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Sample records for cell-cycle checkpoint kinase

  1. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2015-01-01

    Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2ACdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle. PMID:25713391

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

    PubMed

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw; Syljuåsen, Randi G; Lundin, Cecilia; Bartek, Jiri; Helleday, Thomas

    2005-02-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

  4. Cell Cycle Regulation by Checkpoints

    PubMed Central

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

    2016-01-01

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

  5. Cell cycle regulation by checkpoints.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2014-01-01

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

  6. Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

    PubMed Central

    Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

    2013-01-01

    Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL (Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit. PMID:21840391

  7. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

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

    2007-04-01

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

  8. 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

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

    PubMed

    Nakanishi, Makoto; Shimada, Midori; Niida, Hiroyuki

    2006-10-01

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

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

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

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

  11. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.

    PubMed

    Sanchez, Y; Desany, B A; Jones, W J; Liu, Q; Wang, B; Elledge, S J

    1996-01-19

    Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage.

  12. Viral manipulation of DNA repair and cell cycle checkpoints

    PubMed Central

    Chaurushiya, Mira S.; Weitzman, Matthew D.

    2009-01-01

    Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are exquisitely coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation. PMID:19473887

  13. Identification of a novel EGF-sensitive cell cycle checkpoint

    SciTech Connect

    Walker, Francesca . E-mail: francesca.walker@ludwig.edu.au; Zhang Huihua; Burgess, Antony W.

    2007-02-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.

  14. The Dynamical Mechanisms of the Cell Cycle Size Checkpoint

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

  15. 4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway*

    PubMed Central

    Chaudhary, Pankaj; Sharma, Rajendra; Sahu, Mukesh; Vishwanatha, Jamboor K.; Awasthi, Sanjay; Awasthi, Yogesh C.

    2013-01-01

    4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested. PMID:23733185

  16. A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy.

    PubMed

    Cannell, Ian G; Merrick, Karl A; Morandell, Sandra; Zhu, Chang-Qi; Braun, Christian J; Grant, Robert A; Cameron, Eleanor R; Tsao, Ming-Sound; Hemann, Michael T; Yaffe, Michael B

    2015-11-09

    In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

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

    PubMed

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

    2017-03-06

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2015-02-15

    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made. © 2015. Published by The Company of Biologists Ltd.

  20. [Involvement of cyclin-dependent kinase CDK1/CDC28 in regulation of cell cycle].

    PubMed

    Koltovaya, N A

    2013-07-01

    Cyclin-dependent kinases (CDKs) are a family of enzymes essential for the progression of the cells through the cell cycle in eukaryotes. Moreover, genetic stability-maintaining processes, such as checkpoint control and DNA repair, require the phosphorylation of a wide variety of target substrates by CDK. In budding yeast Saccharomyces cerevisiae, the key role in the cell cycle progression is played by CDK1/CDC28 kinase. This enzyme is the most thoroughly investigated. In this review the involvement of CDC28 kinase in regulation of the cell cycle is discussed in the light of newly obtained data.

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

    PubMed

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

    2003-09-18

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

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

    PubMed

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-06-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

  3. Decision for cell fate: deubiquitinating enzymes in cell cycle checkpoint.

    PubMed

    Lim, Key-Hwan; Song, Myoung-Hyun; Baek, Kwang-Hyun

    2016-04-01

    All organs consisting of single cells are consistently maintaining homeostasis in response to stimuli such as free oxygen, DNA damage, inflammation, and microorganisms. The cell cycle of all mammalian cells is regulated by protein expression in the right phase to respond to proliferation and apoptosis signals. Post-translational modifications (PTMs) of proteins by several protein-editing enzymes are associated with cell cycle regulation by their enzymatic functions. Ubiquitination, one of the PTMs, is also strongly related to cell cycle regulation by protein degradation or signal transduction. The importance of deubiquitinating enzymes (DUBs), which have a reversible function for ubiquitination, has recently suggested that the function of DUBs is also important for determining the fate of proteins during cell cycle processing. This article reviews and summarizes the diverse roles of DUBs, including DNA damage, cell cycle processing, and regulation of histone proteins, and also suggests the possibility for therapeutic targets.

  4. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer.

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

    PubMed

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

    2008-12-01

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

  6. 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.

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

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

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

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

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

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

  9. Cell cycle in the fucus zygote parallels a somatic cell cycle but displays a unique translational regulation of cyclin-dependent kinases.

    PubMed

    Corellou, F; Brownlee, C; Detivaud, L; Kloareg, B; Bouget, F Y

    2001-03-01

    In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos, however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression. The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs. Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.

  10. DNA Damage activates A Spatially Distinct Late Cytoplasmic Cell Cycle Checkpoint Network Controlled by MK2-mediated RNA Stabilization

    PubMed Central

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

    2010-01-01

    Summary Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1 pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G2/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of post-transcriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2, phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α mRNA degradation. Gadd45α functions within a positive feedback loop, sustaining the MK2-dependent cytoplasmic sequestration of Cdc25B/C to block mitotic entry in the presence of unrepaired DNA damage. Our findings demonstrate a critical role for the MK2 pathway in the post-transcriptional regulation of gene expression as part of the DNA damage response in cancer cells. PMID:20932473

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

    PubMed Central

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

    2006-01-01

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

  12. Tumor-suppressor Genes, Cell Cycle Regulatory Checkpoints, and the Skin

    PubMed Central

    Velez, Ana Maria Abreu; Howard, Michael S.

    2015-01-01

    The cell cycle (or cell-division cycle) is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs) that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs) or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH) terms “tumor suppressor's genes,” “skin,” and “cell cycle regulatory checkpoints.” We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses. PMID:26110128

  13. Alteration of cell cycle in cervical tumor associated with human papillomavirus: cyclin-dependent kinase inhibitors.

    PubMed

    Cho, Nam Hoon; Kim, Young Tae; Kim, Jae Wook

    2002-12-01

    The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.

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

    PubMed

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

    2010-09-20

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

  15. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

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

    2010-01-01

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

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

    SciTech Connect

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

    2012-10-12

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

  17. The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

    SciTech Connect

    Zhang Weifang; Li Jing; Kanginakudru, Sriramana; Zhao Weiming; Yu Xiuping; Chen, Jason J.

    2010-02-05

    HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

  18. Checkpoint kinase 2 is required for efficient immunoglobulin diversification.

    PubMed

    Davari, Kathrin; Frankenberger, Samantha; Schmidt, Angelika; Tomi, Nils-Sebastian; Jungnickel, Berit

    2014-01-01

    Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.

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

    PubMed

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

    2015-01-05

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

  20. Rapid construction of transgene-amplified CHO cell lines by cell cycle checkpoint engineering.

    PubMed

    Lee, Kyoung Ho; Onitsuka, Masayoshi; Honda, Kohsuke; Ohtake, Hisao; Omasa, Takeshi

    2013-07-01

    The process of establishing high-producing cell lines for the manufacture of therapeutic proteins is usually both time-consuming and laborious due to the low probability of obtaining high-producing clones from a pool of transfected cells and slow cell growth under the strong selective pressure of screening to identify high-producing clones. We present a novel method to rapidly generate more high-producing cells by accelerating transgene amplification. A small interfering RNA (siRNA) expression vector against ataxia telangiectasia and Rad3 related (ATR), a cell cycle checkpoint kinase, was transfected into Chinese hamster ovary (CHO) cells. The influences of ATR downregulation on gene amplification and the productivity were investigated in CHO cells producing green fluorescent protein (GFP) and secreting monoclonal antibody (mAb). The ATR-downregulated cells showed up to a 6-fold higher ratio of GFP-positive cells than that of the control cell pool. Moreover, the downregulated mAb-producing cells had about a 4-fold higher specific production rate and a 3-fold higher volumetric productivity as compared with the mock cells. ATR-downregulated cells showed a much faster increase in transgene copy numbers during the gene amplification process via methotrexate (MTX) treatment in both GFP- and mAb-producing cells. Our results suggest that a pool of high-producing cells can be more rapidly generated by ATR downregulation as compared with conventional gene amplification by MTX treatment. This novel method may be a promising approach to reduce time and labor in the process of cell line development.

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

    PubMed

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

    2012-10-12

    Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1(+), the overexpression of ago1(+) alleviated the cell cycle defect in dcr1Δ. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1(+) is dependent on ptr1(+). Nuclear accumulation of poly(A)(+) RNAs was detected in mutants of ago1(+) and ptr1(+), suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited.

    PubMed

    Uhlmann, Frank; Bouchoux, Céline; López-Avilés, Sandra

    2011-12-27

    The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed 'A quantitative model for cdc2 control of S phase and mitosis in fission yeast'. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications.

  3. A quantitative model for cyclin-dependent kinase control of the cell cycle: revisited

    PubMed Central

    Uhlmann, Frank; Bouchoux, Céline; López-Avilés, Sandra

    2011-01-01

    The eukaryotic cell division cycle encompasses an ordered series of events. Chromosomal DNA is replicated during S phase of the cell cycle before being distributed to daughter cells in mitosis. Both S phase and mitosis in turn consist of an intricately ordered sequence of molecular events. How cell cycle ordering is achieved, to promote healthy cell proliferation and avert insults on genomic integrity, has been a theme of Paul Nurse's research. To explain a key aspect of cell cycle ordering, sequential S phase and mitosis, Stern & Nurse proposed ‘A quantitative model for cdc2 control of S phase and mitosis in fission yeast’. In this model, S phase and mitosis are ordered by their dependence on increasing levels of cyclin-dependent kinase (Cdk) activity. Alternative mechanisms for ordering have been proposed that rely on checkpoint controls or on sequential waves of cyclins with distinct substrate specificities. Here, we review these ideas in the light of experimental evidence that has meanwhile accumulated. Quantitative Cdk control emerges as the basis for cell cycle ordering, fine-tuned by cyclin specificity and checkpoints. We propose a molecular explanation for quantitative Cdk control, based on thresholds imposed by Cdk-counteracting phosphatases, and discuss its implications. PMID:22084384

  4. Cell cycle execution point analysis of ORC function and characterization of the checkpoint response to ORC inactivation in Saccharomyces cerevisiae.

    PubMed

    Gibson, Daniel G; Bell, Stephen P; Aparicio, Oscar M

    2006-06-01

    Chromosomal replication initiates through the assembly of a prereplicative complex (pre-RC) at individual replication origins in the G1-phase, followed by activation of these complexes in the S-phase. In Saccharomyces cerevisiae, the origin recognition complex (ORC) binds replication origins throughout the cell cycle and participates in pre-RC assembly. Whether the ORC plays an additional role subsequent to pre-RC assembly in replication initiation or any other essential cell cycle process is not clear. To study the function of the ORC during defined cell cycle periods, we performed cell cycle execution point analyses with strains containing a conditional mutation in the ORC1, ORC2 or ORC5 subunit of ORC. We found that the ORC is essential for replication initiation, but is dispensable for replication elongation or later cell cycle events. Defective initiation in ORC mutant cells results in incomplete replication and mitotic arrest enforced by the DNA damage and spindle assembly checkpoint pathways. The involvement of the spindle assembly checkpoint implies a defect in kinetochore-spindle attachment or sister chromatid cohesion due to incomplete replication and/or DNA damage. Remarkably, under semipermissive conditions for ORC1 function, the spindle checkpoint alone suffices to block proliferation, suggesting this checkpoint is highly sensitive to replication initiation defects. We discuss the potential significance of these overlapping checkpoints and the impact of our findings on previously postulated role(s) of ORCs in other cell cycle functions.

  5. Gene expression signatures but not cell cycle checkpoint functions distinguish AT carriers from normal individuals

    PubMed Central

    Zhang, Liwen; Simpson, Dennis A.; Innes, Cynthia L.; Chou, Jeff; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

    2013-01-01

    Ataxia telangiectasia (AT) is a rare autosomal recessive disease caused by mutations in the ataxia telangiectasia-mutated gene (ATM). AT carriers with one mutant ATM allele are usually not severely affected although they carry an increased risk of developing cancer. There has not been an easy and reliable diagnostic method to identify AT carriers. Cell cycle checkpoint functions upon ionizing radiation (IR)-induced DNA damage and gene expression signatures were analyzed in the current study to test for differential responses in human lymphoblastoid cell lines with different ATM genotypes. While both dose- and time-dependent G1 and G2 checkpoint functions were highly attenuated in ATM−/− cell lines, these functions were preserved in ATM+/− cell lines equivalent to ATM+/+ cell lines. However, gene expression signatures at both baseline (consisting of 203 probes) and post-IR treatment (consisting of 126 probes) were able to distinguish ATM+/− cell lines from ATM+/+ and ATM−/− cell lines. Gene ontology (GO) and pathway analysis of the genes in the baseline signature indicate that ATM function-related categories, DNA metabolism, cell cycle, cell death control, and the p53 signaling pathway, were overrepresented. The same analyses of the genes in the IR-responsive signature revealed that biological categories including response to DNA damage stimulus, p53 signaling, and cell cycle pathways were overrepresented, which again confirmed involvement of ATM functions. The results indicate that AT carriers who have unaffected G1 and G2 checkpoint functions can be distinguished from normal individuals and AT patients by expression signatures of genes related to ATM functions. PMID:23943852

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2009-04-01

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

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

    PubMed

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

    2016-02-16

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

  9. Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases.

    PubMed Central

    Platz, A.; Sevigny, P.; Norberg, T.; Ring, P.; Lagerlöf, B.; Ringborg, U.

    1996-01-01

    A common characteristic of cancer cells is unrestrained cell division. This may be caused by mutational changes in genes coding for components of cell cycle-controlling networks. Alterations in genes involved in G1 checkpoint control have been registered in many human tumours, and investigations from several laboratories show that such alterations, taken together, are the most frequent changes detected in cancer cells. The present paper describes mutational analysis by polymerase chain reaction-single-strand conformation polymorphism (PCR/SSCP) and nucleotide sequence analysis of the genes coding for the p15, p53 and N-ras proteins in 26 metastases from 25 melanoma patients. The registered mutation frequencies add together with previously registered mutations in p16 in the same patient samples to a substantial total frequency of 44% of patients with mutation in at least one of the investigated genes. These results show the occurrence of heterogeneous defects among components of the cell cycle controlling machinery in a human melanoma tumour sample collection and demonstrate that the total frequency of detected alterations increases with the number of cell cycle controlling genes included in the screening panel. Images Figure 1 PMID:8826861

  10. Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint*

    PubMed Central

    Fukumoto, Yasunori; Morii, Mariko; Miura, Takahito; Kubota, Sho; Ishibashi, Kenichi; Honda, Takuya; Okamoto, Aya; Yamaguchi, Noritaka; Iwama, Atsushi; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination. PMID:24634213

  11. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  12. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  13. SNM1A Acts Downstream of ATM to Promote the G1 Cell Cycle Checkpoint

    PubMed Central

    Akhter, Shamima; Legerski, Randy J.

    2008-01-01

    We have shown previously that SNM1A co-localizes with 53BP1 at sites of double-strand breaks (DSBs) induced by IR, and that these proteins interact with or without DNA damage. However, the role of SNM1A in the DNA damage response has not been elucidated. Here, we show that SNM1A is required for an efficient G1 checkpoint arrest after IR exposure. Interestingly, the localization of SNM1A to sites of DSBs does not require either 53BP1 or H2AX, nor does the localization of 53BP1 require SNM1A. However, the localization of SNM1A does require ATM. Furthermore, SNM1A is shown to be a phosphorylation substrate of ATM in vitro, and to interact with ATM in vivo particularly after exposure of cells to IR. In addition, in the absence of SNM1A the activation of the downstream ATM target p53 is reduced. These findings suggest that SNM1A acts with ATM to promote the G1 cell cycle checkpoint. PMID:18848520

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

    PubMed

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

    2014-04-14

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

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

    PubMed Central

    Dewachter, Liselot; Verstraeten, Natalie; Fauvart, Maarten; Michiels, Jan

    2016-01-01

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

  16. Artemis regulates cell cycle recovery from the S phase checkpoint by promoting degradation of cyclin E.

    PubMed

    Wang, Haiyong; Zhang, Xiaoshan; Geng, Liyi; Teng, Lisong; Legerski, Randy J

    2009-07-03

    Artemis, a member of the SNM1 gene family, is a known phosphorylation target of ATM, ATR, and DNA-PKcs. We have previously identified two serine residues in Artemis (Ser(516) and Ser(645)) that are subject to phosphorylation by ATM and are involved in mediating recovery from the G(2)/M checkpoint in response to ionizing radiation. Here we show that these same sites are also phosphorylated by ATR in response to various types of replication stress including UVC, aphidicolin, and hydroxyurea. We also show that mutation of the Ser(516) and Ser(645) residues causes a prolonged S phase checkpoint recovery after treatment with UV or aphidicolin, and that this delayed recovery process coincides with a prolonged stabilization of cyclin E and down-regulation of Cdk2 kinase activity. Furthermore, we show that Artemis interacts with the F-box protein Fbw7, and that this interaction regulates cyclin E degradation through the SCF(Fbw7) E3 ubiquitin ligase complex. The interaction between Artemis and Fbw7 is regulated by phosphorylation of Ser(516) and Ser(645) sites that occur in response to replication stress. Thus, our findings suggest a novel pathway of recovery from the S phase checkpoint in that in response to replication stress phosphorylation of Artemis by ATR enhances its interaction with Fbw7, which in turn promotes ubiquitylation and the ultimate degradation of cyclin E.

  17. Creatine kinase in cell cycle regulation and cancer.

    PubMed

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK.

  18. Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests

    PubMed Central

    Stanford, Jennifer S.; Ruderman, Joan V.

    2005-01-01

    Entry into mitosis is catalyzed by cdc2 kinase. Previous work identified the cdc2-activating phosphatase cdc25C and the cdc2-inhibitory kinase wee1 as targets of the incomplete replication-induced kinase Chk1. Further work led to the model that checkpoint kinases block mitotic entry by inhibiting cdc25C through phosphorylation on Ser287 and activating wee1 through phosphorylation on Ser549. However, almost all conclusions underlying this idea were drawn from work using recombinant proteins. Here, we report that in the early Xenopus egg cell cycles, phosphorylation of endogenous cdc25C Ser287 is normally high during interphase and shows no obvious increase after checkpoint activation. By contrast, endogenous wee1 Ser549 phosphorylation is low during interphase and increases after activation of either the DNA damage or replication checkpoints; this is accompanied by a slight increase in wee1 kinase activity. Blocking mitotic entry by adding the catalytic subunit of PKA also results in increased wee1 Ser549 phosphorylation and maintenance of cdc25C Ser287 phosphorylation. These results argue that in response to checkpoint activation, endogenous wee1 is indeed a critical responder that functions by repressing the cdc2-cdc25C positive feedback loop. Surprisingly, endogenous wee1 Ser549 phosphorylation is highest during mitosis just after the peak of cdc2 activity. Treatments that block inactivation of cdc2 result in further increases in wee1 Ser549 phosphorylation, suggesting a previously unsuspected role for wee1 in mitosis. PMID:16195348

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

    PubMed

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

    2014-03-28

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

  20. Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

    PubMed Central

    Donnianni, Roberto Antonio; Ferrari, Matteo; Lazzaro, Federico; Clerici, Michela; Tamilselvan Nachimuthu, Benjamin; Plevani, Paolo; Muzi-Falconi, Marco; Pellicioli, Achille

    2010-01-01

    Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends. PMID:20098491

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

    PubMed Central

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

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  2. Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells.

    PubMed

    Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D; Yu, Jun; Zhu, Lihua Julie; Kaufman, Paul D

    2017-09-01

    The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells. Copyright © 2017 American Society for Microbiology.

  3. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints

    PubMed Central

    Zhao, Hui; Watkins, Janis L.; Piwnica-Worms, Helen

    2002-01-01

    Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G2 checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G2 checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks. PMID:12399544

  4. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

    PubMed Central

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. PMID:24955955

  5. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

    PubMed

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

  6. Modulation of cellular response to anticancer treatment by caffeine: inhibition of cell cycle checkpoints, DNA repair and more.

    PubMed

    Sabisz, Michal; Skladanowski, Andrzej

    2008-08-01

    Caffeine and other methylxanthines produce multiple physiologic effects throughout the human body, many of these effects could potentially modulate the activity of anticancer therapy. Caffeine may directly interfere with drug transport to tumor cells by formation of mixed stacking complexes with polyaromatic drugs. If formed in cells, these complexes may also prevent of intercalating drugs from DNA binding and, in this way, lower their antitumor activity. Since many of potent carcinogens are polyaromatic compounds, formation of stacking complexes with carcinogens could be associated with anti-genotoxic activity of caffeine and its use in cancer chemoprevention. Caffeine has also been reported to inhibit ATM and ATR kinases which leads to the disruption of multiple DNA damage-responsive cell cycle checkpoints and greatly sensitizes tumor cells to antitumor agents which induce genotoxic stress. Caffeine may inhibit repair of DNA lesions through a direct interference with DNA-PK activity and other repair enzymes. A number of in vitro and in vivo studies demonstrated that caffeine modulates both innate and adaptive immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase. Finally, another group of effects induced by caffeine is mediated through its inhibitory action on adenosine receptors. This may modulate the stability of HIF1 alpha as well as VEGF and interleukin-8 expression in tumor cells, which could have a direct impact on neovascularization of human tumors. In this review, we present different molecular mechanisms by which caffeine and other methylxanthines may directly or indirectly modulate the effect of antitumor treatment in tumor cells and in cancer patients.

  7. Kaempferol Activates G₂-Checkpoint of the Cell Cycle Resulting in G₂-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells.

    PubMed

    Kim, Ki Yun; Jang, Won Young; Lee, Ji Young; Jun, Do Youn; Ko, Jee Youn; Yun, Young Ho; Kim, Young Ho

    2016-02-01

    The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δpsim loss, and caspase cascade activation.

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

    PubMed Central

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  9. Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5'-nucleotidase I activity in MOLT-4 cells.

    PubMed

    Fyrberg, A; Mirzaee, S; Lotfi, K

    2006-01-01

    Activation of nucleoside analogues is dependent on kinases and 5'-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5'-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5'-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.

  10. Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

    PubMed Central

    Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

    2015-01-01

    Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment

  11. Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein.

    PubMed

    Singh, Rumani; Kalra, Rajkumar S; Hasan, Kamrul; Kaul, Zeenia; Cheung, Caroline T; Huschtscha, Lily; Reddel, Roger R; Kaul, Sunil C; Wadhwa, Renu

    2014-04-01

    CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae

    PubMed Central

    Bierle, Lindsey A.; Reich, Kira L.; Taylor, Braden E.; Blatt, Eliot B.; Middleton, Sydney M.; Burke, Shawnecca D.; Stultz, Laura K.; Hanson, Pamela K.; Partridge, Janet F.; Miller, Mary E.

    2015-01-01

    Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage–including that induced by many anticancer drugs–results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events. PMID:26375390

  13. DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

    PubMed

    Bierle, Lindsey A; Reich, Kira L; Taylor, Braden E; Blatt, Eliot B; Middleton, Sydney M; Burke, Shawnecca D; Stultz, Laura K; Hanson, Pamela K; Partridge, Janet F; Miller, Mary E

    2015-01-01

    Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

  14. Entry into Mitosis in Vertebrate Somatic Cells Is Guarded by a Chromosome Damage Checkpoint That Reverses the Cell Cycle When Triggered during Early but Not Late Prophase

    PubMed Central

    Rieder, Conly L.; Cole, Richard W.

    1998-01-01

    When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity. PMID:9722613

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

    PubMed

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

    2007-08-03

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

  16. 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

  17. Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

    PubMed

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Grollman, Arthur P; Rosenquist, Thomas

    2015-01-01

    Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

  18. Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

    PubMed Central

    Krause, Sue A.; Loupart, Marie-Louise; Vass, Sharron; Schoenfelder, Stefan; Harrison, Steve; Heck, Margarete M. S.

    2001-01-01

    Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes. PMID:11438670

  19. Loss of cell cycle checkpoint control in Drosophila Rfc4 mutants.

    PubMed

    Krause, S A; Loupart, M L; Vass, S; Schoenfelder, S; Harrison, S; Heck, M M

    2001-08-01

    Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.

  20. The mysterious human epidermal cell cycle, or an oncogene-induced differentiation checkpoint

    PubMed Central

    Gandarillas, Alberto

    2012-01-01

    Fifteen years ago, we reported that proto-oncogene MYC promoted differentiation of human epidermal stem cells, a finding that was surprising to the MYC and the skin research communities. MYC was one of the first human oncogenes identified, and it had been strongly associated with proliferation. However, it was later shown that MYC could induce apoptosis under low survival conditions. Currently, the notion that MYC promotes epidermal differentiation is widely accepted, but the cell cycle mechanisms that elicit this function remain unresolved. We have recently reported that keratinocytes respond to cell cycle deregulation and DNA damage by triggering terminal differentiation. This mechanism might constitute a homeostatic protection face to cell cycle insults. Here, I discuss recent and not-so-recent evidence suggesting the existence of a largely unexplored oncogene-induced differentiation response (OID) analogous to oncogene-induced apoptosis (OIA) or senescence (OIS). In addition, I propose a model for the role of the cell cycle in skin homeostasis maintenance and for the dual role of MYC in differentiation. PMID:23114621

  1. RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

    PubMed Central

    Davey, S; Beach, D

    1995-01-01

    We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis. Images PMID:8573795

  2. Targeting lung cancer through inhibition of checkpoint kinases

    PubMed Central

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

    2015-01-01

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

  3. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

  4. Scaffolding during the cell cycle by A-kinase anchoring proteins.

    PubMed

    Han, B; Poppinga, W J; Schmidt, M

    2015-12-01

    Cell division relies on coordinated regulation of the cell cycle. A process including a well-defined series of strictly regulated molecular mechanisms involving cyclin-dependent kinases, retinoblastoma protein, and polo-like kinases. Dysfunctions in cell cycle regulation are associated with disease such as cancer, diabetes, and neurodegeneration. Compartmentalization of cellular signaling is a common strategy used to ensure the accuracy and efficiency of cellular responses. Compartmentalization of intracellular signaling is maintained by scaffolding proteins, such as A-kinase anchoring proteins (AKAPs). AKAPs are characterized by their ability to anchor the regulatory subunits of protein kinase A (PKA), and thereby achieve guidance to different cellular locations via various targeting domains. Next to PKA, AKAPs also associate with several other signaling elements including receptors, ion channels, protein kinases, phosphatases, small GTPases, and phosphodiesterases. Taking the amount of possible AKAP signaling complexes and their diverse localization into account, it is rational to believe that such AKAP-based complexes regulate several critical cellular events of the cell cycle. In fact, several AKAPs are assigned as tumor suppressors due to their vital roles in cell cycle regulation. Here, we first briefly discuss the most important players of cell cycle progression. After that, we will review our recent knowledge of AKAPs linked to the regulation and progression of the cell cycle, with special focus on AKAP12, AKAP8, and Ezrin. At last, we will discuss this specific AKAP subset in relation to diseases with focus on a diverse subset of cancer.

  5. The Protein Kinase Cδ Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint*

    PubMed Central

    LaGory, Edward L.; Sitailo, Leonid A.; Denning, Mitchell F.

    2010-01-01

    Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G2/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G2/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. PMID:19917613

  6. Crystal Structure of Checkpoint Kinase 2 in Complex with Nsc 109555, a Potent and Selective Inhibitor

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Zhang, Di; Jobson, Andrew G.; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2009-03-05

    Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC{sub 50} = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

  7. The Yeast Cyclin-Dependent Kinase Routes Carbon Fluxes to Fuel Cell Cycle Progression.

    PubMed

    Ewald, Jennifer C; Kuehne, Andreas; Zamboni, Nicola; Skotheim, Jan M

    2016-05-19

    Cell division entails a sequence of processes whose specific demands for biosynthetic precursors and energy place dynamic requirements on metabolism. However, little is known about how metabolic fluxes are coordinated with the cell division cycle. Here, we examine budding yeast to show that more than half of all measured metabolites change significantly through the cell division cycle. Cell cycle-dependent changes in central carbon metabolism are controlled by the cyclin-dependent kinase (Cdk1), a major cell cycle regulator, and the metabolic regulator protein kinase A. At the G1/S transition, Cdk1 phosphorylates and activates the enzyme Nth1, which funnels the storage carbohydrate trehalose into central carbon metabolism. Trehalose utilization fuels anabolic processes required to reliably complete cell division. Thus, the cell cycle entrains carbon metabolism to fuel biosynthesis. Because the oscillation of Cdk activity is a conserved feature of the eukaryotic cell cycle, we anticipate its frequent use in dynamically regulating metabolism for efficient proliferation.

  8. The roles of nitric oxide synthase and eIF2alpha kinases in regulation of cell cycle upon UVB-irradiation.

    PubMed

    Wang, Lei; Liu, Yan; Wu, Shiyong

    2010-01-01

    In response to ultraviolet light (UV)-induced damage, cells initiate cellular recovery mechanisms including activation of repair genes and redistribution of cell cycle phases. While most studies have focused on DNA damage-inducible transcriptional regulation of cell cycle checkpoints, translational regulation also plays an important role in control of cell cycle progression upon UV-irradiation. UV-irradiation activates two kinases, PERK and GCN2, which phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and subsequently inhibit protein synthesis. We recently identified an upstream regulator, nitric oxide synthase (NOS), which controls the activation of both PERK and GCN2 upon UVB-irradiation. Our data suggested that UVB induces NOS activation and NO(.) production, which reacts with superoxide (O(2)(*-)) to form peroxynitrite (ONOO(-)) and activate PERK. The NO(*) production also leads to L-Arg depletion and GCN2 activation. The elevation of nitric oxide and activation of PERK/GCN2 have been shown to play roles in regulation of cell cycle upon UVB irradiation. In the present study, we show that the cell cycle phases were redistributed by inhibition of NOS activation or reduction of oxidative stress upon UVB irradiation, indicating the roles of NO(*) and its oxidative products in regulation of cell cycle. We also demonstrate that both PERK and GCN2 were involved in regulation of cell cycle upon UVB-irradiation, but the regulation is independent of eIF2alpha phosphorylation. While the mechanism for UVB-induced cell cycle control is yet to be unraveled, we here discuss the differential roles of NOS, PERK and GCN2 in regulation of cell cycle upon UVB-irradiation.

  9. MK3 Modulation Affects BMI1-Dependent and Independent Cell Cycle Check-Points

    PubMed Central

    Dahlmans, Vivian E. H.; Spaapen, Frank; Salvaing, Juliette; Vanhove, Jolien; Geijselaers, Claudia; Bartels, Stefanie J. J.; Partouns, Iris; Neumann, Dietbert; Speel, Ernst-Jan; Takihara, Yoshihiro; Wouters, Bradly G.; Voncken, Jan Willem

    2015-01-01

    Although the MK3 gene was originally found deleted in some cancers, it is highly expressed in others. The relevance of MK3 for oncogenesis is currently not clear. We recently reported that MK3 controls ERK activity via a negative feedback mechanism. This prompted us to investigate a potential role for MK3 in cell proliferation. We here show that overexpression of MK3 induces a proliferative arrest in normal diploid human fibroblasts, characterized by enhanced expression of replication stress- and senescence-associated markers. Surprisingly, MK3 depletion evokes similar senescence characteristics in the fibroblast model. We previously identified MK3 as a binding partner of Polycomb Repressive Complex 1 (PRC1) proteins. In the current study we show that MK3 overexpression results in reduced cellular EZH2 levels and concomitant loss of epigenetic H3K27me3-marking and PRC1/chromatin-occupation at the CDKN2A/INK4A locus. In agreement with this, the PRC1 oncoprotein BMI1, but not the PCR2 protein EZH2, bypasses MK3-induced senescence in fibroblasts and suppresses P16INK4A expression. In contrast, BMI1 does not rescue the MK3 loss-of-function phenotype, suggesting the involvement of multiple different checkpoints in gain and loss of MK3 function. Notably, MK3 ablation enhances proliferation in two different cancer cells. Finally, the fibroblast model was used to evaluate the effect of potential tumorigenic MK3 driver-mutations on cell proliferation and M/SAPK signaling imbalance. Taken together, our findings support a role for MK3 in control of proliferation and replicative life-span, in part through concerted action with BMI1, and suggest that the effect of MK3 modulation or mutation on M/SAPK signaling and, ultimately, proliferation, is cell context-dependent. PMID:25853770

  10. Elevated lung cancer risk is associated with deficiencies in cell cycle checkpoints: Genotype and phenotype analyses from a case-control study

    PubMed Central

    Zheng, Yun-Ling; Kosti, Ourania; Loffredo, Christopher; Bowman, Elise; Mechanic, Leah; Perlmutter, Donna; Jones, Raymond; Shields, Peter G.; Harris, Curtis

    2010-01-01

    Cell cycle checkpoints play critical roles in the maintenance of genomic integrity and inactivation of checkpoint genes, and are frequently perturbed in most cancers. In a case-control study of 299 non-small cell lung cancer cases and 550 controls in Maryland, we investigated the association between γ-radiation-induced G2/M arrest in cultured blood lymphocytes and lung cancer risk, and examined genotype-phenotype correlations between genetic polymorphisms of 20 genes involving in DNA repair and cell cycle control and γ-radiation-induced G2/M arrest. The study was specifically designed to examine race and gender differences in risk factors. Our data indicated that a less efficient DNA damage-induced G2/M checkpoint was associated with an increased risk of lung cancer in African American women with an adjusted odds ratio (OR) of 2.63 (95% CI = 1.01 – 7.26); there were no statistically significant associations for Caucasians, or African American men. When the African American women were categorized into quartiles, a significant reverse trend of decreased G2/M checkpoint function and increased lung cancer risk was present, with lowest-vs-highest quartile OR of 13.72 (95% CI = 2.30 – 81.92, Ptrend < 0.01). Genotype-phenotype correlation analysis indicated that polymorphisms in ATM, CDC25C, CDKN1A, BRCA2, ERCC6, TP53, and TP53BP1 genes were significantly associated with the γ-radiation-induced G2/M arrest phenotype. This study provides evidence that a less efficient G2/M checkpoint is significantly associated with lung cancer risk in African American women. The data also suggested that the function of G2/M checkpoint is modulated by genetic polymorphisms in genes involved in DNA repair and cell cycle control. PMID:19626602

  11. Elevated lung cancer risk is associated with deficiencies in cell cycle checkpoints: genotype and phenotype analyses from a case-control study.

    PubMed

    Zheng, Yun-Ling; Kosti, Ourania; Loffredo, Christopher A; Bowman, Elise; Mechanic, Leah; Perlmutter, Donna; Jones, Raymond; Shields, Peter G; Harris, Curtis C

    2010-05-01

    Cell cycle checkpoints play critical roles in the maintenance of genomic integrity and inactivation of checkpoint genes are frequently perturbed in most cancers. In a case-control study of 299 non-small cell lung cancer cases and 550 controls in Baltimore, we investigated the association between gamma-radiation-induced G(2)/M arrest in cultured blood lymphocytes and lung cancer risk, and examined genotype-phenotype correlations between genetic polymorphisms of 20 genes involving in DNA repair and cell cycle control and gamma-radiation-induced G(2)/M arrest. The study was specifically designed to examine race and gender differences in risk factors. Our data indicated that a less efficient DNA damage-induced G(2)/M checkpoint was associated with an increased risk of lung cancer in African American women with an adjusted odds ratio (OR) of 2.63 (95% CI = 1.01-7.26); there were no statistically significant associations for Caucasians, or African American men. When the African American women were categorized into quartiles, a significant reverse trend of decreased G(2)/M checkpoint function and increased lung cancer risk was present, with lowest-vs.-highest quartile OR of 13.72 (95% CI = 2.30-81.92, p(trend) < 0.01). Genotype-phenotype correlation analysis indicated that polymorphisms in ATM, CDC25C, CDKN1A, BRCA2, ERCC6, TP53, and TP53BP1 genes were significantly associated with the gamma-radiation-induced G(2)/M arrest phenotype. This study provides evidence that a less efficient G(2)/M checkpoint is significantly associated with lung cancer risk in African American women. The data also suggested that the function of G(2)/M checkpoint is modulated by genetic polymorphisms in genes involved in DNA repair and cell cycle control.

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

    PubMed Central

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

    2016-01-01

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

  13. Cell Cycle Checkpoint Proteins p21 and Hus1 Regulating Intercellular Signaling Induced By Alpha Particle Irradiation

    NASA Astrophysics Data System (ADS)

    Wu, Lijun; Zhao, Ye; Wang, Jun; Hang, Haiying

    In recent years, the attentions for radiation induced bystander effects (RIBE) have been paid on the intercellular signaling events connecting the irradiated and non-irradiated cells. p21 is a member of the Cip/Kip family and plays essential roles in cell cycle progression arrest after cellular irradiation. DNA damage checkpoint protein Hus1 is a member of the Rad9-Rad1-Hus1 complex and functions as scaffold at the damage sites to facilitate the activation of downstream effectors. Using the medium trasfer method and the cells of MEF, MEF (p21-/-), MEF (p21-/-Hus1-/-) as either medium donor or receptor cells, it was found that with 5cGy alpha particle irradiation, the bystander cells showed a significant induction of -H2AX for normal MEFs (p¡0.05). However, the absence of p21 resulted in deficiency in inducing bystander effects. Further results indicated p21 affected the intercellular DNA damage signaling mainly through disrupting the production or release of the damage signals from irradiated cells. When Hus1 and p21 were both knocked out, an obvious induction of -H2AX recurred in bystander cells and the induction of -H2AX was GJIC (gap junction-mediated intercellular communication) dependent, indicating the interrelationship between p21 and Hus1 regulated the production and relay of DNA damage signals from irradiated cells to non-irradiated bystander cells.

  14. Regulation of Cdc28 Cyclin-Dependent Protein Kinase Activity during the Cell Cycle of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Mendenhall, Michael D.; Hodge, Amy E.

    1998-01-01

    The cyclin-dependent protein kinase (CDK) encoded by CDC28 is the master regulator of cell division in the budding yeast Saccharomyces cerevisiae. By mechanisms that, for the most part, remain to be delineated, Cdc28 activity controls the timing of mitotic commitment, bud initiation, DNA replication, spindle formation, and chromosome separation. Environmental stimuli and progress through the cell cycle are monitored through checkpoint mechanisms that influence Cdc28 activity at key cell cycle stages. A vast body of information concerning how Cdc28 activity is timed and coordinated with various mitotic events has accrued. This article reviews that literature. Following an introduction to the properties of CDKs common to many eukaryotic species, the key influences on Cdc28 activity—cyclin-CKI binding and phosphorylation-dephosphorylation events—are examined. The processes controlling the abundance and activity of key Cdc28 regulators, especially transcriptional and proteolytic mechanisms, are then discussed in detail. Finally, the mechanisms by which environmental stimuli influence Cdc28 activity are summarized. PMID:9841670

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

    PubMed

    Bibi, Nousheen; Parveen, Zahida; Rashid, Sajid

    2013-01-01

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

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

    PubMed Central

    Bibi, Nousheen; Parveen, Zahida; Rashid, Sajid

    2013-01-01

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

  17. p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2

    PubMed Central

    Urist, Marshall; Tanaka, Tomoaki; Poyurovsky, Masha V.; Prives, Carol

    2004-01-01

    The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. We report here that in several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. We demonstrate as well that E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance. PMID:15601819

  18. Transient knock down of checkpoint kinase 1 in hematopoietic progenitors is linked to bone marrow toxicity.

    PubMed

    Hu, Wenyue; Zong, Qing; John-Baptiste, Annette; Jessen, Bart

    2011-07-28

    Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2014-03-01

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

  20. Physiological electric fields control the G1/S phase cell cycle checkpoint to inhibit endothelial cell proliferation.

    PubMed

    Wang, Entong; Yin, Yili; Zhao, Min; Forrester, John V; McCaig, Colin D

    2003-03-01

    Vascular endothelial cell (VEC) proliferation is a key event in angiogenesis and is tightly regulated. Electric potential differences exist around the vascular endothelium and give rise to endogenous electric fields (EFs), whether these EFs influence VEC proliferation is unclear. We exposed cultured VECs to applied EFs of physiological strengths for up to 72 h. EF at 50 or 100 mV/mm did not influence cell proliferation, but at 200 mV/mm, cell density, cell growth rate, and mitosis index decreased significantly. EF-induced reduction in VEC proliferation was not due to increased apoptosis, because caspase apoptosis inhibitor Z-VAD-FMK (20 microM), had no effect on this response. Rather, EF responses were mediated via decreased entry of cells into S phase from G1 phase, as shown by flow cytometry. Western blot showed that EFs decreased G1-specific cyclin E expression and increased cyclin/cyclin-dependent kinase complex inhibitor p27kipl expression. Thus EFs controlled VEC proliferation through induction of cell cycle arrest at G1 by down-regulation of cyclin E expression and up-regulation of p27kipl expression, rather than by promoting apoptosis. If control of the cell cycle by endogenous EFs extends beyond VECs, this would be of widespread biological significance in vivo.

  1. A computational platform for robotized fluorescence microscopy (II): DNA damage, replication, checkpoint activation, and cell cycle progression by high-content high-resolution multiparameter image-cytometry.

    PubMed

    Furia, Laura; Pelicci, Pier Giuseppe; Faretta, Mario

    2013-04-01

    Dissection of complex molecular-networks in rare cell populations is limited by current technologies that do not allow simultaneous quantification, high-resolution localization, and statistically robust analysis of multiple parameters. We have developed a novel computational platform (Automated Microscopy for Image CytOmetry, A.M.I.CO) for quantitative image-analysis of data from confocal or widefield robotized microscopes. We have applied this image-cytometry technology to the study of checkpoint activation in response to spontaneous DNA damage in nontransformed mammary cells. Cell-cycle profile and active DNA-replication were correlated to (i) Ki67, to monitor proliferation; (ii) phosphorylated histone H2AX (γH2AX) and 53BP1, as markers of DNA-damage response (DDR); and (iii) p53 and p21, as checkpoint-activation markers. Our data suggest the existence of cell-cycle modulated mechanisms involving different functions of γH2AX and 53BP1 in DDR, and of p53 and p21 in checkpoint activation and quiescence regulation during the cell-cycle. Quantitative analysis, event selection, and physical relocalization have been then employed to correlate protein expression at the population level with interactions between molecules, measured with Proximity Ligation Analysis, with unprecedented statistical relevance.

  2. Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase.

    PubMed Central

    Lin, B T; Gruenwald, S; Morla, A O; Lee, W H; Wang, J Y

    1991-01-01

    The retinoblastoma gene product (RB) is a nuclear protein which has been shown to function as a tumor suppressor. It is phosphorylated from S to M phase of the cell cycle and dephosphorylated in G1. This suggests that the function of RB is regulated by its phosphorylation in the cell cycle. Ten phosphotryptic peptides are found in human RB proteins. The pattern of RB phosphorylation does not change from S to M phases of the cell cycle. Hypophosphorylated RB prepared from insect cells infected with an RB-recombinant baculovirus is used as a substrate for in vitro phosphorylation reactions. Of several protein kinases tested, only cdc2 kinase phosphorylates RB efficiently and all 10 peptides can be phosphorylated by cdc2 in vitro. Removal of cdc2 from mitotic cell extracts by immunoprecipitation causes a concomitant depletion of RB kinase activity. These results indicate that cdc2 or a kinase with similar substrate specificity is involved in the cell cycle-dependent phosphorylation of the RB protein. Images PMID:2009861

  3. Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes.

    PubMed Central

    Tavormina, P A; Burke, D J

    1998-01-01

    The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function. In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10. We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle. The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes. We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function. We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex. PMID:9560388

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

    PubMed

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

    2015-05-08

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

  5. Phosphate-Activated Cyclin-Dependent Kinase Stabilizes G1 Cyclin To Trigger Cell Cycle Entry

    PubMed Central

    Menoyo, S.; Ricco, N.; Bru, S.; Hernández-Ortega, S.; Escoté, X.; Aldea, M.

    2013-01-01

    G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability. PMID:23339867

  6. A Kinase-Phosphatase Switch Transduces Environmental Information into a Bacterial Cell Cycle Circuit

    PubMed Central

    Heinrich, Kristina; Sobetzko, Patrick; Jonas, Kristina

    2016-01-01

    The bacterial cell cycle has been extensively studied under standard growth conditions. How it is modulated in response to environmental changes remains poorly understood. Here, we demonstrate that the freshwater bacterium Caulobacter crescentus blocks cell division and grows to filamentous cells in response to stress conditions affecting the cell membrane. Our data suggest that stress switches the membrane-bound cell cycle kinase CckA to its phosphatase mode, leading to the rapid dephosphorylation, inactivation and proteolysis of the master cell cycle regulator CtrA. The clearance of CtrA results in downregulation of division and morphogenesis genes and consequently a cell division block. Upon shift to non-stress conditions, cells quickly restart cell division and return to normal cell size. Our data indicate that the temporary inhibition of cell division through the regulated inactivation of CtrA constitutes a growth advantage under stress. Taken together, our work reveals a new mechanism that allows bacteria to alter their mode of proliferation in response to environmental cues by controlling the activity of a master cell cycle transcription factor. Furthermore, our results highlight the role of a bifunctional kinase in this process that integrates the cell cycle with environmental information. PMID:27941972

  7. Phosphate-activated cyclin-dependent kinase stabilizes G1 cyclin to trigger cell cycle entry.

    PubMed

    Menoyo, S; Ricco, N; Bru, S; Hernández-Ortega, S; Escoté, X; Aldea, M; Clotet, J

    2013-04-01

    G1 cyclins, in association with a cyclin-dependent kinase (CDK), are universal activators of the transcriptional G1-S machinery during entry into the cell cycle. Regulation of cyclin degradation is crucial for coordinating progression through the cell cycle, but the mechanisms that modulate cyclin stability to control cell cycle entry are still unknown. Here, we show that a lack of phosphate downregulates Cln3 cyclin and leads to G1 arrest in Saccharomyces cerevisiae. The stability of Cln3 protein is diminished in strains with low activity of Pho85, a phosphate-sensing CDK. Cln3 is an in vitro substrate of Pho85, and both proteins interact in vivo. More interestingly, cells that carry a CLN3 allele encoding aspartic acid substitutions at the sites of Pho85 phosphorylation maintain high levels of Cln3 independently of Pho85 activity. Moreover, these cells do not properly arrest in G1 in the absence of phosphate and they die prematurely. Finally, the activity of Pho85 is essential for accumulating Cln3 and for reentering the cell cycle after phosphate refeeding. Taken together, our data indicate that Cln3 is a molecular target of the Pho85 kinase that is required to modulate cell cycle entry in response to environmental changes in nutrient availability.

  8. Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling.

    PubMed

    Khanal, Supreet; Sultana, Hameeda; Catravas, John D; Carlyon, Jason A; Neelakanta, Girish

    2017-01-01

    Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

  9. Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases

    PubMed Central

    Hydbring, Per; Malumbres, Marcos; Sicinski, Piotr

    2016-01-01

    The role of cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), as core components of the machinery that drives cell cycle progression is well established. Increasing evidence indicates that mammalian cyclins and CDKs also carry out important roles in other cellular processes such as transcription, DNA damage repair, the control of cell death, differentiation, the immune response and metabolism. Some of these non-canonical functions are performed by cyclins or by CDKs, independent of their respective cell cycle partners, suggesting a substantial divergence in the function of these proteins during evolution. PMID:27033256

  10. Final checkup of neoplastic DNA replication: evidence for failure in decision-making at the mitotic cell cycle checkpoint G(1)/S.

    PubMed

    Prindull, Gregor

    2008-11-01

    Processing of epigenomic transcriptional information by cell cycle phase G(1) and decision-making at checkpoint G(1)/S are the final organizational steps preceding gene replication in transcriptional reorientation programs (i.e., switches from proliferation to cycle arrest and neoplastic transformation). Further analyses of cycle progression will open up new approaches in antineoplastic therapy. The following bibliographic databases were consulted: Central Medical Library Cologne, PubMed (English), the last search was done on April 23,2008 and key words searched were: cell cycle, cell memory, DNA methylation, embryonal/neoplastic stem cells, enzyme-modulated chromatin, G(1)-G(1)/S checkpoint, genomic/epigenomics, genomic viral DNA, histones, telomere/telomerases, transcription factors, neoplastic transformation, senescence. Gene transcription and epigenomic surveillance form a functional entity. In proliferation programs, transcriptional information is mediated by chromatin and DNA methylation, analyzed and processed in G(1) phase, and converged on the parental checkpoint G(1)/S for final decision-making on DNA replication. Genomic reorientation appears to be associated with transcriptional instability, which normally is corrected, possibly during the G(2)/M phase, to new levels of epigenomic equilibria. We speculate that daughter stem cells inherit persistent neoplasm-specific transcriptional instabilities through failure of the parental G(1)/S checkpoint. Foreign, silenced, potentially oncogenic DNA sequences, i.e. regular components of the human genome such as endogenous retroviruses, could conceivably be activated for expression in neoplastic transformation by epigenomic histone deacetylase/acetyl transferase/histone methyltransferase-mixed lineage leukemia deregulations. Failure of cell cycle G(1)/S decision-making for DNA replication is the final and possibly a major cause in neoplastic transformation. Therefore, further analysis of the dynamics of G(1)-G(1

  11. ALDH1A1 Maintains Ovarian Cancer Stem Cell-Like Properties by Altered Regulation of Cell Cycle Checkpoint and DNA Repair Network Signaling

    PubMed Central

    Meng, Erhong; Mitra, Aparna; Tripathi, Kaushlendra; Finan, Michael A.; Scalici, Jennifer; McClellan, Steve; da Silva, Luciana Madeira; Reed, Eddie; Shevde, Lalita A.; Palle, Komaraiah; Rocconi, Rodney P.

    2014-01-01

    Objective Aldehyde dehydrogenase (ALDH) expressing cells have been characterized as possessing stem cell-like properties. We evaluated ALDH+ ovarian cancer stem cell-like properties and their role in platinum resistance. Methods Isogenic ovarian cancer cell lines for platinum sensitivity (A2780) and platinum resistant (A2780/CP70) as well as ascites from ovarian cancer patients were analyzed for ALDH+ by flow cytometry to determine its association to platinum resistance, recurrence and survival. A stable shRNA knockdown model for ALDH1A1 was utilized to determine its effect on cancer stem cell-like properties, cell cycle checkpoints, and DNA repair mediators. Results ALDH status directly correlated to platinum resistance in primary ovarian cancer samples obtained from ascites. Patients with ALDHHIGH displayed significantly lower progression free survival than the patients with ALDHLOW cells (9 vs. 3 months, respectively p<0.01). ALDH1A1-knockdown significantly attenuated clonogenic potential, PARP-1 protein levels, and reversed inherent platinum resistance. ALDH1A1-knockdown resulted in dramatic decrease of KLF4 and p21 protein levels thereby leading to S and G2 phase accumulation of cells. Increases in S and G2 cells demonstrated increased expression of replication stress associated Fanconi Anemia DNA repair proteins (FANCD2, FANCJ) and replication checkpoint (pS317 Chk1) were affected. ALDH1A1-knockdown induced DNA damage, evidenced by robust induction of γ-H2AX and BAX mediated apoptosis, with significant increases in BRCA1 expression, suggesting ALDH1A1-dependent regulation of cell cycle checkpoints and DNA repair networks in ovarian cancer stem-like cells. Conclusion This data suggests that ovarian cancer cells expressing ALDH1A1 may maintain platinum resistance by altered regulation of cell cycle checkpoint and DNA repair network signaling. PMID:25216266

  12. Design checkpoint kinase 2 inhibitors by pharmacophore modeling and virtual screening techniques.

    PubMed

    Wang, Yen-Ling; Lin, Chun-Yuan; Shih, Kuei-Chung; Huang, Jui-Wen; Tang, Chuan-Yi

    2013-12-01

    Damage to DNA is caused by ionizing radiation, genotoxic chemicals or collapsed replication forks. When DNA is damaged or cells fail to respond, a mutation that is associated with breast or ovarian cancer may occur. Mammalian cells control and stabilize the genome using a cell cycle checkpoint to prevent damage to DNA or to repair damaged DNA. Checkpoint kinase 2 (Chk2) is one of the important kinases, which strongly affects DNA-damage and plays an important role in the response to the breakage of DNA double-strands and related lesions. Therefore, this study concerns Chk2. Its purpose is to find potential inhibitors using the pharmacophore hypotheses (PhModels) and virtual screening techniques. PhModels can identify inhibitors with high biological activities and virtual screening techniques are used to screen the database of the National Cancer Institute (NCI) to retrieve compounds that exhibit all of the pharmacophoric features of potential inhibitors with high interaction energy. Ten PhModels were generated using the HypoGen best algorithm. The established PhModel, Hypo01, was evaluated by performing a cost function analysis of its correlation coefficient (r), root mean square deviation (RMSD), cost difference, and configuration cost, with the values 0.955, 1.28, 192.51, and 16.07, respectively. The result of Fischer's cross-validation test for the Hypo01 model yielded a 95% confidence level, and the correlation coefficient of the testing set (rtest) had a best value of 0.81. The potential inhibitors were then chosen from the NCI database by Hypo01 model screening and molecular docking using the cdocker docking program. Finally, the selected compounds exhibited the identified pharmacophoric features and had a high interaction energy between the ligand and the receptor. Eighty-three potential inhibitors for Chk2 are retrieved for further study.

  13. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

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

  14. Screening of cell cycle fusion proteins to identify kinase signaling networks.

    PubMed

    Trojanowsky, Michelle; Vidovic, Dusica; Simanski, Scott; Penas, Clara; Schurer, Stephan; Ayad, Nagi G

    2015-01-01

    Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27(Kip1). We generated a p27(Kip1)-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27(Kip1)-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27(Kip1) turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.

  15. Mitogen-Activated Protein Kinase Hog1 Mediates Adaptation to G1 Checkpoint Arrest during Arsenite and Hyperosmotic Stress▿

    PubMed Central

    Migdal, Iwona; Ilina, Yulia; Tamás, Markus J.; Wysocki, Robert

    2008-01-01

    Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1 and G2 checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G1 delay but did not cause a persistent G1 arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1 exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G1 arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1 cell cycle arrest. PMID:18552285

  16. Aurora B kinase is required for cell cycle progression in silkworm.

    PubMed

    Gang, Xiaoxu; Qian, Wenliang; Zhang, Tianlei; Yang, Xinxin; Xia, Qingyou; Cheng, Daojun

    2017-01-30

    Aurora B kinase, a member of serine/threonine kinase family, is the catalytic subunit of the chromosomal passenger complex and is essential for chromosome alignment, chromosome segregation, and cytokinesis during mitosis. Here, we cloned the full-length cDNA sequence of silkworm Aurora B (BmAurB) gene and predicted that BmAurB protein contains a conserved S_TKc domain. Phylogenetic analysis between BmAurB and other Aurora kinases indicates that Aurora kinases may have evolved after separation between mammalian and insect, and prior to radiation of either mammalian or insects. RT-PCR examination revealed that the expression of the BmAurB gene was high in mitotic cycling gonads, moderate in mitotic cycling brain, and undetectable in endocycling silk gland during silkworm larval development. RNAi or inhibitor-mediated inhibition of the BmAurB gene in silkworm ovary-derived BmN4-SID1 cells disrupted cell cycle progression during mitosis and induced an accumulation of polyploid cells, cell cycle arrest at G2/M phase, chromosome misalignment, chromosome bridge, and bi-nucleation. Taken together, our results suggest that the BmAurB gene is required for cell cycle progression in silkworm.

  17. miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression.

    PubMed

    Pickering, M T; Stadler, B M; Kowalik, T F

    2009-01-08

    The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

  18. TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: role of the PI3K-Akt pathway.

    PubMed

    Faurschou, Annesofie; Gniadecki, Robert; Calay, Damien; Wulf, Hans Christian

    2008-08-01

    Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and FoxO3a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher proportion of UVB-treated HaCaT cells containing unrepaired cyclobutane pyrimidine dimers (CPDs) escaped the G2/M cell cycle checkpoint in the presence of TNF-alpha (9.5+/-3.3 vs 4.8+/-2.2%). After treatment with the PI3K inhibitor LY294002, only 1.2+/-0.7% of CPD-containing HaCaT cells were actively cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter the cell cycle. The effect of TNF-alpha seems to be dependent on Akt activation and may constitute a relevant mechanism enhancing mutagenesis and tumor development.

  19. The coupling of epidermal growth factor receptor down regulation by 1alpha,25-dihydroxyvitamin D3 to the hormone-induced cell cycle arrest at the G1-S checkpoint in ovarian cancer cells.

    PubMed

    Shen, Zheng; Zhang, Xiaohui; Tang, Jinfu; Kasiappan, Ravi; Jinwal, Umesh; Li, Pengfei; Hann, Shan; Nicosia, Santo V; Wu, Jie; Zhang, Xiaohong; Bai, Wenlong

    2011-05-16

    1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  20. Diallyl disulfide selectively causes checkpoint kinase-1 mediated G2/M arrest in human MGC803 gastric cancer cell line.

    PubMed

    Ling, Hui; Lu, Li-Feng; He, Jie; Xiao, Guo-Hua; Jiang, Hao; Su, Qi

    2014-11-01

    Previous studies have shown that diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, arrested human gastric cancer cells (MGC803) in the G2/M phase of the cell cycle. Due to the importance of cell cycle redistribution in DADS-mediated anticarcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. In the present study, the northern blot analysis showed that mRNA expression of for Chkl and Chk2 was unchanged. Notably, DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, activated phospho-ATR (ATM-RAD3-related gene), and dowregulated CDC25C and cyclin B1 expression. Furthermore, CDC25C was immunoprecipitated by anti-Chk1 but not anti-Chk2. Results of the overexpression and knockdown studies, showed that Chk1 but not Chk2 regulated the DADS-induced G2/M arrest of MGC803 cells. The overexpression of Chk1 resulted in significantly increased DADS-induced G2/M arrest, increased DADS-induced Chk1 phosphorylation and inhibited CDC25C expression. Knockdown of Chk1 reduced DADS‑induced G2/M arrest and blocked the DADS-induced inhibition of CDC25C and cyclin B1 expression. These results suggested that Chk1 is important in DADS‑induced cell cycle G2/M arrest in the human MGC803 gastric cancer cell line. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/CDC25C/cyclin B1.

  1. The cell cycle, cyclin-dependent kinases, and viral infections: new horizons and unexpected connections.

    PubMed

    Schang, Luis M

    2003-01-01

    The genomes of small DNA viruses such as papilloma and polyomaviruses code for few or no DNA replication proteins. Consequently, these viruses depend on cellular DNA replication proteins to replicate their genomes and replicate only when the infected cell progresses into S-phase, when these proteins are active. As a consequence of this strict dependence, the relationship between replication of the small DNA viruses and the cell cycle was obvious from the very early studies. The genomes of larger DNA viruses such as adeno- and herpes-viruses, in contrast, encode many of the proteins required for DNA replication. Some of the larger DNA viruses such as adenoviruses, however, also replicate only in S-phase because expression of viral DNA replication proteins is regulated by cellular factors that are activated in S-phase. Other large DNA viruses such as herpes simplex viruses (HSV) can replicate in arrested cells such as neurons, without inducing progression into S-phase. The relationships between cell cycle and replication of these last viruses are, thus, so subtle that their replication was long thought to be independent from cellular proteins whose activities are regulated in a cell cycle dependent manner. In contrast to this hypothesis, recent studies have shown that replication of HSV and other large DNA viruses requires cellular proteins whose activities are normally regulated in a cell cycle dependent manner, such as the cyclin-dependent kinases (cdks). Many excellent reviews on the interactions between cellular proteins involved in cell cycle regulation and smaller DNA viruses (parvo, papilloma, polyoma and adenoviruses) have been published (for example, see (1, 2)). Many reviews on cell cycle regulation also discuss the interactions between the cell cycle and the smaller DNA viruses (for example, see (3-5)). Herein, we will review these relationships only briefly, while focusing on the interactions between cell cycle proteins such as cdks and herpes-, retro

  2. Regulation of a Rho-associated kinase expression during the corneal epithelial cell cycle.

    PubMed

    Anderson, S C; SundarRaj, N

    2001-04-01

    It has been recognized that an increased expression of the Rho-associated kinase (ROCK-I), a downstream target of Rho (a Ras-related small guanosine triphosphatase [GTPase]), is associated with limbal-to-corneal epithelial transition. The purpose of the present study was to determine whether the expression of ROCK-I is regulated during the cell cycle of corneal epithelial cells. Rabbit corneal epithelial cells in culture were subjected to different culture conditions to enrich them in the G0, G1, and S phases of the cell cycle. Indirect immunofluorescence staining and western blot techniques were used for analyzing the changes in the relative intracellular concentrations of ROCK-I. Northern blot analysis of the isolated cellular RNA was performed to estimate the relative concentrations of ROCK-I mRNA. Serum deprivation did not cause all the corneal epithelial cells in culture to be arrested in the G0 phase of the cell cycle. However, the cells could be arrested in G0 by treating them with culture medium supplemented with transforming growth factor (TGF)-beta1. The relative concentration of ROCK-I in the G0-arrested cells was higher than in the corresponding control untreated cultures. G0-arrested cells were induced to enter G1, followed by the S phase of the cell cycle, by refeeding them with the medium devoid of TGF-beta1. The total intracellular concentration of ROCK-I significantly decreased during the G1 phase of the cell cycle and increased again during the S phase. The decrease in intracellular ROCK-I during the G1 phase was confirmed by arresting the cells in G1 with isoleucine deprivation and thymidine-mimosine treatments. ROCK-I mRNA levels were also found to be decreased during the G1 phase of the cell cycle. The levels of ROCK-I in the corneal epithelial cells were significantly lower in the G1 phase than those in the S and G0 phases of the cell cycle. Therefore, a Rho signaling pathway(s) involving ROCK-I may be regulated during the corneal epithelial

  3. Polo kinase links the stress pathway to cell cycle control and tip growth in fission yeast.

    PubMed

    Petersen, Janni; Hagan, Iain M

    2005-05-26

    Stress-activated mitogen-activated protein kinase cascades instigate a range of changes to enable eukaryotic cells to cope with particular insults. In Schizosaccharomyces pombe these responses include the transcription of specific gene sets and inhibition of entry into mitosis. The S. pombe stress response pathway (SRP) also promotes commitment to mitosis in unperturbed cell cycles to allow cells to match their rate of division with nutrient availability. The nature of this SRP function in cell cycle control is unknown. Entry into mitosis is controlled by mitosis-promoting factor (MPF; Cdc2/cyclin B) activity. Inhibitory phosphorylation of Cdc2 by Wee1 kinase inactivates MPF until Cdc25 removes this phosphate to promote mitosis. The balance between Wee1 and Cdc25 activities is influenced by the recruitment of polo kinase (Plo1) to the spindle pole body (SPB). The SPB component Cut12 mediates this recruitment. Hyper-activating mutations in either cut12 or plo1 enable Cdc25-defective cells to enter mitosis. The hyperactive cut12.s11 mutation suppresses cdc25.22, as it promotes recruitment of active Plo1 to interphase SPBs. Here we show that the SRP promotes phosphorylation of Plo1 on Ser 402. In unperturbed cell cycles, SRP-mediated phosphorylation of Ser 402 promotes Plo1 recruitment to SPBs and thus commitment to mitosis. Ser 402 phosphorylation also ensures efficient reinitiation of cell tip growth and cell division during recovery from particular stresses. Thus, phosphorylation of Plo1 Ser 402 not only enables SRP signalling to modulate the timing of mitotic commitment in response to nutrient status in unperturbed cycles, but also promotes the return to normal cell cycle control after stress.

  4. Escherichia coli cyclomodulin Cif induces G2 arrest of the host cell cycle without activation of the DNA-damage checkpoint-signalling pathway.

    PubMed

    Taieb, Frédéric; Nougayrède, Jean-Philippe; Watrin, Claude; Samba-Louaka, Ascel; Oswald, Eric

    2006-12-01

    The cycle inhibiting factor (Cif) belongs to a family of bacterial toxins and effector proteins, the cyclomodulins, that deregulate the host cell cycle. Upon injection into HeLa cells by the enteropathogenic Escherichia coli (EPEC) type III secretion system, Cif induces a cytopathic effect characterized by the recruitment of focal adhesion plates and the formation of stress fibres, an irreversible cell cycle arrest at the G(2)/M transition, and sustained inhibitory phosphorylation of mitosis inducer, CDK1. Here, we report that the reference typical EPEC strain B171 produces a functional Cif and that lipid-mediated delivery of purified Cif into HeLa cells induces cell cycle arrest and actin stress fibres, implying that Cif is necessary and sufficient for these effects. EPEC infection of intestinal epithelial cells (Caco-2, IEC-6) also induces cell cycle arrest and CDK1 inhibition. The effect of Cif is strikingly similar to that of cytolethal distending toxin (CDT), which inhibits the G(2)/M transition by activating the DNA-damage checkpoint pathway. However, in contrast to CDT, Cif does not cause phosphorylation of histone H2AX, which is associated with DNA double-stranded breaks. Following EPEC infection, the checkpoint effectors ATM/ATR, Chk1 and Chk2 are not activated, the levels of the CDK-activating phosphatases Cdc25B and Cdc25C are not affected, and Cdc25C is not sequestered in host cell cytoplasm. Hence, Cif activates a DNA damage-independent signalling pathway that leads to inhibition of the G(2)/M transition.

  5. Nuclear Localization of Diacylglycerol Kinase Alpha in K562 Cells Is Involved in Cell Cycle Progression.

    PubMed

    Poli, Alessandro; Fiume, Roberta; Baldanzi, Gianluca; Capello, Daniela; Ratti, Stefano; Gesi, Marco; Manzoli, Lucia; Graziani, Andrea; Suh, Pann-Ghill; Cocco, Lucio; Follo, Matilde Y

    2017-09-01

    Phosphatidylinositol (PI) signaling is an essential regulator of cell motility and proliferation. A portion of PI metabolism and signaling takes place in the nuclear compartment of eukaryotic cells, where an array of kinases and phosphatases localize and modulate PI. Among these, Diacylglycerol Kinases (DGKs) are a class of phosphotransferases that phosphorylate diacylglycerol and induce the synthesis of phosphatidic acid. Nuclear DGKalpha modulates cell cycle progression, and its activity or expression can lead to changes in the phosphorylated status of the Retinoblastoma protein, thus, impairing G1/S transition and, subsequently, inducing cell cycle arrest, which is often uncoupled with apoptosis or autophagy induction. Here we report for the first time not only that the DGKalpha isoform is highly expressed in the nuclei of human erythroleukemia cell line K562, but also that its nuclear activity drives K562 cells through the G1/S transition during cell cycle progression. J. Cell. Physiol. 232: 2550-2557, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. 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.

  7. Checkpoint kinase 2 (CHK2) negatively regulates androgen sensitivity and prostate cancer cell growth

    PubMed Central

    Ta, Huy Q; Ivey, Melissa L; Frierson, Henry F; Conaway, Mark R; Dziegielewski, Jaroslaw; Larner, James M; Gioeli, Daniel

    2015-01-01

    Prostate cancer (PCa) is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), but virtually all patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating PCa cell growth led to our discovery that Checkpoint Kinase 2 (CHK2) knockdown dramatically increased PCa growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects PCa proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. Additionally, we provide evidence that CHK2 physically associates with the AR, and that cell cycle inhibition increased this association. Finally, immunohistochemical analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation. PMID:26573794

  8. Kaposi sarcoma herpes virus latency associated nuclear antigen protein release the G2/M cell cycle blocks by modulating ATM/ATR mediated checkpoint pathway.

    PubMed

    Kumar, Amit; Sahu, Sushil Kumar; Mohanty, Suchitra; Chakrabarti, Sudipta; Maji, Santanu; Reddy, R Rajendra; Jha, Asutosh K; Goswami, Chandan; Kundu, Chanakya N; Rajasubramaniam, Shanmugam; Verma, Subhash C; Choudhuri, Tathagata

    2014-01-01

    The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA) plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole) induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block.

  9. Modulation of the G2 cell cycle checkpoint by sesquiterpene lactones psilostachyins A and C isolated from the common ragweed Ambrosia artemisiifolia.

    PubMed

    Sturgeon, Christopher M; Craig, Kyle; Brown, Colleen; Rundle, Natalie T; Andersen, Raymond J; Roberge, Michel

    2005-10-01

    A phenotypic cell-based assay for inhibitors of the G (2) DNA damage checkpoint was used to screen plant extracts from the US National Cancer Institute Natural Products Repository. It revealed activity in a methanol extract from the common ragweed Ambrosia artemisiifolia. Assay-guided fractionation led to the identification of the sesquiterpene lactones psilostachyins A and C as novel checkpoint inhibitors. Elimination of their alpha,beta-unsaturated carbonyl group caused a loss of activity, suggesting that the compounds can bind covalently to target proteins through Michael addition. Psilostachyins A and C also blocked cells in mitosis and caused the formation of aberrant microtubule spindles. However, the compounds did not interfere with microtubule polymerization in vitro. The related sesquiterpene lactones psilostachyin B, paulitin and isopaulitin were also isolated from the same extract but showed no checkpoint inhibition. The identification of the target(s) of psilostachyins A and C may provide further insight into the signalling pathways involved in cell cycle arrest and mitotic progression.

  10. Integrin-Linked Kinase (ILK) Deletion Disrupts Oligodendrocyte Development by Altering Cell Cycle.

    PubMed

    Hussain, Rashad; Macklin, Wendy B

    2017-01-11

    During development, oligodendrocytes are initially specified, after which oligodendrocyte precursor cells (OPCs) migrate and proliferate before differentiating into myelinating cells. Lineage-specific programming of oligodendrocytes results from sensing environmental cues through membrane-bound receptors and related intracellular signaling molecules. Integrin-linked kinase (ILK) is an important protein that is expressed at the inner margins of the plasma membrane and can mediate some of these signals. The current studies demonstrate that ILK deletion reduces the proliferation and differentiation of OPCs in the developing CNS. There was a significant decrease in the number of OPCs and mature oligodendrocytes throughout postnatal development in Olig1Cre(+/-) × ILK(fl/fl) mice. These changes were accompanied by reduced numbers of myelinated axons. Key proteins involved in cell cycle regulation were dysregulated. Cyclin D1/D3 and cyclin-dependent kinase 2/4 (cdc2/cdc4) were downregulated and the cell cycle inhibitor protein p27 Kip1 was upregulated. Therefore, ILK deletion impaired the developmental profile, proliferation, and differentiation of OPCs by altering the expression of regulatory cytoplasmic and nuclear factors. Integrin-linked kinase (ILK) is a scaffolding protein involved in integrating signals from the extracellular environment and communicating those signals to downstream effectors within cells. It has been proposed to regulate aspects of oligodendrocyte process extension and thereby myelination. However, the current studies demonstrate that it has an earlier impact on cells in this lineage. Knocking down ILK in Olig1-Cre-expressing cells reduces the pool of oligodendrocyte progenitor cells (OPCs). This smaller pool of OPCs results from altered cell cycle and reduced cell proliferation. These cells myelinate fewer axons than in wild-type mice and, in corpus callosum, the myelin is thinner than in controls. Interestingly, the smaller pool of spinal cord

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

    PubMed

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

    2007-06-01

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

  12. [Effect of felyanning granule in antagonizing Lewis lung cancer cell proliferation through cell cycle G1/S checkpoint dominating signaling intervention].

    PubMed

    Zheng, Zhan; Wang, Ju-yong; Xu, Zhen-ye

    2009-11-01

    To observe the effect of Feiyanning Granule (FYN) on tumor growth and cell cycle distribution in mice with Lewis lung cancer, as well as its influence on G1/S cell cycle checkpoint dominating signaling RB-E2F1 bio-axis. Modeled C57BL/6 mice were randomly divided into 6 groups: the model group (A), the DDP treated group (B) peritoneally injected with cisplatin 0.1 mg on d1, d3 and d5 after modeling, and the 4 FYN treated groups (C-F), administered via gastrogavage with FYN Decoction, and FYN Granule in small-, median- and high- dose respectively for 14 days. The tumour inhibiting rate, tumour weight, and body weight of mice were observed after treatment; cell cycle distribution was detected by flow cytometry (FCM), RB-E2F1mRNA expressions in tumour tissue were analyzed by RT-PCR, and their protein expressions by Western blot. Tumour weight in the 5 treated groups was lower than that in the model group (P < 0.05, P < 0.01). Body weight in group E was significantly higher than that in group A and B (P < 0.05, P < 0.01). FCM analysis showed the proportion of G0/G1 phase was higher in group E than in group A, B and C (P < 0.01), and cancer cell proliferation index (PI) in group E was lower than in group B (P < 0.05, P < 0.01). RT-PCR showed mRNA level of E2F1 was lower, but that of RB was significantly higher in group E than those in group A, B and C respectively (P < 0.01). Westem blot analysis showed the protein expression of E2F1 was lower in group E and B than that in group A (P < 0.05), while the protein expression of Rb in group E was higher than that in group A, B and C (P < 0.05). The effect of FYN in inhibiting Lewis lung cancer growth was related to its intervention on cancer cell cycle distribution which blocks most tumor cells in G0/G1 phase. Moreover, FYN can reduce MDM2 expression, enhance P53 expression to influence cell cycle G1/S checkpoint dominating signaling, so as to achieve the effect of antagonizing lung cancer cell proliferation.

  13. Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis.

    PubMed

    Kümper, Sandra; Mardakheh, Faraz K; McCarthy, Afshan; Yeo, Maggie; Stamp, Gordon W; Paul, Angela; Worboys, Jonathan; Sadok, Amine; Jørgensen, Claus; Guichard, Sabrina; Marshall, Christopher J

    2016-01-14

    Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.

  14. Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

    PubMed Central

    Kümper, Sandra; Mardakheh, Faraz K; McCarthy, Afshan; Yeo, Maggie; Stamp, Gordon W; Paul, Angela; Worboys, Jonathan; Sadok, Amine; Jørgensen, Claus; Guichard, Sabrina

    2016-01-01

    Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility. DOI: http://dx.doi.org/10.7554/eLife.12203.001 PMID:26765561

  15. The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S-M checkpoint and causes apoptosis in neurons.

    PubMed

    Chen, Y; McPhie, D L; Hirschberg, J; Neve, R L

    2000-03-24

    APP-BP1 binds to the amyloid precursor protein (APP) carboxyl-terminal domain. Recent work suggests that APP-BP1 participates in a novel ubiquitinylation-related pathway involving the ubiquitin-like molecule NEDD8. We show here that, in vivo in mammalian cells, APP-BP1 interacts with hUba3, its presumptive partner in the NEDD8 activation pathway, and that the APP-BP1 binding site for hUba3 is within amino acids 443-479. We also provide evidence that the human APP-BP1 molecule can rescue the ts41 mutation in Chinese hamster cells. This mutation previously has been shown to lead to successive S phases of the cell cycle without intervening G(2), M, and G(1), suggesting that the product of this gene negatively regulates entry into the S phase and positively regulates entry into mitosis. We show that expression of APP-BP1 in ts41 cells drives the cell cycle through the S-M checkpoint and that this function requires both hUba3 and hUbc12. Overexpression of APP-BP1 in primary neurons causes apoptosis via the same pathway. A specific caspase-6 inhibitor blocks this apoptosis. These findings are discussed in the context of abnormalities in the cell cycle that have been observed in Alzheimer's disease.

  16. Live imaging of the Dictyostelium cell cycle reveals widespread S phase during development, a G2 bias in spore differentiation and a premitotic checkpoint.

    PubMed

    Muramoto, Tetsuya; Chubb, Jonathan R

    2008-05-01

    The regulation of the Dictyostelium cell cycle has remained ambiguous owing to difficulties in long-term imaging of motile cells and a lack of markers for defining cell cycle phases. There is controversy over whether cells replicate their DNA during development, and whether spores are in G1 or G2 of the cell cycle. We have introduced a live-cell S-phase marker into Dictyostelium cells that allows us to precisely define cycle phase. We show that during multicellular development, a large proportion of cells undergo nuclear DNA synthesis. Germinating spores enter S phase only after their first mitosis, indicating that spores are in G2. In addition, we demonstrate that Dictyostelium heterochromatin is copied late in S phase and replicates via accumulation of replication factors, rather than recruitment of DNA to pre-existing factories. Analysis of variability in cycle times indicates that regulation of the cycle manifests at a single random transition in G2, and we present the first identified checkpoint in Dictyostelium, which operates at the G2-M transition in response to DNA damage.

  17. The spindle position checkpoint is coordinated by the Elm1 kinase

    PubMed Central

    Moore, Jeffrey K.; Chudalayandi, Prakash; Heil-Chapdelaine, Richard A.

    2010-01-01

    How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother–daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization. PMID:21041444

  18. Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae

    PubMed Central

    Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

    2016-01-01

    The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes. PMID:27782169

  19. snf1lk encodes a protein kinase that may function in cell cycle regulation.

    PubMed

    Stephenson, Angela; Huang, Guo-Ying; Huang, Gui-Yi; Nguyen, Ngoc-Thinh; Reuter, Sean; McBride, Jennifer L; Ruiz, Joseph C

    2004-06-01

    msk, myocardial SNF1-like kinase, was originally isolated in a screen for kinases expressed during early cardiogenesis in the mouse. msk maps to the proximal end of mouse chromosome 17 in a region that is syntenic with human chromosome 21q22.3, where the gene for SNF1LK, a predicted protein that shares 80% identity at the amino acid level with Msk, is located. Accordingly, msk has been redesignated snf1lk. Interestingly, the region encompassing the SNF1LK locus has been implicated in congenital heart defects often observed in patients with Down syndrome. snf1lk is also expressed in skeletal muscle progenitor cells of the somite beginning at 9.5 dpc. These data suggest a more general role for snf1lk in the earliest stages of muscle growth and/or differentiation. Consistent with a role in cell cycling, we observe that Chinese hamster ovary cells that express a tetracycline-inducible SNF1LK kinase domain do not divide, but undergo additional rounds of replication to yield 8N and 16N cells. These data suggest a possible function for SNF1LK in G2/M regulation. We show data that indicate that SNF1LK does not share functional homology with other SNF1-related kinases, but represents a new subclass with novel molecular activities.

  20. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues

    PubMed Central

    Mann, Thomas H.; Seth Childers, W.; Blair, Jimmy A.; Eckart, Michael R.; Shapiro, Lucy

    2016-01-01

    All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation. PMID:27117914

  1. HTLV-I Tax-Mediated Inactivation of Cell Cycle Checkpoints and DNA Repair Pathways Contribute to Cellular Transformation: “A Random Mutagenesis Model”

    PubMed Central

    Nicot, Christophe

    2015-01-01

    To achieve cellular transformation, most oncogenic retroviruses use transduction by proto-oncogene capture or insertional mutagenesis, whereby provirus integration disrupts expression of tumor suppressors or proto-oncogenes. In contrast, the Human T-cell leukemia virus type 1 (HTLV-I) has been classified in a separate class referred to as “transactivating retroviruses”. Current views suggest that the viral encoded Tax protein transactivates expression of cellular genes leading to deregulated growth and transformation. However, if Tax-mediated transactivation was indeed sufficient for cellular transformation, a fairly high frequency of infected cells would eventually become transformed. In contrast, the frequency of transformation by HTLV-I is very low, likely less than 5%. This review will discuss the current understanding and recent discoveries highlighting critical functions of Tax in cellular transformation. HTLV-I Tax carries out essential functions in order to override cell cycle checkpoints and deregulate cellular division. In addition, Tax expression is associated with increased DNA damage and genome instability. Since Tax can inhibit multiple DNA repair pathways and stimulate unfaithful DNA repair or bypass checkpoints, these processes allow accumulation of genetic mutations in the host genome. Given this, a “Random Mutagenesis” transformation model seems more suitable to characterize the oncogenic activities of HTLV-I. PMID:26835512

  2. Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth.

    PubMed

    Ta, Huy Q; Ivey, Melissa L; Frierson, Henry F; Conaway, Mark R; Dziegielewski, Jaroslaw; Larner, James M; Gioeli, Daniel

    2015-12-01

    Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation. ©2015 American Association for Cancer Research.

  3. Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase.

    PubMed

    Yasuhira, Shinji; Saito, Takeshi; Maesawa, Chihaya; Masuda, Tomoyuki

    2012-08-01

    Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans.

    PubMed

    Correia, Inês; Alonso-Monge, Rebeca; Pla, Jesús

    2016-01-01

    Eukaryotic cell cycle progression in response to environmental conditions is controlled via specific checkpoints. Signal transduction pathways mediated by MAPKs play a crucial role in sensing stress. For example, the canonical MAPKs Mkc1 (of the cell wall integrity pathway), and Hog1 (of the HOG pathway), are activated upon oxidative stress. In this work, we have analyzed the effect of oxidative stress induced by hydrogen peroxide on cell cycle progression in Candida albicans. Hydrogen peroxide was shown to induce a transient arrest at the G1 phase of the cell cycle. Specifically, a G1 arrest was observed, although phosphorylation of Mkc1 and Hog1 MAPKs can take place at all stages of the cell cycle. Interestingly, hog1 (but not mkc1) mutants required a longer time compared to wild type cells to resume growth after hydrogen peroxide challenge. Using GFP-labeled cells and mixed cultures of wild type and hog1 cells we were able to show that hog1 mutants progress faster through the cell cycle under standard growth conditions in the absence of stress (YPD at 37°C). Consequently, hog1 mutants exhibited a smaller cell size. The altered cell cycle progression correlates with altered expression of the G1 cyclins Cln3 and Pcl2 in hog1 cells compared to the wild type strain. In addition, Hgc1 (a hypha-specific G1 cyclin) as well as Cln3 displayed a different kinetics of expression in the presence of hydrogen peroxide in hog1 mutants. Collectively, these results indicate that Hog1 regulates the expression of G1 cyclins not only in response to oxidative stress, but also under standard growth conditions. Hydrogen peroxide treated cells did not show fluctuations in the mRNA levels for SOL1, which are observed in untreated cells during cell cycle progression. In addition, treatment with hydrogen peroxide prevented degradation of Sol1, an effect which was enhanced in hog1 mutants. Therefore, in C. albicans, the MAPK Hog1 mediates cell cycle progression in response to oxidative

  5. The Hog1 MAP Kinase Promotes the Recovery from Cell Cycle Arrest Induced by Hydrogen Peroxide in Candida albicans

    PubMed Central

    Correia, Inês; Alonso-Monge, Rebeca; Pla, Jesús

    2017-01-01

    Eukaryotic cell cycle progression in response to environmental conditions is controlled via specific checkpoints. Signal transduction pathways mediated by MAPKs play a crucial role in sensing stress. For example, the canonical MAPKs Mkc1 (of the cell wall integrity pathway), and Hog1 (of the HOG pathway), are activated upon oxidative stress. In this work, we have analyzed the effect of oxidative stress induced by hydrogen peroxide on cell cycle progression in Candida albicans. Hydrogen peroxide was shown to induce a transient arrest at the G1 phase of the cell cycle. Specifically, a G1 arrest was observed, although phosphorylation of Mkc1 and Hog1 MAPKs can take place at all stages of the cell cycle. Interestingly, hog1 (but not mkc1) mutants required a longer time compared to wild type cells to resume growth after hydrogen peroxide challenge. Using GFP-labeled cells and mixed cultures of wild type and hog1 cells we were able to show that hog1 mutants progress faster through the cell cycle under standard growth conditions in the absence of stress (YPD at 37°C). Consequently, hog1 mutants exhibited a smaller cell size. The altered cell cycle progression correlates with altered expression of the G1 cyclins Cln3 and Pcl2 in hog1 cells compared to the wild type strain. In addition, Hgc1 (a hypha-specific G1 cyclin) as well as Cln3 displayed a different kinetics of expression in the presence of hydrogen peroxide in hog1 mutants. Collectively, these results indicate that Hog1 regulates the expression of G1 cyclins not only in response to oxidative stress, but also under standard growth conditions. Hydrogen peroxide treated cells did not show fluctuations in the mRNA levels for SOL1, which are observed in untreated cells during cell cycle progression. In addition, treatment with hydrogen peroxide prevented degradation of Sol1, an effect which was enhanced in hog1 mutants. Therefore, in C. albicans, the MAPK Hog1 mediates cell cycle progression in response to oxidative

  6. Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression

    PubMed Central

    Wang, Peng; Galan, Jacob A.; Normandin, Karine; Bonneil, Éric; Hickson, Gilles R.; Roux, Philippe P.; Thibault, Pierre

    2013-01-01

    Cell division requires the coordination of critical protein kinases and phosphatases. Greatwall (Gwl) kinase activity inactivates PP2A-B55 at mitotic entry to promote the phosphorylation of cyclin B–Cdk1 substrates, but how Gwl is regulated is poorly understood. We found that the subcellular localization of Gwl changed dramatically during the cell cycle in Drosophila. Gwl translocated from the nucleus to the cytoplasm in prophase. We identified two critical nuclear localization signals in the central, poorly characterized region of Gwl, which are required for its function. The Polo kinase associated with and phosphorylated Gwl in this region, promoting its binding to 14-3-3ε and its localization to the cytoplasm in prophase. Our results suggest that cyclin B–Cdk1 phosphorylation of Gwl is also required for its nuclear exclusion by a distinct mechanism. We show that the nucleo-cytoplasmic regulation of Gwl is essential for its functions in vivo and propose that the spatial regulation of Gwl at mitotic entry contributes to the mitotic switch. PMID:23857770

  7. The checkpoint kinase ATM protects against stress-induced elevation of cyclin D1 and potential cell death in neurons.

    PubMed

    Hitomi, Masahiro; Stacey, Dennis W

    2010-06-01

    Quantitative cytometric studies show that cyclin D1 levels must decline during S phase for proper cell cycle progression, and that cyclin D1 decline follows phosphorylation induced by the checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). ATM is mutated in ataxia telangiectasia (AT), a disease characterized by progressive neurodegeneration. Importantly, neurodegeneration in many cases has been linked to the increased expression of cyclin D1 in neurons leading to inappropriate cell cycle entry. These facts prompted us to test the possibility that ATM normally protects against neural degeneration by suppressing cyclin D1 levels, particularly following genotoxic stress. For this purpose, neural stem cells were induced to differentiate into mature neural cells, including neurons. ATM activity in these cultures was inhibited with a specific chemical inhibitor in the presence or absence of hydrogen peroxide treatment, and the effect on cyclin D1 expression was determined by quantitative, single cell cytometric analyses. As predicted, inhibition of ATM did promote elevation of cyclin D1 in differentiated neurons, particularly under conditions of oxidative stress. The survival of differentiated neurons and of neural stem cells was reduced by such treatments. These data support our suggestion that ATM functions to maintain low levels of cyclin D1 expression in differentiated neurons; and may provide important clues in understanding neural degeneration in general. Copyright 2010 International Society for Advancement of Cytometry.

  8. Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

    SciTech Connect

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

    2009-11-10

    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.

  9. Mpg1, a fission yeast protein required for proper septum structure, is involved in cell cycle progression through cell-size checkpoint.

    PubMed

    Donoso, I; Muñoz-Centeno, M C; Sànchez-Durán, M A; Flores, A; Daga, R R; Guevara, C M; Bejarano, E R

    2005-09-01

    Using a yeast two-hybrid screen we isolated a gene from Schizosaccharomyces pombe which corresponds to the previously uncharacterized ORF SPCC1906.01. We have designated this gene as mpg1, based on the putative function of its product as a mannose-1-phosphatase guanyltransferase. Mpg1 shows strong similarity to other GDP-mannose-1-phosphate guanyltransferases involved in the maintenance of cell wall integrity and/or glycosylation. This homology, together with the protein's localization pattern demonstrated in this work, strongly suggests that Mpg1 is involved in cell wall and septum synthesis. Moreover, cells lacking Mpg1 present a defect in glycosylation, are more sensitive to Lyticase, and show an aberrant septum structure from the start of its deposition, indicating that the Mpg1 function is necessary for the correct assembly of the septum. Interestingly, lack of Mpg1 clearly affects cell cycle progression: mpg1 null mutants arrest as septated and bi-nucleated 4C cells, without an actomyosin ring. Wee1 is required for the G2/M arrest induced in the absence of Mpg1, since the blockade is circumvented when Wee1 is inactivated. Wee1 is part of a cell-size checkpoint that prevents entry into mitosis before cells reach a critical size. The results presented in this work demonstrate that the G2/M arrest induced in the absence of Mpg1 is mediated by this cell size checkpoint, since oversized mutant cells enter mitosis. The mpg1 loss-of-function mutant, therefore, provides a good model in which to study how cells coordinate cell growth and cell division.

  10. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4.

    PubMed

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar; Pereira, Gislene

    2011-06-13

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

  11. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

    PubMed Central

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar

    2011-01-01

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2–Bfa1 GAP complex that inhibit the mitotic exit–promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2–Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2–Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB. PMID:21670215

  12. Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells

    PubMed Central

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J.; Baker, Henry V.; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J.; Handfield, Martin

    2010-01-01

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT. PMID:20668524

  13. Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

    PubMed

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J; Baker, Henry V; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J; Handfield, Martin

    2010-07-23

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

  14. The Sinorhizobium meliloti sensor histidine kinase CbrA contributes to free-living cell cycle regulation.

    PubMed

    Sadowski, Craig S; Wilson, Daniel; Schallies, Karla B; Walker, Graham; Gibson, Katherine E

    2013-08-01

    Sinorhizobium meliloti is alternately capable of colonizing the soil as a free-living bacterium or establishing a chronic intracellular infection with its legume host for the purpose of nitrogen fixation. We previously identified the S. meliloti two-component sensor histidine kinase CbrA as playing an important role in regulating exopolysaccharide production, flagellar motility and symbiosis. Phylogenetic analysis of CbrA has highlighted its evolutionary relatedness to the Caulobacter crescentus sensor histidine kinases PleC and DivJ, which are involved in CtrA-dependent cell cycle regulation through the shared response regulator DivK. We therefore became interested in testing whether CbrA plays a role in regulating S. meliloti cell cycle processes. We find the loss of cbrA results in filamentous cell growth accompanied by cells that contain an aberrant genome complement, indicating CbrA plays a role in regulating cell division and possibly DNA segregation. S. meliloti DivK localizes to the old cell pole during distinct phases of the cell cycle in a phosphorylation-dependent manner. Loss of cbrA results in a significantly decreased rate of DivK polar localization when compared with the wild-type, suggesting CbrA helps regulate cell cycle processes by modulating DivK phosphorylation status as a kinase. Consistent with a presumptive decrease in DivK phosphorylation and activity, we also find the steady-state level of CtrA increased in cbrA mutants. Our data therefore demonstrate that CbrA contributes to free-living cell cycle regulation, which in light of its requirement for symbiosis, points to the potential importance of cell cycle regulation for establishing an effective host interaction.

  15. Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

    PubMed Central

    Liu, Dan; Davydenko, Olga

    2012-01-01

    Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint. PMID:22908307

  16. Cyclin-dependent kinase inhibitor roscovitine induces cell cycle arrest and apoptosis in rabbit retinal pigment epithelial cells.

    PubMed

    Wu, Pei-Chang; Tai, Ming-Hong; Hu, Dan-Ning; Lai, Chien-Hsiung; Chen, Yi-Hao; Wu, Yi-Chen; Tsai, Chia-Ling; Shin, Shyi-Jang; Kuo, Hsi-Kung

    2008-02-01

    Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells. Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot. Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine. This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

  17. A novel quantitative model of cell cycle progression based on cyclin-dependent kinases activity and population balances.

    PubMed

    Pisu, Massimo; Concas, Alessandro; Cao, Giacomo

    2015-04-01

    Cell cycle regulates proliferative cell capacity under normal or pathologic conditions, and in general it governs all in vivo/in vitro cell growth and proliferation processes. Mathematical simulation by means of reliable and predictive models represents an important tool to interpret experiment results, to facilitate the definition of the optimal operating conditions for in vitro cultivation, or to predict the effect of a specific drug in normal/pathologic mammalian cells. Along these lines, a novel model of cell cycle progression is proposed in this work. Specifically, it is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases experienced by each cell of the entire population during its own life. The transition between two consecutive cell cycle phases is simulated by taking advantage of the biochemical kinetic model developed by Gérard and Goldbeter (2009) which involves cyclin-dependent kinases (CDKs) whose regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylation-dephosphorylation, and cyclin synthesis or degradation. This biochemical model properly describes the entire cell cycle of mammalian cells by maintaining a sufficient level of detail useful to identify check point for transition and to estimate phase duration required by PB. Specific examples are discussed to illustrate the ability of the proposed model to simulate the effect of drugs for in vitro trials of interest in oncology, regenerative medicine and tissue engineering.

  18. Attenuation of G{sub 2} cell cycle checkpoint control in human tumor cells is associated with increased frequencies of unrejoined chromosome breaks but not increased cytotoxicity following radiation exposure

    SciTech Connect

    Schwartz, J.L.; Cowan, J.; Grdina, D.J.

    1997-08-01

    The contribution of G{sub 2} cell cycle checkpoint control to ionizing radiation responses was examined in ten human tumor cell lines. Most of the delay in cell cycle progression seen in the first cell cycle following radiation exposure was due to blocks in G{sub 2} and there were large cell line-to-cell line variations in the length of the G{sub 2} block. Longer delays were seen in cell lines that had mutations in p53. There was a highly significant inverse correlation between the length of G{sub 2} delay and the frequency of unrejoined chromosome breaks seen as chromosome terminal deletions in mitosis, and observation that supports the hypothesis that the signal for G{sub 2} delay in mammalian cells is an unrejoined chromosome break. There were also an inverse correlation between the length of G{sub 2} delay and the level of chromosome aneuploidy in each cell line, suggesting that the G{sub 2} and mitotic spindel checkpoints may be linked to each other. Attenuation in G{sub 2} checkpoint control was not associated with alterations in either the frequency of induced chromosome rearrangements or cell survival following radiation exposure suggesting that chromosome rearrangements, the major radiation-induced lethal lesion in tumor cells, form before cells enters G{sub 2}. Thus, agents that act solely to override G{sub 2} arrest should produce little radiosensitization in human tumor cells.

  19. A Synthetic Human Kinase Can Control Cell Cycle Progression in Budding Yeast

    PubMed Central

    Davey, Megan J.; Andrighetti, Heather J.; Ma, Xiaoli; Brandl, Christopher J.

    2011-01-01

    The DDK kinase complex, composed of Cdc7 and Dbf4, is required for S-phase progression. The two component proteins show different degrees of sequence conservation between human and yeast. Here, we determine that Saccharomyces cerevisiae bearing human CDC7 and DBF4 grows comparably to cells with yeast DDK under standard growth conditions. HsDrf1 (a second human Dbf4-like protein) does not support growth, suggesting that HsDbf4 is the true ortholog of ScDbf4. Both human subunits are required to complement yeast cdc7Δ or dbf4Δ due to the inability of human Cdc7 or Dbf4 to interact with the corresponding yeast protein. Flow cytometry indicates normal cell cycle progression for yeast containing human DDK. However, yeast containing human DDK is sensitive to long-term exposure to hydroxyurea and fails to sporulate, suggesting that human DDK substitutes for some, but not all, of yeast DDK’s functions. We mapped the region of Cdc7 required for species-specific function of DDK to the C-terminus of Cdc7 by substituting the yeast C-terminal 55 amino acid residues in place of the equivalent human residues. The resulting hybrid protein supported growth of a cdc7Δ strain only in the presence of ScDBF4. The strain supported by the hybrid CDC7 was not sensitive to HU and formed tetrads. Together, our data indicate that DDK’s targeting of its essential substrate is conserved between species, whereas the interactions within DDK are species specific. PMID:22384342

  20. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

    PubMed

    Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

    2012-07-15

    Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that

  1. Checkpoint kinase 1 expression is an adverse prognostic marker and therapeutic target in MYC-driven medulloblastoma

    PubMed Central

    Shah, Monil; Mulcahy Levy, Jean M.; Griesinger, Andrea M.; Alimova, Irina; Harris, Peter S.; Birks, Diane K.; Donson, Andrew M.; Davidson, Nathan; Remke, Marc; Taylor, Michael D.; Handler, Michael H.; Foreman, Nicholas K.; Venkataraman, Sujatha; Vibhakar, Rajeev

    2016-01-01

    Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway. Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, but the role of CHK1 mediated DDR in medulloblastoma is unknown. CHK1, both at the mRNA and protein level, is highly expressed in medulloblastoma and elevated CHK1 expression in Group3 medulloblastoma is an adverse prognostic marker. CHK1 inhibition with the small-molecule drug AZD7762, results in decreased cell growth, increased DNA damage and cell apoptosis. Furthermore, AZD7762 acts in synergy with cisplatin in reducing cell proliferation in medulloblastoma. Similar phenotypic changes were observed with another CHK1 inhibitor, PF477736, as well as genetic knockdown using siRNA against CHK1. Treatments with small-molecule inhibitors of CHK1 profoundly modulated the expression of both upstream and downstream target proteins within the CHK1 signaling pathways. This suggests the presence of a feedback loop in activating CHK1. Overall, our results demonstrate that small-molecule inhibition of CHK1 in combination with, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation. PMID:27449089

  2. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes.

    PubMed Central

    Kim, Y K; Wells, S; Lau, Y F; Lee, A S

    1988-01-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5' flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within -441 to -63 nucleotides from the transcriptional initiation site. This region (-441 to -63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. We conclude that the -441 to -63 sequence within the human TK promoter is important for cell-cycle-dependent expression. Images PMID:3413063

  3. Sequences contained within the promoter of the human thymidine kinase gene can direct cell-cycle regulation of heterologous fusion genes

    SciTech Connect

    Kim, Yongkyu; Wells, S.; Lau, Yunfai Chris; Lee, A.S. )

    1988-08-01

    Recent evidence on the transcriptional regulation of the human thymidine kinase (TK) gene raises the possibility that cell-cycle regulatory sequences may be localized within its promoter. A hybrid gene that combines the TK 5{prime} flanking sequence and the coding region of the bacterial neomycin-resistance gene (neo) has been constructed. Upon transfection into a hamster fibroblast cell line K12, the hybrid gene exhibits cell-cycle-dependent expression. Deletion analysis reveals that the region important for cell-cycle regulation is within {minus}441 to {minus}63 nucleotides from the transcriptional initiation site. This region ({minus}441 to {minus}63) also confers cell-cycle regulation to the herpes simplex virus thymidine kinase (HSVtk) promoter, which is not expressed in a cell-cycle manner. The authors conclude that the {minus}441 to {minus}63 sequence within the human TK promoter is important for cell-cycle-dependent expression.

  4. Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1

    SciTech Connect

    Yu, Eun-Young; Lee, Ju-Hee; Kang, Won-Hwa; Park, Yun-Hee; Kim, Lila; Park, Hee-Moon

    2013-03-01

    Highlights: ► Deletion of lkh1{sup +} made cells pass the G1/S phase faster than the wild type. ► Lkh1 can interact with a cyclin-dependent kinase inhibitor (CKI) Rum1. ► Lkh1 can phosphorylate Rum1 to activate its CKI activity. ► Thr110 was confirmed as the Lkh1-dependent phosphorylation site of Rum1. ► Positive acting mechanism for the Rum1 activation is reported for the first time. - Abstract: In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1{sup T110A} confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

  5. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

    PubMed

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Kumar, Narender; Churchman, Michelle; Larkin, John C; Kwon, Ashley; Lu, Hua

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. © 2016 American Society of Plant Biologists. All Rights Reserved.

  6. 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

  7. 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.

  8. A Feed Forward Loop Involving Protein Kinase C Alpha and MicroRNAs Regulates Tumor Cell Cycle

    PubMed Central

    Cohen, Ezra Eddy Wyssam; Zhu, Hongyan; Lingen, Mark W.; Martin, Leslie E.; Kuo, Wen-Liang; Choi, Eugene A.; Kocherginsky, Masha; Parker, Joel S.; Chung, Christine H.; Rosner, Marsha Rich

    2009-01-01

    Protein Kinase C alpha (PKCα) has been implicated in cancer but the mechanism is largely unknown. Here we show that PKCα promotes head and neck squamous cell carcinoma (SCCHN) by a feed forward network leading to cell cycle deregulation. PKCα inhibitors decrease proliferation in SCCHN cell lines and xenografted tumors. PKCα inhibition or depletion in tumor cells decreases DNA synthesis by suppressing ERK phosphorylation and cyclin E synthesis. Additionally, PKCα down-regulates miR-15a, a microRNA that directly inhibits protein synthesis of cyclin E as well as other cell cycle regulators. Furthermore, both PKCα and cyclin E protein expression are increased in primary tumors, and PKCα inversely correlates with miR15a expression in primary tumors. Finally, PKCα is associated with poor prognosis in SCCHN. These results identify PKCα as a key regulator of HNSCC tumor cell growth by a mechanism involving activation of MAP kinase, an initiator of the cell cycle, and suppression of miR-15a, an inhibitor of DNA synthesis. Although the specific components may be different, this type of feed forward loop network, consisting of a stimulus that activates a positive signal and removes a negative brake, is likely to be a general one that enables induction of DNA synthesis by a variety of growth or oncogenic stimuli. PMID:19117988

  9. Replication Checkpoint Kinase Cds1 Regulates Recombinational Repair Protein Rad60

    PubMed Central

    Boddy, Michael N.; Shanahan, Paul; Hayes McDonald, W.; Lopez-Girona, Antonia; Noguchi, Eishi; Yates III, John R.; Russell, Paul

    2003-01-01

    Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks. PMID:12897162

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

    SciTech Connect

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

    2014-09-26

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

  11. Inhibition of protein kinase B activity induces cell cycle arrest and apoptosis during early G₁ phase in CHO cells.

    PubMed

    van Opstal, Angélique; Bijvelt, José; van Donselaar, Elly; Humbel, Bruno M; Boonstra, Johannes

    2012-04-01

    Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.

  12. ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes.

    PubMed

    Rodríguez, Javier; Calvo, Fernando; González, José M; Casar, Berta; Andrés, Vicente; Crespo, Piero

    2010-11-29

    As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.

  13. ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma–lamin A complexes

    PubMed Central

    Rodríguez, Javier; Calvo, Fernando; José; González, M.; Casar, Berta; Andrés, Vicente

    2010-01-01

    As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation. PMID:21115804

  14. Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase

    SciTech Connect

    Cai, Z.; Chehab, N; Pavletich, N

    2009-01-01

    The CHK2 protein kinase is an important transducer of DNA damage checkpoint signals, and its mutation contributes to hereditary and sporadic cancer. CHK2 activation is triggered by the phosphorylation of Thr68 by the DNA damage-activated ATM kinase. This leads to transient CHK2 dimerization, in part through intermolecular phosphoThr68-FHA domain interactions. Dimerization promotes kinase activation through activation-loop autophosphorylation, but the mechanism of this process has not been clear. The dimeric crystal structure of CHK2, described here, in conjunction with biochemical and mutational data reveals that productive CHK2 dimerization additionally involves intermolecular FHA-kinase domain and FHA-FHA interactions. Ile157, mutated in the Li-Fraumeni cancer-predisposition syndrome, plays a central role in the FHA-kinase domain interface, explaining the lack of dimerization and autophosphorylation of this mutant. In the dimer, the kinase active sites face each other in close proximity, indicating that dimerization may also serve to optimally position the kinase active sites for efficient activation loop transphosphorylation.

  15. Natural flavonoids targeting deregulated cell cycle progression in cancer cells.

    PubMed

    Singh, Rana Pratap; Agarwal, Rajesh

    2006-03-01

    The prolonged duration requiring alteration of multi-genetic and epigenetic molecular events for cancer development provides a strong rationale for cancer prevention, which is developing as a potential strategy to arrest or reverse carcinogenic changes before the appearance of the malignant disease. Cell cycle progression is an important biological event having controlled regulation in normal cells, which almost universally becomes aberrant or deregulated in transformed and neoplastic cells. In this regard, targeting deregulated cell cycle progression and its modulation by various natural and synthetic agents are gaining widespread attention in recent years to control the unchecked growth and proliferation in cancer cells. In fact, a vast number of experimental studies convincingly show that many phytochemicals halt uncontrolled cell cycle progression in cancer cells. Among these phytochemicals, natural flavonoids have been identified as a one of the major classes of natural anticancer agents exerting antineoplastic activity via cell cycle arrest as a major mechanism in various types of cancer cells. This review is focused at the modulatory effects of natural flavonoids on cell cycle regulators including cyclin-dependent kinases and their inhibitors, cyclins, p53, retinoblastoma family of proteins, E2Fs, check-point kinases, ATM/ATR and survivin controlling G1/S and G2/M check-point transitions in cell cycle progression, and discusses how these molecular changes could contribute to the antineoplastic effects of natural flavonoids.

  16. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  17. Efficacy of Combined Histone Deacetylase and Checkpoint Kinase Inhibition in a Preclinical Model of Human Burkitt Lymphoma

    PubMed Central

    Kong, YanGuo; Barisone, Gustavo A; Sidhu, Ranjit S; O’Donnell, Robert T; Tuscano, Joseph M

    2015-01-01

    Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies. PMID:26322845

  18. Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis.

    PubMed

    Fang, Hanwei; Klages, Natacha; Baechler, Bastien; Hillner, Evelyn; Yu, Lu; Pardo, Mercedes; Choudhary, Jyoti; Brochet, Mathieu

    2017-05-08

    Malaria transmission relies on the production of gametes following ingestion by a mosquito. Here, we show that Ca(2+)-dependent protein kinase 4 controls three processes essential to progress from a single haploid microgametocyte to the release of eight flagellated microgametes in Plasmodium berghei. A myristoylated isoform is activated by Ca(2+) to initiate a first genome replication within twenty seconds of activation. This role is mediated by a protein of the SAPS-domain family involved in S-phase entry. At the same time, CDPK4 is required for the assembly of the subsequent mitotic spindle and to phosphorylate a microtubule-associated protein important for mitotic spindle formation. Finally, a non-myristoylated isoform is essential to complete cytokinesis by activating motility of the male flagellum. This role has been linked to phosphorylation of an uncharacterised flagellar protein. Altogether, this study reveals how a kinase integrates and transduces multiple signals to control key cell-cycle transitions during Plasmodium gametogenesis.

  19. Cell cycle regulation of breast cancer cells through estrogen-induced activities of ERK and Akt protein kinases.

    PubMed

    Geffroy, Nancy; Guédin, Aurore; Dacquet, Catherine; Lefebvre, Philippe

    2005-06-15

    The proliferative effect of estrogens on breast cancer cell (BCC) is mainly mediated through estrogen receptors (ER). Non-transcriptional effects of estrogens, exerted through activation of several protein kinases, may also contribute to BCC proliferation. However, the relative contribution of these two responses to BCC proliferation is not known. We characterized a novel estrogenic receptor ligand which possess Akt and ERK activating properties distinct from that of 17beta-estradiol. Early and delayed waves of activation of these kinases were detected upon estrogenic challenge of BCC, but only molecules able to promote a significant, delayed activation of ERK-induced BCC proliferation. Estrogen-induced cell cycle progression was not sensitive to the inhibition of ERK-regulating kinases MEK1 and 2. ERalpha was found to be necessary, but not sufficient for kinases activation. Thus, estrogens elicit a distinct pattern of early and delayed activation of ERK and Akt, and early protein kinase activation is probably not involved in BCC proliferation. Structural variations in the estrogen molecule may confer novel biological properties unrelated to estrogen-dependent transcriptional activation.

  20. High density lipoproteins induce cell cycle entry in vascular smooth muscle cells via mitogen activated protein kinase-dependent pathway.

    PubMed

    Nofer, J R; Junker, R; Pulawski, E; Fobker, M; Levkau, B; von Eckardstein, A; Seedorf, U; Assmann, G; Walter, M

    2001-04-01

    In this study we found that HDL acts as a potent and specific mitogen in vascular smooth muscle cells (VSMC) by stimulating entry into S-phase and DNA synthesis in a time- and concentration-dependent manner, induction of cyclins D1, E, and A, as well as activation of cyclin D-dependent kinases as inferred from phosphorylation of the retinoblastoma protein (pRb). Moreover, HDL induced activation of the mitogen-activated protein kinase pathway including Raf-, MEK-1, and ERK1/2, as well as the expression of proto-oncogen c-fos, which is controlled by ERK1/2. PD98059, an inhibitor of MEK-1 blocked the mitogenic activity of HDL and cyclin D1 expression. HDL-induced VSMC proliferation, cell cycle progression, cyclin D1 expression, and activation of the Raf-1/MEK-1/ERK1/2 cascade were blocked by preincubation of cells with pertussis toxin indicating involvement of trimeric G-protein. By contrast, none of these responses was inhibited by the protein kinase C inhibitor, GF109203X. The mitogenic effects of native HDL were not mimicked by apo A-I, reconstituted HDL containing apo A-I, or cholesterol-containing liposomes. In conclusion, HDL possesses an intrinsic property to induce G-protein- and MAP-kinase-dependent proliferation and cell cycle progression in VSMC. The strong and specific mitogenic effect of HDL should be taken into account, when therapeutic strategies to elevate the plasma level of these lipoproteins are developed.

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

    SciTech Connect

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

    2009-10-23

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

  2. Bacterial cyclomodulin Cif blocks the host cell cycle by stabilizing the cyclin-dependent kinase inhibitors p21 and p27.

    PubMed

    Samba-Louaka, Ascel; Nougayrède, Jean-Philippe; Watrin, Claude; Jubelin, Grégory; Oswald, Eric; Taieb, Frédéric

    2008-12-01

    The cycle inhibiting factor (Cif) is a cyclomodulin produced by enteropathogenic and enterohemorrhagic Escherichia coli. Upon injection into the host cell by the bacterial type III secretion system, Cif inhibits the G2/M transition via sustained inhibition of the mitosis inducer CDK1 independently of the DNA damage response. In this study, we show that Cif induces not only G2, but also G1 cell cycle arrest depending on the stage of cells in the cell cycle during the infection. In various cell lines including differentiated and untransformed enterocytes, the cell cycle arrests are correlated with the accumulation of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1). Cif-induced cyclin-dependent kinase inhibitor accumulation is independent of the p53 pathway but occurs through inhibition of their proteasome-mediated degradation. Our results provide a direct link between the mode of action of Cif and the host cell cycle control.

  3. BRCA2 coordinates the activities of cell-cycle kinases to promote genome stability.

    PubMed

    Yata, Keiko; Bleuyard, Jean-Yves; Nakato, Ryuichiro; Ralf, Christine; Katou, Yuki; Schwab, Rebekka A; Niedzwiedz, Wojciech; Shirahige, Katsuhiko; Esashi, Fumiko

    2014-06-12

    Numerous human genome instability syndromes, including cancer, are closely associated with events arising from malfunction of the essential recombinase Rad51. However, little is known about how Rad51 is dynamically regulated in human cells. Here, we show that the breast cancer susceptibility protein BRCA2, a key Rad51 binding partner, coordinates the activity of the central cell-cycle drivers CDKs and Plk1 to promote Rad51-mediated genome stability control. The soluble nuclear fraction of BRCA2 binds Plk1 directly in a cell-cycle- and CDK-dependent manner and acts as a molecular platform to facilitate Plk1-mediated Rad51 phosphorylation. This phosphorylation is important for enhancing the association of Rad51 with stressed replication forks, which in turn protects the genomic integrity of proliferating human cells. This study reveals an elaborate but highly organized molecular interplay between Rad51 regulators and has significant implications for understanding tumorigenesis and therapeutic resistance in patients with BRCA2 deficiency.

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

    PubMed Central

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

    2016-01-01

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

  5. 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

  6. Mitogen-activated protein kinase kinase activity is required for the G2/M transition of the cell cycle in mammalian fibroblasts

    PubMed Central

    Wright, Jocelyn H.; Munar, Erlynda; Jameson, Damon R.; Andreassen, Paul R.; Margolis, Robert L.; Seger, Rony; Krebs, Edwin G.

    1999-01-01

    The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 (MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G0, the level of MAPKK activity was observed to affect the kinetics of progression through both the G1 and G2 phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G0/G1 progression, was found to also delay progression of cells through G2. In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrested the cells in the following G2 phase. These data demonstrate a novel role for the MAPK cascade in progression from G2 into mitosis in NIH 3T3 cells. PMID:10500177

  7. Mathematical model of the morphogenesis checkpoint in budding yeast

    PubMed Central

    Ciliberto, Andrea; Novak, Bela; Tyson, John J.

    2003-01-01

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next. PMID:14691135

  8. Mathematical model of the morphogenesis checkpoint in budding yeast.

    PubMed

    Ciliberto, Andrea; Novak, Bela; Tyson, John J

    2003-12-22

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

  9. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    SciTech Connect

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.

    2005-06-01

    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  10. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.

    PubMed

    Malumbres, Marcos; Sotillo, Rocío; Santamaría, David; Galán, Javier; Cerezo, Ana; Ortega, Sagrario; Dubus, Pierre; Barbacid, Mariano

    2004-08-20

    Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.

  11. Ipl1/Aurora B kinase coordinates synaptonemal complex disassembly with cell cycle progression and crossover formation in budding yeast meiosis

    PubMed Central

    Jordan, Philip; Copsey, Alice; Newnham, Louise; Kolar, Elizabeth; Lichten, Michael; Hoffmann, Eva

    2009-01-01

    Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. We identified Ipl1/Aurora B kinase as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO, from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase. PMID:19759266

  12. Replication Checkpoint: Tuning and Coordination of Replication Forks in S Phase

    PubMed Central

    Hustedt, Nicole; Gasser, Susan M.; Shimada, Kenji

    2013-01-01

    Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability. PMID:24705211

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

    PubMed Central

    Krenn, Veronica; Musacchio, Andrea

    2015-01-01

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

  14. Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation.

    PubMed

    Wang, Guoliang; Tong, Xiangyan; Weng, Stephanie; Zhou, Huilin

    2012-10-15

    The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

  15. M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe.

    PubMed

    Evison, Benny J; Pastuovic, Mile; Bilardi, Rebecca A; Forrest, Robert A; Pumuye, Paul P; Sleebs, Brad E; Watson, Keith G; Phillips, Don R; Cutts, Suzanne M

    2011-12-01

    Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex.

  16. Cyclin-Dependent Kinase 7 Controls mRNA Synthesis by Affecting Stability of Preinitiation Complexes, Leading to Altered Gene Expression, Cell Cycle Progression, and Survival of Tumor Cells

    PubMed Central

    Kelso, Timothy W. R.; Baumgart, Karen; Eickhoff, Jan; Albert, Thomas; Antrecht, Claudia; Lemcke, Sarah; Klebl, Bert

    2014-01-01

    Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. PMID:25047832

  17. Protein kinase CK2 activates the atypical Rio1p kinase and promotes its cell-cycle phase-dependent degradation in yeast.

    PubMed

    Angermayr, Michaela; Hochleitner, Elisabeth; Lottspeich, Friedrich; Bandlow, Wolfhard

    2007-09-01

    Using co-immunoprecipitation combined with MS analysis, we identified the alpha' subunit of casein kinase 2 (CK2) as an interaction partner of the atypical Rio1 protein kinase in yeast. Co-purification of Rio1p with CK2 from Deltacka1 or Deltacka2 mutant extracts shows that Rio1p preferentially interacts with Cka2p in vitro. The C-terminal domain of Rio1p is essential and sufficient for this interaction. Six C-terminally located clustered serines were identified as the only CK2 sites present in Rio1p. Replacement of all six serine residues by aspartate, mimicking constitutive phosphorylation, stimulates Rio1p kinase activity about twofold in vitro compared with wild-type or the corresponding (S > A)(6) mutant proteins. Both mutant alleles (S > A)(6) or (S > D)(6) complement in vivo, however, growth of the RIO1 (S > A)(6) mutant is greatly retarded and shows a cell-cycle phenotype, whereas the behaviour of the RIO1 (S > D)(6) mutant is indistinguishable from wild-type. This suggests that phosphorylation by protein kinase CK2 leads to moderate activation of Rio1p in vivo and promotes cell proliferation. Physiological studies indicate that phosphorylation by CK2 renders the Rio1 protein kinase susceptible to proteolytic degradation at the G(1)/S transition in the cell-division cycle, whereas the non-phosphorylated version is resistant.

  18. Differences in kinase-mediated regulation of cell cycle progression in normal and transformed cells

    SciTech Connect

    Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Stevenson, A.P.; Kraemer, P.M.; Bustos, L.D.; Dickson, J.A.; Bradbury, E.M. )

    1993-01-01

    Staurosporine (Stsp), a general protein kinase inhibitor, was used to investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation. Low levels of Stsp (1-2nM) prevented nontransformed cells from entering S phase, indicating that protein phosphorylation processes are essential for commitment of DNA replication in normal cells. Cells resumed cycling when Stsp was removed. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 h later than the G0/G1 boundary and extended through the G1/S boundary. The initial block point at 3 h corresponds neither to the serum nor the amino acid restriction point. In contrast, neither low nor high concentrations (100nm) of Stsp affected G1 progression of transformed cells. High drug concentrations blocked normal cells in G1 and G2 but affected only G2-progression in transformed cells. These results indicate that kinase-mediated regulation of DNA replication is lost as a result of neoplastic transformation, but the G2-arrest mechanism remains intact.

  19. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase

    SciTech Connect

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U.

    1996-04-02

    The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33-{Angstrom} resolution and refined to an R{sub factor} of 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2. 36 refs., 4 figs., 2 tabs.

  20. Cell cycle control as a basis for cancer chemoprevention through dietary agents

    PubMed Central

    Meeran, Syed Musthapa; Katiyar, Santosh Kumar

    2008-01-01

    The development of cancer is associated with disorders in the regulation of the cell cycle. The purpose of this review is to briefly summarize the known sequence of events that regulate cell cycle progression with an emphasis on the checkpoints and the mechanisms cell employ to insure DNA stability in the face of genotoxic stress. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinases (CDK) molecules. The cyclins are CDK binding partners which are required for kinase activity and their protein levels are intimately linked to the cell cycle stage. CDK activity can be regulated by other mechanisms, such as phosphorylation events, that may contribute to deregulation of cell cycle and the development of cancer. While fruits and vegetables are recommended for prevention of cancer, their active ingredients and mechanisms of action are less well understood. Here, we briefly present evidence that dietary agents identified from fruits and vegetables can act to modulate the effects of deregulated cell cycle checkpoints, and that this may contribute to the prevention of cancer. The agents include apigenin (celery, parsley), curcumin (turmeric), (−)-epigallocatechin-3-gallate (green tea), resveratrol (red grape, peanuts and berries), genistein (soybean), and silymarin (milk thistle). The teachings of Hippocrates are still true “let food be thy medicine and medicine be thy food”. PMID:17981702

  1. 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

  2. The RNA-Binding Proteins Zfp36l1 and Zfp36l2 Enforce the Thymic β-Selection Checkpoint by Limiting DNA Damage Response Signaling and Cell Cycle Progression.

    PubMed

    Vogel, Katharina U; Bell, Lewis S; Galloway, Alison; Ahlfors, Helena; Turner, Martin

    2016-10-01

    The RNA-binding proteins Zfp36l1 and Zfp36l2 act redundantly to enforce the β-selection checkpoint during thymopoiesis, yet their molecular targets remain largely unknown. In this study, 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. Double-negative 3 thymocytes lacking Zfp36l1/l2 share a gene expression profile with postselected double-negative 3b 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, posttranscriptional control by Zfp36l1/l2 limits DNA damage responses, which are known to promote thymocyte differentiation. Zfp36l1/l2 therefore act as posttranscriptional safeguards against chromosomal instability and replication stress by integrating pre-TCR and IL-7 signaling with DNA damage and cell cycle control. Copyright © 2016 by The American Association of Immunologists, Inc.

  3. Mechanisms of sulindac-induced apoptosis and cell cycle arrest.

    PubMed

    Jung, Barbara; Barbier, Valerie; Brickner, Howard; Welsh, John; Fotedar, Arun; McClelland, Michael

    2005-02-28

    The mechanism underlying the chemopreventive effects of the non-steroidal anti-inflammatory drug sulindac remains unclear. Its active metabolite, sulindac sulfide, induces cell cycle arrest as well as apoptosis in mammalian cell lines. We now show that in murine thymocytes, sulindac sulfide-induced cell death is p53, bax, Fas, and FasL independent. In contrast, bcl2 transgenic thymocytes are resistant to sulindac sulfide-induced apoptosis. In addition, we demonstrate that sulindac sulfide-induced cell cycle arrest in mouse embryonic fibroblasts (MEFs) is partly mediated by the retinoblastoma tumor suppressor protein (Rb) and the cyclin kinase inhibitor p21waf1/cip1. Furthermore, MEFs deficient in p21 or Rb are more susceptible to sulindac sulfide-induced cell death. These results suggest that sulindac may selectively target premalignant cells with cell cycle checkpoint deficits.

  4. Cell cycle regulated D3-type cyclins form active complexes with plant-specific B-type cyclin-dependent kinase in vitro.

    PubMed

    Kawamura, Kazue; Murray, James A H; Shinmyo, Atsuhiko; Sekine, Masami

    2006-05-01

    Tobacco (Nicotiana tabacum L.) cv Bright Yellow-2 (BY-2) cells are the most highly synchronizable plant cell culture, and previously we used them to analyze cell cycle regulation of cyclin-dependent kinases (CDKs) containing the cyclin binding motifs PSTAIRE (CDKA) and PPTA/TLRE (CDKB). Here we describe the analysis of tobacco CycD3 cyclins whose transcripts predominantly accumulate during G2 to M phase, which represents a unique feature of this type of cyclin D in plants. Although protein levels of CycD3s fluctuate with different patterns during the cell cycle, kinase assays revealed that the CycD3-associated kinases phosphorylate histone H1 and the tobacco retinoblastoma related protein (NtRBR1) with two peaks at the G1/S and G2/M boundaries. In vitro pull-down assays revealed that cell cycle-regulated CycD3s bind to CDKA, but more weakly than does CycD3;3, and that they also bind to CDKB and the CDK inhibitor NtKIS1a. Mutations in the cyclin box of the CycD3s showed that two amino acids are required for binding with CDKA and NtKIS1a, but no diminished interaction was observed with CDKB. A reconstituted kinase assay was adapted for use with bacterially produced GST-CycD3s, and kinase activity could be activated by incubation of extracts from exponentially growing BY-2 cells. Such activated complexes contained CDKA and CDKB, and the reconstituted GST-CycD3 mutants, retaining binding ability to CDKB, showed kinase activity, suggesting that these cell cycle-regulated CycD3s form active complexes with both A- and B-type CDKs in vitro.

  5. Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis.

    PubMed

    Kallio, Marko J; McCleland, Mark L; Stukenberg, P Todd; Gorbsky, Gary J

    2002-06-04

    How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.

  6. Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.

    PubMed Central

    Knight, G B; Gudas, J M; Pardee, A B

    1987-01-01

    Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene. Images PMID:3479796

  7. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-06

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

  8. SUMOylation Regulates Polo-like Kinase 1-interacting Checkpoint Helicase (PICH) during Mitosis*

    PubMed Central

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-01-01

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres. PMID:25564610

  9. Possible attenuation of the G2 DNA damage cell cycle checkpoint in HeLa cells by extremely low frequency (ELF) electromagnetic fields

    PubMed Central

    Harris, Paul A; Lamb, Justin; Heaton, Brian; Wheatley, Denys N

    2002-01-01

    Background The issue remains unresolved as to whether low frequency magnetic fields can affect cell behaviour, with the possibility that they may be in part responsible for the increased incidence of leukaemia in parts of the population exposed to them. Methods Combined treatment of HeLa cells with gamma-irradiation (1, 3 and 5 Grays) and extra low frequency magnetic fields of ~50 Hz was carried out under rigorously controlled conditions. Results Synchronised cells progressing from S-phase arrived at mitosis on average marginally ahead of irradiation controls not exposed to ELF. In no instance out of a total of twenty separate experiments did this "double-insult" further delay entry of cells into mitosis, as had been anticipated. Conclusion This apparently "non-genotoxic" agent (ELF) appears to be capable of affecting cells that would normally arrest for longer in G2, suggesting a weakening of the stringency of the late cycle (G2) checkpoint. PMID:12069691

  10. X-ray structures of checkpoint kinase 2 in complex with inhibitors that target its gatekeeper-dependent hydrophobic pocket

    SciTech Connect

    Lountos, George T.; Jobson, Andrew G.; Tropea, Joseph E.; Self, Christopher R.; Zhang, Guangtao; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2012-09-17

    The serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper-dependent hydrophobic pocket located behind the adenine-binding region of the ATP-binding site. These compounds exhibit IC{sub 50} values in the low nanomolar range and are highly selective for Chk2 over Chk1. X-ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding.

  11. Cyclin I-like (CCNI2) is a cyclin-dependent kinase 5 (CDK5) activator and is involved in cell cycle regulation

    PubMed Central

    Liu, Chengcheng; Zhai, Xiaoyan; Zhao, Bin; Wang, Yanfei; Xu, Zhigang

    2017-01-01

    In contrast to conventional cyclin-dependent kinases that are important for mitotic cell division, cyclin-dependent kinase 5 (CDK5) is predominantly activated in post-mitotic cells and is involved in various cellular events. The kinase activity of CDK5 is tightly regulated by specific activators including p35, p39, and cyclin I (CCNI). Here we show that cyclin I-like (CCNI2), a homolog of CCNI, interacts with CDK5 and activates the kinase activity of CDK5. Different from CCNI, which colocalizes with CDK5 in the nuclei in transfected cells, CCNI2 mainly retains CDK5 in the cytoplasm as well as on the cell membrane. Furthermore, although the expression level of CCNI2 mRNA and CCNI2 protein do not change significantly during cell cycle, depletion of CCNI2 with siRNA affects cell cycle progression as well as cell proliferation. In conclusion, our data strongly suggest that CCNI2 is a novel CDK5 activator and is involved in cell cycle regulation. PMID:28112194

  12. Inhibition of p70 S6 Kinase (S6K1) Activity by A77 1726 and Its Effect on Cell Proliferation and Cell Cycle Progress12

    PubMed Central

    Doscas, Michelle E.; Williamson, Ashley J.; Usha, Lydia; Bogachkov, Yedida; Rao, Geetha S.; Xiao, Fei; Wang, Yimin; Ruby, Carl; Kaufman, Howard; Zhou, Jingsong; Williams, James W.; Li, Yi; Xu, Xiulong

    2014-01-01

    Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor–mediated signaling pathway. Invitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726–induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726–induced cell cycle arrest in the S phase. PMID:25379019

  13. Inhibition of p70 S6 kinase (S6K1) activity by A77 1726 and its effect on cell proliferation and cell cycle progress.

    PubMed

    Doscas, Michelle E; Williamson, Ashley J; Usha, Lydia; Bogachkov, Yedida; Rao, Geetha S; Xiao, Fei; Wang, Yimin; Ruby, Carl; Kaufman, Howard; Zhou, Jingsong; Williams, James W; Li, Yi; Xu, Xiulong

    2014-10-01

    Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 μM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.

  14. c-Jun NH(2)-terminal kinase signaling axis regulates diallyl trisulfide-induced generation of reactive oxygen species and cell cycle arrest in human prostate cancer cells.

    PubMed

    Antosiewicz, Jedrzej; Herman-Antosiewicz, Anna; Marynowski, Stanley W; Singh, Shivendra V

    2006-05-15

    We have shown previously that generation of reactive oxygen species (ROS) is a critical event in G(2)-M phase cell cycle arrest caused by diallyl trisulfide (DATS), which is a highly promising anticancer constituent of processed garlic. Using DU145 and PC-3 human prostate cancer cells as a model, we now report a novel mechanism involving c-Jun NH(2)-terminal kinase (JNK) signaling axis, which is known for its role in regulation of cell survival and apoptosis, in DATS-induced ROS production. The DATS-induced ROS generation, G(2)-M phase cell cycle arrest and degradation, and hyperphosphorylation of Cdc25C were significantly attenuated in the presence of EUK134, a combined mimetic of superoxide dismutase and catalase. Interestingly, the DATS-induced ROS generation and G(2)-M phase cell cycle arrest were also inhibited significantly in the presence of desferrioxamine, an iron chelator, but this protection was not observed with iron-saturated desferrioxamine. DATS treatment caused a marked increase in the level of labile iron that was accompanied by degradation of light chain of iron storage protein ferritin. Interestingly, DATS-mediated degradation of ferritin, increase in labile iron pool, ROS generation, and/or cell cycle arrest were significantly attenuated by ectopic expression of a catalytically inactive mutant of JNK kinase 2 and RNA interference of stress-activated protein kinase/extracellular signal-regulated kinase 1 (SEK1), upstream kinases in JNK signal transduction pathway. In conclusion, the present study provides experimental evidence to indicate existence of a novel pathway involving JNK signaling axis in regulation of DATS-induced ROS generation.

  15. Cell cycle-dependent control of polarised development by a cyclin-dependent kinase-like protein in the Fucus zygote.

    PubMed

    Corellou, F; Brownlee, C; Kloareg, B; Bouget, F Y

    2001-11-01

    Although iterative development can be uncoupled from morphogenesis in plant organs, the relationship between the cell cycle and developmental events is not well established in embryos. Zygotes of fucoid algae, including Fucus and Pelvetia are particularly well suited for studying the interaction(s) between cell cycle progression and the early morphogenetic events, as the establishment of polarity and its morphogenetic expression, i.e. germination, and the first cell cycle are concomitant. We have previously demonstrated that, in Fucus zygotes, various aspects of cell cycle progression are tightly controlled by cyclin-dependent kinase (CDK)-like proteins, including two PSTAIRE CDK-like proteins, p34 and p32, which are synthesised after fertilisation. We show that specific inhibition of CDK-like proteins, either with purine derivatives such as olomoucine and amino-purvalanol or by microinjection of the CDK inhibitor p21(cip1), prevents germination and cell division. Whereas direct inhibition of DNA replication by aphidicolin did not affect polarised development, olomoucine, which has previously been shown to prevent entry in S phase, and other purine derivatives also inhibited photopolarisation. Early microinjection of a monoclonal anti-PSTAIRE antibody also prevented germination and cell division. Only p34 had affinity for amino-purvalanol, suggesting that among PSTAIRE CDKs, this protein is the main target of purine derivatives. Models to account for the simultaneous control of early cell cycle progression and polarisation are proposed.

  16. RNAi mediated silencing of cyclin-dependent kinases of G1 phase slows down the cell-cycle progression and reduces apoptosis.

    PubMed

    Sierant, Malgorzata; Piotrzkowska, Danuta; Nawrot, Barbara

    2015-01-01

    One of the hypotheses on the origin of Alzheimer's disease (AD) stems from a close relation between a re-activation of a cell-cycle in post-mitotic neurons and a neural cells death observed in pathologically affected parts of AD brains. In the normal, healthy brain almost all neural cells are terminally differentiated and "locked" in the G0 phase of the cell-cycle. For these cells, the consequence of the re-entry to the cell-cycle is targeting them towards cellular divisions and turning on the apoptotic pathway. We used an RNA interference (RNAi) methodology in neural cells to switch-off genes for two cyclindependent kinases 4 and 6 (cdk4, cdk6), which control the activation of the initial steps of the cell-cycle. As a result, some evidences are delivered that silencing these genes, which are expressed during cell proliferation but inhibited at mature neurons, prevents the stimulation of apoptotic pathways in the neural cells cultured in a oxidative stress conditions and may have a neuroprotective effect. We demonstrate that down-regulation of genes important in the G1 phase of the cell-cycle may play the protective function on the neuronal cells, and can be considered as the promising approach for the potential gene therapy of neurodegenerative diseases.

  17. Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

    PubMed Central

    Wood, Matthew D

    2010-01-01

    The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events. PMID:20716966

  18. The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle.

    PubMed

    Kassardjian, Ari; Rizkallah, Raed; Riman, Sarah; Renfro, Samuel H; Alexander, Karen E; Hurt, Myra M

    2012-01-01

    Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

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

    PubMed Central

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

    2008-01-01

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

  20. Recovery from the DNA Replication Checkpoint.

    PubMed

    Chaudhury, Indrajit; Koepp, Deanna M

    2016-10-28

    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.

  1. 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

  2. Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis1[OPEN

    PubMed Central

    Hamdoun, Safae; Zhang, Chong; Gill, Manroop; Churchman, Michelle; Larkin, John C.

    2016-01-01

    Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions. PMID:26561564

  3. Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

    PubMed Central

    Silva-Santisteban, M. Cris; Westwood, Isaac M.; Boxall, Kathy; Brown, Nathan; Peacock, Sam; McAndrew, Craig; Barrie, Elaine; Richards, Meirion; Mirza, Amin; Oliver, Antony W.; Burke, Rosemary; Hoelder, Swen; Jones, Keith; Aherne, G. Wynne; Blagg, Julian; Collins, Ian; Garrett, Michelle D.; van Montfort, Rob L. M.

    2013-01-01

    Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors. PMID:23776527

  4. Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans.

    PubMed

    Church, D L; Guan, K L; Lambie, E J

    1995-08-01

    In the germline of Caenorhabditis elegans hermaphrodites, meiotic cell cycle progression occurs in spatially restricted regions. Immediately after leaving the distal mitotic region, germ cells enter meiosis and thereafter remain in the pachytene stage of first meiotic prophase for an extended period. At the dorsoventral gonadal flexure, germ cells exit pachytene and subsequently become arrested in diakinesis. We have found that exit from pachytene is dependent on the function of three members of the MAP kinase signaling cascade. One of these genes, mek-2, is a newly identified C. elegans MEK (MAP kinase kinase). The other two genes, mpk-1/sur-1 (MAP kinase) and let-60 ras, were previously identified based on their roles in vulval induction and are shown here to act in combination with mek-2 to permit exit from pachytene. Through genetic mosaic analysis, we demonstrate that the expression of mpk-1/sur-1 is required within the germline to permit exit from pachytene.

  5. Inhibition of Aurora A Kinase by Alisertib Induces Autophagy and Cell Cycle Arrest and Increases Chemosensitivity in Human Hepatocellular Carcinoma HepG2 Cells.

    PubMed

    Zhu, Qiaohua; Yu, Xinfa; Zhou, Zhi-Wei; Zhou, Chengyu; Chen, Xiao-Wu; Zhou, Shu-Feng

    2017-01-01

    Aurora A kinase represent a feasible target in cancer therapy. To evaluate the proteomic response of human liver carcinoma cells to alisertib (ALS) and identify the molecular targets of ALS, we examined the effects of ALS on the proliferation, cell cycle, autophagy, apoptosis, and chemosensitivity in HepG2 cells. The stable-isotope labeling by amino acids in cell culture (SILAC) based quantitative proteomic study was performed to evaluate the proteomic response to ALS. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. Our SILAC proteomic study showed that ALS regulated the expression of 914 proteins, with 407 molecules being up-regulated and 507 molecules being down-regulated in HepG2 cells. Ingenuity pathway analysis (IPA) and KEGG pathway analysis identified 146 and 32 signaling pathways were regulated by ALS, respectively, which were associated with cell survival, programmed cell death, and nutrition-energy metabolism. Subsequently, the verification experiments showed that ALS remarkably arrested HepG2 cells in G2/M phase and led to an accumulation of aneuploidy via regulating the expression of key cell cycle regulators. ALS induced a marked autophagy in a concentration- and time-dependent manner via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Autophagy inhibition promoted the pro-apoptotic effect of ALS, indicating a cyto-protective role of ALS-induced autophagy. ALS increased the chemosensitivity of HepG2 cells to cisplatin and doxorubicin. Taken together, ALS induces autophagy and cell cycle arrest in HepG2 cells via PI3K/Akt/mTOR-mediated pathway. Autophagy inhibition may promote the anticancer effect of ALS and sensitize the chemotherapy in HepG2 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    PubMed

    Szymiczek, A; Carbone, M; Pastorino, S; Napolitano, A; Tanji, M; Minaai, M; Pagano, I; Mason, J M; Pass, H I; Bray, M R; Mak, T W; Yang, H

    2017-07-31

    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. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) 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.Oncogene advance online publication, 31 July 2017; doi:10.1038/onc.2017.266.

  7. Chromosomal Instability Is Associated with Higher Expression of Genes Implicated in Epithelial-Mesenchymal Transition, Cancer Invasiveness, and Metastasis and with Lower Expression of Genes Involved in Cell Cycle Checkpoints, DNA Repair, and Chromatin Maintenance1

    PubMed Central

    Roschke, Anna V; Glebov, Oleg K; Lababidi, Samir; Gehlhaus, Kristen S; Weinstein, John N; Kirsch, Ilan R

    2008-01-01

    Chromosomal instability—a hallmark of epithelial cancers—is an ongoing process that results in aneuploidy and karyotypic heterogeneity of a cancer cell population. Previously, we stratified cancer cell lines in the NCI-60 drug discovery panel based on their karyotypic complexity and heterogeneity. Using this stratification in conjunction with drug response data for the cell lines allowed us to identify classes of chemical compounds whose growthinhibitory activity correlates with karyotypic complexity and chromosomal instability. In this article, we asked the question: What are the biological processes, pathways, or genes associated with chromosomal instability of cancer cells? We found that increased instability of the chromosomal content in a cancer cell population, particularly, persistent gains and losses of chromosomes, is associated with elevated expression of genes involved with aggressive cellular behavior, including invasion- and metastasis-associated changes in cell communication, adhesion, motility, and migration. These same karyotypic features are negatively correlated with the expression of genes involved in cell cycle checkpoints, DNA repair, and chromatin maintenance. PMID:18953431

  8. 5-Aza-2′-deoxycytidine Sensitizes Busulfan-resistant Myeloid Leukemia Cells By Regulating Expression of Genes Involved in Cell Cycle Checkpoint and Apoptosis

    PubMed Central

    Valdez, Benigno C.; Li, Yang; Murray, David; Corn, Paul; Champlin, Richard E.; Andersson, Borje S.

    2009-01-01

    Busulfan (Bu) is a DNA-alkylating drug used in myeloablative pretransplant conditioning therapy for patients with myeloid leukemia (ML). A major obstacle to successful treatment is cellular Bu-resistance. To investigate the possible contribution of DNA hypermethylation to Bu-resistance, we examined the cytotoxic activity of combined 5-aza-2′-deoxycytidine (DAC) and Bu. Exposure of Bu-resistant B5/Bu2506 ML cells to 0.5 μM DAC resulted in G2-arrest and apoptosis. The observed G2-arrest was associated with hypomethylation and subsequent expression of epigenetically controlled genes including p16INK4A, activation of the p53 pathway, and phosphorylation of CDC2. The DAC-mediated apoptosis was partly due to hypomethylation and up-regulation of XAF1, which resulted in down-regulation of the anti-apoptotic proteins XIAP, cIAP1 and cIAP2. The pro-apoptotic PUMA and BNIP3 proteins were up-regulated while pro-survival STAT3 and c-MYC were suppressed. Combination of 0.05 μM DAC and 5 μg/ml Bu resulted in synergistic cytotoxicity, which was associated with PARP1 cleavage and activation of caspases 3 and 8, suggesting induction of an apoptotic response. P53 inhibition in B5/Bu2506 cells using pifithrin-α alleviated these effects, suggesting a role for p53 therein; this observation was supported by the relative resistance of p53-null K562 cells to [DAC+Bu] combinations and by the effects of an anti-p53 shRNA on the OCI-AML3 cell line. We conclude that the synergistic effects of [DAC+Bu] are p53-dependent and involve cell-cycle arrest, apoptosis induction and down-regulation of pro-survival genes. Our results suggest that, depending on tumor p53 status, incorporation of DAC might synergistically improve the cytoreductive efficacy of Bu-based pre-transplant regimen in patients with ML. PMID:19732952

  9. Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1

    PubMed Central

    Greer, Yoshimi Endo; Gao, Bo; Yang, Yingzi; Nussenzweig, Andre; Rubin, Jeffrey S.

    2017-01-01

    Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1. PMID:28125685

  10. [Cell cycle, mitosis and therapeutic applications].

    PubMed

    Levy, Antonin; Albiges-Sauvin, Laurence; Massard, Christophe; Soria, Jean-Charles; Deutsch, Eric

    2011-10-01

    Genomic DNA is constantly under stress of endogenous and exogenous DNA damaging agents. Without proper care, the DNA damage causes an alteration of the genomic structure and can lead to cell death or the occurrence of mutations involved in tumorigenesis. During the process of evolution, organisms have acquired a series of response mechanisms and repair of DNA damage, thereby ensuring the maintenance of genome stability and faithful transmission of genetic information. The checkpoints are the major mechanisms by which a cell can respond to DNA damage, either by actively stopping the cell cycle or by induction of apoptosis. Two parallel signalling pathways, ATM and ATR respond to genotoxic stress by activating their downstream target proteins including the two effectors kinases CHK1 and CHK2. Promising preliminary data render these proteins potential targets for therapeutic development against cancer.

  11. Phase I Study of LY2606368, a Checkpoint Kinase 1 Inhibitor, in Patients With Advanced Cancer

    PubMed Central

    Infante, Jeffrey; Janku, Filip; Jones, Suzanne; Nguyen, Ly M.; Burris, Howard; Naing, Aung; Bauer, Todd M.; Piha-Paul, Sarina; Johnson, Faye M.; Kurzrock, Razelle; Golden, Lisa; Hynes, Scott; Lin, Ji; Lin, Aimee Bence; Bendell, Johanna

    2016-01-01

    Purpose The primary objective was to determine safety, toxicity, and a recommended phase II dose regimen of LY2606368, an inhibitor of checkpoint kinase 1, as monotherapy. Patients and Methods This phase I, nonrandomized, open-label, dose-escalation trial used a 3 + 3 dose-escalation scheme and included patients with advanced solid tumors. Intravenous LY2606368 was dose escalated from 10 to 50 mg/m2 on schedule 1 (days 1 to 3 every 14 days) or from 40 to 130 mg/m2 on schedule 2 (day 1 every 14 days). Safety measures and pharmacokinetics were assessed, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Results Forty-five patients were treated; seven experienced dose-limiting toxicities (all hematologic). The maximum-tolerated doses (MTDs) were 40 mg/m2 (schedule 1) and 105 mg/m2 (schedule 2). The most common related grade 3 or 4 treatment-emergent adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (typically < 5 days). Febrile neutropenia incidence was low (7%). The LY2606368 exposure over the first 72 hours (area under the curve from 0 to 72 hours) at the MTD for each schedule coincided with the exposure in mouse xenografts that resulted in maximal tumor responses. Minor intra- and intercycle accumulation of LY2606368 was observed at the MTDs for both schedules. Two patients (4.4%) had a partial response; one had squamous cell carcinoma (SCC) of the anus and one had SCC of the head and neck. Fifteen patients (33.3%) had a best overall response of stable disease (range, 1.2 to 6.7 months), six of whom had SCC. Conclusion An LY2606368 dose of 105 mg/m2 once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC. PMID:27044938

  12. Effect of positive feedback loops on the robustness of oscillations in the network of cyclin-dependent kinases driving the mammalian cell cycle.

    PubMed

    Gérard, Claude; Gonze, Didier; Goldbeter, Albert

    2012-09-01

    The transitions between the G(1), S, G(2) and M phases of the mammalian cell cycle are driven by a network of cyclin-dependent kinases (Cdks), whose sequential activation is regulated by intertwined negative and positive feedback loops. We previously proposed a detailed computational model for the Cdk network, and showed that this network is capable of temporal self-organization in the form of sustained oscillations, which govern ordered progression through the successive phases of the cell cycle [Gérard and Goldbeter (2009) Proc Natl Acad Sci USA 106, 21643-21648]. We subsequently proposed a skeleton model for the cell cycle that retains the core regulatory mechanisms of the detailed model [Gérard and Goldbeter (2011) Interface Focus 1, 24-35]. Here we extend this skeleton model by incorporating Cdk regulation through phosphorylation/dephosphorylation and by including the positive feedback loops that underlie the dynamics of the G(1)/S and G(2)/M transitions via phosphatase Cdc25 and via phosphatase Cdc25 and kinase Wee1, respectively. We determine the effects of these positive feedback loops and ultrasensitivity in phosphorylation/dephosphorylation on the dynamics of the Cdk network. The multiplicity of positive feedback loops as well as the existence of ultrasensitivity promote the occurrence of bistability and increase the amplitude of the oscillations in the various cyclin/Cdk complexes. By resorting to stochastic simulations, we further show that the presence of multiple, redundant positive feedback loops in the G(2)/M transition of the cell cycle markedly enhances the robustness of the Cdk oscillations with respect to molecular noise. © 2012 The Authors Journal compilation © 2012 FEBS.

  13. AP-1 blockade in breast cancer cells causes cell cycle arrest by suppressing G1 cyclin expression and reducing cyclin-dependent kinase activity.

    PubMed

    Liu, Yongmin; Lu, Chunhua; Shen, Qiang; Munoz-Medellin, Debbie; Kim, Heetae; Brown, Powel H

    2004-10-28

    The AP-1 transcription factor is a central component of signal transduction pathways in many cells, although the exact role of AP-1 in controlling cell growth and malignant transformation is unknown. We have previously shown that AP-1 complexes are activated by peptide and steroid growth factors in both normal and malignant breast cells, and that blocking AP-1 by overexpressing a dominant-negative form of cJun (cJun-DN, TAM67) inhibits breast cancer cell growth both in vivo and in vitro. We hypothesized that TAM67 inhibits cell growth by altering the expression of cell cycle regulatory proteins, thus causing a cell cycle block. In the present study, we used clones of MCF7 breast cancer cells that express TAM67 under the control of an inducible promoter. First, we determined the effect of AP-1 blockade on cell growth, then we performed 3H-thymidine incorporation and flow cytometry assays to investigate whether TAM67 inhibits the cell cycle. We observed that in the presence of serum TAM67 inhibited cell growth and caused a block in the G1 phase of the cell cycle. Next, we performed Western-blotting and CDK kinase assays to determine the effects of TAM67 on retinoblastoma (Rb) phosphorylation, the expression of cell cycle regulatory proteins, and CDK activity. We discovered that TAM67 inhibited Rb phosphorylation and reduced E2F activity. We also found that TAM67 decreased the expression of D and E cyclins, reduced CDK2 and CDK4 activity, and increased the CDK inhibitor p27. The studies of gene expression at the RNA level showed that TAM67 decreased cyclin Ds mRNA expression. Our study suggests that in the presence of serum, TAM67 inhibits breast cancer growth predominantly by inducing inhibitors of cyclin-dependent kinases (such as p27) and by reducing the expression of the cyclins involved in transitioning from G1 into S phase of the cell cycle. These studies lay the foundation for future attempt to develop new agents for the treatment and prevention of breast

  14. Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    PubMed

    Golsteyn, R M; Mundt, K E; Fry, A M; Nigg, E A

    1995-06-01

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

  15. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    SciTech Connect

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

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

    PubMed

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

    2011-08-02

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

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

    PubMed Central

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

    2011-01-01

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother–bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell. PMID:21709215

  18. The alpha-fetoprotein (AFP) third domain: a search for AFP interaction sites of cell cycle proteins.

    PubMed

    Mizejewski, G J

    2016-09-01

    The carboxy-terminal third domain of alpha-fetoprotein (AFP-3D) is known to harbor binding and/or interaction sites for hydrophobic ligands, receptors, and binding proteins. Such reports have established that AFP-3D consists of amino acid (AA) sequence stretches on the AFP polypeptide that engages in protein-to-protein interactions with various ligands and receptors. Using a computer software program specifically designed for such interactions, the present report identified AA sequence fragments on AFP-3D that could potentially interact with a variety of cell cycle proteins. The cell cycle proteins identified were (1) cyclins, (2) cyclin-dependent kinases, (3) cell cycle-associated proteins (inhibitors, checkpoints, initiators), and (4) ubiquitin ligases. Following detection of the AFP-3D to cell cycle protein interaction sites, the computer-derived AFP localization AA sequences were compared and aligned with previously reported hydrophobic ligand and receptor interaction sites on AFP-3D. A literature survey of the association of cell cycle proteins with AFP showed both positive relationships and correlations. Previous reports of experimental AFP-derived peptides effects on various cell cycle proteins served to confirm and verify the present computer cell cycle protein identifications. Cell cycle protein interactions with AFP-CD peptides have been reported in cultured MCF-7 breast cancer cells subjected to mRNA microarray analysis. After 7 days in culture with MCF-7 cells, the AFP-derived peptides were shown to downregulate cyclin E, SKP2, checkpoint suppressors, cyclin-dependent kinases, and ubiquitin ligases that modulate cyclin E/CdK2 transition from the G1 to the S-phase of the cell cycle. Thus, the experimental data on AFP-CD interaction with cell cycle proteins were consistent with the "in silico" findings.

  19. The Botrytis cinerea PAK kinase BcCla4 mediates morphogenesis, growth and cell cycle regulating processes downstream of BcRac.

    PubMed

    Minz-Dub, Anna; Sharon, Amir

    2017-02-06

    Rac proteins are involved in a variety of cellular processes. Effector proteins that interact with active Rac convey the GTPase-generated signal to downstream developmental cascades and processes. Here we report on the analysis of the main effector and signal cascade downstream of BcRac, the Rac homolog of the grey mold fungus Botrytis cinerea. Several lines of evidence highlighted the p21-activated kinase Cla4 as an important effector of Rac in fungi. Analysis of Δbccla4 strains revealed that the BcCla4 protein was sufficient to mediate all of the examined BcRac-driven processes, including hyphal growth and morphogenesis, conidia production and pathogenicity. In addition, the Δbccla4 strains had altered nuclei content, a phenomenon that was previously observed in Δbcrac isolates, thus connecting the BcRac/BcCla4 module with cell cycle control. Further analyses revealed that BcRac/BcCla4 control mitotic entry through changes in phosphorylation status of the cyclin dependent kinase BcCdk1. The complete cascade includes the kinase BcWee1, which is downstream of BcCla4 and upstream of BcCdk1. These results provide a mechanistic insight on the connection of cell cycle, morphogenesis and pathogenicity in fungi, and position BcCla4 as the most essential effector and central regulator of all of these processes downstream of BcRac.

  20. Cell cycle- and protein kinase C-specific effects of resiniferatoxin and resiniferonol 9,13,14-ortho-phenylacetate in intestinal epithelial cells.

    PubMed

    Frey, Mark R; Clark, Jennifer A; Bateman, Nicholas W; Kazanietz, Marcelo G; Black, Adrian R; Black, Jennifer D

    2004-05-15

    We have previously reported that protein kinase C (PKC) signaling can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells, including downregulation of cyclin D1, induction of p21(Waf1/Cip1), and activation of the growth suppressor function of pocket proteins. In the current study, we compared the cell cycle- and PKC-specific effects of the vanilloid resiniferatoxin (RTX), its parent diterpene resiniferonol 9,13,14-ortho-phenylacetate (ROPA), and the PKC agonist PMA in the IEC-18 non-transformed intestinal crypt cell line. ROPA and PMA were found to produce strikingly similar alterations in cell cycle progression and PKC activity in IEC-18 cells, although PMA was approximately 1000-fold more potent in producing these effects. Both agents induced a transient PKC-dependent blockade in G1---> S progression associated with transient downregulation of cyclin D1 and induction of p21(Waf1/Cip1). In contrast, RTX produced a prolonged PKC-independent cell cycle arrest in G(0)/G(1) phase which was maintained for longer than 24h. This arrest was vanilloid receptor-independent and associated with prolonged downregulation of cyclin D1 mRNA and protein, with little effect on levels of p21(Waf1/Cip1). Combined exposure to RTX and ROPA produced a sustained and complete cell cycle blockade in IEC-18 cells, associated with depletion of cyclin D1 and sustained enhancement of p21(Waf1/Cip1) levels. PMA, ROPA, RTX and the RTX/ROPA combination were capable of activating ERK1/2 signaling in IEC-18 cells, albeit with different kinetics. In contrast, only PMA and ROPA activated JNK1/2 and p38 in this system. Notably, some preparations of commercially obtained RTX produced effects indistinguishable from those of the RTX/ROPA combination, suggesting that certain batches of the compound may contain significant amounts of ROPA (or another PKC agonist activity). Together, these data demonstrate that structurally related compounds can produce similar cell cycle

  1. 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.

  2. Fission yeast TOR complex 2 activates the AGC-family Gad8 kinase essential for stress resistance and cell cycle control.

    PubMed

    Ikeda, Kyoko; Morigasaki, Susumu; Tatebe, Hisashi; Tamanoi, Fuyuhiko; Shiozaki, Kazuhiro

    2008-02-01

    Members of the mitogen-activated protein kinase (MAPK) subfamily responsive to environmental stress stimuli are known as SAPKs (stress-activated protein kinases), which are conserved from yeast to humans. In the fission yeast Schizosaccharomyces pombe, Spc1/Sty1 SAPK is activated by diverse forms of stress, such as osmostress, oxidative stress and heat shock, and induces gene expression through the Atf1 transcription factor. Sin1 (SAPK interacting protein 1) was originally isolated as a protein that interacts with Spc1, and its orthologs were also found in diverse eukaryotes. Here we report that Sin1 is not required for the stress gene expression regulated by Spc1 and Atf1, and that Sin1 is an essential component of TOR (target of rapamycin) complex 2 (TORC2). TORC2 is not essential for cell viability in S. pombe but plays important roles in cellular survival of stress conditions through phosphorylation and activation of an AGC-family protein kinase, Gad8. In addition, inactivation of Gad8 results in a synthetic growth defect with cdc25-22, a temperature-sensitive mutation of the Cdc25 phosphatase that activates Cdc2 kinase at G(2)/M. Gad8 also positively regulates expression of the CDK inhibitor gene rum1+, which is essential for cell cycle arrest in G(1) after nitrogen starvation. These results strongly suggest that the TORC2-Gad8 pathway has multiple physiological functions in cellular stress resistance and cell cycle progression at both G(1)/S and G(2)/M transitions.

  3. Phospho-Ser383-Elk-1 is localized to the mitotic spindles during cell cycle and interacts with mitotic kinase Aurora-A.

    PubMed

    Demir, Ozlem; Kurnaz, Isil Aksan

    2013-10-01

    Elk-1 is a member of the E-twenty-six (ETS) domain superfamily of transcription factors and has been traditionally associated with mitogen-induced immediate early gene transcription upon phosphorylation by mitogen activated protein kinases (ERK/MAPK). Elk-1 is not only upregulated but also phosphorylated in brain tumour cells. However, in this study, we show for the first time that S383-phosphorylated Elk-1 (P-S383-Elk-1) is associated with mitotic spindle poles from metaphase through telophase and relocates to the spindle midbody during cytokinesis, while Thr417Ala mutation is associated with DNA throughout mitosis. Serine 383 phosphorylation appears to be important for polar localization of Elk-1, since exogenous protein including serine-to-alanine mutation was seen to be distributed throughout the spindle fibres. We further show that Elk-1 interacts with the cell cycle kinase Aurora-A, and when Aurora inhibitors are used, P-S383-Elk-1 fails to localize to the poles and remains associated with DNA. Apart from one transcriptional repressor molecule, Kaiso, this is the first time a transactivator was shown to possess such mitotic localization and interaction. The functional significance and detailed mechanism of this cell cycle-related localization of Elk-1 are yet to be determined.

  4. Targeted disruption of the murine homeodomain-interacting protein kinase-2 causes growth deficiency in vivo and cell cycle arrest in vitro.

    PubMed

    Trapasso, Francesco; Aqeilan, Rami I; Iuliano, Rodolfo; Visone, Rosa; Gaudio, Eugenio; Ciuffini, Laura; Alder, Hansjuerg; Paduano, Francesco; Pierantoni, Giovanna Maria; Soddu, Silvia; Croce, Carlo M; Fusco, Alfredo

    2009-04-01

    The homeodomain-interacting protein kinase 2 (HIPK2) protein is a member of a recently identified family of nuclear protein kinases that are well conserved in various organisms. HIPK2 can bind to several homeotic factors and to a series of proteins involved in the regulation of cell survival and proliferation in response to morphogenetic and genotoxic signals. Here we report Hipk2-targeted disruption in mouse; Hipk2(-/-) mice are viable and fertile but significantly smaller than their wild-type littermates. This feature is present at birth and retained throughout the mouse adulthood. Mouse embryo fibroblasts from Hipk2(-/-) mice show a reduced proliferation rate, compared to the wild-type counterparts, with accumulation in the G0/G1 phase of the cell cycle and altered levels of the cell cycle regulators cyclin D and CDK6. Restoration of wild-type HIPK2 expression in Hipk2(-/-) cells rescues the normal phenotype supporting a role for HIPK2 in the regulation of cell proliferation.

  5. Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

    PubMed Central

    Nair, Jayasree S.; Ho, Alan L.; Tse, Archie N.; Coward, Jesse; Cheema, Haider; Ambrosini, Grazia; Keen, Nicholas

    2009-01-01

    The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein. PMID:19225156

  6. Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

    PubMed Central

    Krenn, Veronica; Wehenkel, Annemarie; Santaguida, Stefano

    2012-01-01

    The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1. PMID:22331848

  7. Tethering DNA damage checkpoint mediator proteins topoisomerase IIbeta-binding protein 1 (TopBP1) and Claspin to DNA activates ataxia-telangiectasia mutated and RAD3-related (ATR) phosphorylation of checkpoint kinase 1 (Chk1).

    PubMed

    Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

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

    PubMed Central

    Liu, Xiaoqun; Liu, Xiangdong; Qiao, Tiankui; Chen, Wei; Yuan, Sujuan

    2015-01-01

    Objective The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM) kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2) on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909) in human lung adenocarcinoma A549 cells. Methods In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+ X-ray, ATM kinase-small interfering RNA (siRNA)+CpG+X-ray (ATM-siRNA), and Chk2-siRNA+CpG+X-ray (Chk2-siRNA) groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry. Results Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively), though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively) and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01) when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM

  9. The metabolic checkpoint kinase mTOR is essential for interleukin-15 signaling during NK cell development and activation

    PubMed Central

    Marçais, Antoine; Degouve, Sophie; Viel, Sébastien; Fenis, Aurore; Rabilloud, Jessica; Mayol, Katia; Tavares, Armelle; Bienvenu, Jacques; Gangloff, Yann-Gaël; Gilson, Eric; Vivier, Eric; Walzer, Thierry

    2014-01-01

    Interleukin-15 (IL-15) controls both the homeostasis and the peripheral activation of Natural Killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we report that the metabolic checkpoint kinase mTOR is activated and boosts bioenergetic metabolism upon NK cell exposure to high concentrations of IL-15 whereas low doses of IL-15 only triggers the phosphorylation of the transcription factor STAT5. mTOR stimulates NK cell growth and nutrient uptake and positively feeds back onto the IL-15 receptor. This process is essential to sustain NK cell proliferation during development and acquisition of cytolytic potential upon inflammation or virus infection. The mTORC1 inhibitor rapamycin inhibits NK cell cytotoxicity both in mouse and human, which likely contribute to the immunosuppressant activities of this drug in different clinical settings. PMID:24973821

  10. DW-MRI as a Predictive Biomarker of Radiosensitization of GBM through Targeted Inhibition of Checkpoint Kinases.

    PubMed

    Williams, Terence M; Galbán, Stefanie; Li, Fei; Heist, Kevin A; Galbán, Craig J; Lawrence, Theodore S; Holland, Eric C; Thomae, Tami L; Chenevert, Thomas L; Rehemtulla, Alnawaz; Ross, Brian D

    2013-04-01

    The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.

  11. Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

    PubMed

    Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

    2010-04-01

    Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

  12. Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry.

    PubMed Central

    Brunet, A; Roux, D; Lenormand, P; Dowd, S; Keyse, S; Pouysségur, J

    1999-01-01

    Mitogen-activated protein kinase (MAPK) modules, composed of three protein kinases activated by successive phosphorylation, are involved in the signal transduction of a wide range of extracellular agents. In mammalian cells, mitogenic stimulation triggers the translocation of p42/p44MAPK from the cytoplasm to the nucleus, whereas the other protein kinases of the module remain cytosolic. Since MAPK has been shown to phosphorylate and activate nuclear targets, such as the transcription factor Elk1, it has been proposed, but not yet demonstrated, that MAPK nuclear translocation could represent a critical step in signal transduction. In this study, we sequestered p42/p44MAPK in the cytoplasm by the expression of a catalytically inactive form of cytoplasmic MAP kinase phosphatase (MKP-3/Pyst-1). Sequestering MAPK in the cytoplasm did not alter its activation or its ability to phosphorylate cytoplasmic substrates of MAPK (p90RSK1 or an engineered cytoplasmic form of Elk1). In contrast, prevention of MAPK nuclear translocation strongly inhibited Elk1-dependent gene transcription and the ability of cells to reinitiate DNA replication in response to growth factors. Thus the relocalization of MAPK to the nucleus appears to be an important regulatory step for mitogen-induced gene expression and cell cycle re-entry. PMID:9927426

  13. Chloroplast division checkpoint in eukaryotic algae

    PubMed Central

    Sumiya, Nobuko; Fujiwara, Takayuki; Era, Atsuko; Miyagishima, Shin-ya

    2016-01-01

    Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle. PMID:27837024

  14. Cell Cycle Regulation and Melanoma.

    PubMed

    Xu, Wen; McArthur, Grant

    2016-06-01

    Dysregulation of cell cycle control is a hallmark of melanomagenesis. Agents targeting the G1-S and G2-M checkpoints, as well as direct anti-mitotic agents, have all shown promising preclinical activity in melanoma. However, in vivo, standalone single agents targeting cell cycle regulation have only demonstrated modest efficacy in unselected patients. The advent of specific CDK 4/6 inhibitors targeting the G1-S transition, with an improved therapeutic index, is a significant step forward. Potential synergy exists with the combination of CDK4/6 inhibitors with existing therapies targeting the MAPK pathway, particularly in subsets of metastatic melanomas such as NRAS and BRAF mutants. This reviews summaries of the latest developments in both preclinical and clinical data with cell cycle-targeted therapies in melanoma.

  15. Plitidepsin has a dual effect inhibiting cell cycle and inducing apoptosis via Rac1/c-Jun NH2-terminal kinase activation in human melanoma cells.

    PubMed

    Muñoz-Alonso, María J; González-Santiago, Laura; Zarich, Natasha; Martínez, Teresa; Alvarez, Enrique; Rojas, José María; Muñoz, Alberto

    2008-03-01

    Melanoma is the most aggressive skin cancer and a serious health problem worldwide because of its increasing incidence and the lack of satisfactory chemotherapy for late stages of the disease. The marine depsipeptide Aplidin (plitidepsin) is an antitumoral agent under phase II clinical development against several neoplasias, including melanoma. We report that plitidepsin has a dual effect on the human SK-MEL-28 and UACC-257 melanoma cell lines; at low concentrations (cell cycle by inducing G(1) and G(2)/M arrest, whereas at higher concentrations it induces apoptosis as assessed by poly-(ADP-ribose) polymerase cleavage and the appearance of a hypodiploid peak in flow cytometry analyses. Plitidepsin activates Rac1 GTPase and c-Jun NH(2)-terminal kinase (JNK). In addition, it induces AKT and p38 mitogen-activated protein kinase (MAPK) phosphorylation. By using inhibitors, we found that JNK and p38 MAPK activation depends on Rac1 but not on phosphatidylinositol 3-kinase (PI3K), whereas AKT activation is independent of Rac1 but requires PI3K activity. Plitidepsin cytotoxicity diminishes by Rac1 inhibition or by the blockage of JNK and p38 MAPK using 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), but not by PI3K inhibition using wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). It is remarkable that plitidepsin and dacarbazine, the alkylating agent most active for treating metastatic melanoma, show a synergistic antiproliferative effect that was paralleled at the level of JNK activation. These results indicate that Rac1/JNK activation is critical for cell cycle arrest and apoptosis induction by plitidepsin in melanoma cells. They also support the combined use of plitidepsin and dacarbazine in in vivo studies.

  16. Distinct effects of methylseleninic acid versus selenite on apoptosis, cell cycle, and protein kinase pathways in DU145 human prostate cancer cells.

    PubMed

    Jiang, Cheng; Wang, Zaisen; Ganther, Howard; Lü, Junxuan

    2002-10-01

    Selenium has been implicated as a promising chemopreventive agent for prostate cancer. Whereas the anticancer mechanisms have not been clearly defined, one hypothesis relates to selenium metabolites, especially the monomethyl selenium pool, generated under supranutritional selenium supplementation. To explore potential molecular targets for mediating the chemopreventive activity, we contrasted the effects of methylseleninic acid (MSeA), a novel precursor of methylselenol, versus sodium selenite, a representative of the hydrogen selenide metabolite pool, on apoptosis execution, cell cycle distribution, and selected protein kinases in DU145 human prostate cancer cells. Exposure of DU145 cells to 3 microM MSeA led to a profound G1 arrest at 24 h, and exposure to greater concentrations led to not only G1 arrest, but also to DNA fragmentation and caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP), two biochemical hallmarks of apoptosis. Immunobiot analyses indicated that G1 arrest induced by the subapoptogenic doses of MSeA was associated with increased expression of p27kip1 and p21cip1, but apoptosis was accompanied by dose-dependent decreases of phosphorylation of protein kinase AKT and extracellular signal-regulated kinase (ERK1/2) in the absence of any phosphorylation change in p38 mitogen-activated protein kinase (p38MAPK) and c-Jun NH2-terminal kinase (JNK1/2). In contrast, selenite exposure caused S-phase arrest and caspase-independent apoptotic DNA fragmentation, which were associated with decreased expression of p27kip1 and p21cip1 and increased phosphorylation of AKT, JNK1/2, and p38MAPK. Although apoptosis induction by MSeA exposure was not sensitive to superoxide dismutase added into the cell culture medium, cell detachment and DNA nucleosomal fragmentation induced by selenite exposure were greatly attenuated by this enzyme, supporting a chemical mediator role of superoxide for these processes. Despite a temporal relationship of AKT and ERK1

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

    PubMed Central

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

    2009-01-01

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential. PMID:19741151

  18. Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression, and Proliferation in Human Islets

    PubMed Central

    Liu, Hui; Remedi, Maria S.; Pappan, Kirk L.; Kwon, Guim; Rohatgi, Nidhi; Marshall, Connie A.; McDaniel, Michael L.

    2009-01-01

    OBJECTIVE—Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/β-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human β-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS—Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of β-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS—Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of β-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human β-cells. Nuclear translocation of β-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin. CONCLUSIONS—A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human β-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation. PMID:19073772

  19. [Jaridonin, a new diterpenoid from Isodon rubescens, induces cell cycle arrest in gastric cancer cells through activating ataxia telangiectasia mutated kinase].

    PubMed

    Ma, Y C; Su, N; Zhao, N M; Li, Q Y; Zhang, M; Zhao, H W; Liu, H M; Qin, Y H

    2016-04-01

    To study the effects of Jaridonin, a novel diterpenoid from isodon rubescens, on the cell cycle of human gastric cancer cells and its molecular mechanism of action. Flow cytometry was used to analyze the cell cycle distribution and expression of ataxia telangiectasia mutated kinase (ATM) after Jaridonin treatment. Western blot was performed to detect the expression of cell cycle-related proteins. The results of flow cytometry showed that the percentages of MGC-803 cells in G(2)/M phase at 6 hours after 0, 10, 20 μmol/L Jaridonin-treatment were (10.8±2.2)%, (18.2±2.5)%, (27.3±3.2)%, respectively; those at 12 hours after Jaridonin-treatment were (12.0±1.5)%, (24.1±2.0)% and (39.7±5.2)%, respectively, indicating a G2/M phase arrest of MGC-803 cells was resulted in a time- and dose-dependent manner. The expressions of ATM, Chk1, Chk2, phosphorylated Cdc2 and CDK2 were up-regulated in the MGC-803 cells after Jaridonin treatment, while the levels of Cdc2 and CDK2 were decreased. KU-55933, an inhibitor of ATM, reversed the expression of relevant proteins and G(2)/M phase arrest induced by Jaridonin. Jaridonin can significantly induce G(2)/M arrest in gastric cancer MGC-803 cells. Its mechanism may be related to the activation of ATM and Chk1/2, and inactivation of Cdc2 and CDK2 phosphorylation.

  20. Inhibition of Aurora-A kinase induces cell cycle arrest in epithelial ovarian cancer stem cells by affecting NFĸB pathway.

    PubMed

    Chefetz, Ilana; Holmberg, Jennie C; Alvero, Ayesha B; Visintin, Irene; Mor, Gil

    2011-07-01

    Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.

  1. Enterolactone induces G1-phase cell cycle arrest in non-small cell lung cancer cells by down-regulating cyclins and cyclin-dependent kinases

    PubMed Central

    Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M.

    2017-01-01

    Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG) which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anti-cancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study we investigated the anti-cancer effects of EL for several non-small cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The anti-proliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL- decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1-phase. The results suggest that EL inhibits the growth of NSCLC cell lines by down-regulating G1-phase cyclins and CDKs, and up-regulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy. PMID:28323486

  2. Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases.

    PubMed

    Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M

    2017-01-01

    Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21(WAF1/CIP1), a negative regulator of the G1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G1-phase cyclins and CDKs, and upregulating p21(WAF1/CIP1), which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

  3. Insulin-like growth factor-1 promotes G(1)/S cell cycle progression through bidirectional regulation of cyclins and cyclin-dependent kinase inhibitors via the phosphatidylinositol 3-kinase/Akt pathway in developing rat cerebral cortex.

    PubMed

    Mairet-Coello, Georges; Tury, Anna; DiCicco-Bloom, Emanuel

    2009-01-21

    Although survival-promoting effects of insulin-like growth factor-1 (IGF-1) during neurogenesis are well characterized, mitogenic effects remain less well substantiated. Here, we characterize cell cycle regulators and signaling pathways underlying IGF-1 effects on embryonic cortical precursor proliferation in vitro and in vivo. In vitro, IGF-1 stimulated cell cycle progression and increased cell number without promoting cell survival. IGF-1 induced rapid increases in cyclin D1 and D3 protein levels at 4 h and cyclin E at 8 h. Moreover, p27(KIP1) and p57(KIP2) expression were reduced, suggesting downregulation of negative regulators contributes to mitogenesis. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway specifically underlies IGF-1 activity, because blocking this pathway, but not MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal-regulated kinase), prevented mitogenesis. To determine whether mechanisms defined in culture relate to corticogenesis in vivo, we performed transuterine intracerebroventricular injections. Whereas blockade of endogenous factor with anti-IGF-1 antibody decreased DNA synthesis, IGF-1 injection stimulated DNA synthesis and increased the number of S-phase cells in the ventricular zone. IGF-1 treatment increased phospho-Akt fourfold at 30 min, cyclins D1 and E by 6 h, and decreased p27(KIP1) and p57(KIP2) expression. Moreover, blockade of the PI3K/Akt pathway in vivo decreased DNA synthesis and cyclin E, increased p27(KIP1) and p57(KIP2) expression, and prevented IGF-1-induced cyclin E mRNA upregulation. Finally, IGF-1 injection in embryos increased postnatal day 10 brain DNA content by 28%, suggesting a role for IGF-1 in brain growth control. These results demonstrate a mitogenic role for IGF-1 that tightly controls both positive and negative cell cycle regulators, and indicate that the PI3K/Akt pathway mediates IGF-1 mitogenic signaling during corticogenesis.

  4. Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

    SciTech Connect

    Lountos, George T.; Jobson, Andrew G.; Tropea, Joseph E.; Self, Christopher R.; Zhang, Guangtao; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2012-01-20

    Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.

  5. Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

    PubMed

    Linke, Monika; Pham, Ha Thi Thanh; Katholnig, Karl; Schnöller, Thomas; Miller, Anne; Demel, Florian; Schütz, Birgit; Rosner, Margit; Kovacic, Boris; Sukhbaatar, Nyamdelger; Niederreiter, Birgit; Blüml, Stephan; Kuess, Peter; Sexl, Veronika; Müller, Mathias; Mikula, Mario; Weckwerth, Wolfram; Haschemi, Arvand; Susani, Martin; Hengstschläger, Markus; Gambello, Michael J; Weichhart, Thomas

    2017-03-01

    The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

  6. Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

  7. Bortezomib and Fenretinide Induce Synergistic Cytotoxicity in Mantle Cell Lymphoma Via Apoptosis, Cell Cycle Dysregulation, and IκBα Kinase Down-regulation

    PubMed Central

    Cowan, Andrew J.; Frayo, Shani L.; Press, Oliver W.; Palanca-Wessels, Maria C.; Pagel, John M.; Green, Damian J.; Gopal, Ajay K.

    2015-01-01

    Background Mantle cell lymphoma (MCL) remains incurable for most patients and proteasome inhibitors like bortezomib induce responses in a minority of patients with relapsed disease. Fenretinide is a retinoid that has shown preclinical activity in B-cell lymphomas. We hypothesized that these agents could yield augmented anti-tumor activity. Methods Mantle cell lymphoma lines (Granta-519, Jeko-1, Rec-1) were treated with escalating concentrations of bortezomib and fenretinide singly and in combination. Cytotoxicity was assessed using the MTT assay. Flow cytometric methods assessed apoptosis and necrosis with annexin V-FITC/propidium iodide and G1 and G2 cell cycle changes with DAPI staining. Changes in Cyclin D1, Cyclin B, IκBα, and IKKα expression were quantified by Western blot. Results Cytotoxicity was mediated via apoptosis; both agents showed observed vs expected cytotoxicity in Granta-519 of 92.2% vs 55.1%, in Jeko-1 of 87.6% vs 36.3%, and in Rec-1 of 63.2% vs 29.8%. Isobolographic analysis confirmed synergy in Jeko-1 and Rec-1. Bortezomib induced G2 phase arrest with a 1.7 fold-increase over control, and fenretinide resulted in G1 phase arrest, with an increase of 1.3 fold over control. In combination G2 phase arrest predominated, with a 1.4 fold-increase compared to control, and reduced expression of Cyclin D1 to 24%, Cyclin B to 52% and 64%, Cyclin D3 to 25% and 43%, IκBα to 23% and 46%, and IκBα kinase to 34% and 44%. Conclusions Bortezomib and fenretinide exhibit synergistic cytotoxicity against MCL cell lines. This activity is mediated by IκBα kinase modulation, decreased cyclin expression, cell cycle dysregulation, and apoptotic cell death. PMID:26237500

  8. Bortezomib and fenretinide induce synergistic cytotoxicity in mantle cell lymphoma through apoptosis, cell-cycle dysregulation, and IκBα kinase downregulation.

    PubMed

    Cowan, Andrew J; Frayo, Shani L; Press, Oliver W; Palanca-Wessels, Maria C; Pagel, John M; Green, Damian J; Gopal, Ajay K

    2015-10-01

    Mantle cell lymphoma (MCL) remains incurable for most patients, and proteasome inhibitors like bortezomib induce responses in a minority of patients with relapsed disease. Fenretinide is a retinoid that has shown preclinical activity in B-cell lymphomas. We hypothesized that these agents could yield augmented antitumor activity. MCL lines (Granta-519, Jeko-1, and Rec-1) were treated with escalating concentrations of bortezomib and fenretinide singly and in combination. Cytotoxicity was assessed using the MTT assay. Flow cytometric methods were used to assess apoptosis and necrosis, with annexin V-FITC/propidium iodide staining, and G1 and G2 cell-cycle changes were assessed by DAPI staining. Changes in cyclin D1, cyclin B, IκBα, and IKKα expressions were quantified by western blotting. Cytotoxicity was mediated through apoptosis; both agents showed observed versus expected cytotoxicities of 92.2 versus 55.1% in Granta-519, of 87.6 versus 36.3% in Jeko-1, and of 63.2 versus 29.8% in Rec-1. Isobolographic analysis confirmed synergy in Jeko-1 and Rec-1 cell lines. Bortezomib induced G2-phase arrest, with a 1.7-fold increase compared with control, and fenretinide resulted in G1-phase arrest, with an increase of 1.3-fold compared with control. In the combination, G2-phase arrest predominated, with a 1.4-fold increase compared with control, and there was reduced expression of cyclin D1 to 24%, cyclin B to 52 and 64%, cyclin D3 to 25 and 43%, IκBα to 23 and 46%, and IκBα kinase to 34 and 44%. Bortezomib and fenretinide exhibit synergistic cytotoxicity against MCL cell lines. This activity is mediated by IκBα kinase modulation, decreased cyclin expression, cell cycle dysregulation, and apoptotic cell death.

  9. Effects of fucoidan on proliferation, AMP-activated protein kinase, and downstream metabolism- and cell cycle-associated molecules in poorly differentiated human hepatoma HLF cells.

    PubMed

    Kawaguchi, Takumi; Hayakawa, Masako; Koga, Hironori; Torimura, Takuji

    2015-05-01

    Survival rates are low in patients with poorly differentiated hepatocellular carcinoma (HCC). Fucoidan, a sulfated polysaccharide derived from brown seaweed, has anticancer activity; however, the effects of fucoidan on poorly differentiated HCC remain unclear. In this study, we investigated the effects of fucoidan on AMP-activated protein kinase (AMPK), a proliferation regulator, and its downstream metabolism- and cell cycle-related molecules in a poorly differentiated human hepatoma HLF cell line. HLF cells were treated with fucoidan (10, 50, or 100 µg/ml; n=4) or phosphate buffered saline (control; n=4) for 96 h. Proliferation was evaluated by counting cells every 24 h. AMPK, TSC2, mTOR, GSK3β, acetyl-CoA carboxylase (ACC), ATP-citrate lyase, p53, cyclin D1, cyclin-dependent kinase (CDK) 4, and CDK6 expression and/or phosphorylation were examined by immunoblotting 24 h after treatment with 100 µg/ml fucoidan. Cell cycle progression was analyzed by fluorescence-activated cell sorter 48 h after treatment. Treatment with 50 or 100 µg/ml fucoidan significantly and dose- and time-dependently suppressed HLF cell proliferation (P<0.0001). Fucoidan induced AMPK phosphorylation on Ser172 24 h after treatment. Although no differences were seen in expression and phosphorylation levels of TSC2, mTOR, GSK3β, ATP-citrate lyase, and p53 between the control and fucoidan-treated HLF cells, fucoidan induced ACC phosphorylation on Ser79. Moreover, fucoidan decreased cyclin D1, CDK4 and CDK6 expression 24 h after treatment. Furthermore, HLF cells were arrested in the G1/S phase 48 h after fucoidan treatment. We demonstrated that fucoidan suppressed HLF cell proliferation with AMPK phosphorylation. We showed that fucoidan phosphorylated ACC and downregulated cyclin D1, CDK4 and CDK6 expression. Our findings suggest that fucoidan inhibits proliferation through AMPK-associated suppression of fatty acid synthesis and G1/S transition in HLF cells.

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

    PubMed Central

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

    2015-01-01

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

  11. 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.

  12. DNA damage checkpoint recovery and cancer development

    SciTech Connect

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

    2015-06-10

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

  13. 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.

  14. Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression1[OPEN

    PubMed Central

    Weimer, Annika K.; Stoppin-Mellet, Virginie; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin

    2017-01-01

    Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. PMID:27879390

  15. Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression.

    PubMed

    Boruc, Joanna; Weimer, Annika K; Stoppin-Mellet, Virginie; Mylle, Evelien; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Gonzalez, Nathalie; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin; Van Damme, Daniël

    2017-01-01

    Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1-a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants-is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Quercetin and Cisplatin combined treatment altered cell cycle and mitogen activated protein kinase expressions in malignant mesotelioma cells.

    PubMed

    Demiroglu-Zergeroglu, Asuman; Ergene, Emel; Ayvali, Nurettin; Kuete, Victor; Sivas, Hulya

    2016-08-11

    Malignant mesothelioma is a locally aggressive and highly lethal neoplasm of pleural, peritoneal and pericardial mesothelial cells without successful therapy. Previously, we reported that Quercetin in combination with Cisplatin inhibits cell proliferation and activates caspase-9 and -3 enzymes in different malignant mesothelioma cell lines. Moreover, Quercetin + Cisplatin lead to accumulation of both SPC111 and SPC212 cell lines in S phase. In present work, 84 genes involved in cell growth and proliferation have analysed by using RT(2)-PCR array system and protein profile of mitogen activated protein kinase (MAPK) family proteins investigated by western blots. Our results showed that Quercetin and Quercetin + Cisplatin modulated gene expression of cyclins, cyclin dependent kinases and cyclin dependent kinases inhibitors. In addition genes involved in JNK, p38 and MAPK/ERK pathways were up regulated. Moreover, while p38 and JNK phosphorylations were increased, ERK phosphorylations were decreased after using Quercetin + Cisplatin. This research has clarified our previous results and detailed mechanism of anti-carcinogenic potential of Quercetin alone and incombination with Cisplatin on malignant mesothelioma cells.

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

    SciTech Connect

    Yang, Heekyoung; Yoon, Su Jin; Jin, Juyoun; Choi, Seung Ho; Seol, Ho Jun; Lee, Jung-Il; and others

    2011-03-04

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

  18. The Cell Cycle Regulator Phosphorylated Retinoblastoma Protein Is Associated with Tau Pathology in Several Tauopathies

    PubMed Central

    Stone, Jeremy G.; Siedlak, Sandra L.; Tabaton, Massimo; Hirano, Asao; Castellani, Rudy J.; Santocanale, Corrado; Perry, George; Smith, Mark A.; Zhu, Xiongwei; Lee, Hyoung-gon

    2011-01-01

    Retinoblastoma protein (pRb) is a ubiquitous 928 amino acid cell cycle regulatory molecule with diverse biological activities. One critical function of pRb is control of the G1-to-S phase checkpoint of the cell cycle. In the hypophosphorylated state, pRb suppresses the activity of E2F transcription factors thereby inhibiting transcription of cell cycle promoting genes. Upon phosphorylation, primarily by cyclin dependent kinases, phosphorylated pRb dissociates from E2F and permits cell cycle progression. We previously found phosphorylated pRb to be intimately associated with hyperphosphorylated tau-containing neurofibrillary tangles of Alzheimer disease (AD), the pathogenesis of which is believed to involve dysregulation of the cell cycle and marked neuronal death. Here, we used immunohistochemistry to investigate the presence of phosphorylated pRb in other distinct neurodegenerative diseases that share the common characteristic of hyperphosphorylated tau pathology and neuronal loss with AD. We found colocalized labeling of tau pathology and phosphorylated pRb in Pick disease and progressive supranuclear palsy (3 cases each), neurodegeneration with brain iron accumulation type 1 (2 cases) and Parkinson-amyotrophic lateral sclerosis of Guam, subacute sclerosing panencephalitis, frontotemporal dementia and Parkinsonism linked to chromosome 17 and dementia pugilistica (1 case each). These observations further implicate aberrant neuronal cell cycle progression in neurodegenerative diseases, particularly tauopathies, and suggest a novel target for therapeutic intervention. PMID:21666500

  19. Kick-starting the cell cycle: From growth-factor stimulation to initiation of DNA replication

    NASA Astrophysics Data System (ADS)

    Aguda, Baltazar D.

    2001-03-01

    The essential genes, proteins and associated regulatory networks involved in the entry into the mammalian cell cycle are identified, from activation of growth-factor receptors to intracellular signal transduction pathways that impinge on the cell cycle machinery and ultimately on the initiation of DNA replication. Signaling pathways mediated by the oncoproteins Ras and Myc induce the activation of cyclin-dependent kinases CDK4 and CDK2, and the assembly and firing of pre-replication complexes require a collaboration among E2F, CDK2, and Cdc7 kinase. A proposed core mechanism of the restriction point, the major checkpoint prior to commitment to DNA synthesis, involves cyclin E/CDK2, the phosphatase Cdc25A, and the CDK inhibitor p27Kip1.

  20. Genome-Wide Analyses Identify Recurrent Amplifications of Receptor Tyrosine Kinases and Cell-Cycle Regulatory Genes in Diffuse Intrinsic Pontine Glioma

    PubMed Central

    Paugh, Barbara S.; Broniscer, Alberto; Qu, Chunxu; Miller, Claudia P.; Zhang, Junyuan; Tatevossian, Ruth G.; Olson, James M.; Geyer, J. Russell; Chi, Susan N.; da Silva, Nasjla Saba; Onar-Thomas, Arzu; Baker, Justin N.; Gajjar, Amar; Ellison, David W.; Baker, Suzanne J.

    2011-01-01

    Purpose Long-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG. Patients and Methods Single-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas. Results Frequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase–Ras–phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar. Conclusion DIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG. PMID:21931021

  1. The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit.

    PubMed

    Gonzalez, Nathalie; Gévaudant, Frédéric; Hernould, Michel; Chevalier, Christian; Mouras, Armand

    2007-08-01

    Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.

  2. Variation in the checkpoint kinase 2 gene is associated with type 2 diabetes in multiple populations

    PubMed Central

    Franceschini, Nora; Avery, Christy L.; Baird, Lisa; Graff, Mariaelisa; Leppert, Mark; Chung, Jay H.; Zhang, Jinghui; Hanis, Craig; Boerwinkle, Eric; Volcik, Kelly A.; Grove, Megan L.; Mosley, Thomas H.; Gu, Charles; Heiss, Gerardo; Pankow, James S.; Couper, David J.; Ballantyne, Christie M.; Linda Kao, W. H.; Weder, Alan B.; Cooper, Richard S.; Ehret, Georg B.; O'Connor, Ashley A.; Chakravarti, Aravinda; Hunt, Steven C.

    2010-01-01

    Identification and characterization of the genetic variants underlying type 2 diabetes susceptibility can provide important understanding of the etiology and pathogenesis of type 2 diabetes. We previously identified strong evidence of linkage for type 2 diabetes on chromosome 22 among 3,383 Hypertension Genetic Epidemiology Network (HyperGEN) participants from 1,124 families. The checkpoint 2 (CHEK2) gene, an important mediator of cellular responses to DNA damage, is located 0.22 Mb from this linkage peak. In this study, we tested the hypothesis that the CHEK2 gene contains one or more polymorphic variants that are associated with type 2 diabetes in HyperGEN individuals. In addition, we replicated our findings in two other Family Blood Pressure Program (FBPP) populations and in the population-based Atherosclerosis Risk in Communities (ARIC) study. We genotyped 1,584 African-American and 1,531 white HyperGEN participants, 1,843 African-American and 1,569 white GENOA participants, 871 African-American and 1,009 white GenNet participants, and 4,266 African-American and 11,478 white ARIC participants for four single nucleotide polymorphisms (SNPs) in CHEK2. Using additive models, we evaluated the association of CHEK2 SNPs with type 2 diabetes in participants within each study population stratified by race, and in a meta-analysis, adjusting for age, age2, sex, sex-by-age interaction, study center, and relatedness. One CHEK2 variant, rs4035540, was associated with an increased risk of type 2 diabetes in HyperGEN participants, two replication samples, and in the meta-analysis. These results may suggest a new pathway in the pathogenesis of type 2 diabetes that involves pancreatic beta-cell damage and apoptosis. PMID:19855918

  3. Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae.

    PubMed

    Nourikyan, Julien; Kjos, Morten; Mercy, Chryslène; Cluzel, Caroline; Morlot, Cécile; Noirot-Gros, Marie-Francoise; Guiral, Sébastien; Lavergne, Jean-Pierre; Veening, Jan-Willem; Grangeasse, Christophe

    2015-09-01

    Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation

  4. Autophosphorylation of the Bacterial Tyrosine-Kinase CpsD Connects Capsule Synthesis with the Cell Cycle in Streptococcus pneumoniae

    PubMed Central

    Mercy, Chryslène; Cluzel, Caroline; Morlot, Cécile; Noirot-Gros, Marie-Francoise; Guiral, Sébastien; Lavergne, Jean-Pierre; Veening, Jan-Willem; Grangeasse, Christophe

    2015-01-01

    Bacterial capsular polysaccharides (CPS) are produced by a multi-protein membrane complex, in which a particular type of tyrosine-autokinases named BY-kinases, regulate their polymerization and export. However, our understanding of the role of BY-kinases in these processes remains incomplete. In the human pathogen Streptococcus pneumoniae, the BY-kinase CpsD localizes at the division site and participates in the proper assembly of the capsule. In this study, we show that the cytoplasmic C-terminal end of the transmembrane protein CpsC is required for CpsD autophosphorylation and localization at mid-cell. Importantly, we demonstrate that the CpsC/CpsD complex captures the polysaccharide polymerase CpsH at the division site. Together with the finding that capsule is not produced at the division site in cpsD and cpsC mutants, these data show that CPS production occurs exclusively at mid-cell and is tightly dependent on CpsD interaction with CpsC. Next, we have analyzed the impact of CpsD phosphorylation on CPS production. We show that dephosphorylation of CpsD induces defective capsule production at the septum together with aberrant cell elongation and nucleoid defects. We observe that the cell division protein FtsZ assembles and localizes properly although cell constriction is impaired. DAPI staining together with localization of the histone-like protein HlpA further show that chromosome replication and/or segregation is defective suggesting that CpsD autophosphorylation interferes with these processes thus resulting in cell constriction defects and cell elongation. We show that CpsD shares structural homology with ParA-like ATPases and that it interacts with the chromosome partitioning protein ParB. Total internal reflection fluorescence microscopy imaging demonstrates that CpsD phosphorylation modulates the mobility of ParB. These data support a model in which phosphorylation of CpsD acts as a signaling system coordinating CPS synthesis with chromosome segregation

  5. miR-26a plays an important role in cell cycle regulation in ACTH-secreting pituitary adenomas by modulating protein kinase Cδ.

    PubMed

    Gentilin, Erica; Tagliati, Federico; Filieri, Carlo; Molè, Daniela; Minoia, Mariella; Rosaria Ambrosio, Maria; Degli Uberti, Ettore C; Zatelli, Maria Chiara

    2013-05-01

    The functional aftermath of microRNA (miRNA) dysregulation in ACTH-secreting pituitary adenomas has not been demonstrated. miRNAs represent diagnostic and prognostic biomarkers as well as putative therapeutic targets; their investigation may shed light on the mechanisms that underpin pituitary adenoma development and progression. Drugs interacting with such pathways may help in achieving disease control also in the settings of ACTH-secreting pituitary adenomas. We investigated the expression of 10 miRNAs among those that were found as most dysregulated in human pituitary adenoma tissues in the settings of a murine ACTH-secreting pituitary adenoma cell line, AtT20/D16v-F2. The selected miRNAs to be submitted to further investigation in AtT20/D16v-F2 cells represent an expression panel including 5 up-regulated and 5 down-regulated miRNAs. Among these, we selected the most dysregulated mouse miRNA and searched for miRNA targets and their biological function. We found that AtT20/D16v-F2 cells have a specific miRNA expression profile and that miR-26a is the most dysregulated miRNA. The latter is overexpressed in human pituitary adenomas and can control viable cell number in the in vitro model without involving caspase 3/7-mediated apoptosis. We demonstrated that protein kinase Cδ (PRKCD) is a direct target of miR-26a and that miR26a inhibition delays the cell cycle in G1 phase. This effect involves down-regulation of cyclin E and cyclin A expression via PRKCD modulation. miR-26a and related pathways, such as PRKCD, play an important role in cell cycle control of ACTH pituitary cells, opening new therapeutic possibilities for the treatment of persistent/recurrent Cushing's disease.

  6. miR-26a Plays an Important Role in Cell Cycle Regulation in ACTH-Secreting Pituitary Adenomas by Modulating Protein Kinase

    PubMed Central

    Gentilin, Erica; Tagliati, Federico; Filieri, Carlo; Molè, Daniela; Minoia, Mariella; Rosaria Ambrosio, Maria; degli Uberti, Ettore C

    2013-01-01

    The functional aftermath of microRNA (miRNA) dysregulation in ACTH-secreting pituitary adenomas has not been demonstrated. miRNAs represent diagnostic and prognostic biomarkers as well as putative therapeutic targets; their investigation may shed light on the mechanisms that underpin pituitary adenoma development and progression. Drugs interacting with such pathways may help in achieving disease control also in the settings of ACTH-secreting pituitary adenomas. We investigated the expression of 10 miRNAs among those that were found as most dysregulated in human pituitary adenoma tissues in the settings of a murine ACTH-secreting pituitary adenoma cell line, AtT20/D16v-F2. The selected miRNAs to be submitted to further investigation in AtT20/D16v-F2 cells represent an expression panel including 5 up-regulated and 5 down-regulated miRNAs. Among these, we selected the most dysregulated mouse miRNA and searched for miRNA targets and their biological function. We found that AtT20/D16v-F2 cells have a specific miRNA expression profile and that miR-26a is the most dysregulated miRNA. The latter is overexpressed in human pituitary adenomas and can control viable cell number in the in vitro model without involving caspase 3/7-mediated apoptosis. We demonstrated that protein kinase Cδ (PRKCD) is a direct target of miR-26a and that miR26a inhibition delays the cell cycle in G1 phase. This effect involves down-regulation of cyclin E and cyclin A expression via PRKCD modulation. miR-26a and related pathways, such as PRKCD, play an important role in cell cycle control of ACTH pituitary cells, opening new therapeutic possibilities for the treatment of persistent/recurrent Cushing's disease. PMID:23525216

  7. Induction of p21CIP1 protein and cell cycle arrest after inhibition of Aurora B kinase is attributed to aneuploidy and reactive oxygen species.

    PubMed

    Kumari, Geeta; Ulrich, Tanja; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

    2014-06-06

    Cell cycle progression requires a series of highly coordinated events that ultimately lead to faithful segregation of chromosomes. Aurora B is an essential mitotic kinase, which is involved in regulation of microtubule-kinetochore attachments and cytokinesis. Inhibition of Aurora B results in stabilization of p53 and induction of p53-target genes such as p21 to inhibit proliferation. We have previously demonstrated that induction of p21 by p53 after inhibition of Aurora B is dependent on the p38 MAPK, which promotes transcriptional elongation of p21 by RNA Pol II. In this study, we show that a subset of p53-target genes are induced in a p38-dependent manner upon inhibition of Aurora B. We also demonstrate that inhibition of Aurora B results in down-regulation of E2F-mediated transcription and that the cell cycle arrest after Aurora B inhibition depends on p53 and pRB tumor suppressor pathways. In addition, we report that activation of p21 after inhibition of Aurora B is correlated with increased chromosome missegregation and aneuploidy but not with binucleation or tetraploidy. We provide evidence that p21 is activated in aneuploid cells by reactive oxygen species (ROS) and p38 MAPK. Finally, we demonstrate that certain drugs that act on aneuploid cells synergize with inhibitors of Aurora B to inhibit colony formation and oncogenic transformation. These findings provide an important link between aneuploidy and the stress pathways activated by Aurora B inhibition and also support the use of Aurora B inhibitors in combination therapy for treatment of cancer. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

    SciTech Connect

    Gonzales, Andrea J. . E-mail: Andrea.Gonzales@pfizer.com; Fry, David W.

    2005-02-01

    The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 {mu}M) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27{sup KIP1} protein when treated with PD 158780 and increased association of p27{sup KIP1} with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB.

  9. Selective inhibitors of aurora kinases inhibit proliferation, reduce cell viability and impair cell cycle progression in papillary thyroid carcinoma cells.

    PubMed

    Baldini, E; Tuccilli, C; Prinzi, N; Sorrenti, S; Antonelli, A; Fallahi, P; Mian, C; Barollo, S; Catania, A; Morrone, S; Tartaglia, F; Mascagni, D; Coccaro, C; Pepe, M; Filippini, A; D'Armiento, M; Ulisse, S

    2015-01-01

    The three members of the Aurora kinase family, Aurora-A, -B and -C, regulate several aspects of the mitotic process, and their aberrant expression and/or function causes mitotic abnormalities leading either to cell death or aneuploidy. They are found overexpressed in several human malignancies, including the papillary thyroid carcinoma (PTC). In the present study, we sought to establish whether Aurora kinase inhibition could be of any therapeutic value in the treatment of aggressive forms of PTC, enduring to radioactive iodide (RAI) ablation. To this end, the effects of selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) were analyzed on 3 human PTC cell lines expressing either wild-type (K1 and TPC1) or mutant p53 (BCPAP). The two inhibitors were capable of reducing cell proliferation in a time- and dose-dependent manner, with IC₅₀ comprised between 65.4 and 114.9 nM for MLN8237, and between 26.6 and 484.6 nM for AZD1152. Immunofluorescence experiments confirmed that AZD1152 inhibited Aurora-B phosphorylation of histone H3 on Ser10, however, it did not affect Aurora-A autophosphorylation. MLN8237 inhibited Aurora-A autophosphorylation as expected, but at concentrations required to achieve the maximum antiproliferative effects it also abolished H3 (Ser10) phosphorylation. Time-lapse videomicroscopy evidenced that both inhibitors prevented the completion of cytokinesis, and cytofluorimetric analysis showed accumulation of cells in G2/M phase and/or polyploidy. Apoptosis was induced in all the cells by both inhibitors independently from the p53 status. In conclusion, in the present preclinical study MLN8237 and AZD1152 have emerged as promising drug candidates for RAI-insensitive PTC.

  10. Protein kinase C-beta inhibition induces apoptosis and inhibits cell cycle progression in acquired immunodeficiency syndrome-related non-hodgkin lymphoma cells.

    PubMed

    Saba, Nakhle S; Levy, Laura S

    2012-01-01

    Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate, and resistance to chemotherapy. Protein kinase C-beta (PKCβ) targeting showed promising results in preclinical and clinical studies involving a wide variety of cancers, but studies describing the role of PKCβ in AIDS-NHL are primitive if not lacking. In the present study, 3 AIDS-NHL cell lines were examined: 2F7 (AIDS-Burkitt lymphoma), BCBL-1 (AIDS-primary effusion lymphoma), and UMCL01-101 (AIDS-diffuse large B-cell lymphoma). Immunoblot analysis demonstrated expression of PKCβ1 and PKCβ2 in 2F7 and UMCL01-101 cells, and PKCβ1 alone in BCBL-1 cells. The viability of 2F7 and BCBL-1 cells decreased significantly in the presence of PKCβ-selective inhibitor at half-maximal inhibitory concentration of 14 and 15 μmol/L, respectively, as measured by tetrazolium dye reduction assay. In contrast, UMCL01-101 cells were relatively resistant. As determined using flow cytometric deoxynucleotidyl transferase dUTP nick-end labeling assay with propidium iodide staining, the responsiveness of sensitive cells was associated with apoptotic induction and cell cycle inhibition. Protein kinase C-beta-selective inhibition was observed not to affect AKT phosphorylation but to induce a rapid and sustained reduction in the phosphorylation of glycogen synthase kinase-3 beta, ribosomal protein S6, and mammalian target of rapamycin in sensitive cell lines. The results indicate that PKCβ plays an important role in AIDS-related NHL survival and suggest that PKCβ targeting should be considered in a broader spectrum of NHL. The observations in BCBL-1 were unexpected in the absence of PKCβ2 expression and implicate PKCβ1 as a regulator in those cells.

  11. Cell cycle-dependent control of homologous recombination.

    PubMed

    Zhao, Xin; Wei, Chengwen; Li, Jingjing; Xing, Poyuan; Li, Jingyao; Zheng, Sihao; Chen, Xuefeng

    2017-08-01

    DNA double-strand breaks (DSBs) are among the most deleterious type of DNA lesions threatening genome integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are two major pathways to repair DSBs. HR requires a homologous template to direct DNA repair, and is generally recognized as a high-fidelity pathway. In contrast, NHEJ directly seals broken ends, but the repair product is often accompanied by sequence alterations. The choice of repair pathways is strictly controlled by the cell cycle. The occurrence of HR is restricted to late S to G2 phases while NHEJ operates predominantly in G1 phase, although it can act throughout most of the cell cycle. Deregulation of repair pathway choice can result in genotoxic consequences associated with cancers. How the cell cycle regulates the choice of HR and NHEJ has been extensively studied in the past decade. In this review, we will focus on the current progresses on how HR is controlled by the cell cycle in both Saccharomyces cerevisiae and mammals. Particular attention will be given to how cyclin-dependent kinases modulate DSB end resection, DNA damage checkpoint signaling, repair and processing of recombination intermediates. In addition, we will discuss recent findings on how HR is repressed in G1 and M phases by the cell cycle. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

    PubMed

    Mu, Jung-Jung; Wang, Yi; Luo, Hao; Leng, Mei; Zhang, Jinglan; Yang, Tao; Besusso, Dario; Jung, Sung Yun; Qin, Jun

    2007-06-15

    ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

  13. Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei

    SciTech Connect

    Li Ziyin; Tu Xiaoming; Wang, Ching C. . E-mail: ccwang@cgl.ucsf.edu

    2006-11-01

    Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrested in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.

  14. 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

  15. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  16. Gene copy number and cell cycle arrest

    NASA Astrophysics Data System (ADS)

    Ghosh, Bhaswar; Bose, Indrani

    2006-03-01

    The cell cycle is an orderly sequence of events which ultimately lead to the division of a single cell into two daughter cells. In the case of DNA damage by radiation or chemicals, the damage checkpoints in the G1 and G2 phases of the cell cycle are activated. This results in an arrest of the cell cycle so that the DNA damage can be repaired. Once this is done, the cell continues with its usual cycle of activity. We study a mathematical model of the DNA damage checkpoint in the G2 phase which arrests the transition from the G2 to the M (mitotic) phase of the cell cycle. The tumor suppressor protein p53 plays a key role in activating the pathways leading to cell cycle arrest in mammalian systems. If the DNA damage is severe, the p53 proteins activate other pathways which bring about apoptosis, i.e., programmed cell death. Loss of the p53 gene results in the proliferation of cells containing damaged DNA, i.e., in the growth of tumors which may ultimately become cancerous. There is some recent experimental evidence which suggests that the mutation of a single copy of the p53 gene (in the normal cell each gene has two identical copies) is sufficient to trigger the formation of tumors. We study the effect of reducing the gene copy number of the p53 and two other genes on cell cycle arrest and obtain results consistent with experimental observations.

  17. LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

    SciTech Connect

    Wang, Huiling; Li, Ridong; Li, Li; Ge, Zemei; Zhou, Rouli; Li, Runtao

    2015-02-27

    In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{sub 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.

  18. SD-208, a Novel Protein Kinase D Inhibitor, Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

    PubMed Central

    Tandon, Manuj; Salamoun, Joseph M.; Carder, Evan J.; Farber, Elisa; Xu, Shuping; Deng, Fan; Tang, Hua; Wipf, Peter; Wang, Q. Jane

    2015-01-01

    Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment. PMID:25747583

  19. Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells.

    PubMed

    Zeng, Huawei; Botnen, James H; Briske-Anderson, Mary

    2010-01-01

    The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer nutrients. Methylselenol has been hypothesized to be a critical Se metabolite for anticancer activity in vivo. In this study, we demonstrated that both DCA (75-300 micromol/l) and submicromolar methylselenol inhibited colon cancer cell proliferation by up to 64% and 63%, respectively. In addition, DCA and methylselenol each increased colon cancer cell apoptosis rate by up to twofold. Cell cycle analyses revealed that DCA induced an increase in only the G1 fraction with a concomitant drop in G2 and S-phase; in contrast, methylselenol led to an increase in the G1 and G2 fractions with a concomitant drop only in the S-phase. Although both DCA and methylselenol significantly promoted apoptosis and inhibited cell growth, examination of mitogen-activated protein kinase (MAPK) pathway activation showed that DCA, but not methylselenol, induced SAPK/JNK1/2, p38 MAPK, ERK1/2 activation. Thus, our data provide, for the first time, the molecular basis for opposite effects of methylselenol and DCA on colon tumorigenesis.

  20. Ras/cAMP-dependent Protein Kinase (PKA) Regulates Multiple Aspects of Cellular Events by Phosphorylating the Whi3 Cell Cycle Regulator in Budding Yeast*

    PubMed Central

    Mizunuma, Masaki; Tsubakiyama, Ryohei; Ogawa, Takafumi; Shitamukai, Atsunori; Kobayashi, Yoshifumi; Inai, Tomomi; Kume, Kazunori; Hirata, Dai

    2013-01-01

    The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events. PMID:23471970

  1. Combined Treatment of MCF-7 Cells with AICAR and Methotrexate, Arrests Cell Cycle and Reverses Warburg Metabolism through AMP-Activated Protein Kinase (AMPK) and FOXO1.

    PubMed

    Fodor, Tamás; Szántó, Magdolna; Abdul-Rahman, Omar; Nagy, Lilla; Dér, Ádám; Kiss, Borbála; Bai, Peter

    2016-01-01

    Cancer cells are characterized by metabolic alterations, namely, depressed mitochondrial oxidation, enhanced glycolysis and pentose phosphate shunt flux to support rapid cell growth, which is called the Warburg effect. In our study we assessed the metabolic consequences of a joint treatment of MCF-7 breast cancer cells with AICAR, an inducer of AMP-activated kinase (AMPK) jointly with methotrexate (MTX), a folate-analog antimetabolite that blunts de novo nucleotide synthesis. MCF7 cells, a model of breast cancer cells, were resistant to the individual application of AICAR or MTX, however combined treatment of AICAR and MTX reduced cell proliferation. Prolonged joint application of AICAR and MTX induced AMPK and consequently enhanced mitochondrial oxidation and reduced the rate of glycolysis. These metabolic changes suggest an anti-Warburg rearrangement of metabolism that led to the block of the G1/S and the G2/M transition slowing down cell cycle. The slowdown of cell proliferation was abolished when mitotropic transcription factors, PGC-1α, PGC-1β or FOXO1 were silenced. In human breast cancers higher expression of AMPKα and FOXO1 extended survival. AICAR and MTX exerts similar additive antiproliferative effect on other breast cancer cell lines, such as SKBR and 4T1 cells, too. Our data not only underline the importance of Warburg metabolism in breast cancer cells but nominate the AICAR+MTX combination as a potential cytostatic regime blunting Warburg metabolism. Furthermore, we suggest the targeting of AMPK and FOXO1 to combat breast cancer.

  2. Targeting Focal Adhesion Kinase Renders Pancreatic Cancers Responsive to Checkpoint Immunotherapy

    PubMed Central

    Jiang, Hong; Hegde, Samarth; Knolhoff, Brett L.; Zhu, Yu; Herndon, John M.; Meyer, Melissa A.; Nywening, Timothy M.; Hawkins, William G.; Shapiro, Irina M.; Weaver, David T.; Pachter, Jonathan A.; Wang-Gillam, Andrea; DeNardo, David G.

    2016-01-01

    Single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME). Critical obstacles to immunotherapy in PDAC tumors include a high number of tumor-associated immunosuppressive cells and a uniquely desmoplastic stroma that acts as a barrier to T-cell infiltration. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition using the selective FAK inhibitor VS-4718 significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This delay in tumor progression was associated with dramatically reduced tumor fibrosis, and decreased numbers of tumor-infiltrating immunosuppressive cells. We also found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to T cell immunotherapy and PD-1 antagonists. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and renders tumors responsive to immunotherapy. PMID:27376576

  3. Discovery of 1,4-dihydroindeno[1,2-c]pyrazoles as a novel class of potent and selective checkpoint kinase 1 inhibitors.

    PubMed

    Tong, Yunsong; Claiborne, Akiyo; Stewart, Kent D; Park, Chang; Kovar, Peter; Chen, Zehan; Credo, Robert B; Gu, Wen-Zhen; Gwaltney, Stephen L; Judge, Russell A; Zhang, Haiying; Rosenberg, Saul H; Sham, Hing L; Sowin, Thomas J; Lin, Nan-Horng

    2007-04-01

    A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (<10nM) against CHK-1. X-ray co-crystal structures of bound inhibitors demonstrated that two sub-series of this class of compounds, exemplified by 21 and 41, exhibit distinctive hydrogen bonding patterns in the specificity pocket of the active site. Two compounds, 41 and 43, were capable of potentiating doxorubicin and camptothecin, both DNA-damaging agents, in cell proliferation assays (MTS and soft agar assays) and abrogating G2/M checkpoint in a mechanism-based FACS assay.

  4. Artemis links ATM to G2/M checkpoint recovery via regulation of Cdk1-cyclin B.

    PubMed

    Geng, Liyi; Zhang, Xiaoshan; Zheng, Shu; Legerski, Randy J

    2007-04-01

    Artemis is a phospho-protein that has been shown to have roles in V(D)J recombination, nonhomologous end-joining of double-strand breaks, and regulation of the DNA damage-induced G(2)/M cell cycle checkpoint. Here, we have identified four sites in Artemis that are phosphorylated in response to ionizing radiation (IR) and show that ATM is the major kinase responsible for these modifications. Two of the sites, S534 and S538, show rapid phosphorylation and dephosphorylation, and the other two sites, S516 and S645, exhibit rapid and prolonged phosphorylation. Mutation of both of these latter two residues results in defective recovery from the G(2)/M cell cycle checkpoint. This defective recovery is due to promotion by mutant Artemis of an enhanced interaction between unphosphorylated cyclin B and Cdk1, which in turn promotes inhibitory phosphorylation of Cdk1 by the Wee1 kinase. In addition, we show that mutant Artemis prevents Cdk1-cyclin B activation by causing its retention in the centrosome and inhibition of its nuclear import during prophase. These findings show that ATM regulates G(2)/M checkpoint recovery through inhibitory phosphorylations of Artemis that occur soon after DNA damage, thus setting a molecular switch that, hours later upon completion of DNA repair, allows activation of the Cdk1-cyclin B complex. These findings thus establish a novel function of Artemis as a regulator of the cell cycle in response to DNA damage.

  5. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

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

    SciTech Connect

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

    2010-04-05

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

  7. Glucose restriction induces transient G2 cell cycle arrest extending cellular chronological lifespan

    PubMed Central

    Masuda, Fumie; Ishii, Mahiro; Mori, Ayaka; Uehara, Lisa; Yanagida, Mitsuhiro; Takeda, Kojiro; Saitoh, Shigeaki

    2016-01-01

    While glucose is the fundamental source of energy in most eukaryotes, it is not always abundantly available in natural environments, including within the human body. Eukaryotic cells are therefore thought to possess adaptive mechanisms to survive glucose-limited conditions, which remain unclear. Here, we report a novel mechanism regulating cell cycle progression in response to abrupt changes in extracellular glucose concentration. Upon reduction of glucose in the medium, wild-type fission yeast cells undergo transient arrest specifically at G2 phase. This cell cycle arrest is dependent on the Wee1 tyrosine kinase inhibiting the key cell cycle regulator, CDK1/Cdc2. Mutant cells lacking Wee1 are not arrested at G2 upon glucose limitation and lose viability faster than the wild-type cells under glucose-depleted quiescent conditions, suggesting that this cell cycle arrest is required for extension of chronological lifespan. Our findings indicate the presence of a novel cell cycle checkpoint monitoring glucose availability, which may be a good molecular target for cancer therapy. PMID:26804466

  8. Transforming growth factor-β1 induces cell cycle arrest by activating atypical cyclin-dependent kinase 5 through up-regulation of Smad3-dependent p35 expression in human MCF10A mammary epithelial cells.

    PubMed

    Park, Seong Ji; Yang, Sun Woo; Kim, Byung-Chul

    2016-04-08

    Cyclin-dependent kinases (Cdks) play important roles in control of cell division. Cdk5 is an atypical member of Cdk family with non-cyclin-like regulatory subunit, p35, but its role in cell cycle progression is still unclear. In the present study, we investigated the role of Cdk5/p35 on transforming growth factor-β1 (TGF-β1)-induced cell cycle arrest. In human MCF10A mammary epithelial cells, TGF-β1 induced cell cycle arrest at G1 phase and increased p27KIP1 expression. Interestingly, pretreatment with roscovitine, an inhibitor of Cdk5, or transfection with small interfering (si) RNAs specific to Cdk5 and p35 significantly attenuated the TGF-β1-induced p27KIP1 expression and cell cycle arrest. TGF-β1 increased Cdk5 activity via up-regulation of p35 gene at transcriptional level, and these effects were abolished by transfection with Smad3 siRNA or infection of adenovirus carrying Smad3 mutant at the C-tail (3SA). Chromatin immunoprecipitation assay further revealed that wild type Smad3, but not mutant Smad3 (3SA), binds to the region of the p35 promoter region (-1000--755) in a TGF-β1-dependent manner. These results for the first time demonstrate a role of Cdk5/p35 in the regulation of cell cycle progression modulated by TGF-β1. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Chromium-VI arrests cell cycle and decreases granulosa cell proliferation by down-regulating cyclin-dependent kinases (CDK) and cyclins and up-regulating CDK-inhibitors.

    PubMed

    Stanley, Jone A; Lee, JeHoon; Nithy, Thamizh K; Arosh, Joe A; Burghardt, Robert C; Banu, Sakhila K

    2011-07-01

    Environmental contamination with hexavalent chromium (CrVI) has been increasing in the drinking water of the USA and developing countries. CrVI causes various health problems including menstrual disorders and infertility. Recently, we reported that CrVI causes granulosa cell apoptosis through the intrinsic apoptotic pathway. Our previous studies showed that postnatal exposure to CrVI arrests follicle development. In order to explore the underlying mechanism, primary and immortalized granulosa cells from rats were treated with 10 μM potassium dichromate and analyses of the cell cycle, and cell cycle regulatory proteins were performed. CrVI decreased cell proliferation as a result of cell cycle arrest and down-regulated cyclin-dependent kinases (CDK), cyclins, and PCNA while up-regulating CDK-inhibitors and down-regulating FSH receptor and ERβ. Vitamin C mitigated the effects of CrVI. This study shows that CrVI causes cell cycle arrest in granulosa cells by altering cell cycle regulatory proteins with potential intervention by vitamin C. Published by Elsevier Inc.

  10. Chromium-VI arrests cell cycle and decreases granulosa cell proliferation by down-regulating cyclin-dependent kinases (CDK) and cyclins and up-regulating CDK-Inhibitors

    PubMed Central

    Stanley, Jone A.; Lee, JeHoon; Nithy, Thamizh K.; Arosh, Joe A.; Burghardt, Robert C.; Banu, Sakhila K.

    2011-01-01

    Environmental contamination with hexavalent chromium (CrVI) has been increasing in the drinking water of the USA and developing countries. CrVI causes various health problems including menstrual disorders and infertility. Recently, we reported that CrVI causes granulosa cell apoptosis through the intrinsic apoptotic pathway. Our previous studies showed that postnatal exposure to CrVI arrests follicle development. In order to explore the underlying mechanism, primary and immortalized granulosa cells from rats were treated with 10 μM potassium dichromate and analyses of the cell cycle, and cell cycle regulatory proteins were performed. CrVI decreased cell proliferation as a result of cell cycle arrest and down-regulated cyclin-dependent kinases (CDK), cyclins, and PCNA while up-regulating CDK-inhibitors and down-regulating FSH receptor and ERβ. Vitamin C mitigated the effects of CrVI. This study shows that CrVI causes cell cycle arrest in granulosa cells by altering cell cycle regulatory proteins with potential intervention by vitamin C. PMID:21621607

  11. Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling.

    PubMed

    Wang, Yanchang; Shirogane, Takahiro; Liu, Dou; Harper, J Wade; Elledge, Stephen J

    2003-03-07

    In S. cerevisiae cells undergoing anaphase, a ras-related GTPase, Tem1, is located on the spindle pole body that enters the daughter cell and activates a signal transduction pathway, MEN, to allow mitotic exit. MEN activation must be reversed after mitotic exit to reset the cell cycle in G1. We find that daughter cells activate an Antagonist of MEN pathway (AMEN) in part through induction of the Amn1 protein that binds directly to Tem1 and prevents its association with its target kinase Cdc15. Failure of Amn1 function results in defects of both the spindle assembly and nuclear orientation checkpoints and delays turning off Cdc14 in G1. Thus, Amn1 is part of a daughter-specific switch that helps cells exit from mitotic exit and reset the cell cycle.

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

    PubMed

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  13. Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFα)-induced apoptosis through SIRT1 inhibition.

    PubMed

    Dixit, D; Sharma, V; Ghosh, S; Mehta, V S; Sen, E

    2012-02-09

    Glioblastoma multiforme (GBM) are resistant to TNFα-induced apoptosis and blockade of TNFα-induced NF-κB activation sensitizes glioma cells to apoptosis. As Casein kinase-2 (CK2) induces aberrant NF-κB activation and as we observed elevated CK2 levels in GBM tumors, we investigated the potential of CK2 inhibitors (CK2-Is) - DRB and Apigenin in sensitizing glioma cells to TNFα-induced apoptosis. CK2-Is and CK2 small interfering RNA (siRNA) reduced glioma cell viability, inhibited TNFα-mediated NF-κB activation, and sensitized cell to TNFα-induced apoptosis. Importantly, CK2-Is activated p53 function in wild-type but not in p53 mutant cells. Activation of p53 function involved its increased transcriptional activation, DNA-binding ability, increased expression of p53 target genes associated with cell cycle progression and apoptosis. Moreover, CK2-Is decreased telomerase activity and increased senescence in a p53-dependent manner. Apoptotic gene profiling indicated that CK2-Is differentially affect p53 and TNFα targets in p53 wild-type and mutant glioma cells. CK2-I decreased MDM2-p53 association and p53 ubiquitination to enhance p53 levels. Interestingly, CK2-Is downregulated SIRT1 activity and over-expression of SIRT1 decreased p53 transcriptional activity and rescued cells from CK2-I-induced apoptosis. This ability of CK2-Is to sensitize glioma to TNFα-induced death via multiple mechanisms involving abrogation of NF-κB activation, reactivation of wild-type p53 function and SIRT1 inhibition warrants investigation.

  14. XPB and XPD helicases in TFIIH orchestrate DNA duplex opening and damage verification to coordinate repair with transcription and cell cycle via CAK kinase

    PubMed Central

    Fuss, Jill O.; Tainer, John A.

    2011-01-01

    coordinate repair with transcription and cell cycle through CAK signaling. PMID:21571596

  15. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells

    PubMed Central

    Rossi, Alessandra; Schenone, Silvia; Angelucci, Adriano; Cozzi, Martina; Caracciolo, Valentina; Pentimalli, Francesca; Puca, Andrew; Pucci, Biagio; La Montagna, Raffaele; Bologna, Mauro; Botta, Maurizio; Giordano, Antonio

    2010-01-01

    Medulloblastoma is the most common malignant brain tumor in children, and despite improvements in the overall survival rate, it still lacks an effective treatment. Src plays an important role in cancer, and recently high Src activity was documented in medulloblastoma. In this report, we examined the effects of novel pyrazolo-[3,4-d]-pyrimidine derivative Src inhibitors in medulloblastoma. By MTS assay, we showed that the pyrimidine derivatives indicated as S7, S29, and SI163 greatly reduce the growth rate of medulloblastoma cells by inhibiting Src phosphorylation, compared with HT22 non-neoplastic nerve cells. These compounds also halt cells in the G2/M phase, and this effect likely occurs through the regulation of cdc2 and CDC25C phosphorylation, as shown by Western blot. Moreover, the exposure to pyrimidine derivatives induces apoptosis, assayed by the supravital propidium iodide assay, through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in vivo in a mouse model. Notably, S7, S29, and SI163 show major inhibitory effects on medulloblastoma cell growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that S7, S29, and SI163 could be novel attractive candidates for the treatment of medulloblastoma or tumors characterized by high Src activity.—Rossi, A., Schenone, S., Angelucci, A., Cozzi, M., Caracciolo, V., Pentimalli, F., Puca, A., Pucci, B., La Montagna, R., Bologna, M., Botta, M., Giordano, A. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells. PMID:20354138

  16. Combination of ascorbate/epigallocatechin-3-gallate/gemcitabine synergistically induces cell cycle deregulation and apoptosis in mesothelioma cells

    SciTech Connect

    Martinotti, Simona; Ranzato, Elia; Parodi, Monica; Vitale, Massimo; Burlando, Bruno

    2014-01-01

    Malignant mesothelioma (MMe) is a poor-prognosis tumor in need of innovative therapies. In a previous in vivo study, we showed synergistic anti-MMe properties of the ascorbate/epigallocatechin-3-gallate/gemcitabine combination. We have now focused on the mechanism of action, showing the induction of apoptosis and cell cycle arrest through measurements of caspase 3, intracellular Ca{sup 2+}, annexin V, and DNA content. StellArray™ PCR technology and Western immunoblotting revealed DAPK2-dependent apoptosis, upregulation of cell cycle promoters, downregulation of cell cycle checkpoints and repression of NFκB expression. The complex of data indicates that the mixture is synergistic in inducing cell cycle deregulation and non-inflammatory apoptosis, suggesting its possible use in MMe treatment. - Highlights: • Ascorbate/epigallocathechin-gallate/gemcitabine has been tested on mesothelioma cells • A synergistic mechanism has been shown for cell cycle arrest and apoptosis • PCR-array analysis has revealed the de-regulation of apoptosis and cell cycle genes • Maximum upregulation has been found for the Death-Associated Protein Kinase-2 gene • Data suggest that the mixture could be used as a clinical treatment.

  17. Cell cycle transitions: a common role for stoichiometric inhibitors.

    PubMed

    Hopkins, Michael; Tyson, John J; Novák, Béla

    2017-09-20

    The cell division cycle is the process by which eukaryotic cells replicate their chromosomes and partition them to two daughter cells. To maintain the integrity of the genome, proliferating cells must be able to block progression through the division cycle at key transition points (called 'checkpoints'), if there have been problems in the replication of the chromosomes or their biorientation on the mitotic spindle. These checkpoints are governed by protein-interaction networks, composed of phase-specific cell-cycle activators and inhibitors. Examples include Cdk1:Clb5 and its inhibitor Sic1 at the G1/S checkpoint in budding yeast, APC:Cdc20 and its inhibitor MCC at the mitotic checkpoint, and PP2A:B55 and its inhibitor ENSA at the mitotic-exit checkpoint. Each of these inhibitors is a substrate as well as a stoichiometric inhibitor of the cell-cycle activator. Because the production of each inhibitor is promoted by a regulatory protein that is itself inhibited by the cell cycle activator, their interaction network presents a regulatory motif characteristic of a 'feedback-amplified domineering substrate' (FADS). We describe how the FADS motif responds to signals in the fashion of a bistable toggle switch, and then we discuss how this toggle switch accounts for the abrupt and irreversible nature of three specific cell-cycle checkpoints. © 2017 by The American Society for Cell Biology.

  18. Myc and cell cycle control.

    PubMed

    Bretones, Gabriel; Delgado, M Dolores; León, Javier

    2015-05-01

    Soon after the discovery of the Myc gene (c-Myc), it became clear that Myc expression levels tightly correlate to cell proliferation. The entry in cell cycle of quiescent cells upon Myc enforced expression has been described in many models. Also, the downregulation or inactivation of Myc results in the impairment of cell cycle progression. Given the frequent deregulation of Myc oncogene in human cancer it is important to dissect out the mechanisms underlying the role of Myc on cell cycle control. Several parallel mechanisms account for Myc-mediated stimulation of the cell cycle. First, most of the critical positive cell cycle regulators are encoded by genes induced by Myc. These Myc target genes include Cdks, cyclins and E2F transcription factors. Apart from its direct effects on the transcription, Myc is able to hyperactivate cyclin/Cdk complexes through the induction of Cdk activating kinase (CAK) and Cdc25 phosphatases. Moreover, Myc antagonizes the activity of cell cycle inhibitors as p21 and p27 through different mechanisms. Thus, Myc is able to block p21 transcription or to induce Skp2, a protein involved in p27 degradation. Finally, Myc induces DNA replication by binding to replication origins and by upregulating genes encoding proteins required for replication initiation. Myc also regulates genes involved in the mitotic control. A promising approach to treat tumors with deregulated Myc is the synthetic lethality based on the inhibition of Cdks. Thus, the knowledge of the Myc-dependent cell cycle regulatory mechanisms will help to discover new therapeutic approaches directed against malignancies with deregulated Myc. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

    Paek, Andrew L; Weinert, Ted

    2010-11-24

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

  20. 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

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

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

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

  2. Magnolol elicits activation of the extracellular signal-regulated kinase pathway by inducing p27KIP1-mediated G2/M-phase cell cycle arrest in human urinary bladder cancer 5637 cells.

    PubMed

    Lee, Se-Jung; Cho, Young-Hwa; Park, Keerang; Kim, Eun-Jung; Jung, Kyung-Hwan; Park, Sung-Soo; Kim, Wun-Jae; Moon, Sung-Kwon

    2008-06-15

    Magnolol has been reported to play a role in antitumor activity. However, the relevant pathway integrating cell cycle regulation and signaling pathways involved in growth inhibition in cancer cells remains to be identified. In the present study, magnolol treatment of these cells resulted in significant dose-dependent growth inhibition together with apoptosis, G1- and G2/M-phase cell cycle arrest at a 60 microM (IC50) dose in 5637 bladder cancer cells. In addition, magnolol treatment strongly induced p27KIP1 expression, and down-regulated expression of cyclin-dependent kinases (CDKs) and cyclins. Moreover, treatment with magnolol-induced phosphorylation of ERK, p38 MAP kinase, and JNK. Among the pathway inhibitors examined, only PD98059, an ERK-specific inhibitor, blocked magnolol-dependent p27KIP1 expression. Blockade of ERK function consistently reversed magnolol-mediated inhibition of cell proliferation and decreased G2/M cell cycle proteins, but not G1 cell cycle proteins. Furthermore, magnolol treatment increased both Ras and Raf activation. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed magnolol-induced ERK activity and p27KIP1 expression. Finally, the magnolol-induced reduction in cell proliferation and G2/M cell cycle proteins was also abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p27KIP1 induction, leading to a decrease in the levels of cyclin B1/Cdc2 complexes and magnolol-dependent inhibition of cell growth. Overall, these novel findings concerning the molecular mechanisms of magnolol in 5637 bladder cancer cells provide a theoretical basis for therapeutic treatment of malignancies.

  3. Fungal Cell Cycle: A Unicellular versus Multicellular Comparison.

    PubMed

    Dörter, Ilkay; Momany, Michelle

    2016-12-01

    All cells must accurately replicate DNA and partition it to daughter cells. The basic cell cycle machinery is highly conserved among eukaryotes. Most of the mechanisms that control the cell cycle were worked out in fungal cells, taking advantage of their powerful genetics and rapid duplication times. Here we describe the cell cycles of the unicellular budding yeast Saccharomyces cerevisiae and the multicellular filamentous fungus Aspergillus nidulans. We compare and contrast morphological landmarks of G1, S, G2, and M phases, molecular mechanisms that drive cell cycle progression, and checkpoints in these model unicellular and multicellular fungal systems.

  4. Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.

    PubMed

    Sánchez-Martínez, Concepción; Gelbert, Lawrence M; Lallena, María José; de Dios, Alfonso

    2015-09-01

    Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Does a shift to limited glucose activate checkpoint control in fission yeast?

    PubMed

    Saitoh, Shigeaki; Yanagida, Mitsuhiro

    2014-08-01

    Here we review cell cycle control in the fission yeast, Schizosaccharomyces pombe, in response to an abrupt reduction of glucose concentration in culture media. S. pombe arrests cell cycle progression when transferred from media containing 2.0% glucose to media containing 0.1%. After a delay, S. pombe resumes cell division at a surprisingly fast rate, comparable to that observed in 2% glucose. We found that a number of genes, including zinc-finger transcription factor Scr1, CaMKK-like protein kinase Ssp1, and glucose transporter Ght5, enable rapid cell division in low glucose. In this article, we examine whether cell cycle checkpoint-like control operates during the delay and after resumption of cell division in limited-glucose. Using microarray analysis and genetic screening, we identified several candidate genes that may be involved in controlling this low-glucose adaptation. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  6. 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.

  7. Mdt1, a Novel Rad53 FHA1 Domain-Interacting Protein, Modulates DNA Damage Tolerance and G2/M Cell Cycle Progression in Saccharomyces cerevisiae

    PubMed Central

    Pike, Brietta L.; Yongkiettrakul, Suganya; Tsai, Ming-Daw; Heierhorst, Jörg

    2004-01-01

    The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G2/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G2/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways. PMID:15024067

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2013-07-31

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

  10. G2 cell cycle arrest, down-regulation of cyclin B, and induction of mitotic catastrophe by the flavoprotein inhibitor diphenyleneiodonium.

    PubMed

    Scaife, Robin M

    2004-10-01

    Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.

  11. Subcellular Proteomics Reveals a Role for Nucleo-cytoplasmic Trafficking at the DNA Replication Origin Activation Checkpoint

    PubMed Central

    Mulvey, Claire M.; Tudzarova, Slavica; Crawford, Mark; Williams, Gareth H.; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations. PMID:23320540

  12. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  13. Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development

    PubMed Central

    Moser, Sandra C.; von Elsner, Sophie; Büssing, Ingo; Alpi, Arno; Schnabel, Ralf; Gartner, Anton

    2009-01-01

    CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. PMID:19360121

  14. Cell cycle regulation during viral infection.

    PubMed

    Bagga, Sumedha; Bouchard, Michael J

    2014-01-01

    To replicate their genomes in cells and generate new progeny, viruses typically require factors provided by the cells that they have infected. Subversion of the cellular machinery that controls replication of the infected host cell is a common activity of many viruses. Viruses employ different strategies to deregulate cell cycle checkpoint controls and modulate cell proliferation pathways. A number of DNA and RNA viruses encode proteins that target critical cell cycle regulators to achieve cellular conditions that are beneficial for viral replication. Many DNA viruses induce quiescent cells to enter the cell cycle; this is thought to increase pools of deoxynucleotides and thus, facilitate viral replication. In contrast, some viruses can arrest cells in a particular phase of the cell cycle that is favorable for replication of the specific virus. Cell cycle arrest may inhibit early cell death of infected cells, allow the cells to evade immune defenses, or help promote virus assembly. Although beneficial for the viral life cycle, virus-mediated alterations in normal cell cycle control mechanisms could have detrimental effects on cellular physiology and may ultimately contribute to pathologies associated with the viral infection, including cell transformation and cancer progression and maintenance. In this chapter, we summarize various strategies employed by DNA and RNA viruses to modulate the replication cycle of the virus-infected cell. When known, we describe how these virus-associated effects influence replication of the virus and contribute to diseases associated with infection by that specific virus.

  15. Drug-induced cell cycle modulation leading to cell-cycle arrest, nuclear mis-segregation, or endoreplication

    PubMed Central

    2011-01-01

    Background Cancer cell responses to chemotherapeutic agents vary, and this may reflect different defects in DNA repair, cell-cycle checkpoints, and apoptosis control. Cytometry analysis only quantifies dye-incorporation to examine DNA content and does not reflect the biological complexity of the cell cycle in drug discovery screens. Results Using population and time-lapse imaging analyses of cultured immortalized cells expressing a new version of the fluorescent cell-cycle indicator, Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator), we found great diversity in the cell-cycle alterations induced by two anticancer drugs. When treated with etoposide, an inhibitor of DNA topoisomerase II, HeLa and NMuMG cells halted at the G2/M checkpoint. HeLa cells remained there, but NMuMG cells then overrode the checkpoint and underwent nuclear mis-segregation or avoided the checkpoint and entered the endoreplication cycle in a drug concentration dependent manner. In contrast, an inhibitor of Cdk4 led to G1 arrest or endoreplication in NMuMG cells depending upon the initial cell-cycle phase of drug exposure. Conclusions Drug-induced cell cycle modulation varied not only between different cell types or following treatment with different drugs, but also between cells treated with different concentrations of the same drug or following drug addition during different phases of the cell cycle. By combining cytometry analysis with the Fucci probe, we have developed a novel assay that fully integrates the complexity of cell cycle regulation into drug discovery screens. This assay system will represent a powerful drug-discovery tool for the development of the next generation of anti-cancer therapies. PMID:21226962

  16. Topology and Control of the Cell-Cycle-Regulated Transcriptional Circuitry

    PubMed Central

    Haase, Steven B.; Wittenberg, Curt

    2014-01-01

    Nearly 20% of the budding yeast genome is transcribed periodically during the cell division cycle. The precise temporal execution of this large transcriptional program is controlled by a large interacting network of transcriptional regulators, kinases, and ubiquitin ligases. Historically, this network has been viewed as a collection of four coregulated gene clusters that are associated with each phase of the cell cycle. Although the broad outlines of these gene clusters were described nearly 20 years ago, new technologies have enabled major advances in our understanding of the genes comprising those clusters, their regulation, and the complex regulatory interplay between clusters. More recently, advances are being made in understanding the roles of chromatin in the control of the transcriptional program. We are also beginning to discover important regulatory interactions between the cell-cycle transcriptional program and other cell-cycle regulatory mechanisms such as checkpoints and metabolic networks. Here we review recent advances and contemporary models of the transcriptional network and consider these models in the context of eukaryotic cell-cycle controls. PMID:24395825

  17. A microbial avenue to cell cycle control in the plant superkingdom.

    PubMed

    Tulin, Frej; Cross, Frederick R

    2014-10-01

    Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage.

  18. A Microbial Avenue to Cell Cycle Control in the Plant Superkingdom[C][W][OPEN

    PubMed Central

    Tulin, Frej; Cross, Frederick R.

    2014-01-01

    Research in yeast and animals has resulted in a well-supported consensus model for eukaryotic cell cycle control. The fit of this model to early diverging eukaryotes, such as the plant kingdom, remains unclear. Using the green alga Chlamydomonas reinhardtii, we developed an efficient pipeline, incorporating robotics, semiautomated image analysis, and deep sequencing, to molecularly identify >50 genes, mostly conserved in higher plants, specifically required for cell division but not cell growth. Mutated genes include the cyclin-dependent kinases CDKA (resembling yeast and animal Cdk1) and the plant-specific CDKB. The Chlamydomonas cell cycle consists of a long G1 during which cells can grow >10-fold, followed by multiple rapid cycles of DNA replication and segregation. CDKA and CDKB execute nonoverlapping functions: CDKA promotes transition between G1 and entry into the division cycle, while CDKB is essential specifically for spindle formation and nuclear division, but not for DNA replication, once CDKA-dependent initiation has occurred. The anaphase-promoting complex is required for similar steps in the Chlamydomonas cell cycle as in Opisthokonts; however, the spindle assembly checkpoint, which targets the APC in Opisthokonts, appears severely attenuated in Chlamydomonas, based on analysis of mutants affecting microtubule function. This approach allows unbiased integration of the consensus cell cycle control model with innovations specific to the plant lineage. PMID:25336509

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

    PubMed Central

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

    1993-01-01

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

  20. Reverse the curse - the role of deubiquitination in cell cycle control

    PubMed Central

    Song, Ling; Rape, Michael

    2008-01-01

    Reversible protein ubiquitination is a crucial mechanism regulating the progression through the eukaryotic cell cycle. Ubiquitin-dependent signaling is terminated by specific deubiquitinating enzymes (DUBs), which now are known to be integral components of the core cell cycle machinery and cell cycle checkpoints. The importance of DUBs for cell cycle control is underscored by their frequent misregulation in cancer. Here, we discuss the role of deubiquitinating enzymes in controlling proliferation. PMID:18346885

  1. Cordycepin causes p21WAF1-mediated G2/M cell-cycle arrest by regulating c-Jun N-terminal kinase activation in human bladder cancer cells.

    PubMed

    Lee, Se-Jung; Kim, Si-Kwan; Choi, Won-Seok; Kim, Wun-Jae; Moon, Sung-Kwon

    2009-10-15

    Cordycepin (3'-deoxyadenosine), a bioactive compound of Cordyceps militaris, has many pharmacological activities. The present study reveals novel molecular mechanisms for the anti-tumor effects of cordycepin in two different bladder cancer cell lines, 5637 and T-24 cells. Cordycepin treatment, at a dose of 200 microM (IC(50)) during cell-cycle progression resulted in significant and dose-dependent growth inhibition, which was largely due to G2/M-phase arrest, and resulted in an up-regulation of p21WAF1 expression, independent of the p53 pathway. Moreover, treatment with cordycepin-induced phosphorylation of JNK (c-Jun N-terminal kinases). Blockade of JNK function using SP6001259 (JNK-specific inhibitor) and small interfering RNA (si-JNK1) rescued cordycepin-dependent p21WAF1 expression, inhibited cell growth, and decreased cell cycle proteins. These results suggest that cordycepin could be an effective treatment for bladder cancer.

  2. Molecular mechanisms controlling the cell cycle in embryonic stem cells.

    PubMed

    Abdelalim, Essam M

    2013-12-01

    Embryonic stem (ES) cells are originated from the inner cell mass of a blastocyst stage embryo. They can proliferate indefinitely, maintain an undifferentiated state (self-renewal), and differentiate into any cell type (pluripotency). ES cells have an unusual cell cycle structure, consists mainly of S phase cells, a short G1 phase and absence of G1/S checkpoint. Cell division and cell cycle progression are controlled by mechanisms ensuring the accurate transmission of genetic information from generation to generation. Therefore, control of cell cycle is a complicated process, involving several signaling pathways. Although great progress has been made on the molecular mechanisms involved in the regulation of ES cell cycle, many regulatory mechanisms remain unknown. This review summarizes the current knowledge about the molecular mechanisms regulating the cell cycle of ES cells and describes the relationship existing between cell cycle progression and the self-renewal.

  3. p53 controls CDC7 levels to reinforce G1 cell cycle arrest upon genotoxic stress

    PubMed Central

    Tudzarova, Slavica; Dey, Ayona; Stoeber, Kai; Okorokov, Andrei L.; Williams, Gareth H.

    2016-01-01

    ABSTRACT DNA replication initiation is a key event in the cell cycle, which is dependent on 2 kinases - CDK2 and CDC7. Here we report a novel mechanism in which p53 induces G1 checkpoint and cell cycle arrest by downregulating CDC7 kinase in response to genotoxic stress. We demonstrate that p53 controls CDC7 stability post-transcriptionally via miR-192/215 and post-translationally via Fbxw7β E3 ubiquitin ligase. The p53-dependent pathway of CDC7 downregulation is interlinked with the p53-p21-CDK2 pathway, as p21-mediated inhibition of CDK2-dependent phosphorylation of CDC7 on Thr376 is required for GSK3ß-phosphorylation and Fbxw7ß-dependent degradation of CDC7. Notably, sustained oncogenic high levels of active CDC7 exert a negative feedback onto p53, leading to unrestrained S-phase progression and accumulation of DNA damage. Thus, p53-dependent control of CDC7 levels is essential for blocking G1/S cell-cycle transition upon genotoxic stress, thereby safeguarding the genome from instability and thus representing a novel general stress response. PMID:27611229

  4. Current topics of functional links between primary cilia and cell cycle.

    PubMed

    Izawa, Ichiro; Goto, Hidemasa; Kasahara, Kousuke; Inagaki, Masaki

    2015-01-01

    Primary cilia, microtubule-based sensory structures, orchestrate various critical signals during development and tissue homeostasis. In view of the rising interest into the reciprocal link between ciliogenesis and cell cycle, we discuss here several recent advances to understand the molecular link between the individual step of ciliogenesis and cell cycle control. At the onset of ciliogenesis (the transition from centrosome to basal body), distal appendage proteins have been established as components indispensable for the docking of vesicles at the mother centriole. In the initial step of axonemal extension, CP110, Ofd1, and trichoplein, key negative regulators of ciliogenesis, are found to be removed by a kinase-dependent mechanism, autophagy, and ubiquitin-proteasome system, respectively. Of note, their disposal functions as a restriction point to decide that the axonemal nucleation and extension begin. In the elongation step, Nde1, a negative regulator of ciliary length, is revealed to be ubiquitylated and degraded by CDK5-SCF(Fbw7) in a cell cycle-dependent manner. With regard to ciliary length control, it has been uncovered in flagellar shortening of Chlamydomonas that cilia itself transmit a ciliary length signal to cytoplasm. At the ciliary resorption step upon cell cycle re-entry, cilia are found to be disassembled not only by Aurora A-HDAC6 pathway but also by Nek2-Kif24 and Plk1-Kif2A pathways through their microtubule-depolymerizing activity. On the other hand, it is becoming evident that the presence of primary cilia itself functions as a structural checkpoint for cell cycle re-entry. These data suggest that ciliogenesis and cell cycle intimately link each other, and further elucidation of these mechanisms will contribute to understanding the pathology of cilia-related disease including cancer and discovering targets of therapeutic interventions.

  5. The microbial cell cycle

    SciTech Connect

    Nurse, P.; Streiblova, E.

    1984-01-01

    This book concentrates on the major problems of cell cycle control in microorganisms. A wide variety of microorganisms, ranging from bacteria and yeasts to hyphal fungi, algae, and ciliates are analyzed, with emphasis on the basic similarities among the organisms. Different ways of looking at cell cycle control which emphasize aspects of the problem such as circadian rhythms, limit cycle oscillators, and cell size models, are considered. New approaches such as the study of cell cycle mutants, and cloning of cell cycle control genes are also presented.

  6. c-Jun N-terminal kinase 1 is required for cordycepin-mediated induction of G2/M cell-cycle arrest via p21WAF1 expression in human colon cancer cells.

    PubMed

    Lee, Se-Jung; Moon, Gi-Seong; Jung, Kyung-Hwan; Kim, Wun-Jae; Moon, Sung-Kwon

    2010-01-01

    Cordycepin (3'-deoxyadenosine) has many anti-cancer properties. However, neither its molecular mechanism nor its molecular targets are well understood. In the present study, we investigated novel molecular mechanisms for the anti-tumor effects of cordycepin in human colon cancer HCT116 cells. After treatment of cells with cordycepin, dose-dependent cell growth inhibition was observed at an IC(50) value of 200muM. Cordycepin treatment resulted in G2/M-phase cell-cycle arrest, which was associated with increased p21WAF1 levels and reduced amounts of cyclin B1, Cdc2, and Cdc25c in a p53-independent pathway. Moreover, cordycepin treatment induced activation of JNK (c-Jun N-terminal kinases). Pretreatment with SP600125, a JNK-specific inhibitor, abrogated cordycepin-mediated p21WAF1 expression, cell growth inhibition, and reduced cell-cycle proteins. Furthermore, JNK1 inhibition by small interfering RNA (siRNA) produced similar results: suppression of cordycepin-induced p21WAF1 expression, decreased cell growth, and reduced cell-cycle proteins. Together, these results suggest a critical role for JNK1 activation in cordycepin-induced inhibition of cell growth and G2/M-phase arrest in human colon cancer cells. Copyright 2009 Elsevier Ltd. All rights reserved.

  7. Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.

    PubMed

    Heise, Nicole; Palme, Daniela; Misovic, Milan; Koka, Saisudha; Rudner, Justine; Lang, Florian; Salih, Helmut R; Huber, Stephan M; Henke, Guido

    2010-01-01

    Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

  8. Effect of a combination of S-1 and gemcitabine on cell cycle regulation in pancreatic cancer cell lines.

    PubMed

    Morimoto, Yoshihito; Takeuchi, Osamu; Takizawa, Asako; Yoneyama, Hiroshi; Asanuma, Fumiki; Suzuki, Yukio; Atsuda, Koichiro; Yamada, Yoshinori

    2012-06-01

    In a previous study, we showed that a combination of an oral fluoropyrimidine anticancer agent (S-1) and gemcitabine (GEM) had synergistic effects on cell growth and cell cycle arrest in the pancreatic cancer cell line MIA PaCa-2. Therefore, we conducted further mechanistic studies using the pancreatic cancer cell lines MIA PaCa-2 and SUIT-2. The combined effect of S-1 and GEM in SUIT-2 cells was evaluated using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the effects of S-1, GEM and S-1 plus GEM on cell cycle regulation were assessed using flow cytometry. We also examined the expression of several cell cycle regulatory proteins in both MIA PaCa-2 and SUIT-2 cells by western blotting. Classical isobolographic analysis of the MTT assay results showed that the combination of S-1 and GEM had a synergistic effect in SUIT-2 cells, and flow cytometric analysis of the cell cycle showed that the combination of S-1 plus GEM induced S-phase arrest to a greater degree than did either S-1 or GEM alone. Also, the combination of S-1 and GEM resulted in the downregulation of cyclin D1 expression and upregulation of cyclin A, p21 and p27 expression levels. Treatment of MIA PaCa-2 and SUIT-2 cells with a combination of both drugs also led to the increased phosphorylation of checkpoint kinase 1. Combined treatment with S-1 and GEM resulted in more prolonged S-phase arrest than with either treatment alone. This difference is shown to be potentially due to the higher levels of phosphorylated checkpoint kinase 1 in pancreatic cancer cell lines treated with the two agents.

  9. The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization*

    PubMed Central

    Sawicka, Marta; Wanrooij, Paulina H.; Darbari, Vidya C.; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V.; Burgers, Peter M.; Zhang, Xiaodong

    2016-01-01

    The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. PMID:27129217

  10. The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

    PubMed

    Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

    2016-06-24

    The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

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

    PubMed

    Gao, Xiaodong; Han, Liping; Ren, Yujie

    2016-05-05

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

  12. Polo-like kinase 1 inhibitor BI2536 causes mitotic catastrophe following activation of the spindle assembly checkpoint in non-small cell lung cancer cells.

    PubMed

    Choi, Minji; Kim, Wootae; Cheon, Min Gyeong; Lee, Chang Woo; Kim, Ja Eun

    2015-02-28

    Polo-like kinase 1 (PLK1), a critical kinase that regulates multiple steps in mitosis, is overexpressed in diverse human cancers; thus many PLK1 inhibitors have been developed as potential cancer therapeutic agents. One of these compounds, the PLK1-specific inhibitor BI2536, has been investigated as a cytotoxic drug in several cancers, including lung cancer; however, the detailed mechanism by which BI2536 induces defects in cell proliferation of non-small cell lung cancer (NSCLC) has not yet been determined. We found that BI2536 treatment resulted in mitotic arrest due to improper formation of the mitotic spindles and mitotic centrosomes. The unattached kinetochores in BI2536-treated NSCLC cells activated the spindle assembly checkpoint (SAC). The prolonged activation of the SAC led to a type of apoptotic cell death referred to as mitotic catastrophe. Finally, BI2536-treated NSCLC cells show a defect in cell proliferation. Overall, these data indicate that PLK1 inhibition via mitotic disruption represents a potential approach for the treatment of NSCLC. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. Cell cycle gene expression under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  14. PaKRP, a cyclin-dependent kinase inhibitor from avocado, may facilitate exit from the cell cycle during fruit growth.

    PubMed

    Sabag, Michal; Ben Ari, Giora; Zviran, Tali; Biton, Iris; Goren, Moshe; Dahan, Yardena; Sadka, Avi; Irihimovitch, Vered

    2013-12-01

    Previous studies using 'Hass' avocado cultivar showed that its small-fruit (SF) phenotype is limited by cell number. To explore the molecular components affecting avocado cell production, we isolated four cDNAs encoding: an ICK/KRP protein, known to play cell cycle-regulating roles through modulation of CDK function; two CDK proteins and a D-type cyclin, and monitored their expression patterns, comparing NF (normal fruit) versus SF profiles. The accumulation of PaKRP gradually deceased during growth in both fruit populations. Despite these similarities, SF exhibited higher levels of PaKRP accumulation at early stages of growth. Moreover, in NF, augmented PaKRP expression coincided with a decrease in CDK and PaCYCD1 levels, whereas in SF, enhanced PaKPR expression was coupled with an earlier decline of CDK and PaCYCD1 levels. For both NF and SF, enhanced mesocarp PaKRP transcript accumulation, was associated with elevated abscisic acid (ABA) and ABA catabolites content. Nevertheless, the collective ABA levels, including catabolites, were substantially higher in SF tissues, as compared with NF tissues. Finally, additional expression analysis revealed that in cultured cells, PaKRP could be induced by ABA. Together, our data links PaKRP with exit from the fruit cell cycle and suggest a role for ABA in controlling its expression.

  15. A role for homologous recombination proteins in cell cycle regulation.

    PubMed

    Kostyrko, Kaja; Bosshard, Sandra; Urban, Zuzanna; Mermod, Nicolas

    2015-01-01

    Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling.

  16. RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation

    PubMed Central

    2012-01-01

    Introduction In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR. Methods With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor. Results IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis. Conclusion Studies in this report suggest that Rac1 GTPase plays an

  17. From the Cover: Hysteresis drives cell-cycle transitions in Xenopus laevis egg extracts

    NASA Astrophysics Data System (ADS)

    Sha, Wei; Moore, Jonathan; Chen, Katherine; Lassaletta, Antonio D.; Yi, Chung-Seon; Tyson, John J.; Sible, Jill C.

    2003-02-01

    Cells progressing through the cell cycle must commit irreversibly to mitosis without slipping back to interphase before properly segregating their chromosomes. A mathematical model of cell-cycle progression in cell-free egg extracts from frog predicts that irreversible transitions into and out of mitosis are driven by hysteresis in the molecular control system. Hysteresis refers to toggle-like switching behavior in a dynamical system. In the mathematical model, the toggle switch is created by positive feedback in the phosphorylation reactions controlling the activity of Cdc2, a protein kinase bound to its regulatory subunit, cyclin B. To determine whether hysteresis underlies entry into and exit from mitosis in cell-free egg extracts, we tested three predictions of the Novak-Tyson model. (i) The minimal concentration of cyclin B necessary to drive an interphase extract into mitosis is distinctly higher than the minimal concentration necessary to hold a mitotic extract in mitosis, evidence for hysteresis. (ii) Unreplicated DNA elevates the cyclin threshold for Cdc2 activation, indication that checkpoints operate by enlarging the hysteresis loop. (iii) A dramatic "slowing down" in the rate of Cdc2 activation is detected at concentrations of cyclin B marginally above the activation threshold. All three predictions were validated. These observations confirm hysteresis as the driving force for cell-cycle transitions into and out of mitosis.

  18. Effect of sesamin on apoptosis and cell cycle arrest in human breast cancer mcf-7 cells.

    PubMed

    Siao, An-Ci; Hou, Chien-Wei; Kao, Yung-Hsi; Jeng, Kee-Ching

    2015-01-01

    Dietary prevention has been known to reduce breast cancer risk. Sesamin is one of the major components in sesame seeds and has been widely studied and proven to have anti-proliferation and anti-angiogenic effects on cancer cells. In this study, the influence of sesamin was tested in the human breast cancer MCF-7 cell line for cell viability (MTT assay) and cell cycling (flow cytometry). Results showed that sesamin dose-dependently (1, 10 and 50 μM) reduced the cell viability and increased LDH release and apoptosis (TUNEL assay). In addition, there was a significant increase of sub-G1 phase arrest in the cell cycle after sesamin treatment. Furthermore, sesamin increased the expression of apoptotic markers of Bax, caspase-3, and cell cycle control proteins, p53 and checkpoint kinase 2. Taken together, these results suggested that sesamin might be used as a dietary supplement for prevention of breast cancer by modulating apoptotic signal pathways and inhibiting tumor cell growth.

  19. Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21Waf1/Cip1 expression: involvement of p38 mitogen-activated protein kinase.

    PubMed

    Weng, Meng-Shih; Ho, Yuan-Soon; Lin, Jen-Kun

    2005-06-15

    Flavonoids are a broadly distributed class of plant pigments, universally present in plants. They are strong anti-oxidants that can inhibit carcinogenesis in rodents. Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammatory, anti-oxidant properties, promotes cell death, and perturbing cell cycle progression. However, the mechanism by which chrysin inhibits cancer cell growth remains poorly understood. Therefore, we developed an interest in the relationship between MAPK signaling pathways and cell growth inhibition after chrysin treatment in rat C6 glioma cells. Cell viability assay and flow cytometric analysis suggested that chrysin exhibited a dose-dependent and time-dependent ability to block rat C6 glioma cell line cell cycle progression at the G1 phase. Western blotting analysis showed that the levels of Rb phosphorylation in C6 glioma cells exposed to 30 microM chrysin for 24h decreased significantly. We demonstrated the expression of cyclin-dependent kinase inhibitor, p21(Waf1/Cip1), to be significantly increased, but the p53 protein level did not change in chrysin-treated cells. Both cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) kinase activities were reduced by chrysin in a dose-dependent manner. Furthermore, chrysin also inhibited proteasome activity. We further showed that chrysin induced p38-MAPK activation, and using a specific p38-MAPK inhibitor, SB203580, attenuated chrysin-induced p21(Waf1/Cip1) expression. These results suggest that chrysin exerts its growth-inhibitory effects either through activating p38-MAPK leading to the accumulation of p21(Waf1/Cip1) protein or mediating the inhibition of proteasome activity.

  20. The Chlamydomonas cell cycle.

    PubMed

    Cross, Frederick R; Umen, James G

    2015-05-01

    The position of Chlamydomonas within the eukaryotic phylogeny makes it a unique model in at least two important ways: as a representative of the critically important, early-diverging lineage leading to plants; and as a microbe retaining important features of the last eukaryotic common ancestor (LECA) that has been lost in the highly studied yeast lineages. Its cell biology has been studied for many decades and it has well-developed experimental genetic tools, both classical (Mendelian) and molecular. Unlike land plants, it is a haploid with very few gene duplicates, making it ideal for loss-of-function genetic studies. The Chlamydomonas cell cycle has a striking temporal and functional separation between cell growth and rapid cell division, probably connected to the interplay between diurnal cycles that drive photosynthetic cell growth and the cell division cycle; it also exhibits a highly choreographed interaction between the cell cycle and its centriole-basal body-flagellar cycle. Here, we review the current status of studies of the Chlamydomonas cell cycle. We begin with an overview of cell-cycle control in the well-studied yeast and animal systems, which has yielded a canonical, well-supported model. We discuss briefly what is known about similarities and differences in plant cell-cycle control, compared with this model. We next review the cytology and cell biology of the multiple-fission cell cycle of Chlamydomonas. Lastly, we review recent genetic approaches and insights into Chlamydomonas cell-cycle regulation that have been enabled by a new generation of genomics-based tools. © 2015 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  1. Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

    PubMed Central

    MacAuley, Alasdair; Cross, James C.; Werb, Zena

    1998-01-01

    Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle. PMID:9529378

  2. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

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

    2014-01-01

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

  3. Cell cycle regulation by long non-coding RNAs.

    PubMed

    Kitagawa, Masatoshi; Kitagawa, Kyoko; Kotake, Yojiro; Niida, Hiroyuki; Ohhata, Tatsuya

    2013-12-01

    The mammalian cell cycle is precisely controlled by cyclin-dependent kinases (CDKs) and related pathways such as the RB and p53 pathways. Recent research on long non-coding RNAs (lncRNAs) indicates that many lncRNAs are involved in the regulation of critical cell cycle regulators such as the cyclins, CDKs, CDK inhibitors, pRB, and p53. These lncRNAs act as epigenetic regulators, transcription factor regulators, post-transcription regulators, and protein scaffolds. These cell cycle-regulated lncRNAs mainly control cellular levels of cell cycle regulators via various mechanisms, and may provide diversity and reliability to the general cell cycle. Interestingly, several lncRNAs are induced by DNA damage and participate in cell cycle arrest or induction of apoptosis as DNA damage responses. Therefore, deregulations of these cell cycle regulatory lncRNAs may be involved in tumorigenesis, and they are novel candidate molecular targets for cancer therapy and diagnosis.

  4. Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells.

    PubMed

    Bryant, Christopher; Scriven, Kirsten; Massey, Andrew J

    2014-06-10

    Chk1 forms a core component of the DNA damage response and small molecule inhibitors are currently being investigated in the clinic as cytotoxic chemotherapy potentiators. Recent evidence suggests that Chk1 inhibitors may demonstrate significant single agent activity in tumors with specific DNA repair defects, a constitutively activated DNA damage response or oncogene induced replicative stress. Growth inhibition induced by the small molecule Chk1 inhibitor V158411 was assessed in a panel of human leukemia and lymphoma cell lines and compared to cancer cell lines derived from solid tumors. The effects on cell cycle and DNA damage response markers were further evaluated. Leukemia and lymphoma cell lines were identified as particularly sensitive to the Chk1 inhibitor V158411 (mean GI50 0.17 μM) compared to colon (2.8 μM) or lung (6.9 μM) cancer cell lines. Chk1 inhibition by V158411 in the leukemia and lymphoma cell lines induced DNA fragmentation and cell death that was both caspase dependent and independent, and prevented cells undergoing mitosis. An analysis of in vitro pharmacodynamic markers identified a dose dependent decrease in Chk1 and cyclin B1 protein levels and Cdc2 Thr15 phosphorylation along with a concomitant increase in H2AX phosphorylation at Ser139 following V158411 treatment. These data support the further evaluation of Chk1 inhibitors in hematopoietic cancers as single agents as well as in combination with standard of care cytotoxic drugs.

  5. The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

    PubMed

    Masrouha, Nisrine; Yang, Long; Hijal, Sirine; Larochelle, Stéphane; Suter, Beat

    2003-03-01

    Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

  6. The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

    PubMed Central

    Masrouha, Nisrine; Yang, Long; Hijal, Sirine; Larochelle, Stéphane; Suter, Beat

    2003-01-01

    Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system. PMID:12663536

  7. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

    2000-01-01

    Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

  8. Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway

    NASA Technical Reports Server (NTRS)

    Chakravarthy, M. V.; Abraha, T. W.; Schwartz, R. J.; Fiorotto, M. L.; Booth, F. W.

    2000-01-01

    Interest is growing in methods to extend replicative life span of non-immortalized stem cells. Using the insulin-like growth factor I (IGF-I) transgenic mouse in which the IGF-I transgene is expressed during skeletal muscle development and maturation prior to isolation and during culture of satellite cells (the myogenic stem cells of mature skeletal muscle fibers) as a model system, we elucidated the underlying molecular mechanisms of IGF-I-mediated enhancement of proliferative potential of these cells. Satellite cells from IGF-I transgenic muscles achieved at least five additional population doublings above the maximum that was attained by wild type satellite cells. This IGF-I-induced increase in proliferative potential was mediated via activation of the phosphatidylinositol 3'-kinase/Akt pathway, independent of mitogen-activated protein kinase activity, facilitating G(1)/S cell cycle progression via a down-regulation of p27(Kip1). Adenovirally mediated ectopic overexpression of p27(Kip1) in exponentially growing IGF-I transgenic satellite cells reversed the increase in cyclin E-cdk2 kinase activity, pRb phosphorylation, and cyclin A protein abundance, thereby implicating an important role for p27(Kip1) in promoting satellite cell senescence. These observations provide a more complete dissection of molecular events by which increased local expression of a growth factor in mature skeletal muscle fibers extends replicative life span of primary stem cells than previously known.

  9. Cdc28 provides a molecular link between Hsp90, morphogenesis, and cell cycle progression in Candida albicans

    PubMed Central

    Senn, Heather; Shapiro, Rebecca S.; Cowen, Leah E.

    2012-01-01

    The trimorphic fungus Candida albicans is the leading cause of systemic candidiasis, a disease with poor prognosis affecting immunocompromised individuals. The capacity of C. albicans to transition between morphological states is a key determinant of its ability to cause life-threatening infection. Recently the molecular chaperone heat shock protein 90 (Hsp90) was implicated as a major regulator of temperature-dependent C. albicans morphogenesis; compromising Hsp90 function induces filamentation and relieves repression of Ras1–protein kinase A (PKA) signaling, although the mechanism involved remains unknown. Here we demonstrate that filaments generated by compromise of Hsp90 function are neither pseudohyphae nor hyphae but closely resemble filaments formed in response to cell cycle arrest. Closer examination revealed that these filaments exhibit a delay in mitotic exit mediated by the checkpoint protein Bub2. Furthermore, Hsp90 inhibition also led to a distinct morphology with defects in cytokinesis. We found that the cyclin-dependent kinase Cdc28 was destabilized in response to depletion of Hsp90 and that Cdc28 physically interacts with Hsp90, implicating this major cell cycle regulator as a novel Hsp90 client protein in C. albicans. Taken together, our results suggest that Hsp90 is instrumental in the regulation of cell division during yeast-form growth in C. albicans and exerts its major effects during late cell cycle events. PMID:22090345

  10. Cell cycle-dependent localization of CHK2 at centrosomes during mitosis

    PubMed Central

    2013-01-01

    Background Centrosomes function primarily as microtubule-organizing centres and play a crucial role during mitosis by organizing the bipolar spindle. In addition to this function, centrosomes act as reaction centers where numerous key regulators meet to control cell cycle progression. One of these factors involved in genome stability, the checkpoint kinase CHK2, was shown to localize at centrosomes throughout the cell cycle. Results Here, we show that CHK2 only localizes to centrosomes during mitosis. Using wild-type and CHK2−/− HCT116 human colon cancer cells and human osteosarcoma U2OS cells depleted for CHK2 with small hairpin RNAs we show that several CHK2 antibodies are non-specific and cross-react with an unknown centrosomal protein(s) by immunofluorescence. To characterize the localization of CHK2, we generated cells expressing inducible GFP-CHK2 and Flag-CHK2 fusion proteins. We show that CHK2 localizes to the nucleus in interphase cells but that a fraction of CHK2 associates with the centrosomes in a Polo-like kinase 1-dependent manner during mitosis, from early mitotic stages until cytokinesis. Conclusion Our findings demonstrate that a subpopulation of CHK2 localizes at the centrosomes in mitotic cells but not in interphase. These results are consistent with previous reports supporting a role for CHK2 in the bipolar spindle formation and the timely progression of mitosis. PMID:23680298

  11. Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells.

    PubMed

    Kuffer, Christian; Kuznetsova, Anastasia Yurievna; Storchová, Zuzana

    2013-08-01

    Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.

  12. Mps1 is SUMO-modified during the cell cycle

    PubMed Central

    Chen, Changyan; Lu, Lou; Dai, Wei

    2016-01-01

    Mps1 is a dual specificity protein kinase that regulates the spindle assembly checkpoint and mediates proper microtubule attachment to chromosomes during mitosis. However, the molecular mechanism that controls Mps1 protein level and its activity during the cell cycle remains unclear. Given that sumoylation plays an important role in mitotic progression, we investigated whether Mps1 was SUMO-modified and whether sumoylation affects its activity in mitosis. Our results showed that Mps1 was sumoylated in both asynchronized and mitotic cell populations. Mps1 was modified by both SUMO-1 and SUMO-2. Our further studies revealed that lysine residues including K71, K287, K367 and K471 were essential for Mps1 sumoylation. Sumoylation appeared to play a role in mediating kinetochore localization of Mps1, thus affecting normal mitotic progression. Furthermore, SUMO-resistant mutants of Mps1 interacted with BubR1 more efficiently than it did with the wild-type control. Combined, our results indicate that Mps1 is SUMO-modified that plays an essential role in regulating Mps1 functions during mitosis. PMID:26675261

  13. Targeting cell cycle regulation in cancer therapy.

    PubMed

    Diaz-Moralli, Santiago; Tarrado-Castellarnau, Míriam; Miranda, Anibal; Cascante, Marta

    2013-05-01

    Cell proliferation is an essential mechanism for growth, development and regeneration of eukaryotic organisms; however, it is also the cause of one of the most devastating diseases of our era: cancer. Given the relevance of the processes in which cell proliferation is involved, its regulation is of paramount importance for multicellular organisms. Cell division is orchestrated by a complex network of interactions between proteins, metabolism and microenvironment including several signaling pathways and mechanisms of control aiming to enable cell proliferation only in response to specific stimuli and under adequate conditions. Three main players have been identified in the coordinated variation of the many molecules that play a role in cell cycle: i) The cell cycle protein machinery including cyclin-dependent kinases (CDK)-cyclin complexes and related kinases, ii) The metabolic enzymes and related metabolites and iii) The reactive-oxygen species (ROS) and cellular redox status. The role of these key players and the interaction between oscillatory and non-oscillatory species have proved essential for driving the cell cycle. Moreover, cancer development has been associated to defects in all of them. Here, we provide an overview on the role of CDK-cyclin complexes, metabolic adaptations and oxidative stress in regulating progression through each cell cycle phase and transitions between them. Thus, new approaches for the design of innovative cancer therapies targeting crosstalk between cell cycle simultaneous events are proposed. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Methylselenol, a selenium metabolite, induces cell cycle arrest in G1 phase and apoptosis via the extracellular-regulated kinase 1/2 pathway and other cancer signaling genes.

    PubMed

    Zeng, Huawei; Wu, Min; Botnen, James H

    2009-09-01

    Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo, and our previous study demonstrated that submicromolar methylselenol generated by incubating methionase with seleno-l-methionine inhibits the migration and invasive potential of HT1080 tumor cells. However, little is known about the association between cancer signal pathways and methylselenol's inhibition of tumor cell invasion. In this study, we demonstrated that methylselenol exposure inhibited cell growth and we used a cancer signal pathway-specific array containing 15 different signal transduction pathways involved in oncogenesis to study the effect of methylselenol on cellular signaling. Using real-time RT-PCR, we confirmed that cellular mRNA levels of cyclin-dependent kinase inhibitor 1C (CDKN1C), heme oxygenase 1, platelet/endothelial cell adhesion molecule, and PPARgamma genes were upregulated to 2.8- to 5.7-fold of the control. BCL2-related protein A1, hedgehog interacting protein, and p53 target zinc finger protein genes were downregulated to 26-52% of the control, because of methylselenol exposure. These genes are directly related to the regulation of cell cycle and apoptosis. Methylselenol increased apoptotic cells up to 3.4-fold of the control and inhibited the extracellular-regulated kinase 1/2 (ERK1/2) signaling and cellular myelocytomatosis oncogene (c-Myc) expression. Taken together, our studies identify 7 novel methylselenol responsive genes and demonstrate that methylselenol inhibits ERK1/2 pathway activation and c-Myc expression. The regulation of these genes is likely to play a key role in G1 cell cycle arrest and apoptosis, which may contribute to the inhibition of tumor cell invasion.

  15. A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines.

    PubMed

    Brandi, Giorgio; Paiardini, Mirko; Cervasi, Barbara; Fiorucci, Chiara; Filippone, Paolino; De Marco, Cinzia; Zaffaroni, Nadia; Magnani, Mauro

    2003-07-15

    Indole-3-carbinol (I3C), autolysis product of glucosinolates present in cruciferous vegetables, has been indicated as a promising agent in preventing the development and progression of breast cancer. I3C has been shown to inhibit the growth of human cancer cells in vitro and possesses anticarcinogenic activity in vivo. Because I3C is unstable and may be converted into many polymeric products in the digestive tract, it is not yet clear whether the biological activity observed can be attributed to I3C or some of its polymeric products. In this study we synthesized a stable I3C cyclic tetrameric derivative and investigated its effects on a panel of human breast cancer cell lines. The I3C tetramer suppressed the growth of both estrogen receptor (ER) -positive (MCF-7, 734B, and BT474) and ER-negative (BT20, MDA-MB-231, and BT539) human breast cancer cell lines, and it was found to induce G(1) cell cycle arrest in a dose-dependent manner without evidence of apoptosis, suggesting a growth arrest via a cytostatic mechanism. At the molecular level, the tetramer inhibited cyclin-dependent kinase (CDK) 6 expression and activity, induced an increase in the level of p27(kip1), and reduced the level of retinoblastoma protein expression. Contrarily to CDK6, the level of CDK4, the other kinase involved in the G(1) phase of the cell cycle, remains unchanged. Interestingly, the tetramer resulted about five times more active than I3C in suppressing the growth of human breast cancer cells. On the whole, our data suggest that the I3C tetrameric derivative is a novel lead inhibitor of breast cancer cell growth that may be a considered a new, promising therapeutic agent for both ER+ and ER- breast cancer.

  16. Glyphosate-based pesticides affect cell cycle regulation.

    PubMed

    Marc, Julie; Mulner-Lorillon, Odile; Bellé, Robert

    2004-04-01

    Cell-cycle dysregulation is a hallmark of tumor cells and human cancers. Failure in the cell-cycle checkpoints leads to genomic instability and subsequent development of cancers from the initial affected cell. A worldwide used product Roundup 3plus, based on glyphosate as the active herbicide, was suggested to be of human health concern since it induced cell cycle dysfunction as judged from analysis of the first cell division of sea urchin embryos, a recognized model for cell cycle studies. Several glyphosate-based pesticides from different manufacturers were assayed in comparison with Roundup 3plus for their ability to interfere with the cell cycle regulation. All the tested products, Amega, Cargly, Cosmic, and Roundup Biovert induced cell cycle dysfunction. The threshold concentration for induction of cell cycle dysfunction was evaluated for each product and suggests high risk by inhalation for people in the vicinity of the pesticide handling sprayed at 500 to 4000 times higher dose than the cell-cycle adverse concentration.

  17. The adaptor-like protein ROG-1 is required for activation of the Ras-MAP kinase pathway and meiotic cell cycle progression in Caenorhabditis elegans.

    PubMed

    Matsubara, Yosuke; Kawasaki, Ichiro; Urushiyama, Seiichi; Yasuda, Tomoharu; Shirakata, Masaki; Iino, Yuichi; Shibuya, Hiroshi; Yamanashi, Yuji

    2007-03-01

    The Ras-MAP kinase pathway regulates varieties of fundamental cellular events. In Caenorhabditis elegans, this pathway is required for oocyte development; however, the nature of its up-stream regulators has remained elusive. Here, we identified a C. elegans gene, rog-1, which encodes the only protein having the IRS-type phosphotyrosine-binding (PTB) domain in the worms. ROG-1 has no obvious domain structure aside from the PTB domain, suggesting that it could serve as an adaptor down-stream of protein-tyrosine kinases (PTKs). RNA interference (RNAi)-mediated down-regulation of rog-1 mRNA significantly decreased brood size. rog-1(tm1031) truncation mutants showed a severe disruption in progression of developing oocytes from pachytene to diakinesis, as was seen in worms carrying a loss-of-function mutation in the let-60 Ras or mpk-1 MAP kinase gene. Furthermore, let-60 Ras-regulated activation of MPK-1 in the gonad is undetectable in rog-1(tm1031) mutants. Conversely, a gain-of-function mutation in the let-60 Ras gene rescues the brood size reduction and germ cell abnormality in rog-1(tm1031) worms. Consistently, rog-1 is preferentially expressed in the germ cells and its expression in the gonad is essential for oocyte development. Thus, ROG-1 is a key positive regulator of the Ras-MAP kinase pathway that permits germ cells to exit from pachytene.

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

    PubMed

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

    2012-02-07

    Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c(+) cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8(+) and CD103(+) DC subsets in lymphoid and nonlymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Prosurvival signals from Toll-like receptors, CD40 and receptor activator of nuclear factor-κB (RANK) are integrated by TAK1 in DCs, which in turn mediated activation of downstream NF-κB and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T-cell homeostasis, and prevented effective T-cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a prosurvival checkpoint in DCs that affects the homeostasis and function of the immune system.

  19. SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis.

    PubMed

    Sridharan, Vinidhra; Azuma, Yoshiaki

    2016-08-17

    Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.

  20. SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

    PubMed Central

    Sridharan, Vinidhra; Azuma, Yoshiaki

    2016-01-01

    ABSTRACT Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres. PMID:27230136

  1. Lipin1 Regulates Skeletal Muscle Differentiation through Extracellular Signal-regulated Kinase (ERK) Activation and Cyclin D Complex-regulated Cell Cycle Withdrawal*

    PubMed Central

    Jiang, Weihua; Zhu, Jing; Zhuang, Xun; Zhang, Xiping; Luo, Tao; Esser, Karyn A.; Ren, Hongmei

    2015-01-01

    Lipin1, an intracellular protein, plays critical roles in controlling lipid synthesis and energy metabolism through its enzymatic activity and nuclear transcriptional functions. Several mouse models of skeletal muscle wasting are associated with lipin1 mutation or altered expression. Recent human studies have suggested that children with homozygous null mutations in the LPIN1 gene suffer from rhabdomyolysis. However, the underlying pathophysiologic mechanism is still poorly understood. In the present study we examined whether lipin1 contributes to regulating muscle regeneration. We characterized the time course of skeletal muscle regeneration in lipin1-deficient fld mice after injury. We found that fld mice exhibited smaller regenerated muscle fiber cross-sectional areas compared with wild-type mice in response to injury. Our results from a series of in vitro experiments suggest that lipin1 is up-regulated and translocated to the nucleus during myoblast differentiation and plays a key role in myogenesis by regulating the cytosolic activation of ERK1/2 to form a complex and a downstream effector cyclin D3-mediated cell cycle withdrawal. Overall, our study reveals a previously unknown role of lipin1 in skeletal muscle regeneration and expands our understanding of the cellular and molecular mechanisms underlying skeletal muscle regeneration. PMID:26296887

  2. Robust cell size checkpoint from spatiotemporal positive feedback loop in fission yeast.

    PubMed

    Yan, Jie; Ni, Xin; Yang, Ling

    2013-01-01

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

  3. Genome-wide inhibitory impact of the AMPK activator metformin on [kinesins, tubulins, histones, auroras and polo-like kinases] M-phase cell cycle genes in human breast cancer cells.

    PubMed

    Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Menendez, Javier A

    2009-05-15

    Prompted by the ever-growing scientific rationale for examining the antidiabetic drug metformin as a potential antitumor agent in breast cancer disease, we recently tested the hypothesis that the assessment of metformin-induced global changes in gene expression-as identified using 44 K (double density) Agilent's whole human genome arrays-could reveal gene-expression signatures that would allow proper selection of breast cancer patients who should be considered for metformin-based clinical trials. Using Database for Annotation, Visualization and Integrated Discovery bioinformatics (DAVID) resources we herein reveal that, at doses that lead to activation of the AMP-activated protein kinase (AMPK), metformin not only downregulates genes coding for ribosomal proteins (i.e., protein and macromolecule biosynthesis) but unexpectedly suppresses numerous mitosis-related gene families including kinesins, tubulins, histones, auroras and polo-like kinases. This is, to our knowledge, the first genome-scale evidence of a mitotic core component in the transcriptional response of human breast cancer cells to metformin. These findings further support a tight relationship between the activation status of AMPK and the chromosomal and cytoskeletal checkpoints of cell mitosis at the transcriptional level.

  4. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway

    USDA-ARS?s Scientific Manuscript database

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser33 in p53, which is associated with decreased p53...

  5. Virus manipulation of cell cycle.

    PubMed

    Nascimento, R; Costa, H; Parkhouse, R M E

    2012-07-01

    Viruses depend on host cell resources for replication and access to those resources may be limited to a particular phase of the cell cycle. Thus manipulation of cell cycle is a commonly employed strategy of viruses for achieving a favorable cellular environment. For example, viruses capable of infecting nondividing cells induce S phase in order to activate the host DNA replication machinery and provide the nucleotide triphosphates necessary for viral DNA replication (Flemington in J Virol 75:4475-4481, 2001; Sullivan and Pipas in Microbiol Mol Biol Rev 66:179-202, 2002). Viruses have developed several strategies to subvert the cell cycle by association with cyclin and cyclin-dependent kinase complexes and molecules that regulate their activity. Viruses tend to act on cellular proteins involved in a network of interactions in a way that minimal protein-protein interactions lead to a major effect. The complex and interactive nature of intracellular signaling pathways controlling cell division affords many opportunities for virus manipulation strategies. Taking the maxim "Set a thief to catch a thief" as a counter strategy, however, provides us with the very same virus evasion strategies as "ready-made tools" for the development of novel antivirus therapeutics. The most obvious are attenuated virus vaccines with critical evasion genes deleted. Similarly, vaccines against viruses causing cancer are now being successfully developed. Finally, as viruses have been playing chess with our cell biology and immune responses for millions of years, the study of their evasion strategies will also undoubtedly reveal new control mechanisms and their corresponding cellular intracellular signaling pathways.

  6. A role for the p34cdc2 kinase and phosphatases in the regulation of phosphorylation and disassembly of lamin B2 during the cell cycle.

    PubMed Central

    Lüscher, B; Brizuela, L; Beach, D; Eisenman, R N

    1991-01-01

    While the p34cdc2 kinase is considered to be a critical regulator of mitosis, its function has not yet been directly linked to one of the key events during the onset of mitosis: nuclear envelope breakdown. Here we show that a major structural protein of the nuclear envelope, lamin B2, is phosphorylated by p34cdc2. Results from two-dimensional phosphopeptide mapping experiments demonstrate that the p34cdc2-specific phosphopeptides represent both mitotic and interphase specific phosphorylations of lamin B2 and include the major interphase phosphorylation site. In mitotic cells we detected two distinct forms of lamin B2 which differ in electrophoretic mobility and in degree of phosphorylation. The phosphorylation pattern of lamin B2 generated in vitro by p34cdc2 was more closely related to the less phosphorylated mitotic lamin B2, suggesting that another kinase(s) in addition to p34cdc2 is involved in generating the mitotic phosphorylation pattern. In addition, we show that treatment of interphase cells with okadaic acid, a potent phosphatase inhibitor, leads to the acquisition of mitosis-specific phosphopeptides and can reversibly increase the detergent-solubility of lamin B2. However, the M-phase-like phosphorylation of lamin B2 in itself is not sufficient to induce its disassembly from the nuclear lamina suggesting that an additional event(s) besides phosphorylation is required. Images PMID:1849074

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

    PubMed

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

    2016-08-17

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

  8. Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

    PubMed Central

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T.C.; Fu, Li; Xu, Bo

    2011-01-01

    Summary The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically-activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. PMID:22099307

  9. Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

    PubMed

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T C; Fu, Li; Xu, Bo

    2011-11-18

    The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Cell cycle regulated synthesis of stable mouse thymidine kinase mRNA is mediated by a sequence within the cDNA.

    PubMed Central

    Hofbauer, R; Müllner, E; Seiser, C; Wintersberger, E

    1987-01-01

    The cDNA for mouse thymidine kinase (TK) was isolated from a cDNA library in lambda-gt11 and sequenced. It was used as a probe to follow the time course of TK mRNA expression in growth stimulated mouse fibroblasts. Linked to the HSV-TK promoter the cDNA was able to transform LTK-cells to the TK+ phenotype. The transformed cells expressed the TK mRNA and enzyme activity in a growth dependent fashion suggesting that the regulatory element is localized on the cDNA. Images PMID:3822814

  11. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    PubMed

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  12. Regulation of the Embryonic Cell Cycle During Mammalian Preimplantation Development.

    PubMed

    Palmer, N; Kaldis, P

    2016-01-01

    The preimplantation development stage of mammalian embryogenesis consists of a series of highly conserved, regulated, and predictable cell divisions. This process is essential to allow the rapid expansion and differentiation of a single-cell zygote into a multicellular blastocyst containing cells of multiple developmental lineages. This period of development, also known as the germinal stage, encompasses several important developmental transitions, which are accompanied by dramatic changes in cell cycle profiles and dynamics. These changes are driven primarily by differences in the establishment and enforcement of cell cycle checkpoints, which must be bypassed to facilitate the completion of essential cell cycle events. Much of the current knowledge in this area has been amassed through the study of knockout models in mice. These mouse models are powerful experimental tools, which have allowed us to dissect the relative dependence of the early embryonic cell cycles on various aspects of the cell cycle machinery and highlight the extent of functional redundancy between members of the same gene family. This chapter will explore the ways in which the cell cycle machinery, their accessory proteins, and their stimuli operate during mammalian preimplantation using mouse models as a reference and how this allows for the usually well-defined stages of the cell cycle to be shaped and transformed during this unique and critical stage of development. © 2016 Elsevier Inc. All rights reserved.

  13. Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

    PubMed

    Tran, Kalvin Q; Tin, Antony S; Firestone, Gary L

    2014-03-01

    Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

  14. Interplay between the cell cycle and double-strand break response in mammalian cells.

    PubMed

    Beishline, Kate; Azizkhan-Clifford, Jane

    2014-01-01

    The cell cycle is intimately associated with the ability of cells to sense and respond to and repair DNA damage. Understanding how cell cycle progression, particularly DNA replication and cell division, are regulated and how DNA damage can affect these processes has been the subject of intense research. Recent evidence suggests that the repair of DNA damage is regulated by the cell cycle, and that cell cycle factors are closely associated with repair factors and participate in cellular decisions regarding how to respond to and repair damage. Precise regulation of cell cycle progression in the presence of DNA damage is essential to maintain genomic stability and avoid the accumulation of chromosomal aberrations that can promote tumor formation. In this review, we discuss the current understanding of how mammalian cells induce cell cycle checkpoints in response to DNA double-strand breaks. In addition, we discuss how cell cycle factors modulate DNA repair pathways to facilitate proper repair of DNA lesions.

  15. Cell cycle control across the eukaryotic kingdom.

    PubMed

    Harashima, Hirofumi; Dissmeyer, Nico; Schnittger, Arp

    2013-07-01

    Almost two billion years of evolution have generated a vast and amazing variety of eukaryotic life with approximately 8.7 million extant species. Growth and reproduction of all of these organisms depend on faithful duplication and distribution of their chromosomes to the newly forming daughter cells in a process called the cell cycle. However, most of what is known today about cell cycle control comes from a few model species that belong to the unikonts; that is, to only one of five 'supergroups' that comprise the eukaryotic kingdom. Recently, analyzing species from distantly related clades is providing insights into general principles of cell cycle regulation and shedding light on its evolution. Here, referring to animal and fungal as opposed to non-unikont systems, especially flowering plants from the archaeplastid supergroup, we compare the conservation of central cell cycle regulator functions, the structure of network topologies, and the evolutionary dynamics of substrates of core cell cycle kinases. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Hexavalent chromium induces energy metabolism disturbance and p53-dependent cell cycle arrest via reactive oxygen species in L-02 hepatocytes.

    PubMed

    Xiao, Fang; Feng, Xiaotao; Zeng, Ming; Guan, Lan; Hu, Qingqing; Zhong, Caigao

    2012-12-01

    Hexavalent chromium [Cr(VI)] has become a non-negligible pollutant in the world. Cr(VI) exposure leads to severe damage to the liver, but the mechanisms involved in Cr(VI)-mediated toxicity in the liver are unclear. The present study aimed to explore whether Cr(VI) induces energy metabolism disturbance and cell cycle arrest in human L-02 hepatocytes. We showed that Cr(VI) inhibited state 3 respiration, respiratory control rate (RCR), and subsequently induced energy metabolism disturbance with decreased ATP production. Interestingly, cell cycle analysis by flow cytometry and protein expression analysis by western blotting revealed that low dose of Cr(VI) (4 uM) exposure induced S phase cell cycle arrest with decreased mediator of replication checkpoint 1 (Mrc1) and cyclin-dependent kinase 2 (CDK2), while higher doses of Cr(VI) (16, 32 uM) exposure resulted in G2/M phase arrest with decreased budding uninhibited by benzimidazoles-related 1 (BubR1) and cell division cycle 25 (CDC25). Mechanism study revealed that Cr(VI) decreased the activities of mitochondrial respiratory chain complex (MRCC) I and II, thus leading to ROS accumulation. Moreover, inhibiting ROS production by antioxidant N-acetyl-L-cysteine (NAC) rescued Cr(VI)-induced ATP depletion and cell cycle arrest. ROS-mediated p53 activation was found to involve in Cr(VI)-induced cell cycle arrest, and p53 inhibitor Pifithrin-α (PFT-α) rescued Cr(VI)-induced reduction of check point proteins Mrc1 and BubR1, thus inhibiting cell cycle arrest. In summary, the present study provides experimental evidence that Cr(VI) leads to energy metabolism disturbance and p53-dependent cell cycle arrest via ROS in L-02 hepatocytes.

  17. A Bioinformatics Approach Identifies Signal Transducer and Activator of Transcription-3 and Checkpoint Kinase 1 as Upstream Regulators of Kidney Injury Molecule-1 after Kidney Injury

    PubMed Central

    Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J.; Frank, David A.

    2014-01-01

    Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention. PMID:24158981

  18. A bioinformatics approach identifies signal transducer and activator of transcription-3 and checkpoint kinase 1 as upstream regulators of kidney injury molecule-1 after kidney injury.

    PubMed

    Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J; Frank, David A; Vaidya, Vishal S

    2014-01-01

    Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention.

  19. Minichromosome Maintenance Complex is Required for Checkpoint Kinase 2 Chromatin Loading and its Phosphorylation to DNA Damage Response in SCC-4 Cells.

    PubMed

    Li, Liang; Feng, Yi; Luo, Rui

    2017-01-01

    Checkpoint kinase 2 (Chk2) is a significant mediator of diverse responses to DNA damage. The present study was aimed to identify possible interactive proteins of Chk2 and try to clarify the underlying mechanism regarding Chk2 chromatin loading and its phosphorylation to DNA damage response in oral squamous cell carcinoma (OSCC). Differently tagged Chk2 and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were overexpressed into SCC-4 cells. After 48 h of transfection cell fractionation was performed to localize proteins. In addition, immunoreactive species were detected by immunoprecipitation (IP) and immunoblot (IB) analysis, and protein-protein interaction between Chk2 and MCM complex was ensured by glutathione S-transferase (GST) pull-down assay. Expression of MCM2 and MCM6 was downregulated by small interfering RNA (siRNA), and the chromatin and non-chromatin fraction were analyzed. The expression of Chk2 phosphorylation (pT68-Chk2) was measured after administration of different dosages of siMCM2 (0.5 μg, 1 μg, and 2.5 μg) and camptothecin (CPT). Our results showed that Chk2 directly interacts with MCM2, MCM3, MCM5, and MCM6 in SCC-4 cells. Downregulation of MCM2 and MCM6 markedly reduced Chk2 chromatin fraction, and downregulation of MCM2 decreased the expression of pT68-Chk2 to DNA damage response in a dose manner. Our results suggest that the interaction between Chk2 and MCM complex is required for Chk2 chromatin loading and its phosphorylation to DNA damage response in SCC-4 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Neuronal cell cycle: the neuron itself and its circumstances.

    PubMed

    Frade, José M; Ovejero-Benito, María C

    2015-01-01

    Neurons are usually regarded as postmitotic cells that undergo apoptosis in response to cell cycle reactivation. Nevertheless, recent evidence indicates the existence of a defined developmental program that induces DNA replication in specific populations of neurons, which remain in a tetraploid state for the rest of their adult life. Similarly, de novo neuronal tetraploidization has also been described in the adult brain as an early hallmark of neurodegeneration. The aim of this review is to integrate these recent developments in the context of cell cycle regulation and apoptotic cell death in neurons. We conclude that a variety of mechanisms exists in neuronal cells for G1/S and G2/M checkpoint regulation. These mechanisms, which are connected with the apoptotic machinery, can be modulated by environmental signals and the neuronal phenotype itself, thus resulting in a variety of outcomes ranging from cell death at the G1/S checkpoint to full proliferation of differentiated neurons.

  1. 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

  2. Down-regulation of cyclin-dependent kinase-4 and MAPK through estrogen receptor mediated cell cycle arrest in human breast cancer induced by gold nanoparticle tagged toxin protein NKCT1.

    PubMed

    Bhowmik, Tanmoy; Gomes, Antony

    2017-04-25

    The aim of this study was to determine whether gold nanoparticles conjugated cytotoxic protein NKCT1 (GNP-NKCT1) acted through the estrogen receptor mediated pathway in MCF-7 cells and to establish the MAPK and PI3k/Akt signal transduction pathway. Apoptosis was done by flow cytometry. BrdU incorporation and nuclear proliferating antigen was measured by flow cytometry. Wound healing assay along with matrigel chamber invasion and migration was done. Expression of MMP9 was checked by flow cytometry and also by gelatin zymography. To analyze the regulation of signaling protein, western blot was done. MTT assay was done to evaluate the ligand receptor pathway using the estrogen receptor negative cell line (MDA-MB-231) for inhibitor effects. Treatment of GNP-NKCT1 (3.9 μg/ml) exhibited 38.04% early apoptosis and 4.29% late apoptotic cell. GNP-NKCT1 significantly inhibited both cell migration and invasion with suppressed expression of MMP9. In addition, treatment of cultured human breast cancer MCF7 cells with GNP-NKCT1 reversely suppressed the incorporation of BrdU, with reduced expression of Ki-67. The western blot analysis showed that GNP-NKCT1 arrested cell cycle progression through upregulation of the kinase inhibitor protein p21 and inactivation of G1-cylin dependent kinase (CDK4). GNP-NKCT1 suppressed nuclear translocation of nuclear factor kappa B (NF-κB) and also abrogated the phosphorylation of p38 mitogen activated protein kinase (MAPK), phosphatidylinositide-3-kinase (PI3k), Akt and extracellular regulated kinase (ERK1/2). MTT assay indicated that GNP-NKCT1 reduced proliferation in the estrogen receptor induced ER negative breast cancer cell line (MDA-MB-231). Addition of, ER inhibitor (tamoxifen) and PI3K inhibitor (wortmannin) to cells resulted in reduced expression of Ki-67 and MMP-9. The data suggested that GNP-NKCT1 induced MCF7 cell inhibition may occur through estrogen receptor pathway via inactivation of CDK4 and inactivation of PI3K/Akt, ERK1

  3. Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression.

    PubMed

    Soliman, Elwy M; Rodrigues, Michele Angela; Gomes, Dawidson Assis; Sheung, Nina; Yu, Jin; Amaya, Maria Jimina; Nathanson, Michael H; Dranoff, Jonathan A

    2009-03-01

    Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca(2+) signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca(2+) signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca(2+) chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca(2+) signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca(2+) signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca(2+) signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca(2+) signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca(2+) are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis.

  4. Transformed and nontransformed cells differ in stability and cell cycle regulation of a binding activity to the murine thymidine kinase promoter.

    PubMed Central

    Bradley, D W; Dou, Q P; Fridovich-Keil, J L; Pardee, A B

    1990-01-01

    A DNA binding activity to an upstream region of the murine thymidine kinase gene is regulated differently in a transformed and nontransformed cell line pair. Differences in regulation were observed (i) after serum levels were reduced, (ii) when serum levels were returned to initial high levels, and (iii) while protein synthesis was inhibited. After reduction of serum levels, the binding activity was unstable in nontransformed BALB/c 3T3 clone A31 cells but was significantly more stable in benzo[a]pyrene-transformed BALB/c 3T3 cells. After serum concentration was returned to high levels, the kinetic pattern of the binding activity differed between nontransformed and transformed cells. While protein synthesis was inhibited, the binding activity was unstable in nontransformed cells and stable in transformed cells. Partial inhibition of protein synthesis--a more stringent condition to test instability--prevented the induction of the binding activity in nontransformed cells. Previously, the labile protein hypothesis set forth the criterion that a protein regulating the onset of DNA synthesis should be unstable in nontransformed cells and stable in transformed cells. The DNA binding activity described here satisfies this criterion. Images PMID:2251273

  5. Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation

    PubMed Central

    Klotz-Noack, Kathleen; McIntosh, Debbie; Schurch, Nicholas; Pratt, Norman; Blow, J. Julian

    2012-01-01

    Summary To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell cycle regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2/M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. In contrast, when Cdt1 is overexpressed or is stabilised by the Neddylation inhibitor MLN4924, re-replication can occur throughout S phase. PMID:22366459

  6. Cell cycle arrest during S or M phase generates polarized growth via distinct signals in Candida albicans.

    PubMed

    Bachewich, Catherine; Nantel, Andre; Whiteway, Malcolm

    2005-08-01

    Treatments that perturb DNA synthesis or mitosis will activate checkpoints that prevent cell cycle progression and cell proliferation. In yeast-form cells of the fungal pathogen Candida albicans, exposure to hydroxyurea (HU) or shutting off expression of the polo-like kinase CaCDC5 blocked nuclear division and spindle elongation, but activated a highly polarized growth mode. We have used transcription profiling both to characterize the initiation and progression of this polar growth pattern and to determine how cell elongation may be linked to the cell cycle in C. albicans. Different gene expression patterns during early stages of cell elongation support the concept that CaCdc5p-depleted and HU-exposed cells were blocked at different stages of the cell cycle, and suggest that different signals may generate the common polarized growth phenotype. Consistent with this, BUB2 expression was modulated in CaCdc5p-depleted cells, and absence of BUB2 prevented the maintenance of cell polarization, resulting in multibudded, pseudohyphal cells with constrictions. In contrast, HU-induced filaments did not modulate or require BUB2, but were dependent on the GTPase Ras1p. However, at later stages of cell elongation, transcription profiles were more similar, and comparisons with serum-induced hyphae revealed that the cell cycle-arrested filaments expressed several targets of the hyphal signalling pathways. Thus, arresting the yeast cell cycle in S or M phase generates a polarized growth pattern through different mechanisms in C. albicans, and maintenance of the polar growth mode can ultimately lead to the expression of hyphal-associated cell wall and virulence-related factors, in the absence of any external stimuli.

  7. Polycomb protein SCML2 regulates the cell cycle by binding and modulating CDK/CYCLIN/p21 complexes.

    PubMed

    Lecona, Emilio; Rojas, Luis Alejandro; Bonasio, Roberto; Johnston, Andrew; Fernández-Capetillo, Oscar; Reinberg, Danny

    2013-12-01

    Polycomb group (PcG) proteins are transcriptional repressors of genes involved in development and differentiation, and also maintain repression of key genes involved in the cell cycle, indirectly regulating cell proliferation. The human SCML2 gene, a mammalian homologue of the Drosophila PcG protein SCM, encodes two protein isoforms: SCML2A that is bound to chromatin and SCML2B that is predominantly nucleoplasmic. Here, we purified SCML2B and found that it forms a stable complex with CDK/CYCLIN/p21 and p27, enhancing the inhibitory effect of p21/p27. SCML2B participates in the G1/S checkpoint by stabilizing p21 and favoring its interaction with CDK2/CYCE, resulting in decreased kinase activity and inhibited progression through G1. In turn, CDK/CYCLIN complexes phosphorylate SCML2, and the interaction of SCML2B with CDK2 is regulated through the cell cycle. These findings highlight a direct crosstalk between the Polycomb system of cellular memory and the cell-cycle machinery in mammals.

  8. Heparin selectively inhibits a protein kinase C-dependent mechanism of cell cycle progression in calf aortic smooth muscle cells [published erratum appears in J Cell Biol 1990 Mar;110(3):863

    PubMed Central

    1989-01-01

    The proliferation of arterial smooth muscle cells (SMCs) plays a critical role in the pathogenesis of arteriosclerosis. Previous studies have indicated that the glycosaminoglycan heparin specifically inhibited the growth of vascular SMCs in vivo and in culture, although the precise mechanism(s) of action have not been elucidated. In this study, we have examined the ability of specific mitogens (PDGF, EGF, heparin-binding growth factors, phorbol esters, and insulin) to stimulate SMC proliferation. Our results indicate that SMCs derived from different species and vascular sources respond differently to these growth factors. We next examined the ability of heparin to inhibit the proliferative responses to these mitogens. In calf aortic SMCs, heparin inhibits a protein kinase C-dependent pathway for mitogenesis. Detailed cell cycle analysis revealed several new features of the effects of heparin on SMCs. For example, heparin has two effects on the Go----S transition: it delays entry into S phase and also reduces the number of cells entering the cycle from Go. Using two separate experimental approaches, we found that heparin must be present during the last 4 h before S phase, suggesting a mid-to-late G1 heparin block. In addition, our data indicate that heparin-treated SMCs, while initially blocked in mid-to-late G1, slowly move back into a quiescent growth state in the continued presence of heparin. These results suggest that heparin may have multiple targets for its antiproliferative effect. PMID:2592420

  9. Cell cycle alterations in the blastoid variant of mantle cell lymphoma (MCL-BV) as detected by gene expression profiling of mantle cell lymphoma (MCL) and MCL-BV.

    PubMed

    de Vos, Sven; Krug, Utz; Hofmann, Wolf-Karsten; Pinkus, Geraldine S; Swerdlow, Steven H; Wachsman, William; Grogan, Thomas M; Said, Jonathan W; Koeffler, H Phillip

    2003-03-01

    Overexpression of cyclin D1 is necessary but by itself insufficient for the development of mantle cell lymphoma (MCL). To identify pathways in the pathogenesis of MCL and the blastoid variant (MLC-BV), we compared the gene-expression profiles of microdissected normal mantle cells, MCL, and MCL-BV by oligonucleotide microarrays and quantitative reverse transcriptase PCR (QRT-PCR). We identified and confirmed the overexpression of several genes in MCL-BV that are involved in the cell cycle control at the G1/S and G2/M checkpoints or inhibit apoptotic cell death. The highly expressed cyclin dependent kinase 4 (CDK4) is a cell cycle kinase that associates with cyclin D1 for the progression through the G1/S checkpoint, whereas overexpression of cdc28 protein kinase 1 (CKS1) blocks the inhibition of the cyclin D1/CDK4 complex by the CDK inhibitor p27/Kip1. Other highly expressed genes in MCL-BV that promote the cells through the G1/S-checkpoint include the oncogenes B-Myb, PIM1, and PIM2, and passage through the G2/M-checkpoint is enhanced by high levels of cdc25B. Furthermore, two highly expressed genes that inhibit apoptosis are defender against cell death (DAD1) and RSK1. In summary, our microarray and QRT-PCR analyses identified several candidate genes whose expression increased when comparing normal follicular mantles with MCL and MCLBV, suggesting a potential pathogenic role in the evolution of MCL-BV.

  10. Interplay between cell growth and cell cycle in plants.

    PubMed

    Sablowski, Robert; Carnier Dornelas, Marcelo

    2014-06-01

    The growth of organs and whole plants depends on both cell growth and cell-cycle progression, but the interaction between both processes is poorly understood. In plants, the balance between growth and cell-cycle progression requires coordinated regulation of four different processes: macromolecular synthesis (cytoplasmic growth), turgor-driven cell-wall extension, mitotic cycle, and endocycle. Potential feedbacks between these processes include a cell-size checkpoint operating before DNA synthesis and a link between DNA contents and maximum cell size. In addition, key intercellular signals and growth regulatory genes appear to target at the same time cell-cycle and cell-growth functions. For example, auxin, gibberellin, and brassinosteroid all have parallel links to cell-cycle progression (through S-phase Cyclin D-CDK and the anaphase-promoting complex) and cell-wall functions (through cell-wall extensibility or microtubule dynamics). Another intercellular signal mediated by microtubule dynamics is the mechanical stress caused by growth of interconnected cells. Superimposed on developmental controls, sugar signalling through the TOR pathway has recently emerged as a central control point linking cytoplasmic growth, cell-cycle and cell-wall functions. Recent progress in quantitative imaging and computational modelling will facilitate analysis of the multiple interconnections between plant cell growth and cell cycle and ultimately will be required for the predictive manipulation of plant growth.

  11. Calcium signaling and cell cycle: Progression or death.

    PubMed

    Humeau, Juliette; Bravo-San Pedro, José Manuel; Vitale, Ilio; Nuñez, Lucia; Villalobos, Carlos; Kroemer, Guido; Senovilla, Laura

    2017-07-25

    Cytosolic Ca(2+) concentration levels fluctuate in an ordered manner along the cell cycle, in line with the fact that Ca(2+) is involved in the regulation of cell proliferation. Cell proliferation should be an error-free process, yet is endangered by mistakes. In fact, a complex network of proteins ensures that cell cycle does not progress until the previous phase has been successfully completed. Occasionally, errors occur during the cell cycle leading to cell cycle arrest. If the error is severe, and the cell cycle checkpoints work perfectly, this results into cellular demise by activation of apoptotic or non-apoptotic cell death programs. Cancer is characterized by deregulated proliferation and resistance against cell death. Ca(2+) is a central key to these phenomena as it modulates signaling pathways that control oncogenesis and cancer progression. Here, we discuss how Ca(2+) participates in the exogenous and endogenous signals controlling cell proliferation, as well as in the mechanisms by which cells die if irreparable cell cycle damage occurs. Moreover, we summarize how Ca(2+) homeostasis remodeling observed in cancer cells contributes to deregulated cell proliferation and resistance to cell death. Finally, we discuss the possibility to target specific components of Ca(2+) signal pathways to obtain cytostatic or cytotoxic effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Wogonoside induces growth inhibition and cell cycle arrest via promoting the expression and binding activity of GATA-1 in chronic myelogenous leukemia cells.

    PubMed

    Li, Hui; Hui, Hui; Xu, Jingyan; Yang, Hao; Zhang, Xiaoxiao; Liu, Xiao; Zhou, Yuxin; Li, Zhiyu; Guo, Qinglong; Lu, Na

    2016-06-01

    GATA-1, a zinc finger transcription factor, has been demonstrated to play a key role in the progression of leukemia. In this study, we investigate the effects of wogonoside, a naturally bioactive flavonoid derived from Scutellaria baicalensis Georgi, on cell growth and cell cycle in chronic myeloid leukemia (CML) cells, and uncover its underlying mechanisms. The experimental design comprised CML cell lines K562, imatinib-resistant K562 (K562r) cells, and primary CML cells, treated in vitro or in vivo, respectively, with wogonoside; growth and cell cycle were then evaluated. We found that wogonoside could induce growth inhibition and G0/G1 cell cycle arrest in both normal and K562r cells. Wogonoside promotes the expression of GATA-1 and facilitates the binding to methyl ethyl ketone (MEK) and p21 promoter, thus inhibiting MEK/extracellular signal-regulated kinase signaling and cell cycle checkpoint proteins, including CDK2, CDK4, cyclin A, and cyclin D1, and increasing p21 expression. Furthermore, in vivo studies showed that administration of wogonoside decreased CML cells and prolonged survival in NOD/SCID mice with CML cell xenografts. In conclusion, these results clearly revealed the inhibitory effect of wogonoside on the growth in CML cells and suggested that wogonoside may act as a promising drug for the treatment of imatinib-resistant CML.

  13. Grow₂: the HIF system, energy homeostasis and the cell cycle.

    PubMed

    Moniz, Sónia; Biddlestone, John; Rocha, Sónia

    2014-05-01

    Cell cycle progression is an energy demanding process and requires fine-tuned metabolic regulation. Cells must overcome an energy restriction checkpoint before becoming committed to progress through the cell cycle. Aerobic organisms need oxygen for the metabolic conversion of nutrients into energy. As such, environmental oxygen is a critical signalling molecule regulating cell fate. The Hypoxia Inducible Factors (HIFs) are a family of transcription factors that respond to changes in environmental oxygen and cell energy and coordinate a transcriptional program which forms an important part of the cellular response to a hostile environment. A significant proportion of HIF-dependent transcriptional target genes, code for proteins that are involved in energy homeostasis. In this review we discuss the role of the HIF system in the regulation of energy homeostasis in response to changes in environmental oxygen and the impact on cell cycle control, and address the implications of the deregulation of this effect in cancer.

  14. Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells.

    PubMed

    Yuan, Hebao; Chiang, C-Y Ason; Cheng, Jun; Salzmann, Viktoria; Yamashita, Yukiko M

    2012-01-01

    Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. We recently demonstrated that GSCs possess a checkpoint ("the centrosome orientation checkpoint") that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. Here, we show that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. We propose that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint.

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

    PubMed Central

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

    2017-01-01

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

  16. Phosphorylation network dynamics in the control of cell cycle transitions.

    PubMed

    Fisher, Daniel; Krasinska, Liliana; Coudreuse, Damien; Novák, Béla

    2012-10-15

    Fifteen years ago, it was proposed that the cell cycle in fission yeast can be driven by quantitative changes in the activity of a single protein kinase complex comprising a cyclin - namely cyclin B - and cyclin dependent kinase 1 (Cdk1). When its activity is low, Cdk1 triggers the onset of S phase; when its activity level exceeds a specific threshold, it promotes entry into mitosis. This model has redefined our understanding of the essential functional inputs that organize cell cycle progression, and its main principles now appear to be applicable to all eukaryotic cells. But how does a change in the activity of one kinase generate ordered progression through the cell cycle in order to separate DNA replication from mitosis? To answer this question, we must consider the biochemical processes that underlie the phosphorylation of Cdk1 substrates. In this Commentary, we discuss recent findings that have shed light on how the threshold levels of Cdk1 activity that are required for progression through each phase are determined, how an increase in Cdk activity generates directionality in the cell cycle, and why cell cycle transitions are abrupt rather than gradual. These considerations lead to a general quantitative model of cell cycle control, in which opposing kinase and phosphatase activities have an essential role in ensuring dynamic transitions.

  17. Targeting cell cycle regulators in hematologic malignancies

    PubMed Central

    Aleem, Eiman; Arceci, Robert J.

    2015-01-01

    Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed. PMID:25914884

  18. Targeting cell cycle regulators in hematologic malignancies.

    PubMed

    Aleem, Eiman; Arceci, Robert J

    2015-01-01

    Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

  19. Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis.

    PubMed

    Ahmad, N; Feyes, D K; Agarwal, R; Mukhtar, H

    1998-06-09

    Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.

  20. Cell cycle arrest induced by MPPa-PDT in MDA-MB-231 cells

    NASA Astrophysics Data System (ADS)

    Liang, Liming; Bi, Wenxiang; Tian, Yuanyuan

    2016-05-01

    Photodynamic therapy (PDT) is a medical treatment using a photosensitizing agent and light source to treat cancers. Pyropheophorbidea methyl ester (MPPa), a derivative of chlorophyll, is a novel potent photosensitizer. To learn more about this photosensitizer, we examined the cell cycle arrest in MDA-MB-231. Cell cycle and apoptosis were measured by flow cytometer. Checkpoints of the cell cycle were measured by western blot. In this study, we found that the expression of Cyclin D1 was obviously decreased, while the expression of Chk2 and P21 was increased after PDT treatment. This study showed that MPPa-PDT affected the checkpoints of the cell cycle and led the cells to apoptosis.

  1. Cell cycle control and seed development

    PubMed Central

    Dante, Ricardo A.; Larkins, Brian A.; Sabelli, Paolo A.

    2014-01-01

    Seed development is a complex process that requires coordinated integration of many genetic, metabolic, and physiological pathways and environmental cues. Different cell cycle types, such as asymmetric cell division, acytokinetic mitosis, mitotic cell division, and endoreduplication, frequently occur in sequential yet overlapping manner during the development of the embryo and the endosperm, seed structures that are both products of double fertilization. Asymmetric cell divisions in the embryo generate polarized daughter cells with different cell fates. While nuclear and cell division cycles play a key role in determining final seed cell numbers, endoreduplication is often associated with processes such as cell enlargement and accumulation of storage metabolites that underlie cell differentiation and growth of the different seed compartments. This review focuses on recent advances in our understanding of different cell cycle mechanisms operating during seed development and their impact on the growth, development, and function of seed tissues. Particularly, the roles of core cell cycle regulators, such as cyclin-dependent-kinases and their inhibitors, the Retinoblastoma-Related/E2F pathway and the proteasome-ubiquitin system, are discussed in the contexts of different cell cycle types that characterize seed development. The contributions of nuclear and cellular proliferative cycles and endoreduplication to cereal endosperm development are also discussed. PMID:25295050

  2. Cell cycle control and seed development.

    PubMed

    Dante, Ricardo A; Larkins, Brian A; Sabelli, Paolo A

    2014-01-01

    Seed development is a complex process that requires coordinated integration of many genetic, metabolic, and physiological pathways and environmental cues. Different cell cycle types, such as asymmetric cell division, acytokinetic mitosis, mitotic cell division, and endoreduplication, frequently occur in sequential yet overlapping manner during the development of the embryo and the endosperm, seed structures that are both products of double fertilization. Asymmetric cell divisions in the embryo generate polarized daughter cells with different cell fates. While nuclear and cell division cycles play a key role in determining final seed cell numbers, endoreduplication is often associated with processes such as cell enlargement and accumulation of storage metabolites that underlie cell differentiation and growth of the different seed compartments. This review focuses on recent advances in our understanding of different cell cycle mechanisms operating during seed development and their impact on the growth, development, and function of seed tissues. Particularly, the roles of core cell cycle regulators, such as cyclin-dependent-kinases and their inhibitors, the Retinoblastoma-Related/E2F pathway and the proteasome-ubiquitin system, are discussed in the contexts of different cell cycle types that characterize seed development. The contributions of nuclear and cellular proliferative cycles and endoreduplication to cereal endosperm development are also discussed.

  3. Enhanced radiation-induced cytotoxic effect by 2-ME in glioma cells is mediated by induction of cell cycle arrest and DNA damage via activation of ATM pathways.

    PubMed

    Zou, Huichao; Zhao, Shiguang; Zhang, Jianhua; Lv, Gongwei; Zhang, Xu; Yu, Hongwei; Wang, Huibo; Wang, Ligang

    2007-12-14

    Glioblastoma multiform is the most common malignant primary brain tumor in adults, but there remains no effective therapeutic approach. 2-methoxyestradiol (2-ME), which is a naturally occurring metabolite of 17beta-estradiol, was shown to enhance radiotherapeutic effect in certain tumors; however, whether 2-ME can also enhance the sensitivity of glioma cells to radiotherapy remains unknown. The present study, therefore, was to address this issue using two human glioma cell lines (T98G and U251MG). These cells were irradiated with and without 2-ME and then clonogenic assay, apoptosis assay, DNA damage, and cell cycle change were examined. Results showed that 2-ME significantly enhances radiation-induced cell death in both glioma cells, shown by decreasing cell viability and increasing apoptotic cell death. No such radiosensitizing effect was observed if cells pre-treated with Estrodiol, suggesting the specifically radiosensitizing effect of 2-ME rather than a general effect of estrodials. The enhanced radio-cytotoxic effect in glioma cells by 2-ME was found to be associated with its enhancement of G(2)/M arrest and DNA damage, and phosphorylated ATM protein kinases as well as cell cycle checkpoint protein Chk2. Furthermore, inhibition of ATM by ATM inhibitor abolished 2-ME-activated Chk2 and enhanced radio-cytotoxic effects. These results suggest that 2-ME enhancement of the sensitivity of glioma cell lines to radiotherapy is mediated by induction of G2/M cell cycle arrest and increased DNA damage via activation of ATM kinases.

  4. Molecular regulation of the diatom cell cycle.

    PubMed

    Huysman, Marie J J; Vyverman, Wim; De Veylder, Lieven

    2014-06-01

    Accounting for almost one-fifth of the primary production on Earth, the unicellular eukaryotic group of diatoms plays a key ecological and biogeochemical role in our contemporary oceans. Furthermore, as producers of various lipids and pigments, and characterized by their finely ornamented silica cell wall, diatoms hold great promise for different industrial fields, including biofuel production, nanotechnology, and pharmaceutics. However, in spite of their major ecological importance and their high commercial value, little is known about the mechanisms that control the diatom life and cell cycle. To date, both microscopic and genomic analyses have revealed that diatoms exhibit specific and unique mechanisms of cell division compared with those found in the classical model organisms. Here, we review the structural peculiarities of diatom cell proliferation, highlight the regulation of their major cell cycle checkpoints by environmental factors, and discuss recent progress in molecular cell division research. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Inhibition of histone deacetylase activity increases chromosomal instability by the aberrant regulation of mitotic checkpoint activation.

    PubMed

    Shin, Hyun-Jin; Baek, Kwan-Hyuck; Jeon, Ae-Hwa; Kim, So-Jung; Jang, Kyung-Lib; Sung, Young-Chul; Kim, Chang-Min; Lee, Chang-Woo

    2003-06-19

    Histone modification through acetylation and deacetylation is a key process in transcription, DNA replication, and chromosome segregation. During mitosis, histones are highly acetylated and chromatin is condensed. Here, we investigate the mechanistic involvement of histone deacetylase (HDAC) activity in the regulation of mitotic checkpoint activation. Inhibition of HDAC activity was found to cause the improper kinetochore localization of the mitotic checkpoint proteins, and to prolong mitotic arrest, and thus to lead to chromosomal instability due to aberrant exit from the mitotic cell cycle arrest. In addition, treatment with HDAC inhibitor attenuated the activations of p38 and ERK kinases, and increased the expression levels of cIAP-1, suggesting that the observed increased adaptation and chromosomal instability induced by inhibiting HDAC activity might be directly connected with the activations of cell survival and/or antiapoptotic signals. Moreover, the treatment of cells with mitotic defects with HDAC inhibitor increased their susceptibility to chromosomal instability. These results support the notion that HDAC activity plays an important role in the regulation of mitotic checkpoint activation, and thus the aberrant control of HDAC activity contributes to chromosomal instability.

  6. The cell cycle rallies the transcription cycle: Cdc28/Cdk1 is a cell cycle-regulated transcriptional CDK.

    PubMed

    Chymkowitch, Pierre; Enserink, Jorrit M

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases (CDKs) Kin28, Bur1 and Ctk1 regulate basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. However, very little is known about the involvement of the cell cycle CDK Cdc28 in the transcription process. We have recently shown that, upon cell cycle entry, Cdc28 kinase activity boosts transcription of a subset of genes by directly stimulating the basal transcription machinery. Here, we discuss the biological significance of this finding and give our view of the kinase-dependent role of Cdc28 in regulation of RNA polymerase II.

  7. Overexpression of hepatocyte nuclear factor-4α initiates cell cycle entry, but is not sufficient to promote β-cell expansion in human islets.

    PubMed

    Rieck, Sebastian; Zhang, Jia; Li, Zhaoyu; Liu, Chengyang; Naji, Ali; Takane, Karen K; Fiaschi-Taesch, Nathalie M; Stewart, Andrew F; Kushner, Jake A; Kaestner, Klaus H

    2012-09-01

    The transcription factor HNF4α (hepatocyte nuclear factor-4α) is required for increased β-cell proliferation during metabolic stress in vivo. We hypothesized that HNF4α could induce proliferation of human β-cells. We employed adenoviral-mediated overexpression of an isoform of HNF4α (HNF4α8) alone, or in combination with cyclin-dependent kinase (Cdk)6 and Cyclin D3, in human islets. Heightened HNF4α8 expression led to a 300-fold increase in the number of β-cells in early S-phase. When we overexpressed HNF4α8 together with Cdk6 and Cyclin D3, β-cell cycle entry was increased even further. However, the punctate manner of bromodeoxyuridine incorporation into HNF4α(High) β-cells indicated an uncoupling of the mechanisms that control the concise timing and execution of each cell cycle phase. Indeed, in HNF4α8-induced bromodeoxyuridine(+,punctate) β-cells we observed signs of dysregulated DNA synthesis, cell cycle arrest, and activation of a double stranded DNA damage-associated cell cycle checkpoint mechanism, leading to the initiation of loss of β-cell lineage fidelity. However, a substantial proportion of β-cells stimulated to enter the cell cycle by Cdk6 and Cyclin D3 alone also exhibited a DNA damage response. HNF4α8 is a mitogenic signal in the human β-cell but is not sufficient for completion of the cell cycle. The DNA damage response is a barrier to efficient β-cell proliferation in vitro, and we suggest its evaluation in all attempts to stimulate β-cell replication as an approach to diabetes treatment.

  8. Overexpression of Hepatocyte Nuclear Factor-4α Initiates Cell Cycle Entry, but Is not Sufficient to Promote β-Cell Expansion in Human Islets

    PubMed Central

    Rieck, Sebastian; Zhang, Jia; Li, Zhaoyu; Liu, Chengy