior 4D-CT images, and solves the deformation field based on deformation models and data fidelity constraint. LIVE reaches a checkpoint after a limited-angle scan, and reconstructs 4D-CBCT for intrafractionmore » verification at the checkpoint. In adaptive reconstruction strategy, a larger scanning angle of 30° is used for the first checkpoint, and smaller scanning angles of 15° are used for subsequent checkpoints. The onboard images reconstructed at the previous adjacent checkpoint are used as the prior images for reconstruction at the current checkpoint. As the algorithm only needs to reconstruct the small deformation occurred between adjacent checkpoints, projections from a smaller scan angle provide enough information for the reconstruction. XCAT was used to simulate tumor motion baseline drift of 2mm along sup-inf direction at every subsequent checkpoint, which are 15° apart. Adaptive reconstruction strategy was used to reconstruct the images at each checkpoint using orthogonal 15° kV and MV projections. Results: Results showed that LIVE reconstructed the tumor volumes accurately using orthogonal 15° kV-MV projections. Volume percentage differences (VPDs) were within 5% and center of mass shifts (COMS) were within 1mm for reconstruction at all checkpoints. Conclusion: It's feasible to use an adaptive reconstruction strategy to further reduce the scan angle needed by LIVE to allow faster and more frequent intrafraction verification to minimize the treatment errors in lung cancer treatments. Grant from Varian Medical System.« less

Overexpression of Mps1 in colon cancer cells attenuates the spindle assembly checkpoint and increases aneuploidy.

PubMed

Ling, Youguo; Zhang, Xiaojuan; Bai, Yuanyuan; Li, Ping; Wei, Congwen; Song, Ting; Zheng, Zirui; Guan, Kai; Zhang, Yanhong; Zhang, Buchang; Liu, Xuedong; Ma, Runlin Z; Cao, Cheng; Zhong, Hui; Xu, Quanbin

2014-08-08

The spindle assembly checkpoint kinase Mps1 is highly expressed in several types of cancers, but its cellular involvement in tumorigenesis is less defined. Herein, we confirm that Mps1 is overexpressed in colon cancer tissues. Further, we find that forced expression of Mps1 in the colon cancer cell line SW480 enables cells to become resistant to both Mps1 inhibition-induced checkpoint depletion and cell death. Overexpression of Mps1 also increases genome instability in tumor cells owing to a weakened spindle assembly checkpoint. Collectively, our findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint. Copyright © 2014 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.

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

An overactivated ATR/CHK1 pathway is responsible for the prolonged G2 accumulation in irradiated AT cells

NASA Technical Reports Server (NTRS)

Wang, Xiang; Khadpe, Jay; Hu, Baocheng; Iliakis, George; Wang, Ya

2003-01-01

Induction of checkpoint responses in G1, S, and G2 phases of the cell cycle after exposure of cells to ionizing radiation (IR) is essential for maintaining genomic integrity. Ataxia telangiectasia mutated (ATM) plays a key role in initiating this response in all three phases of the cell cycle. However, cells lacking functional ATM exhibit a prolonged G2 arrest after IR, suggesting regulation by an ATM-independent checkpoint response. The mechanism for this ataxia telangiectasia (AT)-independent G2-checkpoint response remains unknown. We report here that the G2 checkpoint in irradiated human AT cells derives from an overactivation of the ATR/CHK1 pathway. Chk1 small interfering RNA abolishes the IR-induced prolonged G2 checkpoint and radiosensitizes AT cells to killing. These results link the activation of ATR/CHK1 with the prolonged G2 arrest in AT cells and show that activation of this G2 checkpoint contributes to the survival of AT cells.

A smart checkpointing scheme for improving the reliability of clustering routing protocols.

PubMed

Min, Hong; Jung, Jinman; Kim, Bongjae; Cho, Yookun; Heo, Junyoung; Yi, Sangho; Hong, Jiman

2010-01-01

In wireless sensor networks, system architectures and applications are designed to consider both resource constraints and scalability, because such networks are composed of numerous sensor nodes with various sensors and actuators, small memories, low-power microprocessors, radio modules, and batteries. Clustering routing protocols based on data aggregation schemes aimed at minimizing packet numbers have been proposed to meet these requirements. In clustering routing protocols, the cluster head plays an important role. The cluster head collects data from its member nodes and aggregates the collected data. To improve reliability and reduce recovery latency, we propose a checkpointing scheme for the cluster head. In the proposed scheme, backup nodes monitor and checkpoint the current state of the cluster head periodically. We also derive the checkpointing interval that maximizes reliability while using the same amount of energy consumed by clustering routing protocols that operate without checkpointing. Experimental comparisons with existing non-checkpointing schemes show that our scheme reduces both energy consumption and recovery latency.

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

DOE PAGES

Cuellar-Hengartner, Leticia; Watkins, Daniel; Kubicek, Deborah A.; ...

2015-09-01

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

Analyzing checkpointing trends for applications on the IBM Blue Gene/P system.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Naik, H.; Gupta, R.; Beckman, P.

Current petascale systems have tens of thousands of hardware components and complex system software stacks, which increase the probability of faults occurring during the lifetime of a process. Checkpointing has been a popular method of providing fault tolerance in high-end systems. While considerable research has been done to optimize checkpointing, in practice the method still involves a high-cost overhead for users. In this paper, we study the checkpointing overhead seen by applications running on leadership-class machines such as the IBM Blue Gene/P at Argonne National Laboratory. We study various applications and design a methodology to assist users in understanding andmore » choosing checkpointing frequency and reducing the overhead incurred. In particular, we study three popular applications -- the Grid-Based Projector-Augmented Wave application, the Carr-Parrinello Molecular Dynamics application, and a Nek5000 computational fluid dynamics application -- and analyze their memory usage and possible checkpointing trends on 32,768 processors of the Blue Gene/P system.« less

A Smart Checkpointing Scheme for Improving the Reliability of Clustering Routing Protocols

PubMed Central

Min, Hong; Jung, Jinman; Kim, Bongjae; Cho, Yookun; Heo, Junyoung; Yi, Sangho; Hong, Jiman

2010-01-01

In wireless sensor networks, system architectures and applications are designed to consider both resource constraints and scalability, because such networks are composed of numerous sensor nodes with various sensors and actuators, small memories, low-power microprocessors, radio modules, and batteries. Clustering routing protocols based on data aggregation schemes aimed at minimizing packet numbers have been proposed to meet these requirements. In clustering routing protocols, the cluster head plays an important role. The cluster head collects data from its member nodes and aggregates the collected data. To improve reliability and reduce recovery latency, we propose a checkpointing scheme for the cluster head. In the proposed scheme, backup nodes monitor and checkpoint the current state of the cluster head periodically. We also derive the checkpointing interval that maximizes reliability while using the same amount of energy consumed by clustering routing protocols that operate without checkpointing. Experimental comparisons with existing non-checkpointing schemes show that our scheme reduces both energy consumption and recovery latency. PMID:22163389

G1 arrest induction represents a critical determinant for cisplatin cytotoxicity in G1 checkpoint-retaining human cancers.

PubMed

Un, Frank

2007-04-01

Cisplatin has been used effectively to treat various human cancer types; yet, the precise mechanism underlying its cytotoxicity remains unknown. In eukaryotes, progression through G1 is monitored by a checkpoint, which executes G1 arrest in the event of DNA damage to allow time for repair before initiating DNA replication. The retinoblastoma tumor suppressor gene is an integral component of the mammalian G1 checkpoint. The utility of the retinoblastoma gene as a therapeutic for human cancers has been investigated. Intriguingly, the cytotoxicity profile of the retinoblastoma gene therapy closely parallels the clinical targets of cisplatin. It prompted an investigation into the potential role of the checkpoint-induced G1 arrest in cisplatin cytotoxicity. Here, the evidence that G1 arrest induction represents a critical step in cisplatin-induced lytic path is presented. First, cisplatin-treated human cancer cells undergo a prolonged G1 arrest before dying. Second, triggering G1 arrest via infection with a recombinant adenovirus expressing the human retinoblastoma gene is sufficient to potentiate lethality in the absence of cisplatin. Third, the extent of the lethality induced correlates with the G1-arresting potential of the ectopically expressed human retinoblastoma polypeptide. Fourth, human cancer cells resistant to cisplatin do not undergo G1 arrest despite cisplatin treatment. The above mechanism may be exploited to develop therapeutics that preserve the efficacy of cisplatin yet bypass its mutagenicity associated with the formation of secondary tumors.

Increased PD-1+ and TIM-3+ TILs during cetuximab therapy inversely correlates with response in head and neck cancer patients

PubMed Central

Jie, Hyun-Bae; Srivastava, Raghvendra M.; Argiris, Athanassios; Bauman, Julie E.; Kane, Lawrence P.; Ferris, Robert L.

2017-01-01

Despite emerging appreciation for the important role of immune checkpoint receptors in regulating the effector functions of T cells, it is unknown whether their expression is involved in determining the clinical outcome in response to cetuximab therapy. We examined the expression patterns of immune checkpoint receptors (including PD-1, CTLA-4, and TIM-3) and cytolytic molecules (including granzyme B and perforin) of CD8+ tumor-infiltrating lymphocytes (TILs) and compared them to those of peripheral blood T lymphocytes (PBLs) in patients with head and neck cancer (HNSCC) during cetuximab therapy. The frequency of PD-1 and TIM-3 expression was significantly increased in CD8+ TILs compared to CD8+ PBLs (P = 0.008 and P = 0.02, respectively). This increased CD8+ TIL population co-expressed granzyme B/perforin and PD-1/TIM-3, which suggests a regulatory role for these immune checkpoint receptors in cetuximab-promoting cytolytic activities of CD8+ TIL. Indeed, the increased frequency of PD-1+ and TIM-3+ CD8+ TILs was inversely correlated with clinical outcome of cetuximab therapy. These findings support the use of PD-1 and TIM-3 as biomarkers to reflect immune status of CD8+ T cells in the tumor microenvironment during cetuximab therapy. Blockade of these immune checkpoint receptors might enhance cetuximab-based cancer immunotherapy to reverse CD8+ TIL dysfunction, thus potentially improving clinical outcomes of HNSCC patients. PMID:28408386

P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

PubMed Central

Lee, Kyunghee; Kenny, Alison E.

2010-01-01

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends. PMID:20462950

FANCA safeguards interphase and mitosis during hematopoiesis in vivo

PubMed Central

Abdul-Sater, Zahi; Cerabona, Donna; Sierra Potchanant, Elizabeth; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz

2015-01-01

Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in non-hematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material (PCM) to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA cross-linking and antimitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that the FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers. PMID:26366677

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

PubMed

Benoit, Beatrice; He, Chun Hua; Zhang, Fan; Votruba, Sarah M; Tadros, Wael; Westwood, J Timothy; Smibert, Craig A; Lipshitz, Howard D; Theurkauf, William E

2009-03-01

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies.

PubMed

Friedman, Jay; Morisada, Megan; Sun, Lillian; Moore, Ellen C; Padget, Michelle; Hodge, James W; Schlom, Jeffrey; Gameiro, Sofia R; Allen, Clint T

2018-06-21

Natural killer (NK) cells recognize and lyse target tumor cells in an MHC-unrestricted fashion and complement antigen- and MHC-restricted killing by T-lymphocytes. NK cells and T-lymphocytes mediate early killing of targets through a common granzyme B-dependent mechanism. Tumor cell resistance to granzyme B and how this alters NK cell killing is not clearly defined. Tumor cell sensitivity to cultured murine KIL and human high affinity NK (haNK) cells in the presence or absence of AZD1775, a small molecule inhibitor of WEE1 kinase, was assessed via real time impedance analysis. Mechanisms of enhanced sensitivity to NK lysis were determined and in vivo validation via adoptive transfer of KIL cells into syngeneic mice was performed. Cultured murine KIL cells lyse murine oral cancer 2 (MOC2) cell targets more efficiently than freshly isolated peripheral murine NK cells. MOC2 sensitivity to granzyme B-dependent KIL cell lysis was enhanced by inhibition of WEE1 kinase, reversing G2/M cell cycle checkpoint activation and resulting in enhanced DNA damage and apoptosis. Treatment of MOC2 tumor-bearing wild-type C57BL/6 mice with AZD1775 and adoptively transferred KIL cells resulted in enhanced tumor growth control and survival over controls or either treatment alone. Validating these findings in human models, WEE1 kinase inhibition sensitized two human head and neck cancer cell lines to direct lysis by haNK cells. Further, WEE1 kinase inhibition sensitized these cell lines to antibody-dependent cell-mediated cytotoxicity when combined with the anti-PD-L1 IgG1 mAb Avelumab. Tumor cell resistance to granzyme B-induced cell death can be reversed through inhibition of WEE1 kinase as AZD1775 sensitized both murine and human head and neck cancer cells to NK lysis. These data provide the pre-clinical rationale for the combination of small molecules that reverse cell cycle checkpoint activation and NK cellular therapies.

The CD47-SIRPα signaling axis as an innate immune checkpoint in cancer.

PubMed

Matlung, Hanke L; Szilagyi, Katka; Barclay, Neil A; van den Berg, Timo K

2017-03-01

Immune checkpoint inhibitors, including those targeting CTLA-4/B7 and the PD-1/PD-L1 inhibitory pathways, are now available for clinical use in cancer patients, with other interesting checkpoint inhibitors being currently in development. Most of these have the purpose to promote adaptive T cell-mediated immunity against cancer. Here, we review another checkpoint acting to potentiate the activity of innate immune cells towards cancer. This innate immune checkpoint is composed of what has become known as the 'don't-eat me' signal CD47, which is a protein broadly expressed on normal cells and often overexpressed on cancer cells, and its counter-receptor, the myeloid inhibitory immunoreceptor SIRPα. Blocking CD47-SIRPα interactions has been shown to promote the destruction of cancer cells by phagocytes, including macrophages and neutrophils. Furthermore, there is growing evidence that targeting of the CD47-SIRPα axis may also promote antigen-presenting cell function and thereby stimulate adaptive T cell-mediated anti-cancer immunity. The development of CD47-SIRPα checkpoint inhibitors and the potential side effects that these may have are discussed. Collectively, this identifies the CD47-SIRPα axis as a promising innate immune checkpoint in cancer, and with data of the first clinical studies with CD47-SIRPα checkpoint inhibitors expected within the coming years, this is an exciting and rapidly developing field. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Immunotherapy: a new treatment paradigm in bladder cancer

PubMed Central

Davarpanah, Nicole N.; Yuno, Akira; Trepel, Jane B.; Apolo, Andrea B.

2017-01-01

Purpose of review T-cell checkpoint blockade has become a dynamic immunotherapy for bladder cancer. In 2016, atezolizumab, an immune checkpoint inhibitor, became the first new drug approved in metastatic urothelial carcinoma (mUC) in over 30 years. In 2017, nivolumab was also approved for the same indication. This overview of checkpoint inhibitors in clinical trials focuses on novel immunotherapy combinations, predictive biomarkers including mutational load and neoantigen identification, and an evaluation of the future of bladder cancer immunotherapy. Recent findings Programed cell death protein 1/programed death-ligand 1 (PD-1/PD-L1) checkpoint inhibitors have achieved durable clinical responses in a subset of previously treated and treatment-naïve patients with mUC. The combination of PD-1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) has successfully improved response rates in multiple malignancies, and combination studies are underway in many tumor types, including bladder cancer, combining T-cell checkpoint blockade with other checkpoint agents and immunomodulatory therapies. Strong tumor responses to checkpoint blockade have been reported to be positively associated with expression of PD-L1 on tumor and tumor-infiltrating immune cells and with increased mutation-associated neoantigen load, which may lead to the development of predictive biomarkers. Summary Recent clinical evidence suggests that mUC is susceptible to T-cell checkpoint blockade. A global effort is underway to achieve higher response rates and more durable remissions, accelerate the development of immunotherapies, employ combination therapies, and test novel immune targets. PMID:28306559

Immune checkpoint therapy in liver cancer.

PubMed

Xu, Feng; Jin, Tianqiang; Zhu, Yuwen; Dai, Chaoliu

2018-05-29

Immune checkpoints include stimulatory and inhibitory checkpoint molecules. In recent years, inhibitory checkpoints, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein-1 (PD-1), and programmed cell death ligand 1 (PD-L1), have been identified to suppress anti-tumor immune responses in solid tumors. Novel drugs targeting immune checkpoints have succeeded in cancer treatment. Specific PD-1 blockades were approved for treatment of melanoma in 2014 and for treatment of non-small-cell lung cancer in 2015 in the United States, European Union, and Japan. Preclinical and clinical studies show immune checkpoint therapy provides survival benefit for greater numbers of patients with liver cancer, including hepatocellular carcinoma and cholangiocarcinoma, two main primary liver cancers. The combination of anti-PD-1/PD-L1 with anti-CTLA-4 antibodies is being evaluated in phase 1, 2 or 3 trials, and the results suggest that an anti-PD-1 antibody combined with locoregional therapy or other molecular targeted agents is an effective treatment strategy for HCC. In addition, studies on activating co-stimulatory receptors to enhance anti-tumor immune responses have increased our understanding regarding this immunotherapy in liver cancer. Epigenetic modulations of checkpoints for improving the tumor microenvironment also expand our knowledge of potential therapeutic targets in improving the tumor microenvironment and restoring immune recognition and immunogenicity. In this review, we summarize current knowledge and recent developments in immune checkpoint-based therapies for the treatment of hepatocellular carcinoma and cholangiocarcinoma and attempt to clarify the mechanisms underlying its effects.

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

PubMed

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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Aviation security : vulnerabilities still exist in the aviation security system

DOT National Transportation Integrated Search

2000-04-06

The testimony today discusses the Federal Aviation Administration's (FAA) efforts to implement and improve security in two key areas: air traffic control computer systems and airport passenger screening checkpoints. Computer systems-and the informati...

Telomere shortening triggers a feedback loop to enhance end protection

PubMed Central

Yang, Chia-Wei; Tseng, Shun-Fu; Yu, Chia-Jung; Chung, Chia-Yu; Chang, Cheng-Yen; Pobiega, Sabrina

2017-01-01

Abstract Telomere homeostasis is controlled by both telomerase machinery and end protection. Telomere shortening induces DNA damage sensing kinases ATM/ATR for telomerase recruitment. Yet, whether telomere shortening also governs end protection is poorly understood. Here we discover that yeast ATM/ATR controls end protection. Rap1 is phosphorylated by Tel1 and Mec1 kinases at serine 731, and this regulation is stimulated by DNA damage and telomere shortening. Compromised Rap1 phosphorylation hampers the interaction between Rap1 and its interacting partner Rif1, which thereby disturbs the end protection. As expected, reduction of Rap1–Rif1 association impairs telomere length regulation and increases telomere–telomere recombination. These results indicate that ATM/ATR DNA damage checkpoint signal contributes to telomere protection by strengthening the Rap1–Rif1 interaction at short telomeres, and the checkpoint signal oversees both telomerase recruitment and end capping pathways to maintain telomere homeostasis. PMID:28575419

THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

PubMed Central

González Besteiro, Marina A.; Gottifredi, Vanesa

2014-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. 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. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

The tumor suppressor CDKN3 controls mitosis

PubMed Central

Nalepa, Grzegorz; Barnholtz-Sloan, Jill; Enzor, Rikki; Dey, Dilip; He, Ying; Gehlhausen, Jeff R.; Lehmann, Amalia S.; Park, Su-Jung; Yang, Yanzhu; Yang, Xianlin; Chen, Shi; Guan, Xiaowei; Chen, Yanwen; Renbarger, Jamie; Yang, Feng-Chun; Parada, Luis F.

2013-01-01

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2pThr-161 at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics. PMID:23775190

The Scalable Checkpoint/Restart Library

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moody, A.

The Scalable Checkpoint/Restart (SCR) library provides an interface that codes may use to worite our and read in application-level checkpoints in a scalable fashion. In the current implementation, checkpoint files are cached in local storage (hard disk or RAM disk) on the compute nodes. This technique provides scalable aggregate bandwidth and uses storage resources that are fully dedicated to the job. This approach addresses the two common drawbacks of checkpointing a large-scale application to a shared parallel file system, namely, limited bandwidth and file system contention. In fact, on current platforms, SCR scales linearly with the number of compute nodes.more » It has been benchmarked as high as 720GB/s on 1094 nodes of Atlas, which is nearly two orders of magnitude faster thanthe parallel file system.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko

2009-10-23

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

Hallmarks of response to immune checkpoint blockade

PubMed Central

Cogdill, Alexandria P; Andrews, Miles C; Wargo, Jennifer A

2017-01-01

Unprecedented advances have been made in the treatment of cancer through the use of immune checkpoint blockade, with approval of several checkpoint blockade regimens spanning multiple cancer types. However, responses to this form of therapy are not universal, and insights are clearly needed to identify optimal biomarkers of response and to combat mechanisms of therapeutic resistance. A working knowledge of the hallmarks of cancer yields insight into responses to immune checkpoint blockade, although the focus of this is rather tumour-centric and additional factors are pertinent, including host immunity and environmental influences. Herein, we describe the foundation for pillars and hallmarks of response to immune checkpoint blockade, with a discussion of their relevance to immune monitoring and mechanisms of resistance. Evolution of this understanding will ultimately help guide treatment strategies to enhance therapeutic responses. PMID:28524159

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

PubMed

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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM

PubMed Central

Bower, Jacquelyn J.; Zhou, Yingchun; Zhou, Tong; Simpson, Dennis A.; Arlander, Sonnet J.; Paules, Richard S.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in CHEK2, but no appreciable phosphorylation of Ser139 on H2AX. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage. PMID:20372057

Neuro-Oncology Branch

MedlinePlus

... Neurological Disorders and Stroke (NINDS) to develop novel experimental therapeutics for children and adults with tumors of ... most promising of these therapeutic approaches to larger groups to do large-scale ... checkpoint inhibitor, can improve control of cancer for patients with several types of ...

Chemical genetic inhibition of Mps1 in stable human cell lines reveals novel aspects of Mps1 function in mitosis.

PubMed

Sliedrecht, Tale; Zhang, Chao; Shokat, Kevan M; Kops, Geert J P L

2010-04-22

Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1. We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as) that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells. Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability.

Immune Checkpoint Inhibitors for Patients With Advanced Non-Small-Cell Lung Cancer: A Systematic Review.

PubMed

Ellis, Peter M; Vella, Emily T; Ung, Yee C

2017-09-01

Second-line treatment options are limited for patients with advanced non-small-cell lung cancer (NSCLC). Standard therapy includes the cytotoxic agents docetaxel and pemetrexed, and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors erlotinib and gefitinib. Immune checkpoint inhibitors are a new class of treatment that have shown durable overall radiologic response rates and have been well tolerated. The objective of this systematic review was to investigate the efficacy of immune checkpoint inhibitors compared with other chemotherapies in patients with advanced NSCLC. Medline, Embase, and PubMed were searched for randomized controlled trials comparing treatment with immune checkpoint inhibitors against treatment with chemotherapy in patients with stage IIIB or IV NSCLC. Nine randomized controlled trials with 15 publications were included. A significant overall survival benefit of second-line nivolumab (nonsquamous: hazard ratio [HR] = 0.72, 95% confidence interval [CI], 0.60-0.77; P < .001; squamous: HR = 0.59, 95% CI, 0.44-0.79; P < .001) or second-line atezolizumab (HR = 0.73, 95% CI, 0.62-0.87; P = .0003) or second-line pembrolizumab (in patients with programmed cell death ligand 1 [PD-L1]-positive tumors) (pembrolizumab 2 mg/kg HR = 0.71, 95% CI, 0.58-0.88; P = .0008; pembrolizumab 10 mg/kg HR = 0.61, 95% CI, 0.49-0.75; P < .0001) or first-line pembrolizumab (HR = 0.60, 95% CI, 0.41-0.89; P = .005) compared with chemotherapy was found. The adverse effects were mainly higher in the chemotherapy arms. For patients with advanced stage IIIB/IV NSCLC, the improvement in overall survival outweighed the harms and supported the use of first-line pembrolizumab (in patients with ≥ 50% PD-L1-positive tumors) or second-line nivolumab, atezolizumab, or pembrolizumab (in patients with PD-L1-positive tumors). Copyright © 2017 Elsevier Inc. All rights reserved.

Intellectual property issues of immune checkpoint inhibitors

PubMed Central

Storz, Ulrich

2016-01-01

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

Message Efficient Checkpointing and Rollback Recovery in Heterogeneous Mobile Networks

NASA Astrophysics Data System (ADS)

Jaggi, Parmeet Kaur; Singh, Awadhesh Kumar

2016-06-01

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

Toward innovative combinational immunotherapy: A systems biology perspective.

PubMed

Li, Xue-Tao; Yang, Jin-Ji; Wu, Yi-Long; Hou, Jun

2018-05-08

The treatment of non-small-cell lung cancer (NSCLC) has advanced significantly in the last decades. Especially immune checkpoint inhibitors have shown inconceivable effect on enhancing host anti-tumor activity in NSCLC. However, the limitation of checkpoint blockade monotherapy seems unavoidable in most of the NSCLC patients and only ∼20% of them achieved response to monotherapy with immune checkpoint inhibitors. Thus combining immune checkpoint inhibitors with other agents with different action mechanisms holds a promise to revitalize NSCLC treatment, such as the combination of checkpoint inhibitors with angiogenesis inhibitors, or with chemotherapy, as well as the combination of two checkpoint inhibitors. Recently, various combinational strategies have been explored to setup promising combination regimens and to understand the action mechanisms. In this review, we summarize the suspected synergistic mechanisms of several combinational approaches by reviewing the available preclinical and clinical data. Then we discuss in light of the current knowledge of cancer biology and systems biology the important facets to be examined when setting up a framework for developing immunotherapy-based combination strategies. Copyright © 2018. Published by Elsevier Ltd.

Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells

PubMed Central

Janssen, Aniek; Kops, Geert J. P. L.; Medema, René H.

2009-01-01

The mitotic checkpoint has evolved to prevent chromosome mis-segregations by delaying mitosis when unattached chromosomes are present. Inducing severe chromosome segregation errors by ablating the mitotic checkpoint causes cell death. Here we have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in four tumor cell lines caused mild chromosome mis-segregations, but no lethality. These cells were, however, remarkably more sensitive to low doses of taxol, which enhanced the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing levels of Mps1 or BubR1, proteins having dual roles in checkpoint activation and chromosome alignment, but not by reducing Mad2, functioning solely in the mitotic checkpoint. Moreover, we find that untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sublethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells by enhancing chromosome mis-segregations. PMID:19855003

Berkeley lab checkpoint/restart (BLCR) for Linux clusters

DOE PAGES

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 instancemore » 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. © 2006 IOP Publishing Ltd.« less

Musculoskeletal and rheumatic diseases induced by immune checkpoint inhibitors: a review of the literature.

PubMed

Benfaremo, Devis; Manfredi, Lucia; Luchetti, Michele Maria; Gabrielli, Armando

2018-05-08

Immune checkpoint inhibitors are a new promising class of antitumor drugs that have been associated to a number of immune-related adverse events (AEs), including musculoskeletal and rheumatic disease. We searched Medline reviewing reports of musculoskeletal and rheumatic AEs induced by immune checkpoint inhibitors. Several musculoskeletal and rheumatic AEs associated with immune checkpoint inhibitors treatment are reported in literature. In particular, arthralgia and myalgia were the most common reported AEs, whereas the prevalence of arthritis, myositis and vasculitis is less characterized and mainly reported in case series and case reports. Other occasionally described AEs are sicca syndrome, polymyalgia rheumatica, systemic lupus erythematosus and sarcoidosis. Newly induced musculoskeletal and rheumatic diseases are a frequent adverse event associated with immune checkpoint inhibitors treatment. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Combination Controversies: Checkpoint Inhibition Alone or in Combination for the Treatment of Melanoma?

PubMed

Warner, Allison Betof; Postow, Michael A

2018-05-15

The immune checkpoint inhibitors ipilimumab, nivolumab, and pembrolizumab have dramatically improved outcomes for patients with metastatic melanoma; however, not all patients benefit from monotherapy with these agents. To address this issue, complementary combinations of immunotherapy are increasingly being explored as a strategy to improve outcomes. However, combinatorial approaches come with heightened risk of toxicity. In this review, we highlight combinations for which there are prospective data from clinical trials. The combinations discussed include ipilimumab plus anti-programmed death 1 agents, ipilimumab plus granulocyte-macrophage colony-stimulating factor, checkpoint inhibitor plus talimogene laherparepvec, ipilimumab plus chemotherapy, checkpoint inhibitor plus BRAF/MEK targeted therapy, and checkpoint inhibition plus radiation therapy. We discuss data regarding the efficacy and toxicity of combination therapy, and we identify clinical scenarios that may favor treatment with combination therapy.

As we age: Does slippage of quality control in the immune system lead to collateral damage?

PubMed

Müller, Ludmila; Pawelec, Graham

2015-09-01

The vertebrate adaptive immune system is remarkable for its possession of a very broad range of antigen receptors imbuing the system with exquisite specificity, in addition to the phagocytic and inflammatory cells of the innate system shared with invertebrates. This system requires strict control both at the level of the generation the cells carrying these receptors and at the level of their activation and effector function mediation in order to avoid autoimmunity and mitigate immune pathology. Thus, quality control checkpoints are built into the system at multiple nodes in the response, relying on clonal selection and regulatory networks to maximize pathogen-directed effects and minimize collateral tissue damage. However, these checkpoints are compromised with age, resulting in poorer immune control manifesting as tissue-damaging autoimmune and inflammatory phenomena which can cause widespread systemic disease, paradoxically compounding the problems associated with increased susceptibility to infectious disease and possibly cancer in the elderly. Better understanding the reasons for slippage of immune control will pave the way for developing rational strategies for interventions to maintain appropriate immunity while reducing immunopathology. Copyright © 2015 Elsevier B.V. All rights reserved.

Next generation of immune checkpoint therapy in cancer: new developments and challenges.

PubMed

Marin-Acevedo, Julian A; Dholaria, Bhagirathbhai; Soyano, Aixa E; Knutson, Keith L; Chumsri, Saranya; Lou, Yanyan

2018-03-15

Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body's immunological activity against tumors. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clinical activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or molecules targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clinical trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Riesen, Rolf E.; Bridges, Patrick G.; Stearley, Jon R.

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 systemsmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko

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 processmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holmfeldt, Per; Sellin, Mikael E.; Gullberg, Martin, E-mail: Martin.Gullberg@molbiol.umu.se

2010-07-15

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

Emerging growth factor receptor antagonists for the treatment of renal cell carcinoma.

PubMed

Zahoor, Haris; Rini, Brian I

2016-12-01

The landscape of systemic treatment for metastatic renal cell carcinoma (RCC) has dramatically changed with the introduction of targeted agents including vascular endothelial growth factor (VEGF) inhibitors. Recently, multiple new agents including growth factor receptor antagonists and a checkpoint inhibitor were approved for the treatment of refractory metastatic RCC based on encouraging benefit shown in clinical trials. Areas covered: The background and biological rationale of existing treatment options including a brief discussion of clinical trials which led to their approval, is presented. This is followed by reviewing the limitations of these therapeutic options, medical need to develop new treatments and major goals of ongoing research. We then discuss two recently approved growth factor receptor antagonists i.e. cabozantinib and lenvatinib, and a recently approved checkpoint inhibitor, nivolumab, and issues pertaining to drug development, and future directions in treatment of metastatic RCC. Expert opinion: Recently approved growth factor receptor antagonists have shown encouraging survival benefit but associated drug toxicity is a major issue. Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, has similarly shown survival benefit and is well tolerated. With multiple options now available in this patient population, the right sequence of these agents remains to be determined.

Control Points in School Business Management, Presenting General Observations, Specific Control Points, and a Series of Checkpoints for the Practicing School Business Administrator.

ERIC Educational Resources Information Center

Association of School Business Officials, Chicago, IL.

This revised outline of a 1962 address contains general guidelines considered still useful for practicing school business administrators. Introductory "general observations" and a preamble give advice about good personal qualities, policy and planning practices, and relations with school boards, community, staff, and fellow professional…

Programme development of drug abuse control in Baluchistan, Pakistan.

PubMed

Zaidi, S M; Ashraf, S M; Afridi, A A

1982-01-01

The drug abuse control programme in Baluchistan is inter-disciplinary and progressive. Its main thrust consists of constant vigilance on border check-points by law enforcement agencies, developing in-patient and out-patient facilities for treatment and rehabilitation of addicted persons, and a preventive education programme using the mass media and students from higher educational institutions.

78 FR 27472 - Notice of Passenger Facility Charge (PFC) Approvals and Disapprovals

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-10

... Approved For Collection And Use: Airfield lighting and vault. Runway 21 extension--preliminary design...--80 facility modification design and build-out. Access control enhancements. Security fence replacement. Airfield pavement survey. Jet bridge refurbishment. Security checkpoint modification. Friction...

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

ERIC Educational Resources Information Center

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

2012-01-01

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

The Geography of Deterrence: Exploring the Small Area Effects of Sobriety Checkpoints on Alcohol-Impaired Collision Rates within a City

ERIC Educational Resources Information Center

Nunn, Samuel; Newby, William

2011-01-01

This article examines alcohol-impaired collision metrics around nine sobriety checkpoint locations in Indianapolis, Indiana, before and after implementation of 22 checkpoints, using a pre/post examination, a pre/post nonequivalent comparison group analysis, and an interrupted time series approach. Traffic safety officials used geographical…

Characterization of a Putative Spindle Assembly Checkpoint Kinase Mps1, Suggests Its Involvement in Cell Division, Morphogenesis and Oxidative Stress Tolerance in Candida albicans

PubMed Central

Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus. PMID:25025778

Characterization of a putative spindle assembly checkpoint kinase Mps1, suggests its involvement in cell division, morphogenesis and oxidative stress tolerance in Candida albicans.

PubMed

Kamthan, Mohan; Nalla, Vijaya Kumar; Ruhela, Deepa; Kamthan, Ayushi; Maiti, Protiti; Datta, Asis

2014-01-01

In Saccharomyces cerevisiae MPS1 is one of the major protein kinase that governs the spindle checkpoint pathway. The S. cerevisiae structural homolog of opportunistic pathogen Candida albicans CaMPS1, is indispensable for the cell viability. The essentiality of Mps1 was confirmed by Homozygote Trisome test. To determine its biological function in this pathogen conditional mutant was generated through regulatable MET3 promoter. Examination of heterozygous and conditional (+Met/Cys) mps1 mutants revealed a mitosis specific arrest phenotype, where mutants showed large buds with undivided nuclei. Flowcytometry analysis revealed abnormal ploidy levels in mps1 mutant. In presence of anti-microtubule drug Nocodazole, mps1 mutant showed a dramatic loss of viability suggesting a role of Mps1 in Spindle Assembly Checkpoint (SAC) activation. These mutants were also defective in microtubule organization. Moreover, heterozygous mutant showed defective in-vitro yeast to hyphae morphological transition. Growth defect in heterozygous mutant suggest haploinsufficiency of this gene. qRT PCR analysis showed around 3 fold upregulation of MPS1 in presence of serum. This expression of MPS1 is dependent on Efg1 and is independent of other hyphal regulators like Ras1 and Tpk2. Furthermore, mps1 mutants were also sensitive to oxidative stress. Heterozygous mps1 mutant did not undergo morphological transition and showed 5-Fold reduction in colony forming units in response to macrophage. Thus, the vital checkpoint kinase, Mps1 besides cell division also has a role in morphogenesis and oxidative stress tolerance, in this pathogenic fungus.

Detecting concealed objects at a checkpoint

DOEpatents

McMakin, Douglas L [Richland, WA; Hall, Thomas E [Kennewick, WA; Sheen, David M [Richland, WA; Severtsen, Ronald H [Richland, WA

2008-07-29

Disclosed are systems, methods, devices, and apparatus to interrogate a clothed individual with electromagnetic radiation to determine if a concealed object is being carried. This determination includes establishing data corresponding to an image of the individual with a pair of opposed, semi-cylindrical array panels each configured to interrogate the individual with electromagnetic radiation in the 200 MHz to 1 THz range.

Immune checkpoint failures in inflammatory myopathies: An overview.

PubMed

Herbelet, Sandrine; De Bleecker, Jan L

2018-06-06

Dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), immune mediated necrotizing myopathy (IMNM) and overlap myositis (OM) are classified as inflammatory myopathies (IM) with involvement of autoimmune features such as autoreactive lymphocytes and autoantibodies. Autoimmunity can be defined as a loss in self-tolerance and attack of autoantigens by the immune system. Self-tolerance is achieved by a group of immune mechanisms occurring in central and periphal lymphoid organs and tissues, called immune checkpoints, that work in synergy to protect the body from harmful immune reactions. Autoimmune disorders appear when immune checkpoints fail. In this review, the different immune checkpoint failures are discussed in DM, PM, IBM and IMNM. Exploring research contribution in each of these immune checkpoints might help to highlight research perspectives in the field and obtain a more complete picture of IM disease pathology. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565

Architecture and method for a burst buffer using flash technology

DOEpatents

Tzelnic, Percy; Faibish, Sorin; Gupta, Uday K.; Bent, John; Grider, Gary Alan; Chen, Hsing-bung

2016-03-15

A parallel supercomputing cluster includes compute nodes interconnected in a mesh of data links for executing an MPI job, and solid-state storage nodes each linked to a respective group of the compute nodes for receiving checkpoint data from the respective compute nodes, and magnetic disk storage linked to each of the solid-state storage nodes for asynchronous migration of the checkpoint data from the solid-state storage nodes to the magnetic disk storage. Each solid-state storage node presents a file system interface to the MPI job, and multiple MPI processes of the MPI job write the checkpoint data to a shared file in the solid-state storage in a strided fashion, and the solid-state storage node asynchronously migrates the checkpoint data from the shared file in the solid-state storage to the magnetic disk storage and writes the checkpoint data to the magnetic disk storage in a sequential fashion.

B cell expression of the inhibitory Fc gamma receptor is unchanged in early MS.

PubMed

Comabella, Manuel; Montalban, Xavier; Kakalacheva, Kristina; Osman, Deeqa; Nimmerjahn, Falk; Tintoré, Mar; Lünemann, Jan D

2010-06-01

Expression of the inhibitory Fcgamma receptor IIB (FcgammaRIIB) has emerged as a late checkpoint during peripheral B cell development which prevents autoreactive memory B lymphocytes from becoming long-lived plasma cells. Decreased expression of FcgammaRIIB or non-functional FcgammaRIIB variants are associated with the development of autoimmune tissue inflammation. We determined the expression profile of FcgammaRIIB in peripheral blood cells in treatment-naïve patients with early MS. Twenty-five patients with clinically isolated syndrome (CIS) who converted to clinically definite MS (CDMS) and 25 demographically matched healthy donors were included in the study. Frequencies of peripheral blood monocytes and B cell subsets as well as FcgammaRIIB expression profile was determined by flow cytometry. FcgammaRIIB expression levels were higher in B cells compared to monocytes (p<0.0001) and higher in memory B cells compared to their naïve counterparts (p<0.0001). However, FcgammaRIIB expression in naïve and memory B cells as well as monocytes was unchanged in patients with early MS at onset of symptoms as well as after conversion to CDMS compared to controls. No significant correlations were found between FcgammaRIIB expression levels and brain MRI-derived metrics or EDSS progression during follow-up. These data indicate that FcgammaRIIB expression, a critical late B cell differentiation checkpoint preventing the occurrence of autoreactive long-lived plasma cells, is not impaired in treatment-naïve patients with MS, at least in the early phases of the disease. Copyright 2010 Elsevier B.V. All rights reserved.

DNA Damage Response Genes and the Development of Cancer Metastasis

PubMed Central

Broustas, Constantinos G.; Lieberman, Howard B.

2014-01-01

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

Terrorist Watchlist Checks and Air Passenger Prescreening

DTIC Science & Technology

2009-12-30

U.S. port of entry or at airport security checkpoints prior U.S. air carrier flights. For these purposes, CBP administers the Automated Targeting...Passenger Screening at Airport Security Checkpoints ................................. 14 9/11 Commission Recommendations and CAPPS II...individuals at either international ports of entries upon arrival at a U.S. port of entry or at airport security checkpoints prior U.S. air carrier

Experimental evaluation of multiprocessor cache-based error recovery

NASA Technical Reports Server (NTRS)

Janssens, Bob; Fuchs, W. K.

1991-01-01

Several variations of cache-based checkpointing for rollback error recovery in shared-memory multiprocessors have been recently developed. By modifying the cache replacement policy, these techniques use the inherent redundancy in the memory hierarchy to periodically checkpoint the computation state. Three schemes, different in the manner in which they avoid rollback propagation, are evaluated. By simulation with address traces from parallel applications running on an Encore Multimax shared-memory multiprocessor, the performance effect of integrating the recovery schemes in the cache coherence protocol are evaluated. The results indicate that the cache-based schemes can provide checkpointing capability with low performance overhead but uncontrollable high variability in the checkpoint interval.

Evaluating the evidence for non-monotonic dose-response relationships: A systematic literature review and (re-)analysis of in vivo toxicity data in the area of food safety.

PubMed

Varret, C; Beronius, A; Bodin, L; Bokkers, B G H; Boon, P E; Burger, M; De Wit-Bos, L; Fischer, A; Hanberg, A; Litens-Karlsson, S; Slob, W; Wolterink, G; Zilliacus, J; Beausoleil, C; Rousselle, C

2018-01-15

This study aims to evaluate the evidence for the existence of non-monotonic dose-responses (NMDRs) of substances in the area of food safety. This review was performed following the systematic review methodology with the aim to identify in vivo studies published between January 2002 and February 2015 containing evidence for potential NMDRs. Inclusion and reliability criteria were defined and used to select relevant and reliable studies. A set of six checkpoints was developed to establish the likelihood that the data retrieved contained evidence for NMDR. In this review, 49 in vivo studies were identified as relevant and reliable, of which 42 were used for dose-response analysis. These studies contained 179 in vivo dose-response datasets with at least five dose groups (and a control group) as fewer doses cannot provide evidence for NMDR. These datasets were extracted and analyzed using the PROAST software package. The resulting dose-response relationships were evaluated for possible evidence of NMDRs by applying the six checkpoints. In total, 10 out of the 179 in vivo datasets fulfilled all six checkpoints. While these datasets could be considered as providing evidence for NMDR, replicated studies would still be needed to check if the results can be reproduced to rule out that the non-monotonicity was caused by incidental anomalies in that specific study. This approach, combining a systematic review with a set of checkpoints, is new and appears useful for future evaluations of the dose response datasets regarding evidence of non-monotonicity. Published by Elsevier Inc.

Checkpoint inhibitors in advanced melanoma: effect on the field of immunotherapy.

PubMed

O'reilly, Aine; Larkin, James

2017-07-01

The success of the immune checkpoint inhibitors in melanoma has reinvigorated the field of immunotherapy. Immune checkpoint inhibitors are now the standard of care in multiple cancer types including lung cancer, head and neck cancer, urothelial cancer and renal cell cancer. The field of immunotherapy is currently expanding rapidly and will be a focus of research and development for decades to come. Areas covered: This review covers the early development of immune checkpoint inhibitors and the changes that occurred in the drug development paradigm to facilitate the development of immunotherapy. The review will summarise the areas into which immune checkpoint inhibitors have been adopted and will review the data that supported this. Furthermore, we will discuss future developments in immunotherapy and the current landscape regarding maximising the potential of immunotherapy in clinical practice. Expert commentary: In the author's opinion, the potential of immunotherapy is vast. To date immune checkpoint inhibition has already delivered durable responses in a proportion of patients with cancer types which were previously universally lethal. The future of immunotherapy will rely upon the intelligent application of translational research to clinical practice, such that immunotherapy can be effective for a wider population and maintain its current growth.

Natural Loss of Mps1 Kinase in Nematodes Uncovers a Role for Polo-like Kinase 1 in Spindle Checkpoint Initiation.

PubMed

Espeut, Julien; Lara-Gonzalez, Pablo; Sassine, Mélanie; Shiau, Andrew K; Desai, Arshad; Abrieu, Ariane

2015-07-07

The spindle checkpoint safeguards against chromosome loss during cell division by preventing anaphase onset until all chromosomes are attached to spindle microtubules. Checkpoint signal is generated at kinetochores, the primary attachment site on chromosomes for spindle microtubules. Mps1 kinase initiates checkpoint signaling by phosphorylating the kinetochore-localized scaffold protein Knl1 to create phospho-docking sites for Bub1/Bub3. Mps1 is widely conserved but is surprisingly absent in many nematode species. Here, we show that PLK-1, which targets a substrate motif similar to that of Mps1, functionally substitutes for Mps1 in C. elegans by phosphorylating KNL-1 to direct BUB-1/BUB-3 kinetochore recruitment. This finding led us to re-examine checkpoint initiation in human cells, where we found that Plk1 co-inhibition significantly reduced Knl1 phosphorylation and Bub1 kinetochore recruitment relative to Mps1 inhibition alone. Thus, the finding that PLK-1 functionally substitutes for Mps1 in checkpoint initiation in C. elegans uncovered a role for Plk1 in species that have Mps1. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

FAP positive fibroblasts induce immune checkpoint blockade resistance in colorectal cancer via promoting immunosuppression.

PubMed

Chen, Lingling; Qiu, Xiangting; Wang, Xinhua; He, Jian

2017-05-20

Immune checkpoint blockades that significantly prolonged survival of melanoma patients have been less effective on colorectal cancer (CRC) patients. Growing evidence suggested that fibroblast activation protein-alpha (FAP) on cancer associate fibroblasts (CAFs) has critical roles in regulating antitumor immune response by inducing tumor-promoting inflammation. In this study, we explored the roles of FAP in regulating the tumor immunity and immune checkpoint blockades resistance in CRC experimental systems. We found that CAFs with high FAP expression could induce immune checkpoint blockade resistance in CRC mouse model. Mechanistically, CAFs with high FAP expression promoted immunosuppression in the CRC tumor immune microenvironment by up-regulating CCL2 secretion, recruiting myeloid cells, and decreasing T-cell activity. In human CRC samples, FAP expression was proportional to myeloid cells number, but inversely related to T-cell number. High FAP expression also predicted poor survival of CRC patients. Taken together, our study suggested that high FAP expression in CAFs is one reason leading to immune checkpoint blockades resistance in CRC patients and FAP is an optional target for reversing immune checkpoint blockades resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of the spindle assembly checkpoint kinase Mps-1 as a novel therapeutic strategy in malignant mesothelioma

PubMed Central

Szymiczek, Agata; Carbone, Michele; Pastorino, Sandra; Napolitano, Andrea; Tanji, Mika; Minaai, Michael; Pagano, Ian; Mason, Jacqueline M.; Pass, Harvey I.; Bray, Mark R.; Mak, Tak W.; Yang, Haining

2017-01-01

Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. TTK/Mps-1 (monopolar spindle 1 kinase) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients’ outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients. PMID:28759042

Therapeutic vaccines in HBV: lessons from HCV.

PubMed

Barnes, Eleanor

2015-02-01

Currently, millions of people infected with hepatitis B virus (HBV) are committed to decades of treatment with anti-viral therapy to control viral replication. However, new tools for immunotherapy that include both viral vectors and molecular checkpoint inhibitors are now available. This has led to a resurgence of interest in new strategies to develop immunotherapeutic strategies with the aim of inducing HBeAg seroconversion--an end-point that has been associated with a decrease in the rates of disease progression. Ultimately, a true cure will involve the elimination of covalently closed circular DNA which presents a greater challenge for immunotherapy. In this manuscript, I describe the development of immunotherapeutic strategies for HBV that are approaching or currently in clinical studies, and draw on observations of T cell function in natural infection supported by recent animal studies that may lead to additional rational vaccine strategies using checkpoint inhibitors. I also draw on our recent experience in developing potent vaccines for HCV prophylaxis based on simian adenoviral and MVA vectors used in prime-boost strategies in both healthy volunteers and HCV infected patients. I have shown that the induction of T cell immune responses is markedly attenuated when administered to people with persistent HCV viremia. These studies and recently published animal studies using the woodchuck model suggest that potent vaccines based on DNA or adenoviral vectored vaccination represent a rational way forward. However, combining these with drugs to suppress viral replication, alongside checkpoint inhibitors may be required to induce long-term immune control.

The RNA-binding proteins Zfp36l1 and Zfp36l2 enforce the thymic β-selection checkpoint by limiting DNA damage response signaling and cell cycle progression

PubMed Central

Galloway, Alison; Ahlfors, Helena; Turner, Martin

2016-01-01

The RNA binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. Here, we identify these targets on a genome wide scale in primary mouse thymocytes and show that Zfp36l1/l2 regulate DNA damage response and cell cycle transcripts to ensure proper β-selection. DN3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with post-selected DN3b cells despite the absence of intracellular TCRβ and reduced IL-7 signaling. Our findings show that in addition to controlling the timing of proliferation at β-selection post-transcriptional control by Zfp36l1/l2 limits DNA damage responses which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as post-transcriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control. PMID:27566829

Synergy of Immune Checkpoint Blockade with a Novel Synthetic Consensus DNA Vaccine Targeting TERT.

PubMed

Duperret, Elizabeth K; Wise, Megan C; Trautz, Aspen; Villarreal, Daniel O; Ferraro, Bernadette; Walters, Jewell; Yan, Jian; Khan, Amir; Masteller, Emma; Humeau, Laurent; Weiner, David B

2018-02-07

Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet + /CD44 + effector CD8 + T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Tumor cell-associated immune checkpoint molecules - Drivers of malignancy and stemness.

PubMed

Marcucci, Fabrizio; Rumio, Cristiano; Corti, Angelo

2017-12-01

Inhibitory or stimulatory immune checkpoint molecules are expressed on a sizeable fraction of tumor cells in different tumor types. It was thought that the main function of tumor cell-associated immune checkpoint molecules would be the modulation (down- or upregulation) of antitumor immune responses. In recent years, however, it has become clear that the expression of immune checkpoint molecules on tumor cells has important consequences on the biology of the tumor cells themselves. In particular, a causal relationship between the expression of these molecules and the acquisition of malignant traits has been demonstrated. Thus, immune checkpoint molecules have been shown to promote the epithelial-mesenchymal transition of tumor cells, the acquisition of tumor-initiating potential and resistance to apoptosis and antitumor drugs, as well as the propensity to disseminate and metastasize. Herein, we review this evidence, with a main focus on PD-L1, the most intensively investigated tumor cell-associated immune checkpoint molecule and for which most information is available. Then, we discuss more concisely other tumor cell-associated immune checkpoint molecules that have also been shown to induce the acquisition of malignant traits, such as PD-1, B7-H3, B7-H4, Tim-3, CD70, CD28, CD137, CD40 and CD47. Open questions in this field as well as some therapeutic approaches that can be derived from this knowledge, are also addressed. Copyright © 2017 Elsevier B.V. All rights reserved.

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis.

PubMed

Lee, Seung Joon; Langhans, Sigrid A

2012-01-26

Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin.

Anaphase-promoting complex/cyclosome protein Cdc27 is a target for curcumin-induced cell cycle arrest and apoptosis

PubMed Central

2012-01-01

Background Curcumin (diferuloylmethane), the yellow pigment in the Asian spice turmeric, is a hydrophobic polyphenol from the rhizome of Curcuma longa. Because of its chemopreventive and chemotherapeutic potential with no discernable side effects, it has become one of the major natural agents being developed for cancer therapy. Accumulating evidence suggests that curcumin induces cell death through activation of apoptotic pathways and inhibition of cell growth and proliferation. The mitotic checkpoint, or spindle assembly checkpoint (SAC), is the major cell cycle control mechanism to delay the onset of anaphase during mitosis. One of the key regulators of the SAC is the anaphase promoting complex/cyclosome (APC/C) which ubiquitinates cyclin B and securin and targets them for proteolysis. Because APC/C not only ensures cell cycle arrest upon spindle disruption but also promotes cell death in response to prolonged mitotic arrest, it has become an attractive drug target in cancer therapy. Methods Cell cycle profiles were determined in control and curcumin-treated medulloblastoma and various other cancer cell lines. Pull-down assays were used to confirm curcumin binding. APC/C activity was determined using an in vitro APC activity assay. Results We identified Cdc27/APC3, a component of the APC/C, as a novel molecular target of curcumin and showed that curcumin binds to and crosslinks Cdc27 to affect APC/C function. We further provide evidence that curcumin preferably induces apoptosis in cells expressing phosphorylated Cdc27 usually found in highly proliferating cells. Conclusions We report that curcumin directly targets the SAC to induce apoptosis preferably in cells with high levels of phosphorylated Cdc27. Our studies provide a possible molecular mechanism why curcumin induces apoptosis preferentially in cancer cells and suggest that phosphorylation of Cdc27 could be used as a biomarker to predict the therapeutic response of cancer cells to curcumin. PMID:22280307

Three Drugs Approved for Urothelial Carcinoma by FDA.

PubMed

2017-07-01

The FDA has approved one PD-1 checkpoint inhibitor, pembrolizumab, and two PD-L1 checkpoint inhibitors, avelumab and durvalumab, to treat metastatic urothelial carcinoma in patients whose disease continues to progress despite platinum-based chemotherapy. This brings the total number of checkpoint inhibitors for the disease to five, prompting questions about how best to use them. ©2017 American Association for Cancer Research.

A pathway of targeted autophagy is induced by DNA damage in budding yeast

PubMed Central

Eapen, Vinay V.; Waterman, David P.; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G.; Loewith, Robbie J.; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J.; Haber, James E.

2017-01-01

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response. PMID:28154131

A pathway of targeted autophagy is induced by DNA damage in budding yeast.

PubMed

Eapen, Vinay V; Waterman, David P; Bernard, Amélie; Schiffmann, Nathan; Sayas, Enrich; Kamber, Roarke; Lemos, Brenda; Memisoglu, Gonen; Ang, Jessie; Mazella, Allison; Chuartzman, Silvia G; Loewith, Robbie J; Schuldiner, Maya; Denic, Vladimir; Klionsky, Daniel J; Haber, James E

2017-02-14

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

Telomere shortening triggers a feedback loop to enhance end protection.

PubMed

Yang, Chia-Wei; Tseng, Shun-Fu; Yu, Chia-Jung; Chung, Chia-Yu; Chang, Cheng-Yen; Pobiega, Sabrina; Teng, Shu-Chun

2017-08-21

Telomere homeostasis is controlled by both telomerase machinery and end protection. Telomere shortening induces DNA damage sensing kinases ATM/ATR for telomerase recruitment. Yet, whether telomere shortening also governs end protection is poorly understood. Here we discover that yeast ATM/ATR controls end protection. Rap1 is phosphorylated by Tel1 and Mec1 kinases at serine 731, and this regulation is stimulated by DNA damage and telomere shortening. Compromised Rap1 phosphorylation hampers the interaction between Rap1 and its interacting partner Rif1, which thereby disturbs the end protection. As expected, reduction of Rap1-Rif1 association impairs telomere length regulation and increases telomere-telomere recombination. These results indicate that ATM/ATR DNA damage checkpoint signal contributes to telomere protection by strengthening the Rap1-Rif1 interaction at short telomeres, and the checkpoint signal oversees both telomerase recruitment and end capping pathways to maintain telomere homeostasis. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

PD-1-PD-L1 immune-checkpoint blockade in malignant lymphomas.

PubMed

Wang, Yi; Wu, Ling; Tian, Chen; Zhang, Yizhuo

2018-02-01

Tumor cells can evade immune surveillance through overexpressing the ligands of checkpoint receptors on tumor cells or adjacent cells, leading T cells to anergy or exhaustion. Growing evidence of the interaction between tumor cells and microenvironment promoted the emergence of immune-checkpoint blockade. By targeting programmed cell death-1 (PD-1) pathway, cytotoxic activity of T cell is enhanced significantly and tumor cell lysis is induced subsequently. Currently, various antibodies against PD-1 and programmed death-ligand 1 (PD-L1) are under clinical studies in lymphomas. In this review, we outline the rationale for investigation of PD-1-PD-L1 immune-checkpoint blockade in lymphomas and discuss their prospect of applications in clinical treatment.

Immune checkpoint inhibitors: basics and challenges.

PubMed

Li, Bin; Chan, Ho Lam; Chen, Pingping

2017-08-04

Cancer is one of the most deadly diseases in modern world. The last decade has witnessed dramatic advances in the cancer treatment through immunotherapy. One extremely promising means to achieve anti-caner immunity is to block the immune checkpoint pathways, which mechanism was adopted by cancer cells to disguise themselves as regular components of human body. While checkpoint blockade is universally effective against a broad spectrum of cancer types and mostly unrestricted by certain gene mutation status, only a minority of patients achieved a complete response to such treatment. In this review we summarize the basic principles of immune checkpoint inhibitors and discuss potential mechanisms of resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Error recovery in shared memory multiprocessors using private caches

NASA Technical Reports Server (NTRS)

Wu, Kun-Lung; Fuchs, W. Kent; Patel, Janak H.

1990-01-01

The problem of recovering from processor transient faults in shared memory multiprocesses systems is examined. A user-transparent checkpointing and recovery scheme using private caches is presented. Processes can recover from errors due to faulty processors by restarting from the checkpointed computation state. Implementation techniques using checkpoint identifiers and recovery stacks are examined as a means of reducing performance degradation in processor utilization during normal execution. This cache-based checkpointing technique prevents rollback propagation, provides rapid recovery, and can be integrated into standard cache coherence protocols. An analytical model is used to estimate the relative performance of the scheme during normal execution. Extensions to take error latency into account are presented.

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

PubMed

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Pseudoprogression and hyperprogression after checkpoint blockade.

PubMed

Wang, Qiaohong; Gao, Jingze; Wu, Xia

2018-05-01

Immune checkpoint inhibitors appear to be one of the most promising immunotherapies with significant clinical benefits and durable responses in multiple tumor types. A heterogeneity of responses appears in patients receiving checkpoint blockade, including pseudoprogression where the tumor burden or number of tumor lesions increases initially before decreasing. Another special response observed after checkpoint blockade is hyperprogression, a phenomenon reflecting a very rapid tumor progression following immunotherapy, suggesting that checkpoint blockade could impact detrimentally on a small subset of patients. As immunotherapeutics, especially anti-PD-1/PD-L1 agents, become more widely available, evaluating the efficacy of these novel drugs poses a major challenge to clinicians, who aim to avoid either premature withdrawal of the treatment or prolonging ineffective treatment. Although the mechanism and recognition of pseudoprogression have gradually come to light, the incidence, basis, identification and predictive biomarkers of hyperprogression have been largely unknown, and this review documents the existing research findings and points out the areas where further studies are badly needed. Copyright © 2018 Elsevier B.V. All rights reserved.

Nutrient-Dependent Endocycling in Steroidogenic Tissue Dictates Timing of Metamorphosis in Drosophila melanogaster

PubMed Central

Ohhara, Yuya; Kobayashi, Satoru

2017-01-01

Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation. PMID:28121986

A p53-independent damage-sensing mechanism that functions as a checkpoint at the G1/S transition in Chinese hamster ovary cells

PubMed Central

Lee, Hoyun; Larner, James M.; Hamlin, Joyce L.

1997-01-01

In response to a moderate dose of radiation, asynchronous mammalian cell populations rapidly and transiently down-regulate the rate of DNA synthesis to ≈50% of preirradiation values. We show here that only half of the reduction in overall replication rate can be accounted for by direct inhibition of initiation at origins in S-phase cells. The other half results from the operation of a newly defined cell cycle checkpoint that functions at the G1/S transition. This checkpoint senses damage incurred at any time during the last 2 hr of G1 and effectively prevents entry into the S period. The G1/S and S-phase checkpoints are both p53-independent and, unlike the p53-mediated G1 checkpoint, respond rapidly to radiation, suggesting that they may represent major damage-sensing mechanisms connecting the replication machinery with DNA repair pathways. PMID:9012817

Structure and substrate recruitment of the human spindle checkpoint kinase Bub1.

PubMed

Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M; Tomchick, Diana R; Machius, Mischa; Yu, Hongtao

2008-11-07

In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

Myasthenia triggered by immune checkpoint inhibitors: New case and literature review.

PubMed

Gonzalez, Natalia L; Puwanant, Araya; Lu, Angela; Marks, Stanley M; Živković, Saša A

2017-03-01

Immune checkpoint molecules are potent regulators of immunologic homeostasis that prevent the development of autoimmunity while maintaining self-tolerance. Inhibitors of immune checkpoint molecules are used as immunotherapy in the treatment of melanoma and different types of refractory cancer, and can trigger various autoimmune complications including myositis and myasthenia gravis. We describe a case of generalized myasthenia gravis induced by pembrolizumab and review 11 other cases. Five patients also had elevated serum CK levels ranging from 1200 to 8729 IU/L, and biopsy showed myositis in one. Severity was highly variable as symptoms normalized spontaneously in one patient, but three others developed myasthenic crisis (including two with fatal outcomes). Steroids have been recommended as a preferred treatment of autoimmune complications of immune-checkpoint inhibitors. Myasthenia gravis should be considered when weakness, diplopia or bulbar symptoms are seen after treatment with immune checkpoint inhibitors, and additional studies are needed to characterize association with hyperCKemia. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of immune checkpoints in T cells of esophageal cancer patients.

PubMed

Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

2016-09-27

Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer.

Immune-related neurological toxicities among solid tumor patients treated with immune checkpoint inhibitors: a systematic review.

PubMed

Eltobgy, Mostafa; Oweira, Hani; Petrausch, Ulf; Helbling, Daniel; Schmidt, Jan; Mehrabi, Arianeb; Schöb, Othmar; Giryes, Anwar; Decker, Michael; Abdel-Rahman, Omar

2017-07-01

Immune-related neurologic toxicities are uncommon but serious adverse events that may be associated with the use of immune checkpoint inhibitors. The objective of this review is to assess the incidence and risk of neurologic toxicities which are potentially immune-related and occur with immune checkpoint treatment of solid tumors. Areas covered: PubMed database has been searched till January 2017. Clinical trials, case series and case reports reporting the occurrence of immune-related neurologic toxicities in solid tumor patients treated with immune checkpoint inhibitors were included. Eighteen trials with 4469 participants were included. The most common neurologic toxicities reported with these agents included sensory and motor peripheral neuropathies. Moreover, 17 case reports describing immune-related neurological events occurring with 22 patients were included. Expert commentary: Immune-related neurological toxicities occur uncommonly in cancer patients treated immune checkpoint inhibitors. Further studies are needed to better describe the course of these events (i.e. time to onset, time to resolution and responsiveness to different immunosuppressives).

[Immune checkpoints inhibitors: Recent data from ASCO's meeting 2017 and perspectives].

PubMed

Kfoury, Maria; Disdero, Valentine; Vicier, Cécilé; Le Saux, Olivia; Gougis, Paul; Sajous, Christophe; Vignot, Stéphane

2018-06-19

Immune checkpoint inhibitors anti-PD-1, anti-PD-L1 and anti-CTLA-4 have been in development in several indications and have changed the face of cancer patients' management. Cancer immunotherapy was central in ASCO's meeting 2017. The identification of patients who could benefit most from immune checkpoint inhibitors is essential. The predictive value of PD-L1 status remains insufficient to select patients who could respond to immunotherapy. An extended search for new biomarkers predictive of response (INF-γ, mutational load) is ongoing, in order to better select responders. Immune checkpoint inhibitors have mainly been developed as monotherapy. However, the low response rate, between 10 and 30%, and the occurrence of resistance, contributes to the increment of new therapeutic strategies. This review summarizes the results of combination trials of two immune checkpoint inhibitors, combination of immunotherapy with conventional chemotherapy, radiotherapy or targeted therapies active on the oncogenic addiction pathway. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Mechanisms to Control Rereplication and Implications for Cancer

PubMed Central

Hook, Sara S.; Lin, Jie Jessie; Dutta, Anindya

2007-01-01

Recent advances in the replication field have highlighted how the replication initiator proteins are negatively regulated by inhibitor proteins and ubiquitin-mediated degradation in mammalian cells to prevent rereplication. When these regulatory pathways go awry, uncontrolled rereplication ensues and a G2/M checkpoint is evoked to prevent cellular death. Many components of the checkpoints activated by rereplicaton are important for cancer prevention by facilitating DNA damage repair processes. The pathways that prevent rereplication themselves have also recently been implicated in preventing tumorigenesis. Studies from patient tumors, genetically altered mice, and mammalian cell culture suggest that deregulation of replication licensing proteins results in an increase in aneuploidy, chromosomal fusions, and DNA breaks. These studies provide a framework to address how regulators of replication function to maintain genomic stability. PMID:18053699

Mammalian Homologs of Yeast Checkpoint Genes

DTIC Science & Technology

2002-07-01

pathway is sensitive to various forms of DNA damage Developmental Biology throughout the cell cycle . The DNA replication check- Yale University point...components would be ordered into pathways for mammalian checkpoint function, with emphasis on p53 regulation, cell cycle regulation, and complementation...structurally related to the human tumor suppressor ATM. MEC1 and RAD53, two essential genes, play a central role in DNA damage checkpoints at all cell cycle

Expression of checkpoint molecules on myeloid-derived suppressor cells.

PubMed

Ballbach, Marlene; Dannert, Angelika; Singh, Anurag; Siegmund, Darina M; Handgretinger, Rupert; Piali, Luca; Rieber, Nikolaus; Hartl, Dominik

2017-12-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population expanded in cancer, infection and autoimmunity capable of suppressing T-cell functions. Checkpoint inhibitors have emerged as a key therapeutic strategy in immune-oncology. While checkpoint molecules were initially associated with T cell functions, recent evidence suggests a broader expression and function in innate myeloid cells. Previous studies provided first evidence for a potential role for checkpoints on MDSCs, yet the human relevance remained poorly understood. Therefore, we investigated the expression and functional relevance of checkpoint molecules in human MDSC-T-cell interactions. Our studies demonstrate that programmed death-ligand 1 (PD-L1) is expressed on granulocytic MDSCs upon co-culture with T cells. Transwell experiments showed that cell-to-cell contact was required for MDSC-T-cell interactions and antibody blocking studies showed that targeting PD-L1 partially impaired MDSC-mediated T-cell suppression. Collectively, these studies suggest a role for PD-L1 in human MDSC function and thereby expand the functionality of this checkpoint beyond T cells, which could pave the way for further understanding and therapeutic targeting of PD-1/PD-L1 in innate immune-mediated diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy

PubMed Central

Faisal, Amir; Mak, Grace W Y; Gurden, Mark D; Xavier, Cristina P R; Anderhub, Simon J; Innocenti, Paolo; Westwood, Isaac M; Naud, Sébastien; Hayes, Angela; Box, Gary; Valenti, Melanie R; De Haven Brandon, Alexis K; O'Fee, Lisa; Schmitt, Jessica; Woodward, Hannah L; Burke, Rosemary; vanMontfort, Rob L M; Blagg, Julian; Raynaud, Florence I; Eccles, Suzanne A; Hoelder, Swen; Linardopoulos, Spiros

2017-01-01

Background: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. Methods: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. Results: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. Conclusions: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850. PMID:28334731

Characterisation of CCT271850, a selective, oral and potent MPS1 inhibitor, used to directly measure in vivo MPS1 inhibition vs therapeutic efficacy.

PubMed

Faisal, Amir; Mak, Grace W Y; Gurden, Mark D; Xavier, Cristina P R; Anderhub, Simon J; Innocenti, Paolo; Westwood, Isaac M; Naud, Sébastien; Hayes, Angela; Box, Gary; Valenti, Melanie R; De Haven Brandon, Alexis K; O'Fee, Lisa; Schmitt, Jessica; Woodward, Hannah L; Burke, Rosemary; vanMontfort, Rob L M; Blagg, Julian; Raynaud, Florence I; Eccles, Suzanne A; Hoelder, Swen; Linardopoulos, Spiros

2017-04-25

The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.

Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline.

PubMed

Fan, S; Smith, M L; Rivet, D J; Duba, D; Zhan, Q; Kohn, K W; Fornace, A J; O'Connor, P M

1995-04-15

The possibility that appropriately designed chemotherapy could act selectively against p53-defective tumor cells was explored in MCF-7 human breast cancer cells. These cells were chosen because they have normal p53 function but are representative of a tumor cell type that does not readily undergo p53-dependent apoptosis. Two sublines (MCF-7/E6 and MCF-7/mu-p53) were established in which p53 function was disrupted by transfection with either the human papillomavirus type-16 E6 gene or a dominant-negative mutant p53 gene. p53 function in MCF-7/E6 and MCF-7/mu-p53 cells was defective relative to control cells in that there were no increases in p53 or p21Waf1/Cip1 protein levels and no G1 arrest following exposure to ionizing radiation. Survival assays showed that p53 disruption sensitized MCF-7 cells to cisplatin (CDDP) but not to several other DNA-damaging agents. CDDP sensitization was not limited to MCF-7 cells since p53 disruption in human colon carcinoma RKO cells also enhanced sensitivity to CDDP. Contrary to the other DNA-damaging agents tested, CDDP-induced DNA lesions are repaired extensively by nucleotide excision, and in agreement with a defect in this process, MCF-7/E6 and MCF-7/mu-p53 cells exhibited a reduced ability to repair a CDDP-damaged chloramphenicol acetyltransferase-reporter plasmid transfected into the cells. Therefore, we attributed the increased CDDP sensitivity of MCF-7 cells with disrupted p53 to defects in G1 checkpoint control, nucleotide excision repair, or both. The G2 checkpoint inhibitor pentoxifylline exhibited synergism with CDDP in killing MCF-7/E6 cells but did not affect sensitivity of the control cells. Moreover, pentoxifylline inhibited G2 checkpoint function to a greater extent in MCF-7/E6 than in the parental cells. These results suggested that, in the absence of p53 function, cancer cells are more vulnerable to G2 checkpoint abrogators. Our results show that a combination of CDDP and pentoxifylline is capable of synergistic and preferential killing of p53-defective tumor cells that do not readily undergo apoptosis.

A Slowed Cell Cycle Stabilizes the Budding Yeast Genome.

PubMed

Vinton, Peter J; Weinert, Ted

2017-06-01

During cell division, aberrant DNA structures are detected by regulators called checkpoints that slow division to allow error correction. In addition to checkpoint-induced delay, it is widely assumed, though rarely shown, that merely slowing the cell cycle might allow more time for error detection and correction, thus resulting in a more stable genome. Fidelity by a slowed cell cycle might be independent of checkpoints. Here we tested the hypothesis that a slowed cell cycle stabilizes the genome, independent of checkpoints, in the budding yeast Saccharomyces cerevisiae We were led to this hypothesis when we identified a gene ( ERV14 , an ER cargo membrane protein) that when mutated, unexpectedly stabilized the genome, as measured by three different chromosome assays. After extensive studies of pathways rendered dysfunctional in erv14 mutant cells, we are led to the inference that no particular pathway is involved in stabilization, but rather the slowed cell cycle induced by erv14 stabilized the genome. We then demonstrated that, in genetic mutations and chemical treatments unrelated to ERV14 , a slowed cell cycle indeed correlates with a more stable genome, even in checkpoint-proficient cells. Data suggest a delay in G2/M may commonly stabilize the genome. We conclude that chromosome errors are more rarely made or are more readily corrected when the cell cycle is slowed (even ∼15 min longer in an ∼100-min cell cycle). And, some chromosome errors may not signal checkpoint-mediated responses, or do not sufficiently signal to allow correction, and their correction benefits from this "time checkpoint." Copyright © 2017 by the Genetics Society of America.

Effects of Selective Checkpoint Kinase 1 Inhibition on Cytarabine Cytotoxicity in Acute Myelogenous Leukemia Cells in Vitro

PubMed Central

Schenk, Erin L.; Koh, Brian D.; Flatten, Karen S.; Peterson, Kevin L.; Parry, David; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Karnitz, Larry M.; Kaufmann, Scott H.

2012-01-01

Purpose Previous studies have demonstrated that the replication checkpoint, which involves the kinases ATR and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical AML during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results Immunoblotting demonstrated increased Chk1 phosphorylation, a marker of checkpoint activation, in over half of Chk1-containing AMLs after 48 h of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S phase arrest, but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the anti-proliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions These results not only provide evidence for cytarabine-induced S phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. PMID:22869869

Optimal management of immune-related adverse events resulting from treatment with immune checkpoint inhibitors: a review and update.

PubMed

Nagai, Hiroki; Muto, Manabu

2018-06-01

Over the last two decades, molecular-targeted agents have become mainstream treatment for many types of malignancies and have improved the overall survival of patients. However, most patients eventually develop resistance to these targeted therapies. Recently, immunotherapies such as immune checkpoint inhibitors have revolutionized the treatment paradigm for many types of malignancies. Immune checkpoint inhibitors have been approved for treatment of melanoma, non-small cell lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, Hodgkin's lymphoma, bladder cancer and gastric cancer. However, oncologists have been faced with immune-related adverse events caused by immune checkpoint inhibitors; these are generally mild but can be fatal in some cases. Because immune checkpoint inhibitors have distinct toxicity profiles from those of chemotherapy or targeted therapy, many oncologists are not familiar with the principles for optimal management of immune-related adverse events, which require early recognition and appropriate treatment without delay. To achieve this, oncologists must educate patients and health-care workers, develop checklists of appropriate tests for immune-related adverse events and collaborate closely with organ specialists. Clinical questions that remain include whether immune checkpoint inhibitors should be administered to patients with autoimmune disease and whether patients for whom immune-related adverse events lead to delays in immunotherapy should be retreated. In addition, the predicted use of combination immunotherapies in the near future means that oncologists will face a higher incidence and severity of immune-related adverse events. This review provides an overview of the optimal management of immune-related adverse events attributed to immune checkpoint inhibitors.

Immunotherapy in Gynecologic Cancers: Are We There Yet?

PubMed

Pakish, Janelle B; Jazaeri, Amir A

2017-08-24

Immune-targeted therapies have demonstrated durable responses in many tumor types with limited treatment options and poor overall prognosis. This has led to enthusiasm for expanding such therapies to other tumor types including gynecologic malignancies. The use of immunotherapy in gynecologic malignancies is in the early stages and is an active area of ongoing clinical research. Both cancer vaccines and immune checkpoint inhibitor therapy continue to be extensively studied in gynecologic malignancies. Immune checkpoint inhibitors, in particular, hold promising potential in specific subsets of endometrial cancer that express microsatellite instability. The key to successful treatment with immunotherapy involves identification of the subgroup of patients that will derive benefit. The number of ongoing trials in cervical, ovarian, and endometrial cancer will help to recognize these patients and make treatment more directed. Additionally, a number of studies are combining immunotherapy with standard treatment options and will help to determine combinations that will enhance responses to standard therapy. Overall, there is much enthusiasm for immunotherapy approaches in gynecologic malignancies. However, the emerging data shows that with the exception of microsatellite unstable tumors, the use of single-agent immune checkpoint inhibitors is associated with response rates of 10-15%. More effective and likely combinatorial approaches are needed and will be informed by the findings of ongoing trials.

Host-Pathogen Checkpoints and Population Bottlenecks in Persistent and Intracellular Uropathogenic E. coli Bladder Infection

PubMed Central

Hannan, Thomas J.; Totsika, Makrina; Mansfield, Kylie J.; Moore, Kate H.; Schembri, Mark A.; Hultgren, Scott J.

2013-01-01

Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multi-drug resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic E. coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the Quiescent Intracellular Reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection: QIR, ASB, or chronic cystitis, is determined within the first 24 hours of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies. PMID:22404313

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

PubMed

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.

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

PubMed Central

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

2016-01-01

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

Beyond CTLA-4 and PD-1, the Generation Z of Negative Checkpoint Regulators.

PubMed

Le Mercier, Isabelle; Lines, J Louise; Noelle, Randolph J

2015-01-01

In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy.

Beyond CTLA-4 and PD-1, the Generation Z of Negative Checkpoint Regulators

PubMed Central

Le Mercier, Isabelle; Lines, J. Louise; Noelle, Randolph J.

2015-01-01

In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy. PMID:26347741

Method and apparatus for offloading compute resources to a flash co-processing appliance

DOEpatents

Tzelnic, Percy; Faibish, Sorin; Gupta, Uday K.; Bent, John; Grider, Gary Alan; Chen, Hsing -bung

2015-10-13

Solid-State Drive (SSD) burst buffer nodes are interposed into a parallel supercomputing cluster to enable fast burst checkpoint of cluster memory to or from nearby interconnected solid-state storage with asynchronous migration between the burst buffer nodes and slower more distant disk storage. The SSD nodes also perform tasks offloaded from the compute nodes or associated with the checkpoint data. For example, the data for the next job is preloaded in the SSD node and very fast uploaded to the respective compute node just before the next job starts. During a job, the SSD nodes perform fast visualization and statistical analysis upon the checkpoint data. The SSD nodes can also perform data reduction and encryption of the checkpoint data.

Induction of Mitotic Cell Death by Overriding G2/M Checkpoint in Endometrial Cancer Cells with Non-functional p53

PubMed Central

Meng, Xiangbing; Laidler, Laura L.; Kosmacek, Elizabeth A.; Yang, Shujie; Xiong, Zhi; Zhu, Danlin; Wang, Xinjun; Dai, Donghai; Zhang, Yuping; Wang, Xiaofang; Brachova, Pavla; Albitar, Lina; Liu, Dawei; Ianzini, Fiorenza; Mackey, Michael A.; Leslie, Kimberly K.

2012-01-01

Objective Endometrial tumors with non-functional p53, such as serous uterine endometrial carcinomas, are aggressive malignancies with a poor outcome, yet they have an Achilles’ heel: due to loss of p53 function, these tumors may be sensitive to treatments which abrogate the G2/M checkpoint. Our objective was to exploit this weakness to induce mitotic cell death using two strategies: (1) EGFR inhibitor gefitinib combined with paclitaxel to arrest cells at mitosis, or (2) BI2536, an inhibitor of polo-like kinase 1 (PLK1), to block PLK1 activity. Methods We examined the impact of combining gefitinib and paclitaxel or PLK1 inhibitor on expression of G2/M checkpoint controllers, cell viability, and cell cycle progression in endometrial cancer cells with mutant p53. Results In cells lacking normal p53 activity, each treatment activated CDC25C and inactivated Wee1, which in turn activated cdc2 and sent cells rapidly through the G2/M checkpoint and into mitosis. Live cell imaging demonstrated irreversible mitotic arrest and eventual cell death. Combinatorial therapy with paclitaxel and gefitinib was highly synergistic and resulted in a 10-fold reduction in the IC50 for paclitaxel, from 14 nM as a single agent to 1.3 nM in the presence of gefitinib. However, BI2536 alone at low concentrations (5 nM) was the most effective treatment and resulted in massive mitotic cell death. In a xenograft mouse model with p53-deficient cells, low dose BI2536 significantly inhibited tumor growth. Conclusions These findings reveal induction of mitotic cell death as a therapeutic strategy for endometrial tumors lacking functional p53. PMID:23146687

Mutant p53 perturbs DNA replication checkpoint control through TopBP1 and Treslin.

PubMed

Liu, Kang; Lin, Fang-Tsyr; Graves, Joshua D; Lee, Yu-Ju; Lin, Weei-Chin

2017-05-09

Accumulating evidence supports the gain-of-function of mutant forms of p53 (mutp53s). However, whether mutp53 directly perturbs the DNA replication checkpoint remains unclear. Previously, we have demonstrated that TopBP1 forms a complex with mutp53s and mediates their gain-of-function through NF-Y and p63/p73. Akt phosphorylates TopBP1 and induces its oligomerization, which inhibits its ATR-activating function. Here we show that various contact and conformational mutp53s bypass Akt to induce TopBP1 oligomerization and attenuate ATR checkpoint response during replication stress. The effect on ATR response caused by mutp53 can be exploited in a synthetic lethality strategy, as depletion of another ATR activator, DNA2, in mutp53-R273H-expressing cancer cells renders cells hypersensitive to cisplatin. Expression of mutp53-R273H also makes cancer cells more sensitive to DNA2 depletion or DNA2 inhibitors. In addition to ATR-activating function during replication stress, TopBP1 interacts with Treslin in a Cdk-dependent manner to initiate DNA replication during normal growth. We find that mutp53 also interferes with TopBP1 replication function. Several contact, but not conformational, mutp53s enhance the interaction between TopBP1 and Treslin and promote DNA replication despite the presence of a Cdk2 inhibitor. Together, these data uncover two distinct mechanisms by which mutp53 enhances DNA replication: ( i ) Both contact and conformational mutp53s can bind TopBP1 and attenuate the checkpoint response to replication stress, and ( ii ) during normal growth, contact (but not conformational) mutp53s can override the Cdk2 requirement to promote replication by facilitating the TopBP1/Treslin interaction.

Contribution of market value chain to the control of African swine fever in Zambia.

PubMed

Siamupa, C; Saasa, N; Phiri, A M

2018-01-01

African swine fever (ASF) is a worldwide disease of pigs endemic in most sub-Saharan African countries. Zambia has been experiencing outbreaks of ASF for many years because the disease is endemic in the eastern part of the country, with incursion into the central part of Lusaka Province. The latest outbreaks of ASF in Lusaka occurred in 2013 with substantial pig mortalities, loss in trade, and cost of control measures and compensation of affected farmers. The aims of the study were to identify market value chain-related factors that were associated with ASF outbreaks and assess why these outbreaks are becoming frequent despite control measures being put in place. Using a mixed-method design, participants involved in the value chain were purposively sampled. Some pig farmers were included using a respondent-driven technique. Farmers came from Lusaka, Chilanga, Kafue, and Chongwe districts. Other participants included district veterinary officers, veterinary assistants, police officers, and veterinary staff manning veterinary checkpoints, abattoir and processing plant managers, meat inspectors, market chairpersons, and traders. Semi-structured questionnaires, in-depth interviews, and direct observations were used to collect data to come up with narrations, tables, and flow charts. In assessing the contribution of the value chain in ASF, aspects of ASF screening, market availability and procedures, knowledge on ASF transmission, occurrence of ASF outbreak, and regulation of pig movement were investigated. Despite government ASF control measures being applied, the following were noted: (1) low awareness levels of ASF transmission among pig farmers and traders; (2) only 50% of farmers had their animals screened for ASF before sale; (3) all the markets did not have the pork inspected; (4) laxity in enforcing livestock movement control because of inadequate police and veterinary staff manning checkpoints; (5) lack of enforcement of meat inspection and food safety regulations at pig markets; and (6) inadequate and bureaucratic ASF screening. Improving biosecurity; sensitizing farmers, traders, and all stakeholders in the pig value chain on ASF prevention and control; reinforcement of staff at checkpoints; and regulation of pig markets are some of the ways in which future outbreaks can be prevented.

Systemic Therapy for Stage IV Non-Small-Cell Lung Cancer: American Society of Clinical Oncology Clinical Practice Guideline Update.

PubMed

Hanna, Nasser; Johnson, David; Temin, Sarah; Baker, Sherman; Brahmer, Julie; Ellis, Peter M; Giaccone, Giuseppe; Hesketh, Paul J; Jaiyesimi, Ishmael; Leighl, Natasha B; Riely, Gregory J; Schiller, Joan H; Schneider, Bryan J; Smith, Thomas J; Tashbar, Joan; Biermann, William A; Masters, Gregory

2017-10-20

Purpose Provide evidence-based recommendations updating the 2015 ASCO guideline on systemic therapy for patients with stage IV non-small-cell lung cancer (NSCLC). Methods The ASCO NSCLC Expert Panel made recommendations based on a systematic review of randomized controlled trials from February 2014 to December 2016 plus the Cancer Care Ontario Program in Evidence-Based Care's update of a previous ASCO search. Results This guideline update reflects changes in evidence since the previous guideline update. Fourteen randomized controlled trials provide the evidence base; earlier phase trials also informed recommendation development. Recommendations New or revised recommendations include the following. Regarding first-line treatment for patients with non-squamous cell carcinoma or squamous cell carcinoma (without positive markers, eg, EGFR/ALK /ROS1), if the patient has high programmed death ligand 1 (PD-L1) expression, pembrolizumab should be used alone; if the patient has low PD-L1 expression, clinicians should offer standard chemotherapy. All other clinical scenarios follow 2015 recommendations. Regarding second-line treatment in patients who received first-line chemotherapy, without prior immune checkpoint therapy, if NSCLC tumor is positive for PD-L1 expression, clinicians should use single-agent nivolumab, pembrolizumab, or atezolizumab; if tumor has negative or unknown PD-L1 expression, clinicians should use nivolumab or atezolizumab. All immune checkpoint therapy is recommended alone plus in the absence of contraindications. For patients who received a prior first-line immune checkpoint inhibitor, clinicians should offer standard chemotherapy. For patients who cannot receive immune checkpoint inhibitor after chemotherapy, docetaxel is recommended; in patients with nonsquamous NSCLC, pemetrexed is recommended. In patients with a sensitizing EGFR mutation, disease progression after first-line epidermal growth factor receptor tyrosine kinase inhibitor therapy, and T790M mutation, osimertinib is recommended; if NSCLC lacks the T790M mutation, then chemotherapy is recommended. Patients with ROS1 gene rearrangement without prior crizotinib may be offered crizotinib, or if they previously received crizotinib, they may be offered chemotherapy.

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

NASA Astrophysics Data System (ADS)

Besnard, Sophie; Claverie, Vincent; Blanc, Frédérique

2015-04-01

The Emodnet Checkpoint projects aim is to assess the cost-effectiveness, reliability and utility of the existing monitoring at the sea basin level. This involves the development of monitoring system indicators and a GIS Platform to perform the assessment and make it available. Assessment or production of Check-Point information is made by developing targeted products based on the monitoring data and determining whether the products are meeting the needs of industry and public authorities. Check-point users are the research community, the 'institutional' policy makers for IMP and MSFD implementation, the 'intermediate users', i.e., users capable to understand basic raw data but that benefit from seeing the Checkpoint targeted products and the assessment of the fitness for purpose. We define assessment criteria aimed to characterize/depict the input datasets in terms of 3 territories capable to show performance and gaps of the present monitoring system, appropriateness, availability and fitness for purpose. • Appropriateness: What is made available to users? What motivate/decide them to select this observation rather than this one. • Availability: How this is made available to the user? Place to understand the readiness and service performance of the EU infrastructure • Fitness for use / fitness for purpose: Ability for non-expert user to appreciate the data exploitability (feedback on efficiency & reliability of marine data) For each territory (appropriateness, Availability and Fitness for purpose / for use), we define several indicators. For example, for Availability we define Visibility, Accessibility and Performance. And Visibility is itself defined by "Easily found" and "EU service". So these indicators can be classified according to their territory and sub-territory as seen above, but also according to the complexity to build them. Indicators are built from raw descriptors in 3 stages:  Stage 1: to give a neutral and basic status directly computed from the raw checkpoint descriptors.  Stage 2: to get a more sectorial status, aggregating level 1 results for instance depending on processing level.  Stage 3: to synthesize and focus view at characteristic level for decision making and actions plan. They are computed from stage 2 indicators. To produce this checkpoint information, we describe upstream data as input data sets which are uniquely identified as a combination of (variable, dataset, intended use) or of (geographical feature, dataset, intended use) depending on their nature. The information is called descriptors. The descriptors cover 8 sections: 1. Characteristics (= What) 2. Data sources (= From) 3. Overview elements (= Why for) 4. Spatial coverage (= Where) 5. Temporal coverage (= When) 6. Accessibility (= How) 7. Quality elements (= ISO 19113 quality elements) 8. Other information (= for administration/management needs) Check-Point services should be permanent services, because: • Monitoring systems will evolve and every few years there is need to re-assess; • Different/more use cases of monitoring are required to really show gaps and complementarities in the monitoring system components; • Monitoring system evolution for the European Sea and the global ocean require constant upgrade of the assessment indicators and descriptors; • Need to establish strong & permanent links with intermediate and end users from industry to public authorities and 'regional' approach is appropriate and feasible. • Need to maintain the process, methodology used and to be applied.

The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse

PubMed Central

Vasileva, Ana; Hopkins, Kevin M.; Wang, Xiangyuan; Weisbach, Melissa M.; Friedman, Richard A.; Wolgemuth, Debra J.; Lieberman, Howard B.

2013-01-01

Summary In mitotic cells, RAD9A functions in repairing DNA double-strand breaks (DSBs) by homologous recombination and facilitates the process by cell cycle checkpoint control in response to DNA damage. DSBs occur naturally in the germline during meiosis but whether RAD9A participates in repairing such breaks is not known. In this study, we determined that RAD9A is indeed expressed in the male germ line with a peak of expression in late pachytene and diplotene stages, and the protein was found associated with the XY body. As complete loss of RAD9A is embryonic lethal, we constructed and characterized a mouse strain with Stra8-Cre driven germ cell-specific ablation of Rad9a beginning in undifferentiated spermatogonia in order to assess its role in spermatogenesis. Adult mutant male mice were infertile or sub-fertile due to massive loss of spermatogenic cells. The onset of this loss occurs during meiotic prophase, and there was an increase in the numbers of apoptotic spermatocytes as determined by TUNEL. Spermatocytes lacking RAD9A usually arrested in meiotic prophase, specifically in pachytene. The incidence of unrepaired DNA breaks increased, as detected by accumulation of γH2AX and DMC1 foci on the axes of autosomal chromosomes in pachytene spermatocytes. The DNA topoisomerase IIβ-binding protein 1 (TOPBP1) was still localized to the sex body, albeit with lower intensity, suggesting that RAD9A may be dispensable for sex body formation. We therefore show for the first time that RAD9A is essential for male fertility and for repair of DNA DSBs during meiotic prophase I. PMID:23788429

Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

NASA Technical Reports Server (NTRS)

Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

2000-01-01

The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

Extraocular Muscle Enlargement and Thyroid Eye Disease-like Orbital Inflammation Associated with Immune Checkpoint Inhibitor Therapy in Cancer Patients.

PubMed

Sagiv, Oded; Kandl, Thomas J; Thakar, Sudip D; Thuro, Bradley A; Busaidy, Naifa L; Cabanillas, Maria; Jimenez, Camilo; Dadu, Ramona; Graham, Paul H; Debnam, J Matthew; Esmaeli, Bita

2018-06-19

To describe thyroid eye disease (TED)-like orbital inflammatory syndrome in 3 cancer patients treated with immune checkpoint inhibitors. All consecutive patients treated by the senior author who were receiving immune checkpoint inhibitors and developed TED-like orbital inflammation were included. Three cancer patients treated with immune checkpoint inhibitors developed orbital inflammation. The first patient was treated with a combination of a cytotoxic T-lymphocyte antigen-4 inhibitor and a programmed cell death protein 1 inhibitor and developed TED-like orbital inflammation with normal thyroid function and antibody levels. The second patient had a previous diagnosis of Graves disease without TED, and developed TED soon after initiating treatment with a programmed cell death protein 1 inhibitor. The third patient developed acute hyperthyroidism with symptomatic TED following treatment with an investigational cytotoxic T-lymphocyte antigen-4 inhibitor agent. All 3 patients were managed with either systemic steroids or observation, with resolution of their symptoms and without the need to halt immune checkpoint inhibitor treatment for their cancer. TED-like orbital inflammation may occur as a side effect of immune checkpoint inhibitor therapy with anti-cytotoxic T-lymphocyte antigen-4 or anti-PD-1 inhibitors. To the best of their knowledge, this is the first reported case of TED as a result of programmed cell death protein 1 inhibitor monotherapy. All 3 patients were treated with systemic steroids and responded quickly while continuing treatment with immune checkpoint inhibitors for their cancer. With increasing use of this class of drugs, clinicians should be familiar with the clinical manifestations and treatments for this adverse reaction.

The therapeutic potential of cell cycle targeting in multiple myeloma.

PubMed

Maes, Anke; Menu, Eline; Veirman, Kim De; Maes, Ken; Vand Erkerken, Karin; De Bruyne, Elke

2017-10-27

Proper cell cycle progression through the interphase and mitosis is regulated by coordinated activation of important cell cycle proteins (including cyclin-dependent kinases and mitotic kinases) and several checkpoint pathways. Aberrant activity of these cell cycle proteins and checkpoint pathways results in deregulation of cell cycle progression, which is one of the key hallmarks of cancer. Consequently, intensive research on targeting these cell cycle regulatory proteins identified several candidate small molecule inhibitors that are able to induce cell cycle arrest and even apoptosis in cancer cells. Importantly, several of these cell cycle regulatory proteins have also been proposed as therapeutic targets in the plasma cell malignancy multiple myeloma (MM). Despite the enormous progress in the treatment of MM the past 5 years, MM still remains most often incurable due to the development of drug resistance. Deregulated expression of the cyclins D is observed in virtually all myeloma patients, emphasizing the potential therapeutic interest of cyclin-dependent kinase inhibitors in MM. Furthermore, other targets have also been identified in MM, such as microtubules, kinesin motor proteins, aurora kinases, polo-like kinases and the anaphase promoting complex/cyclosome. This review will provide an overview of the cell cycle proteins and checkpoint pathways deregulated in MM and discuss the therapeutic potential of targeting proteins or protein complexes involved in cell cycle control in MM.

Caffeine stabilizes Cdc25 independently of Rad3 in S chizosaccharomyces pombe contributing to checkpoint override

PubMed Central

Alao, John P; Sjölander, Johanna J; Baar, Juliane; Özbaki-Yagan, Nejla; Kakoschky, Bianca; Sunnerhagen, Per

2014-01-01

Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both S chizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S . pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S . pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25. PMID:24666325

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

PubMed

Morin, Violeta; Prieto, Susana; Melines, Sabrina; Hem, Sonia; Rossignol, Michel; Lorca, Thierry; Espeut, Julien; Morin, Nathalie; Abrieu, Ariane

2012-02-21

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached. Copyright © 2012 Elsevier Ltd. All rights reserved.

Localization of spindle checkpoint proteins in cells undergoing mitosis with unreplicated genomes.

PubMed

Johnson, Mary Kathrine; Cooksey, Amanda M; Wise, Dwayne A

2008-11-01

CHO cells can be arrested with hydoxyurea at the beginning of the DNA synthesis phase of the cell cycle. Subsequent treatment with the xanthine, caffeine, induces cells to bypass the S-phase checkpoint and enter unscheduled mitosis [Schlegel and Pardee,1986, Science 232:1264-1266]. These treated cells build a normal spindle and distribute kinetochores, unattached to chromosomes, to their daughter cells [Brinkley et al.,1988, Nature 336:251-254; Zinkowski et al.,1991, J Cell Biol 113:1091-1110; Wise and Brinkley,1997, Cell Motil Cytoskeleton 36:291-302; Balczon et al.,2003, Chromosoma 112:96-102]. To investigate how these cells distribute kinetochores to daughter cells, we analyzed the spindle checkpoint components, Mad2, CENP-E, and the 3F3 phosphoepitope, using immunofluorescence and digital microscopy. Even though the kinetochores were unpaired and DNA was fragmented, the tension, alignment, and motor components of the checkpoint were found to be present and localized as predicted in prometaphase and metaphase. This unusual mitosis proves that a cell can successfully localize checkpoint proteins and divide even when kinetochores are unpaired and fragmented. (c) 2008 Wiley-Liss, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher

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 inmore » 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. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

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

DOE PAGES

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher; ...

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 inmore » 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. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

PubMed Central

Hematulin, Arunee; Meethang, Sutiwan; Utapom, Kitsana; Wongkham, Sopit; Sagan, Daniel

2018-01-01

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint. PMID:29541168

Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

EPA Pesticide Factsheets

Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints.

PubMed

Bezawork-Geleta, Ayenachew; Wen, He; Dong, LanFeng; Yan, Bing; Vider, Jelena; Boukalova, Stepana; Krobova, Linda; Vanova, Katerina; Zobalova, Renata; Sobol, Margarita; Hozak, Pavel; Novais, Silvia Magalhaes; Caisova, Veronika; Abaffy, Pavel; Naraine, Ravindra; Pang, Ying; Zaw, Thiri; Zhang, Ping; Sindelka, Radek; Kubista, Mikael; Zuryn, Steven; Molloy, Mark P; Berridge, Michael V; Pacak, Karel; Rohlena, Jakub; Park, Sunghyouk; Neuzil, Jiri

2018-06-07

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CII low , serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CII low leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CII low is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.

Targeting ornithine decarboxylase in Myc-induced lymphomagenesis prevents tumor formation.

PubMed

Nilsson, Jonas A; Keller, Ulrich B; Baudino, Troy A; Yang, Chunying; Norton, Sara; Old, Jennifer A; Nilsson, Lisa M; Neale, Geoffrey; Kramer, Debora L; Porter, Carl W; Cleveland, John L

2005-05-01

Checkpoints that control Myc-mediated proliferation and apoptosis are bypassed during tumorigenesis. Genes encoding polyamine biosynthetic enzymes are overexpressed in B cells from E mu-Myc transgenic mice. Here, we report that disabling one of these Myc targets, Ornithine decarboxylase (Odc), abolishes Myc-induced suppression of the Cdk inhibitors p21(Cip1) and p27(Kip1), thereby impairing Myc's proliferative, but not apoptotic, response. Moreover, lymphoma development was markedly delayed in E mu-Myc;Odc(+/-) transgenic mice and in E mu-Myc mice treated with the Odc inhibitor difluoromethylornithine (DFMO). Strikingly, tumors ultimately arising in E mu-Myc;Odc(+/-) transgenics lacked deletions of Arf, suggesting that targeting Odc forces other routes of transformation. Therefore, Odc is a critical Myc transcription target that regulates checkpoints that guard against tumorigenesis and is an effective target for cancer chemoprevention.

Genetic Control of the Trigger for the G2/M Checkpoint

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 findingsmore » 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 system, the engagement of the G2/M checkpoint only occurs at doses where most of the cells are bound for mitotic catastrophe. Further, compound haploinsufficiency of various radiosensitizing genes does not impact the threshold of activation. The experiments confirm a threshold of activation for the G2/M checkpoint, hinting at two separate radiation response programs acting below and above this threshold. Small RNA transfer in bystander effect biology: Small regulatory RNA molecules have now risen in prominence and utility. Specific examples are small interfering RNAs (siRNA) which are employed in cell level expression ablation projects and micro-RNAs (miRNA) which are a pool of short transcription products which serve to modulate the expression of other transcripts emerging from the genome in a meta-regulatory fine tuning of gene expression. The existing tenets of bystander effect radiation biology involve the communication of inflammatory mediators or direct intercellular communication of reactive oxygen/nitrogen species in cell-to-cell communicative organelles called gap junctions. By ablating gap junctions, reducing the ROS/inflammatory cytokine expression one can attenuate bystander effect signaling in cell culture systems. We hypothesized that miRNAs are a competent intercellular communication molecule and therefore a possible component of the bystander response. This view is supported by the observation that miRNA are secreted from cells in exosomes found in the circulation. This circulating pool reports disease type and severity in humans. We proposed use of microbeam irradiation technology at our facilities and enhancement of this capability with a new sorting technology which would allow us to sort irradiated and non-irradiated cells with absolute fidelity. Pursuing direct quantitative transfer assessment, we succeeded in designing and constructing a new add-on sorting appliance which harmonized with our existing instruments. The sorter allowed us to gently sort single fluorescently labeled cells. The plans for this appliance were published and are now available for use in other laboratories for single-cell analyses. Our microfluidic cell sorting modality is being integrated into subsequent microbeam irradiation experiments that are planned and ongoing. We generated and irradiated pools of specially engineered Donor-Recipient cell lines in co-culture that would report a small RNA transfer event by modulation of fluorescent protein expression. Both induction and reciprocal silencing designs were tested. We observed elevation of miRNA/siRNA transfer in response to radiation at doses of 5Gy in experiments to date. The reproducibility of these findings has not been good. Future studies will involve refinement of the reporting systems and a decrease in acute dose of radiation used to determine the lowest dose at which miRNA transfer between cells contributes to radiation bystander effect biology.« less

United States Air Force Academy: A Bibliography 2011-2015

DTIC Science & Technology

2016-11-23

Tactical Arms Training with the Cadet Combat Shooting Team." Checkpoints 39.4 (March 2011): 18-23. Print. 703. Do, James J., et al. "Gender Bias and...34 Checkpoints 41.3 (December 2012): 30-35. Print. 753. ---. "The Line of Fire: Tactical Arms Training with the Cadet Combat Shooting Team." Checkpoints...June 2013): 18-22. Print. 831. Thomas, Bill. " Hearts and Bones: There Was a Problem with the Last Burial in the Tomb of the Unknowns: The Soldier

Exploring the Underlying Mechanisms of the Xenopus laevis Embryonic Cell Cycle.

PubMed

Zhang, Kun; Wang, Jin

2018-05-31

The cell cycle is an indispensable process in proliferation and development. Despite significant efforts, global quantification and physical understanding are still challenging. In this study, we explored the mechanisms of the Xenopus laevis embryonic cell cycle by quantifying the underlying landscape and flux. We uncovered the Mexican hat landscape of the Xenopus laevis embryonic cell cycle with several local basins and barriers on the oscillation path. The local basins characterize the different phases of the Xenopus laevis embryonic cell cycle, and the local barriers represent the checkpoints. The checkpoint mechanism of the cell cycle is revealed by the landscape basins and barriers. While landscape shape determines the stabilities of the states on the oscillation path, the curl flux force determines the stability of the cell cycle flow. Replication is fundamental for biology of living cells. We quantify the input energy (through the entropy production) as the thermodynamic requirement for initiation and sustainability of single cell life (cell cycle). Furthermore, we also quantify curl flux originated from the input energy as the dynamical requirement for the emergence of a new stable phase (cell cycle). This can provide a new quantitative insight for the origin of single cell life. In fact, the curl flux originated from the energy input or nutrition supply determines the speed and guarantees the progression of the cell cycle. The speed of the cell cycle is a hallmark of cancer. We characterized the quality of the cell cycle by the coherence time and found it is supported by the flux and energy cost. We are also able to quantify the degree of time irreversibility by the cross correlation function forward and backward in time from the stochastic traces in the simulation or experiments, providing a way for the quantification of the time irreversibility and the flux. Through global sensitivity analysis upon landscape and flux, we can identify the key elements for controlling the cell cycle speed. This can help to design an effective strategy for drug discovery against cancer.

Effects of the selective MPS1 inhibitor MPS1-IN-3 on glioblastoma sensitivity to antimitotic drugs.

PubMed

Tannous, Bakhos A; Kerami, Mariam; Van der Stoop, Petra M; Kwiatkowski, Nicholas; Wang, Jinhua; Zhou, Wenjun; Kessler, Almuth F; Lewandrowski, Grant; Hiddingh, Lotte; Sol, Nik; Lagerweij, Tonny; Wedekind, Laurine; Niers, Johanna M; Barazas, Marco; Nilsson, R Jonas A; Geerts, Dirk; De Witt Hamer, Philip C; Hagemann, Carsten; Vandertop, W Peter; Van Tellingen, Olaf; Noske, David P; Gray, Nathanael S; Würdinger, Thomas

2013-09-04

Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival. Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Effects of the Selective MPS1 Inhibitor MPS1-IN-3 on Glioblastoma Sensitivity to Antimitotic Drugs

PubMed Central

2013-01-01

Background Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest. Methods We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3–7 mice/group). MPS1 expression levels were examined in relation to patient survival. Results Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity. Conclusions Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs. PMID:23940287

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

PubMed

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

2017-01-01

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

Checkpoint repair for high-performance out-of-order execution machines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwu, W.M.W.; Patt, Y.N.

Out-or-order execution and branch prediction are two mechanisms that can be used profitably in the design of supercomputers to increase performance. Proper exception handling and branch prediction miss handling in an out-of-order execution machine to require some kind of repair mechanism which can restore the machine to a known previous state. In this paper the authors present a class of repair mechanisms using the concept of checkpointing. The authors derive several properties of checkpoint repair mechanisms. In addition, they provide algorithms for performing checkpoint repair that incur little overhead in time and modest cost in hardware, which also require nomore » additional complexity or time for use with write-back cache memory systems than they do with write-through cache memory systems, contrary to statements made by previous researchers.« less

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

PubMed Central

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

2014-01-01

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

Initial characterization of a low-molecular-weight factor enhancing the checkpoint response.

PubMed

Fan, Xiaoxiang; Cheong, Nge; Iliakis, George

2010-10-01

In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

Immune checkpoint inhibitor colitis: the flip side of the wonder drugs.

PubMed

Assarzadegan, Naziheh; Montgomery, Elizabeth; Anders, Robert A

2018-01-01

Immune checkpoint inhibitors block the co-inhibitory receptors on T cells to activate their cytotoxic immune function and are rapidly being explored for the treatment of various advanced-stage malignancies. These novel drugs have already significantly increased survival rates. The first available immune checkpoint inhibitors were cytotoxic T lymphocyte antigen 4 (CTLA-4) inhibitors (such as ipilimumab), followed by programmed cell death protein 1 (PD-1) and programmed cell death protein ligand 1 (PD-L1) inhibitors (such as pembrolizumab and nivolumab). Anti-PD-1 and anti-PD-L1 therapies have demonstrated better efficacy and tolerability and less severe adverse effects compared to anti-CTLA-4 agents. Idelalisib, a PI3Kδ isoform inhibitor, is another immunotherapeutic agent that is often classified separately and is currently used in treatment of chronic lymphocytic leukemia and non-Hodgkin lymphomas. Despite successful therapeutic responses, immune-related adverse events have been reported with the use of these agents. The gastrointestinal side effects, particularly diarrhea, are among the most commonly reported symptoms. The histologic features of immune checkpoint inhibitor-associated colitis show a spectrum of patterns of injury among various drug classes. There is significant overlap between immune checkpoint inhibitor-associated colitis and other colitides, making the differential diagnosis difficult-especially in the absence of clinical history. The histopathology data on immune checkpoint inhibitor-associated colitis are limited. Here we review clinical features as well as various histologic patterns of colitis associated with these groups of medications.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

The fidelity of synaptonemal complex assembly is regulated by a signaling mechanism that controls early meiotic progression.

PubMed

Silva, Nicola; Ferrandiz, Nuria; Barroso, Consuelo; Tognetti, Silvia; Lightfoot, James; Telecan, Oana; Encheva, Vesela; Faull, Peter; Hanni, Simon; Furger, Andre; Snijders, Ambrosius P; Speck, Christian; Martinez-Perez, Enrique

2014-11-24

Proper chromosome segregation during meiosis requires the assembly of the synaptonemal complex (SC) between homologous chromosomes. However, the SC structure itself is indifferent to homology, and poorly understood mechanisms that depend on conserved HORMA-domain proteins prevent ectopic SC assembly. Although HORMA-domain proteins are thought to regulate SC assembly as intrinsic components of meiotic chromosomes, here we uncover a key role for nuclear soluble HORMA-domain protein HTP-1 in the quality control of SC assembly. We show that a mutant form of HTP-1 impaired in chromosome loading provides functionality of an HTP-1-dependent checkpoint that delays exit from homology search-competent stages until all homolog pairs are linked by the SC. Bypassing of this regulatory mechanism results in premature meiotic progression and licensing of homology-independent SC assembly. These findings identify nuclear soluble HTP-1 as a regulator of early meiotic progression, suggesting parallels with the mode of action of Mad2 in the spindle assembly checkpoint. Copyright © 2014 Elsevier Inc. All rights reserved.

Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity

PubMed Central

Warenius, H M; Jones, M; Gorman, T; McLeish, R; Seabra, L; Barraclough, R; Rudland, P

2000-01-01

The tumour suppressor gene, p53, and genes coding for positive signal transduction factors can influence transit through cell-cycle checkpoints and modulate radiosensitivity. Here we examine the effects of RAF1 protein on the rate of exit from a G2/M block induced by γ-irradiation in relation to intrinsic cellular radiosensitivity in human cell lines expressing wild-type p53 (wtp53) protein as compared to mutant p53 (mutp53) protein. Cell lines which expressed mutp53 protein were all relatively radioresistant and exhibited no relationship between RAF1 protein and cellular radiosensitivity. Cell lines expressing wtp53 protein, however, showed a strong relationship between RAF1 protein levels and the radiosensitivity parameter SF2. In addition, when post-irradiation perturbation of G2/M transit was compared using the parameter T50 (time after the peak of G2/M delay at which 50% of the cells had exited from a block induced by 2 Gy of irradiation), RAF1 was related to T50 in wtp53, but not mutp53, cell lines. Cell lines which expressed wtp53 protein and high levels of RAF1 had shorter T50s and were also more radiosensitive. These results suggest a cooperative role for wtp53 and RAF1 protein in determining cellular radiosensitivity in human cells, which involves control of the G2/M checkpoint. © 2000 Cancer Research Campaign PMID:10993658

Topoisomerase IIα maintains genomic stability through decatenation G2 checkpoint signaling

PubMed Central

Bower, Jacquelyn J.; Karaca, Gamze F.; Zhou, Yingchun; Simpson, Dennis A.; Cordeiro-Stone, Marila; Kaufmann, William K.

2010-01-01

Topoisomerase IIα (topoIIα) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G2 checkpoint response, which delays mitotic entry due to insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIα and topoIIβ on decatenatory activity in cell extracts, DNA damage and decatenation G2 checkpoint function, and the frequencies of p16INK4A allele loss and gain. In diploid human fibroblast lines, depletion of topoIIα by siRNA was associated with severely reduced decatenatory activity, delayed progression from G2 into mitosis, and insensitivity to G2 arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIα-depleted cells displayed increased frequencies of losses and gains of the tumor suppressor genetic locus p16INK4A. This study demonstrates that the topoIIα protein is required for decatenation G2 checkpoint function, and inactivation of decatenation and the decatenation G2 checkpoint leads to abnormal chromosome segregation and genomic instability. PMID:20562910

Endocrinological side-effects of immune checkpoint inhibitors.

PubMed

Torino, Francesco; Corsello, Salvatore M; Salvatori, Roberto

2016-07-01

Three mAbs targeting immune checkpoint proteins are available for the treatment of patients with melanoma, lung, and kidney cancer, and their use will likely expand in the future to additional tumor types. We here update the literature on the incidence and pathophysiology of endocrine toxicities induced by these agents, and discuss management guidance. Immune checkpoint inhibition may trigger autoimmune syndromes involving different organs, including several endocrine glands (pituitary, thyroid, adrenals, and endocrine pancreas). Hypophysitis is more frequently associated with ipilimumab, whereas the incidence of thyroid dysfunction is higher with nivolumab/pembrolizumab. Primary adrenal insufficiency can rarely occur with either treatment. Autoimmune diabetes is very rare. As hypophysitis and adrenalitis may be life-threatening, endocrinological evaluation is essential particularly in patients developing fatigue and other symptoms consistent with adrenal insufficiency. Corticosteroids should be promptly used when hypophysitis-induced adrenal insufficiency or adrenalitis are diagnosed, but not in thyroiditis or diabetes. No impact of corticosteroids on the efficacy/activity of immune checkpoint-inhibiting drugs is reported. Hormonal deficiencies are often permanent. In absence of predicting factors, accurate information to patients provided by the oncology care team is essential for early diagnosis and to limit the consequences of checkpoint inhibition-related endocrine toxicity.

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint

PubMed Central

Cescutti, Rachele; Negrini, Simona; Kohzaki, Masaoki; Halazonetis, Thanos D

2010-01-01

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double-strand breaks (DSBs), but only in the G1-phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1–2 and 7–8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1–2 and 4–5. The BRCT domains 4–5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S-phase, we observed a checkpoint defect after siRNA-mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1. PMID:20871591

TopBP1 functions with 53BP1 in the G1 DNA damage checkpoint.

PubMed

Cescutti, Rachele; Negrini, Simona; Kohzaki, Masaoki; Halazonetis, Thanos D

2010-11-03

TopBP1 is a checkpoint protein that colocalizes with ATR at sites of DNA replication stress. In this study, we show that TopBP1 also colocalizes with 53BP1 at sites of DNA double-strand breaks (DSBs), but only in the G1-phase of the cell cycle. Recruitment of TopBP1 to sites of DNA replication stress was dependent on BRCT domains 1-2 and 7-8, whereas recruitment to sites of DNA DSBs was dependent on BRCT domains 1-2 and 4-5. The BRCT domains 4-5 interacted with 53BP1 and recruitment of TopBP1 to sites of DNA DSBs in G1 was dependent on 53BP1. As TopBP1 contains a domain important for ATR activation, we examined whether it contributes to the G1 cell cycle checkpoint. By monitoring the entry of irradiated G1 cells into S-phase, we observed a checkpoint defect after siRNA-mediated depletion of TopBP1, 53BP1 or ATM. Thus, TopBP1 may mediate the checkpoint function of 53BP1 in G1.

Immune checkpoint inhibitors in lung cancer: current status and future directions.

PubMed

Fan, Yun; Mao, Weimin

2017-04-01

Recently, the immune checkpoint inhibitors that target programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) have made a breakthrough in treating advanced non-small cell lung cancer (NSCLC) with the efficacy of approximately 20%; among which, nivolumab has acquired treatment indications in lung squamous cell carcinoma. The inhibitors targeting cytotoxic T lymphocyte associated antigen 4 (CTLA-4) are also undergoing clinical trials. Researches on immune checkpoint inhibitors have been rapidly implemented in a variety of different types of lung cancer, such as small cell lung cancer (SCLC) and locally advanced NSCLC, and these inhibitors began to be applied in combination with some established treatments, including chemotherapy, targeting therapy and radiotherapy. Undoubtedly, the immune checkpoint inhibitors have become a hot spot in the research and treatment of lung cancer. However, many problems wait to be solved, such as searching for ideal biomarkers, constituting the best criteria for curative effect evaluation, exploring different combination treatment models, and clearly understanding the mechanisms of primary or secondary drug resistance. Along with these problems to be successfully solved, the immune checkpoint inhibitors will have more broad applications in lung cancer therapy.

Deficiency of the Arabidopsis Helicase RTEL1 Triggers a SOG1-Dependent Replication Checkpoint in Response to DNA Cross-Links

PubMed Central

Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

2015-01-01

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. PMID:25595823

Somatic Mutations and Neoepitope Homology in Melanomas Treated with CTLA-4 Blockade.

PubMed

Nathanson, Tavi; Ahuja, Arun; Rubinsteyn, Alexander; Aksoy, Bulent Arman; Hellmann, Matthew D; Miao, Diana; Van Allen, Eliezer; Merghoub, Taha; Wolchok, Jedd D; Snyder, Alexandra; Hammerbacher, Jeff

2017-01-01

Immune checkpoint inhibitors are promising treatments for patients with a variety of malignancies. Toward understanding the determinants of response to immune checkpoint inhibitors, it was previously demonstrated that the presence of somatic mutations is associated with benefit from checkpoint inhibition. A hypothesis was posited that neoantigen homology to pathogens may in part explain the link between somatic mutations and response. To further examine this hypothesis, we reanalyzed cancer exome data obtained from our previously published study of 64 melanoma patients treated with CTLA-4 blockade and a new dataset of RNA-Seq data from 24 of these patients. We found that the ability to accurately predict patient benefit did not increase as the analysis narrowed from somatic mutation burden, to inclusion of only those mutations predicted to be MHC class I neoantigens, to only including those neoantigens that were expressed or that had homology to pathogens. The only association between somatic mutation burden and response was found when examining samples obtained prior to treatment. Neoantigen and expressed neoantigen burden were also associated with response, but neither was more predictive than somatic mutation burden. Neither the previously described tetrapeptide signature nor an updated method to evaluate neoepitope homology to pathogens was more predictive than mutation burden. Cancer Immunol Res; 5(1); 84-91. ©2016 AACR. ©2016 American Association for Cancer Research.

C/EBPα Expression is Partially Regulated by C/EBPβ in Response to DNA Damage and C/EBPα Deficient Fibroblasts Display an Impaired G1 Checkpoint

PubMed Central

Ranjan, Rakesh; Thompson, Elizabeth A.; Yoon, Kyungsil; Smart, Robert C.

2009-01-01

We observed that C/EBPα is highly inducible in primary fibroblasts by DNA damaging agents that induce strand breaks, alkylate and crosslink DNA as well as those that produce bulky DNA lesions. Fibroblasts deficient in C/EBPα (C/EBPα-/-) display an impaired G1 checkpoint as evidenced by inappropriate entry into S-phase in response to DNA damage and these cells also display an enhanced G1 to S transition in response to mitogens. The induction of C/EBPα by DNA damage in fibroblasts does not require p53. EMSA analysis of nuclear extracts prepared from UVB- and MNNG-treated fibroblasts revealed increased binding of C/EBPβ to a C/EBP consensus sequence and ChIP analysis revealed increased C/EBPβ binding to the C/EBPα promoter. To determine whether C/EBPβ has a role in the regulation of C/EBPα we treated C/EBPβ-/- fibroblasts with UVB or MNNG. We observed C/EBPα induction was impaired in both UVB- and MNNG- treated C/EBPβ-/- fibroblasts. Our study reveals a novel role for C/EBPβ in the regulation of C/EBPα in response to DNA damage and provides definitive genetic evidence that C/EBPα has a critical role in the DNA damage G1 checkpoint. PMID:19581927

Rituximab does not reset defective early B cell tolerance checkpoints

PubMed Central

Chamberlain, Nicolas; Massad, Christopher; Oe, Tyler; Cantaert, Tineke; Herold, Kevan C.; Meffre, Eric

2015-01-01

Type 1 diabetes (T1D) patients show abnormalities in early B cell tolerance checkpoints, resulting in the accumulation of large numbers of autoreactive B cells in their blood. Treatment with rituximab, an anti-CD20 mAb that depletes B cells, has been shown to preserve β cell function in T1D patients and improve other autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. However, it remains largely unknown how anti–B cell therapy thwarts autoimmunity in these pathologies. Here, we analyzed the reactivity of Abs expressed by single, mature naive B cells from 4 patients with T1D before and 52 weeks after treatment to determine whether rituximab resets early B cell tolerance checkpoints. We found that anti–B cell therapy did not alter the frequencies of autoreactive and polyreactive B cells, which remained elevated in the blood of all patients after rituximab treatment. Moreover, the limited proliferative history of autoreactive B cells after treatment revealed that these clones were newly generated B cells and not self-reactive B cells that had escaped depletion and repopulated the periphery through homeostatic expansion. We conclude that anti–B cell therapy may provide a temporary dampening of autoimmune processes through B cell depletion. However, repletion with autoreactive B cells may explain the relapse that occurs in many autoimmune patients after anti–B cell therapy. PMID:26642366

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

PubMed

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

PubMed Central

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

sLORETA current source density analysis of evoked potentials for spatial updating in a virtual navigation task

PubMed Central

Nguyen, Hai M.; Matsumoto, Jumpei; Tran, Anh H.; Ono, Taketoshi; Nishijo, Hisao

2014-01-01

Previous studies have reported that multiple brain regions are activated during spatial navigation. However, it is unclear whether these activated brain regions are specifically associated with spatial updating or whether some regions are recruited for parallel cognitive processes. The present study aimed to localize current sources of event related potentials (ERPs) associated with spatial updating specifically. In the control phase of the experiment, electroencephalograms (EEGs) were recorded while subjects sequentially traced 10 blue checkpoints on the streets of a virtual town, which were sequentially connected by a green line, by manipulating a joystick. In the test phase of the experiment, the checkpoints and green line were not indicated. Instead, a tone was presented when the subjects entered the reference points where they were then required to trace the 10 invisible spatial reference points corresponding to the checkpoints. The vertex-positive ERPs with latencies of approximately 340 ms from the moment when the subjects entered the unmarked reference points were significantly larger in the test than in the control phases. Current source density analysis of the ERPs by standardized low-resolution brain electromagnetic tomography (sLORETA) indicated activation of brain regions in the test phase that are associated with place and landmark recognition (entorhinal cortex/hippocampus, parahippocampal and retrosplenial cortices, fusiform, and lingual gyri), detecting self-motion (posterior cingulate and posterior insular cortices), motor planning (superior frontal gyrus, including the medial frontal cortex), and regions that process spatial attention (inferior parietal lobule). The present results provide the first identification of the current sources of ERPs associated with spatial updating, and suggest that multiple systems are active in parallel during spatial updating. PMID:24624067

Cache-based error recovery for shared memory multiprocessor systems

NASA Technical Reports Server (NTRS)

Wu, Kun-Lung; Fuchs, W. Kent; Patel, Janak H.

1989-01-01

A multiprocessor cache-based checkpointing and recovery scheme for of recovering from transient processor errors in a shared-memory multiprocessor with private caches is presented. New implementation techniques that use checkpoint identifiers and recovery stacks to reduce performance degradation in processor utilization during normal execution are examined. This cache-based checkpointing technique prevents rollback propagation, provides for rapid recovery, and can be integrated into standard cache coherence protocols. An analytical model is used to estimate the relative performance of the scheme during normal execution. Extensions that take error latency into account are presented.

A Job Pause Service under LAM/MPI+BLCR for Transparent Fault Tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Chao; Mueller, Frank; Engelmann, Christian

2007-01-01

Checkpoint/restart (C/R) has become a requirement for long-running jobs in large-scale clusters due to a meantime- to-failure (MTTF) in the order of hours. After a failure, C/R mechanisms generally require a complete restart of an MPI job from the last checkpoint. A complete restart, however, is unnecessary since all but one node are typically still alive. Furthermore, a restart may result in lengthy job requeuing even though the original job had not exceeded its time quantum. In this paper, we overcome these shortcomings. Instead of job restart, we have developed a transparent mechanism for job pause within LAM/MPI+BLCR. This mechanismmore » allows live nodes to remain active and roll back to the last checkpoint while failed nodes are dynamically replaced by spares before resuming from the last checkpoint. Our methodology includes LAM/MPI enhancements in support of scalable group communicationwith fluctuating number of nodes, reuse of network connections, transparent coordinated checkpoint scheduling and a BLCR enhancement for job pause. Experiments in a cluster with the NAS Parallel Benchmark suite show that our overhead for job pause is comparable to that of a complete job restart. A minimal overhead of 5.6% is only incurred in case migration takes place while the regular checkpoint overhead remains unchanged. Yet, our approach alleviates the need to reboot the LAM run-time environment, which accounts for considerable overhead resulting in net savings of our scheme in the experiments. Our solution further provides full transparency and automation with the additional benefit of reusing existing resources. Executing continues after failures within the scheduled job, i.e., the application staging overhead is not incurred again in contrast to a restart. Our scheme offers additional potential for savings through incremental checkpointing and proactive diskless live migration, which we are currently working on.« less

Prospects for TIM3-Targeted Antitumor Immunotherapy.

PubMed

Ngiow, Shin Foong; Teng, Michele W L; Smyth, Mark J

2011-11-01

New insights into the control of T-cell activation and proliferation have led to the identification of checkpoint proteins that either up- or downmodulate T-cell reactivity. Monoclonal antibody immunotherapies that are reactive with cytotoxic T lymphocyte antigen 4 or programmed death receptor 1 have shown promising therapeutic outcomes in mice and humans with established cancer, highlighting the fact that cancer immunotherapy using T-cell checkpoint inhibitors is one of the most promising new therapeutic approaches. T-cell immunoglobulin and mucin domain 3 (TIM3) is one of many similar inhibitory molecules that are gaining attention as targets, but it remains relatively poorly studied in oncology. This review discusses our recent probing of the mechanism of action of anti-TIM3 antibody against established spontaneous and experimental tumors in mice, in the context of the exciting possibility of rationally combining agents that promote tumor-specific T-cell activation, proliferation, effector function, and survival. ©2011 AACR.

In-field and abscopal response after short-course radiation therapy in patients with metastatic Merkel cell carcinoma progressing on PD-1 checkpoint blockade: a case series.

PubMed

Xu, Melody J; Wu, Susan; Daud, Adil I; Yu, Siegrid S; Yom, Sue S

2018-05-30

Patients with metastatic Merkel cell carcinoma (mMCC) who experience disease progression on immunotherapy have limited additional standard options. Given evidence of synergism between radiation therapy (RT) and immunotherapy, two patients progressing on PD-1 inhibition were referred for short-course RT. Two patients were found to have progressive mMCC on PD-1 inhibitor therapy and were treated with single-fraction palliative RT. Both patients were observed to have local control at irradiated regions, as well as durable abscopal response at unirradiated, out-of-field, sites of metastatic disease. Short-course RT is a compelling strategy that could be a means to augment response in patients with mMCC who show progression on immune checkpoint blockade. Ongoing clinical trials are investigating the relationship between RT and immunotherapy in mMCC.

Sperm nuclear protamines: A checkpoint to control sperm chromatin quality.

PubMed

Steger, Klaus; Balhorn, Rod

2018-05-23

Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint during sperm development rendering protamines as suitable biomarkers for the estimation of sperm quality. This review presents an overview of our current knowledge on protamines comparing gene and protein structures between different mammalian species with particular consideration given to man, mouse and stallion. At last, recent insights into the possible role of inherited sperm histones for early embryo development are provided. © 2018 Blackwell Verlag GmbH.

What’s the Damage? The Impact of Pathogens on Pathways that Maintain Host Genome Integrity

PubMed Central

Weitzman, Matthew D.; Weitzman, Jonathan B.

2014-01-01

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs. PMID:24629335

Targeting Tumor-Associated Macrophages as a Potential Strategy to Enhance the Response to Immune Checkpoint Inhibitors.

PubMed

Cassetta, Luca; Kitamura, Takanori

2018-01-01

Inhibition of immune checkpoint pathways in CD8 + T cell is a promising therapeutic strategy for the treatment of solid tumors that has shown significant anti-tumor effects and is now approved by the FDA to treat patients with melanoma and lung cancer. However the response to this therapy is limited to a certain fraction of patients and tumor types, for reasons still unknown. To ensure success of this treatment, CD8 + T cells, the main target of the checkpoint inhibitors, should exert full cytotoxicity against tumor cells. However recent studies show that tumor-associated macrophages (TAM) can impede this process by different mechanisms. In this mini-review we will summarize recent studies showing the effect of TAM targeting on immune checkpoint inhibitors efficacy. We will also discuss on the limitations of the current strategies as well on the future scientific challenges for the progress of the tumor immunology field.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

PubMed

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2014-06-30

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

SCFFBXW7α modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability

PubMed Central

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

2014-01-01

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint. PMID:24970797

Cell cycle gene expression networks discovered using systems biology: Significance in carcinogenesis

PubMed Central

Scott, RE; Ghule, PN; Stein, JL; Stein, GS

2015-01-01

The early stages of carcinogenesis are linked to defects in the cell cycle. A series of cell cycle checkpoints are involved in this process. The G1/S checkpoint that serves to integrate the control of cell proliferation and differentiation is linked to carcinogenesis and the mitotic spindle checkpoint with the development of chromosomal instability. This paper presents the outcome of systems biology studies designed to evaluate if networks of covariate cell cycle gene transcripts exist in proliferative mammalian tissues including mice, rats and humans. The GeneNetwork website that contains numerous gene expression datasets from different species, sexes and tissues represents the foundational resource for these studies (www.genenetwork.org). In addition, WebGestalt, a gene ontology tool, facilitated the identification of expression networks of genes that co-vary with key cell cycle targets, especially Cdc20 and Plk1 (www.bioinfo.vanderbilt.edu/webgestalt). Cell cycle expression networks of such covariate mRNAs exist in multiple proliferative tissues including liver, lung, pituitary, adipose and lymphoid tissues among others but not in brain or retina that have low proliferative potential. Sixty-three covariate cell cycle gene transcripts (mRNAs) compose the average cell cycle network with p = e−13 to e−36. Cell cycle expression networks show species, sex and tissue variability and they are enriched in mRNA transcripts associated with mitosis many of which are associated with chromosomal instability. PMID:25808367

Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function.

PubMed

Lee, Young-Hee; Martin-Orozco, Natalia; Zheng, Peilin; Li, Jing; Zhang, Peng; Tan, Haidong; Park, Hyun Jung; Jeong, Mira; Chang, Seon Hee; Kim, Byung-Seok; Xiong, Wei; Zang, Wenjuan; Guo, Li; Liu, Yang; Dong, Zhong-Jun; Overwijk, Willem W; Hwu, Patrick; Yi, Qing; Kwak, Larry; Yang, Zhiying; Mak, Tak W; Li, Wei; Radvanyi, Laszlo G; Ni, Ling; Liu, Dongfang; Dong, Chen

2017-08-01

The interaction between tumor and the immune system is still poorly understood. Significant clinical responses have been achieved in cancer patients treated with antibodies against the CTLA4 and PD-1/PD-L1 checkpoints; however, only a small portion of patients responded to the therapies, indicating a need to explore additional co-inhibitory molecules for cancer treatment. B7-H3, a member of the B7 superfamily, was previously shown by us to inhibit T-cell activation and autoimmunity. In this study, we have analyzed the function of B7-H3 in tumor immunity. Expression of B7-H3 was found in multiple tumor lines, tumor-infiltrating dendritic cells, and macrophages. B7-H3-deficient mice or mice treated with an antagonistic antibody to B7-H3 showed reduced growth of multiple tumors, which depended on NK and CD8 + T cells. With a putative receptor expressed by cytotoxic lymphocytes, B7-H3 inhibited their activation, and its deficiency resulted in increased cytotoxic lymphocyte function in tumor-bearing mice. Combining blockades of B7-H3 and PD-1 resulted in further enhanced therapeutic control of late-stage tumors. Taken together, our results indicate that the B7-H3 checkpoint may serve as a novel target for immunotherapy against cancer.

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

PubMed Central

Warenius, H. M.; Jones, M.; Jones, M. D.; Browning, P. G.; Seabra, L. A.; Thompson, C. C.

1998-01-01

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009). PMID:9579826

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma

PubMed Central

Zeng, Ling; Beggs, Reena R.; Cooper, Tiffiny S.; N.Weaver, Alice; S.Yang, Eddy

2017-01-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo, suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). PMID:28138028

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

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. PMID:21437275

Checkpoint inhibitors in endometrial cancer: preclinical rationale and clinical activity.

PubMed

Mittica, Gloria; Ghisoni, Eleonora; Giannone, Gaia; Aglietta, Massimo; Genta, Sofia; Valabrega, Giorgio

2017-10-27

Treatment of advanced and recurrent endometrial cancer (EC) is still an unmet need for oncologists and gynecologic oncologists. The Cancer Genome Atlas Research Network (TCGA) recently provided a new genomic classification, dividing EC in four subgroups. Two types of EC, the polymerase epsilon (POLE)-ultra-mutated and the microsatellite instability-hyper-mutated (MSI-H), are characterized by a high mutation rate providing the rationale for a potential activity of checkpoint inhibitors. We analyzed all available evidence supporting the role of tumor microenvironment (TME) in EC development and the therapeutic implications offered by immune checkpoint inhibitors in this setting. We performed a review on Pubmed with Mesh keywords 'endometrial cancer' and the name of each checkpoint inhibitor discussed in the article. The same search was operated on clinicaltrial.gov to identify ongoing clinical trials exploring PD-1/PD-L1 and CTLA-4 axis in EC, particularly focusing on POLE-ultra-muted and MSI-H cancer types. POLE-ultra-mutated and MSI-H ECs showed an active TME expressing high number of neo-antigens and an elevated amount of tumor infiltrating lymphocytes (TILs). Preliminary results from a phase-1 clinical trial (KEYNOTE-028) demonstrated antitumor activity of Pembrolizumab in EC. Moreover, both Pembrolizumab and Nivolumab reported durable clinical responses in POLE-ultra-mutated patients. Immune checkpoint inhibitors are an attractive option in POLE-ultra-mutated and MSI-H ECs. Future investigations in these subgroups include combinations of checkpoints inhibitors with chemotherapy and small tyrosine kinase inhibitors (TKIs) to enhance a more robust intra-tumoral immune response.

Counterinsurgency After Afghanistan: A Concept in Crisis

DTIC Science & Technology

2011-12-01

in strategy was undeniably stabilizing. This also explains the concrete walls, barriers, check- points, and other population-control measures...partnering with Sunni community leaders, Dagger Brigade was able to interpose itself—with concrete barriers, combined outposts, and checkpoints...local forces work together toward the same end. Also important in this regard are the aforementioned check- points, concrete barriers, and other

Washington’s target zero teams project : reduction in fatalities during year one.

DOT National Transportation Integrated Search

2012-11-01

Highly visible sobriety checkpoint programs have : been shown to be an effective method of reducing : alcohol-related driving crashes (Elder et al., 2002). : However, the use of checkpoints is prohibited in 12 : States, including Washington. These St...

CD8+T cells expressing both PD-1 and TIGIT but not CD226 are dysfunctional in acute myeloid leukemia (AML) patients.

PubMed

Wang, Mengjie; Bu, Jin; Zhou, Maohua; Sido, Jessica; Lin, Yu; Liu, Guanfang; Lin, Qiwen; Xu, Xiuzhang; Leavenworth, Jianmei W; Shen, Erxia

2018-05-01

Acute myeloid leukemia (AML) is one of the most common types of leukemia among adults with an overall poor prognosis and very limited treatment management. Immune checkpoint blockade of PD-1 alone or combined with other immune checkpoint blockade has gained impressive results in murine AML models by improving anti-leukemia CD8 + T cell function, which has greatly promoted the strategy to utilize combined immune checkpoint inhibitors to treat AML patients. However, the expression profiles of these immune checkpoint receptors, such as co-inhibitory receptors PD-1 and TIGIT and co-stimulatory receptor CD226, in T cells from AML patients have not been clearly defined. Here we have defined subsets of CD8 + and CD4 + T cells in the peripheral blood (PB) from newly diagnosed AML patients and healthy controls (HCs). We have observed increased frequencies of PD-1- and TIGIT- expressing CD8 + T cells but decreased occurrence of CD226-expressing CD8 + T cells in AML patients. Further analysis of these CD8 + T cells revealed a unique CD8 + T cell subset that expressed PD-1 and TIGIT but displayed lower levels of CD226 was associated with failure to achieve remission after induction chemotherapy and FLT3-ITD mutations which predict poor clinical prognosis in AML patients. Importantly, these PD-1 + TIGIT + CD226 - CD8 + T cells are dysfunctional with lower expression of intracellular IFN-γ and TNF-α than their counterparts in HCs. Therefore, our studies revealed that an increased frequency of a unique CD8 + T cell subset, PD-1 + TIGIT + CD226 - CD8 + T cells, is associated with CD8 + T cell dysfunction and poor clinical prognosis of AML patients, which may reveal critical diagnostic or prognostic biomarkers and direct more efficient therapeutic strategies. Copyright © 2017. Published by Elsevier Inc.

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

PubMed Central

2013-01-01

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 Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway. PMID:23347679

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

PubMed

Pushpavalli, Sreerangam N C V L; Sarkar, Arpita; Ramaiah, M Janaki; Chowdhury, Debabani Roy; Bhadra, Utpal; Pal-Bhadra, Manika

2013-01-24

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. 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. 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 Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway.

Checkpoint and restart procedures for single and multi-stage structural model analysis in NASTRAN/COSMIC on a CDC 176

NASA Technical Reports Server (NTRS)

Camp, George H.; Fallon, Dennis J.

1987-01-01

The Underwater Explosions Research Division (UERD) of the David Taylor Naval Ship Research and Development Center makes extensive use of NASTRAN/COSMIC on a CDC 176 to evaluate the structural response of ship structures subjected to underwater explosion shock loadings in the time domain. As relatively new users, UERD engineers have experienced difficulties with the checkpoint/restart feature because of the vague instructions in the user manual. Working procedures for the application of the checkpoint/restart feature to the transient analysis using NASTRAN/COSMIC are illustrated.

Immune mediated neuropathy following checkpoint immunotherapy.

PubMed

Gu, Yufan; Menzies, Alexander M; Long, Georgina V; Fernando, S L; Herkes, G

2017-11-01

Checkpoint immunotherapy has revolutionised cancer therapy and is now standard treatment for many malignancies including metastatic melanoma. Acute inflammatory neuropathies, often labelled as Guillain-Barre syndrome, are an uncommon but potentially severe complication of checkpoint immunotherapy with individual cases described but never characterised as a group. We describe a case of acute sensorimotor and autonomic neuropathy following a single dose of combination ipilimumab and nivolumab for metastatic melanoma. A literature search was performed, identifying 14 other cases of acute neuropathy following checkpoint immunotherapy, with the clinical, electrophysiological and laboratory features summarised. Most cases described an acute sensorimotor neuropathy (92%) with hyporeflexia (92%) that could occur from induction up till many weeks after the final dose of therapy. In contrast to Guillain-Barre syndrome, the cerebrospinal fluid (CSF) analysis often shows a lymphocytic picture (50%) and the electrophysiology showed an axonal pattern (55%). Treatment was variable and often in combination. 11 cases received steroid therapy with only 1 death within this group, whereas of the 4 patients who did not receive steroid therapy there were 3 deaths. In conclusion checkpoint immunotherapy - induced acute neuropathies are distinct from and progress differently to Guillain-Barre syndrome. As with other immunotherapy related adverse events corticosteroid therapy should be initiated in addition to usual therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

CHECKPOINT INHIBITOR IMMUNE THERAPY: Systemic Indications and Ophthalmic Side Effects.

PubMed

Dalvin, Lauren A; Shields, Carol L; Orloff, Marlana; Sato, Takami; Shields, Jerry A

2018-06-01

To review immune checkpoint inhibitor indications and ophthalmic side effects. A literature review was performed using a PubMed search for publications between 1990 and 2017. Immune checkpoint inhibitors are designed to treat system malignancies by targeting one of three ligands, leading to T-cell activation for attack against malignant cells. These ligands (and targeted drug) include cytotoxic T-lymphocyte antigen-4 (CTLA-4, ipilimumab), programmed death protein 1 (PD-1, pembrolizumab, nivolumab), and programmed death ligand-1 (PD-L1, atezolizumab, avelumab, durvalumab). These medications upregulate the immune system and cause autoimmune-like side effects. Ophthalmic side effects most frequently manifest as uveitis (1%) and dry eye (1-24%). Other side effects include myasthenia gravis (n = 19 reports), inflammatory orbitopathy (n = 11), keratitis (n = 3), cranial nerve palsy (n = 3), optic neuropathy (n = 2), serous retinal detachment (n = 2), extraocular muscle myopathy (n = 1), atypical chorioretinal lesions (n = 1), immune retinopathy (n = 1), and neuroretinitis (n = 1). Most inflammatory side effects are managed with topical or periocular corticosteroids, but advanced cases require systemic corticosteroids and cessation of checkpoint inhibitor therapy. Checkpoint inhibitors enhance the immune system by releasing inhibition on T cells, with risk of autoimmune-like side effects. Ophthalmologists should include immune-related adverse events in their differential when examining cancer patients with new ocular symptoms.

Endocrine-related adverse events associated with immune checkpoint blockade and expert insights on their management.

PubMed

Sznol, Mario; Postow, Michael A; Davies, Marianne J; Pavlick, Anna C; Plimack, Elizabeth R; Shaheen, Montaser; Veloski, Colleen; Robert, Caroline

2017-07-01

Agents that modulate immune checkpoint proteins, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death receptor-1 (PD-1), have become a mainstay in cancer treatment. The clinical benefit afforded by immune checkpoint inhibitors can be accompanied by immune-related adverse events (irAE) that affect the skin, gastrointestinal tract, liver, and endocrine system. The types of irAEs associated with immune checkpoint inhibitors are generally consistent across tumor types. Immune-related endocrine events can affect the pituitary, thyroid, and adrenal glands, as well as other downstream target organs. These events are unique when compared with other irAEs because the manifestations are often irreversible. Immune-related endocrine events are typically grade 1/2 in severity and often present with non-specific symptoms, making them difficult to diagnose. The mechanisms underlying immune-related target organ damage in select individuals remain mostly undefined. Management includes close patient monitoring, appropriate laboratory testing for endocrine function, replacement of hormones, and consultation with an endocrinologist when appropriate. An awareness of the symptoms and management of immune-related endocrine events may aid in the safe and appropriate use of immune checkpoint inhibitors in clinical practice. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Modeling the temporal evolution of the spindle assembly checkpoint and role of Aurora B kinase

PubMed Central

Mistry, Hitesh B.; MacCallum, David E.; Jackson, Robert C.; Chaplain, Mark A. J.; Davidson, Fordyce A.

2008-01-01

Faithful separation of chromosomes prior to cell division at mitosis is a highly regulated process. One family of serine/threonine kinases that plays a central role in regulation is the Aurora family. Aurora B plays a role in the spindle assembly checkpoint, in part, by destabilizing the localization of BubR1 and Mad2 at centrosomes and responds to changes in tension caused by aberrant microtubule kinetochore attachments. Aurora B is overexpressed in a subset of cancers and is required for mitosis, making it an attractive anticancer target. Here, we use mathematical modeling to extend a current model of the spindle assembly checkpoint to incorporate all signaling kinetochores within a cell rather than just one and the role of Aurora B within the resulting model. We find that the current model of the spindle assembly checkpoint is robust to variation in its key diffusion-limited parameters. Furthermore, when Aurora B inhibition is considered within the model, for a certain range of inhibitor concentrations, a prolonged prometaphase/metaphase is observed. This level of inhibitor concentrations has not yet been studied experimentally, to the authors' best knowledge. Therefore, experimental verification of the results discussed here could provide a deeper understanding of how kinetochores and Aurora B cooperate in the spindle assembly checkpoint. PMID:19091947

Distinct chromosome segregation roles for spindle checkpoint proteins.

PubMed

Warren, Cheryl D; Brady, D Michelle; Johnston, Raymond C; Hanna, Joseph S; Hardwick, Kevin G; Spencer, Forrest A

2002-09-01

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.

Distinct Chromosome Segregation Roles for Spindle Checkpoint Proteins

PubMed Central

Warren, Cheryl D.; Brady, D. Michelle; Johnston, Raymond C.; Hanna, Joseph S.; Hardwick, Kevin G.; Spencer, Forrest A.

2002-01-01

The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage. PMID:12221113

Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores.

PubMed

London, Nitobe; Ceto, Steven; Ranish, Jeffrey A; Biggins, Sue

2012-05-22

Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that copurifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores, and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein. Copyright © 2012 Elsevier Ltd. All rights reserved.

Expected Paradigm Shift in Brain Metastases Therapy-Immune Checkpoint Inhibitors.

PubMed

Jindal, Vishal; Gupta, Sorab

2018-01-30

Brain metastasis (BM) is one of the dreadful complications of malignancies. The prognosis after BM is extremely poor and life expectancy is meager. Currently, our treatment modalities are limited to radiotherapy and surgical resection, which also has poor outcomes and leads to various neurological deficits and affects the quality of life of patients. New treatment modality, i.e., immune checkpoint inhibitors, has brought revolution in management of melanoma, renal cancer, and non-small cell lung cancer (NSCLC). Immune checkpoint inhibitors basically enhance the immune response of the body to fight against cancers. Immune response in the brain is highly regulated; therefore, it is challenging to use immune-modulator drugs in BM. The microenvironment of BM is rich in cytotoxic T lymphocytes and which is the target of immune checkpoint inhibitors. Few studies have shown some hope regarding use of immune checkpoint inhibitors in management of BM. It works through inhibiting immune check point gates, i.e., CTLA-4 (cytotoxic T-lymphocyte-associated protein) and PD-1/PD-L1 (programmed cell death protein-1/program death ligand-1). This article explains the basic mechanism of immune check point inhibitors, rationale behind their usage in BM, and some of the clinical studies which have shown the efficacy of immune check point inhibitors in BM.

Stop and go: hematopoietic cell transplantation in the era of chimeric antigen receptor T cells and checkpoint inhibitors.

PubMed

Ghosh, Arnab; Politikos, Ioannis; Perales, Miguel-Angel

2017-11-01

For several decades, hematopoietic cell transplantation (HCT) has been considered the standard curative therapy for many patients with hematological malignancies. In addition to the cytotoxic effects of the chemotherapy and radiation used in the conditioning regimen, the benefits of HCT are derived from a reset of the immune system and harnessing the ability of donor T cells to eliminate malignant cells. With the dawn of the era of immunotherapies in the form of checkpoint inhibitors and chimeric antigen receptor (CAR) T cells, the role of HCT has evolved. Immunotherapy with checkpoint inhibitors is increasingly being used for relapsed Hodgkin and non-Hodgkin lymphoma after autologous HCT. Checkpoint inhibitors are also being tested after allogeneic HCT with observable benefits in treating hematological malignancies, but with a potential risk of increased graft versus host disease and transplant-related mortality. Immunotherapy with Cluster of differentiation 19 CAR T cells are powerful options with aggressive B-cell malignancies both for therapy and as induction leading to allogeneic HCT. Although immunotherapies with checkpoint inhibition and CAR T cells are increasingly being used to treat hematological malignancies, HCT remains a standard of care for most of the diseases with the best chance of cure. Combination of these therapies with HCT has the potential to more effectively treat hematological malignancies.

Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

PubMed

Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

2015-01-01

To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. © 2015 American Society of Plant Biologists. All rights reserved.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria.

PubMed

Dulin, David; Bauer, David L V; Malinen, Anssi M; Bakermans, Jacob J W; Kaller, Martin; Morichaud, Zakia; Petushkov, Ivan; Depken, Martin; Brodolin, Konstantin; Kulbachinskiy, Andrey; Kapanidis, Achillefs N

2018-04-16

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Pilot test of selected DWI detection procedures for use at sobriety checkpoints

DOT National Transportation Integrated Search

1985-04-01

This report presents the results of a study designed to evaluate a variety of potential screening procedures police officers could use at the brief initial stop at a sobriety checkpoint to discriminate between impaired and sober drivers. The potentia...

Epithelial-mesenchymal transition is associated with a distinct tumor microenvironment including elevation of inflammatory signals and multiple immune checkpoints in lung adenocarcinoma

PubMed Central

Lou, Yanyan; Diao, Lixia; Cuentas, Edwin Roger Parra; Denning, Warren L.; Chen, Limo; Fan, Youhong; Byers, Lauren A.; Wang, Jing; Papadimitrakopoulou, Vassiliki; Behrens, Carmen; Rodriguez, Jaime Canales; Hwu, Patrick; Wistuba, Ignacio I.; Heymach, John V.; Gibbons, Don L.

2016-01-01

Purpose Promising results in the treatment of NSCLC have been seen with agents targeting immune checkpoints, such as PD-1 or PD-L1. However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas (TCGA) of lung adenocarcinoma and two datasets from MD Anderson Cancer Center, Profiling of Resistance patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse phase protein array (RPPA), immunohistochemistry and correlation with clinical data were performed. Results Epithelial-mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation co-existent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. PMID:26851185

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

PubMed Central

Wu, Xiaoming; Shell, Steven M.; Yang, Zhengguan; Zou, Yue

2006-01-01

DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATR-dependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation of XPA at Ser196 on UV irradiation. PMID:16540648

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

PubMed Central

Palii, Stela S.; Cui, Yuxia; Innes, Cynthia L.; Paules, Richard S.

2013-01-01

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G₂/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G₂/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G₂/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G₂/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments. PMID:23462183

5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint.

PubMed

Luciani, M Gloria; Campregher, Christoph; Fortune, John M; Kunkel, Thomas A; Gasche, Christoph

2007-01-01

Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.

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

PubMed

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

2017-04-24

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. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Immune checkpoint inhibitor-related myocarditis.

PubMed

Tajiri, Kazuko; Aonuma, Kazutaka; Sekine, Ikuo

2018-01-01

Immune checkpoint inhibitors have demonstrated significant clinical benefit in many cancers. The clinical benefit afforded by these treatments can be accompanied by a unique and distinct spectrum of adverse events. Recently, several fatal cases of immune checkpoint inhibitor-related myocarditis were reported. Although its frequency is comparatively lower than that of other immune-related adverse events, myocarditis can lead to circulatory collapse and lethal ventricular arrhythmia. Immune checkpoints, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), play important roles in establishing peripheral tolerance to the heart. Evidence from studies using genetically engineered mouse models suggests that CTLA-4 signaling terminates proliferation and promotes anergy during the primary response to cardiac self-peptide recognition. PD-1 signaling restrains autoreactive T cells that enter the peripheral tissues and recognize cardiac-peptide, maintaining them in an anergic state. Patients affected by immune checkpoint inhibitor-related myocarditis often experience rapid onset of profound hemodynamic compromise progressing to cardiogenic shock. Early diagnosis is mandatory to address specific therapy and correct the timing of circulatory support. However, the diagnosis of myocarditis is challenging due to the heterogeneity of clinical presentations. Owing to its early onset, nonspecific symptomatology and fulminant progression, especially when these drugs are used in combination, oncologists should be vigilant for immune checkpoint inhibitor-related myocarditis. With many questions yet to be answered, from basic immune biology to clinical management, future research should aim to optimize the use of these drugs by identifying predictive biomarkers of either a response to therapy or the risks of myocarditis development. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cellular abundance of Mps1 and the role of its carboxyl terminal tail in substrate recruitment.

PubMed

Sun, Tingting; Yang, Xiaomei; Wang, Wei; Zhang, Xiaojuan; Xu, Quanbin; Zhu, Songcheng; Kuchta, Robert; Chen, Guanjun; Liu, Xuedong

2010-12-03

Mps1 is a protein kinase that regulates normal mitotic progression and the spindle checkpoint in response to spindle damage. The levels of Mps1 are relatively low in cells during interphase but elevated in mitosis or upon activation of the spindle checkpoint, although the dynamic range of Mps1 expression and the Mps1 catalytic mechanism have not been carefully characterized. Our recent structural studies of the Mps1 kinase domain revealed that the carboxyl-terminal tail region of Mps1 is unstructured, raising the question of whether this region has any functional role in Mps1 catalysis. Here we first determined the cellular abundance of Mps1 during cell cycle progression and found that Mps1 levels vary between 60,000 per cell in early G(1) and 110,000 per cell during mitosis. We studied phosphorylation of a number of Mps1 substrates in vitro and in culture cells. Unexpectedly, we found that the unstructured carboxyl-terminal region of Mps1 plays an essential role in substrate recruitment. Kinetics studies using the purified recombinant wild type and mutant kinases indicate that the carboxyl-terminal tail is largely dispensable for autophosphorylation of Mps1 but critical for trans-phosphorylation of substrates in vitro and in cultured cells. Mps1 mutant without the unstructured tail region is defective in mediating spindle assembly checkpoint activation. Our results underscore the importance of the unstructured tail region of Mps1 in kinase activation.

C/EBPα expression is downregulated in human nonmelanoma skin cancers and inactivation of C/EBPα confers susceptibility to UVB-induced skin squamous cell carcinomas.

PubMed

Thompson, Elizabeth A; Zhu, Songyun; Hall, Jonathan R; House, John S; Ranjan, Rakesh; Burr, Jeanne A; He, Yu-Ying; Owens, David M; Smart, Robert C

2011-06-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G(1) checkpoint, and diminished or ablated expression of C/EBPα results in G(1) checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB.

C/EBPα Expression Is Downregulated in Human Nonmelanoma Skin Cancers and Inactivation of C/EBPα Confers Susceptibility to UVB-Induced Skin Squamous Cell Carcinomas

PubMed Central

Thompson, Elizabeth A.; Zhu, Songyun; Hall, Jonathan R.; House, John S.; Ranjan, Rakesh; Burr, Jeanne A.; He, Yu-Ying; Owens, David M.; Smart, Robert C.

2012-01-01

Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. PMID:21346772

Affinity-aware checkpoint restart

DOE PAGES

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

2014-12-08

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

SFT: Scalable Fault Tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrini, Fabrizio; Nieplocha, Jarek; Tipparaju, Vinod

2006-04-15

In this paper we will present a new technology that we are currently developing within the SFT: Scalable Fault Tolerance FastOS project which seeks to implement fault tolerance at the operating system level. Major design goals include dynamic reallocation of resources to allow continuing execution in the presence of hardware failures, very high scalability, high efficiency (low overhead), and transparency—requiring no changes to user applications. Our technology is based on a global coordination mechanism, that enforces transparent recovery lines in the system, and TICK, a lightweight, incremental checkpointing software architecture implemented as a Linux kernel module. TICK is completely user-transparentmore » and does not require any changes to user code or system libraries; it is highly responsive: an interrupt, such as a timer interrupt, can trigger a checkpoint in as little as 2.5μs; and it supports incremental and full checkpoints with minimal overhead—less than 6% with full checkpointing to disk performed as frequently as once per minute.« less

A tumor suppressor role of the Bub3 spindle checkpoint protein after apoptosis inhibition

PubMed Central

Moutinho-Santos, Tatiana

2013-01-01

Most solid tumors contain aneuploid cells, indicating that the mitotic checkpoint is permissive to the proliferation of chromosomally aberrant cells. However, mutated or altered expression of mitotic checkpoint genes accounts for a minor proportion of human tumors. We describe a Drosophila melanogaster tumorigenesis model derived from knocking down spindle assembly checkpoint (SAC) genes and preventing apoptosis in wing imaginal discs. Bub3-deficient tumors that were also deficient in apoptosis displayed neoplastic growth, chromosomal aneuploidy, and high proliferative potential after transplantation into adult flies. Inducing aneuploidy by knocking down CENP-E and preventing apoptosis does not induce tumorigenesis, indicating that aneuploidy is not sufficient for hyperplasia. In this system, the aneuploidy caused by a deficient SAC is not driving tumorigenesis because preventing Bub3 from binding to the kinetochore does not cause hyperproliferation. Our data suggest that Bub3 has a nonkinetochore-dependent function that is consistent with its role as a tumor suppressor. PMID:23609535

Release of Mps1 from kinetochores is crucial for timely anaphase onset.

PubMed

Jelluma, Nannette; Dansen, Tobias B; Sliedrecht, Tale; Kwiatkowski, Nicholas P; Kops, Geert J P L

2010-10-18

Mps1 kinase activity is required for proper chromosome segregation during mitosis through its involvements in microtubule-chromosome attachment error correction and the mitotic checkpoint. Mps1 dynamically exchanges on unattached kinetochores but is largely removed from kinetochores in metaphase. Here we show that Mps1 promotes its own turnover at kinetochores and that removal of Mps1 upon chromosome biorientation is a prerequisite for mitotic checkpoint silencing. Inhibition of Mps1 activity increases its half-time of recovery at unattached kinetochores and causes accumulation of Mps1 protein at these sites. Strikingly, preventing dissociation of active Mps1 from kinetochores delays anaphase onset despite normal chromosome attachment and alignment, and high interkinetochore tension. This delay is marked by continued recruitment of Mad1 and Mad2 to bioriented chromosomes and is attenuated by Mad2 depletion, indicating chronic engagement of the mitotic checkpoint in metaphase. We propose that release of Mps1 from kinetochores is essential for mitotic checkpoint silencing and a fast metaphase-to-anaphase transition.

Affinity-aware checkpoint restart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saini, Ajay; Rezaei, Arash; Mueller, Frank

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

Checkpoint Inhibition in Hodgkin Lymphoma - a Review.

PubMed

Bröckelmann, Paul J; Engert, Andreas

2017-01-01

Physiological immune checkpoint pathways are important to regulate self-tolerance, limit immune reactions, and moderate autoimmunity. Various cancers are commonly exploiting these mechanisms to evade the host immune system by restraining a durable, efficient anti-tumor immune response. Immune checkpoints include, but are not limited to, the programmed death 1 (PD1) and the cytotoxic T-lymphocyte-associated protein-4 (CTLA4) axis, which are both druggable by monoclonal antibodies referred to as checkpoint inhibitors (CIs). To date, the anti-PD1 antibodies nivolumab and pembrolizumab are approved for relapsed or refractory classical Hodgkin lymphoma (cHL) due to high response rates with a favorable yet distinct safety profile, and other agents are under investigation. This review summarizes the available preclinical and clinical data including the toxicity and efficacy of different CIs in cHL. It also provides future perspectives based on ongoing clinical trials, potentially synergistic combinatory approaches, and their fit in the therapeutic landscape in cHL. © 2017 S. Karger GmbH, Freiburg.

Profile of pembrolizumab in the treatment of head and neck squamous cell carcinoma: design development and place in therapy

PubMed Central

Haque, Sulsal; Yellu, Mahender; Randhawa, Jaskirat; Hashemi-Sadraei, Nooshin

2017-01-01

Head and neck squamous cell cancer (HNSCC) is the sixth most common malignancy worldwide, and despite advances in cytotoxic, surgical and radiation techniques, outcomes are still poor in those with both locally advanced and metastatic diseases. The need for development of better therapeutics along with a greater understanding of the relationship between the immune system and malignancies has led to a new therapeutic modality, immune modulators, particularly checkpoint inhibitors in HNSCC. It is now well recognized that HNSCC circumvents crucial pathways utilized by the immune system to escape surveillance. These hijacked pathways include impairing tumor antigen presentation machinery and co-opting checkpoint receptors. This understanding has led to the development of monoclonal antibodies targeting checkpoint receptors and has resulted in promising outcomes in HNSCC. This article describes the mechanisms that HNSCC utilizes to escape immune surveillance, clinical impact of checkpoint inhibitors (with a focus on pembrolizumab), ongoing studies, and future directions. PMID:28919706

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

PubMed

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

2016-09-29

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

In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

PubMed Central

Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

2012-01-01

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.

PubMed

Lindsey-Boltz, Laura A; Reardon, Joyce T; Wold, Marc S; Sancar, Aziz

2012-10-19

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation*

PubMed Central

Han, Xiangzi; Aslanian, Aaron; Fu, Kang; Tsuji, Toshiya; Zhang, Youwei

2014-01-01

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage. PMID:25049228

Lithologic and hydrologic controls of mixed alluvial-bedrock channels in flood-prone fluvial systems: bankfull and macrochannels in the Llano River watershed, central Texas, USA

USGS Publications Warehouse

Heitmuller, Frank T.; Hudson, Paul F.; Asquith, William H.

2015-01-01

The rural and unregulated Llano River watershed located in central Texas, USA, has a highly variable flow regime and a wide range of instantaneous peak flows. Abrupt transitions in surface lithology exist along the main-stem channel course. Both of these characteristics afford an opportunity to examine hydrologic, lithologic, and sedimentary controls on downstream changes in channel morphology. Field surveys of channel topography and boundary composition are coupled with sediment analyses, hydraulic computations, flood-frequency analyses, and geographic information system mapping to discern controls on channel geometry (profile, pattern, and shape) and dimensions along the mixed alluvial-bedrock Llano River and key tributaries. Four categories of channel classification in a downstream direction include: (i) uppermost ephemeral reaches, (ii) straight or sinuous gravel-bed channels in Cretaceous carbonate sedimentary zones, (iii) straight or sinuous gravel-bed or bedrock channels in Paleozoic sedimentary zones, and (iv) straight, braided, or multithread mixed alluvial–bedrock channels with sandy beds in Precambrian igneous and metamorphic zones. Principal findings include: (i) a nearly linear channel profile attributed to resistant bedrock incision checkpoints; (ii) statistically significant correlations of both alluvial sinuosity and valley confinement to relatively high f (mean depth) hydraulic geometry values; (iii) relatively high b (width) hydraulic geometry values in partly confined settings with sinuous channels upstream from a prominent incision checkpoint; (iv) different functional flow categories including frequently occurring events (< 1.5-year return periods) that mobilize channel-bed material and less frequent events that determine bankfull channel (1.5- to 3-year return periods) and macrochannel (10- to 40-year return periods) dimensions; (v) macrochannels with high f values (most ≤ 0.45) that develop at sites with unit stream power values in excess of 200 watts per square meter (W/m2); and (vi) downstream convergence of hydraulic geometry exponents for bankfull and macrochannels, explained by co-increases of flood magnitude and noncohesive sandy sediments that collectively minimize development of alluvial bankfull indicators. Collectively, these findings indicate that mixed alluvial–bedrock channels exhibit first-order lithologic controls (lithologic resistance and valley confinement) of channel geometry, second-order hydrologic (flow regime) control of channel dimensions, and third-order sedimentary controls that exert subsidiary influence on channel shape and bed configuration.

A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress.

PubMed

Abroudi, Ali; Samarasinghe, Sandhya; Kulasiri, Don

2017-09-21

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model, comprehensive details of cell cycle dynamics under normal and DNA damage conditions revealing the role and value of the added new modules and elements, (ii) assess, through a global sensitivity analysis, the most influential sub-systems, modules and parameters on system response, such as G1-S and G2-M transitions, and (iii) probe deeply into the relationship between DNA damage and cell cycle progression and test the biological evidence that G1-S is relatively inefficient in arresting damaged cells compared to G2-M checkpoint. To perform sensitivity analysis, Self-Organizing Map with Correlation Coefficient Analysis (SOMCCA) is developed which shows that Growth Factor and G1-S Checkpoint sub-systems and 13 parameters in the modules within them are crucial for G1-S and G2-M transitions. To study the relative efficiency of DNA damage checkpoints, a Checkpoint Efficiency Evaluator (CEE) is developed based on perturbation studies and statistical Type II error. Accordingly, cell cycle is about 96% efficient in arresting damaged cells with G2-M checkpoint being more efficient than G1-S. Further, both checkpoint systems are near perfect (98.6%) in passing healthy cells. Thus this study has shown the efficacy of the proposed systems approach to gain a better understanding of different aspects of mammalian cell cycle system separately and as an integrated system that will also be useful in investigating targeted therapy in future cancer treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

PubMed Central

2014-01-01

Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

PubMed

Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

2014-01-01

The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

Immunohistochemical and Image Analysis-Based Study Shows That Several Immune Checkpoints are Co-expressed in Non-Small Cell Lung Carcinoma Tumors.

PubMed

Parra, Edwin Roger; Villalobos, Pamela; Zhang, Jiexin; Behrens, Carmen; Mino, Barbara; Swisher, Stephen; Sepesi, Boris; Weissferdt, Annika; Kalhor, Neda; Heymach, John Victor; Moran, Cesar; Zhang, Jianjun; Lee, Jack; Rodriguez-Canales, Jaime; Gibbons, Don; Wistuba, Ignacio I

2018-06-01

The understanding of immune checkpoint molecules' co-expression in non-small cell lung carcinoma (NCLC) is important to potentially design combinatorial immunotherapy approaches. We studied 225 formalin-fixed, paraffin-embedded tumor tissues from stage I-III NCLCs - 142 adenocarcinomas (ADCs) and 83 squamous cell carcinomas (SCCs) - placed in tissue microarrays. Nine immune checkpoint markers were evaluated; four (programmed death ligand 1 [PD-L1], B7-H3, B7-H4, and indoleamine 2,3-dioxygenase 1 [IDO-1]) expressed predominantly in malignant cells (MCs) and five (inducible T cell costimulator, V-set immunoregulatory receptor, T-cell immunoglobulin mucin family member 3, lymphocyte activating 3, and OX40) expressed mostly in stromal tumor-associated inflammatory cells (TAICs). All markers were examined using a quantitative image analysis and correlated with clinicopathologic features, TAICs, and molecular characteristics. Using above the median value as positive expression in MCs and high density of TAICs expressing those markers, we identified higher expression of immune checkpoints in SCC than ADC. Common simultaneous expression by MCs was PD-L1 + B7-H3 + IDO-1 in ADC and PD-L1 + B7-H3, or B7-H3 + B7-H4, in SCC. TAICs expressing checkpoint were significantly higher in current smokers than in never smokers. Almost all the immune checkpoint markers showed positive correlation with TAICs expressing inflammatory cell markers. KRAS-mutant ADC specimens showed higher expression of PD-L1 in MCs and of B7-H3, T-cell immunoglobulin mucin family member 3, and IDO-1 in TAICs than wild type. Kaplan-Meier survival curves showed worse prognosis in ADC patients with higher B7-H4 expression by MCs. We found frequent immunohistochemical co-expression of immune checkpoints in surgically resected NCLC tumors and correlated with tumor histology, smoking history, tumor size, and the density of inflammatory cells and tumor mutational status. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Centriole assembly and the role of Mps1: defensible or dispensable?

PubMed

Pike, Amanda N; Fisk, Harold A

2011-04-14

The Mps1 protein kinase is an intriguing and controversial player in centriole assembly. Originally shown to control duplication of the budding yeast spindle pole body, Mps1 is present in eukaryotes from yeast to humans, the nematode C. elegans being a notable exception, and has also been shown to regulate the spindle checkpoint and an increasing number of cellular functions relating to genomic stability. While its function in the spindle checkpoint appears to be both universally conserved and essential in most organisms, conservation of its originally described function in spindle pole duplication has proven controversial, and it is less clear whether Mps1 is essential for centrosome duplication outside of budding yeast. Recent studies of Mps1 have identified at least two distinct functions for Mps1 in centriole assembly, while simultaneously supporting the notion that Mps1 is dispensable for the process. However, the fact that at least one centrosomal substrate of Mps1 is conserved from yeast to humans down to the phosphorylation site, combined with evidence demonstrating the exquisite control exerted over centrosomal Mps1 levels suggest that the notion of being essential may not be the most important of distinctions.

A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae

PubMed Central

Val, Marie-Eve; Marbouty, Martial; de Lemos Martins, Francisco; Kennedy, Sean P.; Kemble, Harry; Bland, Michael J.; Possoz, Christophe; Koszul, Romain; Skovgaard, Ole; Mazel, Didier

2016-01-01

Bacteria with multiple chromosomes represent up to 10% of all bacterial species. Unlike eukaryotes, these bacteria use chromosome-specific initiators for their replication. In all cases investigated, the machineries for secondary chromosome replication initiation are of plasmid origin. One of the important differences between plasmids and chromosomes is that the latter replicate during a defined period of the cell cycle, ensuring a single round of replication per cell. Vibrio cholerae carries two circular chromosomes, Chr1 and Chr2, which are replicated in a well-orchestrated manner with the cell cycle and coordinated in such a way that replication termination occurs at the same time. However, the mechanism coordinating this synchrony remains speculative. We investigated this mechanism and revealed that initiation of Chr2 replication is triggered by the replication of a 150-bp locus positioned on Chr1, called crtS. This crtS replication–mediated Chr2 replication initiation mechanism explains how the two chromosomes communicate to coordinate their replication. Our study reveals a new checkpoint control mechanism in bacteria, and highlights possible functional interactions mediated by contacts between two chromosomes, an unprecedented observation in bacteria. PMID:27152358

Suspended animation in C. elegans requires the spindle checkpoint.

PubMed

Nystul, Todd G; Goldmark, Jesse P; Padilla, Pamela A; Roth, Mark B

2003-11-07

In response to environmental signals such as anoxia, many organisms enter a state of suspended animation, an extreme form of quiescence in which microscopically visible movement ceases. We have identified a gene, san-1, that is required for suspended animation in Caenorhabditis elegans embryos. We show that san-1 functions as a spindle checkpoint component in C. elegans. During anoxia-induced suspended animation, embryos lacking functional SAN-1 or a second spindle checkpoint component, MDF-2, failed to arrest the cell cycle, exhibited chromosome missegregation, and showed reduced viability. These data provide a model for how a dynamic biological process is arrested in suspended animation.

Validate Mitotic Checkpoint and Kinetochore Motor Proteins in Breast Cancer Cells as Targets for the Development of Novel Anti-Mitotic Drugs

DTIC Science & Technology

2004-07-01

checkpoint pathway remains to MAD1 MAD1 xMADI be clarified, it is dear that all of them MAD2 MAD2 xMAD2 are essential for cells to arrest in mitosis MPS1 ...TrK in response to unattached kineto- chores. Given that MPS1 , BUB1 and (G) Structural Proteins/Unknown Functions the Mad3-related BUBR1 are all pro...BUB3, MADI, MAD2, MAD3, and MPS1 have been shown to be essential for establishing the checkpoint response in all eukaryotes examined to date (Abrieu et

Interprofessional Collaboration with Immune Checkpoint Inhibitor Therapy: the Roles of Gastroenterology, Endocrinology and Neurology.

PubMed

Seery, Virginia

2017-11-01

To discuss immune checkpoint inhibitor therapy and identify opportunities for interprofessional collaboration in the management of toxicities in the areas of gastroenterology, endocrinology, and neurology. Published research and education articles in oncology, nursing, and various specialties. The use of immune checkpoint inhibitors is expanding; timely management of toxicity is critical for positive patient outcomes. There are many opportunities for interprofessional collaboration in the diagnosis and treatment of immune-related adverse events. Nurses play key roles in recognizing immune-related adverse events, providing patient education, and helping to facilitate interprofessional collaboration. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimal message log reclamation for independent checkpointing

NASA Technical Reports Server (NTRS)

Wang, Yi-Min; Fuchs, W. Kent

1993-01-01

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

Low-cost Cognitive Electronics Technology for Enhanced Communications and Situational Awareness for Networks of Small Unmanned Aerial Vehicles (UAV)

DTIC Science & Technology

2013-03-01

series of checkpoints in a complex route network,” while observing standard traffic etiquette and regulations [17]. The rules for the 2012 RoboCup...structure or protocols above the PHY. To support AVEP operation, we developed a packet structure based on the transmission control protocol (TCP...Control Protocol .” 1981. [37] F. Ge, Q. Chen, Y. Wang, C. W. Bostian, T. W. Rondeau, and B. Le, “Cognitive radio: from spectrum sharing to adaptive

Checkpoint Inhibitors Hold Promise for Rare Melanoma

Cancer.gov

Patients with a rare form of melanoma, called desmoplastic melanoma, may be particularly likely to benefit from immune checkpoint inhibitors, a new study shows. As this Cancer Currents post explains, an NCI-sponsored clinical trial is already testing one such drug, pembrolizumab (Keytruda) in patients with this cancer.

Treatment-related Death in Cancer Patients Treated with Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis.

PubMed

Abdel-Rahman, O; Helbling, D; Schmidt, J; Petrausch, U; Giryes, A; Mehrabi, A; Schöb, O; Mannhart, M; Oweira, H

2017-04-01

We carried out a meta-analysis to determine the risk of treatment-related death associated with immune checkpoint inhibitor use in cancer patients. We examined data from the Medline and Google Scholar databases. We also examined original studies and review articles for cross-references. Eligible studies included randomised phase II and phase III trials of patients with cancer treated with ipilimumab, pembrolizumab; nivolumab; tremelimumab and atezolizumab. The authors extracted relevant information on participants, characteristics, treatment-related death and information on the methodology of the studies. After exclusion of ineligible records, 18 clinical trials were included in the analysis. The odds ratio for treatment-related death for CTLA-4 inhibitors (ipilimumab and tremelimumab) was 1.80 (95% confidence interval 1.25, 2.59; P=0.002) and for PD-1/PD-L1 inhibitors (nivolumab, pembrolizumab and atezolizumab) was 0.63 (95% confidence interval 0.31, 1.30; P=0.22). Treated cancer seems to have no effect on the risk of treatment-related death. Analysis of our data showed that CTLA-4 inhibitors (ipilimumab and tremelimumab) in a higher dose (10 mg/kg) seem to be associated with a higher risk of treatment-related death compared with control regimens, whereas PD-1/PD-L1 inhibitors (nivolumab, pembrolizumab and atezolizumab) do not cause the same risk. Clinicians have to be fully aware of these differential risks and council their patients appropriately. Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

DNA-dependent protein kinase (DNA-PK)-deficient human glioblastoma cells are preferentially sensitized by Zebularine

PubMed Central

Meador, Jarah A.; Su, Yanrong; Ravanat, Jean-Luc; Balajee, Adayabalam S.

2010-01-01

Brain tumor cells respond poorly to radiotherapy and chemotherapy due to inherently efficient anti-apoptotic and DNA repair mechanisms. This necessitates the development of new strategies for brain cancer therapy. Here, we report that the DNA-demethylating agent Zebularine preferentially sensitizes the killing of human glioblastomas deficient in DNA-dependent protein kinase (DNA-PK). In contrast to DNA-PK-proficient human glioblastoma cells (MO59K), cytotoxicity assay with increasing Zebularine concentrations up to 300 μM resulted in a specific elevation of cell killing in DNA-PK-deficient MO59J cells. Further, an elevated frequency of polyploid cells observed in MO59J cells after Zebularine treatment pointed out a deficiency in mitotic checkpoint control. Existence of mitotic checkpoint deficiency in MO59J cells was confirmed by the abnormal centrosome number observed in Zebularine-treated MO59J cells. Although depletion of DNA methyltransferase 1 by Zebularine occurred at similar levels in both cell lines, MO59J cells displayed increased extent of DNA demethylation detected both at the gene promoter-specific level and at the genome overall level. Consistent with increased sensitivity, deoxy-Zebularine adduct level in the genomic DNA was 3- to 6-fold higher in MO59J than in MO59K cells. Elevated micronuclei frequency observed after Zebularine treatment in MO59J cells indicates the impairment of DNA repair response in MO59J cells. Collectively, our study suggests that DNA-PK is the major determining factor for cellular response to Zebularine. PMID:19933707

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

PubMed

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

2017-07-01

A major mechanism of tumor development and progression is silencing of the patient's immune response to cancer-specific antigens. Defects in the so-called cancer immunity cycle may occur at any stage of tumor development. Within the tumor microenvironment, aberrant expression of immune checkpoint molecules with activating or inhibitory effects on T lymphocytes induces immune tolerance and cellular immune escape. Targeting immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and its ligand PD-L1 with specific antibodies has proven to be a major advance in the treatment of several types of cancer. Another way to therapeutically influence the tumor microenvironment is by modulating the levels of microRNAs (miRNAs), small noncoding RNAs that shuttle bidirectionally between malignant and tumor microenvironmental cells. These small RNA transcripts have two features: (a) their expression is quite specific to distinct tumors, and (b) they are involved in early regulation of immune responses. Consequently, miRNAs may be ideal molecules for use in cancer therapy. Many miRNAs are aberrantly expressed in human cancer cells, opening new opportunities for cancer therapy, but the exact functions of these miRNAs and their interactions with immune checkpoint molecules have yet to be investigated. This review summarizes recently reported findings about miRNAs as modulators of immune checkpoint molecules and their potential application as cancer therapeutics in clinical practice. © 2017 Federation of European Biochemical Societies.

Deconstructing Ras Signaling in the Thymus

PubMed Central

Kortum, Robert L.; Sommers, Connie L.; Pinski, John M.; Alexander, Clayton P.; Merrill, Robert K.; Li, Wenmei; Love, Paul E.

2012-01-01

Thymocytes must transit at least two distinct developmental checkpoints, governed by signals that emanate from either the pre-T cell receptor (pre-TCR) or the TCR to the small G protein Ras before emerging as functional T lymphocytes. Recent studies have shown a role for the Ras guanine exchange factor (RasGEF) Sos1 at the pre-TCR checkpoint. At the second checkpoint, the quality of signaling through the TCR is interrogated to ensure the production of an appropriate T cell repertoire. Although RasGRP1 is the only confirmed RasGEF required at the TCR checkpoint, current models suggest that the intensity and character of Ras activation, facilitated by both Sos and RasGRP1, will govern the boundary between survival (positive selection) and death (negative selection) at this stage. Using mouse models, we have assessed the independent and combined roles for the RasGEFs Sos1, Sos2, and RasGRP1 during thymocyte development. Although Sos1 was the dominant RasGEF at the pre-TCR checkpoint, combined Sos1/RasGRP1 deletion was required to effectively block development at this stage. Conversely, while RasGRP1 deletion efficiently blocked positive selection, combined RasGRP1/Sos1 deletion was required to block negative selection. This functional redundancy in RasGEFs during negative selection may act as a failsafe mechanism ensuring appropriate central tolerance. PMID:22586275

Cell-autonomous mechanisms of chronological aging in the yeast Saccharomyces cerevisiae.

PubMed

Arlia-Ciommo, Anthony; Leonov, Anna; Piano, Amanda; Svistkova, Veronika; Titorenko, Vladimir I

2014-05-27

A body of evidence supports the view that the signaling pathways governing cellular aging - as well as mechanisms of their modulation by longevity-extending genetic, dietary and pharmacological interventions - are conserved across species. The scope of this review is to critically analyze recent advances in our understanding of cell-autonomous mechanisms of chronological aging in the budding yeast Saccharomyces cerevisiae . Based on our analysis, we propose a concept of a biomolecular network underlying the chronology of cellular aging in yeast. The concept posits that such network progresses through a series of lifespan checkpoints. At each of these checkpoints, the intracellular concentrations of some key intermediates and products of certain metabolic pathways - as well as the rates of coordinated flow of such metabolites within an intricate network of intercompartmental communications - are monitored by some checkpoint-specific "master regulator" proteins. The concept envisions that a synergistic action of these master regulator proteins at certain early-life and late-life checkpoints modulates the rates and efficiencies of progression of such processes as cell metabolism, growth, proliferation, stress resistance, macromolecular homeostasis, survival and death. The concept predicts that, by modulating these vital cellular processes throughout lifespan (i.e., prior to an arrest of cell growth and division, and following such arrest), the checkpoint-specific master regulator proteins orchestrate the development and maintenance of a pro- or anti-aging cellular pattern and, thus, define longevity of chronologically aging yeast.

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

PubMed

Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

2010-08-03

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

Development of small-molecule immune checkpoint inhibitors of PD-1/PD-L1 as a new therapeutic strategy for tumour immunotherapy.

PubMed

Li, Kui; Tian, Hongqi

2018-02-20

Cancer immunotherapy has been increasingly utilised to treat advanced malignancies. The signalling network of immune checkpoints has attracted considerable attention. Immune checkpoint inhibitors are revolutionising the treatment options and expectations for patients with cancer. The reported clinical success of targeting the T-cell immune checkpoint receptors PD-1/PD-L1 has demonstrated the importance of immune modulation. Indeed, antibodies binding to PD-1 or PD-L1 have shown remarkable efficacy. However, antibody drugs have many disadvantages, such as their production cost, stability, and immunogenicity and, therefore, small-molecule inhibitors of PD-1 and its ligand PD-L1 are being introduced. Small-molecule inhibitors could offer inherent advantages in terms of pharmacokinetics and druggability, thereby providing additional methods for cancer treatment and achieving better therapeutic effects. In this review, we first discuss how PD-1/PD-L1-targeting inhibitors modulate the relationship between immune cells and tumour cells in tumour immunotherapy. Second, we discuss how the immunomodulatory potential of these inhibitors can be exploited via rational combinations with immunotherapy and targeted therapy. Third, this review is the first to summarise the current clinical and preclinical evidence regarding small-molecule inhibitors of the PD-1/PD-L1 immune checkpoint, considering features and responses related to the tumours and to the host immune system.

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

PubMed

Vialard, J E; Gilbert, C S; Green, C M; Lowndes, N F

1998-10-01

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

Finite-Time Performance of Local Search Algorithms: Theory and Application

DTIC Science & Technology

2010-06-10

security devices deployed at airport security checkpoints are used to detect prohibited items (e.g., guns, knives, explosives). Each security device...security devices are deployed, the practical issue of determining how to optimally use them can be difficult. For an airport security system design...checked baggage), explosive detection systems (designed to detect explosives in checked baggage), and detailed hand search by an airport security official

Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma.

PubMed

Zeng, Ling; Beggs, Reena R; Cooper, Tiffiny S; Weaver, Alice N; Yang, Eddy S

2017-04-01

EGFR inhibition and radiotherapy are potent inducers of DNA damage. Checkpoint kinases 1 and 2 (Chk1/2) are critical regulators of the DNA-damage response, controlling cell-cycle checkpoints that may permit recovery from therapy-associated genomic stress. We hypothesized that Chk1/2 inhibition (CHKi) with prexasertib may enhance cytotoxicity from EGFR inhibition plus radiotherapy in head and neck squamous cell carcinoma (HNSCC). In this study, we found that the addition of CHKi to the EGFR inhibitor cetuximab with and without radiotherapy significantly decreased cell proliferation and survival fraction in human papillomavirus virus (HPV)-positive and HPV-negative HNSCC cell lines. Reduced proliferation was accompanied by decreased checkpoint activation, induced S-phase accumulation, persistent DNA damage, and increased caspase cleavage and apoptosis. Importantly, a significant tumor growth delay was observed in vivo in both HPV-positive and HPV-negative cell line xenografts receiving triple combination therapy with CHKi, cetuximab, and radiotherapy without a concomitant increase in toxicity as assessed by mouse body weight. Taken together, the combination of CHKi with cetuximab plus irradiation displayed significant antitumor effects in HNSCCs both in vitro and in vivo , suggesting that this combination therapy may increase clinical benefit. A clinical trial to test this treatment for patients with head and neck cancer is currently ongoing (NCT02555644). Mol Cancer Ther; 16(4); 591-600. ©2017 AACR . ©2017 American Association for Cancer Research.

Effects of enforcement intensity on alcohol impaired driving crashes.

PubMed

Fell, James C; Waehrer, Geetha; Voas, Robert B; Auld-Owens, Amy; Carr, Katie; Pell, Karen

2014-12-01

Research measuring levels of enforcement has investigated whether increases in police activities (e.g., checkpoints, driving-while-intoxicated [DWI] special patrols) above some baseline level are associated with reduced crashes and fatalities. Little research, however, has attempted to quantitatively measure enforcement efforts and relate different enforcement levels to specific levels of the prevalence of alcohol-impaired driving. The objective of this study was to investigate the effects of law-enforcement intensity in a sample of communities on the rate of crashes involving a drinking driver. We analyzed the influence of different enforcement strategies and measures: (1) specific deterrence - annual number of driving-under-the-influence (DUI) arrests per capita; (2) general deterrence - frequency of sobriety checkpoint operations; (3) highly visible traffic enforcement - annual number of traffic stops per capita; (4) enforcement presence - number of sworn officers per capita; and (5) overall traffic enforcement - the number of other traffic enforcement citations per capita (i.e., seat belt citations, speeding tickets, and other moving violations and warnings) in each community. We took advantage of nationwide data on the local prevalence of impaired driving from the 2007 National Roadside Survey (NRS), measures of DUI enforcement activity provided by the police departments that participated in the 2007 NRS, and crashes from the General Estimates System (GES) in the same locations as the 2007 NRS. We analyzed the relationship between the intensity of enforcement and the prevalence of impaired driving crashes in 22-26 communities with complete data. Log-linear regressions were used throughout the study. A higher number of DUI arrests per 10,000 driving-aged population was associated with a lower ratio of drinking-driver crashes to non-drinking-driver crashes (p=0.035) when controlling for the percentage of legally intoxicated drivers on the roads surveyed in the community from the 2007 NRS. Results indicate that a 10% increase in the DUI arrest rate is associated with a 1% reduction in the drinking driver crash rate. Similar results were obtained for an increase in the number of sworn officers per 10,000 driving-age population. While a higher DUI arrest rate was associated with a lower drinking-driver crash rate, sobriety checkpoints did not have a significant relationship to drinking-driver crashes. This appeared to be due to the fact that only 3% of the on-the-road drivers were exposed to frequent sobriety checkpoints (only 1 of 36 police agencies where we received enforcement data conducted checkpoints weekly). This low-use strategy is symptomatic of the general decline in checkpoint use in the U.S. since the 1980s and 1990s when the greatest declines in alcohol-impaired-driving fatal crashes occurred. The overall findings in this study may help law enforcement agencies around the country adjust their traffic enforcement intensity in order to reduce impaired driving in their community. Copyright © 2014 Elsevier Ltd. All rights reserved.

p21 stability: linking chaperones to a cell cycle checkpoint.

PubMed

Liu, Geng; Lozano, Guillermina

2005-02-01

Progression through the cell cycle is regulated by numerous proteins, one of which is the cyclin-dependent kinase inhibitor, p21. A new study identifies a novel protein complex that stabilizes p21. The stability of this complex is critical in effecting the p53-mediated cell cycle checkpoint.

CHEK2 gene alterations independently increase the risk of death from breast cancer in Bulgarian patients.

PubMed

Angelova, S G; Krasteva, M E; Gospodinova, Z I; Georgieva, E I

2012-01-01

Checkpoint kinase 2 (CHEK2) is a DNA damage-activated protein kinase implicated in cell cycle checkpoint control. The significance of CHEK2 alterations for breast cancer incidence and clinical behavior is not clear. In this study we determined the mutational spectrum and the level of promoter hypermethylation of CHEK2 gene in a group of 145 Bulgarian patients with breast cancer. A special emphasis was put on the clinical impact of CHEK2 alterations for breast cancerogenesis. PCR-SSCP-sequencing analysis of the entire coding sequence of CHEK2 gene was performed to estimate the mutational profile of tumor samples. Methylation-sensitive SSCP was applied to determine the methylation status in CpG clusters implicated in CHEK2 silencing. Clinical significance of CHEK2 alterations was evaluated using standard statistical methods. Mutations in CHEK2 were identified in 9.65 % of the patients. Two novel missense substitutions Thr476Met (C >T) and Ala507Gly (C>G), and a novel silent variant Glu79Glu (A>G) were registered. However, hypermethylation was not found in any of the studied cases. Comparison with clinical characteristics showed that CHEK2 positive women have predominantly lobular type of breast carcinoma (р=0.04) and PR+ status (p=0.092). CHEK2 mutations correlated significantly with ATM+ status (p=0.046). All patients with the Glu79Glu variant were progesterone receptor positive (p=0.004). A decrease in overall survival (p = 0.6301) and a threefold increased independent risk of death (HR = 3.295, 95%CI 0.850-12.778, p = 0.085) in CHEK2+patients was found. Our data indicate the significance of CHEK2 gene alterations in contrast to promoter hypermethylation in breast cancerogenesis. Specificity of CHEK2 mutational profile for the Bulgarian population was found. Though CHEK2 mutational status correlated with more favorable clinical characteristics, including positive progesterone receptor and lobular histological type, it independently increased the risk of death in these patients.

ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

DOE PAGES

Caballe, Anna; Wenzel, Dawn M.; Agromayor, Monica; ...

2015-05-26

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1,more » an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.« less

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

PubMed

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-05-02

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Coinhibitory molecules in cancer biology and therapy.

PubMed

Mocellin, Simone; Benna, Clara; Pilati, Pierluigi

2013-04-01

The adaptive immune response is controlled by checkpoints represented by coinhibitory molecules, which are crucial for maintaining self-tolerance and minimizing collateral tissue damage under physiological conditions. A growing body of preclinical evidence supports the hypothesis that unleashing this immunological break might be therapeutically beneficial in the fight against cancer, as it would elicit an effective antitumor immune response. Remarkably, recent clinical trials have demonstrated that this novel strategy can be highly effective in the treatment of patients with cancer, as shown by the paradigmatic case of ipilimumab (a monoclonal antibody blocking the coinhibitory molecule cytotoxic T lymphocyte associated antigen-4 [CTLA4]) that is opening a new era in the therapeutic approach to a chemoresistant tumor such as cutaneous melanoma. In this review we summarize the biology of coinhibitory molecules, overview the experimental and clinical attempts to interfere with these immune checkpoints to treat cancer and critically discuss the challenges posed by such a promising antitumor modality. Copyright © 2013 Elsevier Ltd. All rights reserved.

ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caballe, Anna; Wenzel, Dawn M.; Agromayor, Monica

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1,more » an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations.« less

ULK3 regulates cytokinetic abscission by phosphorylating ESCRT-III proteins

PubMed Central

Caballe, Anna; Wenzel, Dawn M; Agromayor, Monica; Alam, Steven L; Skalicky, Jack J; Kloc, Magdalena; Carlton, Jeremy G; Labrador, Leticia; Sundquist, Wesley I; Martin-Serrano, Juan

2015-01-01

The endosomal sorting complexes required for transport (ESCRT) machinery mediates the physical separation between daughter cells during cytokinetic abscission. This process is regulated by the abscission checkpoint, a genome protection mechanism that relies on Aurora B and the ESCRT-III subunit CHMP4C to delay abscission in response to chromosome missegregation. In this study, we show that Unc-51-like kinase 3 (ULK3) phosphorylates and binds ESCRT-III subunits via tandem MIT domains, and thereby, delays abscission in response to lagging chromosomes, nuclear pore defects, and tension forces at the midbody. Our structural and biochemical studies reveal an unusually tight interaction between ULK3 and IST1, an ESCRT-III subunit required for abscission. We also demonstrate that IST1 phosphorylation by ULK3 is an essential signal required to sustain the abscission checkpoint and that ULK3 and CHMP4C are functionally linked components of the timer that controls abscission in multiple physiological situations. DOI: http://dx.doi.org/10.7554/eLife.06547.001 PMID:26011858

The Role of the Transcriptional Response to DNA Replication Stress

PubMed Central

Herlihy, Anna E.; de Bruin, Robertus A.M.

2017-01-01

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

ATM-like kinases and regulation of telomerase: lessons from yeast and mammals

PubMed Central

Sabourin, Michelle; Zakian, Virginia A.

2008-01-01

Telomeres, the essential structures at the ends of eukaryotic chromosomes, are composed of G-rich DNA and asociated proteins. These structures are crucial for the integrity of the genome, because they protect chromosome ends from degradation and distinguish natural ends from chromosomal breaks. The complete replication of telomeres requires a telomere-dedicated reverse transcriptase called telomerase. Paradoxically, proteins that promote the very activities against which telomeres protect, namely DNA repair, recombination and checkpoint activation, are integral to both telomeric chromatin and telomere elongation. This review focuses on recent findings that shed light on the roles of ATM-like kinases and other checkpoint and repair proteins in telomere maintenance, replication and checkpoint signaling. PMID:18502129

The Role of the Transcriptional Response to DNA Replication Stress.

PubMed

Herlihy, Anna E; de Bruin, Robertus A M

2017-03-02

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

Analysis of hRad1, a Human G2 Checkpoint Control Gene

DTIC Science & Technology

2002-03-01

kinases chk1(+) and cds1(+). Genetics, 1999. 152(4): p. 1501-12. 39. Fritz , E., A.A. Friedl, R.M. Zwacka, F. Eckardt-Schupp, and M.S. Meyn, The yeast...Schiripo, M.M. Jorczak, D.C. Sgroi, J.E. Garber, F.P. Li, K.E. Nichols, J.M. Varley, A.K. Godwin, K.M. Shannon, E. Harlow, and D.A. Haber

Estimating the drink driving attributable fraction of road traffic deaths in Mexico.

PubMed

Santoyo-Castillo, Dzoara; Pérez-Núñez, Ricardo; Borges, Guilherme; Híjar, Martha

2018-05-01

To estimate the Drink Driving Attributable Fraction (DDAF) of road traffic injury mortality in car occupants in Mexico during 2010-13. A case-control study was conducted to examine the presence of alcohol in analysed body fluids of car occupants killed in fatal crashes (cases) compared with car drivers tested in alcohol-testing checkpoints who were not involved in a fatal collision (controls). Two data sets were used for the period 2010-13: the forensic module of the Epidemiological Surveillance System on Addictions that included car occupants killed in a collision (cases) and a data set from alcohol-testing at police checkpoints available for matching municipalities (controls). Mexico. The analysed study sample included 1718 car occupants killed in a traffic collision and 80 656 drivers tested at alcohol police checkpoints, all from 10 municipalities. Unadjusted and adjusted odds ratios (OR) of presence of alcohol in body fluids were obtained stratified by sex and age groups and the interaction with these two variables were assessed. The ORs were used to calculate the DDAF. It was estimated that 19.5% of car occupants' deaths due to road traffic injuries were attributable to alcohol consumption [95% confidence interval (CI) = 19.1-19.9]. The adjusted OR of presence of alcohol was 6.84 (95% CI = 6.06-7.71) overall. For males it was 7.21 (95% CI = 6.35-8.18) and for females it was 4.45 (95% CI = 3.01-6.60). The ORs were similar across younger age bands (10-19 years: 9.61, 95% CI = 6.72-13.73; 20-29 years: 7.70, 95% CI = 6.28-9.4; and 30-49 years: 7.21, 95% CI = 5.98-8.70); and lower but still elevated among older people (50+ years: 3.19, 95% CI = 2.19-4.65). An estimated 19.5% of car occupant deaths in Mexico may have been caused by alcohol in 2010-13. © 2017 Society for the Study of Addiction.

CrossTalk: The Journal of Defense Software Engineering. Volume 18, Number 4

DTIC Science & Technology

2005-04-01

older automated cost- estimating tools are no longer being actively marketed but are still in use such as CheckPoint, COCOMO, ESTIMACS, REVIC, and SPQR ...estimation tools: SPQR /20, Checkpoint, and Knowl- edgePlan. These software estimation tools pioneered the use of function point metrics for sizing and

The checkpoint ordering problem

PubMed Central

Hungerländer, P.

2017-01-01

Abstract We suggest a new variant of a row layout problem: Find an ordering of n departments with given lengths such that the total weighted sum of their distances to a given checkpoint is minimized. The Checkpoint Ordering Problem (COP) is both of theoretical and practical interest. It has several applications and is conceptually related to some well-studied combinatorial optimization problems, namely the Single-Row Facility Layout Problem, the Linear Ordering Problem and a variant of parallel machine scheduling. In this paper we study the complexity of the (COP) and its special cases. The general version of the (COP) with an arbitrary but fixed number of checkpoints is NP-hard in the weak sense. We propose both a dynamic programming algorithm and an integer linear programming approach for the (COP) . Our computational experiments indicate that the (COP) is hard to solve in practice. While the run time of the dynamic programming algorithm strongly depends on the length of the departments, the integer linear programming approach is able to solve instances with up to 25 departments to optimality. PMID:29170574

Mec1/ATR, the Program Manager of Nucleic Acids Inc.

PubMed

Feng, Wenyi

2016-12-28

Eukaryotic cells are equipped with surveillance mechanisms called checkpoints to ensure proper execution of cell cycle events. Among these are the checkpoints that detect DNA damage or replication perturbations and coordinate cellular activities to maintain genome stability. At the forefront of damage sensing is an evolutionarily conserved molecule, known respectively in budding yeast and humans as Mec1 (Mitosis entry checkpoint 1) and ATR (Ataxia telangiectasia and Rad3-related protein). Through phosphorylation, Mec1/ATR activates downstream components of a signaling cascade to maintain nucleotide pool balance, protect replication fork integrity, regulate activation of origins of replication, coordinate DNA repair, and implement cell cycle delay. This list of functions continues to expand as studies have revealed that Mec1/ATR modularly interacts with various protein molecules in response to different cellular cues. Among these newly assigned functions is the regulation of RNA metabolism during checkpoint activation and the coordination of replication-transcription conflicts. In this review, I will highlight some of these new functions of Mec1/ATR with a focus on the yeast model organism.

New Parallel computing framework for radiation transport codes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kostin, M.A.; /Michigan State U., NSCL; Mokhov, N.V.

A new parallel computing framework has been developed to use with general-purpose radiation transport codes. The framework was implemented as a C++ module that uses MPI for message passing. The module is significantly independent of radiation transport codes it can be used with, and is connected to the codes by means of a number of interface functions. The framework was integrated with the MARS15 code, and an effort is under way to deploy it in PHITS. Besides the parallel computing functionality, the framework offers a checkpoint facility that allows restarting calculations with a saved checkpoint file. The checkpoint facility canmore » be used in single process calculations as well as in the parallel regime. Several checkpoint files can be merged into one thus combining results of several calculations. The framework also corrects some of the known problems with the scheduling and load balancing found in the original implementations of the parallel computing functionality in MARS15 and PHITS. The framework can be used efficiently on homogeneous systems and networks of workstations, where the interference from the other users is possible.« less

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

NASA Astrophysics Data System (ADS)

Min, Wookee; Bruhn, Christopher; Grigaravicius, Paulius; Zhou, Zhong-Wei; Li, Fu; Krüger, Anja; Siddeek, Bénazir; Greulich, Karl-Otto; Popp, Oliver; Meisezahl, Chris; Calkhoven, Cornelis F.; Bürkle, Alexander; Xu, Xingzhi; Wang, Zhao-Qi

2013-12-01

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

TAM receptor tyrosine kinases as emerging targets of innate immune checkpoint blockade for cancer therapy.

PubMed

Akalu, Yemsratch T; Rothlin, Carla V; Ghosh, Sourav

2017-03-01

Cancer immunotherapy utilizing T-cell checkpoint inhibitors has shown tremendous clinical success. Yet, this mode of treatment is effective in only a subset of patients. Unresponsive patients tend to have non-T-cell-inflamed tumors that lack markers associated with the activation of adaptive anti-tumor immune responses. Notably, elimination of cancer cells by T cells is critically dependent on the optimal activity of innate immune cells. Therefore, identifying new targets that regulate innate immune cell function and promote the engagement of adaptive tumoricidal responses is likely to lead to the development of improved therapies against cancer. Here, we review the TAM receptor tyrosine kinases-TYRO3, AXL, and MERTK-as an emerging class of innate immune checkpoints that participate in key steps of anti-tumoral immunity. Namely, TAM-mediated efferocytosis, negative regulation of dendritic cell activity, and dysregulated production of chemokines collectively favor the escape of malignant cells. Hence, disabling TAM signaling may promote engagement of adaptive immunity and complement T-cell checkpoint blockade. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching

DOE PAGES

Ye, Qiaozhen; Rosenberg, Scott C.; Moeller, Arne; ...

2015-04-28

The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from amore » signaling-active ‘closed’ conformer to an inactive ‘open’ conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination.« less

Top2 and Sgs1-Top3 Act Redundantly to Ensure rDNA Replication Termination

PubMed Central

Fredsøe, Jacob; Nielsen, Ida; Pedersen, Jakob Madsen; Bentsen, Iben Bach; Lisby, Michael; Bjergbaek, Lotte; Andersen, Anni H

2015-01-01

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time. PMID:26630413

Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

PubMed Central

Ahn, Brian J.; Pollack, Ian F.; Okada, Hideho

2013-01-01

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. PMID:24202450

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

PubMed Central

Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

2015-01-01

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)2 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO3)2-treated cells, indicative of membrane rupture by Pb(NO3)2 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO3)2 exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO3)2 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO3)2 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO3)2 exposure and its associated adverse health effects. PMID:26703663

DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

PubMed

Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

2015-12-22

In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

PubMed

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

PubMed Central

LUCIANI, M. GLORIA; CAMPREGHER, CHRISTOPH; FORTUNE, JOHN M.; KUNKEL, THOMAS A.; GASCHE, CHRISTOPH

2007-01-01

Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. PMID:17241873

[Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

PubMed

Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

2007-01-01

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.

The p53-p21WAF1 checkpoint pathway plays a protective role in preventing DNA rereplication induced by abrogation of FOXF1 function

PubMed Central

Lo, Pang-Kuo; Lee, Ji Shin; Sukumar, Saraswati

2011-01-01

We previously identified FOXF1 as a potential tumor suppressor gene with an essential role in preventing DNA rereplication to maintain genomic stability, which is frequently inactivated in breast cancer through the epigenetic mechanism. Here we further addressed the role of the p53-p21WAF1 checkpoint pathway in DNA rereplication induced by silencing of FOXF1. Knockdown of FOXF1 by small interference RNA (siRNA) rendered colorectal p53-null and p21WAF1-null HCT116 cancer cells more susceptible to rereplication and apoptosis than the wild-type parental cells. In parental HCT116 cells with a functional p53 checkpoint, the p53-p21WAF1 checkpoint pathway was activated upon FOXF1 knockdown, which was concurrent with suppression of the CDK2-Rb cascade and induction of G1 arrest. In contrast, these events were not observed in FOXF1-depleted HCT116-p53−/− and HCT116-p21−/− cells, indicating the p53-dependent checkpoint function is vital for inhibiting CDK2 to induce G1 arrest and protect cells from rereplication. The pharmacologic inhibitor (caffeine) of Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases abolished activation of the p53-p21WAF1 pathway upon FOXF1 knockdown, suggesting that suppression of FOXF1 function triggered the ATM/ATR-mediated DNA damage response. Cosilencing of p53 by siRNA synergistically enhanced the effect of FOXF1 depletion on stimulation of DNA rereplication and apoptosis in wild-type HCT116. Finally, we show that FOXF1 expression is predominantly silenced in breast and colorectal cancer cell lines with inactive p53. Our study demonstrated that the p53-p21WAF1 checkpoint pathway is an intrinsically protective mechanism to prevent DNA rereplication induced by silencing of FOXF1. PMID:21964066

Comparative Analysis of Immune Checkpoint Molecules and Their Potential Role in the Transmissible Tasmanian Devil Facial Tumor Disease

PubMed Central

Flies, Andrew S.; Blackburn, Nicholas B.; Lyons, Alan Bruce; Hayball, John D.; Woods, Gregory M.

2017-01-01

Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil (Sarcophilus harrisii) facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology. PMID:28515726

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas.

PubMed

Zhou, Guoying; Sprengers, Dave; Boor, Patrick P C; Doukas, Michail; Schutz, Hannah; Mancham, Shanta; Pedroza-Gonzalez, Alexander; Polak, Wojciech G; de Jonge, Jeroen; Gaspersz, Marcia; Dong, Haidong; Thielemans, Kris; Pan, Qiuwei; IJzermans, Jan N M; Bruno, Marco J; Kwekkeboom, Jaap

2017-10-01

Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell-mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non-small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4 + and CD8 + T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8 + and CD4 + T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8 + and CD4 + T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8 + TIL, compared with other CD8 + TIL. Compared with TIL that did not express these inhibitory receptors, CD8 + and CD4 + TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8 + and CD4 + TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Finding Order in Randomness: Single-Molecule Studies Reveal Stochastic RNA Processing | Center for Cancer Research

Cancer.gov

Producing a functional eukaryotic messenger RNA (mRNA) requires the coordinated activity of several large protein complexes to initiate transcription, elongate nascent transcripts, splice together exons, and cleave and polyadenylate the 3’ end. Kinetic competition between these various processes has been proposed to regulate mRNA maturation, but this model could lead to multiple, randomly determined, or stochastic, pathways or outcomes. Regulatory checkpoints have been suggested as a means of ensuring quality control. However, current methods have been unable to tease apart the contributions of these processes at a single gene or on a time scale that could provide mechanistic insight. To begin to investigate the kinetic relationship between transcription and splicing, Daniel Larson, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues employed a single-molecule RNA imaging approach to monitor production and processing of a human β-globin reporter gene in living cells.

Multiple treatment comparison of seven new drugs for patients with advanced malignant melanoma: a systematic review and health economic decision model in a Norwegian setting

PubMed Central

Pike, Eva; Hamidi, Vida; Saeterdal, Ingvil; Odgaard-Jensen, Jan; Klemp, Marianne

2017-01-01

Objective To assess the relative effectiveness and cost-effectiveness of seven new drugs (cobimetinib, dabrafenib, ipilimumab, nivolumab, pembrolizumab, trametinib and vemurafenib) used for treatment of patients with advanced malignant melanoma in the Norwegian setting. Design A multiple technology assessment. Patients Patients with advanced malignant melanoma aged 18 or older. Data sources A systematic search for randomised controlled trials in relevant bibliographic databases. Methods We performed network meta-analyses using both direct and indirect evidence with dacarbazine as a common comparator. We ranked the different treatments in terms of their likelihood of leading to the best results for each endpoint. The cost-utility analysis was based on a probabilistic discrete-time Markov cohort model. The model calculated the costs and quality-adjusted life years (QALYs) with different treatment strategies from a healthcare perspective. Sensitivity analysis was performed by means of Monte Carlo simulation. Results Monotherapies with a programmed cell death 1 (PD-1) immune-checkpoint-inhibitor had a higher probability of good performance for overall survival than monotherapies with ipilimumab or BRAF/MEK inhibitors. The combination treatments had all similar levels of effectiveness to the PD-1 immune-checkpoint-inhibitors. PD-1 immune-checkpoint-inhibitors are more effective and more costly compared with ipilimumab in monotherapy. Nivolumab in combination with ipilimumab had higher costs and the same level of effectiveness as the PD-1 immune-checkpoint-inhibitors in monotherapy. BRAF/MEK inhibitor combinations (dabrafenib and trametinib or vemurafenib and cobimetinib) had both similar effectiveness and cost-effectiveness; however, the combination therapies are more likely to give higher quality adjusted life year gains than BRAF or MEK inhibitor monotherapies, but to a higher cost. Conclusions None of the drugs investigated can be considered cost-effective at what has normally been considered a reasonable willingness-to-pay (WTP) in Norway. Price reductions (from the official list prices) in the region of 63%–84% would be necessary for these drugs to be cost-effective at a WTP of €55 850 per QALY. PMID:28827234

Epigenetic modifiers in immunotherapy: a focus on checkpoint inhibitors.

PubMed

Terranova-Barberio, Manuela; Thomas, Scott; Munster, Pamela N

2016-06-01

Immune surveillance should be directed to suppress tumor development and progression, involving a balance of coinhibitory and costimulatory signals that amplify immune response without overwhelming the host. Immunotherapy confers durable clinical benefit in 'immunogenic tumors', whereas in other tumors the responses are modest. Thus, immune checkpoint inhibitors may need to be combined with strategies to boost immune response or increase the tumor immune profile. Epigenetic aberrations contribute significantly to carcinogenesis. Recent findings suggest that epigenetic drugs prime the immune response by increasing expression of tumor-associated antigens and immune-related genes, as well as modulating chemokines and cytokines involved in immune system activation. This review describes our current understanding regarding epigenetic and immunotherapy combination, focusing on immune response priming to checkpoint blockade.

Immune checkpoint inhibitor toxicity review for the palliative care clinician.

PubMed

Hansen, Eric D; Wang, Xiao; Case, Amy A; Puzanov, Igor; Smith, Tom

2018-05-21

Immune checkpoint inhibitors (ICI) have opened an exciting chapter in the treatment of patients with advanced cancer. For the palliative care clinician, however, ICI present several new challenges, including new ways to define treatment success, as well as treatment-related toxicities which differ in nature and timing from traditional chemotherapy. In this article, we review the mechanism of action of immune checkpoint inhibitors, as well as selected published data supporting the efficacy of ICI in patients with advanced cancer. In addition, we summarize existing data of ICI toxicity prevalence, patterns of severity and timing of onset. Finally, we briefly review key principles from published guidelines on the management of ICI toxicities. Copyright © 2018. Published by Elsevier Inc.

The Mechanism of Nucleotide Excision Repair-Mediated UV-Induced Mutagenesis in Nonproliferating Cells

PubMed Central

Kozmin, Stanislav G.; Jinks-Robertson, Sue

2013-01-01

Following the irradiation of nondividing yeast cells with ultraviolet (UV) light, most induced mutations are inherited by both daughter cells, indicating that complementary changes are introduced into both strands of duplex DNA prior to replication. Early analyses demonstrated that such two-strand mutations depend on functional nucleotide excision repair (NER), but the molecular mechanism of this unique type of mutagenesis has not been further explored. In the experiments reported here, an ade2 adeX colony-color system was used to examine the genetic control of UV-induced mutagenesis in nondividing cultures of Saccharomyces cerevisiae. We confirmed a strong suppression of two-strand mutagenesis in NER-deficient backgrounds and demonstrated that neither mismatch repair nor interstrand crosslink repair affects the production of these mutations. By contrast, proteins involved in the error-prone bypass of DNA damage (Rev3, Rev1, PCNA, Rad18, Pol32, and Rad5) and in the early steps of the DNA-damage checkpoint response (Rad17, Mec3, Ddc1, Mec1, and Rad9) were required for the production of two-strand mutations. There was no involvement, however, for the Pol η translesion synthesis DNA polymerase, the Mms2-Ubc13 postreplication repair complex, downstream DNA-damage checkpoint factors (Rad53, Chk1, and Dun1), or the Exo1 exonuclease. Our data support models in which UV-induced mutagenesis in nondividing cells occurs during the Pol ζ-dependent filling of lesion-containing, NER-generated gaps. The requirement for specific DNA-damage checkpoint proteins suggests roles in recruiting and/or activating factors required to fill such gaps. PMID:23307894

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast.

PubMed

Kume, Kazunori; Hashimoto, Tomoyo; Suzuki, Masashi; Mizunuma, Masaki; Toda, Takashi; Hirata, Dai

2017-09-30

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca 2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca 2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca 2+ -CN signaling plays a central role in cell polarity control by checkpoint activation. Copyright © 2017 Elsevier Inc. All rights reserved.

NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

PubMed

Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

2016-09-01

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

Checkpoints for vesicular traffic?

PubMed

Fiset, A; Faure, R

2001-01-01

During interphase the transport of material between different intracellular organelles requires accurate regulation of fusiogenic domains. Recent studies on hepatic endosomes indicated that compartmentalized Cdk2-cyclin E complexes act by braking fusion events. These Cdk2 complexes integrate tyrosine phosphorylation and dephosphorylation inputs, resulting in the control of the number of rounds of fusion at discrete domains. This leads to changes in the intracellular location of internalized receptors and ultimately their biological response.

Induction of the tumor-suppressor p16(INK4a) within regenerative epithelial crypts in ulcerative colitis.

PubMed

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-06-01

p16(INK4a) is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)-a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an "emergency brake" in cells experiencing sustained replicative stress.

The mechanistic effects of the dioxonaphthoimidazolium analog YM155 in renal cell carcinoma cell cycling and apoptosis.

PubMed

Sim, Mei Yi; Go, Mei Lin; Yuen, John Shyi Peng

2018-06-15

To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC). Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting. Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP. Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC. Copyright © 2018 Elsevier Inc. All rights reserved.

Immunotherapy in melanoma: Recent advances and future directions.

PubMed

Franklin, C; Livingstone, E; Roesch, A; Schilling, B; Schadendorf, D

2017-03-01

Malignant melanoma contributes the majority of skin cancer related deaths and shows an increasing incidence in the past years. Despite all efforts of early diagnosis, metastatic melanoma still has a poor prognosis and remains a challenge for treating physicians. In recent years, improved knowledge of the pathophysiology and a better understanding of the role of the immune system in tumour control have led to the development and approval of several immunotherapies. Monoclonal antibodies against different immune checkpoints have been revolutionizing the treatment of metastatic and unresectable melanoma. Ipilimumab, a monoclonal antibody against the cytotoxic T-lymphocyte antigen 4 (CTLA-4) as well as nivolumab and pembrolizumab which target the programmed cell death protein 1 (PD-1) have been shown to prolong overall survival in patients with advanced melanoma. The latter substances seem to have an increased response rate and more tolerable safety profile compared to ipilimumab. The combination of a CTLA-4 and a PD-1 inhibitor seems to be superior to the monotherapies, especially in patients with PD-L1 negative tumours. Checkpoint inhibitors are currently being tested in the adjuvant setting with initial data for ipilimumab suggesting efficacy in this context. Talimogene laherparepvec (TVEC) is the first oncolytic virus approved in the therapy of metastatic melanoma offering a treatment option especially for patients with limited disease. In this review, data on these recently developed and approved immunotherapies are presented. However, further studies are necessary to determine the optimal duration, sequencing and combinations of immunotherapies to further improve the outcome of patients with advanced melanoma. Copyright © 2016 Elsevier Ltd, BASO ~ The Association for Cancer Surgery, and the European Society of Surgical Oncology. All rights reserved.

Keeping Tumors in Check: A Mechanistic Review of Clinical Response and Resistance to Immune Checkpoint Blockade in Cancer.

PubMed

Borcherding, Nicholas; Kolb, Ryan; Gullicksrud, Jodi; Vikas, Praveen; Zhu, Yuwen; Zhang, Weizhou

2018-07-06

Immune checkpoints are a diverse set of inhibitory signals to the immune system that play a functional role in adaptive immune response and self-tolerance. Dysregulation of these pathways is a vital mechanism in the avoidance of immune destruction by tumor cells. Immune checkpoint blockade (ICB) refers to targeted strategies to disrupt the tumor co-opted immune suppression to enhance anti-tumor immunity. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) are two immune checkpoints that have the widest range of antibody-based therapies. These therapies have gone from promising approaches to Food and Drug Administration-approved first- and second-line agents for a number of immunogenic cancers. The burgeoning investigations of ICB efficacy in blood and solid cancers have underscored the importance of identifying the predictors of response and resistance to ICB. Identification of response correlates is made complicated by the observations of mixed reactions, or different responses in multiple lesions from the same patient, and delayed responses that can occur over a year after the induction therapy. Factors that can influence response and resistance in ICB can illuminate underlying molecular mechanisms of immune activation and suppression. These same response predictors can guide the identification of patients who would benefit from ICB, reduce off-target immune-relate adverse events, and facilitate the use of combinatorial therapies to increase efficacy. Here we review the underlying principles of immune checkpoint therapy and results of single-agent ICB clinical trials, and summarize the predictors of response and resistance. Copyright © 2018 Elsevier Ltd. All rights reserved.

[Use of the Six Sigma methodology for the preparation of parenteral nutrition mixtures].

PubMed

Silgado Bernal, M F; Basto Benítez, I; Ramírez García, G

2014-04-01

To use the tools of the Six Sigma methodology for the statistical control in the elaboration of parenteral nutrition mixtures at the critical checkpoint of specific density. Between August of 2010 and September of 2013, specific density analysis was performed to 100% of the samples, and the data were divided in two groups, adults and neonates. The percentage of acceptance, the trend graphs, and the sigma level were determined. A normality analysis was carried out by using the Shapiro Wilk test and the total percentage of mixtures within the specification limits was calculated. The specific density data between August of 2010 and September of 2013 comply with the normality test (W = 0.94) and show improvement in sigma level through time, reaching 6/6 in adults and 3.8/6 in neonates. 100% of the mixtures comply with the specification limits for adults and neonates, always within the control limits during the process. The improvement plans together with the Six Sigma methodology allow controlling the process, and warrant the agreement between the medical prescription and the content of the mixture. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

Modern Languages for Communication. Teaching the Curriculum: Checkpoint A, Grades K-6. Topics, Objectives, Activities in French, German, Italian, and Spanish.

ERIC Educational Resources Information Center

Yonkers City School District, NY.

The modern language curriculum guide for grades K-6 is designed to correlate with Checkpoint A of the New York State Syllabus. It presents major topics, listing instructional objectives, functions, skill areas, suggested instructional materials, suggested activities, cultural content, and games, songs, and puzzles. Introductory sections outline…

Memory management and compiler support for rapid recovery from failures in computer systems

NASA Technical Reports Server (NTRS)

Fuchs, W. K.

1991-01-01

This paper describes recent developments in the use of memory management and compiler technology to support rapid recovery from failures in computer systems. The techniques described include cache coherence protocols for user transparent checkpointing in multiprocessor systems, compiler-based checkpoint placement, compiler-based code modification for multiple instruction retry, and forward recovery in distributed systems utilizing optimistic execution.

Fault Tolerant Frequent Pattern Mining

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shohdy, Sameh; Vishnu, Abhinav; Agrawal, Gagan

FP-Growth algorithm is a Frequent Pattern Mining (FPM) algorithm that has been extensively used to study correlations and patterns in large scale datasets. While several researchers have designed distributed memory FP-Growth algorithms, it is pivotal to consider fault tolerant FP-Growth, which can address the increasing fault rates in large scale systems. In this work, we propose a novel parallel, algorithm-level fault-tolerant FP-Growth algorithm. We leverage algorithmic properties and MPI advanced features to guarantee an O(1) space complexity, achieved by using the dataset memory space itself for checkpointing. We also propose a recovery algorithm that can use in-memory and disk-based checkpointing,more » though in many cases the recovery can be completed without any disk access, and incurring no memory overhead for checkpointing. We evaluate our FT algorithm on a large scale InfiniBand cluster with several large datasets using up to 2K cores. Our evaluation demonstrates excellent efficiency for checkpointing and recovery in comparison to the disk-based approach. We have also observed 20x average speed-up in comparison to Spark, establishing that a well designed algorithm can easily outperform a solution based on a general fault-tolerant programming model.« less

Silencing of human DNA polymerase λ causes replication stress and is synthetically lethal with an impaired S phase checkpoint

PubMed Central

Zucca, Elisa; Bertoletti, Federica; Wimmer, Ursula; Ferrari, Elena; Mazzini, Giuliano; Khoronenkova, Svetlana; Grosse, Nicole; van Loon, Barbara; Dianov, Grigory; Hübscher, Ulrich; Maga, Giovanni

2013-01-01

Human DNA polymerase (pol) λ functions in base excision repair and non-homologous end joining. We have previously shown that DNA pol λ is involved in accurate bypass of the two frequent oxidative lesions, 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine during the S phase. However, nothing is known so far about the relationship of DNA pol λ with the S phase DNA damage response checkpoint. Here, we show that a knockdown of DNA pol λ, but not of its close homologue DNA pol β, results in replication fork stress and activates the S phase checkpoint, slowing S phase progression in different human cancer cell lines. We furthermore show that DNA pol λ protects cells from oxidative DNA damage and also functions in rescuing stalled replication forks. Its absence becomes lethal for a cell when a functional checkpoint is missing, suggesting a DNA synthesis deficiency. Our results provide the first evidence, to our knowledge, that DNA pol λ is required for cell cycle progression and is functionally connected to the S phase DNA damage response machinery in cancer cells. PMID:23118481

Immune checkpoint inhibitors for metastatic bladder cancer.

PubMed

Massari, Francesco; Di Nunno, Vincenzo; Cubelli, Marta; Santoni, Matteo; Fiorentino, Michelangelo; Montironi, Rodolfo; Cheng, Liang; Lopez-Beltran, Anto; Battelli, Nicola; Ardizzoni, Andrea

2018-03-01

Chemotherapy has represented the standard therapy for unresectable or metastatic urothelial carcinoma for more than 20 years. The growing knowledge of the interaction between tumour and immune system has led to the advent of new classes of drugs, the immune-checkpoints inhibitors, which are intended to change the current scenario. To date, immunotherapy is able to improve the overall responses and survival. Moreover, thanks to its safety profile immune-checkpoint inhibitors could be proposed also to patients unfit for standard chemotherapy. No doubts that these agents have started a revolution expected for years, but despite this encouraging results it appears clear that not all subjects respond to these agents and requiring the development of reliable predictive response factors able to isolate patients who can more benefit from these treatments as well as new strategies aimed to improve immunotherapy clinical outcome. In this review we describe the active or ongoing clinical trials involving Programmed Death Ligand 1 (PD-L1), Programmed Death receptor 1 (PD-1) and Cytotoxic-T Lymphocyte Antigen 4 (CTLA 4) inhibitors in urothelial carcinoma focusing our attention on the developing new immune-agents and combination strategies with immune-checkpoint inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

TRIO: Burst Buffer Based I/O Orchestration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Teng; Oral, H Sarp; Pritchard, Michael

The growing computing power on leadership HPC systems is often accompanied by ever-escalating failure rates. Checkpointing is a common defensive mechanism used by scientific applications for failure recovery. However, directly writing the large and bursty checkpointing dataset to parallel filesystem can incur significant I/O contention on storage servers. Such contention in turn degrades the raw bandwidth utilization of storage servers and prolongs the average job I/O time of concurrent applications. Recently burst buffer has been proposed as an intermediate layer to absorb the bursty I/O traffic from compute nodes to storage backend. But an I/O orchestration mechanism is still desiredmore » to efficiently move checkpointing data from bursty buffers to storage backend. In this paper, we propose a burst buffer based I/O orchestration framework, named TRIO, to intercept and reshape the bursty writes for better sequential write traffic to storage severs. Meanwhile, TRIO coordinates the flushing orders among concurrent burst buffers to alleviate the contention on storage server bandwidth. Our experimental results reveal that TRIO can deliver 30.5% higher bandwidth and reduce the average job I/O time by 37% on average for data-intensive applications in various checkpointing scenarios.« less

Gaining ground on a cure through synergy: combining checkpoint inhibitors with cancer vaccines.

PubMed

Vreeland, T J; Clifton, G T; Herbert, G S; Hale, D F; Jackson, D O; Berry, J S; Peoples, G E

2016-12-01

The approval of multiple checkpoint inhibitors (CPIs) for the treatment of advanced malignancies has sparked an explosion of research in the field of cancer immunotherapy. Despite the success of these medications, a large number of patients with advanced malignancy do not benefit from therapy. Early research indicates that a therapeutic combination of cancer vaccines with checkpoint inhibitors may lead to synergistic effects and higher response rates than monotherapy. Areas covered: This paper summarizes the previously completed and ongoing research on this exciting combination, including the use of the tumor lysate, particle-loaded dendritic cell (TLPLDC) vaccine combined with checkpoint inhibitors in advanced melanoma. Expert commentary: Increasing experience with CPIs has led to improved understanding of which patients may benefit and it is increasingly clear that the presence of a pre-existing immune response to the tumor, along with tumor-infiltrating lymphocytes, is key to the success of CPIs. One exciting possibility for the future is the addition of a cancer vaccine to CPI therapy, eliciting these crucial T cells, which can then be augmented and protected by the CPI. A number of current and future studies are addressing this very exciting combination therapy.

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

DOEpatents

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.

Incorporation of Immune Checkpoint Blockade into Chimeric Antigen Receptor T Cells (CAR-Ts): Combination or Built-In CAR-T

PubMed Central

Yoon, Dok Hyun; Osborn, Mark J.; Tolar, Jakub; Kim, Chong Jai

2018-01-01

Chimeric antigen receptor (CAR) T cell therapy represents the first U.S. Food and Drug Administration approved gene therapy and these engineered cells function with unprecedented efficacy in the treatment of refractory CD19 positive hematologic malignancies. CAR translation to solid tumors is also being actively investigated; however, efficacy to date has been variable due to tumor-evolved mechanisms that inhibit local immune cell activity. To bolster the potency of CAR-T cells, modulation of the immunosuppressive tumor microenvironment with immune-checkpoint blockade is a promising strategy. The impact of this approach on hematological malignancies is in its infancy, and in this review we discuss CAR-T cells and their synergy with immune-checkpoint blockade. PMID:29364163

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

Phenotypic Checkpoints Regulate Neuronal Development

PubMed Central

Ben-Ari, Yehezkel; Spitzer, Nicholas C.

2010-01-01

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 may constitute presymptomatic signatures of neurological disorders when they go awry. PMID:20864191

Role of BRCA1 in Controlling Mitotic Arrest in Ovarian Cystadenoma Cells

PubMed Central

Yu, Vanessa M.; Marion, Christine M.; Austria, Theresa M.; Yeh, Jennifer; Schönthal, Axel H.; Dubeau, Louis

2011-01-01

Cancers that develop in BRCA1 mutation carriers are usually near tetraploid/polyploid. This led us to hypothesize that BRCA1 controls the mitotic checkpoint complex, as loss of such control could lead to mitotic errors resulting in tetraploidy/polyploidy with subsequent aneuploidy. We used an in vitro system mimicking pre-malignant conditions, consisting of cell strains derived from the benign counterparts of serous ovarian carcinomas (cystadenomas) and expressing SV40 large T antigen, conferring the equivalent of a p53 mutation. We previously showed that such cells undergo one or several doublings of their DNA content as they age in culture and approach the phenomenon of in vitro crisis. Here we show that such increase in DNA content reflects a cell cycle arrest possibly at the anaphase promoting complex, as evidenced by decreased BrdU incorporation and increased expression of the mitotic checkpoint complex. Down-regulation of BRCA1 in cells undergoing crisis leads to activation of the anaphase promoting complex and resumption of growth kinetics similar to those seen in cells before they reach crisis. Cells recovering from crisis after BRCA1 down-regulation become multinucleated, suggesting that reduced BRCA1 expression may lead to initiation of a new cell cycle without completion of cytokinesis. This is the first demonstration that BRCA1 controls a physiological arrest at the M phase apart from its established role in DNA damage response, a role that could represent an important mechanism for acquisition of aneuploidy during tumor development. This may be particularly relevant to cancers that have a near tetraploid/polyploid number of chromosomes. PMID:21792894

Collective synchronization of divisions in Drosophila development

NASA Astrophysics Data System (ADS)

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.

Soldiers of Misfortune?

DTIC Science & Technology

2014-05-01

Iraq established procedures for convoys approaching checkpoints and made sure PMSC personnel had the correct phone numbers to contact military...collection of information if it does not display a currently valid OMB control number . 1. REPORT DATE MAY 2014 2. REPORT TYPE 3. DATES COVERED 00-00-2014...to 00-00-2014 4. TITLE AND SUBTITLE Soldiers of Misfortune? 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

PubMed Central

Sheu, Yi-Jun; Kinney, Justin B.; Stillman, Bruce

2016-01-01

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability. PMID:26733669

Discovery of a novel class of triazolones as checkpoint kinase inhibitors--hit to lead exploration.

PubMed

Oza, Vibha; Ashwell, Susan; Brassil, Patrick; Breed, Jason; Deng, Chun; Ezhuthachan, Jay; Haye, Heather; Horn, Candice; Janetka, James; Lyne, Paul; Newcombe, Nicholas; Otterbien, Ludo; Pass, Martin; Read, Jon; Roswell, Sian; Su, Mei; Toader, Dorin; Yu, Dingwei; Yu, Yan; Valentine, Anna; Webborn, Peter; White, Ann; Zabludoff, Sonya; Zheng, Xiaolan

2010-09-01

Checkpoint Kinase-1 (Chk1, CHK1, CHEK1) is a Ser/Thr protein kinase that mediates cellular responses to DNA-damage. A novel class of Chk1 inhibitors, triazoloquinolones/triazolones (TZ's) was identified by high throughput screening. The optimization of these hits to provide a lead series is described. Copyright 2010 Elsevier Ltd. All rights reserved.

Slip slidin’ away of mitosis with CRL2Zyg11

PubMed Central

2016-01-01

The spindle assembly checkpoint arrests mitotic cells by preventing degradation of cyclin B1 by the anaphase-promoting complex/cyclosome, but some cells evade this checkpoint and slip out of mitosis. Balachandran et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601083) show that the E3 ligase CRL2ZYG11 degrades cyclin B1, allowing mitotic slippage. PMID:27810907

Implementing forward recovery using checkpointing in distributed systems

NASA Technical Reports Server (NTRS)

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

1991-01-01

The paper describes the implementation of a forward recovery scheme using checkpoints and replicated tasks. The implementation is based on the concept of lookahead execution and rollback validation. In the experiment, two tasks are selected for the normal execution and one for rollback validation. It is shown that the recovery strategy has nearly error-free execution time and an average redundancy lower than TMR.

Cytotoxic Effects of Temozolomide and Radiation are Additive- and Schedule-Dependent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chalmers, Anthony J., E-mail: a.j.chalmers@sussex.ac.u; Genome Damage and Stability Centre, University of Sussex, Falmer; Ruff, Elliot M.

2009-12-01

Purpose: Despite aggressive therapy comprising radical radiation and temozolomide (TMZ) chemotherapy, the prognosis for patients with glioblastoma multiforme (GBM) remains poor, particularly if tumors express O{sup 6}-methylguanine-DNA-methyltransferase (MGMT). The interactions between radiation and TMZ remain unclear and have important implications for scheduling and for developing strategies to improve outcomes. Methods and Materials: Factors determining the effects of combination therapy on clonogenic survival, cell-cycle checkpoint signaling and DNA repair were investigated in four human glioma cell lines (T98G, U373-MG, UVW, U87-MG). Results: Combining TMZ and radiation yielded additive cytotoxicity, but only when TMZ was delivered 72 h before radiation. Radiosensitization wasmore » not observed. TMZ induced G2/M cell-cycle arrest at 48-72 h, coincident with phosphorylation of Chk1 and Chk2. Additive G2/M arrest and Chk1/Chk2 phosphorylation was only observed when TMZ preceded radiation by 72 h. The ataxia-telangiectasia mutated (ATM) inhibitor KU-55933 increased radiation sensitivity and delayed repair of radiation-induced DNA breaks, but did not influence TMZ effects. The multiple kinase inhibitor caffeine enhanced the cytotoxicity of chemoradiation and exacerbated DNA damage. Conclusions: TMZ is not a radiosensitizing agent but yields additive cytotoxicity in combination with radiation. Our data indicate that TMZ treatment should commence at least 3 days before radiation to achieve maximum benefit. Activation of G2/M checkpoint signaling by TMZ and radiation has a cytoprotective effect that can be overcome by dual inhibition of ATM and ATR. More specific inhibition of checkpoint signaling will be required to increase treatment efficacy without exacerbating toxicity.« less

Proactive Fault Tolerance for HPC with Xen Virtualization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagarajan, Arun Babu; Mueller, Frank; Engelmann, Christian

2007-01-01

with thousands of processors. At such large counts of compute nodes, faults are becoming common place. Current techniques to tolerate faults focus on reactive schemes to recover from faults and generally rely on a checkpoint/restart mechanism. Yet, in today's systems, node failures can often be anticipated by detecting a deteriorating health status. Instead of a reactive scheme for fault tolerance (FT), we are promoting a proactive one where processes automatically migrate from “unhealthy” nodes to healthy ones. Our approach relies on operating system virtualization techniques exemplied by but not limited to Xen. This paper contributes an automatic and transparent mechanismmore » for proactive FT for arbitrary MPI applications. It leverages virtualization techniques combined with health monitoring and load-based migration. We exploit Xen's live migration mechanism for a guest operating system (OS) to migrate an MPI task from a health-deteriorating node to a healthy one without stopping the MPI task during most of the migration. Our proactive FT daemon orchestrates the tasks of health monitoring, load determination and initiation of guest OS migration. Experimental results demonstrate that live migration hides migration costs and limits the overhead to only a few seconds making it an attractive approach to realize FT in HPC systems. Overall, our enhancements make proactive FT a valuable asset for long-running MPI application that is complementary to reactive FT using full checkpoint/ restart schemes since checkpoint frequencies can be reduced as fewer unanticipated failures are encountered. In the context of OS virtualization, we believe that this is the rst comprehensive study of proactive fault tolerance where live migration is actually triggered by health monitoring.« less

PD-L1 and HLA Class I Antigen Expression and Clinical Course of the Disease in Intrahepatic Cholangiocarcinoma

PubMed Central

Sabbatino, Francesco; Villani, Vincenzo; Yearley, Jennifer H.; Deshpande, Vikram; Cai, Lei; Konstantinidis, Ioannis T.; Moon, Christina; Nota, Sjoerd; Wang, Yangyang; Al-Sukaini, Ahmad; Zhu, Andrew X.; Goyal, Lipika; Ting, David T.; Bardeesy, Nabeel; Hong, Theodore S.; Castillo, Carlos Fernandez-del; Tanabe, Kenneth K.; Lillemoe, Keith D.; Ferrone, Soldano; Ferrone, Cristina R.

2017-01-01

Purpose More effective therapy is needed for intrahepatic cholangiocarcinoma (ICC). The encouraging clinical results obtained with checkpoint molecule-specific monoclonal antibodies (mAb) have prompted us to investigate whether this type of immunotherapy may be applicable to ICC. The aims of this study were to determine whether (i) patients mount a T-cell immune response to their ICC, (ii) checkpoint molecules are expressed on both T cells and tumor cells, and (iii) tumor cells are susceptible to recognition by cognate T cells. Experimental Design Twenty-seven ICC tumors were analyzed for (i) lymphocyte infiltrate, (ii) HLA class I and HLA class II expression, and (iii) PD-1 and PD-L1 expression by T cells and ICC cells, respectively. The results of this analysis were correlated with the clinicopathologic characteristics of the patients investigated. Results Lymphocyte infiltrates were identified in all tumors. PD-L1 expression and HLA class I antigen expression by ICC cells was observed in 8 and 11, respectively, of the 27 tumors analyzed. HLA class I antigen expression correlated with CD8+ T-cell infiltrate. Furthermore, positive HLA class I antigen expression in combination with negative/rare PD-L1 expression was associated with favorable clinical course of the disease. Conclusions ICC patients are likely to mount a T-cell immune response against their own tumors. Defects in HLA class I antigen expression in combination with PD-L1 expression by ICC cells provide them with an immune escape mechanism. This mechanism justifies the implementation of immunotherapy with checkpoint molecule-specific mAbs in patients bearing ICC tumors without defects in HLA class I antigen expression. PMID:26373575

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation.

PubMed

Conde, Carlos; Osswald, Mariana; Barbosa, João; Moutinho-Santos, Tatiana; Pinheiro, Diana; Guimarães, Sofia; Matos, Irina; Maiato, Helder; Sunkel, Claudio E

2013-06-12

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

PubMed Central

Moura, Margarida; Osswald, Mariana; Leça, Nelson; Barbosa, João; Pereira, António J; Maiato, Helder; Sunkel, Claudio E; Conde, Carlos

2017-01-01

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit. DOI: http://dx.doi.org/10.7554/eLife.25366.001 PMID:28463114

Drosophila Polo regulates the spindle assembly checkpoint through Mps1-dependent BubR1 phosphorylation

PubMed Central

Conde, Carlos; Osswald, Mariana; Barbosa, João; Moutinho-Santos, Tatiana; Pinheiro, Diana; Guimarães, Sofia; Matos, Irina; Maiato, Helder; Sunkel, Claudio E

2013-01-01

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase-promoting complex/cyclosome-inhibitory complexes to levels that ensure long-term SAC activity. We propose a model in which Polo controls Mps1-dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function. PMID:23685359

Akt interaction with PLC(gamma) regulates the G(2)/M transition triggered by FGF receptors from MDA-MB-231 breast cancer cells.

PubMed

Browaeys-Poly, Edith; Perdereau, Dominique; Lescuyer, Arlette; Burnol, Anne-Françoise; Cailliau, Katia

2009-12-01

Estrogen-independent breast cancer cell growth is under the control of fibroblast growth factors receptors (FGFRs), but the role of phospholipase C gamma (PLC(gamma)) and Akt, the downstream effectors activated by FGFRs, in cell proliferation is still unresolved. FGFRs from highly invasive MDA-MB-231 cells were expressed in Xenopus oocyte, a powerful model system to assess the G(2)/M checkpoint regulation. Under FGF1 stimulation, an analysis of the progression in the M-phase of the cell cycle and of the Akt signaling cascades were performed using the phosphatidylinositol-3-kinase inhibitor, LY294002, and a mimetic peptide of the SH3 domain of PLC(gamma). Activated Akt binds and phosphorylates PLC(gamma) before Akt targets the tumor suppressor Chfr. Disruption of the Akt-PLC(gamma) interaction directs Akt binding to Chfr and accelerates the alleviation of the G(2)/M checkpoint. The PLC(gamma)-Akt interaction, triggered by FGF receptors from estrogen-independent breast cancer cells MDA-MB-231, regulates progression in the M-phase of the cell cycle.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

PubMed Central

Lee, Ji-Hoon; Mand, Michael R.; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W.; Richards, Alicia L.; Coon, Joshua J.; Paull, Tanya T.

2018-01-01

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes Ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. Here, we genetically separated DNA damage activation of ATM from oxidative activation using separation-of-function mutations. We found that deficiency in ATM activation by Mre11-Rad50-Nbs1 and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of ATM lacking oxidative activation generates widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicates ATM in the control of protein homeostasis. PMID:29317520

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore

PubMed Central

Marquardt, Joseph R.; Perkins, Jennifer L.; Beuoy, Kyle J.; Fisk, Harold A.

2016-01-01

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus. PMID:27339139

Modular elements of the TPR domain in the Mps1 N terminus differentially target Mps1 to the centrosome and kinetochore.

PubMed

Marquardt, Joseph R; Perkins, Jennifer L; Beuoy, Kyle J; Fisk, Harold A

2016-07-12

Faithful segregation of chromosomes to two daughter cells is regulated by the formation of a bipolar mitotic spindle and the spindle assembly checkpoint, ensuring proper spindle function. Here we show that the proper localization of the kinase Mps1 (monopolar spindle 1) is critical to both these processes. Separate elements in the Mps1 N-terminal extension (NTE) and tetratricopeptide repeat (TPR) domains govern localization to either the kinetochore or the centrosome. The third TPR (TPR3) and the TPR-capping helix (C-helix) are each sufficient to target Mps1 to the centrosome. TPR3 binds to voltage-dependent anion channel 3, but although this is sufficient for centrosome targeting of Mps1, it is not necessary because of the presence of the C-helix. A version of Mps1 lacking both elements cannot localize to or function at the centrosome, but maintains kinetochore localization and spindle assembly checkpoint function, indicating that TPR3 and the C-helix define a bipartite localization determinant that is both necessary and sufficient to target Mps1 to the centrosome but dispensable for kinetochore targeting. In contrast, elements required for kinetochore targeting (the NTE and first two TPRs) are dispensable for centrosomal localization and function. These data are consistent with a separation of Mps1 function based on localization determinants within the N terminus.

Reproductive health and rights in East Jerusalem: the effects of militarisation and biopolitics on the experiences of pregnancy and birth of Palestinians living in the Kufr 'Aqab neighbourhood.

PubMed

Hamayel, Layaly; Hammoudeh, Doaa; Welchman, Lynn

2017-10-01

Research with marginalised communities points to the need to understand political determinants of reproductive health. For residents of Kufr 'Aqab neighbourhood, Israeli biopolitics in East Jerusalem can be barriers to access to maternal health. This is manifested in women having to cross military checkpoints to give birth in hospitals located in Jerusalem to make their children eligible for "permanent residency", a document required for Palestinians to live in Jerusalem. A basic qualitative design is utilised, and semi-structured in-depth interviews with 27 women and 20 men were conducted and thematic analysis was used to extract themes and subthemes. Women reported exposure to risky conditions during pregnancy and worries of giving birth at checkpoints. Social support was restricted for some women due to inability of the husband/family to reach the hospital at the time of birth. Men reported distress related to inability to attend birth. Giving birth in a Jerusalem hospital, as part of passing residency to children, was perceived as reaffirming Palestinian presence in the City and transforming sites of suffering to sites of resistance. Israeli residency policies and segregation of Jerusalem affect Kufr 'Aqab residents' pregnancy and birth on physical, social and psychological levels. Results indicate the importance of incorporating political determinants of access to maternal care and safe pregnancy in the conceptualisation of reproductive rights.

Perfect Detection of Spikes in the Linear Sub-threshold Dynamics of Point Neurons

PubMed Central

Krishnan, Jeyashree; Porta Mana, PierGianLuca; Helias, Moritz; Diesmann, Markus; Di Napoli, Edoardo

2018-01-01

Spiking neuronal networks are usually simulated with one of three main schemes: the classical time-driven and event-driven schemes, and the more recent hybrid scheme. All three schemes evolve the state of a neuron through a series of checkpoints: equally spaced in the first scheme and determined neuron-wise by spike events in the latter two. The time-driven and the hybrid scheme determine whether the membrane potential of a neuron crosses a threshold at the end of the time interval between consecutive checkpoints. Threshold crossing can, however, occur within the interval even if this test is negative. Spikes can therefore be missed. The present work offers an alternative geometric point of view on neuronal dynamics, and derives, implements, and benchmarks a method for perfect retrospective spike detection. This method can be applied to neuron models with affine or linear subthreshold dynamics. The idea behind the method is to propagate the threshold with a time-inverted dynamics, testing whether the threshold crosses the neuron state to be evolved, rather than vice versa. Algebraically this translates into a set of inequalities necessary and sufficient for threshold crossing. This test is slower than the imperfect one, but can be optimized in several ways. Comparison confirms earlier results that the imperfect tests rarely miss spikes (less than a fraction 1/108 of missed spikes) in biologically relevant settings. PMID:29379430

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

PubMed

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.

Comprehensive analysis of cancers of unknown primary for the biomarkers of response to immune checkpoint blockade therapy.

PubMed

Gatalica, Zoran; Xiu, Joanne; Swensen, Jeff; Vranic, Semir

2018-05-01

Cancer of unknown primary (CUP) accounts for approximately 3% of all malignancies. Avoiding immune destruction is a major cancer characteristic and therapies aimed at immune checkpoint blockade are in use for several specific cancer types. A comprehensive survey of predictive biomarkers to immune checkpoint blockade in CUP were explored in this study. About 389 cases of CUP were analysed for mutations in 592 genes and 52 gene fusions using a massively parallel DNA sequencing platform (next-generation sequencing [NGS]). Total mutational load (TML) and microsatellite instability (MSI) were calculated from NGS data. PD-L1 expression was explored using immunohistochemistry (with 5% cutoff value). High TML was seen in 11.8% (46/389) of tumours. MSI-high (MSI-H) was detected in 7/384 (1.8%) of tumours. Tumour PD-L1 expression was detected in 80/362 CUP (22%). A small proportion of CUP cases harboured genetic alterations of negative predictive biomarkers to immune checkpoint inhibitors (predictors to hyperprogression) including MDM2 gene amplification (2%) and loss of function JAK2 gene mutations (1%). Amplifications of CD274 (PD-L1) and PDCD1LG2 (PD-L2) genes were also rare (1.4% and 0.8%, respectively). The most frequently mutated genes were TP53 (54%), KRAS (22%), ARID1A (13%), PIK3CA (9%), CDKN2A (8%), SMARCA4 (7%) and PBRM1, STK11, APC, RB1 (5%, respectively). Using a multiplex testing approach, 28% of CUP carried one or more predictive biomarkers (MSI-H, PD-L1 and/or TML-H) to the immune checkpoint blockade, providing a novel option for treatment in patients with CUP. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Analysis of Drug Development Paradigms for Immune Checkpoint Inhibitors.

PubMed

Jardim, Denis L; de Melo Gagliato, Débora; Giles, Francis J; Kurzrock, Razelle

2018-04-15

Immune checkpoint inhibitors have unique toxicities and response kinetics compared with cytotoxic and gene-targeted anticancer agents. We investigated the impact of innovative/accelerated immunotherapy drug development/approval models on the accuracy of safety and efficacy assessments by searching the FDA website. Initial phase I trials for each agent were reviewed and safety and efficacy data compared with that found in later trials leading to regulatory approvals of the same agents. As of June 2017, the FDA approved six checkpoint inhibitors for a variety of cancer types. All checkpoint inhibitors received a priority review status and access to at least two additional FDA special access programs, more often breakthrough therapy designation and accelerated approval. Median clinical development time (investigational new drug application to approval) was 60.77 months [avelumab had the shortest timeline (52.33 months)]. Response rates during early phase I trials (median = 16%) are higher than for phase I trials of other agents (with the exception of gene-targeted agents tested with a biomarker). Doses approved were usually not identical to doses recommended on phase I trials. Approximately 50% of types of immune-related and 43% of types of clinically relevant toxicities from later trials were identified in early-phase trials. Even so, treatment-related mortality remains exceedingly low in later studies (0.33% of patients). In conclusion, efficacy and safety of immune checkpoint inhibitors appear to be reasonably predicted from the dose-finding portion of phase I trials, indicating that the fast-track development of these agents is safe and justified. Clin Cancer Res; 24(8); 1785-94. ©2017 AACR . ©2017 American Association for Cancer Research.

Profiling the dynamic expression of checkpoint molecules on cytokine-induced killer cells from non-small-cell lung cancer patients.

PubMed

Zhang, Lin; Wang, Jian; Wei, Feng; Wang, Kaiyuan; Sun, Qian; Yang, Fan; Jin, Hao; Zheng, Yu; Zhao, Hua; Wang, Limei; Yu, Wenwen; Zhang, Xiying; An, Yang; Yang, Lili; Zhang, Xinwei; Ren, Xiubao

2016-07-12

Immune checkpoints associate with dysfunctional T cells, which have a reduced ability to clear pathogens or cancer cells. T-cell checkpoint blockade may improve patient survival. However, checkpoint molecules on cytokine-induced killer (CIK) cell, a non-specific adoptive immunotherapy, remain unknown. In present study, we detected the dynamic expression of eight major checkpoint molecules (CTLA-4, PD-1, PD-L1, TIM- 3, CEACAM-1, LAG-3, TIGIT and BTLA) on CIK cells from NSCLC patients. The majority of these molecules, except BTLA, were sharply elevated during the early stage of CIK cell culture. Thereafter, PD-1 and TIGIT expressions decreased gradually towards the initial level (day 0). Moreover, CTLA-4 faded away during the later stage of CIK culture. LAG-3 expression decreased but was still significantly higher than the initial level. Of note, PD-L1 remained stably upregulated during CIK culture compared with PD-1, indicating that PD-L1 might act as an inhibitory molecule on CIK cells instead of PD-1. Furthermore, TIM-3 and CEACAM1 were strongly expressed simultaneously during long-term CIK culture and showed a significant and mutually positive correlation. BTLA displayed a distinct pattern, and its expression gradually decreased throughout the CIK culture. These observations suggested that CIK cells might be partly exhausted before clinical transfusion, characterized by the high expression of PD-L1, LAG-3, TIM- 3, and CEACAM-1 and the low expression of TIGIT, BTLA, PD-1, and CTLA-4 compared with initial culture. Our results imply that implementing combined treatment on CIK cells before transfusion via antibodies targeting PD-L1, LAG-3, TIM-3, and CEACAM-1 might improve the efficiency of CIK therapy for NSCLC patients.

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Checkpointing Shared Memory Programs at the Application-level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bronevetsky, G; Schulz, M; Szwed, P

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 focusedmore » 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.« less

Impaired driving enforcement practices among state and local law enforcement agencies in the United States.

PubMed

Eichelberger, Angela H; McCartt, Anne T

2016-09-01

Alcohol-impaired driving (DUI) persists as a substantial problem, yet detailed data on DUI enforcement practices are rarely collected. The present study surveyed state and local law enforcement agencies about their DUI enforcement activities. Telephone interviews were conducted with law enforcement liaisons in state highway safety offices. Officers from a nationally representative sample of municipal, county, and state law enforcement agencies were also interviewed about their agency's DUI enforcement activities, including the types of enforcement, frequency of use, and whether activities were publicized. Response rates were 100% among law enforcement liaisons, 86% among county agencies, 93% among municipal agencies, and 98% among state agencies. Based on the highway safety office survey, 38 states conducted sobriety checkpoints in 2011. Nationally, 58% of law enforcement agencies reported that they conducted or helped conduct sobriety checkpoints during 2011-12, with 14% of all agencies conducting them monthly or more frequently. The vast majority (87%) of agencies reported conducting dedicated DUI patrols. However, dedicated DUI patrols were less likely to be publicized than checkpoints. Less than a quarter of agencies reported using passive alcohol sensors to improve detection of alcohol-impaired drivers. Results show that 38 states conducted sobriety checkpoints in 2011, little changed from a previous survey in 2000. Despite evidence of effectiveness, many agencies do not conduct frequent, publicized DUI enforcement or use passive alcohol sensors. The survey suggests that there are several areas in which impaired driving enforcement could be improved: increasing the frequency of special enforcement, such as sobriety checkpoints and/or dedicated patrols; publicizing these efforts to maximize deterrent effects; and using passive alcohol sensors to improve detection of alcohol-impaired drivers. Copyright © 2016 Elsevier Ltd and National Safety Council. All rights reserved.

[Clinical Development of Immune Checkpoint Inhibitors in Patients with Small Cell Lung Cancer].

PubMed

Zhang, Shuang; Liu, Jingjing; Cheng, Ying

2017-09-20

Small cell lung cancer (SCLC) is a poorly differentiated high-grade neuroendocrine tumor, accounts for approximately 14% of all lung cancers. SCLC is characterized by rapid growth, early metastasis without effective treatments after recurrence. It is urgently need to improve the therapy of patients with SCLC. In recent years Tumor immunotherapy has shown promising efficacy, especially in immune checkpoints including inhibitors programmed cell-death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). These immune checkpoint inhibitors of the researches are changing the clinical practice of many kinds of solid tumor. SCLC is a potential ideal type of tumor immunotherapy for tobacco exposure and the highest mutational load. In this report, the authors review the current state of the immunotherapy in SCLC, to discussing the problems, challenge and application development prospect.

[Adoptive Cell Therapy with Immune Checkpoint Blockade].

PubMed

Aruga, Atsushi

2017-09-01

Cancer immunotherapy are taking a leading role of cancer therapy due to the development of the immune checkpoint blockade. To date, however, only about 20% of patients have clinical responses and the cancer-specific T cells in cancer site are required to obtain beneficial effects. There has been an innovative development in the field of adoptive cell therapy, especially receptor gene-modified T cells in recent years. The effector cells mostly express PD-1, therefore the cytotoxic reactivity of the effector cells are inhibited by PD-L1. The combination of the adoptive cell therapy and the immune checkpoint blockade is expected to enhance efficacy. On the other hand, the immune-related adverse events may also be enhanced, therefore, it is needed to develop the combination therapy carefully, improving the cancer antigen-specificity or dealing with the cytokine release syndrome.

Kinetochore localized Mad2 and Cdc20 is itself insufficient for triggering the mitotic checkpoint when Mps1 is low in Drosophila melanogaster neuroblasts.

PubMed

Herriott, Ashleigh; Sweeney, Michele; Whitaker, Michael; Taggart, Michael; Huang, Jun-Yong

2012-12-15

The relationships between the kinetochore and checkpoint control remain unresolved. Here, we report the characterization of the in vivo behavior of Cdc20 and Mad2 and the relevant spindle assembly checkpoint (SAC) functions in the neuroblasts of a Drosophila Mps1 weak allele (ald (B4-2) ). ald (B4-2) third instar larvae brain samples contain only around 16% endogenous Mps1 protein, and the SAC function is abolished. However, this does not lead to rapid anaphase onset and mitotic exit, in contrast to the loss of Mad2 alone in a mad2 (EY) mutant. The level of GFP-Cdc20 recruitment to the kinetochore is unaffected in ald (B4-2) neuroblasts, while the level of GFP-Mad2 is reduced to just about 20%. Cdc20 and Mad2 display only monophasic exponential kinetics at the kinetochores. The ald (B4-2) heterozygotes expressed approximately 65% of normal Mps1 protein levels, and this is enough to restore the SAC function. The kinetochore recruitment of GFP-Mad2 in response to SAC activation increases by around 80% in heterozygotes, compared with just about 20% in ald (B4-2) mutant. This suggests a correlation between Mps1 levels and Mad2 kinetochore localization and perhaps the existence of a threshold level at which Mps1 is fully functional. The failure to arrest the mitotic progression in ald (B4-2) neuroblasts in response to colchicine treatment suggests that when Mps1 levels are low, approximately 20% of normal GFP-Mad2, alongside normal levels of GFP-Cdc20 kinetochore recruitments, is insufficient for triggering SAC signal propagation.

Immune checkpoint inhibitors and targeted therapies for metastatic melanoma: A network meta-analysis.

PubMed

Pasquali, Sandro; Chiarion-Sileni, Vanna; Rossi, Carlo Riccardo; Mocellin, Simone

2017-03-01

Immune checkpoint inhibitors and targeted therapies, two new class of drugs for treatment of metastatic melanoma, have not been compared in randomized controlled trials (RCT). We quantitatively summarized the evidence and compared immune and targeted therapies in terms of both efficacy and toxicity. A comprehensive search for RCTs of immune checkpoint inhibitors and targeted therapies was conducted to August 2016. Using a network meta-analysis approach, treatments were compared with each other and ranked based on their effectiveness (as measured by the impact on progression-free survival [PFS]) and acceptability (the inverse of high grade toxicity). Twelve RCTs enrolling 6207 patients were included. Network meta-analysis generated 15 comparisons. Combined BRAF and MEK inhibitors were associated with longer PFS as compared to anti-CTLA4 (HR: 0.22; 95% confidence interval [CI]: 0.12-0.41) and anti-PD1 antibodies alone (HR: 0.38; CI: 0.20-0.72). However, anti-PD1 monoclonal antibodies were less toxic than anti-CTLA4 monoclonal antibodies (RR: 0.65; CI: 0.40-0.78) and their combination significantly increased toxicity compared to either single agent anti-CTLA4 (RR: 2.06; CI: 1.45-2.93) or anti-PD1 monoclonal antibodies (RR: 3.67; CI: 2.27-5.96). Consistently, ranking analysis suggested that the combination of targeted therapies is the most effective strategy, whereas single agent anti-PD1 antibodies have the best acceptability. The GRADE level of evidence quality for these findings was moderate to low. The simultaneous inhibition of BRAF and MEK appears the most effective treatment for melanomas harboring BRAF V600 mutation, although anti-PD1 antibodies appear to be less toxic. Further research is needed to increase the quality of evidence. Copyright © 2017 Elsevier Ltd. All rights reserved.

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos.

PubMed

Garner, M; van Kreeveld, S; Su, T T

2001-10-16

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus. dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16). Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16). We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16. This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.

Role of NADPH oxidases in inducing a selective increase of oxidant stress and cyclin D1 and checkpoint 1 over-expression during progression to human gastric adenocarcinoma.

PubMed

Montalvo-Javé, Eduardo E; Olguín-Martínez, Marisela; Hernández-Espinosa, Diego R; Sánchez-Sevilla, Lourdes; Mendieta-Condado, Edgar; Contreras-Zentella, Martha L; Oñate-Ocaña, Luis F; Escalante-Tatersfield, Tomás; Echegaray-Donde, Agustín; Ruiz-Molina, Juan M; Herrera, Miguel F; Morán, Julio; Hernández-Muñoz, Rolando

2016-04-01

Gastric cancer is one of the main causes of global mortality. Here, reactive oxygen species (ROS) could largely contribute to gastric carcinogenesis. Hence, the present work was aimed to assess the role of ROS, oxidant status, NADPH oxidases (NOXs) expression, during human gastric adenocarcinoma. We obtained subcellular fraction from samples of gastric mucosa taken from control subjects (n = 20), and from 40 patients with gastric adenocarcinoma, as well as samples of distant areas (tumour-free gastric mucosa). Parameters indicative of lipid peroxidation and cell proliferation were selectively increased in both tumour-free and in cancerous gastric mucosa, despite of glutathione (GSH) content, glutathione reductase (GR) and superoxide dismutase (SOD) activities were increased in the adenocarcinoma. These high levels of antioxidant defences inversely correlated with down-regulated expression for NOX2 and 4; however, over-expression of NOX1 occurred with increased caspase-3 activity and overexpressed checkpoint 1 (MDC1) and cyclin D1 proteins. In the tumour-free mucosa an oxidant stress took place, without changing total GSH but with decreased activities for GR and mitochondrial SOD; moreover, over-expression of checkpoint 1 (MDC1) correlated with lower NOX2 and 4 expression in this mucosa. Chronically injured gastric mucosa increases lipoperoxidative events and cell proliferation. In the adenocarcinoma, cell proliferation was further enhanced, oxidant stress decreased which seemed to be linked to NOX1, MDC1 and cyclin D1 over-expression, but with a lower NOXs activity leading a 'low tone' of ROS formation. Therefore, our results could be useful for early detection and treatment of gastric adenocarcinoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

Leptin Regulation of Gonadotrope Gonadotropin-Releasing Hormone Receptors As a Metabolic Checkpoint and Gateway to Reproductive Competence

PubMed Central

Odle, Angela K.; Akhter, Noor; Syed, Mohsin M.; Allensworth-James, Melody L.; Beneš, Helen; Melgar Castillo, Andrea I.; MacNicol, Melanie C.; MacNicol, Angus M.; Childs, Gwen V.

2018-01-01

The adipokine leptin signals the body’s nutritional status to the brain, and particularly, the hypothalamus. However, leptin receptors (LEPRs) can be found all throughout the body and brain, including the pituitary. It is known that leptin is permissive for reproduction, and mice that cannot produce leptin (Lep/Lep) are infertile. Many studies have pinpointed leptin’s regulation of reproduction to the hypothalamus. However, LEPRs exist at all levels of the hypothalamic–pituitary–gonadal axis. We have previously shown that deleting the signaling portion of the LEPR specifically in gonadotropes impairs fertility in female mice. Our recent studies have targeted this regulation to the control of gonadotropin releasing hormone receptor (GnRHR) expression. The hypotheses presented here are twofold: (1) cyclic regulation of pituitary GnRHR levels sets up a target metabolic checkpoint for control of the reproductive axis and (2) multiple checkpoints are required for the metabolic signaling that regulates the reproductive axis. Here, we emphasize and explore the relationship between the hypothalamus and the pituitary with regard to the regulation of GnRHR. The original data we present strengthen these hypotheses and build on our previous studies. We show that we can cause infertility in 70% of female mice by deleting all isoforms of LEPR specifically in gonadotropes. Our findings implicate activin subunit (InhBa) mRNA as a potential leptin target in gonadotropes. We further show gonadotrope-specific upregulation of GnRHR protein (but not mRNA levels) following leptin stimulation. In order to try and understand this post-transcriptional regulation, we tested candidate miRNAs (identified with in silico analysis) that may be binding the Gnrhr mRNA. We show significant upregulation of one of these miRNAs in our gonadotrope-Lepr-null females. The evidence provided here, combined with our previous work, lay the foundation for metabolically regulated post-transcriptional control of the gonadotrope. We discuss possible mechanisms, including miRNA regulation and the involvement of the RNA binding protein, Musashi. We also demonstrate how this regulation may be vital for the dynamic remodeling of gonadotropes in the cycling female. Finally, we propose that the leptin receptivity of both the hypothalamus and the pituitary are vital for the body’s ability to delay or slow reproduction during periods of low nutrition. PMID:29354094

Central Tolerance Blockade to Augment Checkpoint Immunotherapy in Melanoma

DTIC Science & Technology

2017-09-01

unique mode of action . By itself, anti- RANKL antibody improves the survival of mice injected with melanoma cells. Because anti-RANKL antibody and...anti-RANKL antibody or anti-CTLA-4 antibody alone. These findings are of potential clinical importance because it may pave the way to testing the...combination. If anti-RANKL 7 antibody increases the effectiveness of checkpoint inhibitors, this could potentially have a major impact on how

The United States Air Force Academy: A Bibliography 2001-2005

DTIC Science & Technology

2006-01-01

Faldons. WEEKLY COMPILATION OF PRESIDENTIAL DOCUMENTS May 16, 2003: 612-613. PERIODICAL ARTICLES 31. Academy names new women’s basketball coach...succeed. CHECKPOINTS Fall 2003: 50-51. 382. Popovich, 󈨊, NBA Coach of the Year. CHECKPOINTS Spring 2003: 50. 383. Rauschkolb, Dick...178 185 Basketball 31 38 45 Battlebots Tournament 64 Beer, Heather 128 Bell, Luke 65 Benn, Elizabeth 245 Biancur, Andrew W 314 Big Brothers

Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress

PubMed Central

Basile, Giorgia; Leuzzi, Giuseppe; Pichierri, Pietro; Franchitto, Annapaola

2014-01-01

Werner syndrome (WS) is a human chromosomal instability disorder associated with cancer predisposition and caused by mutations in the WRN gene. WRN helicase activity is crucial in limiting breakage at common fragile sites (CFS), which are the preferential targets of genome instability in precancerous lesions. However, the precise function of WRN in response to mild replication stress, like that commonly used to induce breaks at CFS, is still missing. Here, we establish that WRN plays a role in mediating CHK1 activation under moderate replication stress. We provide evidence that phosphorylation of CHK1 relies on the ATR-mediated phosphorylation of WRN, but not on WRN helicase activity. Analysis of replication fork dynamics shows that loss of WRN checkpoint mediator function as well as of WRN helicase activity hamper replication fork progression, and lead to new origin activation to allow recovery from replication slowing upon replication stress. Furthermore, bypass of WRN checkpoint mediator function through overexpression of a phospho-mimic form of CHK1 restores fork progression and chromosome stability to the wild-type levels. Together, these findings are the first demonstration that WRN regulates the ATR-checkpoint activation upon mild replication stress, preventing chromosome fragility. PMID:25352544

Immune checkpoint inhibitors in small cell lung cancer.

PubMed

Pakkala, Suchita; Owonikoko, Taofeek K

2018-02-01

Small cell lung cancer (SCLC) is a rapidly progressive cancer that often debilitates patients within months of detection and quickly becomes refractory to the limited options of therapy. While SCLC is not generally considered an immunogenic tumor, clinical experience suggests that patients with robust immune response manifesting as paraneoplastic syndrome are more likely to present with limited stage of the disease and tend to have a better prognosis. Monoclonal antibodies targeting critical negative regulators of immune response, so called immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death 1 (PD-1) have expanded the application of immune-based therapies to increasing number of advanced stage cancers. These agents overcome the inhibitory immune signals leading to a heightened immune response against cancer cells. These immune checkpoint inhibitors have established efficacy leading to regulatory approval for their use in many cancer types including non-small cell lung cancer (NSCLC). Evaluation of the CTLA-4 inhibitor, ipilimumab and PD-1 inhibitors, nivolumab and pembrolizumab in SCLC have shown encouraging signal but definitive studies are still ongoing. In this review, we discuss the rationale behind the use of checkpoint inhibitors in SCLC, contextualize the results of early trials of immunotherapy agents in SCLC and project the future evolution of this strategy.

Sarcoidosis Following Anti-PD-1 and Anti-CTLA-4 Therapy for Metastatic Melanoma.

PubMed

Reddy, Swathi B; Possick, Jennifer D; Kluger, Harriet M; Galan, Anjela; Han, Dale

2017-10-01

Immune checkpoint inhibitors represent the newest treatment for stage IV melanoma. These agents are generally well tolerated, however severe immune-related adverse effects have been noted in a small, but clinically significant percentage of patients. Specifically, sarcoidosis is a known potential complication following anti-CTLA-4 therapy. We present 2 cases of pulmonary and cutaneous sarcoidosis developing in patients with stage IV melanoma. Both patients were treated with ipilimumab and anti-PD-1 therapy, and both experienced good oncologic responses to treatment; neither had evidence of preexisting sarcoidosis. Of note, both patients developed sarcoidosis only after undergoing immune checkpoint inhibitor therapy. In 1 patient, sarcoidosis developed after initiation of anti-PD-1 therapy, 3 months after the last dose of anti-CTLA-4 monotherapy, suggesting a synergistic immune dysmodulating effect of both checkpoint inhibitors. Ultimately, both patients' symptoms and radiologic findings resolved with corticosteroid treatment, and both patients have tolerated retreatment with PD-1 inhibitors. Sarcoidosis is a rare complication of immune checkpoint inhibitors and can manifest with severe pulmonary manifestations. However, sarcoidosis in this setting is responsive to corticosteroids and does not necessarily recur with retreatment. It is yet unclear whether the development of sarcoidosis in these patients represents unmasking of preexisting autoimmune tendencies or is a marker of oncologic response.

The effect of tributyltin chloride on Caenorhabditis elegans germline is mediated by a conserved DNA damage checkpoint pathway.

PubMed

Cheng, Zhe; Tian, Huimin; Chu, Hongran; Wu, Jianjian; Li, Yingying; Wang, Yanhai

2014-03-21

Tributyltin (TBT), one of the environmental pollutants, has been shown to impact the reproduction of animals. However, due to the lack of appropriate animal model, analysis of the affected molecular pathways in germ cells is lagging and has been particularly challenging. In the present study, we investigated the effects of tributyltin chloride (TBTCL) on the nematode Caenorhabditis elegans germline. We show that exposure of C. elegans to TBTCL causes significantly elevated level of sterility and embryonic lethality. TBTCL exposure results in an increased number of meiotic DNA double-strand breaks in germ cells, subsequently leading to activated DNA damage checkpoint. Exposing C. elegans to TBTCL causes dose- and time-dependent germline apoptosis. This apoptotic response was blocked in loss-of-function mutants of hus-1 (op241), mrt-2 (e2663) and p53/cep-1 (gk138), indicating that checkpoints and p53 are essential for mediating TBTCL-induced germ cell apoptosis. Moreover, TBTCL exposure can inhibit germ cell proliferation, which is also mediated by the conserved checkpoint pathway. We thereby propose that TBT exhibits its effects on the germline by inducing DNA damage and impaired maintenance of genomic integrity. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perumalla, Kalyan S.; Yoginath, Srikanth B.

Problems such as fault tolerance and scalable synchronization can be efficiently solved using reversibility of applications. Making applications reversible by relying on computation rather than on memory is ideal for large scale parallel computing, especially for the next generation of supercomputers in which memory is expensive in terms of latency, energy, and price. In this direction, a case study is presented here in reversing a computational core, namely, Basic Linear Algebra Subprograms, which is widely used in scientific applications. A new Reversible BLAS (RBLAS) library interface has been designed, and a prototype has been implemented with two modes: (1) amore » memory-mode in which reversibility is obtained by checkpointing to memory in forward and restoring from memory in reverse, and (2) a computational-mode in which nothing is saved in the forward, but restoration is done entirely via inverse computation in reverse. The article is focused on detailed performance benchmarking to evaluate the runtime dynamics and performance effects, comparing reversible computation with checkpointing on both traditional CPU platforms and recent GPU accelerator platforms. For BLAS Level-1 subprograms, data indicates over an order of magnitude better speed of reversible computation compared to checkpointing. For BLAS Level-2 and Level-3, a more complex tradeoff is observed between reversible computation and checkpointing, depending on computational and memory complexities of the subprograms.« less

Cytokinesis Failure Leading to Chromosome Instability in v-Src-Induced Oncogenesis.

PubMed

Nakayama, Yuji; Soeda, Shuhei; Ikeuchi, Masayoshi; Kakae, Keiko; Yamaguchi, Naoto

2017-04-12

v-Src, an oncogene found in Rous sarcoma virus, is a constitutively active variant of c-Src. Activation of Src is observed frequently in colorectal and breast cancers, and is critical in tumor progression through multiple processes. However, in some experimental conditions, v-Src causes growth suppression and apoptosis. In this review, we highlight recent progress in our understanding of cytokinesis failure and the attenuation of the tetraploidy checkpoint in v-Src-expressing cells. v-Src induces cell cycle changes-such as the accumulation of the 4N cell population-and increases the number of binucleated cells, which is accompanied by an excess number of centrosomes. Time-lapse analysis of v-Src-expressing cells showed that cytokinesis failure is caused by cleavage furrow regression. Microscopic analysis revealed that v-Src induces delocalization of cytokinesis regulators including Aurora B and Mklp1. Tetraploid cell formation is one of the causes of chromosome instability; however, tetraploid cells can be eliminated at the tetraploidy checkpoint. Interestingly, v-Src weakens the tetraploidy checkpoint by inhibiting the nuclear exclusion of the transcription coactivator YAP, which is downstream of the Hippo pathway and its nuclear exclusion is critical in the tetraploidy checkpoint. We also discuss the relationship between v-Src-induced chromosome instability and growth suppression in v-Src-induced oncogenesis.

Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells

PubMed Central

Zhou, Yunying; Zhang, Qishu; Gao, Ge; Zhang, Xiaoli; Liu, Yafei; Yuan, Shoudao

2016-01-01

ABSTRACT The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. PMID:27099318

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

PubMed

Chou, Chao-Kai; Huang, Po-Jung; Tsou, Pei-Hsiang; Wei, Yongkun; Lee, Heng-Huan; Wang, Ying-Nai; Liu, Yen-Liang; Shi, Colin; Yeh, Hsin-Chih; Kameoka, Jun; Hung, Mien-Chie

2018-05-29

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. Copyright © 2018 Elsevier B.V. All rights reserved.

Induction of the Tumor-Suppressor p16INK4a within Regenerative Epithelial Crypts in Ulcerative Colitis1

PubMed Central

Furth, Emma E; Gustafson, Karen S; Dai, Charlotte Y; Gibson, Steven L; Menard-Katcher, Paul; Chen, Tina; Koh, Jim; Enders, Greg H

2006-01-01

Abstract p16INK4a is a major tumor-suppressor protein, but its regulation and settings of fuction remain poorly understood. To explore the notion that p16 is induced in vivo in response to replicative stress, we examined p16 expression in tissues from human ulcerative colitis (UC; n = 25) and normal controls (n = 20). p16 was expressed strongly in UC-associated neoplasms (n = 17), as seen previously in sporadic colonic neoplasms. In non-neoplastic UC epithelium, p16 was expressed in 33% of crypts (the proliferative compartment) compared to < 1% of normal controls. p16 expression did not correlate with degree of inflammation but did correlate with the degree of crypt architecture distortion (P = .002)—a reflection of epithelial regeneration. In coimmunofluorescence studies with Ki67, p16 expression was associated with cell cycle arrest (P < .001). Both UC and normal crypts displayed evidence for the activation of the DNA damage checkpoint pathway, and p16 was induced in primary cultures of normal epithelial cells by ionizing irradiation (IR). However, induction by IR displayed delayed kinetics, implying that p16 is not an immediate target of the checkpoint pathway. These findings support a model in which p16 is induced as an “emergency brake” in cells experiencing sustained replicative stress. PMID:16820088

Ionizing radiation and cell cycle progression in ataxia telangiectasia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beamish, H.; Khanna, K.K.; Lavin, M.F.

1994-04-01

Exposure of mammalian cells to ionizing radiation causes delay in normal progress through the cell cycle at a number of different checkpoints. Abnormalities in these checkpoints have been described for ataxia telangiectasia cells after irradiation. In this report we show that these abnormalities occur at different phases in the cell cycle in several ataxia telangiectasia lymphoblastoid cells. Ataxia telangiectasia cells, synchronized in late G{sub 1} phase with either mimosine or aphidicolin and exposed to radiation, showed a reduced delay in entering S phase compared to irradiated control cells. Failure to exhibit G{sub 1}-phase delay in ataxia telangiectasia cells is accompaniedmore » by a reduced ability of radiation to activate the product of the tumor suppressor gene p53, a protein involved in G{sub 1}/S-phase delay. When the progress of irradiated G{sub 1}-phase cells was followed into the subsequent G{sub 2} and G{sub 1} phases ataxia telangiectasia cells showed a more pronounced accumulation in G{sub 2} phase than control cells. When cells were irradiated in S phase and extent of delay was more evident in G{sub 2} phase and ataxia telangiectasia cells were delayed to a greater extent. These results suggest that the lack of initial delay in both G{sub 1} and S phases to the radiosensitivity observed in this syndrome. 26 refs., 3 figs., 2 tabs.« less

A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors

PubMed Central

Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G.; Nimmagadda, Sridhar

2016-01-01

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings. PMID:26848870

Multiple treatment comparison of seven new drugs for patients with advanced malignant melanoma: a systematic review and health economic decision model in a Norwegian setting.

PubMed

Pike, Eva; Hamidi, Vida; Saeterdal, Ingvil; Odgaard-Jensen, Jan; Klemp, Marianne

2017-08-21

To assess the relative effectiveness and cost-effectiveness of seven new drugs (cobimetinib, dabrafenib, ipilimumab, nivolumab, pembrolizumab, trametinib and vemurafenib) used for treatment of patients with advanced malignant melanoma in the Norwegian setting. A multiple technology assessment. Patients with advanced malignant melanoma aged 18 or older. A systematic search for randomised controlled trials in relevant bibliographic databases. We performed network meta-analyses using both direct and indirect evidence with dacarbazine as a common comparator. We ranked the different treatments in terms of their likelihood of leading to the best results for each endpoint. The cost-utility analysis was based on a probabilistic discrete-time Markov cohort model. The model calculated the costs and quality-adjusted life years (QALYs) with different treatment strategies from a healthcare perspective. Sensitivity analysis was performed by means of Monte Carlo simulation. Monotherapies with a programmed cell death 1 (PD-1) immune-checkpoint-inhibitor had a higher probability of good performance for overall survival than monotherapies with ipilimumab or BRAF/MEK inhibitors. The combination treatments had all similar levels of effectiveness to the PD-1 immune-checkpoint-inhibitors.PD-1 immune-checkpoint-inhibitors are more effective and more costly compared with ipilimumab in monotherapy. Nivolumab in combination with ipilimumab had higher costs and the same level of effectiveness as the PD-1 immune-checkpoint-inhibitors in monotherapy.BRAF/MEK inhibitor combinations (dabrafenib and trametinib or vemurafenib and cobimetinib) had both similar effectiveness and cost-effectiveness; however, the combination therapies are more likely to give higher quality adjusted life year gains than BRAF or MEK inhibitor monotherapies, but to a higher cost. None of the drugs investigated can be considered cost-effective at what has normally been considered a reasonable willingness-to-pay (WTP) in Norway. Price reductions (from the official list prices) in the region of 63%-84% would be necessary for these drugs to be cost-effective at a WTP of €55 850 per QALY. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

T-cell-based Immunotherapy: Adoptive Cell Transfer and Checkpoint Inhibition.

PubMed

Houot, Roch; Schultz, Liora Michal; Marabelle, Aurélien; Kohrt, Holbrook

2015-10-01

Tumor immunotherapy has had demonstrable efficacy in patients with cancer. The most promising results have been with T-cell-based therapies. These include adoptive cell transfer of tumor-infiltrating lymphocytes, genetically engineered T cells, and immune checkpoint inhibitor antibodies. In this review, we describe the different T-cell-based strategies currently in clinical trials and put their applications, present and future, into perspective. ©2015 American Association for Cancer Research.

Mammalian Homologs of Yeast Checkpoint Genes

DTIC Science & Technology

2001-07-01

previous cycle we developed systems and reagents for expression and analysis of all of the pertinent proteins, and are made headway on association of Chk2...function, with emphasis on p53 regulation, cell cycle regulation, and complementation of ATM defects. Saccharomyces Schizosaceharomy Homo sapiens...RAD53, two essential genes, play a central role in DNA damage checkpoints at all cell cycle stages. Our lab showed that Rad9 is a regulator coupling DNA

Covering Resilience: A Recent Development for Binomial Checkpointing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walther, Andrea; Narayanan, Sri Hari Krishna

In terms of computing time, adjoint methods offer a very attractive alternative to compute gradient information, required, e.g., for optimization purposes. However, together with this very favorable temporal complexity result comes a memory requirement that is in essence proportional with the operation count of the underlying function, e.g., if algorithmic differentiation is used to provide the adjoints. For this reason, checkpointing approaches in many variants have become popular. This paper analyzes an extension of the so-called binomial approach to cover also possible failures of the computing systems. Such a measure of precaution is of special interest for massive parallel simulationsmore » and adjoint calculations where the mean time between failure of the large scale computing system is smaller than the time needed to complete the calculation of the adjoint information. We describe the extensions of standard checkpointing approaches required for such resilience, provide a corresponding implementation and discuss first numerical results.« less

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

PubMed

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.

Immunostimulatory monoclonal antibodies for hepatocellular carcinoma therapy. Trends and perspectives.

PubMed

Mazzolini, Guillermo D; Malvicini, Mariana

2018-01-01

Hepatocellular carcinoma (HCC) is the second cause of cancer-related death in the world and is the main cause of death in cirrhotic patients. Unfortunately, the incidence of HCC has grown significantly in the last decade. Curative treatments such as surgery, liver transplantation or percutaneous ablation can only be applied in less than 30% of cases. The multikinase inhibitor sorafenib is the first line therapy for advanced HCC. Regorafenib is the standard of care for second-line patients. However, novel and more specific potent therapeutic approaches for advanced HCC are still needed. The liver constitutes a unique immunological microenvironment, although anti-tumor immunity seems to be feasible with the use of checkpoint inhibitors such as nivolumab. Efficacy may be further increased by combining checkpoint inhibitors or by applying loco-regional treatments. The success of immune checkpoint blockade has renewed interest in immunotherapy in HCC.

A case of pembrolizumab-induced type-1 diabetes mellitus and discussion of immune checkpoint inhibitor-induced type 1 diabetes.

PubMed

Chae, Young Kwang; Chiec, Lauren; Mohindra, Nisha; Gentzler, Ryan; Patel, Jyoti; Giles, Francis

2017-01-01

Immune checkpoint inhibitors such as pembrolizumab, ipilimumab, and nivolumab, now FDA-approved for use in treating several types of cancer, have been associated with immune-related adverse effects. Specifically, the antibodies targeting the programmed-cell death-1 immune checkpoint, pembrolizumab and nivolumab, have been rarely reported to induce the development of type 1 diabetes mellitus. Here we describe a case of a patient who developed antibody-positive type 1 diabetes mellitus following treatment with pembrolizumab in combination with systemic chemotherapy for metastatic adenocarcinoma of the lung. We will also provide a brief literature review of other rarely reported cases of type 1 diabetes presenting after treatment with pembrolizumab and nivolumab, as well as discussion regarding potential mechanisms of this adverse effect and its importance as these drugs continue to become even more widespread.

Granulomatous and lichenoid dermatitis after IgG4 anti-PD-1 monoclonal antibody therapy for advanced cancer.

PubMed

Diaz-Perez, Julio A; Beveridge, Mara G; Victor, Thomas A; Cibull, Thomas L

2018-06-01

Nivolumab is a fully human IgG4 monoclonal antibody directed against programmed cell death protein 1 (PD-1). PD-1 inhibition allows T-cell activation and recruitment to destroy cancer cells. Checkpoint inhibitors have shown significant survival advantage and relatively low side-effects in comparison with conventional chemotherapy in several types of advanced cancer. Granulomatous cutaneous reactions have been reported showing sarcoidal and panniculitic morphology. Here we present a case of drug-induced lichenoid and granulomatous dermatitis after checkpoint inhibitor therapy observed in a 63-year-old male treated with nivolumab for advanced glioblastoma. This morphology has not been previously reported. We documented a high number of CD8+ T-cells within the lesions. Additionally, we review the side-effects observed with the use of checkpoint inhibitors, with special focus on cutaneous manifestations. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Checkpoint-based forward recovery using lookahead execution and rollback validation in parallel and distributed systems. Ph.D. Thesis, 1992

NASA Technical Reports Server (NTRS)

Long, Junsheng

1994-01-01

This thesis studies a forward recovery strategy using checkpointing and optimistic execution in parallel and distributed systems. The approach uses replicated tasks executing on different processors for forwared recovery and checkpoint comparison for error detection. To reduce overall redundancy, this approach employs a lower static redundancy in the common error-free situation to detect error than the standard N Module Redundancy scheme (NMR) does to mask off errors. For the rare occurrence of an error, this approach uses some extra redundancy for recovery. To reduce the run-time recovery overhead, look-ahead processes are used to advance computation speculatively and a rollback process is used to produce a diagnosis for correct look-ahead processes without rollback of the whole system. Both analytical and experimental evaluation have shown that this strategy can provide a nearly error-free execution time even under faults with a lower average redundancy than NMR.

Endocrine toxicity of immune checkpoint inhibitors: essential crosstalk between endocrinologists and oncologists.

PubMed

Illouz, Frédéric; Briet, Claire; Cloix, Lucie; Le Corre, Yannick; Baize, Nathalie; Urban, Thierry; Martin, Ludovic; Rodien, Patrice

2017-08-01

Two types of immune checkpoint inhibitors, both antibodies that target cytotoxic T-lymphocyte antigen-4 and those that target programmed cell death-protein 1, have been approved for use in melanoma, non-small-cell lung cancer, and renal cell carcinoma as first-line or second-line therapy. Their adverse events are primarily regarded as immune-related adverse events. We felt it was important to pinpoint and discuss certain preconceptions or misconceptions regarding thyroid dysfunction, hypophysitis, and diabetes induced by immune checkpoint inhibitors. We have identified areas of uncertainty and unmet requirements, including essential interaction between endocrinologists and oncologists. Five issues have been identified for discussion: (1) diagnosis of endocrine toxicity, (2) assessment of toxicity severity, (3) treatment of toxicity, (4) withdrawal or continuation of immunotherapy, (5) preventive action. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

ATM-Mediated Transcriptional and Developmental Responses to γ-rays in Arabidopsis

PubMed Central

Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

2007-01-01

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of γ-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed. PMID:17487278

ATM-mediated transcriptional and developmental responses to gamma-rays in Arabidopsis.

PubMed

Ricaud, Lilian; Proux, Caroline; Renou, Jean-Pierre; Pichon, Olivier; Fochesato, Sylvain; Ortet, Philippe; Montané, Marie-Hélène

2007-05-09

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.

BMAL1 and CLOCK proteins in regulating UVB-induced apoptosis and DNA damage responses in human keratinocytes.

PubMed

Sun, Yang; Wang, Peiling; Li, Hongyu; Dai, Jun

2018-06-26

A diverse array of biological processes are under circadian controls. In mouse skin, ultraviolet ray (UVR)-induced apoptosis and DNA damage responses are time-of-day dependent, which are controlled by core clock proteins. This study investigates the roles of clock proteins in regulating UVB responses in human keratinocytes (HKCs). We found that the messenger RNA expression of brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) genes is altered by low doses (5 mJ/cm 2 ) of UVB in the immortalized HaCat HKCs cell line. Although depletion of BMAL1 or CLOCK has no effect on the activation of Rad3-related protein kinases-checkpoint kinase 1-p53 mediated DNA damage checkpoints, it leads to suppression of UVB-stimulated apoptotic responses, and downregulation of UVB-elevated expression of DNA damage marker γ-H2AX and cell cycle inhibitor p21. Diminished apoptotic responses are also observed in primary HKCs depleted of BMAL1 or CLOCK after UVB irradiation. While CLOCK depletion shows a suppressive effect on UVB-induced p53 protein accumulation, depletion of either clock gene triggers early keratinocyte differentiation of HKCs at their steady state. These results suggest that UVB-induced apoptosis and DNA damage responses are controlled by clock proteins, but via different mechanisms in the immortalized human adult low calcium temperature and primary HKCs. Given the implication of UVB in photoaging and photocarcinogenesis, mechanistic elucidation of circadian controls on UVB effects in human skin will be critical and beneficial for prevention and treatment of skin cancers and other skin-related diseases. © 2018 Wiley Periodicals, Inc.

EMODnet MedSea Checkpoint for sustainable Blue Growth

NASA Astrophysics Data System (ADS)

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

2016-04-01

The EMODNET checkpoint is a wide monitoring system assessment activity aiming to support the sustainable Blue Growth at the scale of the European Sea Basins by: 1) Clarifying the observation landscape of all compartments of the marine environment including Air, Water, Seabed, Biota and Human activities, pointing out to the existing programs, national, European and international 2) Evaluating fitness for use indicators that will show the accessibility and usability of observation and modeling data sets and their roles and synergies based upon selected applications by the European Marine Environment Strategy 3) Prioritizing the needs to optimize the overall monitoring Infrastructure (in situ and satellite data collection and assembling, data management and networking, modeling and forecasting, geo-infrastructure) and release recommendations for evolutions to better meet the application requirements in view of sustainable Blue Growth The assessment is designed for : - Institutional stakeholders for decision making on observation and monitoring systems - Data providers and producers to know how their data collected once for a given purpose could fit other user needs - End-users interested in a regional status and possible uses of existing monitoring data Selected end-user applications are of paramount importance for: (i) the blue economy sector (offshore industries, fisheries); (ii) marine environment variability and change (eutrophication, river inputs and ocean climate change impacts); (iii) emergency management (oil spills); and (iv) preservation of natural resources and biodiversity (Marine Protected Areas). End-user applications generate innovative products based on the existing observation landscape. The fitness for use assessment is made thanks to the comparison of the expected product specifications with the quality of the product derived from the selected data. This involves the development of checkpoint information and indicators based on Data quality and Metadata standards for geographic information (ISO 19157 and ISO 19115 respectively). The fitness for use of the input datasets are assessed using 2 categories of criteria to determine how these datasets fits the user requirements which drive them to select a data source rather than another one and to show performance and gaps of the present monitoring systems : • Data appropriateness : what is made available to the user ?. • Data availability : how it is made available to the user? All information are stored in a GIS platform and made available with two types of interfaces: - Front-end interfaces with users, to present the input data used by all challenges, the innovative products generated by challenges and the assessment indicators. - Back-end interfaces to partners, to store the checkpoint descriptors of input data, specification to generate targeted products, catalogue information of products with associated checkpoint indicators linked to the input data The validation of the records is done at three levels, at technical level (GIS), at challenge level (use), and at sea basin level (synthesis of monitoring data adequacy including expert comments) to end with the production of a yearly Data Adequacy Report.

Atomic structure of the APC/C and its mechanism of protein ubiquitination

PubMed Central

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe

PubMed Central

Duhig, Trevor; Nam, Miyoung; Palmer, Georgia; Han, Sangjo; Jeffery, Linda; Baek, Seung-Tae; Lee, Hyemi; Shim, Young Sam; Lee, Minho; Kim, Lila; Heo, Kyung-Sun; Noh, Eun Joo; Lee, Ah-Reum; Jang, Young-Joo; Chung, Kyung-Sook; Choi, Shin-Jung; Park, Jo-Young; Park, Youngwoo; Kim, Hwan Mook; Park, Song-Kyu; Park, Hae-Joon; Kang, Eun-Jung; Kim, Hyong Bai; Kang, Hyun-Sam; Park, Hee-Moon; Kim, Kyunghoon; Song, Kiwon; Song, Kyung Bin; Nurse, Paul; Hoe, Kwang-Lae

2014-01-01

SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth. PMID:20473289

Development of new immunotherapy treatments in different cancer types.

PubMed

Stanculeanu, D L; Daniela, Zob; Lazescu, A; Bunghez, R; Anghel, R

2016-01-01

Cancer immunotherapy involves the use of therapeutic modalities that determine a manipulation of the immune system by using immune agents such as cytokines, vaccines, cell therapies and humoral, transfection agents. Immunotherapy of cancer has to stimulate the host's anti-tumor response by increasing the effector cell number and the production of soluble mediators and decrease the host's suppressor mechanisms by inducing tumor killing environment and by modulating immune checkpoints. Immunotherapy seems to work better in more immunogenic tumors. Making a review of literature, the article presents the new immunologic treatments in cancers less presented in the latest conferences, cancers in which, immunotherapy is still under investigation. Bladder cancer was the first indication for which immunotherapy was used in 1970. A promising clinical research in bladder cancer is the use of immune checkpoint inhibitors. Although breast cancer is considered immunologically silent, several preclinical and clinical studies suggested that immunotherapy has the potential to improve the clinical outcomes for patients with breast cancer. Cervical cancer, brain cancer, head and neck cancer and colorectal and esophageal cancers are cancer types for which new immune-based cancer treatments are currently under development. Recent agents used in clinical trials will be described in before mentioned cancers.

Association between alcohol-impaired driving enforcement-related strategies and alcohol-impaired driving.

PubMed

Sanem, Julia R; Erickson, Darin J; Rutledge, Patricia C; Lenk, Kathleen M; Nelson, Toben F; Jones-Webb, Rhonda; Toomey, Traci L

2015-05-01

All states in the U.S. prohibit alcohol-impaired driving but active law enforcement is necessary for effectively reducing this behavior. Sobriety checkpoints, saturation patrols, open container laws, and media campaigns related to enforcement efforts are all enforcement-related strategies for reducing alcohol-impaired driving. We conducted surveys of all state patrol agencies and a representative sample of local law enforcement agencies to assess their use of alcohol-impaired driving enforcement-related strategies and to determine the relationship between these enforcement-related strategies and self-reported alcohol-impaired driving behavior obtained from the Behavioral Risk Factor Surveillance System. We found that sobriety checkpoints, saturation patrols, and enforcement of open container laws were associated with a lower prevalence of alcohol-impaired driving but, more importantly, a combination of enforcement-related strategies was associated with a greater decrease in alcohol-impaired driving than any individual enforcement-related activity. In addition, alcohol-impaired driving enforcement-related strategies were associated with decreased alcohol-impaired driving above and beyond their association with decreased binge drinking. Results suggest law enforcement agencies should give greater priority to using a combination of strategies rather than relying on any one individual enforcement activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Complete intracranial response to talimogene laherparepvec (T-Vec), pembrolizumab and whole brain radiotherapy in a patient with melanoma brain metastases refractory to dual checkpoint-inhibition.

PubMed

Blake, Zoë; Marks, Douglas K; Gartrell, Robyn D; Hart, Thomas; Horton, Patti; Cheng, Simon K; Taback, Bret; Horst, Basil A; Saenger, Yvonne M

2018-04-06

Immunotherapy, in particular checkpoint blockade, has changed the clinical landscape of metastatic melanoma. Nonetheless, the majority of patients will either be primary refractory or progress over follow up. Management of patients progressing on first-line immunotherapy remains challenging. Expanded treatment options with combination immunotherapy has demonstrated efficacy in patients previously unresponsive to single agent or alternative combination therapy. We describe the case of a patient with diffusely metastatic melanoma, including brain metastases, who, despite being treated with stereotactic radiosurgery and dual CTLA-4/PD-1 blockade (ipilimumab/nivolumab), developed systemic disease progression and innumerable brain metastases. This patient achieved a complete CNS response and partial systemic response with standard whole brain radiation therapy (WBRT) combined with Talimogene laherparepvec (T-Vec) and pembrolizumab. Patients who do not respond to one immunotherapy combination may respond during treatment with an alternate combination, even in the presence of multiple brain metastases. Biomarkers are needed to assist clinicians in evidence based clinical decision making after progression on first line immunotherapy to determine whether response can be achieved with second line immunotherapy.

Development of new immunotherapy treatments in different cancer types

PubMed Central

Stanculeanu, DL; Daniela, Zob; Lazescu, A; Bunghez, R; Anghel, R

2016-01-01

Cancer immunotherapy involves the use of therapeutic modalities that determine a manipulation of the immune system by using immune agents such as cytokines, vaccines, cell therapies and humoral, transfection agents. Immunotherapy of cancer has to stimulate the host’s anti-tumor response by increasing the effector cell number and the production of soluble mediators and decrease the host’s suppressor mechanisms by inducing tumor killing environment and by modulating immune checkpoints. Immunotherapy seems to work better in more immunogenic tumors. Making a review of literature, the article presents the new immunologic treatments in cancers less presented in the latest conferences, cancers in which, immunotherapy is still under investigation. Bladder cancer was the first indication for which immunotherapy was used in 1970. A promising clinical research in bladder cancer is the use of immune checkpoint inhibitors. Although breast cancer is considered immunologically silent, several preclinical and clinical studies suggested that immunotherapy has the potential to improve the clinical outcomes for patients with breast cancer. Cervical cancer, brain cancer, head and neck cancer and colorectal and esophageal cancers are cancer types for which new immune-based cancer treatments are currently under development. Recent agents used in clinical trials will be described in before mentioned cancers. PMID:27974927

The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity.

PubMed

Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

2016-03-01

The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes-UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Calibration and Validation of the Checkpoint Model to the Air Force Electronic Systems Center Software Database

DTIC Science & Technology

1997-09-01

Illinois Institute of Technology Research Institute (IITRI) calibrated seven parametric models including SPQR /20, the forerunner of CHECKPOINT. The...a semicolon); thus, SPQR /20 was calibrated using SLOC sizing data (IITRI, 1989: 3-4). The results showed only slight overall improvements in accuracy...even when validating the calibrated models with the same data sets. The IITRI study demonstrated SPQR /20 to be one of two models that were most

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation.

PubMed

Minemoto, Yuzuru; Uchida, Sanae; Ohtsubo, Motoaki; Shimura, Mari; Sasagawa, Toshiyuki; Hirata, Masato; Nakagama, Hitoshi; Ishizaka, Yukihito; Yamashita, Katsumi

2003-04-01

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

PubMed Central

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

2013-01-01

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. PMID:23564203

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

PubMed

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.

Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases*

PubMed Central

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L.; Landry, Christian R.; Bolanos-Garcia, Victor M.; Elowe, Sabine

2012-01-01

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region. PMID:22187426

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

PubMed

Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L; Landry, Christian R; Bolanos-Garcia, Victor M; Elowe, Sabine

2012-02-17

Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR.

PubMed Central

Laurençon, Anne; Purdy, Amanda; Sekelsky, Jeff; Hawley, R Scott; Su, Tin Tin

2003-01-01

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule. PMID:12807779

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation.

PubMed

Ikegami, Yosuke; Goto, Hidemasa; Kiyono, Tohru; Enomoto, Masato; Kasahara, Kousuke; Tomono, Yasuko; Tozawa, Keiichi; Morita, Akimichi; Kohri, Kenjiro; Inagaki, Masaki

2008-12-26

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

Checkpoint kinase 1-induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis.

PubMed

Li, Zhe; Li, Xueyan; Nai, Shanshan; Geng, Qizhi; Liao, Ji; Xu, Xingzhi; Li, Jing

2017-12-01

Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O- linked β- N -acetylglucosamine ( O -GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an si OGT- based approach. Lastly, expression of plasmids resistant to si OGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stripling, H. F.; Adams, M. L.; Hawkins, W. D.

2013-07-01

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

Towards reversible basic linear algebra subprograms: A performance study

DOE PAGES

Perumalla, Kalyan S.; Yoginath, Srikanth B.

2014-12-06

Problems such as fault tolerance and scalable synchronization can be efficiently solved using reversibility of applications. Making applications reversible by relying on computation rather than on memory is ideal for large scale parallel computing, especially for the next generation of supercomputers in which memory is expensive in terms of latency, energy, and price. In this direction, a case study is presented here in reversing a computational core, namely, Basic Linear Algebra Subprograms, which is widely used in scientific applications. A new Reversible BLAS (RBLAS) library interface has been designed, and a prototype has been implemented with two modes: (1) amore » memory-mode in which reversibility is obtained by checkpointing to memory in forward and restoring from memory in reverse, and (2) a computational-mode in which nothing is saved in the forward, but restoration is done entirely via inverse computation in reverse. The article is focused on detailed performance benchmarking to evaluate the runtime dynamics and performance effects, comparing reversible computation with checkpointing on both traditional CPU platforms and recent GPU accelerator platforms. For BLAS Level-1 subprograms, data indicates over an order of magnitude better speed of reversible computation compared to checkpointing. For BLAS Level-2 and Level-3, a more complex tradeoff is observed between reversible computation and checkpointing, depending on computational and memory complexities of the subprograms.« less

PARP Inhibitors Synergize With Loss of Checkpoint Control to Kill Mammary Carcinoma Cells

DTIC Science & Technology

2011-06-01

from three studies S.E.M. B, MCF7 breast cancer and PANC -1 and MiaPaca2 pancreatic cancer cells were plated in triplicate and treated with vehicle...inhibitors to kill pancreatic carcinoma cells PANC -1 (pancreatic) and MiaPaca2 (pancreatic) carcinoma cells were plated as single cells (250–2000 cells...231 and PANC -1. Simian virus 40 large T antigen-transformed fibroblasts that are not tu- morigenic in mice were also sensitive to the drug schedule