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Sample records for activate cre-dependent cyclin

  1. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    PubMed

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells. PMID:27128150

  2. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    PubMed Central

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit; Gantier, Michael P.

    2016-01-01

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  3. Cre-dependent DNA recombination activates a STING-dependent innate immune response.

    PubMed

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W Samantha N; Cain, Jason E; Behlke, Mark A; Gough, Daniel J; G Williams, Bryan R; Hornung, Veit; Gantier, Michael P

    2016-06-20

    Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell-cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  4. Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice.

    PubMed

    Zhu, Hu; Aryal, Dipendra K; Olsen, Reid H J; Urban, Daniel J; Swearingen, Amanda; Forbes, Stacy; Roth, Bryan L; Hochgeschwender, Ute

    2016-08-01

    DREADDs, designer receptors exclusively activated by designer drugs, are engineered G protein-coupled receptors (GPCR) which can precisely control GPCR signaling pathways (for example, Gq, Gs, and Gi). This chemogenetic technology for control of GPCR signaling has been successfully applied in a variety of in vivo studies, including in mice, to remotely control GPCR signaling, for example, in neurons, glia cells, pancreatic β-cells, or cancer cells. In order to fully explore the in vivo applications of the DREADD technology, we generated hM3Dq and hM4Di strains of mice which allow for Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. With the many Cre driver lines now available, these DREADD lines will be applicable to studying a wide array of research and preclinical questions. genesis 54:439-446, 2016. © 2016 Wiley Periodicals, Inc. PMID:27194399

  5. Imaging Activity in Neurons and Glia with a Polr2a-based and Cre-dependent GCaMP5G-IRES-tdTomato Reporter Mouse

    PubMed Central

    Gee, J. Michael; Smith, Nathan A.; Fernandez, Fernando R.; Economo, Michael N.; Brunert, Daniela; Rothermel, Markus; Morris, S. Craig; Talbot, Amy; Palumbos, Sierra; Ichida, Jennifer M.; Shepherd, Jason D.; West, Peter J.; Wachowiak, Matt; Capecchi, Mario R.; Wilcox, Karen S.; White, John A.; Tvrdik, Petr

    2014-01-01

    SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments. PMID:25155958

  6. Cyclin activation of p34cdc2.

    PubMed

    Solomon, M J; Glotzer, M; Lee, T H; Philippe, M; Kirschner, M W

    1990-11-30

    The gradual accumulation of cyclin in the frog egg induces an abrupt and concerted activation of p34cdc2 that initiates mitosis. Activation is delayed even after the accumulation of cyclin to a critical threshold concentration. We have reproduced these unusual kinetic properties of p34cdc2 activation in vitro using bacterially expressed cyclin proteins and extracts derived from Xenopus eggs. Abrupt activation follows a lag period, the length of which is independent of the concentration of cyclin. The threshold concentration of cyclin and the length of the lag period are regulated by INH, an inhibitor of MPF activation in oocytes recently identified as a type 2A protein phosphatase. Binding to cyclin induces both tyrosine and threonine phosphorylation of the previously unphosphorylated p34cdc2, rendering it inactivated. The concerted transition into mitosis involves both a reduction in the rate of p34cdc2 phosphorylation on tyrosine and an increase in its rate of dephosphorylation. PMID:2147872

  7. Hyposmotic stress induces cell growth arrest via proteasome activation and cyclin/cyclin-dependent kinase degradation.

    PubMed

    Tao, Guo-Zhong; Rott, Lusijah S; Lowe, Anson W; Omary, M Bishr

    2002-05-31

    Ordered cell cycle progression requires the expression and activation of several cyclins and cyclin-dependent kinases (Cdks). Hyperosmotic stress causes growth arrest possibly via proteasome-mediated degradation of cyclin D1. We studied the effect of hyposmotic conditions on three colonic (Caco2, HRT18, HT29) and two pancreatic (AsPC-1 and PaCa-2) cell lines. Hyposmosis caused reversible cell growth arrest of the five cell lines in a cell cycle-independent fashion, although some cell lines accumulated at the G(1)/S interface. Growth arrest was followed by apoptosis or by formation of multinucleated giant cells, which is consistent with cell cycle catastrophe. Hyposmosis dramatically decreased Cdc2, Cdk2, Cdk4, cyclin B1, and cyclin D3 expression in a time-dependent fashion, in association with an overall decrease in cellular protein synthesis. However, some protein levels remained unaltered, including cyclin E and keratin 8. Selective proteasome inhibition prevented Cdk and cyclin degradation and reversed hyposmotic stress-induced growth arrest, whereas calpain and lysosome enzyme inhibitors had no measurable effect on cell cycle protein degradation. Therefore, hyposmotic stress inhibits cell growth and, depending on the cell type, causes cell cycle catastrophe with or without apoptosis. The growth arrest is due to decreased protein synthesis and proteasome activation, with subsequent degradation of several cyclins and Cdks. PMID:11897780

  8. Cyclin D1 transcriptional activation in MCL.

    PubMed

    Beà, Sílvia

    2014-03-27

    In this issue of Blood, Allinne et al propose the nucleolin-dependent activation of the translocated CCND1 allele in mantle cell lymphoma (MCL) because of its relocalization to a transcriptionally favorable area in the perinucleolar region. PMID:24677400

  9. A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo

    PubMed Central

    Zariwala, Hatim A.; Borghuis, Bart G.; Hoogland, Tycho M.; Madisen, Linda; Tian, Lin; De Zeeuw, Chris I.; Zeng, Hongkui; Looger, Loren L.; Svoboda, Karel; Chen, Tsai-Wen

    2012-01-01

    Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and non-stationary expression. Here we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex and cerebellum. In the primary visual cortex, visually-evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared to virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3. PMID:22378886

  10. A Cre-dependent GCaMP3 reporter mouse for neuronal imaging in vivo.

    PubMed

    Zariwala, Hatim A; Borghuis, Bart G; Hoogland, Tycho M; Madisen, Linda; Tian, Lin; De Zeeuw, Chris I; Zeng, Hongkui; Looger, Loren L; Svoboda, Karel; Chen, Tsai-Wen

    2012-02-29

    Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and nonstationary expression. Here, we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex, and cerebellum. In the primary visual cortex, visually evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared with virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3. PMID:22378886

  11. Sequences within the conserved cyclin box of human cyclin A are sufficient for binding to and activation of cdc2 kinase.

    PubMed Central

    Lees, E M; Harlow, E

    1993-01-01

    Cyclins are pivotal in the coordinate regulation of the cell cycle. By physical association, they are able to activate at least one of the cyclin-dependent kinases, cdc2. How this association between the catalytic moiety and cyclins leads to subsequent activation of the kinase remains unclear. In this report, we describe experiments to investigate this event at a physical level. Our approach was to map the regions required on the cyclin A molecule for interaction with cdc2. We have mapped the contact regions to two small noncontiguous stretches of amino acids, residues 189 to 241 and 275 to 320, both located within the conserved cyclin box domain of the protein. We have further shown that this region not only represents a contact site for cdc2 but apparently represents an intact functional domain with respect to cdc2 activation. This region alone is sufficient to stimulate maturation when injected into immature Xenopus laevis oocytes. This observation implies that events leading to the activation of cdc2 kinase can be mediated through small regions of the cyclin molecule that are located in the cyclin box. These regions contain some of the most highly conserved residues found between all the cyclin members so far identified. This suggests that the cyclin family members may have conserved a similar mechanism to bind and activate cyclin-dependent kinases. Images PMID:8423786

  12. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

    PubMed Central

    Neuman, E; Ladha, M H; Lin, N; Upton, T M; Miller, S J; DiRenzo, J; Pestell, R G; Hinds, P W; Dowdy, S F; Brown, M; Ewen, M E

    1997-01-01

    Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor. PMID:9271411

  13. The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.

    PubMed Central

    Sudakin, V; Ganoth, D; Dahan, A; Heller, H; Hershko, J; Luca, F C; Ruderman, J V; Hershko, A

    1995-01-01

    The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles. Images PMID:7787245

  14. The Notch intracellular domain represses CRE-dependent transcription.

    PubMed

    Hallaq, Rania; Volpicelli, Floriana; Cuchillo-Ibanez, Inmaculada; Hooper, Claudie; Mizuno, Keiko; Uwanogho, Dafe; Causevic, Mirsada; Asuni, Ayodeji; To, Alvina; Soriano, Salvador; Giese, K Peter; Lovestone, Simon; Killick, Richard

    2015-03-01

    Members of the cyclic-AMP response-element binding protein (CREB) transcription factor family regulate the expression of genes needed for long-term memory formation. Loss of Notch impairs long-term, but not short-term, memory in flies and mammals. We investigated if the Notch-1 (N1) exerts an effect on CREB-dependent gene transcription. We observed that N1 inhibits CREB mediated activation of cyclic-AMP response element (CRE) containing promoters in a γ-secretase-dependent manner. We went on to find that the γ-cleaved N1 intracellular domain (N1ICD) sequesters nuclear CREB1α, inhibits cAMP/PKA-mediated neurite outgrowth and represses the expression of specific CREB regulated genes associated with learning and memory in primary cortical neurons. Similar transcriptional effects were observed with the N2ICD, N3ICD and N4ICDs. Together, these observations indicate that the effects of Notch on learning and memory are, at least in part, via an effect on CREB-regulated gene expression. PMID:25479589

  15. Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

    PubMed Central

    Gautier, J; Maller, J L

    1991-01-01

    Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase. Images PMID:1824935

  16. Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells

    PubMed Central

    Perez-Neut, Mathew; Shum, Andrew; Cuevas, Bruce D.; Miller, Richard; Gentile, Saverio

    2015-01-01

    Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators. PMID:25596745

  17. Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells.

    PubMed

    Perez-Neut, Mathew; Shum, Andrew; Cuevas, Bruce D; Miller, Richard; Gentile, Saverio

    2015-01-30

    Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators. PMID:25596745

  18. A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase.

    PubMed Central

    Diehl, J A; Sherr, C J

    1997-01-01

    Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting

  19. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle.

    PubMed

    Amon, A; Irniger, S; Nasmyth, K

    1994-07-01

    It is thought that DNA replication and mitosis in yeast are triggered by oscillations in the level of G1-specific (CLN1 and CLN2) and G2-specific (CLB1-CLB4) cyclins, which determine the substrate specificity of the CDC28 protein kinase. It is not understood how the time and order of appearance of different cyclin types are determined. We show here that CLB2 proteolysis, which is important for transition from mitosis to G1, is not confined to a narrow window at the end of mitosis as previously thought but continues until reactivation of CDC28 by CLN cyclins toward the end of the subsequent G1 period. Thus, cell cycle-regulated proteolysis prevents accumulation of G2-specific CLB cyclins during G1 and thereby ensures that the CLN-associated forms of the CDC28 kinase are activated without interference from CLB cyclins. Accumulation of CLN cyclins leads to inactivation of CLB cyclin proteolysis, which is a precondition for subsequent activation of G2-specific B-type cyclins. PMID:8020094

  20. Structural Basis for the Modulation of CDK-Dependent/Independent Activity of Cyclin D1

    PubMed Central

    Ferrer, Jean-Luc; Dupuy, Jérôme; Borel, Franck; Jacquamet, Lilian; Noel, Joseph P.; Dulic, Vjekoslav

    2010-01-01

    D-type cyclins are key regulators of the cell division cycle. In association with Cyclin Dependent Kinases (CDK) 2/4/6, they control the G1/S-phase transition in part by phosphorylation and inactivation of tumor suppressor of retinoblastoma family. Defective regulation of the G1/S transition is a well-known cause of cancer, making the cyclin D1-CDK4/6 complex a promising therapeutic target. Our objective is to develop inhibitors that would block the formation or the activation of the cyclin D1-CDK4/6 complex, using in silico docking experiments on a structural homology model of the cyclin D1-CDK4/6 complex. To this end we focused on the cyclin subunit in three different ways: (1) targeting the part of the cyclin D1 facing the N-terminal domain of CDK4/6, in order to prevent the dimer formation; (2) targeting the part of the cyclin D1 facing the C-terminal domain of CDK4/6, in order to prevent the activation of CDK4/6 by blocking the T-loop in an inactive conformation, and also to destabilize the dimer; (3) targeting the groove of cyclin D1 where p21 binds, in order to mimic its inhibition mode by preventing binding of cyclin D1-CDK4/6 complex to its targets. Our strategy, and the tools we developed, will provide a computational basis to design lead compounds for novel cancer therapeutics, targeting a broad range of proteins involved in the regulation of the cell cycle. PMID:17172845

  1. Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.

    PubMed

    Chassé, Héloïse; Mulner-Lorillon, Odile; Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

    2016-01-01

    The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

  2. Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

    PubMed Central

    Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

    2016-01-01

    The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

  3. Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

    PubMed

    Grigoroudis, Asterios I; McInnes, Campbell; Premnath, Padmavathy Nandha; Kontopidis, George

    2015-09-01

    Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove. PMID:25956535

  4. Reversible phosphorylation controls the activity of cyclosome-associated cyclin-ubiquitin ligase.

    PubMed Central

    Lahav-Baratz, S; Sudakin, V; Ruderman, J V; Hershko, A

    1995-01-01

    Cyclin B/cdc2 is responsible both for driving cells into mitosis and for activating the ubiquitin-dependent degradation of mitotic cyclins near the end of mitosis, an event required for the completion of mitosis and entry into interphase of the next cell cycle. Previous work with cell-free extracts of rapidly dividing clam embryos has identified two specific components required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase that purifies as part of a approximately 1500-kDa complex, termed the cyclosome, and which is active only near the end of mitosis. Here, we have separated the cyclosome from its ultimate upstream activator, cdc2. The mitotic, active form of the cyclosome can be inactivated by incubation with a partially purified, endogenous okadaic acid-sensitive phosphatase; addition of cdc2 restores activity to the cyclosome after a lag that reproduces that seen previously in intact cells and in crude extracts. These results demonstrate that activity of cyclin-ubiquitin ligase is controlled by reversible phosphorylation of the cyclosome complex. Images Fig. 3 PMID:7568122

  5. Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

    PubMed Central

    Haccard, Olivier; Jessus, Catherine

    2006-01-01

    Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant. PMID:16374506

  6. Binding of HTm4 to cyclin-dependent kinase (Cdk)-associated phosphatase (KAP).Cdk2.cyclin A complex enhances the phosphatase activity of KAP, dissociates cyclin A, and facilitates KAP dephosphorylation of Cdk2.

    PubMed

    Chinami, Masanobu; Yano, Yoshihiko; Yang, Xing; Salahuddin, Saira; Moriyama, Kosei; Shiroishi, Mitsunori; Turner, Helen; Shirakawa, Taro; Adra, Chaker N

    2005-04-29

    Cyclin-dependent kinase 2 (cdk2) activation requires phosphorylation of Thr160 and dissociation from cyclin A. The T-loop of cdk2 contains a regulatory phosphorylation site at Thr160. An interaction between cdc-associated phosphatase (KAP) and cdk2 compromises the interaction between cdk2 and cyclin A, which permits access of KAP, a Thr160-directed phosphatase, to its substrate, cdk2. We have reported that KAP is bound and activated by a nuclear membrane protein, HTm4. Here, we present in vitro data showing the direct interaction between the HTm4 C terminus and KAP Tyr141. We show that this interaction not only facilitates access of KAP to Thr160 and accelerates KAP kinetics, but also forces exclusion of cyclin A from the KAP.cdk2 complex. PMID:15671017

  7. Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus

    PubMed Central

    Wall, Nicholas R.; Wickersham, Ian R.; Cetin, Ali; De La Parra, Mauricio; Callaway, Edward M.

    2010-01-01

    We describe a powerful system for revealing the direct monosynaptic inputs to specific cell types in Cre-expressing transgenic mice through the use of Cre-dependent helper virus and a modified rabies virus. We generated helper viruses that target gene expression to Cre-expressing cells, allowing us to control initial rabies virus infection and subsequent monosynaptic retrograde spread. Investigators can use this system to elucidate the connections onto a desired cell type in a high-throughput manner, limited only by the availability of Cre mouse lines. This method allows for identification of circuits that would be extremely tedious or impossible to study with other methods and can be used to build subcircuit maps of inputs onto many different types of cells within the same brain region. Furthermore, by expressing various transgenes from the rabies genome, this system also has the potential to allow manipulation of targeted neuronal circuits without perturbing neighboring cells. PMID:21115815

  8. Telomerase activates transcription of cyclin D1 gene through an interaction with NOL1.

    PubMed

    Hong, Juyeong; Lee, Ji Hoon; Chung, In Kwon

    2016-04-15

    Telomerase is a ribonucleoprotein enzyme that is required for the maintenance of telomere repeats. Although overexpression of telomerase in normal human somatic cells is sufficient to overcome replicative senescence, the ability of telomerase to promote tumorigenesis requires additional activities that are independent of its role in telomere extension. Here, we identify proliferation-associated nucleolar antigen 120 (NOL1, also known as NOP2) as a telomerase RNA component (TERC)-binding protein that is found in association with catalytically active telomerase. Although NOL1 is highly expressed in the majority of human tumor cells, the molecular mechanism by which NOL1 contributes to tumorigenesis remained unclear. We show that NOL1 binds to the T-cell factor (TCF)-binding element of the cyclin D1 promoter and activates its transcription. Interestingly, telomerase is also recruited to the cyclin D1 promoter in a TERC-dependent manner through the interaction with NOL1, further enhancing transcription of the cyclin D1 gene. Depletion of NOL1 suppresses cyclin D1 promoter activity, thereby leading to induction of growth arrest and altered cell cycle distributions. Collectively, our findings suggest that NOL1 represents a new route by which telomerase activates transcription of cyclin D1 gene, thus maintaining cell proliferation capacity. PMID:26906424

  9. Targeting the cyclin-binding groove site to inhibit the catalytic activity of CDK2/cyclin A complex using p27(KIP1)-derived peptidomimetic inhibitors.

    PubMed

    Karthiga, Arumugasamy; Tripathi, Sunil Kumar; Shanmugam, Ramasamy; Suryanarayanan, Venkatesan; Singh, Sanjeev Kumar

    2015-01-01

    Functionally activated cyclin-dependent kinase 2 (CDK2)/cyclin A complex has been validated as an interesting therapeutic target to develop the efficient antineoplastic drug based on the cell cycle arrest. Cyclin A binds to CDK2 and activates the kinases as well as recruits the substrate and inhibitors using a hydrophobic cyclin-binding groove (CBG). Blocking the cyclin substrate recruitment on CBG is an alternative approach to override the specificity hurdle of the currently available ATP site targeting CDK2 inhibitors. Greater understanding of the interaction of CDK2/cyclin A complex with p27 (negative regulator) reveals that the Leu-Phe-Gly (LFG) motif region of p27 binds with the CBG site of cyclin A to arrest the malignant cell proliferation that induces apoptosis. In the present study, Replacement with Partial Ligand Alternatives through Computational Enrichment (REPLACE) drug design strategies have been applied to acquire LFG peptide-derived peptidomimetics library. The peptidomimetics function is equivalent with respect to substrate p27 protein fashion but does not act as an ATP antagonist. The combined approach of molecular docking, molecular dynamics (MD), and molecular electrostatic potential and ADME/T prediction were carried out to evaluate the peptidomimetics. Resultant interaction and electrostatic potential maps suggested that smaller substituent is desirable at the position of phenyl ring to interact with Trp217, Arg250, and Gln254 residues in the active site. The best docked poses were refined by the MD simulations which resulted in conformational changes. After equilibration, the structure of the peptidomimetic and receptor complex was stable. The results revealed that the various substrate protein-derived peptidomimetics could serve as perfect leads against CDK2 protein. PMID:25584078

  10. NF-{kappa}B p65 represses {beta}-catenin-activated transcription of cyclin D1

    SciTech Connect

    Hwang, Injoo; Choi, Yong Seok; Jeon, Mi-Ya; Jeong, Sunjoo

    2010-12-03

    Research highlights: {yields} Cyclin D1 transcription is directly activated by {beta}-catenin; however, {beta}-catenin-induced cyclin D1 transcription is reduced by NF-{kappa}B p65. {yields} Protein-protein interaction between NF-{kappa}B p65 and {beta}-catenin might be responsible for p65-mediated repression of cyclin D1. {yields} One of five putative binding sites, located further upstream of other sites, is the major {beta}-catenin binding site in the cyclin D1 promoter. {yields} NF-{kappa}B binding site in cyclin D1 is occupied not only by p65 but also by {beta}-catenin, which is dynamically regulated by the signal. -- Abstract: Signaling crosstalk between the {beta}-catenin and NF-{kappa}B pathways represents a functional network. To test whether the crosstalk also occurs on their common target genes, the cyclin D1 promoter was used as a model because it contains binding sites for both proteins. {beta}-catenin activated transcription from the cyclin D1 promoter, while co-expression of NF-{kappa}B p65 reduced {beta}-catenin-induced transcription. Chromatin immunoprecipitation revealed lithium chloride-induced binding of {beta}-catenin on one of the T-cell activating factor binding sites. More interestingly, {beta}-catenin binding was greatly reduced by NF-{kappa}B p65, possibly by the protein-protein interaction between the two proteins. Such a dynamic and complex binding of {beta}-catenin and NF-{kappa}B on promoters might contribute to the regulated expression of their target genes.

  11. Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2

    PubMed Central

    Tessari, Michela A.; Gostissa, Monica; Altamura, Sandro; Sgarra, Riccardo; Rustighi, Alessandra; Salvagno, Clio; Caretti, Giuseppina; Imbriano, Carol; Mantovani, Roberto; Del Sal, Giannino; Giancotti, Vincenzo; Manfioletti, Guidalberto

    2003-01-01

    The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120E4F, interfering with p120E4F binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation. PMID:14645522

  12. Ral GTPases Contribute to Regulation of Cyclin D1 through Activation of NF-κB

    PubMed Central

    Henry, Dale O.; Moskalenko, Serge A.; Kaur, Kiran J.; Fu, Maofu; Pestell, Richard G.; Camonis, Jacques H.; White, Michael A.

    2000-01-01

    Ral GTPases have been implicated as mediators of Ras-induced signal transduction from observations that Ral-specific guanine nucleotide exchange factors associate with Ras and are activated by Ras. The cellular role of Ral family proteins is unclear, as is the contribution that Ral may make to Ras-dependent signaling. Here we show that expression of activated Ral in quiescent rodent fibroblasts is sufficient to induce activation of NF-κB-dependent gene expression and cyclin D1 transcription, two key convergence points for mitogenic and survival signaling. The regulation of cyclin D1 transcription by Ral is dependent on NF-κB activation and is mediated through an NF-κB binding site in the cyclin D1 promoter. Ral activation of these responses is likely through an as yet uncharacterized effector pathway, as we find activation of NF-κB and the cyclin D1 promoter by Ral is independent of association of Ral with active phospholipase D1 or Ral-binding protein 1, two proteins proposed to mediate Ral function in cells. PMID:11027278

  13. New structural insights into phosphorylation-free mechanism for full cyclin-dependent kinase (CDK)-cyclin activity and substrate recognition.

    PubMed

    Zheng, Fei; Quiocho, Florante A

    2013-10-18

    Pho85 is a versatile cyclin-dependent kinase (CDK) found in budding yeast that regulates a myriad of eukaryotic cellular functions in concert with 10 cyclins (called Pcls). Unlike cell cycle CDKs that require phosphorylation of a serine/threonine residue by a CDK-activating kinase (CAK) for full activation, Pho85 requires no phosphorylation despite the presence of an equivalent residue. The Pho85-Pcl10 complex is a key regulator of glycogen metabolism by phosphorylating the substrate Gsy2, the predominant, nutritionally regulated form of glycogen synthase. Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATPγS. The structure solidified the mechanism for bypassing CDK phosphorylation to achieve full catalytic activity. An aspartate residue, invariant in all Pcls, acts as a surrogate for the phosphoryl adduct of the phosphorylated, fully activated CDK2, the prototypic cell cycle CDK, complexed with cyclin A. Unlike the canonical recognition motif, SPX(K/R), of phosphorylation sites of substrates of several cell cycle CDKs, the motif in the Gys2 substrate of Pho85-Pcl10 is SPXX. CDK5, an important signal transducer in neural development and the closest known functional homolog of Pho85, does not require phosphorylation either, and we found that in its crystal structure complexed with p25 cyclin a water/hydroxide molecule remarkably plays a similar role to the phosphoryl or aspartate group. Comparison between Pho85-Pcl10, phosphorylated CDK2-cyclin A, and CDK5-p25 complexes reveals the convergent structural characteristics necessary for full kinase activity and the variations in the substrate recognition mechanism. PMID:24022486

  14. A novel inhibitor of cyclin-Cdk activity detected in transforming growth factor beta-arrested epithelial cells.

    PubMed Central

    Slingerland, J M; Hengst, L; Pan, C H; Alexander, D; Stampfer, M R; Reed, S I

    1994-01-01

    Transforming growth factor beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Cyclins E and A in association with Cdk2 have been shown to play a role in the G1-to-S phase transition in mammalian cells. We have studied the effects of TGF-beta-mediated growth arrest on G1/S cyclins E and A. Inhibition of cyclin A-associated kinase by TGF-beta is primarily due to a decrease in cyclin A mRNA and protein. By contrast, while TGF-beta inhibits accumulation of cyclin E mRNA, the reduction in cyclin E protein is minimal. Instead, we find that the activation of cyclin E-associated kinase that normally accompanies the G1-to-S phase transition is inhibited. A novel inhibitor of cyclin-Cdk complexes was detected in TGF-beta-treated cell lysates. Inhibition is mediated by a heat-stable protein that targets both Cdk2 and Cdc2 kinases. In G0-arrested cells, a similar inhibitor of Cdk2 kinase was detected. These data suggest the existence of an inhibitor of cyclin-dependent kinases induced under different conditions to mediate antiproliferative responses. Images PMID:8196612

  15. Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

    PubMed Central

    Lukas, J; Bartkova, J; Rohde, M; Strauss, M; Bartek, J

    1995-01-01

    To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle. PMID:7739541

  16. An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

    SciTech Connect

    Rovnak, Joel; Hronek, Brett W.; Ryan, Sean O.; Cai, Sumin; Quackenbush, Sandra L. . E-mail: sandra.quackenbush@colostate.edu

    2005-11-25

    Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pulldown is lost by mutation of the activation domain.

  17. KSHV viral cyclin interferes with T-cell development and induces lymphoma through Cdk6 and Notch activation in vivo

    PubMed Central

    Pekkonen, Pirita; Järviluoma, Annika; Zinovkina, Nadezhda; Cvrljevic, Anna; Prakash, Sonam; Westermarck, Jukka; Evan, Gerard I; Cesarman, Ethel; Verschuren, Emmy W; Ojala, Päivi M

    2014-01-01

    Kaposi's sarcoma herpesvirus (KSHV)-encoded v-cyclin, a homolog of cellular cyclin D2, activates cellular CDK6, promotes G1-S transition of the cell cycle, induces DNA damage, apoptosis, autophagy and is reported to have oncogenic potential. Here we show that in vivo expression of v-cyclin in the B- and T-cell lymphocyte compartments results in a markedly low survival due to high penetrance of early-onset T-cell lymphoma and pancarditis. The v-cyclin transgenic mice have smaller pre-tumorigenic lymphoid organs, showing decreased cellularity, and increased proliferation and apoptosis. Furthermore, v-cyclin expression resulted in decreased amounts of CD3-expressing mature T-cells in the secondary lymphoid organs concurrent with alterations in the T-cell subpopulations of the thymus. This suggests that v-cyclin interferes with normal T-cell development. As the Notch pathway is recognized for its role in both T-cell development and lymphoma initiation, we addressed the role of Notch in the v-cyclin-induced alterations. Fittingly, we demonstrate induction of Notch3 and Hes1 in the pre-tumorigenic thymi and lymphomas of v-cyclin expressing mice, and show that lymphoma growth and viability are dependent on activated Notch signaling. Notch3 transcription and growth of the lymphomas was dependent on CDK6, as determined by silencing of CDK6 expression or chemical inhibition, respectively. Our work here reveals a viral cyclin-CDK6 complex as an upstream regulator of Notch receptor, suggesting that cyclins can play a role in the initiation of Notch-dependent lymphomagenesis. PMID:25483078

  18. Brk/Protein tyrosine kinase 6 phosphorylates p27KIP1, regulating the activity of cyclin D-cyclin-dependent kinase 4.

    PubMed

    Patel, Priyank; Asbach, Benedikt; Shteyn, Elina; Gomez, Cindy; Coltoff, Alexander; Bhuyan, Sadia; Tyner, Angela L; Wagner, Ralf; Blain, Stacy W

    2015-05-01

    Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27(Kip1) is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity. PMID:25733683

  19. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16

    PubMed Central

    Shehata, Saifeldin N.; Deak, Maria; Morrice, Nicholas A.; Ohta, Eriko; Hunter, Roger W.; Kalscheuer, Vera M.; Sakamoto, Kei

    2015-01-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser12 and Ser336) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser12 as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser336, but not Ser12, as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser336 may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser100 and Ser326) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  20. Cyclin Y phosphorylation- and 14-3-3-binding-dependent activation of PCTAIRE-1/CDK16.

    PubMed

    Shehata, Saifeldin N; Deak, Maria; Morrice, Nicholas A; Ohta, Eriko; Hunter, Roger W; Kalscheuer, Vera M; Sakamoto, Kei

    2015-08-01

    PCTAIRE-1 [also known as cyclin-dependent kinase 16 (CDK16)] is implicated in various physiological processes such as neurite outgrowth and vesicle trafficking; however, its molecular regulation and downstream targets are largely unknown. Cyclin Y has recently been identified as a key interacting/activating cyclin for PCTAIRE-1; however, the molecular mechanism by which it activates PCTAIRE-1 is undefined. In the present study, we initially performed protein sequence analysis and identified two candidate phosphorylation sites (Ser(12) and Ser(336)) on cyclin Y that might be catalysed by PCTAIRE-1. Although in vitro peptide analysis favoured Ser(12) as the candidate phosphorylation site, immunoblot analysis of cell lysates that had been transfected with wild-type (WT) or kinase-inactive (KI) PCTAIRE-1 together with WT or phospho-deficient mutants of cyclin Y suggested Ser(336), but not Ser(12), as a PCTAIRE-1-dependent phosphorylation site. Monitoring phosphorylation of Ser(336) may provide a useful read-out to assess cellular activity of PCTAIRE-1 in vivo; however, a phospho-deficient S336A mutant displayed normal interaction with PCTAIRE-1. Unbiased mass spectrometry and targeted mutagenesis analysis of cyclin Y identified key phosphorylation sites (Ser(100) and Ser(326)) required for 14-3-3 binding. Recombinant WT cyclin Y, but not a S100A/S326A mutant, prepared in COS-1 cells co-purified with 14-3-3 and was able to activate bacterially expressed recombinant PCTAIRE-1 in cell-free assays. Finally, we observed that recently identified PCTAIRE-1 variants found in patients with intellectual disability were unable to interact with cyclin Y, and were inactive enzymes. Collectively, the present work has revealed a new mechanistic insight into activation of PCTAIRE-1, which is mediated through interaction with the phosphorylated form of cyclin Y in complex with 14-3-3. PMID:26205494

  1. Mitotic checkpoint slippage in humans occurs via cyclin B destruction in the presence of an active checkpoint.

    PubMed

    Brito, Daniela A; Rieder, Conly L

    2006-06-20

    In the presence of unattached/weakly attached kinetochores, the spindle assembly checkpoint (SAC) delays exit from mitosis by preventing the anaphase-promoting complex (APC)-mediated proteolysis of cyclin B, a regulatory subunit of cyclin-dependent kinase 1 (Cdk1). Like all checkpoints, the SAC does not arrest cells permanently, and escape from mitosis in the presence of an unsatisfied SAC requires that cyclin B/Cdk1 activity be inhibited. In yeast , and likely Drosophila, this occurs through an "adaptation" process involving an inhibitory phosphorylation on Cdk1 and/or activation of a cyclin-dependent kinase inhibitor (Cdki). The mechanism that allows vertebrate cells to escape mitosis when the SAC cannot be satisfied is unknown. To explore this issue, we conducted fluorescence microscopy studies on rat kangaroo (PtK) and human (RPE1) cells dividing in the presence of nocodazole. We find that in the absence of microtubules (MTs), escape from mitosis occurs in the presence of an active SAC and requires cyclin B destruction. We also find that cyclin B is progressively destroyed during the block by a proteasome-dependent mechanism. Thus, vertebrate cells do not adapt to the SAC. Rather, our data suggest that in normal cells, the SAC cannot prevent a slow but continuous degradation of cyclin B that ultimately drives the cell out of mitosis. PMID:16782009

  2. c-Jun induces apoptosis of starved BM2 monoblasts by activating cyclin A-CDK2

    SciTech Connect

    Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor; Kozubik, Alois; Hampl, Ales; Smarda, Jan . E-mail: smarda@sci.muni.cz

    2007-02-02

    c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.

  3. Protocatechualdehyde possesses anti-cancer activity through downregulating cyclin D1 and HDAC2 in human colorectal cancer cells

    SciTech Connect

    Jeong, Jin Boo; Lee, Seong-Ho

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Protocatechualdehyde (PCA) suppressed cell proliferation and induced apoptosis in human colorectal cancer cells. Black-Right-Pointing-Pointer PCA enhanced transcriptional downregulation of cyclin D1 gene. Black-Right-Pointing-Pointer PCA suppressed HDAC2 expression and activity. Black-Right-Pointing-Pointer These findings suggest that anti-cancer activity of PCA may be mediated by reducing HDAC2-derived cyclin D1 expression. -- Abstract: Protocatechualdehyde (PCA) is a naturally occurring polyphenol found in barley, green cavendish bananas, and grapevine leaves. Although a few studies reported growth-inhibitory activity of PCA in breast and leukemia cancer cells, the underlying mechanisms are still poorly understood. Thus, we performed in vitro study to investigate if treatment of PCA affects cell proliferation and apoptosis in human colorectal cancer cells and define potential mechanisms by which PCA mediates growth arrest and apoptosis of cancer cells. Exposure of PCA to human colorectal cancer cells (HCT116 and SW480 cells) suppressed cell growth and induced apoptosis in dose-dependent manner. PCA decreased cyclin D1 expression in protein and mRNA level and suppressed luciferase activity of cyclin D1 promoter, indicating transcriptional downregulation of cyclin D1 gene by PCA. We also observed that PCA treatment attenuated enzyme activity of histone deacetylase (HDAC) and reduced expression of HDAC2, but not HDAC1. These findings suggest that cell growth inhibition and apoptosis by PCA may be a result of HDAC2-mediated cyclin D1 suppression.

  4. p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

    PubMed Central

    Woo, M S; Sánchez, I; Dynlacht, B D

    1997-01-01

    The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases. PMID:9199292

  5. The CDK4/CDK6 inhibitor PD0332991 paradoxically stabilizes activated cyclin D3-CDK4/6 complexes.

    PubMed

    Paternot, Sabine; Colleoni, Bianca; Bisteau, Xavier; Roger, Pierre P

    2014-01-01

    CDK4 and CDK6 bound to D-type cyclins are master integrators of G1 phase cell cycle regulations by initiating the inactivating phosphorylation of the central oncosuppressor pRb. Because of their frequent deregulation in cancer, cyclin D-CDK4/6 complexes are emerging as especially promising therapeutic targets. The specific CDK4/6 inhibitor PD0332991 is currently tested in a growing number of phase II/III clinical trials against a variety of pRb-proficient chemotherapy-resistant cancers. We have previously shown that PD0332991 inhibits not only CDK4/6 activity but also the activation by phosphorylation of the bulk of cyclin D-CDK4 complexes stabilized by p21 binding. Here we show that PD0332991 has either a positive or a negative impact on the activation of cyclin D-CDK4/6 complexes, depending on their binding to p21. Indeed, whereas PD0332991 inhibits the phosphorylation and activity of p21-bound CDK4/6, it specifically stabilized activated cyclin D3-CDK4/6 complexes devoid of p21 and p27. After elimination of PD0332991, these activated cyclin D3-CDK4/6 complexes persisted for at least 24 h, resulting in paradoxical cell cycle entry in the absence of a mitogenic stimulation. This unsuspected positive effect of PD0332991 on cyclin D3-CDK4/6 activation should be carefully assessed in the clinical evaluation of PD0332991, which until now only involves discontinuous administration protocols. PMID:25486476

  6. Suppression of cell cycle progression by a fungal lectin: activation of cyclin-dependent kinase inhibitors.

    PubMed

    Liua, W; Ho, J C; Ng, T

    2001-01-01

    The antiproliferative activity of a fungal lectin (VVL) isolated from the mushroom, Volvariella volvacea, was studied using a battery of cultured tumor cell lines. It was revealed that [(3)H]thymidine incorporation into the cell lines was markedly reduced at 0.32 microM VVL. When S180 mouse sarcoma cells were incubated for 48 hr with doses of VVL ranging from 0.32 to 0.8 microM, prominent blebs on the cell surface and large vacuoles in the cytoplasm, but not apoptotic bodies, were observed under a fluorescence microscopy. VVL did not exert ribosome-inactivating activity or induce any changes in the expression of cyclins A, D1, and E. However, it did activate the expression of cyclin kinase inhibitors, namely p21, p27, p53, and Rb, in a dose-dependent manner. Flow cytometric analysis demonstrated an accumulation of cells in the G2/M phase in a time- and dose-dependent manner, indicating that VVL arrested cell proliferation by blocking cell cycle progression in the G2/M phase. PMID:11137706

  7. Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells

    PubMed Central

    Wang, Zhu A.; Kalderon, Daniel

    2009-01-01

    Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycEWX protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycEWX at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycEWX FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells. PMID:19966222

  8. Specific Inhibition of Cyclin-dependent Kinase 5 Activity Induces Motor Neuron Development in vivo

    PubMed Central

    Kanungo, Jyotshnabala; Zheng, Ya-Li; Amin, Niranjana D.; Kaur, Sukhbir; Ramchandran, Ramani; Pant, Harish C.

    2009-01-01

    Cyclin-dependent kinase 5 (cdk5) is a ubiquitous protein activated by specific activators, p35 and p39. Cdk5 regulates neuronal migration, differentiation, axonogenesis, synaptic transmission and apoptosis. However, its role in motor neuron development remains unexplored. Here, using gain and loss-of-function analyses in developing zebrafish embryos, we report that cdk5 plays a critical role in spinal and cranial motor neuron development. Cdk5 knockdown results in supernumerary spinal and cranial motor neurons. While a dominant negative, kinase-dead cdk5 promotes the generation of supernumerary motor neurons; over-expression of cdk5 suppresses motor neuron development. Thus, modulating cdk5 activity seems promising in inducing motor neuron development in vivo. PMID:19523926

  9. Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast.

    PubMed Central

    Strich, Randy; Mallory, Michael J; Jarnik, Michal; Cooper, Katrina F

    2004-01-01

    Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute. PMID:15342503

  10. Activity of cyclin B1 in HL-60 cells treated with etoposide.

    PubMed

    Żuryń, Agnieszka; Krajewski, Adrian; Szulc, Dawid; Litwiniec, Anna; Grzanka, Alina

    2016-06-01

    Cyclin B1 triggers G2/M phase transition phosphorylating with its catalytical partner - Cdc2 many of the molecular targets essential for cell cycle progression. Human leukemia cell line HL-60 were treated with increasing doses of etoposide (ETP) (0.5; 0.75; 1μM) to investigate how the drug affects cell morphology, viability, cell cycle distribution and expression of cyclin B1. To achieve this aim we applied light and transmission electron microscopy to observe morphological and ultra structural changes, image-based cytometry for apoptosis evaluation and cell cycle analysis, and then we conducted immunohistochemical and immunofluorescence staining to visualize cyclin localization and expression. Quantitive data about cyclin B1 expression were obtained from flow cytometry. Etoposide caused decrease in cell viability, induced apoptosis and G2/M arrest accompanied by enhanced expression of cyclin B1. Changes in expression and localization of cyclin B1 may constitute a part of the mechanism responsible for resistance of HL-60 cells to etoposide. Our results may reflect involvement of cyclin B1 in opposite processes - apoptosis induction and maintenance of cell viability in leukemia cells. We hypothesized possible roles and pathways by which cyclin B1 takes part in drug treatment response and chemosensitivity. PMID:27297620

  11. Adapalene inhibits the activity of cyclin-dependent kinase 2 in colorectal carcinoma

    PubMed Central

    SHI, XI-NAN; LI, HONGJIAN; YAO, HONG; LIU, XU; LI, LING; LEUNG, KWONG-SAK; KUNG, HSIANG-FU; LIN, MARIE CHIA-MI

    2015-01-01

    Cyclin-dependent kinase 2 (CDK2) has been reported to be overexpressed in human colorectal cancer; it is responsible for the G1-to-S-phase transition in the cell cycle and its deregulation is a hallmark of cancer. The present study was the first to use idock, a free and open-source protein-ligand docking software developed by our group, to identify potential CDK2 inhibitors from 4,311 US Food and Drug Administration-approved small molecular drugs with a re-purposing strategy. Among the top compounds identified by idock score, nine were selected for further study. Among them, adapalene (ADA; CD271,6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphtoic acid) exhibited the highest anti-proliferative effects in LoVo and DLD1 human colon cancer cell lines. Consistent with the expected properties of CDK2 inhibitors, the present study demonstrated that ADA significantly increased the G1-phase population and decreased the expression of CDK2, cyclin E and retinoblastoma protein (Rb), as well as the phosphorylation of CDK2 (on Thr-160) and Rb (on Ser-795). Furthermore, the anti-cancer effects of ADA were examined in vivo on xenograft tumors derived from DLD1 human colorectal cancer cells subcutaneously inoculated in BALB/C nude mice. ADA (20 mg/kg orally) exhibited marked anti-tumor activity, comparable to that of oxaliplatin (40 mg/kg), and dose-dependently inhibited tumor growth (P<0.05), while combined administration of ADA and oxaliplatin produced the highest therapeutic effect. To the best of our knowledge, the present study was the first to indicate that ADA inhibits CDK2 and is a potential candidate drug for the treatment of human colorectal cancer. PMID:26398439

  12. Preclinical activity of P276-00, a novel small-molecule cyclin-dependent kinase inhibitor in the therapy of multiple myeloma.

    PubMed

    Raje, N; Hideshima, T; Mukherjee, S; Raab, M; Vallet, S; Chhetri, S; Cirstea, D; Pozzi, S; Mitsiades, C; Rooney, M; Kiziltepe, T; Podar, K; Okawa, Y; Ikeda, H; Carrasco, R; Richardson, P G; Chauhan, D; Munshi, N C; Sharma, S; Parikh, H; Chabner, B; Scadden, D; Anderson, K C

    2009-05-01

    Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM. PMID:19151776

  13. Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis.

    PubMed Central

    Hoffmann, I; Clarke, P R; Marcote, M J; Karsenti, E; Draetta, G

    1993-01-01

    We have investigated the mechanisms responsible for the sudden activation of the cdc2-cyclin B protein kinase before mitosis. It has been found previously that cdc25 is the tyrosine phosphatase responsible for dephosphorylating and activating cdc2-cyclin B. In Xenopus eggs and early embryos a cdc25 homologue undergoes periodic phosphorylation and activation. Here we show that the catalytic activity of human cdc25-C phosphatase is also activated directly by phosphorylation in mitotic cells. Phosphorylation of cdc25-C in mitotic HeLa extracts or by cdc2-cyclin B increases its catalytic activity. cdc25-C is not a substrate of the cyclin A-associated kinases. cdc25-C is able to activate cdc2-cyclin B1 in Xenopus egg extracts and to induce Xenopus oocyte maturation, but only after stable thiophosphorylation. This demonstrates that phosphorylation of cdc25-C is required for the activation of cdc2-cyclin B and entry into M-phase. Together, these studies offer a plausible explanation for the rapid activation of cdc2-cyclin B at the onset of mitosis and the self-amplification of MPF observed in vivo. Images PMID:8428594

  14. CK2 regulates in vitro the activity of the yeast cyclin-dependent kinase inhibitor Sic1.

    PubMed

    Barberis, Matteo; Pagano, Mario A; Gioia, Luca De; Marin, Oriano; Vanoni, Marco; Pinna, Lorenzo A; Alberghina, Lilia

    2005-11-01

    We have previously demonstrated that the cyclin-dependent kinase inhibitor (Cki) Sic1 of Saccharomyces cerevisiae is phosphorylated in vitro by the CK2 kinase on Ser(201) residue. Moreover, we have collected evidence showing that Sic1 is functionally and structurally related to mammalian Cki p27(Kip1) and binds to the mammalian Cdk2/cyclin A complex with a similar mode of inhibition. In this paper, we use SPR analysis to investigate the binding of Sic1 to the catatytic and regulatory subunits of CK2. Evidence is presented showing that phosphorylation of Sic1 at the CK2 consensus site QES(201)EDEED increases the binding of a Sic1-derived peptide to the Cdk2/cyclin A complex, a functional homologue of the yeast Cdk1/Clb5,6. Moreover, Sic1 fully phosphorylated in vitro on Ser(201) by CK2 is shown to be a stronger inhibitor of the Cdk/cyclin complexes than the unphosphorylated protein. Taken together, these data disclose the possibility that CK2 plays a role in the regulation of Sic1 activity. PMID:16168390

  15. Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activity

    PubMed Central

    Yamasaki, Fumiyuki; Zhang, Dongwei; Bartholomeusz, Chandra; Sudo, Tamotsu; Hortobagyi, Gabriel N.; Kurisu, Kaoru; Ueno, Naoto T.

    2008-01-01

    Inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinases,such as erlotinib and gefitinib, have not been very effective in the treatment of breast cancer although many breast cancer cells express EGFR. To address this apparent paradox, we examined possible predictors of the sensitivity of 10 breast cancer cell lines to erlotinib in light of cyclin-dependent kinase 2 (CDK2), considered the farthest downstream kinase that controls cell cycling in the EGFR signaling pathway. Expression of EGFR and HER2 were not associated with sensitivity to erlotinib. Expression of phosphorylated (p-)tyrosine, p-Akt, phosphorylated extracellular signal-regulated kinase (p-ERK) 1/ERK2 (p42/p44), and p27 after treatment of erlotinib was not associated with erlotinib sensitivity. However, suppression of CDK2 activity after erlotinib treatment correlated with erlotinib sensitivity (P < 0.0001). Restoration of CDK2 activity partially restored proliferation and induced erlotinib resistance in erlotinib-sensitive cell lines, indicating that sensitivity to erlotinib in these breast cancer cells depends, at least in part, on CDK2 activity. p27, an inhibitor of CDK2, was not translocated into the nucleus in erlotinib-resistant cell lines. Knocking down p27 protein partially blocked erlotinib-induced cell death and cell cycle arrest. These findings indicate that the ability of erlotinib to suppress CDK2 activity is critical for cellular sensitivity to erlotinib, regardless of EGFR expression level, and that the presence of p27 in the cytoplasm also participates in erlotinib resistance. PMID:17671085

  16. CARMA3 is overexpressed in colon cancer and regulates NF-{kappa}B activity and cyclin D1 expression

    SciTech Connect

    Miao, Zhifeng; Zhao, Tingting; Wang, Zhenning; Xu, Yingying; Song, Yongxi; Wu, Jianhua; Xu, Huimian

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CARMA3 expression is elevated in colon cancers. Black-Right-Pointing-Pointer CARMA3 promotes proliferation and cell cycle progression in colon cancer cells. Black-Right-Pointing-Pointer CARMA3 upregulates cyclinD1 through NF-{kappa}B activation. -- Abstract: CARMA3 was recently reported to be overexpressed in cancers and associated with the malignant behavior of cancer cells. However, the expression of CARMA3 and its biological roles in colon cancer have not been reported. In the present study, we analyzed the expression pattern of CARMA3 in colon cancer tissues and found that CARMA3 was overexpressed in 30.8% of colon cancer specimens. There was a significant association between CARMA3 overexpression and TNM stage (p = 0.0383), lymph node metastasis (p = 0.0091) and Ki67 proliferation index (p = 0.0035). Furthermore, knockdown of CARMA3 expression in HT29 and HCT116 cells with high endogenous expression decreased cell proliferation and cell cycle progression while overexpression of CARMA3 in LoVo cell line promoted cell proliferation and facilitated cell cycle transition. Further analysis showed that CARMA3 knockdown downregulated and its overexpression upregulated cyclin D1 expression and phospho-Rb levels. In addition, we found that CARMA3 depletion inhibited p-I{kappa}B levels and NF-{kappa}B activity and its overexpression increased p-I{kappa}B expression and NF-{kappa}B activity. NF-{kappa}B inhibitor BAY 11-7082 reversed the role of CARMA3 on cyclin D1 upregulation. In conclusion, our study found that CARMA3 is overexpressed in colon cancers and contributes to malignant cell growth by facilitating cell cycle progression through NF-{kappa}B mediated upregulation of cyclin D1.

  17. Involvement of aberrant cyclin-dependent kinase 5/p25 activity in experimental traumatic brain injury.

    PubMed

    Yousuf, Mohammad A; Tan, Chunfeng; Torres-Altoro, Melissa I; Lu, Fang-Min; Plautz, Erik; Zhang, Shanrong; Takahashi, Masaya; Hernandez, Adan; Kernie, Steven G; Plattner, Florian; Bibb, James A

    2016-07-01

    Traumatic brain injury (TBI) is associated with adverse effects on brain functions, including sensation, language, emotions and/or cognition. Therapies for improving outcomes following TBI are limited. A better understanding of the pathophysiological mechanisms of TBI may suggest novel treatment strategies to facilitate recovery and improve treatment outcome. Aberrant activation of cyclin-dependent kinase 5 (Cdk5) has been implicated in neuronal injury and neurodegeneration. Cdk5 is a neuronal protein kinase activated via interaction with its cofactor p35 that regulates numerous neuronal functions, including synaptic remodeling and cognition. However, conversion of p35 to p25 via Ca(2+) -dependent activation of calpain results in an aberrantly active Cdk5/p25 complex that is associated with neuronal damage and cell death. Here, we show that mice subjected to controlled cortical impact (CCI), a well-established experimental TBI model, exhibit increased p25 levels and consistently elevated Cdk5-dependent phosphorylation of microtubule-associated protein tau and retinoblastoma (Rb) protein in hippocampal lysates. Moreover, CCI-induced neuroinflammation as indicated by increased astrocytic activation and number of reactive microglia. Brain-wide conditional Cdk5 knockout mice (Cdk5 cKO) subjected to CCI exhibited significantly reduced edema, ventricular dilation, and injury area. Finally, neurophysiological recordings revealed that CCI attenuated excitatory post-synaptic potential field responses in the hippocampal CA3-CA1 pathway 24 h after injury. This neurophysiological deficit was attenuated in Cdk5 cKO mice. Thus, TBI induces increased levels of p25 generation and aberrant Cdk5 activity, which contributes to pathophysiological processes underlying TBI progression. Hence, selectively preventing aberrant Cdk5 activity may be an effective acute strategy to improve recovery from TBI. Traumatic brain injury (TBI) increases astrogliosis and microglial activation

  18. Retinofugal Projections from Melanopsin-Expressing Retinal Ganglion Cells Revealed by Intraocular Injections of Cre-Dependent Virus

    PubMed Central

    Delwig, Anton; Larsen, DeLaine D.; Yasumura, Douglas; Yang, Cindy F.; Shah, Nirao M.; Copenhagen, David R.

    2016-01-01

    To understand visual functions mediated by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs), it is important to elucidate axonal projections from these cells into the brain. Initial studies reported that melanopsin is expressed only in retinal ganglion cells within the eye. However, recent studies in Opn4-Cre mice revealed Cre-mediated marker expression in multiple brain areas. These discoveries complicate the use of melanopsin-driven genetic labeling techniques to identify retinofugal projections specifically from mRGCs. To restrict labeling to mRGCs, we developed a recombinant adeno-associated virus (AAV) carrying a Cre-dependent reporter (human placental alkaline phosphatase) that was injected into the vitreous of Opn4-Cre mouse eyes. The labeling observed in the brain of these mice was necessarily restricted specifically to retinofugal projections from mRGCs in the injected eye. We found that mRGCs innervate multiple nuclei in the basal forebrain, hypothalamus, amygdala, thalamus and midbrain. Midline structures tended to be bilaterally innervated, whereas the lateral structures received mostly contralateral innervation. As validation of our approach, we found projection patterns largely corresponded with previously published results; however, we have also identified a few novel targets. Our discovery of projections to the central amygdala suggests a possible direct neural pathway for aversive responses to light in neonates. In addition, projections to the accessory optic system suggest that mRGCs play a direct role in visual tracking, responses that were previously attributed to other classes of retinal ganglion cells. Moreover, projections to the zona incerta raise the possibility that mRGCs could regulate visceral and sensory functions. However, additional studies are needed to investigate the actual photosensitivity of mRGCs that project to the different brain areas. Also, there is a concern of "overlabeling" with very

  19. KSHV vCyclin Counters the Senescence/G1 Arrest Response Triggered by NF-κB Hyper-activation

    PubMed Central

    Zhi, Huijun; Zahoor, Muhammad Atif; Druck Shudofsky, Abigail M.; Giam, Chou-Zen

    2014-01-01

    Many oncogenic viruses activate NF-κB as a part of their replicative cycles. We have shown recently that persistent and potentially oncogenic activation of NF-κB by the human T-lymphotropic virus 1 (HTLV-1) oncoprotein Tax immediately triggers a host senescence response mediated by cyclin-dependent kinase inhibitors: p21CIP1/WAF1 (p21) and p27Kip1 (p27) Here we demonstrate that RelA/NF-κB activation by Kaposi sarcoma herpesvirus (KSHV) latency protein vFLIP also leads to p21/p27 up-regulation and G1 cell cycle arrest. Remarkably, KSHV vCyclin, another latency protein co-expressed with vFLIP from a bicistronic latency-specific mRNA, was found to prevent the senescence and G1 arrest induced by HTLV-1 Tax and vFLIP respectively. This is due to the known ability of vCyclin/CDK6 complex to resist p21 and p27 inhibition and cause p27 degradation23. In KSHV-transformed BCBL-1 cells, sustained vFLIP expression with shRNA-mediated vCyclin depletion resulted in G1 arrest. The functional interdependence of vFLIP and vCyclin explains why they are co-translated from the same viral mRNA. Importantly, deregulation of the G1 cyclin-dependent kinase can facilitate chronic IKK/NF-κB activation. PMID:24469036

  20. Oct-1 Potentiates CREB-Driven Cyclin D1 Promoter Activation via a Phospho-CREB- and CREB Binding Protein-Independent Mechanism

    PubMed Central

    Boulon, Séverine; Dantonel, Jean-Christophe; Binet, Virginie; Vié, Annick; Blanchard, Jean-Marie; Hipskind, Robert A.; Philips, Alexandre

    2002-01-01

    Cyclin D1, the regulatory subunit for mid-G1 cyclin-dependent kinases, controls the expression of numerous cell cycle genes. A cyclic AMP-responsive element (CRE), located upstream of the cyclin D1 mRNA start site, integrates mitogenic signals that target the CRE-binding factor CREB, which can recruit the transcriptional coactivator CREB-binding protein (CBP). We describe an alternative mechanism for CREB-driven cyclin D1 induction that involves the ubiquitous POU domain protein Oct-1. In the breast cancer cell line MCF-7, overexpression of Oct-1 or its POU domain strongly increases transcriptional activation of cyclin D1 and GAL4 reporter genes that is specifically dependent upon CREB but independent of Oct-1 DNA binding. Gel retardation and chromatin immunoprecipitation assays confirm that POU forms a complex with CREB bound to the cyclin D1 CRE. In solution, CREB interaction with POU requires the CREB Q2 domain and, notably, occurs with CREB that is not phosphorylated on Ser 133. Accordingly, Oct-1 also potently enhances transcriptional activation mediated by a Ser133Ala CREB mutant. Oct-1/CREB synergy is not diminished by the adenovirus E1A 12S protein, a repressor of CBP coactivator function. In contrast, E1A strongly represses CBP-enhanced transactivation by CREB phosphorylated on Ser 133. Our observation that Oct-1 potentiates CREB-dependent cyclin D1 transcriptional activity independently of Ser 133 phosphorylation and E1A-sensitive coactivator function offers a new paradigm for the regulation of cyclin D1 induction by proliferative signals. PMID:12391146

  1. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    PubMed

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma. PMID:27203461

  2. Association of Bcl-2 with cyclin a/Cdk-2 complex and its effects on Cdk-2 activity.

    PubMed

    Crescenzi, Elvira; Sannino, Maria; Tonziello, Gilda; Palumbo, Giuseppe

    2002-11-01

    In the human endometrial carcinoma cell line HEC1B, the overexpression of Bcl-2 leads to a G2/M cell cycle arrest. The experiments presented in this work suggest a direct interaction between the antiapoptotic protein Bcl-2 and the cyclin-dependent kinase Cdk-2 and suggest that such interaction is cell cycle dependent. While further experiments are currently under way in our laboratory to elucidate the significance of Cdk-2/Bcl-2 complexes in PCD and cell cycle regulation, we demonstrate also a specific inhibitory function of Bcl-2 on the activity of the coprecipitated kinase. PMID:12485875

  3. Cyclin C is a haploinsufficient tumor suppressor

    PubMed Central

    Li, Na; Fassl, Anne; Chick, Joel; Inuzuka, Hiroyuki; Li, Xiaoyu; Mansour, Marc R.; Liu, Lijun; Wang, Haizhen; King, Bryan; Shaik, Shavali; Gutierrez, Alejandro; Ordureau, Alban; Otto, Tobias; Kreslavsky, Taras; Baitsch, Lukas; Bury, Leah; Meyer, Clifford A.; Ke, Nan; Mulry, Kristin A.; Kluk, Michael J.; Roy, Moni; Kim, Sunkyu; Zhang, Xiaowu; Geng, Yan; Zagozdzon, Agnieszka; Jenkinson, Sarah; Gale, Rosemary E.; Linch, David C.; Zhao, Jean J.; Mullighan, Charles G.; Harper, J. Wade; Aster, Jon C.; Aifantis, Iannis; von Boehmer, Harald; Gygi, Steven P.; Wei, Wenyi; Look, A. Thomas; Sicinski, Piotr

    2014-01-01

    Cyclin C was cloned as a growth-promoting G1 cyclin, and was also shown to regulate gene transcription. Here we report that in vivo cyclin C acts as a haploinsufficient tumor suppressor, by controlling Notch1 oncogene levels. Cyclin C activates an “orphan” CDK19 kinase, as well as CDK8 and CDK3. These cyclin C-CDK complexes phosphorylate Notch1 intracellular domain (ICN1) and promote ICN1 degradation. Genetic ablation of cyclin C blocks ICN1 phosphorylation in vivo, thereby elevating ICN1 levels in cyclin C-knockout mice. Cyclin C ablation or heterozygosity collaborate with other oncogenic lesions and accelerate development of T-cell-acute lymphoblastic leukemia (T-ALL). Furthermore, the cyclin C gene is heterozygously deleted in a significant fraction of human T-ALL, and these tumors express reduced cyclin C levels. We also describe point mutations in human T-ALL that render cyclin C-CDK unable to phosphorylate ICN1. Hence, tumor cells may develop different strategies to evade cyclin C inhibitory function. PMID:25344755

  4. Huanglian, A chinese herbal extract, inhibits cell growth by suppressing the expression of cyclin B1 and inhibiting CDC2 kinase activity in human cancer cells.

    PubMed

    Li, X K; Motwani, M; Tong, W; Bornmann, W; Schwartz, G K

    2000-12-01

    Huanglian is an herb that is widely used in China for the treatment of gastroenteritis. We elected to determine whether huanglian could inhibit tumor cell growth by modulating molecular events directly associated with the cell cycle. Huanglian inhibited tumor growth and colony formation of gastric, colon, and breast cancer cell lines in a time- and dose-dependent manner. Cell growth was completely inhibited after 3 days of continuous drug exposure to 10 microg/ml of herb. This degree of growth inhibition was significantly greater than that observed with berberine, the major constituent of the herb. The inhibition of cell growth by huanglian was associated with up to 8-fold suppression of cyclin B1 protein. This resulted in complete inhibition of cdc2 kinase activity and accumulation of cells in G(2). The mRNA expression of cyclin B1 was not changed after huanglian treatment. There was no change in the protein expression of cyclins A or E. Therefore, the effect of huanglian on inhibiting tumor growth seems to be mediated by the selective suppression of cyclin B1, which results in the inhibition of cdc2 kinase activity. Inhibition of cyclin dependent kinase (cdk) activity is emerging as an attractive target for cancer chemotherapy. Huanglian represents a class of agents that can inhibit tumor cell growth by directly suppressing the expression of a cyclin subunit that is critical for cell cycle progression. These results indicate that traditional Chinese herbs may represent a new source of agents designed for selective inhibition of cyclin dependent kinases in cancer therapy. PMID:11093765

  5. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

    PubMed Central

    Abrieu, A; Dorée, M; Picard, A

    1997-01-01

    The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation. Images PMID:9190205

  6. A western blot assay to measure cyclin dependent kinase activity in cells or in vitro without the use of radioisotopes.

    PubMed

    Lewis, Cody W; Taylor, Ryan G; Kubara, Philip M; Marshall, Kris; Meijer, Laurent; Golsteyn, Roy M

    2013-09-17

    We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 μM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity. PMID:23954627

  7. Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4

    PubMed Central

    2011-01-01

    Background Cyclin-dependent kinases 2, 4 and 6 (Cdk2, Cdk4, Cdk6) are closely structurally homologous proteins which are classically understood to control the transition from the G1 to the S-phases of the cell cycle by combining with their appropriate cyclin D or cyclin E partners to form kinase-active holoenzymes. Deregulation of Cdk4 is widespread in human cancer, CDK4 gene knockout is highly protective against chemical and oncogene-mediated epithelial carcinogenesis, despite the continued presence of CDK2 and CDK6; and overexpresssion of Cdk4 promotes skin carcinogenesis. Surprisingly, however, Cdk4 kinase inhibitors have not yet fulfilled their expectation as 'blockbuster' anticancer agents. Resistance to inhibition of Cdk4 kinase in some cases could potentially be due to a non-kinase activity, as recently reported with epidermal growth factor receptor. Results A search for a potential functional site of non-kinase activity present in Cdk4 but not Cdk2 or Cdk6 revealed a previously-unidentified loop on the outside of the C'-terminal non-kinase domain of Cdk4, containing a central amino-acid sequence, Pro-Arg-Gly-Pro-Arg-Pro (PRGPRP). An isolated hexapeptide with this sequence and its cyclic amphiphilic congeners are selectively lethal at high doses to a wide range of human cancer cell lines whilst sparing normal diploid keratinocytes and fibroblasts. Treated cancer cells do not exhibit the wide variability of dose response typically seen with other anticancer agents. Cancer cell killing by PRGPRP, in a cyclic amphiphilic cassette, requires cells to be in cycle but does not perturb cell cycle distribution and is accompanied by altered relative Cdk4/Cdk1 expression and selective decrease in ATP levels. Morphological features of apoptosis are absent and cancer cell death does not appear to involve autophagy. Conclusion These findings suggest a potential new paradigm for the development of broad-spectrum cancer specific therapeutics with a companion diagnostic

  8. Cyclin-dependent kinases

    PubMed Central

    2014-01-01

    Summary Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  9. Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

    PubMed Central

    Bártová, Iveta; Otyepka, Michal; Kříž, Zdeněk; Koča, Jaroslav

    2004-01-01

    Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data. PMID:15133164

  10. Ectopic expression of cyclin D3 corrects differentiation of DM1 myoblasts through activation of RNA CUG-binding protein, CUGBP1

    SciTech Connect

    Salisbury, Elizabeth; Sakai, Keiko; Schoser, Benedikt; Huichalaf, Claudia; Schneider-Gold, Christiane; Nguyen, Heather; Wang, Gou-Li; Albrecht, Jeffrey H.; Timchenko, Lubov T.

    2008-07-01

    Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBP{beta} mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.

  11. Overexpression of cyclin D1-CDK4 in silica-induced transformed cells is due to activation of ERKs, JNKs/AP-1 pathway.

    PubMed

    Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Du, Hongju; You, Baorong; Ye, Meng; Huang, Chuanshu; Shi, Xianglin

    2006-01-25

    Silica has been known to be a factor inducing acute injury and chronic pulmonary fibrosis. Silica has also been listed as a human carcinogen in 1996 by International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved its pathologic effects are not well understood. In these studies, we found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica could cause increases in activation of extracellular signal-regulated kinases (ERKs), p38K, and c-Jun NH2-terminal amino kinases (JNKs), and HELF transformation. Interestingly, silica-induced transformation of HELF (S-HELF) led to increases in activated levels of ERKs and p46 of JNKs, and decrease in p38K activation, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Further studies showed that there are differential effects of ERKs, JNKs and p38K, as well as their downstream transcription factor AP-1, in regulation of expression of cyclin D1 and CDK4 and cell cycle alternations induced by silica. Cyclin D1 and CDK4 were increased in S-HELF as compared with those in HELF. Inhibition of ERKs activation by AG126, JNK by SP600125, and AP-1 by curcumin could reduced the induction of cyclin D1 and CDK4. There is no significant difference for cell cycle distribution between groups. These results demonstrate that ERKs and JNKs, but not p38K is responsible for induction of cyclin D1 and CDK4 in S-HELF, suggesting that overexpression of cyclin D1 and CDK4 caused by silica is mediated by ERK, JNK/AP-1signaling pathway. PMID:16125882

  12. Involvement of Cyclin-Dependent Kinase 1 during Postovulatory Aging-Mediated Abortive Spontaneous Egg Activation in Rat Eggs Cultured In Vitro.

    PubMed

    Prasad, Shilpa; Koch, Biplob; Chaube, Shail K

    2016-04-01

    Freshly ovulated rat eggs do not remain arrested at metaphase II (MII) and undergo exit from MII arrest with initiation of extrusion of the second polar body (PBII), a characteristic feature of abortive spontaneous egg activation (SEA). The biochemical and molecular changes during postovulatory aging-mediated abortive SEA remain poorly understood. We investigated the morphological, cellular, and molecular changes during postovulatory aging-mediated abortive SEA in eggs cultured in vitro. Our results suggest that postovulatory egg aging in vitro induced initiation of PBII extrusion in a time-dependent manner. Postovulatory aging increased Wee1 kinase and Thr-14/Tyr-15 phosphorylated cyclin-dependent kinase 1 (Cdk1) levels, whereas Thr-161 phosphorylated Cdk1 and cyclin B1 levels were significantly decreased in eggs cultured in vitro. The early mitotic inhibitor 2 (Emi2) level was significantly reduced, but anaphase promoting complex/cyclosome (APC/C) and mitotic arrest deficient protein (MAD2) levels were increased initially and then reduced during a later period of in vitro culture. These results suggest that an increased Wee1 kinase level modulated the specific phosphorylation status of Cdk1, increased Cdk1 activity, and decreased the cyclin B1 level. Furthermore, the decreased Emi2 level was associated with an increased level of APC/C and decreased level of cyclin B1, which resulted in maturation promoting factor (MPF) destabilization and finally led to postovulatory aging-mediated abortive SEA in rat eggs cultured in vitro. PMID:26982431

  13. Baicalein induces G1 arrest in oral cancer cells by enhancing the degradation of cyclin D1 and activating AhR to decrease Rb phosphorylation

    SciTech Connect

    Cheng, Ya-Hsin; Li, Lih-Ann; Lin, Pinpin; Cheng, Li-Chuan; Hung, Chein-Hui; Chang, Nai Wen; Lin, Chingju

    2012-09-15

    Baicalein is a flavonoid, known to have anti-inflammatory and anti-cancer effects. As an aryl hydrocarbon receptor (AhR) ligand, baicalein at high concentrations blocks AhR-mediated dioxin toxicity. Because AhR had been reported to play a role in regulating the cell cycle, we suspected that the anti-cancer effect of baicalein is associated with AhR. This study investigated the molecular mechanism involved in the anti-cancer effect of baicalein in oral cancer cells HSC-3, including whether such effect would be AhR-mediated. Results revealed that baicalein inhibited cell proliferation and increased AhR activity in a dose-dependent manner. Cell cycle was arrested at the G1 phase and the expression of CDK4, cyclin D1, and phosphorylated retinoblastoma (pRb) was decreased. When the AhR was suppressed by siRNA, the reduction of pRb was partially reversed, accompanied by a decrease of cell population at G1 phase and an increase at S phase, while the reduction of cyclin D1 and CDK4 did not change. This finding suggests that the baicalein activation of AhR is indeed associated with the reduction of pRb, but is independent of the reduction of cyclin D1 and CDK4. When cells were pre-treated with LiCl, the inhibitor of GSK-3β, the decrease of cyclin D1 was blocked and the reduction of pRb was recovered. The data indicates that in HSC-3 the reduction of pRb is both mediated by baicalein through activation of AhR and facilitation of cyclin D1 degradation, which causes cell cycle arrest at the G1 phase, and results in the inhibition of cell proliferation. -- Highlights: ► Baicalein causes the G1 phase arrest by decreasing Rb phosphorylation. ► Baicalein modulates AhR-mediated cell proliferation. ► Both AhR activation and cyclin D1 degradation results in hypophosphorylation of Rb. ► Baicalein facilitates cyclin D1 degradation by signalling the GSK-3β pathway.

  14. c-Myc Regulates Cyclin D-Cdk4 and -Cdk6 Activity but Affects Cell Cycle Progression at Multiple Independent Points

    PubMed Central

    Mateyak, Maria K.; Obaya, Alvaro J.; Sedivy, John M.

    1999-01-01

    c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc−/− cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin–cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27KIP1 and decreased expression of Cdk7 in c-myc−/− cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions. PMID:10373516

  15. CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription

    PubMed Central

    Aregger, Michael; Kaskar, Aneesa; Varshney, Dhaval; Fernandez-Sanchez, Maria Elena; Inesta-Vaquera, Francisco A.; Weidlich, Simone; Cowling, Victoria H.

    2016-01-01

    Summary The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine. We report that CDK1-cyclin B1 phosphorylates the RNMT regulatory domain on T77 during G2/M phase of the cell cycle. RNMT T77 phosphorylation activates the enzyme both directly and indirectly by inhibiting interaction with KPNA2, an RNMT inhibitor. RNMT T77 phosphorylation results in elevated m7G cap methyltransferase activity at the beginning of G1 phase, coordinating mRNA capping with the burst of transcription that occurs following nuclear envelope reformation. RNMT T77 phosphorylation is required for the production of cohort of proteins, and inhibiting T77 phosphorylation reduces the cell proliferation rate. PMID:26942677

  16. The Requirement for Cyclin D Function in Tumor Maintenance

    PubMed Central

    Choi, Yoon Jong; Li, Xiaoyu; Hydbring, Per; Sanda, Takaomi; Stefano, Joanna; Christie, Amanda L.; Signoretti, Sabina; Look, A. Thomas; Kung, Andrew L.; von Boehmer, Harald; Sicinski, Piotr

    2012-01-01

    SUMMARY D-cyclins represent components of cell cycle machinery. To test the efficacy of targeting D-cyclins in cancer treatment, we engineered mouse strains which allow acute and global ablation of individual D-cyclins in a living animal. Ubiquitous shutdown of cyclin D1 or inhibition of cyclin D-associated kinase activity in mice bearing ErbB2-driven mammary carcinomas triggered tumor cell senescence, without compromising the animals’ health. Ablation of cyclin D3 in mice bearing Notch1-driven T-cell acute lymphoblastic leukemias (T-ALL) triggered tumor cell apoptosis. Such selective killing of leukemic cells can also be achieved by inhibiting cyclin D-associated kinase activity in mouse and human T-ALL models. Inhibition of cyclin D-kinase activity represents a highly-selective anti-cancer strategy that specifically targets cancer cells without significantly affecting normal tissues. PMID:23079655

  17. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration–proliferation dichotomy

    PubMed Central

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L.; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

    2015-01-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously—a phenomenon called “migration–proliferation dichotomy.” We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration–proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  18. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration-proliferation dichotomy.

    PubMed

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L; Garcia-Marcos, Mikel; Farquhar, Marilyn G; Ghosh, Pradipta

    2015-09-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  19. BAFF induces spleen CD4{sup +} T cell proliferation by down-regulating phosphorylation of FOXO3A and activates cyclin D2 and D3 expression

    SciTech Connect

    Ji, Fang; Chen, Rongjing; Liu, Baojun; Zhang, Xiaoping; Han, Junli; Wang, Haining; Shen, Gang; Tao, Jiang

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer Firstly analyze the mechanism of BAFF and anti-CD3 co-stimulation on purified mouse splenic CD4{sup +} T cells. Black-Right-Pointing-Pointer Carrying out siRNA technology to study FOXO3A protein function. Black-Right-Pointing-Pointer Helpful to understand the T cell especially CD4{sup +} T cell's role in immunological reaction. -- Abstract: The TNF ligand family member 'B cell-activating factor belonging to the TNF family' (BAFF, also called BLyS, TALL-1, zTNF-4, and THANK) is an important survival factor for B and T cells. In this study, we show that BAFF is able to induce CD4{sup +} spleen T cell proliferation when co-stimulated with anti-CD3. Expression of phosphorylated FOXO3A was notably down-regulated and cyclins D2 and D3 were up-regulated and higher in the CD4{sup +} T cells when treated with BAFF and anti-CD3, as assessed by Western blotting. Furthermore, after FOXO3A was knocked down, expression of cyclin D1 was unchanged, compared with control group levels, but the expression of cyclins D2 and D3 increased, compared with the control group. In conclusion, our results suggest that BAFF induced CD4{sup +} spleen T cell proliferation by down-regulating the phosphorylation of FOXO3A and then activating cyclin D2 and D3 expression, leading to CD4{sup +} T cell proliferation.

  20. Selective activation of p38alpha and p38gamma by hypoxia. Role in regulation of cyclin D1 by hypoxia in PC12 cells.

    PubMed

    Conrad, P W; Rust, R T; Han, J; Millhorn, D E; Beitner-Johnson, D

    1999-08-13

    Hypoxic/ischemic trauma is a primary factor in the pathology of a multitude of disease states. The effects of hypoxia on the stress- and mitogen-activated protein kinase signaling pathways were studied in PC12 cells. Exposure to moderate hypoxia (5% O(2)) progressively stimulated phosphorylation and activation of p38gamma in particular, and also p38alpha, two stress-activated protein kinases. In contrast, hypoxia had no effect on enzyme activity of p38beta, p38beta(2), p38delta, or on c-Jun N-terminal kinase, another stress-activated protein kinase. Prolonged hypoxia also induced phosphorylation and activation of p42/p44 mitogen-activated protein kinase, although this activation was modest compared with nerve growth factor- and ultraviolet light-induced activation. Hypoxia also dramatically down-regulated immunoreactivity of cyclin D1, a gene that is known to be regulated negatively by p38 at the level of gene expression (Lavoie, J. N., L'Allemain, G., Brunet, A., Muller, R., and Pouyssegur, J. (1996) J. Biol. Chem. 271, 20608-20616). This effect was partially blocked by SB203580, an inhibitor of p38alpha but not p38gamma. Overexpression of a kinase-inactive form of p38gamma was also able to reverse in part the effect of hypoxia on cyclin D1 levels, suggesting that p38alpha and p38gamma converge to regulate cyclin D1 during hypoxia. These studies demonstrate that an extremely typical physiological stress (hypoxia) causes selective activation of specific p38 signaling elements; and they also identify a downstream target of these pathways. PMID:10438538

  1. Quercetin reduces cyclin D1 activity and induces G1 phase arrest in HepG2 cells

    PubMed Central

    ZHOU, JIN; LI, LU; FANG, LI; XIE, HUA; YAO, WENXIU; ZHOU, XIANG; XIONG, ZHUJUAN; WANG, LI; LI, ZHIXI; LUO, FENG

    2016-01-01

    Quercetin is able to inhibit proliferation of malignant tumor cells; however, the exact mechanism involved in this biological process remains unclear. The current study utilized a quantitative proteomic analysis to explore the antitumor mechanisms of quercetin. The leucine of HepG2 cells treated with quercetin was labeled as d3 by stable isotope labeling by amino acids in cell culture (SILAC). The isotope peaks of control HepG2 cells were compared with the d3-labeled HepG2 cells by mass spectrometry (MS) to identify significantly altered proteins. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses were subsequently employed to verify the results of the MS analysis. A flow cytometry assay was designed to observe the influence of various quercetin treatment concentrations on the cell cycle distribution of HepG2 cells. The results indicated that quercetin is able to substantially inhibit proliferation of HepG2 cells and induce an obvious morphological alteration of cells. According to the MS results, the 70 credibly-changed proteins that were identified may play important roles in multiple cellular processes, including protein synthesis, signaling, cytoskeletal processes and metabolism. Among these functional proteins, the expression of cyclin D1 (CCND1) was found to be significantly decreased. RT-PCR and western blot analyses verified the SILAC-MS results of decreased CCND1 expression. In summary, flow cytometry revealed that quercetin is able to induce G1 phase arrest in HepG2 cells. Based on the aforementioned observations, it is suggested that quercetin exerts antitumor activity in HepG2 cells through multiple pathways, including interfering with CCND1 gene expression to disrupt the cell cycle and proliferation of HepG2 cells. In the future, we aim to explore this effect in vivo. PMID:27347174

  2. Chenodeoxycholic acid through a TGR5-dependent CREB signaling activation enhances cyclin D1 expression and promotes human endometrial cancer cell proliferation.

    PubMed

    Casaburi, Ivan; Avena, Paola; Lanzino, Marilena; Sisci, Diego; Giordano, Francesca; Maris, Pamela; Catalano, Stefania; Morelli, Catia; Andò, Sebastiano

    2012-07-15

    Endometrial cancer exhibits a strong incidence in western developed countries mainly due to fat-rich diet and obesity. Processing of dietary lipids is triggered by bile acids, amphipathic detergents that are synthesized in the liver and stored in the gallbladder. In addition to their well-recognized role in dietary lipid absorption and cholesterol homeostasis, bile acids can also act as signaling molecules with systemic endocrine functions. In the present study we investigated the biological effects of the primary bile chenodeoxycholic acid (CDCA) on a human endometrial cancer cell line, Ishikawa. Low concentrations of CDCA are able to stimulate Ishikawa cell growth by inducing a significant increase in Cyclin D1 protein and mRNA expression through the activation of the membrane G protein-coupled receptor (TGR5)-dependent pathway. Dissecting the molecular mechanism underlying this effect by mutagenesis, EMSA and ChIP analysis revealed that CDCA-induced Cyclin D1 expression requires the enhanced recruitment of the transcription factor CREB on the cyclic AMP-responsive element motif within the Cyclin D1 gene proximal promoter. Our results suggest a novel molecular mechanism explaining the potential contribution of high-fat diet and obesity to endometrial cancer growth and progression opening the rationale for strategies to prevent the risk of this obesity-related cancer in women. PMID:22751440

  3. Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

    PubMed Central

    Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

    2015-01-01

    Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell

  4. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  5. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  6. Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins.

    PubMed

    Zi, X; Feyes, D K; Agarwal, R

    1998-04-01

    There is an increasing interest in identifying potent cancer preventive and therapeutic agents against breast cancer. Silymarin, a flavonoid antioxidant isolated from milk thistle, exerts exceptionally high to complete anticarcinogenic effects in tumorigenesis models of epithelial origin. In this study, we investigated the anticarcinogenic effect of silymarin and associated molecular mechanisms, using human breast carcinoma cells MDA-MB 468. Silymarin treatment resulted in a significantly high to complete inhibition of both anchorage-dependent and anchorage-independent cell growth in a dose- and time-dependent manner. The inhibitory effects of silymarin on cell growth and proliferation were associated with a G1 arrest in cell cycle progression concomitant with an induction of up to 19-fold in the protein expression of cyclin-dependent kinase (CDK) inhibitor Cip1/p21. Following silymarin treatment of cells, an incremental binding of Cip1/p21 with CDK2 and CDK6 paralleled a significant decrease in CDK2-, CDK6-, cyclin D1-, and cyclin E-associated kinase activity with no change in CDK2 and CDK6 expression but a decrease in G1 cyclins D1 and E. Taken together, these results suggest that silymarin may exert a strong anticarcinogenic effect against breast cancer and that this effect possibly involves an induction of Cip1/p21 by silymarin, which inhibits the threshold kinase activities of CDKs and associated cyclins, leading to a G1 arrest in cell cycle progression. PMID:9563902

  7. Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer.

    PubMed

    Karakas, Cansu; Biernacka, Anna; Bui, Tuyen; Sahin, Aysegul A; Yi, Min; Akli, Said; Schafer, Jolie; Alexander, Angela; Adjapong, Opoku; Hunt, Kelly K; Keyomarsi, Khandan

    2016-07-01

    Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer. PMID:27182644

  8. 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. PMID:25601491

  9. Two new competing pathways establish the threshold for cyclin-B–Cdk1 activation at the meiotic G2/M transition

    PubMed Central

    Aono, Ryota; Hanada, Shin-ichiro; Okumura, Eiichi; Kishimoto, Takeo

    2016-01-01

    ABSTRACT Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K–Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B–Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B–Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K–Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries. PMID:27390173

  10. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

    PubMed Central

    Lee, Kuan-Han; Ho, Wen-Yueh; Wu, Shu-Jing; Omar, Hany A.; Huang, Po-Jui; Wang, Clay C. C.; Hung, Jui-Hsiang

    2014-01-01

    Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy. PMID:24406729

  11. Two new competing pathways establish the threshold for cyclin-B-Cdk1 activation at the meiotic G2/M transition.

    PubMed

    Hiraoka, Daisaku; Aono, Ryota; Hanada, Shin-Ichiro; Okumura, Eiichi; Kishimoto, Takeo

    2016-08-15

    Extracellular ligands control biological phenomena. Cells distinguish physiological stimuli from weak noise stimuli by establishing a ligand-concentration threshold. Hormonal control of the meiotic G2/M transition in oocytes is essential for reproduction. However, the mechanism for threshold establishment is unclear. In starfish oocytes, maturation-inducing hormones activate the PI3K-Akt pathway through the Gβγ complex of heterotrimeric G-proteins. Akt directly phosphorylates both Cdc25 phosphatase and Myt1 kinase, resulting in activation of cyclin-B-Cdk1, which then induces meiotic G2/M transition. Here, we show that cyclin-B-Cdk1 is partially activated after subthreshold hormonal stimuli, but this triggers negative feedback, resulting in dephosphorylation of Akt sites on Cdc25 and Myt1, thereby canceling the signal. We also identified phosphatase activity towards Akt substrates that exists independent of stimuli. In contrast to these negative regulatory activities, an atypical Gβγ-dependent pathway enhances PI3K-Akt-dependent phosphorylation. Based on these findings, we propose a model for threshold establishment in which hormonal dose-dependent competition between these new pathways establishes a threshold; the atypical Gβγ-pathway becomes predominant over Cdk-dependent negative feedback when the stimulus exceeds this threshold. Our findings provide a regulatory connection between cell cycle and signal transduction machineries. PMID:27390173

  12. AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition.

    PubMed

    Santo, L; Vallet, S; Hideshima, T; Cirstea, D; Ikeda, H; Pozzi, S; Patel, K; Okawa, Y; Gorgun, G; Perrone, G; Calabrese, E; Yule, M; Squires, M; Ladetto, M; Boccadoro, M; Richardson, P G; Munshi, N C; Anderson, K C; Raje, N

    2010-04-22

    Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM. PMID:20101221

  13. Sangivamycin-Like Molecule 6 (SLM6) exhibits potent anti-multiple myeloma activity through inhibition of cyclin-dependent kinase-9 (CDK9)

    PubMed Central

    Dolloff, Nathan G.; Allen, Joshua E.; Dicker, David T.; Aqui, Nicole; Vogl, Dan; Malysz, Jozef; Talamo, Giampaolo; El-Deiry, Wafik S.

    2012-01-01

    Despite significant treatment advances over the past decade, multiple myeloma (MM) remains largely incurable. In this study we found that MM cells were remarkably sensitive to the death-inducing effects of a new class of sangivamycin-like molecules (SLMs). A panel of structurally related SLMs selectively induced apoptosis in MM cells but not other tumor or non-malignant cell lines at sub-micromolar concentrations. SLM6 was the most active compound in vivo, where it was well-tolerated and significantly inhibited growth and induced apoptosis of MM tumors. We determined that the anti-MM activity of SLM6 was mediated by direct inhibition of cyclin-dependent kinase 9 (CDK9), which resulted in transcriptional repression of oncogenes that are known to drive MM progression (c-Maf, cyclin D1, and c-Myc). Furthermore, SLM6 demonstrated superior in vivo anti-MM activity over the CDK inhibitor flavopiridol, which is currently in clinical trials for MM. These findings demonstrate that SLM6 is a novel CDK9 inhibitor with promising preclinical activity as an anti-MM agent. PMID:22964485

  14. Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts.

    PubMed Central

    Clarke, P R; Leiss, D; Pagano, M; Karsenti, E

    1992-01-01

    Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation. Images PMID:1316271

  15. Dixdc1 targets CyclinD1 and p21 via PI3K pathway activation to promote Schwann cell proliferation after sciatic nerve crush.

    PubMed

    Wu, Weijie; Liu, Qingqing; Liu, Yuxi; Yu, Zhaohui; Wang, Youhua

    2016-09-16

    Dixdc1 (DIX domain containing-1), the mammalian homolog of Ccd1 (Coiled-coil-Dishevelled-Axin1), is a protein containing a coiled-coil domain and a Dishevelled-Axin (DIX) domain. As a novel component of the Wnt pathway, Dixdc1 has been reported to be able to promote neural progenitor proliferation and neuronal differentiation via Wnt/β-catenin signaling. But there still remains something unknown about Dixdc1 distribution and functions in the lesion and regeneration of the peripheral nervous system (PNS), so we tried to investigate dynamic changes of Dixdc1 expression in a rat sciatic nerve crush (SNC) model in this study. First of all, we detected SNC-induced increased levels of Dixdc1 in Schwann cells and interestingly identified parallel expression of PCNA (proliferation cell nuclear antigen) with Dixdc1. Besides, we observed up-regulated Dixdc1 during the process of TNF-α-induced Schwann cell proliferation. Also, we discovered that Dixdc1 could promote G1-S phase transition accompanied with the up-regulation of CyclinD1 and down-regulation of p21. More importantly, enhanced effects of Dixdc1 on cell proliferation were confirmed to be associated with PI3K activation. Not only blocking of the PI3K but Dixdc1 knockdown led to significantly decreased ability for proliferation, as well as down-regulation of CyclinD1 and up-regulation of p21. In summary, these data demonstrated that Dixdc1 might participate in Schwann cell proliferation by targeting CyclinD1 and p21 at least partially through the PI3K/AKT activation. PMID:27521891

  16. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS.

    PubMed

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y; Wiedemeyer, W Ruprecht

    2015-01-20

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  17. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    PubMed Central

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2015-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  18. Variation in growth rate between Arabidopsis ecotypes is correlated with cell division and A-type cyclin-dependent kinase activity.

    PubMed

    Beemster, Gerrit T S; De Vusser, Kristof; De Tavernier, Evelien; De Bock, Kirsten; Inzé, Dirk

    2002-06-01

    We used a kinematic analysis to investigate the growth processes responsible for variation in primary root growth between 18 ecotypes of Arabidopsis. Root elongation rate differed 4-fold between the slowest (Landsberg erecta, 71 microm h(-1)) and fastest growing line (Wassilewskija [Ws]; 338 microm h(-1)). This difference was contributed almost equally by variations in mature cortical cell length (84 microm [Landsberg erecta] to 237 microm [Ws]) and rate of cell production (0.63 cell h(-1) [NW108] to 1.83 cell h(-1) [Ws]). Cell production, in turn, was determined by variation in cell cycle duration (19 h [Tsu] to 48 h [NW108]) and, to a lesser extent, by differences in the number of dividing cells (32 [Weiningen] to 61 [Ws]). We found no correlation between mature cell size and endoreduplication, refuting the hypothesis that the two are linked. However, there was a strong correlation between cell production rates and the activity of the cyclin-dependent kinase (CDKA). The level of the protein could explain 32% of the variation in CDKA. Therefore, it is likely that regulators of CDKA, such as cyclins and inhibitors, are also involved. These data provide a functional link between cell cycle regulation and whole-plant growth rate as affected by genetic differences. PMID:12068124

  19. Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin

    SciTech Connect

    Nigam, Nidhi Prasad, Sahdeo; George, Jasmine; Shukla, Yogeshwer

    2009-04-03

    Lupeol, present in fruits and medicinal plants, is a biologically active compound that has been shown to have various pharmacological properties in experimental studies. In the present study, we demonstrated the modulatory effect of lupeol on 7,12-dimethylbenz[a]anthracene (DMBA)-induced alterations on cell proliferation in the skin of Swiss albino mice. Lupeol treatment showed significant (p < 0.05) preventive effects with marked inhibition at 48, 72, and 96 h against DMBA-mediated neoplastic events. Cell-cycle analysis showed that lupeol-induced G2/M-phase arrest (16-37%) until 72 h, and these inhibitory effects were mediated through inhibition of the cyclin-B-regulated signaling pathway involving p53, p21/WAF1, cdc25C, cdc2, and cyclin-B gene expression. Further lupeol-induced apoptosis was observed, as shown by an increased sub-G1 peak (28%) at 96 h, with upregulation of bax and caspase-3 genes and downregulation of anti-apoptotic bcl-2 and survivin genes. Thus, our results indicate that lupeol has novel anti-proliferative and apoptotic potential that may be helpful in designing strategies to fight skin cancer.

  20. Fisetin inhibits the activities of cyclin-dependent kinases leading to cell cycle arrest in HT-29 human colon cancer cells.

    PubMed

    Lu, Xianghua; Jung, Jae in; Cho, Han Jin; Lim, Do Young; Lee, Hyun Sook; Chun, Hyang Sook; Kwon, Dae Young; Park, Jung Han Yoon

    2005-12-01

    Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 micromol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 +/- 1% decrease in cell number observed 72 h after the addition of 60 micromol/L fisetin. Perturbed cell cycle progression from the G(1) to S phase was observed at 8 h with 60 micromol/L fisetin treatment, whereas a G(2)/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 micromol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21(CIP1/WAF1) levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities. PMID:16317137

  1. Retroviral Cyclin Controls Cyclin-Dependent Kinase 8-Mediated Transcription Elongation and Reinitiation

    PubMed Central

    Birkenheuer, Claire H.; Brewster, Connie D.; Quackenbush, Sandra L.

    2015-01-01

    ABSTRACT Walleye dermal sarcoma virus (WDSV) infection is associated with the seasonal development and regression of walleye dermal sarcoma. Previous work showed that the retroviral cyclin (RV-cyclin), encoded by WDSV, has separable cyclin box and transcription activation domains. It binds to cyclin-dependent kinase 8 (CDK8) and enhances its kinase activity. CDK8 is evolutionarily conserved and is frequently overexpressed in human cancers. It is normally activated by cyclin C and is required for transcription elongation of the serum response genes (immediate early genes [IEGs]) FOS, EGR1, and cJUN. The IEGs drive cell proliferation, and their expression is brief and highly regulated. Here we show that constitutive expression of RV-cyclin in the HCT116 colon cancer cell line significantly increases the level of IEG expression in response to serum stimulation. Quantitative reverse transcription-PCR (RT-PCR) and nuclear run-on assays provide evidence that RV-cyclin does not alter the initiation of IEG transcription but does enhance the overall rate of transcription elongation and maintains transcription reinitiation. RV-cyclin does not increase activating phosphorylation events in the mitogen-activated protein kinase pathway and does not inhibit decay of IEG mRNAs. At the EGR1 gene locus, RV-cyclin increases and maintains RNA polymerase II (Pol II) occupancy after serum stimulation, in conjunction with increased and extended EGR1 gene expression. The RV-cyclin increases CDK8 occupancy at the EGR1 gene locus before and after serum stimulation. Both of RV-cyclin's functional domains, i.e., the cyclin box and the activation domain, are necessary for the overall enhancement of IEG expression. RV-cyclin presents a novel and ancient mechanism of retrovirus-induced oncogenesis. IMPORTANCE The data reported here are important to both virology and cancer biology. The novel mechanism pinpoints CDK8 in the development of walleye dermal sarcoma and sheds light on CDK8's role in

  2. Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

    SciTech Connect

    Chhipa, Rishi Raj; Kumari, Ratna; Upadhyay, Ankur Kumar; Bhat, Manoj Kumar

    2007-11-15

    The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.

  3. Cyclin-dependent kinase inhibition by the KLF6 tumor suppressor protein through interaction with cyclin D1.

    PubMed

    Benzeno, Sharon; Narla, Goutham; Allina, Jorge; Cheng, George Z; Reeves, Helen L; Banck, Michaela S; Odin, Joseph A; Diehl, J Alan; Germain, Doris; Friedman, Scott L

    2004-06-01

    Kruppel-like factor 6 (KLF6) is a tumor suppressor gene inactivated in prostate and colon cancers, as well as in astrocytic gliomas. Here, we establish that KLF6 mediates growth inhibition through an interaction with cyclin D1, leading to reduced phosphorylation of the retinoblastoma protein (Rb) at Ser(795). Furthermore, introduction of KLF6 disrupts cyclin D1-cyclin-dependent kinase (cdk) 4 complexes and forces the redistribution of p21(Cip/Kip) onto cdk2, which promotes G(1) cell cycle arrest. Our data suggest that KLF6 converges with the Rb pathway to inhibit cyclin D1/cdk4 activity, resulting in growth suppression. PMID:15172998

  4. PPARgamma ligands suppress the feedback loop between E2F2 and cyclin-E1.

    PubMed

    Komatsu, Yoko; Ito, Ichiaki; Wayama, Mitsutoshi; Fujimura, Akiko; Akaogi, Kensuke; Machida, Hikaru; Nakajima, Yuka; Kuroda, Takao; Ohmori, Kazuji; Murayama, Akiko; Kimura, Keiji; Yanagisawa, Junn

    2008-05-23

    PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein. PMID:18355447

  5. Cyclin C mediates stress-induced mitochondrial fission and apoptosis

    PubMed Central

    Wang, Kun; Yan, Ruilan; Cooper, Katrina F.; Strich, Randy

    2015-01-01

    Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C–Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells. PMID:25609094

  6. Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis

    PubMed Central

    Panda, Amaresh C.; Abdelmohsen, Kotb; Martindale, Jennifer L.; Di Germanio, Clara; Yang, Xiaoling; Grammatikakis, Ioannis; Noh, Ji Heon; Zhang, Yongqing; Lehrmann, Elin; Dudekula, Dawood B.; De, Supriyo; Becker, Kevin G.; White, Elizabeth J.; Wilson, Gerald M.; de Cabo, Rafael; Gorospe, Myriam

    2016-01-01

    Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3′ untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation. PMID:26819411

  7. Regulation of cyclin E stability in Xenopus laevis embryos

    NASA Astrophysics Data System (ADS)

    Brandt-(Webb), Yekaterina

    Cyclin-Cdk complexes positively regulate cell cycle progression. Cyclins are regulatory subunits that bind to and activate cyclin-dependent kinases or Cdks. Cyclin E associates with Cdk2 to mediate G1/S phase transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how this protein is downregulated during development in order to determine if these mechanisms are disrupted during tumorigenesis, leading to its overexpression. Using Xenopus laevis embryos as a model, we have shown previously that during the first 12 embryonic cell cycles Cyclin E levels remain constant yet Cdk2 activity oscillates twice per cell cycle. Cyclin E is abruptly destabilized by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT). Based on work our work and work by others, we have hypothesized that differential phosphorylation and a change in localization result in Cyclin E degradation by the 26S proteasome at the MBT. To test this, we generated a series of point mutations in conserved threonine/serine residues implicated in degradation of human Cyclin E. Using Western blot analysis, we show that similarly to human Cyclin E, mutation of these residues to unphosphorylatable alanine stabilizes Cyclin E past the MBT when they are expressed in vivo. Cyclin E localization was studied by immunofluorescence analysis of endogenous and exogenous protein in pre-MBT, MBT, and post-MBT embryos. In addition, we developed a novel method of conjugating recombinant His6-tagged Cyclin E to fluorescent (CdSe)ZnS nanoparticles (quantum dots) capped with dihydrolipoic acid. Confocal microscopy was used to visualize His6Cyclin E-quantum dot complexes inside embryo cells in real time. We found that re-localization at the MBT from the cytoplasm to the nucleus

  8. Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1

    PubMed Central

    Zhuang, Yongxian; Miskimins, W Keith

    2008-01-01

    Background The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood. Results In this study, metformin was found to inhibit proliferation of most cultured breast cancer cell lines. This was independent of estrogen receptor, HER2, or p53 status. Inhibition of cell proliferation was associated with arrest within G0/G1 phase of the cell cycle. As in previous studies, metformin treatment led to activation of (AMPK) and downregulation of cyclin D1. However, these events were not sufficient for cell cycle arrest because they were also observed in the MDA-MB-231 cell line, which is not sensitive to growth arrest by metformin. In sensitive breast cancer lines, the reduction in cyclin D1 led to release of sequestered CDK inhibitors, p27Kip1 and p21Cip1, and association of these inhibitors with cyclin E/CDK2 complexes. The metformin-resistant cell line MDA-MB-231 expresses significantly lower levels of p27Kip1 and p21Cip1 than the metformin-sensitive cell line, MCF7. When p27Kip1 or p21Cip1 were overexpressed in MDA-MB-231, the cells became sensitive to cell cycle arrest in response to metformin. Conclusion Cell cycle arrest in response to metformin requires CDK inhibitors in addition to AMPK activation and cyclin D1 downregulation. This is of interest because many cancers are associated with loss or downregulation of CDK inhibitors and the results may be relevant to the development of anti-tumor reagents that target the AMPK pathway. PMID:19046439

  9. Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase

    PubMed Central

    Yu, Lu; Qi, Maosong; Sheff, Mark A.

    2008-01-01

    Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis. PMID:18256288

  10. p21 blocks irradiation-induced apoptosis downstream of mitochondria by inhibition of cyclin-dependent kinase-mediated caspase-9 activation.

    PubMed

    Sohn, Dennis; Essmann, Frank; Schulze-Osthoff, Klaus; Jänicke, Reiner U

    2006-12-01

    The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria. PMID:17145870

  11. Both SCF(Cdc4alpha) and SCF(Cdc4gamma) are required for cyclin E turnover in cell lines that do not overexpress cyclin E.

    PubMed

    Sangfelt, Olle; Cepeda, Diana; Malyukova, Alena; van Drogen, Frank; Reed, Steven I

    2008-04-15

    The ubiquitin-mediated turnover of cyclin E is regulated by phosphorylation and the activity of the ubiquitin ligase SCF(Cdc4) (also known as SCF(Fbw7)). In 293A cells, SCF complexes containing two different Cdc4 isoforms, alpha and gamma, are required for efficient cyclin E ubiquitylation. Whereas SCF(Cdc4gamma) ubiquitylates cyclin E directly, SCF(Cdc4alpha) serves as a cofactor for Pin1-mediated prolyl isomerization of the cyclin E phosphodegron, essential to potentiate ubiquitylation. In the current study, we show that the requirement for both Cdc4alpha and gamma is general, except in cell lines where cyclin E is expressed at an elevated level. Under these circumstances, Cdc4alpha is sufficient for cyclin E turnover. Furthermore, the requirement for Cdc4gamma can be bypassed by ectopic overexpression of cyclin E. PMID:18414042

  12. Interactions between human cyclin T, Tat, and the transactivation response element (TAR) are disrupted by a cysteine to tyrosine substitution found in mouse cyclin T

    PubMed Central

    Fujinaga, Koh; Taube, Ran; Wimmer, Jörg; Cujec, Thomas P.; Peterlin, B. Matija

    1999-01-01

    The transcriptional transactivator Tat from HIV binds to the transactivation response element (TAR) RNA to increase rates of elongation of viral transcription. Human cyclin T supports these interactions between Tat and TAR. In this study, we report the sequence of mouse cyclin T and identify the residues from positions 1 to 281 in human cyclin T that bind to Tat and TAR. Mouse cyclin T binds to Tat weakly and is unable to facilitate interactions between Tat and TAR. Reciprocal exchanges of the cysteine and tyrosine at position 261 in human and mouse cyclin T proteins also render human cyclin T inactive and mouse cyclin T active. These findings reveal the molecular basis for the restriction of Tat transactivation in rodent cells. PMID:9990016

  13. Role of the mTORC1 Complex in Satellite Cell Activation by RNA-Induced Mitochondrial Restoration: Dual Control of Cyclin D1 through MicroRNAs

    PubMed Central

    Jash, Sukanta; Dhar, Gunjan; Ghosh, Utpalendu

    2014-01-01

    During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP-activated protein kinase (AMPK) and the mammalian Target of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3′ untranslated region (UTR) of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis. PMID:25047835

  14. Cyclin-dependent kinase-5 and p35/p25 activators in schizophrenia and major depression prefrontal cortex: basal contents and effects of psychotropic medications.

    PubMed

    Ramos-Miguel, Alfredo; Meana, J Javier; García-Sevilla, Jesús A

    2013-04-01

    Cyclin-dependent kinase-5 (CDK5) and p35/p25 activators, interacting with the exocytotic machinery (e.g. munc18-1 and syntaxin-1A), play critical roles in neurosecretion. The basal status of CDK5/p35/p25 and the effect of psychotropic drugs (detected in blood/urine samples) were investigated in post-mortem prefrontal cortex (PFC)/Brodmann's area 9 of schizophrenia (SZ) and major depression (MD) subjects. In SZ (all subjects, n = 24), CDK5 and p35, but not p25, were reduced (-28 to -58%) compared to controls. In SZ antipsychotic-free (n = 12), activator p35 was decreased (-52%). In SZ antipsychotic-treated (n = 12), marked reductions of CDK5 (-47%), p35 (-76%) and p25 (-36%) were quantified. In MD (n = 13), including antidepressant-free/treated subgroups, CDK5, p35 and p25 were unaltered. In SZ (n = 24), CDK5, p35 or p25 correlated with munc18-1a, but not with syntaxin-1A. The results demonstrate reduced p35 basal content and down-regulation of CDK5/p35/p25 by antipsychotics in SZ. The suggested CDK5/munc18-1a functional interaction may lead to dysregulated neurosecretion in SZ PFC. PMID:22964075

  15. BAI, a novel cyclin-dependent kinase inhibitor induces apoptosis in A549 cells through activation of caspases and inactivation of Akt.

    PubMed

    Kim, Shin; Lee, Jinho; Jang, Byeong-Churl; Kwon, Taeg Kyu; Park, Jong-Wook

    2013-02-01

    Previously, we have synthesized a novel cyclin-dependent kinase (CDK) inhibitor, 2-[1,1'biphenyl]-4-yl-N-[5-(1,1-dioxo-1λ(6) -isothiazolidin-2-yl)-1H-indazol-3-yl]acetamide (BAI) and reported its anti-cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20-60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti-cancer potency. We show that BAI induced a dose-dependent apoptotic cell death in these human cancer cells, as measured by fluorescence-activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20 nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C-γ1 (PLC-γ1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z-VAD-fmk, a pan caspase inhibitor strongly blocked the BAI-induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI-induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI-induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt. PMID:22887215

  16. ATM is required for rapid degradation of cyclin D1 in response to {gamma}-irradiation

    SciTech Connect

    Choo, Dong Wan; Baek, Hye Jung; Motoyama, Noboru; Cho, Kwan Ho; Kim, Hye Sun; Kim, Sang Soo

    2009-01-23

    The cellular response to DNA damage induced by {gamma}-irradiation activates cell-cycle arrest to permit DNA repair and to prevent replication. Cyclin D1 is the key molecule for transition between the G1 and S phases of the cell-cycle, and amplification or overexpression of cyclin D1 plays pivotal roles in the development of several human cancers. To study the regulation of cyclin D1 in the DNA-damaged condition, we analyzed the proteolytic regulation of cyclin D1 expression upon {gamma}-irradiation. Upon {gamma}-irradiation, a rapid reduction in cyclin D1 levels was observed prior to p53 stabilization, indicating that the stability of cyclin D1 is controlled in a p53-independent manner. Further analysis revealed that irradiation facilitated ubiquitination of cyclin D1 and that a proteasome inhibitor blocked cyclin D1 degradation under the same conditions. Interestingly, after mutation of threonine residue 286 of cyclin D1, which is reported to be the GSK-3{beta} phosphorylation site, the mutant protein showed resistance to irradiation-induced proteolysis although inhibitors of GSK-3{beta} failed to prevent cyclin D1 degradation. Rather, ATM inhibition markedly prevented cyclin D1 degradation induced by {gamma}-irradiation. Our data indicate that communication between ATM and cyclin D1 may be required for maintenance of genomic integrity achieved by rapid arrest of the cell-cycle, and that disruption of this crosstalk may increase susceptibility to cancer.

  17. Integrin signaling at the M/G1 transition induces expression of cyclin E.

    PubMed

    Hulleman, E; Bijvelt, J J; Verkleij, A J; Verrips, C T; Boonstra, J

    1999-12-15

    The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21(cip1/Waf1) and p27(Kip1) is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment. PMID:10585265

  18. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes*

    PubMed Central

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A.

    2015-01-01

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  19. Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes.

    PubMed

    Ramos Gomes, Fernanda; Romaniello, Vincenzo; Sánchez, Araceli; Weber, Claudia; Narayanan, Pratibha; Psol, Maryna; Pardo, Luis A

    2015-12-18

    KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy. PMID:26518875

  20. Activation of the p38 MAPK/Akt/ERK1/2 signal pathways is required for the protein stabilization of CDC6 and cyclin D1 in low-dose arsenite-induced cell proliferation.

    PubMed

    Liu, Youhong; Hock, Janet M; Sullivan, Con; Fang, Geying; Cox, Allison J; Davis, Kathleen T; Davis, Bruce H; Li, Xiong

    2010-12-15

    Arsenic trioxide (ATO) is a first-line anti-cancer agent for acute promyelocytic leukemia, and induces apoptosis in other solid cancer cell lines including breast cancer cells. However, as with arsenites found in drinking water and used as raw materials for wood preservatives, insecticides, and herbicides, low doses of ATO can induce carcinogenesis after long-term exposure. At 24 h after exposure, ATO (0.01-1 µM) significantly increased cell proliferation and promoted cell cycle progression from the G1 to S/G2 phases in the non-tumorigenic MCF10A breast epithelial cell line. The expression of 14 out of 96 cell-cycle-associated genes significantly increased, and seven of these genes including cell division cycle 6 (CDC6) and cyclin D1 (CCND1) were closely related to cell cycle progression from G1 to S phase. Low-dose ATO steadily increased gene transcript and protein levels of both CDC6 and cyclin D1 in a dose- and time-dependent manner. Low-dose ATO produced reactive oxygen species (ROS), and activated the p38 MAPK, Akt, and ERK1/2 pathways at different time points within 60 min. Small molecular inhibitors and siRNAs inhibiting the activation of p38 MAPK, Akt, and ERK1/2 decreased the ATO-increased expression of CDC6 protein. Inhibiting the activation of Akt and ERK1/2, but not p38 MAPK, decreased the ATO-induced expression of cyclin D1 protein. This study reports for the first time that p38 MAPK/Akt/ERK1/2 activation is required for the protein stabilization of CDC6 in addition to cyclin D1 in ATO-induced cell proliferation and cell cycle modulation from G1 to S phase. PMID:20862710

  1. MiR-23b Regulates CDK-activating Kinase complex through Cyclin H Repression to Modulate Endothelial Transcription and Growth under Flow

    PubMed Central

    Wang, Kuei-Chun; Nguyen, Phu; Weiss, Anna; Yeh, Yi-Ting; Chien, Hou Su; Lee, Alicia; Teng, Dayu; Subramaniam, Shankar; Li, Yi-Shuan; Chien, Shu

    2014-01-01

    Objective The site-specificity of endothelial phenotype is attributable to the local hemodynamic forces. The flow regulation of microRNAs (miRNAs) in endothelial cells (ECs) plays a significant role invascular homeostasis and diseases. The objective of this study is to elucidate the molecular mechanism by which the pulsatile shear flow (PS)-induced miR-23b exerts anti-proliferative effects on ECs. Approach and Results We used a combination of a cell perfusion system and experimental animals to examine the flow regulation of miR-23b in modulating EC proliferation. Our results demonstrated that PS induces the transcription factor KLF2 to promote miR-23b biosynthesis; the increase in miR-23b then represses cyclin H to impair the activity and integrity of CDK-activating kinase complex (CAK). The inhibitory effect of miR-23b on CAK exerts dual actions to (1) suppress cell cycle progression, and (2) reduce basal transcription by deactivating RNA polymerase II. While PS regulates the miR-23b/CAK pathway to exert anti-proliferative effects on ECs, oscillatory shear flow (OS) has little effect on the miR-23b/CAK pathway and hence does not cause EC growth arrest. Such flow pattern-dependent phenomena are validated with an in vivo model on rat carotid artery: the flow disturbance induced by partial carotid ligation led to a lower expression of miR-23b and a higher EC proliferation in comparison to the pulsatile flow regions of the unligated vessels. Local delivery of miR-23b mitigated the proliferative EC phenotype in partially ligated vessels. Conclusions Our findings unveil a novel mechanism by which hemodynamic forces modulate EC proliferative phenotype through the miR-23b/CAK pathway. PMID:24855060

  2. Mechanisms and regulation of the degradation of cyclin B.

    PubMed Central

    Hershko, A

    1999-01-01

    The degradation of the cyclin B subunit of protein kinase Cdk1/cyclin B is required for inactivation of the kinase and exit from mitosis. Cyclin B is degraded by the ubiquitin pathway, a system involved in most selective protein degradation in eukaryotic cells. In this pathway, proteins are targeted for degradation by ligation to ubiquitin, a process carried out by the sequential action of three enzymes: the ubiquitin-activating enzyme E1, a ubiquitin-carrier protein E2 and a ubiquitin-protein ligase E3. In the system responsible for cyclin B degradation, the E3-like function is carried out by a large complex called cyclosome or anaphase-promoting complex (APC). In the early embryonic cell cycles, the cyclosome is inactive in the interphase, but becomes active at the end of mitosis. Activation requires phosphorylation of the cyclosome/APC by protein kinase Cdk1/cyclin B. The lag kinetics of cyclosome activation may be explained by Suc1-assisted multiple phosphorylations of partly phosphorylated complex. The presence of a Fizzy/Cdc20-like protein is necessary for maximal activity of the mitotic form of cyclosome/APC in cyclin-ubiquitin ligation. PMID:10582242

  3. MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: Correlation with cyclin D1 expression.

    PubMed

    Huth, Hugo W; Albarnaz, Jonas D; Torres, Alice A; Bonjardim, Claudio A; Ropert, Catherine

    2016-09-01

    The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis where MEK2 was essential for the phosphorylation of MKK3/MKK6 and p38 MAPK that directly impacted on cyclin D1 expression. Importantly, the MEK1 inhibitor PD98059, like MEK1 silencing, induced an augment of cyclin D1 expression that correlated with an increase of MDA-MB-231 cell proliferation suggesting that MEK1 may play a regulatory role in these cells. In sum, the crucial role of MEK2 in MDA-MB-231 cell viability and the unknown relationship between MEK2 and MKK3/MKK6-p38 axis here revealed may open new therapeutic strategies for aggressive breast cancer. PMID:27181679

  4. The kinetics of G2 and M transitions regulated by B cyclins.

    PubMed

    Huang, Yehong; Sramkoski, R Michael; Jacobberger, James W

    2013-01-01

    B cyclins regulate G2-M transition. Because human somatic cells continue to cycle after reduction of cyclin B1 (cycB1) or cyclin B2 (cycB2) by RNA interference (RNAi), and because cycB2 knockout mice are viable, the existence of two genes should be an optimization. To explore this idea, we generated HeLa BD™ Tet-Off cell lines with inducible cyclin B1- or B2-EGFP that were RNAi resistant. Cultures were treated with RNAi and/or doxycycline (Dox) and bromodeoxyuridine. We measured G2 and M transit times and 4C cell accumulation. In the absence of ectopic B cyclin expression, knockdown (kd) of either cyclin increased G2 transit. M transit was increased by cycB1 kd but decreased by cycB2 depletion. This novel difference was further supported by time-lapse microscopy. This suggests that cycB2 tunes mitotic timing, and we speculate that this is through regulation of a Golgi checkpoint. In the presence of endogenous cyclins, expression of active B cyclin-EGFPs did not affect G2 or M phase times. As previously shown, B cyclin co-depletion induced G2 arrest. Expression of either B cyclin-EGFP completely rescued knockdown of the respective endogenous cyclin in single kd experiments, and either cyclin-EGFP completely rescued endogenous cyclin co-depletion. Most of the rescue occurred at relatively low levels of exogenous cyclin expression. Therefore, cycB1 and cycB2 are interchangeable for ability to promote G2 and M transition in this experimental setting. Cyclin B1 is thought to be required for the mammalian somatic cell cycle, while cyclin B2 is thought to be dispensable. However, residual levels of cyclin B1 or cyclin B2 in double knockdown experiments are not sufficient to promote successful mitosis, yet residual levels are sufficient to promote mitosis in the presence of the dispensible cyclin B2. We discuss a simple model that would explain most data if cyclin B1 is necessary. PMID:24324638

  5. A novel partner for D-type cyclins: protein kinase A-anchoring protein AKAP95.

    PubMed Central

    Arsenijevic, Tatjana; Degraef, Chantal; Dumont, Jacques E; Roger, Pierre P; Pirson, Isabelle

    2004-01-01

    Using a yeast interaction screen to search for proteins that interact with cyclin D3 in thyroid gland, we identified the cAMP-dependent AKAP95 (protein kinase A-anchoring protein 95). AKAP95 is a scaffolding protein that primarily co-fractionates with the nuclear matrix, whereas a minor fraction associates with chromatin in interphase cells. In co-transfected Chinese-hamster ovary cells, AKAP95 strongly interacted with the three D-type cyclins, but not with CDK4 (cyclin-dependent kinase 4) or with p27kip1. CDK4 displaced the interaction between cyclin D3 and AKAP95, suggesting that AKAP95 could not be the elusive bridging adaptor between D-type cyclins and CDK4 or play a role in the regulation of cyclin D3-CDK4 activity. Interaction between endogenous AKAP95 and cyclin D3 or cyclin D1 was detected in canine thyrocytes, human fibroblasts and NIH-3T3 cells. As both AKAP95 and cyclins D were recently reported to associate with minichromosome maintenance proteins [Eide, Tasken, Carlson, Williams, Jahnsen, Tasken and Collas (2003) J. Biol. Chem. 278, 26750-26756; Gladden and Diehl (2003) J. Biol. Chem. 278, 9754-9760], we hypothesize that the interaction between AKAP95 and D-type cyclins might serve to facilitate the emerging regulatory role of cyclin D-CDK4 in the formation of the prereplication complex at the DNA replication origins. PMID:14641107

  6. Potent and cellularly active 4-aminoimidazole inhibitors of cyclin-dependent kinase 5/p25 for the treatment of Alzheimer's disease.

    PubMed

    Helal, Christopher J; Kang, Zhijun; Lucas, John C; Gant, Thomas; Ahlijanian, Michael K; Schachter, Joel B; Richter, Karl E G; Cook, James M; Menniti, Frank S; Kelly, Kristin; Mente, Scot; Pandit, Jay; Hosea, Natalie

    2009-10-01

    Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E. PMID:19700321

  7. D-type cyclins and G1 progression during liver development in the rat

    SciTech Connect

    Boylan, Joan M. . E-mail: Joan_Boylan@brown.edu; Gruppuso, Philip A. . E-mail: Philip_Gruppuso@brown.edu

    2005-05-13

    Initiation and progression through G1 requires the activity of signaling complexes containing cyclins (D- or E-type) and cyclin-dependent kinases (CDK4/6 and CDK2, respectively). We set out to identify the G1-phase cyclins and CDKs that are operative during late gestation liver development in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of the mitogenic signaling pathways that are functional in mature hepatocytes. RNase protection assay and Western immunoblotting indicated that cyclin D1 is expressed at similar levels in fetal and adult liver. When cyclin D1 was induced after partial hepatectomy, its predominant CDK-binding partner was CDK4. In contrast, cyclins D2 and D3 predominated in fetal liver and were complexed with both CDK4 and CDK6. Little CDK6 protein was expressed in quiescent or regenerating adult liver. Cyclins E1 and E2 were both transcriptionally up-regulated in fetal liver. Activity of complexes containing cyclins E1 and E2 was higher in fetal liver, as was content of the cell cycle regulator, Rb. In fetal liver, Rb was highly phosphorylated at both cyclin D- and cyclin E-dependent sites. In conclusion, liver development is associated with a switch from cyclin D2/D3-containing complexes to cyclin D1:CDK4 complexes. We speculate that the switch in D-type cyclins may be associated with the dependence on mitogenic signaling that develops as hepatocytes mature.

  8. Foci of cyclin A2 interact with actin and RhoA in mitosis

    PubMed Central

    Loukil, Abdelhalim; Izard, Fanny; Georgieva, Mariya; Mashayekhan, Shaereh; Blanchard, Jean-Marie; Parmeggiani, Andrea; Peter, Marion

    2016-01-01

    Cyclin A2 is a key player in the regulation of the cell cycle. Its degradation in mid-mitosis depends primarily on the ubiquitin-proteasome system (UPS), while autophagy also contributes. However, a fraction of cyclin A2 persists beyond metaphase. In this work, we focus on cyclin A2-rich foci detected in mitosis by high resolution imaging and analyse their movements. We demonstrate that cyclin A2 interacts with actin and RhoA during mitosis, and that cyclin A2 depletion induces a dramatic decrease in active RhoA in mitosis. Our data suggest cyclin A2 participation in RhoA activation in late mitosis. PMID:27279564

  9. Silymarin induces cyclin D1 proteasomal degradation via its phosphorylation of threonine-286 in human colorectal cancer cells.

    PubMed

    Eo, Hyun Ji; Park, Gwang Hun; Song, Hun Min; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Man Hyo; Lee, Jeong Rak; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells. PMID:25479723

  10. Hepatitis B Virus X Protein Stabilizes Cyclin D1 and Increases Cyclin D1 Nuclear Accumulation through ERK-Mediated Inactivation of GSK-3β.

    PubMed

    Chen, Xiangmei; Zhang, Ling; Zheng, Sujun; Zhang, Ting; Li, Meng; Zhang, Xiaolei; Zeng, Zhenzhen; McCrae, Malcolm A; Zhao, Jingmin; Zhuang, Hui; Lu, Fengmin

    2015-05-01

    The Hepatitis B virus X protein (HBx) contributes centrally to the pathogenesis of hepatocellular carcinoma (HCC). It has been suggested that the transcriptional activation of cyclin D1 by HBx is implicated in the development of HCC. However, numerous studies have shown that overexpression of cyclin D1 alone is not sufficient to drive oncogenic transformation. Herein, we investigated whether HBx can stabilize cyclin D1 and induce cyclin D1 protein nuclear accumulation, and thereby accelerate hepatocarcinogenesis. The effects of HBx on cyclin D1 stabilization were assessed in cell-based transfection, Western blot, immunoprecipitation, immunocytofluorescence staining, and flow-cytometric assays. The results demonstrated that ectopic expression of HBx in HCC cells could extend the half-life of cyclin D1 protein from 40-60 minutes to 80-110 minutes. HBx stabilized cyclin D1 primarily in the S phase of the cell cycle, in a manner dependent on the inactivation of GSK-3β, which was mediated by ERK activation. HBx also prompted the nuclear accumulation of cyclin D1, and cotransfection of the constitutively active mutant of GSK-3β along with HBx could reverse the nuclear accumulation and subsequent cell proliferation induced by HBx. Further, a positive correlation between HBx and nuclear cyclin D1 level was established in HCC specimens detected by an immunohistochemical assay. Taken together, our results indicated that HBx could stabilize and increase cyclin D1 nuclear accumulation through ERK-mediated inactivation of GSK-3β. This HBx-induced cyclin D1 upregulation might play an important role in HCC development and progression. PMID:25712050

  11. Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6-methoxyphenyl)methanone (R547), A Potent and Selective Cyclin-Dependent Kinase Inhibitor with Significiant in Vivo Antitumor Activity

    SciTech Connect

    Chu,X.; DePinto, W.; Bartkovitz, D.; So, S.; Vu, B.; Packman, K.; Lukacs, C.; Ding, Q.; Jiang, N.; et al.

    2006-01-01

    The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K{sub i} > 10 {mu}M). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K{sub i} = 0.001, 0.003, and 0.001 M for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC{sub 50} = 0.08 {mu}M). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

  12. Cytoplasmic sequestration of cyclin D1 associated with cell cycle withdrawal of neuroblastoma cells

    SciTech Connect

    Sumrejkanchanakij, Piyamas; Eto, Kazuhiro; Ikeda, Masa-Aki . E-mail: mikeda.emb@tmd.ac.jp

    2006-02-03

    The regulation of D-type cyclin-dependent kinase activity is critical for neuronal differentiation and apoptosis. We recently showed that cyclin D1 is sequestered in the cytoplasm and that its nuclear localization induces apoptosis in postmitotic primary neurons. Here, we further investigated the role of the subcellular localization of cyclin D1 in cell cycle withdrawal during the differentiation of N1E-115 neuroblastoma cells. We show that cyclin D1 became predominantly cytoplasmic after differentiation. Targeting cyclin D1 expression to the nucleus induced phosphorylation of Rb and cdk2 kinase activity. Furthermore, cyclin D1 nuclear localization promoted differentiated N1E-115 cells to reenter the cell cycle, a process that was inhibited by p16{sup INK4a}, a specific inhibitor of D-type cyclin activity. These results indicate that cytoplasmic sequestration of cyclin D1 plays a role in neuronal cell cycle withdrawal, and suggests that the abrogation of machinery involved in monitoring aberrant nuclear cyclin D1 activity contributes to neuronal tumorigenesis.

  13. Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability

    SciTech Connect

    Zhang, Jingjie; Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wang, York; Li, Xuejun; Huang, Chuanshu

    2012-09-01

    Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95 h to 2.57 h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells. -- Highlights: ► SAHA inhibits cell transformation in Cl41 cells. ► SAHA suppresses Cyclin D1 protein expression. ► SAHA decreases cyclin D1 mRNA stability.

  14. A novel function for Cyclin A2: Control of cell invasion via RhoA signaling

    PubMed Central

    Arsic, Nikola; Bendris, Nawal; Peter, Marion; Begon-Pescia, Christina; Rebouissou, Cosette; Gadéa, Gilles; Bouquier, Nathalie; Bibeau, Frédéric

    2012-01-01

    Cyclin A2 plays a key role in cell cycle regulation. It is essential in embryonic cells and in the hematopoietic lineage yet dispensable in fibroblasts. In this paper, we demonstrate that Cyclin A2–depleted cells display a cortical distribution of actin filaments and increased migration. These defects are rescued by restoration of wild-type Cyclin A2, which directly interacts with RhoA, or by a Cyclin A2 mutant unable to associate with Cdk. In vitro, Cyclin A2 potentiates the exchange activity of a RhoA-specific guanine nucleotide exchange factor. Consistent with this, Cyclin A2 depletion enhances migration of fibroblasts and invasiveness of transformed cells via down-regulation of RhoA activity. Moreover, Cyclin A2 expression is lower in metastases relative to primary colon adenocarcinoma in matched human tumors. All together, these data show that Cyclin A2 negatively controls cell motility by promoting RhoA activation, thus demonstrating a novel Cyclin A2 function in cytoskeletal rearrangements and cell migration. PMID:22232705

  15. Characterization of a Pyrazolo[4,3-d]pyrimidine Inhibitor of Cyclin-Dependent Kinases 2 and 5 and Aurora A With Pro-Apoptotic and Anti-Angiogenic Activity In Vitro.

    PubMed

    Řezníčková, Eva; Weitensteiner, Sabine; Havlíček, Libor; Jorda, Radek; Gucký, Tomáš; Berka, Karel; Bazgier, Václav; Zahler, Stefan; Kryštof, Vladimír; Strnad, Miroslav

    2015-12-01

    Selective inhibitors of kinases that regulate the cell cycle, such as cyclin-dependent kinases (CDKs) and aurora kinases, could potentially become powerful tools for the treatment of cancer. We prepared and studied a series of 3,5,7-trisubstituted pyrazolo[4,3-d]pyrimidines, a new CDK inhibitor scaffold, to assess their CDK2 inhibitory and antiproliferative activities. A new compound, 2i, which preferentially inhibits CDK2, CDK5, and aurora A was identified. Both biochemical and cellular assays indicated that treatment with compound 2i caused the downregulation of cyclins A and B, the dephosphorylation of histone H3 at Ser10, and the induction of mitochondrial apoptosis in the HCT-116 colon cancer cell line. It also reduced migration as well as tube and lamellipodia formation in human endothelial cells. The kinase inhibitory profile of compound 2i suggests that its anti-angiogenic activity is linked to CDK5 inhibition. This dual mode of action involving apoptosis induction in cancer cells and the blocking of angiogenesis-like activity in endothelial cells offers possible therapeutic potential. PMID:26198005

  16. Rsf-1 is overexpressed in non-small cell lung cancers and regulates cyclinD1 expression and ERK activity

    SciTech Connect

    Li, Qingchang; Dong, Qianze; Wang, Enhua

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Rsf-1 expression is elevated in non-small cell lung cancers. Black-Right-Pointing-Pointer Rsf-1 depletion inhibits proliferation and increased apoptosis in lung cancer cells. Black-Right-Pointing-Pointer Rsf-1 depletion decreases the level of cyclinD1 and phosphor-ERK expression. -- Abstract: Rsf-1 (HBXAP) was recently reported to be overexpressed in various cancers and associated with the malignant behavior of cancer cells. However, the expression of Rsf-1 in primary lung cancer and its biological roles in non-small cell lung cancer (NSCLC) have not been reported. The molecular mechanism of Rsf-1 in cancer aggressiveness remains ambiguous. In the present study, we analyzed the expression pattern of Rsf-1 in NSCLC tissues and found that Rsf-1 was overexpressed at both the mRNA and protein levels. There was a significant association between Rsf-1 overexpression and TNM stage (p = 0.0220) and poor differentiation (p = 0.0013). Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression resulted in a decrease of colony formation ability and inhibition of cell cycle progression. Rsf-1 knockdown also induced apoptosis in these cell lines. Further analysis showed that Rsf-1 knockdown decreased cyclin D1 expression and phospho-ERK levels. In conclusion, Rsf-1 is overexpressed in NSCLC and contributes to malignant cell growth by cyclin D1 and ERK modulation, which makes Rsf-1 a candidate therapeutic target in lung cancer.

  17. Cyclin E Is Stabilized in Response to Replication Fork Barriers Leading to Prolonged S Phase Arrest*

    PubMed Central

    Lu, Xiaoyan; Liu, Jia; Legerski, Randy J.

    2009-01-01

    Cyclin E is a regulator of cyclin-dependent protein kinases (Cdks) and is involved in mediating the cell cycle transition from G1 to S phase. Here, we describe a novel function for cyclin E in the long term maintenance of checkpoint arrest in response to replication barriers. Exposure of cells to mitomycin C or UV irradiation, but not ionizing radiation, induces stabilization of cyclin E. Stabilization of cyclin E reduces the activity of Cdk2-cyclin A, resulting in a slowing of S phase progression and arrest. In addition, cyclin E is shown to be required for stabilization of Cdc6, which is required for activation of Chk1 and the replication checkpoint pathway. Furthermore, the stabilization of cyclin E in response to replication fork barriers depends on ATR, but not Nbs1 or Chk1. These results indicate that in addition to its well studied role in promoting cell cycle progression, cyclin E also has a role in regulating cell cycle arrest in response to DNA damage. PMID:19812034

  18. Salusin-β induces smooth muscle cell proliferation by regulating cyclins D1 and E expression through MAPKs signaling pathways.

    PubMed

    Xu, Xiao-Le; Zeng, Yi; Zhao, Cheng; He, Meng-Zi; Wang, Fei; Zhang, Wei

    2015-04-01

    Despite the clear mitogenic effect of salusin-β on vascular smooth muscle cells (VSMCs), which contributes to its proatherosclerotic effects, additional studies are needed to explore its underlying mechanisms. The aim of this study was to investigate the mechanism of salusin-β's effects on VSMCs cell cycle regulation and the possible signal pathways. Salusin-β accelerated the G1/S phase transition in VSMCs and increased the expression levels of cyclins D1 and E. Silencing either cyclin D1 or cyclin E gene inhibited salusin-β-induced VSMCs proliferation, cell cycle progression, phosphorylation of the Rb protein, and dissociation of the E2F-Rb complex. Importantly, expression of cyclin E was also induced by cyclin D1. Next, we found that salusin-β increased the protein expressions of activator protein-1 (AP-1) subunits c-Jun and c-Fos, and enhanced binding of AP-1 to the promoter region of cyclin D1. In addition, inhibition of AP-1 activity could lead to significant suppression of salusin-β-induced cyclin D1 expression. Furthermore, MPAKs pathways were found to mediate salusin-β-induced VSMCs proliferation, cyclin D1, cyclin E, c-Jun, and c-Fos expression. These results suggest that salusin-β promotes cell cycle progression of VSMCs by upregulating the cyclin D1 and cyclin E, in an AP-1-dependent manner through mitogen-activated protein kinases signaling pathways. PMID:25551321

  19. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

    SciTech Connect

    Sun Maoyun; Wei Yuanyan; Yao Luyang; Xie Jianhui; Chen Xiaoning; Wang Hanzhou; Jiang Jianhai; Gu Jianxin . E-mail: jxgu@shmu.edu.cn

    2006-02-03

    Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.

  20. A minimal cascade model for the mitotic oscillator involving cyclin and cdc2 kinase.

    PubMed Central

    Goldbeter, A

    1991-01-01

    A minimal model for the mitotic oscillator is presented. The model, built on recent experimental advances, is based on the cascade of post-translational modification that modulates the activity of cdc2 kinase during the cell cycle. The model pertains to the situation encountered in early amphibian embryos, where the accumulation of cyclin suffices to trigger the onset of mitosis. In the first cycle of the bicyclic cascade model, cyclin promotes the activation of cdc2 kinase through reversible dephosphorylation, and in the second cycle, cdc2 kinase activates a cyclin protease by reversible phosphorylation. That cyclin activates cdc2 kinase while the kinase triggers the degradation of cyclin has suggested that oscillations may originate from such a negative feedback loop [Félix, M. A., Labbé, J. C., Dorée, M., Hunt, T. & Karsenti, E. (1990) Nature (London) 346, 379-382]. This conjecture is corroborated by the model, which indicates that sustained oscillations of the limit cycle type can arise in the cascade, provided that a threshold exists in the activation of cdc2 kinase by cyclin and in the activation of cyclin proteolysis by cdc2 kinase. The analysis shows how miototic oscillations may readily arise from time lags associated with these thresholds and from the delayed negative feedback provided by cdc2-induced cyclin degradation. A mechanism for the origin of the thresholds is proposed in terms of the phenomenon of zero-order ultrasensitivity previously described for biochemical systems regulated by covalent modification. PMID:1833774

  1. Cytotoxicity of diacetoxyscirpenol is associated with apoptosis by activation of caspase-8 and interruption of cell cycle progression by down-regulation of cdk4 and cyclin B1 in human Jurkat T cells

    SciTech Connect

    Jun, Do Youn; Kim, Jun Seok; Park, Hae Sun; Song, Woo Sun; Bae, Young Seuk; Kim, Young Ho . E-mail: ykim@knu.ac.kr

    2007-07-15

    To understand the mechanism underlying T-cell toxicity of diacetoxyscirpenol (DAS) from Fusarium sambucinum, its apoptogenic as well as growth retardation activity was investigated in human Jurkat T cells. Exposure to DAS (0.01-0.15 {mu}M) caused apoptotic DNA fragmentation along with caspase-8 activation, Bid cleavage, mitochondrial cytochrome c release, activation of caspase-9 and caspase-3, and PARP degradation, without any alteration in the levels of Fas or FasL. Under these conditions, necrosis was not accompanied. The cytotoxicity of DAS was not blocked by the anti-Fas neutralizing antibody ZB-4. Although the DAS-induced apoptotic events were completely prevented by overexpression of Bcl-xL, the cells overexpressing Bcl-xL were unable to divide in the presence of DAS, resulting from the failure of cell cycle progression possibly due to down-regulation in the protein levels of cdk4 and cyclin B1. The DAS-mediated apoptosis and activation of caspase-8, -9, and -3 were abrogated by either pan-caspase inhibitor (z-VAD-fmk) or caspase-8 inhibitor (z-IETD-fmk). While the DAS-mediated apoptosis and activation of caspase-9 and caspase-3 were slightly suppressed by the mitochondrial permeability transition pore inhibitor (CsA), both caspase-8 activation and Bid cleavage were not affected by CsA. The activated normal peripheral T cells possessed a similar susceptibility to the cytotoxicity of DAS. These results demonstrate that the T-cell toxicity of DAS is attributable to not only apoptosis initiated by caspase-8 activation and subsequent mitochondrion-dependent or -independent activation of caspase cascades, which can be regulated by Bcl-xL, but also interruption of cell cycle progression caused by down-regulation of cdk4 and cyclin B1 proteins.

  2. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    SciTech Connect

    Woo, Sang Hyeok; Seo, Sung-Keum; An, Sungkwan; Choe, Tae-Boo; Hong, Seok-Il; Lee, Yun-Han; Park, In-Chul

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  3. Interactions between Equine Cyclin T1, Tat, and TAR Are Disrupted by a Leucine-to-Valine Substitution Found in Human Cyclin T1

    PubMed Central

    Taube, Ran; Fujinaga, Koh; Irwin, Dan; Wimmer, Jörg; Geyer, Matthias; Peterlin, B. Matija

    2000-01-01

    Transcriptional transactivators (Tat) from human immunodeficiency and equine infectious anemia viruses (HIV and EIAV) interact with their transactivation response elements (TAR) to increase the rates of viral transcription. Whereas the human cyclin T1 is required for the binding of Tat to TAR from HIV, it is unknown how Tat from EIAV interacts with its TAR. Furthermore, Tat from EIAV functions in equine and canine cells but not in human cells. In this study, we present sequences of cyclins T1 from horse and dog and demonstrate that their N-terminal 300 residues rescue the transactivation of Tat from EIAV in human cells. Although human and equine cyclins T1 bind to this Tat, only the equine cyclin T1 supports the binding of Tat to TAR from EIAV. Finally, a reciprocal exchange of the valine for the leucine at position 29 in human and equine cyclins T1, respectively, renders the human cyclin T1 active and the equine cyclin T1 inactive for Tat transactivation from EIAV. Thus, the collaboration between a specific cyclin T1 and Tat for their high-affinity interaction with TAR is a common theme of lentiviral transactivation. PMID:10623752

  4. Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen

    SciTech Connect

    Wang Jianling; Wang Gangduo; Ma Huaxian; Khan, M. Firoze

    2011-01-15

    Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in

  5. E2-C, a cyclin-selective ubiquitin carrier protein required for the destruction of mitotic cyclins.

    PubMed Central

    Aristarkhov, A; Eytan, E; Moghe, A; Admon, A; Hershko, A; Ruderman, J V

    1996-01-01

    Ubiquitin-dependent proteolysis of the mitotic cyclins A and B is required for the completion of mitosis and entry into the next cell cycle. This process is catalyzed by the cyclosome, an approximately 22S particle that contains a cyclin-selective ubiquitin ligase activity, E3-C, that requires a cyclin-selective ubiquitin carrier protein (UBC) E2-C. Here we report the purification and cloning of E2-C from clam oocytes. The deduced amino acid sequence of E2-C indicates that it is a new UBC family member. Bacterially expressed recombinant E2-C is active in in vitro cyclin ubiquitination assays, where it exhibits the same substrate specificities seen with native E2-C. These results demonstrate that E2-C is not a homolog of UBC4 or UBC9, proteins previously suggested to be involved in cyclin ubiquitination, but is a new UBC family member with unique properties. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8633058

  6. Inhibition of cyclin-dependent kinases by p21.

    PubMed Central

    Harper, J W; Elledge, S J; Keyomarsi, K; Dynlacht, B; Tsai, L H; Zhang, P; Dobrowolski, S; Bai, C; Connell-Crowley, L; Swindell, E

    1995-01-01

    p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression. Images PMID:7626805

  7. A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

    PubMed Central

    Jaspersen, Sue L.; Charles, Julia F.; Tinker-Kulberg, Rachel L.; Morgan, David O.

    1998-01-01

    Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1. PMID:9763445

  8. Tumor-Specific Proteolytic Processing of Cyclin E Generates Hyperactive Lower-Molecular-Weight Forms

    PubMed Central

    Porter, Donald C.; Zhang, Ning; Danes, Christopher; McGahren, Mollianne J.; Harwell, Richard M.; Faruki, Shamsa; Keyomarsi, Khandan

    2001-01-01

    Cyclin E is a G1 cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G1-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G2/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein. PMID:11509668

  9. The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex.

    PubMed Central

    Sterner, D E; Lee, J M; Hardin, S E; Greenleaf, A L

    1995-01-01

    Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro. PMID:7565723

  10. miR-340 inhibits glioblastoma cell proliferation by suppressing CDK6, cyclin-D1 and cyclin-D2

    SciTech Connect

    Li, Xuesong; Gong, Xuhai; Chen, Jing; Zhang, Jinghui; Sun, Jiahang; Guo, Mian

    2015-05-08

    Glioblastoma development is often associated with alteration in the activity and expression of cell cycle regulators, such as cyclin-dependent kinases (CKDs) and cyclins, resulting in aberrant cell proliferation. Recent studies have highlighted the pivotal roles of miRNAs in controlling the development and growth of glioblastoma. Here, we provide evidence for a function of miR-340 in the inhibition of glioblastoma cell proliferation. We found that miR-340 is downregulated in human glioblastoma tissue samples and several established glioblastoma cell lines. Proliferation and neurosphere formation assays revealed that miR-340 plays an oncosuppressive role in glioblastoma, and that its ectopic expression causes significant defect in glioblastoma cell growth. Further, using bioinformatics, luciferase assay and western blot, we found that miR-340 specifically targets the 3′UTRs of CDK6, cyclin-D1 and cyclin-D2, leading to the arrest of glioblastoma cells in the G0/G1 cell cycle phase. Confirming these results, we found that re-introducing CDK6, cyclin-D1 or cyclin-D2 expression partially, but significantly, rescues cells from the suppression of cell proliferation and cell cycle arrest mediated by miR-340. Collectively, our results demonstrate that miR-340 plays a tumor-suppressive role in glioblastoma and may be useful as a diagnostic biomarker and/or a therapeutic avenue for glioblastoma. - Highlights: • miR-340 is downregulated in glioblastoma samples and cell lines. • miR-340 inhibits glioblastoma cell proliferation. • miR-340 directly targets CDK6, cyclin-D1, and cyclin-D2. • miR-340 regulates glioblastoma cell proliferation via CDK6, cyclin-D1 and cyclin-D2.

  11. 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. PMID:26115571

  12. Identification of E2F-1/Cyclin A antagonists.

    PubMed

    Sharma, S K; Ramsey, T M; Chen, Y N; Chen, W; Martin, M S; Clune, K; Sabio, M; Bair, K W

    2001-09-17

    A simple method for the synthesis of a rationally designed (S,S)-[Pro-Leu]-spirolactam scaffold is described. This was expanded to a small biased library of compounds mimicking the 'ZRXL' motif in order to identify E2F-1/Cyclin A antagonists. The synthesized compounds were evaluated in an E2F-1/Cyclin A binding assay and moderately active analogues were identified. In addition, the critical roles of Phe, Leu, Lys, and Arg residues of the identified motif were determined. PMID:11549444

  13. Cyclin D1 blocks the anti-proliferative function of RUNX3 by interfering with RUNX3-p300 interaction

    SciTech Connect

    Iwatani, Kazunori; Fujimoto, Tetsuhiro; Ito, Takaaki

    2010-09-24

    Research highlights: {yields} Cyclin D1 interacts with RUNX3 and inhibits the interaction and collaboration of RUNX3 with coactivator p300. {yields} Cyclin D1 blocks the ability of RUNX3 to induce the expression of cdk inhibitor p21. {yields} Cyclin D1 releases cancer cells from the inhibition of proliferation induced by RUNX3. -- Abstract: Transcriptional function of cyclin D1, whose deregulation is frequently observed in human cancers, has been suggested to contribute to cancer formation. In the present study, we show that cyclin D1 protein inhibits RUNX3 activity by directly binding to it and interfering with its interaction with p300 interaction in lung cancer cells. Cyclin D1 inhibits p300-dependent RUNX3 acetylation and negatively regulates cyclin-dependent kinase (cdk) inhibitor p21 expression. These transcriptional effects of cyclin D1 do not require cdk4/6 kinase activation. We propose that cyclin D1 provides a transcriptional switch that allows the tumor suppressor activity of RUNX3 to be repressed in cancer cells. Since RUNX3 plays tumor suppressive roles in a wide range of cancers, a non-canonical cyclin D1 function may be critical for neoplastic transformation of the epithelial cells in which RUNX3 regulates proliferation.

  14. Cyclin E overexpression impairs progression through mitosis by inhibiting APCCdh1

    PubMed Central

    Keck, Jamie M.; Summers, Matthew K.; Tedesco, Donato; Ekholm-Reed, Susanna; Chuang, Li-Chiou; Jackson, Peter K.; Reed, Steven I.

    2007-01-01

    Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APCCdh1) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APCCdh1 substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E–mediated mitotic phenotype. PMID:17664332

  15. Dietary myo-inositol modulates immunity through antioxidant activity and the Nrf2 and E2F4/cyclin signalling factors in the head kidney and spleen following infection of juvenile fish with Aeromonas hydrophila.

    PubMed

    Jiang, Wei-Dan; Hu, Kai; Liu, Yang; Jiang, Jun; Wu, Pei; Zhao, Juan; Zhang, Yong-An; Zhou, Xiao-Qiu; Feng, Lin

    2016-02-01

    This study was conducted to investigate the effects of the dietary vitamin myo-inositol (MI), on the immunity and structural integrity of the head kidney and spleen following infection of fish with the major freshwater pathogen bacterial Aeromonas hydrophila. The results demonstrated for the first time that MI deficiency depressed the lysozyme and acid phosphatase (ACP) activities and the complement 3 (C3) and C4 contents in the head kidney and spleen compared with the optimal MI levels, indicating that MI deficiency decreased the immunity of these important fish immune organs. The depression in immunity due to MI deficiency was partially related to oxidative damage [indicated by increases in the malondialdehyde (MDA) and protein carbonyl (PC) contents] that was in turn partially due to the decreased glutathione (GSH) content and the disturbances in antioxidant enzyme activities [total superoxide dismutase (T-SOD), CuZnSOD, MnSOD, catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)]. MI deficiency inhibited the antioxidant-related gene transcription [CuZnSOD, MnSOD, CAT, GPx1a, GR and NF-E2-related factor 2 (Nrf2)] in the head kidney and spleen following infection of the fish with A. hydrophila. The oxidative damage due to MI deficiency also resulted in the inhibition of proliferation-associated signalling (cyclin D1, cyclin A, cyclin E and E2F4). Thus, MI deficiency partially inhibited damage repair. Excessive MI exhibited negative effects that were similar to MI deficiency, whereas the optimal MI content reversed those indicators. These observations indicated that an MI deficiency or excess could cause depression of the immune system that might be partially related to oxidative damage, antioxidant disturbances, and the inhibition of the proliferation-associated signalling in the head kidney and spleen following infection of fish with A. hydrophila. Finally, the optimal MI levels were 660.7 (based on ACP) and 736.8 mg kg(-1) diet (based

  16. Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

    SciTech Connect

    Guo, Zheng; Zhou, Yuning; Evers, B. Mark; Wang, Qingding

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Rictor associates with FBXW7 to form an E3 complex. Black-Right-Pointing-Pointer Knockdown of rictor decreases ubiquitination of c-Myc and cylin E. Black-Right-Pointing-Pointer Knockdown of rictor increases protein levels of c-Myc and cylin E. Black-Right-Pointing-Pointer Overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Black-Right-Pointing-Pointer Rictor regulation of c-Myc and cyclin E requires FBXW7. -- Abstract: Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.

  17. Induced ICER I{gamma} down-regulates cyclin A expression and cell proliferation in insulin-producing {beta} cells

    SciTech Connect

    Inada, Akari; Weir, Gordon C.; Bonner-Weir, Susan . E-mail: susan.bonner-weir@joslin.harvard.edu

    2005-04-15

    We have previously found that cyclin A expression is markedly reduced in pancreatic {beta}-cells by cell-specific overexpression of repressor inducible cyclic AMP early repressor (ICER I{gamma}) in transgenic mice. Here we further examined regulatory effects of ICER I{gamma} on cyclin A gene expression using Min6 cells, an insulin-producing cell line. The cyclin A promoter luciferase assay showed that ICER I{gamma} directly repressed cyclin A gene transcription. In addition, upon ICER I{gamma} overexpression, cyclin A mRNA levels markedly decreased, thereby confirming an inhibitory effect of ICER I{gamma} on cyclin A expression. Suppression of cyclin A results in inhibition of BrdU incorporation. Under normal culture conditions endogenous cyclin A is abundant in these cells, whereas ICER is hardly detectable. However, serum starvation of Min6 cells induces ICER I{gamma} expression with a concomitant very low expression level of cyclin A. Cyclin A protein is not expressed unless the cells are in active DNA replication. These results indicate a potentially important anti-proliferative effect of ICER I{gamma} in pancreatic {beta} cells. Since ICER I{gamma} is greatly increased in diabetes as well as in FFA- or high glucose-treated islets, this effect may in part exacerbate diabetes by limiting {beta}-cell proliferation.

  18. Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm.

    PubMed

    Jackman, Mark; Kubota, Yumiko; den Elzen, Nicole; Hagting, Anja; Pines, Jonathon

    2002-03-01

    Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif. PMID:11907280

  19. The multiple roles of cyclin E1 in controlling cell cycle progression and cellular morphology of Trypanosoma brucei.

    PubMed

    Gourguechon, Stéphane; Savich, Jason M; Wang, Ching C

    2007-05-11

    Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro glutathione-S-transferase pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was impaired significantly only in cyclin E1+CRK1-depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if only CRK3 was depleted. Taken together, these data suggest multiple functions of cyclin E1: it forms a complex with CRK1 in promoting G1/S phase transition; it forms a complex with CRK2 in controlling the posterior morphogenesis during G1/S transition; and it forms a complex with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome. PMID:17376478

  20. THE MULTIPLE ROLES OF CYCLIN E1 IN CONTROLLING CELL CYCLE PROGRESSION AND CELLULAR MORPHOLOGY OF TRYPANOSOMA BRUCEI

    PubMed Central

    Gourguechon, Stéphane; Savich, Jason M.; Wang, Ching C.

    2009-01-01

    SUMMARY Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases. Previous RNA interference (RNAi) experiments in Trypanosoma brucei indicated that cyclin E1, cdc2-related kinase (CRK)1 and CRK2 are involved in regulating G1/S transition, whereas cyclin B2 and CRK3 play a pivotal role in controlling the G2/M checkpoint. To search for potential interactions between the other cyclins and CRKs that may not have been revealed by the RNAi assays, we used the yeast two-hybrid system and an in vitro GST pulldown assay and observed interactions between cyclin E1 and CRK1, CRK2 and CRK3. Cyclins E1-E4 are homologues of yeast Pho80 cyclin. But yeast complementation assays indicated that none of them possesses a Pho80-like function. Analysis of cyclin E1+CRK1 and cyclin E1+CRK2 double knockdowns in the procyclic form of T. brucei indicated that the cells were arrested more extensively in the G1 phase beyond the cumulative effect of individual knockdowns. But BrdU incorporation was significantly impaired only in cyclin E1+CRK1 depleted cells, whereas a higher percentage of cyclin E1+CRK2 knockdown cells assumed a grossly elongated posterior end morphology. A double knockdown of cyclin E1 and CRK3 arrested cells in G2/M much more efficiently than if CRK3 was depleted alone. Taken together, these data suggest multiple functions of cyclin E1. It forms a complex with CRK1 in promoting G1/S phase transition, with CRK2 in controlling the posterior morphogenesis during G1/S transition and with CRK3 in promoting passage across the G2/M checkpoint in the trypanosome. PMID:17376478

  1. Cyclin D1 expression in prostate carcinoma

    PubMed Central

    Pereira, R.A.; Ravinal, R.C.; Costa, R.S.; Lima, M.S.; Tucci, S.; Muglia, V.F.; Reis, R.B. Dos; Silva, G.E.B.

    2014-01-01

    The purpose of this study was to investigate the relationship between cyclin D1 expression and clinicopathological parameters in patients with prostate carcinoma. We assessed cyclin D1 expression by conventional immunohistochemistry in 85 patients who underwent radical prostatectomy for prostate carcinoma and 10 normal prostate tissue samples retrieved from autopsies. We measured nuclear immunostaining in the entire tumor area and based the results on the percentage of positive tumor cells. The preoperative prostate-specific antigen (PSA) level was 8.68±5.16 ng/mL (mean±SD). Cyclin D1 staining was positive (cyclin D1 expression in >5% of tumor cells) in 64 cases (75.4%) and negative (cyclin D1 expression in ≤5% of tumor cells) in 21 cases (including 15 cases with no immunostaining). Normal prostate tissues were negative for cyclin D1. Among patients with a high-grade Gleason score (≥7), 86% of patients demonstrated cyclin D1 immunostaining of >5% (P<0.05). In the crude analysis of cyclin D1 expression, the high-grade Gleason score group showed a mean expression of 39.6%, compared to 26.9% in the low-grade Gleason score group (P<0.05). Perineural invasion tended to be associated with cyclin D1 expression (P=0.07), whereas cyclin D1 expression was not associated with PSA levels or other parameters. Our results suggest that high cyclin D1 expression could be a potential marker for tumor aggressiveness. PMID:24820071

  2. Cyclin K and cyclin D1b are oncogenic in myeloma cells

    PubMed Central

    2010-01-01

    Background Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. Results To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. Conclusions Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions. PMID:20459741

  3. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain*

    PubMed Central

    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P.; Ahn, Jinwoo; Skowronski, Jacek

    2015-01-01

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. PMID:25847232

  4. CyclinA2-Cyclin-dependent Kinase Regulates SAMHD1 Protein Phosphohydrolase Domain.

    PubMed

    Yan, Junpeng; Hao, Caili; DeLucia, Maria; Swanson, Selene; Florens, Laurence; Washburn, Michael P; Ahn, Jinwoo; Skowronski, Jacek

    2015-05-22

    SAMHD1 is a nuclear deoxyribonucleoside triphosphate triphosphohydrolase that contributes to the control of cellular deoxyribonucleoside triphosphate (dNTP) pool sizes through dNTP hydrolysis and modulates the innate immune response to viruses. CyclinA2-CDK1/2 phosphorylates SAMHD1 at Thr-592, but how this modification controls SAMHD1 functions in proliferating cells is not known. Here, we show that SAMHD1 levels remain relatively unchanged during the cell division cycle in primary human T lymphocytes and in monocytic cell lines. Inactivation of the bipartite cyclinA2-CDK-binding site in the SAMHD1 C terminus described herein abolished SAMHD1 phosphorylation on Thr-592 during S and G2 phases thus interfering with DNA replication and progression of cells through S phase. The effects exerted by Thr-592 phosphorylation-defective SAMHD1 mutants were associated with activation of DNA damage checkpoint and depletion of dNTP concentrations to levels lower than those seen upon expression of wild type SAMHD1 protein. These disruptive effects were relieved by either mutation of the catalytic residues of the SAMHD1 phosphohydrolase domain or by a Thr-592 phosphomimetic mutation, thus linking the Thr-592 phosphorylation state to the control of SAMHD1 dNTPase activity. Our findings support a model in which phosphorylation of Thr-592 by cyclinA2-CDK down-modulates, but does not inactivate, SAMHD1 dNTPase in S phase, thereby fine-tuning SAMHD1 control of dNTP levels during DNA replication. PMID:25847232

  5. Cyclin D1-Cdk4 controls glucose metabolism independently of cell cycle progression.

    PubMed

    Lee, Yoonjin; Dominy, John E; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I; Puigserver, Pere

    2014-06-26

    Insulin constitutes a principal evolutionarily conserved hormonal axis for maintaining glucose homeostasis; dysregulation of this axis causes diabetes. PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) links insulin signalling to the expression of glucose and lipid metabolic genes. The histone acetyltransferase GCN5 (general control non-repressed protein 5) acetylates PGC-1α and suppresses its transcriptional activity, whereas sirtuin 1 deacetylates and activates PGC-1α. Although insulin is a mitogenic signal in proliferative cells, whether components of the cell cycle machinery contribute to its metabolic action is poorly understood. Here we report that in mice insulin activates cyclin D1-cyclin-dependent kinase 4 (Cdk4), which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high-throughput chemical screen, we identify a Cdk4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK-3β (glycogen synthase kinase 3-beta) signalling induces cyclin D1 protein stability by sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 messenger RNA transcripts. Activated cyclin D1-Cdk4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycaemia. In diabetic models, cyclin D1-Cdk4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycaemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division. PMID:24870244

  6. Crystal structure of human cyclin K, a positive regulator of cyclin-dependent kinase 9.

    PubMed

    Baek, Kyuwon; Brown, Raymond S; Birrane, Gabriel; Ladias, John A A

    2007-02-16

    Cyclin K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 A resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27(Kip1). Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes. PMID:17169370

  7. Crystal Structure of Human Cyclin K, a Positive Regulator of Cyclin-dependent Kinase 9

    PubMed Central

    Baek, Kyuwon; Brown, Raymond S.; Birrane, Gabriel; Ladias, John A.A.

    2007-01-01

    Summary Cyclin K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, collectively referred to as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 Å resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell cycle inhibitor p27Kip1. Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K as a template reveals that the two proteins have similar structures, as expected from their high sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9–cyclin K and CDK9–cyclin T1 complexes. PMID:17169370

  8. The proteolysis of mitotic cyclins in mammalian cells persists from the end of mitosis until the onset of S phase.

    PubMed

    Brandeis, M; Hunt, T

    1996-10-01

    We have studied how the cell cycle-specific oscillations of mitotic B-type cyclins are generated in mouse fibroblasts. A reporter enzyme comprising the N-terminus of a B-type cyclin fused to bacterial chloramphenicol acetyl transferase (CAT) was degraded at the end of mitosis like endogenous cyclins. Point mutations in the destruction box of this construct completely abolished its mitotic instability. When the destructible reporter was driven by the cyclin B2 promoter, CAT activity mimicked the oscillations in the level of the endogenous cyclin B2. These oscillations were largely conserved when the reporter was transcribed constitutively from the SV40 promoter. Pulse-chase experiments or addition of the proteasome inhibitors lactacystin and ALLN showed that cyclin synthesis continued after the end of mitosis. The destruction box-specific degradation of cyclins normally ceases at the onset of S phase, and is active in fibroblasts arrested in G0 and in differentiated C2 myoblasts. We were able to reproduce this proteolysis in vitro in extracts of synchronized cells. Extracts of G1 cells degraded cyclin B1 whereas p27Kip1 was stable, in contrast, cyclin B1 remained stable and p27Kip1 was degraded in extracts of S phase cells. PMID:8895573

  9. Anti-Proliferative Effect of Naringenin through p38-Dependent Downregulation of Cyclin D1 in Human Colorectal Cancer Cells

    PubMed Central

    Song, Hun Min; Park, Gwang Hun; Eo, Hyun Ji; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Jeong Rak; Lee, Man Hyo; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. However, more detailed mechanism by which NAR exerts anti-cancer properties still remains unanswered. Thus, in this study, we have shown that NAR down-regulates the level of cyclin D1 in human colorectal cancer cell lines, HCT116 and SW480. NAR inhibited the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. Inhibition of proteasomal degradation by MG132 blocked NAR-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with NAR. In addition, NAR increased the phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine blocked cyclin D1 downregulation by NAR. p38 inactivation attenuated cyclin D1 downregulation by NAR. From these results, we suggest that NAR-mediated cyclin D1 downregulation may result from proteasomal degradation through p38 activation. The current study provides new mechanistic link between NAR, cyclin D1 downregulation and cell growth in human colorectal cancer cells. PMID:26157550

  10. CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome.

    PubMed

    Guen, Vincent J; Gamble, Carly; Flajolet, Marc; Unger, Sheila; Thollet, Aurélie; Ferandin, Yoan; Superti-Furga, Andrea; Cohen, Pascale A; Meijer, Laurent; Colas, Pierre

    2013-11-26

    Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome. PMID:24218572

  11. Analysis of substrate specificity and cyclin Y binding of PCTAIRE-1 kinase

    PubMed Central

    Shehata, Saifeldin N.; Hunter, Roger W.; Ohta, Eriko; Peggie, Mark W.; Lou, Hua Jane; Sicheri, Frank; Zeqiraj, Elton; Turk, Benjamin E.; Sakamoto, Kei

    2012-01-01

    PCTAIRE-1 (cyclin-dependent kinase [CDK] 16) is a highly conserved serine/threonine kinase that belongs to the CDK family of protein kinases. Little is known regarding PCTAIRE-1 regulation and function and no robust assay exists to assess PCTAIRE-1 activity mainly due to a lack of information regarding its preferred consensus motif and the lack of bona fide substrates. We used positional scanning peptide library technology and identified the substrate-specificity requirements of PCTAIRE-1 and subsequently elaborated a peptide substrate termed PCTAIRE-tide. Recombinant PCTAIRE-1 displayed vastly improved enzyme kinetics on PCTAIRE-tide compared to a widely used generic CDK substrate peptide. PCTAIRE-tide also greatly improved detection of endogenous PCTAIRE-1 activity. Similar to other CDKs, PCTAIRE-1 requires a proline residue immediately C-terminal to the phosphoacceptor site (+ 1) for optimal activity. PCTAIRE-1 has a unique preference for a basic residue at + 4, but not at + 3 position (a key characteristic for CDKs). We also demonstrate that PCTAIRE-1 binds to a novel cyclin family member, cyclin Y, which increased PCTAIRE-1 activity towards PCTAIRE-tide > 100-fold. We hypothesised that cyclin Y binds and activates PCTAIRE-1 in a way similar to which cyclin A2 binds and activates CDK2. Point mutants of cyclin Y predicted to disrupt PCTAIRE-1-cyclin Y binding severely prevented complex formation and activation of PCTAIRE-1. We have identified PCTAIRE-tide as a powerful tool to study the regulation of PCTAIRE-1. Our understanding of the molecular interaction between PCTAIRE-1 and cyclin Y further facilitates future investigation of the functions of PCTAIRE-1 kinase. PMID:22796189

  12. Opposing action of estrogen receptors alpha and beta on cyclin D1 gene expression.

    PubMed

    Liu, Meng-Min; Albanese, Chris; Anderson, Carol M; Hilty, Kristin; Webb, Paul; Uht, Rosalie M; Price, Richard H; Pestell, Richard G; Kushner, Peter J

    2002-07-01

    Induction of cyclin D1 gene transcription by estrogen receptor alpha (ERalpha) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERalpha has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERbeta at the cyclin D1 promoter is unknown, although evidence suggests that ERbeta may inhibit the proliferative action of ERalpha. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERalpha, ERalphaK206A (a derivative that is superactive at alternative response elements), or ERbeta. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERbeta occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERalpha, inhibit expression with ERbeta. Strikingly, the presence of ERbeta completely inhibits cyclin D1 gene activation by estrogen and ERalpha or even by estrogen and the superactive ERalphaK206A. The observation of the opposing action and dominance of ERbeta over ERalpha in activation of cyclin D1 gene expression has implications for the postulated role of ERbeta as a modulator of the proliferative effects of estrogen. PMID:11986316

  13. Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.

    PubMed

    Karaiskou, A; Perez, L H; Ferby, I; Ozon, R; Jessus, C; Nebreda, A R

    2001-09-21

    Cyclin-dependent kinases (Cdks) are key regulators of the eukaryotic cell division cycle. Cdk1 (Cdc2) and Cdk2 should be bound to regulatory subunits named cyclins as well as phosphorylated on a conserved Thr located in the T-loop for full enzymatic activity. Cdc2- and Cdk2-cyclin complexes can be inactivated by phosphorylation on the catalytic cleft-located Thr-14 and Tyr-15 residues or by association with inhibitory subunits such as p21(Cip1). We have recently identified a novel Cdc2 regulator named RINGO that plays an important role in the meiotic cell cycle of Xenopus oocytes. RINGO can bind and activate Cdc2 but has no sequence homology to cyclins. Here we report that, in contrast with Cdc2- cyclin complexes, the phosphorylation of Thr-161 is not required for full activation of Cdc2 by RINGO. We also show that RINGO can directly stimulate the kinase activity of Cdk2 independently of Thr-160 phosphorylation. Moreover, RINGO-bound Cdc2 and Cdk2 are both less susceptible to inhibition by p21(Cip1), whereas the Thr-14/Tyr-15 kinase Myt1 can negatively regulate the activity of Cdc2-RINGO with reduced efficiency. Our results indicate that Cdk-RINGO complexes may be active under conditions in which cyclin-bound Cdks are inhibited and can therefore play different regulatory roles. PMID:11461916

  14. Nuclear accumulation of cyclin E/Cdk2 triggers a concentration-dependent switch for the destruction of p27Xic1

    PubMed Central

    Swanson, Craig; Ross, John; Jackson, Peter K.

    2000-01-01

    The action of cyclin-dependent kinases (CDKs) is regulated by phosphorylation, cyclin levels, the abundance of CDK inhibitors, and, as recently has been shown for cyclin B/cdc2, their localization. It is unclear how localization regulates the action of cyclin E/Cdk2 and its inhibitors. Here, we show that the closest known Xenopus laevis homolog of mammalian Cdk2 inhibitors p27Kip1 and p21CIP1, Xic1, is concentrated, ubiquitinated, and destroyed in the nucleus. Furthermore, Xic1 destruction requires nuclear import, but not nuclear export, and requires the formation of a transport-competent nuclear envelope, but not interactions between the lamina and chromatin. We show that (i) cyclin E/Cdk2 and Xic1 are transported into the nucleus as a complex and that Xic1 destruction requires the activity of cyclin E, (ii) that phosphorylation of Xic1 by cyclin E/Cdk2 bypasses the requirement for nuclear formation, and (iii) that the phosphorylation of Xic1 by cyclin E/Cdk2 is concentration dependent and likely realized through second-order interactions between stable cyclin E/Cdk2/Xic1 ternary complexes. Based on these results we propose a model wherein nuclear accumulation of the cyclin E/Cdk2/Xic1 complex triggers a concentration-dependent switch that promotes the phosphorylation of Xic1 and, consequently, its ubiquitination and destruction, thus allowing subsequent activation of cyclin E/Cdk2. PMID:10884410

  15. Expression of cyclin D1 correlates with malignancy in human ovarian tumours.

    PubMed Central

    Barbieri, F.; Cagnoli, M.; Ragni, N.; Pedullà, F.; Foglia, G.; Alama, A.

    1997-01-01

    Cyclin D1 is a cell cycle regulator of G1 progression that has been suggested to play a relevant role in the pathogenesis of several human cancer types. In the current study, the expression of cyclin D1 has been investigated in a series of 33 patients, with benign (10 patients), borderline (five patients) and malignant (18 patients) ovarian disease. Cyclin D1 protein and mRNA content were analysed by Western blotting and reverse transcriptase polymerase chain reaction respectively. The levels of cyclin D1 protein were undetectable in patients with benign disease, detectable in the majority of patients with borderline disease and elevated in those with ovarian carcinomas, being significantly related to the degree of malignancy (carcinoma vs benign, P = 0.0001; benign vs borderline, P = 0.0238). A significant relationship between cyclin D1 expression and tumour proliferative activity was also found (P = 0.000001). Moreover, eight benign lesions, two borderline tumours and 11 carcinomas proved to be suitable for the analysis of cyclin D1 transcript, and emerging data demonstrated significant agreement between protein abundance and mRNA expression. Results from the current study suggest that cyclin D1 expression is associated with the degree of transformation and most probably plays a role in the early development of ovarian malignancy. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:9155044

  16. Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

    PubMed

    de la Fuente, Cynthia; Maddukuri, Anil; Kehn, Kylene; Baylor, Shanese Y; Deng, Longwen; Pumfery, Anne; Kashanchi, Fatah

    2003-04-01

    Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment. PMID:15043199

  17. Crystal Structure of Human Cyclin K, A Positive Regulator of Cyclin-Dependent Kinase 9

    SciTech Connect

    Baek,K.; Brown, R.; Birrane, G.; Ladias, J.

    2007-01-01

    K and the closely related cyclins T1, T2a, and T2b interact with cyclin-dependent kinase 9 (CDK9) forming multiple nuclear complexes, referred to collectively as positive transcription elongation factor b (P-TEFb). Through phosphorylation of the C-terminal domain of the RNA polymerase II largest subunit, distinct P-TEFb species regulate the transcriptional elongation of specific genes that play central roles in human physiology and disease development, including cardiac hypertrophy and human immunodeficiency virus-1 pathogenesis. We have determined the crystal structure of human cyclin K (residues 11-267) at 1.5 {angstrom} resolution, which represents the first atomic structure of a P-TEFb subunit. The cyclin K fold comprises two typical cyclin boxes with two short helices preceding the N-terminal box. A prominent feature of cyclin K is an additional helix (H4a) in the first cyclin box that obstructs the binding pocket for the cell-cycle inhibitor p27{sup Kip1}. Modeling of CDK9 bound to cyclin K provides insights into the structural determinants underlying the formation and regulation of this complex. A homology model of human cyclin T1 generated using the cyclin K structure as a template reveals that the two proteins have similar structures, as expected from their high level of sequence identity. Nevertheless, their CDK9-interacting surfaces display significant structural differences, which could potentially be exploited for the design of cyclin-targeted inhibitors of the CDK9-cyclin K and CDK9-cyclin T1 complexes.

  18. The chromatin-remodeling protein Osa interacts with CyclinE in Drosophila eye imaginal discs.

    PubMed

    Baig, Jawaid; Chanut, Francoise; Kornberg, Thomas B; Klebes, Ansgar

    2010-03-01

    Coordinating cell proliferation and differentiation is essential during organogenesis. In Drosophila, the photoreceptor, pigment, and support cells of the eye are specified in an orchestrated wave as the morphogenetic furrow passes across the eye imaginal disc. Cells anterior of the furrow are not yet differentiated and remain mitotically active, while most cells in the furrow arrest at G(1) and adopt specific ommatidial fates. We used microarray expression analysis to monitor changes in transcription at the furrow and identified genes whose expression correlates with either proliferation or fate specification. Some of these are members of the Polycomb and Trithorax families that encode epigenetic regulators. Osa is one; it associates with components of the Drosophila SWI/SNF chromatin-remodeling complex. Our studies of this Trithorax factor in eye development implicate Osa as a regulator of the cell cycle: Osa overexpression caused a small-eye phenotype, a reduced number of M- and S-phase cells in eye imaginal discs, and a delay in morphogenetic furrow progression. In addition, we present evidence that Osa interacts genetically and biochemically with CyclinE. Our results suggest a dual mechanism of Osa function in transcriptional regulation and cell cycle control. PMID:20008573

  19. Cyclin A2: At the crossroads of cell cycle and cell invasion

    PubMed Central

    Loukil, Abdelhalim; Cheung, Caroline T; Bendris, Nawal; Lemmers, Bénédicte; Peter, Marion; Blanchard, Jean Marie

    2015-01-01

    Cyclin A2 is an essential regulator of the cell division cycle through the activation of kinases that participate to the regulation of S phase as well as the mitotic entry. However, whereas its degradation by the proteasome in mid mitosis was thought to be essential for mitosis to proceed, recent observations show that a small fraction of cyclin A2 persists beyond metaphase and is degraded by autophagy. Its implication in the control of cytoskeletal dynamics and cell movement has unveiled its role in the modulation of RhoA activity. Since this GTPase is involved in both cell rounding early in mitosis and later, in the formation of the cleavage furrow, this suggests that cyclin A2 is a novel actor in cytokinesis. Taken together, these data point to this cyclin as a potential mediator of cell-niche interactions whose dysregulation could be taken as a hallmark of metastasis. PMID:26629317

  20. Complexes of D-type cyclins with CDKs during maize germination.

    PubMed

    Godínez-Palma, Silvia K; García, Elpidio; Sánchez, María de la Paz; Rosas, Fernando; Vázquez-Ramos, Jorge M

    2013-12-01

    The importance of cell proliferation in plant growth and development has been well documented. The majority of studies on basic cell cycle mechanisms in plants have been at the level of gene expression and much less knowledge has accumulated in terms of protein interactions and activation. Two key proteins, cyclins and cyclin-dependent kinases (CDKs) are fundamental for cell cycle regulation and advancement. Our aim has been to understand the role of D-type cyclins and type A and B CDKs in the cell cycle taking place during a developmental process such as maize seed germination. Results indicate that three maize D-type cyclins-D2;2, D4;2, and D5;3-(G1-S cyclins by definition) bind and activate two different types of CDK-A and B1;1-in a differential way during germination. Whereas CDKA-D-type cyclin complexes are more active at early germination times than at later times, it was surprising to observe that CDKB1;1, a supposedly G2-M kinase, bound in a differential way to all D-type cyclins tested during germination. Binding to cyclin D2;2 was detectable at all germination times, forming a complex with kinase activity, whereas binding to D4;2 and D5;3 was more variable; in particular, D5;3 was only detected at late germination times. Results are discussed in terms of cell cycle advancement and its importance for seed germination. PMID:24127516

  1. Cyclin D2 induces proliferation of cardiac myocytes and represses hypertrophy

    SciTech Connect

    Busk, Peter K. . E-mail: pkbu@novonordisk.com; Hinrichsen, Rebecca; Bartkova, Jirina; Hansen, Ane H.; Christoffersen, Tue E.H.; Bartek, Jiri; Haunso, Stig

    2005-03-10

    The myocytes of the adult mammalian heart are considered unable to divide. Instead, mitogens induce cardiomyocyte hypertrophy. We have investigated the effect of adenoviral overexpression of cyclin D2 on myocyte proliferation and morphology. Cardiomyocytes in culture were identified by established markers. Cyclin D2 induced DNA synthesis and proliferation of cardiomyocytes and impaired hypertrophy induced by angiotensin II and serum. At the molecular level, cyclin D2 activated CDK4/6 and lead to pRB phosphorylation and downregulation of the cell cycle inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}. Expression of the CDK4/6 inhibitor p16 inhibited proliferation and cyclin D2 overexpressing myocytes became hypertrophic under such conditions. Inhibition of hypertrophy by cyclin D2 correlated with downregulation of p27{sup Kip1}. These data show that hypertrophy and proliferation are highly related processes and suggest that cardiomyocyte hypertrophy is due to low amounts of cell cycle activators unable to overcome the block imposed by cell cycle inhibitors. Cell cycle entry upon hypertrophy may be converted to cell division by increased expression of activators such as cyclin D2.

  2. Cyclin A2 modulates EMT via β-catenin and phospholipase C pathways.

    PubMed

    Cheung, Caroline T; Bendris, Nawal; Paul, Conception; Hamieh, Abdallah; Anouar, Youssef; Hahne, Michael; Blanchard, Jean-Marie; Lemmers, Bénédicte

    2015-08-01

    We have previously demonstrated that Cyclin A2 is involved in cytoskeletal dynamics, epithelial-mesenchymal transition (EMT) and metastasis. This phenotype was potentiated by activated oncogenic H-Ras. However, the mechanisms governing EMT in these cells have not yet been elucidated. Here, we dissected the pathways that are responsible for EMT in cells deficient for Cyclin A2. In Cyclin A2-depleted normal murine mammary gland (NMuMG) cells expressing RasV12, we found that β-catenin was liberated from the cell membrane and cell-cell junctions and underwent nuclear translocation and activation. Components of the canonical wingless (WNT) pathway, including WNT8b, WNT10a, WNT10b, frizzled 1 and 2 and TCF4 were upregulated at the messenger RNA and protein levels following Cyclin A2 depletion. However, suppression of the WNT pathway using the acetyltransferase porcupine inhibitor C59 did not reverse EMT whereas a dominant negative form of TCF4 as well as inhibition of phospholipase C using U73122 were able to do so. This suggests that a WNT-independent mechanism of β-catenin activation via phospholipase C is involved in the EMT induced by Cyclin A2 depletion. Our findings will broaden our knowledge on how Cyclin A2 contributes to EMT and metastasis. PMID:25993989

  3. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.

    PubMed Central

    Minshull, J; Golsteyn, R; Hill, C S; Hunt, T

    1990-01-01

    Cyclins play a key role in the induction of mitosis. In this paper we report the isolation of a cyclin A cDNA clone from Xenopus eggs. Its cognate mRNA encodes a protein that shows characteristic accumulation and destruction during mitotic cell cycles. The cyclin A polypeptide is associated with a protein that cross-reacts with an antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the cyclin A-cdc2 complex exhibits protein kinase activity that oscillates with the cell cycle. This kinase activity rises more smoothly than that of the cyclin B-cdc2 complexes and reaches a peak earlier in the cell cycle; indeed, cyclin A is destroyed before nuclear envelope breakdown. None of the cyclin-cdc2 complexes show simple relationships between the concentration of the cyclin moiety and the kinase activity. All three cyclin associated kinases (A, B1 and B2) phosphorylate identical sites on histones with the consensus XSPXK/R, although they show significant differences in their substrate preferences. We discuss possible models for the different roles of the A- and B-type cyclins in the control of cell division. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2143983

  4. Cyclin-dependent kinases and cell-cycle transitions: does one fit all?

    PubMed

    Hochegger, Helfrid; Takeda, Shunichi; Hunt, Tim

    2008-11-01

    Cell-cycle transitions in higher eukaryotes are regulated by different cyclin-dependent kinases (CDKs) and their activating cyclin subunits. Based on pioneering findings that a dominant-negative mutation of CDK1 blocks the cell cycle at G2-M phase, whereas dominant-negative CDK2 inhibits the transition into S phase, a model of cell-cycle control has emerged in which each transition is regulated by a specific subset of CDKs and cyclins. Recent work with gene-targeted mice has led to a revision of this model. We discuss cell-cycle control in light of overlapping and essential functions of the different CDKs and cyclins. PMID:18813291

  5. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    SciTech Connect

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C. . E-mail: mary-horne@uiowa.edu

    2006-12-10

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G{sub 1}/S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles

  6. Greatwall kinase and cyclin B-Cdk1 are both critical constituents of M-phase-promoting factor

    PubMed Central

    Hara, Masatoshi; Abe, Yusuke; Tanaka, Toshiaki; Yamamoto, Takayoshi; Okumura, Eiichi; Kishimoto, Takeo

    2012-01-01

    Maturation/M-phase-promoting factor is the universal inducer of M-phase in eukaryotic cells. It is currently accepted that M-phase-promoting factor is identical to the kinase cyclin B–Cdk1. Here we show that cyclin B–Cdk1 and M-phase-promoting factor are not in fact synonymous. Instead, M-phase-promoting factor contains at least two essential components: cyclin B–Cdk1 and another kinase, Greatwall kinase. In the absence of Greatwall kinase, the M-phase-promoting factor is undetectable in oocyte cytoplasm even though cyclin B–Cdk1 is fully active, whereas M-phase-promoting factor activity is restored when Greatwall kinase is added back. Although the excess amount of cyclin B–Cdk1 alone, but not Greatwall kinase alone, can induce nuclear envelope breakdown, spindle assembly is abortive. Addition of Greatwall kinase greatly reduces the amount of cyclin B–Cdk1 required for nuclear envelope breakdown, resulting in formation of the spindle with aligned chromosomes. M-phase-promoting factor is thus a system consisting of one kinase (cyclin B–Cdk1) that directs mitotic entry and a second kinase (Greatwall kinase) that suppresses the protein phosphatase 2A-B55 which opposes cyclin B–Cdk1. PMID:22968705

  7. Cyclin D1-CDK4 Controls Glucose Metabolism Independently of Cell Cycle Progression

    PubMed Central

    Lee, Yoonjin; Dominy, John E.; Choi, Yoon Jong; Jurczak, Michael; Tolliday, Nicola; Camporez, Joao Paulo; Chim, Helen; Lim, Ji-Hong; Ruan, Hai-Bin; Yang, Xiaoyong; Vazquez, Francisca; Sicinski, Piotr; Shulman, Gerald I.; Puigserver, Pere

    2014-01-01

    Insulin constitutes a major evolutionarily conserved hormonal axis for maintaining glucose homeostasis1-3; dysregulation of this axis causes diabetes2,4. PGC-1α links insulin signaling to the expression of glucose and lipid metabolic genes5-7. GCN5 acetylates PGC-1α and suppresses its transcriptional activity, whereas SIRT1 deacetylates and activates PGC-1α8,9. Although insulin is a mitogenic signal in proliferative cells10,11, whether components of the cell cycle machinery contribute to insulin’s metabolic action is poorly understood. Herein, we report that insulin activates cyclin D1-CDK4, which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high throughput chemical screen, we identified a CDK4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK3β signaling induces cyclin D1 protein stability via sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 mRNA transcripts. Activated cyclin D1-CDK4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycemia. In diabetic models, cyclin D1-CDK4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division. PMID:24870244

  8. Cerebellar Cortical Lamination and Foliation Require Cyclin A2

    PubMed Central

    Otero, José Javier; Kalaszczynska, Ilona; Michowski, Wojciech; Wong, Michael; Gygli, Patrick Edwin; Gokozan, Hamza Numan; Griveau, Amélie; Odajima, Junko; Czeisler, Catherine; Catacutan, Fay Patsy; Murnen, Alice; Schüler, Ulrich; Sicinski, Piotr; Rowitch, David

    2014-01-01

    The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function. PMID:24184637

  9. 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. PMID:24296733

  10. Epstein-Barr Virus Nuclear Antigen 3C Facilitates G1-S Transition by Stabilizing and Enhancing the Function of Cyclin D1

    PubMed Central

    Saha, Abhik; Halder, Sabyasachi; Upadhyay, Santosh K.; Lu, Jie; Kumar, Pankaj; Murakami, Masanao; Cai, Qiliang; Robertson, Erle S.

    2011-01-01

    EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the present study, we provide evidence which demonstrates that EBNA3C forms a complex with Cyclin D1 in human cells. Detailed mapping experiments show that a small N-terminal region which lies between amino acids 130–160 of EBNA3C binds to two different sites of Cyclin D1- the N-terminal pRb binding domain (residues 1–50), and C-terminal domain (residues 171–240), known to regulate Cyclin D1 stability. Cyclin D1 is short-lived and ubiquitin-mediated proteasomal degradation has been targeted as a means of therapeutic intervention. Here, we show that EBNA3C stabilizes Cyclin D1 through inhibition of its poly-ubiquitination, and also increases its nuclear localization by blocking GSK3β activity. We further show that EBNA3C enhances the kinase activity of Cyclin D1/CDK6 which enables subsequent ubiquitination and degradation of pRb. EBNA3C together with Cyclin D1-CDK6 complex also efficiently nullifies the inhibitory effect of pRb on cell growth. Moreover, an sh-RNA based strategy for knock-down of both cyclin D1 and EBNA3C genes in EBV transformed lymphoblastoid cell lines (LCLs) shows a significant reduction in cell-growth. Based on these results, we propose that EBNA3C can stabilize as well as enhance the functional activity of Cyclin D1 thereby facilitating the G1-S transition in EBV transformed lymphoblastoid cell lines. PMID:21347341

  11. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb{sup 2+}-induced neuronal death in cultured hippocampal neurons

    SciTech Connect

    Li Chenchen Xing Tairan Tang Mingliang Yong Wu Yan Dan Deng Hongmin Wang Huili Wang Ming Chen Jutao Ruan Diyun

    2008-06-15

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb{sup 2+} causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb{sup 2+}. Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb{sup 2+}-induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb{sup 2+} treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 {mu}M) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb{sup 2+}. And that Pb{sup 2+}-elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb{sup 2+} and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death.

  12. Altered Subcellular Localization of Tumor-Specific Cyclin E Isoforms Affects Cyclin-Dependent Kinase 2 Complex Formation and Proteasomal Regulation

    PubMed Central

    Delk, Nikki A.; Hunt, Kelly K.; Keyomarsi, Khandan

    2009-01-01

    In tumors, alternative translation and posttranslational proteolytic cleavage of full-length cyclin E (EL) produces tumorigenic low molecular weight cyclin E (LMW-E) isoforms that lack a portion of the EL amino-terminus containing a nuclear localization sequence. Therefore, we hypothesized that LMW-E isoforms have altered subcellular localization. To explore our hypothesis, we compared EL versus LMW-E localization in cell lysates and in vivo using fractionation and protein complementation assays. Our results reveal that LMW-E isoforms preferentially accumulate in the cytoplasm where they bind the cyclin E kinase partner, cyclin-dependent kinase 2 (Cdk2), and have associated kinase activity. The nuclear ubiquitin ligase Fbw7 targets Cdk2-bound cyclin E for degradation; thus, we examined if altered subcellular localization affected LMW-E degradation. We found that cytoplasmic LMW-E/Cdk2 was less susceptible to Fbw7-mediated degradation. One implication of our findings is that altered LMW-E and LMW-E/Cdk2 subcellular localization may lead to aberrant LMW-E protein interactions, regulation, and activity, ultimately contributing to LMW-E tumorigenicity. PMID:19318554

  13. The cyclin-dependent kinase inhibitor SNS-032 induces apoptosis in breast cancer cells via depletion of Mcl-1 and X-linked inhibitor of apoptosis protein and displays antitumor activity in vivo.

    PubMed

    Xie, Gui'e; Tang, Hongping; Wu, Shaoqing; Chen, Jingsong; Liu, Jiangwen; Liao, Can

    2014-08-01

    Inhibitors of cyclin-dependent kinases (Cdks) have been reported to have activities in many types of cancer cells by inhibiting Cdk7 and Cdk9, which control transcription. SNS-032 is a potent and selective inhibitor of Cdk2, Cdk7 and Cdk9 and has emerged in clinical trials. Here, we examined the viability of MCF-7 and MDA-MB-435 breast cancer cells in the presence of SNS-032 and observed a dose-dependent inhibition of cellular proliferation in both cell lines. SNS-032 had a direct apoptosis-inducing effect through both the extrinsic and intrinsic apoptotic pathways in breast cancer cells as shown by a dose-dependent increase in Annexin V-positive cells and terminal deoxynucleotidyl transferase-mediated dUTP nick?end labeling (TUNEL)-positive cells, as well as activation of caspase-8, -9 and poly(ADP-ribose) polymerase (PARP). At the molecular level, SNS-032 induced a marked dephosphorylation of serine 2 and 5 of RNA polymerase (RNA Pol) II and blocked RNA synthesis. Consistent with the inherently rapid turnover rates of their transcripts and proteins, the anti-apoptotic proteins Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP) were rapidly reduced on exposure to SNS-032. Our results also indicated that SNS-032 suppressed the growth of breast cancer xenografts in mice. These data demonstrate that the use of SNS-032 may be a rational and novel therapeutic strategy for human breast cancer and warrants further clinical investigation. PMID:24865236

  14. Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells

    PubMed Central

    Baldassarre, Gustavo; Belletti, Barbara; Bruni, Paola; Boccia, Angelo; Trapasso, Francesco; Pentimalli, Francesca; Barone, Maria Vittoria; Chiappetta, Gennaro; Vento, Maria Teresa; Spiezia, Stefania; Fusco, Alfredo; Viglietto, Giuseppe

    1999-01-01

    The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27–cyclin D3–Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A–E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells. PMID:10510327

  15. Targeted overexpression of tumor necrosis factor-α increases cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons.

    PubMed

    Rozas, Pablo; Lazcano, Pablo; Piña, Ricardo; Cho, Andrew; Terse, Anita; Pertusa, Maria; Madrid, Rodolfo; Gonzalez-Billault, Christian; Kulkarni, Ashok B; Utreras, Elias

    2016-06-01

    We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain, increases the activity of Cdk5, a key kinase involved in brain development and function and recently found to be involved in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TGs), we engineered a transgenic mouse model (TNF) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNF mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, which suggests that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity; therefore, this mouse model would be valuable for investigating the mechanism of TNF-α involved in orofacial pain. PMID:26894912

  16. BRCA1-IRIS regulates cyclin D1 expression in breast cancer cells

    SciTech Connect

    Nakuci, Enkeleda; Mahner, Sven; DiRenzo, James; ElShamy, Wael M. . E-mail: wael_elshamy@dfci.harvard.edu

    2006-10-01

    The regulator of cell cycle progression, cyclin D1, is up-regulated in breast cancer cells; its expression is, in part, dependent on ER{alpha} signaling. However, many ER{alpha}-negative tumors and tumor cell lines (e.g., SKBR3) also show over-expression of cyclin D1. This suggests that, in addition to ER{alpha} signaling, cyclin D1 expression is under the control of other signaling pathways; these pathways may even be over-expressed in the ER{alpha}-negative cells. We previously noticed that both ER{alpha}-positive and -negative cell lines over-express BRCA1-IRIS mRNA and protein. Furthermore, the level of over-expression of BRCA1-IRIS in ER{alpha}-negative cell lines even exceeded its over-expression level in ER{alpha}-positive cell lines. In this study, we show that: (1) BRCA1-IRIS forms complex with two of the nuclear receptor co-activators, namely, SRC1 and SRC3 (AIB1) in an ER{alpha}-independent manner. (2) BRCA1-IRIS alone, or in connection with co-activators, is recruited to the cyclin D1 promoter through its binding to c-Jun/AP1 complex; this binding activates the cyclin D1 expression. (3) Over-expression of BRCA1-IRIS in breast cells over-activates JNK/c-Jun; this leads to the induction of cyclin D1 expression and cellular proliferation. (4) BRCA1-IRIS activation of JNK/c-Jun/AP1 appears to account for this, because in cells that were depleted from BRCA1-IRIS, JNK remained inactive. However, depletion of SRC1 or SRC3 instead reduced c-Jun expression. Our data suggest that this novel signaling pathway links BRCA1-IRIS to cellular proliferation through c-Jun/AP1 nuclear pathway; finally, this culminates in the increased expression of the cyclin D1 gene.

  17. Identification of the cyclin D1b mRNA variant in mouse

    PubMed Central

    Wu, Jack; Wu, Si-hung; Bollig, Aliccia; Thakur, Archana

    2013-01-01

    Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and over-expressed in many cancers. The cyclin D1b mRNA variant was established in human cells and recent functional analyses revealed that its protein product harbors unique activities in human cancer cells. By performing reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) experiments, we identified the cyclin D1b mRNA variant in mouse. Similar to its human counterpart, the mouse cyclin D1b transcript consists of exon 1, 2, 3, 4 and part of intron 4, and contains a long open reading frame (ORF). The predicted peptide from this ORF is 34-amino acid longer than the human cyclin D1b. The expression of this mouse mRNA variant was investigated. It appears to be expressed ubiquitously and differentially in various mouse cell lines and tissues and its level might be proportional to that of the canonical endogenous cyclin D1a mRNA. PMID:18446443

  18. Cooperation between Dmp1 Loss and Cyclin D1 Overexpression in Breast Cancer

    PubMed Central

    Zhu, Sinan; Mott, Ryan T.; Fry, Elizabeth A.; Taneja, Pankaj; Kulik, George; Sui, Guangchao; Inoue, Kazushi

    2014-01-01

    Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1–cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1–induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1+/+;MMTV-cyclin D1 and Dmp1+/+;MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer. PMID:23938323

  19. Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells.

    PubMed

    Kalaszczynska, Ilona; Geng, Yan; Iino, Tadafumi; Mizuno, Shin-ichi; Choi, Yoon; Kondratiuk, Ilona; Silver, Daniel P; Wolgemuth, Debra J; Akashi, Koichi; Sicinski, Piotr

    2009-07-23

    Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation. PMID:19592082

  20. Caenorhabditis elegans cyclin D/CDK4 and cyclin E/CDK2 induce distinct cell cycle re-entry programs in differentiated muscle cells.

    PubMed

    Korzelius, Jerome; The, Inge; Ruijtenberg, Suzan; Prinsen, Martine B W; Portegijs, Vincent; Middelkoop, Teije C; Groot Koerkamp, Marian J; Holstege, Frank C P; Boxem, Mike; van den Heuvel, Sander

    2011-11-01

    Cell proliferation and differentiation are regulated in a highly coordinated and inverse manner during development and tissue homeostasis. Terminal differentiation usually coincides with cell cycle exit and is thought to engage stable transcriptional repression of cell cycle genes. Here, we examine the robustness of the post-mitotic state, using Caenorhabditis elegans muscle cells as a model. We found that expression of a G1 Cyclin and CDK initiates cell cycle re-entry in muscle cells without interfering with the differentiated state. Cyclin D/CDK4 (CYD-1/CDK-4) expression was sufficient to induce DNA synthesis in muscle cells, in contrast to Cyclin E/CDK2 (CYE-1/CDK-2), which triggered mitotic events. Tissue-specific gene-expression profiling and single molecule FISH experiments revealed that Cyclin D and E kinases activate an extensive and overlapping set of cell cycle genes in muscle, yet failed to induce some key activators of G1/S progression. Surprisingly, CYD-1/CDK-4 also induced an additional set of genes primarily associated with growth and metabolism, which were not activated by CYE-1/CDK-2. Moreover, CYD-1/CDK-4 expression also down-regulated a large number of genes enriched for catabolic functions. These results highlight distinct functions for the two G1 Cyclin/CDK complexes and reveal a previously unknown activity of Cyclin D/CDK-4 in regulating metabolic gene expression. Furthermore, our data demonstrate that many cell cycle genes can still be transcriptionally induced in post-mitotic muscle cells, while maintenance of the post-mitotic state might depend on stable repression of a limited number of critical cell cycle regulators. PMID:22102824

  1. An okadaic acid-sensitive phosphatase negatively controls the cyclin degradation pathway in amphibian eggs.

    PubMed Central

    Lorca, T; Fesquet, D; Zindy, F; Le Bouffant, F; Cerruti, M; Brechot, C; Devauchelle, G; Dorée, M

    1991-01-01

    Inhibition of okadaic acid-sensitive phosphatases released the cyclin degradation pathway from its inhibited state in extracts prepared from unfertilized Xenopus eggs arrested at the second meiotic metaphase. It also switched on cyclin protease activity in a permanent fashion in interphase extracts prepared from activated eggs. Even after cdc2 kinase inactivation, microinjection of okadaic acid-treated interphase extracts pushed G2-arrested recipient oocytes into the M phase, suggesting that the phosphatase inhibitor stabilizes the activity of an unidentified factor which shares in common with cdc2 kinase the maturation-promoting factor activity. Images PMID:1846666

  2. Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.

    PubMed

    Di Renzo, María Agostina; Laverrière, Marc; Schenkman, Sergio; Wehrendt, Diana Patricia; Tellez-Iñón, María Teresa; Potenza, Mariana

    2016-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi. PMID:26709077

  3. Cyclin E/Cdk2-dependent phosphorylation of Mcl-1 determines its stability and cellular sensitivity to BH3 mimetics

    PubMed Central

    Choudhary, Gaurav S.; Tat, Trinh T.; Misra, Saurav; Hill, Brian T.; Smith, Mitchell R.; Almasan, Alexandru; Mazumder, Suparna

    2015-01-01

    Cyclin E/Cdk2 kinase activity is frequently deregulated in human cancers, resulting in impaired apoptosis. Here, we show that cyclin E/Cdk2 phosphorylates and stabilizes the pro-survival Bcl-2 family protein Mcl-1, a key cell death resistance determinant to the small molecule Bcl-2 family inhibitors ABT-199 and ABT-737, mimetics of the Bcl-2 homology domain 3 (BH3). Cyclin E levels were elevated and there was increased association of cyclin E/Cdk2 with Mcl-1 in ABT-737-resistant compared to parental cells. Cyclin E depletion in various human tumor cell-lines and cyclin E−/− mouse embryo fibroblasts showed decreased levels of Mcl-1 protein, with no change in Mcl-1 mRNA levels. In the absence of cyclin E, Mcl-1 ubiquitination was enhanced, leading to decreased protein stability. Studies with Mcl-1 phosphorylation mutants show that cyclin E/Cdk2-dependent phosphorylation of Mcl-1 residues on its PEST domain resulted in increased Mcl-1 stability (Thr92, and Thr163) and Bim binding (Ser64). Cyclin E knock-down restored ABT-737 sensitivity to acquired and inherently resistant Mcl-1-dependent tumor cells. CDK inhibition by dinaciclib resulted in Bim release from Mcl-1 in ABT-737-resistant cells. Dinaciclib in combination with ABT-737 and ABT-199 resulted in robust synergistic cell death in leukemic cells and primary chronic lymphocytic leukemia patient samples. Collectively, our findings identify a novel mechanism of cyclin E-mediated Mcl-1 regulation that provides a rationale for clinical use of Bcl-2 family and Cdk inhibitors for Mcl-1-dependent tumors. PMID:26219338

  4. Myb controls G2/M progression by inducing cyclin B expression in the Drosophila eye imaginal disc

    PubMed Central

    Okada, Masahiro; Akimaru, Hiroshi; Hou, De-Xing; Takahashi, Tomomi; Ishii, Shunsuke

    2002-01-01

    The c-myb proto-oncogene product (c-Myb) is a transcriptional activator. Vertebrate c-Myb is a key regulator of the G1/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G2/M transition. Here we report that dMyb induces expression of cyclin B, a critical regulator of the G2/M transition, in Drosophila eye imaginal disc. In the wild-type eye disc, dmyb mRNA was expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C-terminal-truncated dMyb in the eye disc caused ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlated with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression was lost in dmyb-deficient clones. In Schneider cells, the activity of the cyclin B promoter was dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolished the promoter activity. These results indicate that dMyb regulates the G2/M transition by inducing cyclin B expression via binding to its promoter. PMID:11847115

  5. A nuclear factor required for specific translation of cyclin B may control the timing of first meiotic cleavage in starfish oocytes.

    PubMed Central

    Galas, S; Barakat, H; Dorée, M; Picard, A

    1993-01-01

    In most animals, the rate of cyclin B synthesis increases after nuclear envelope breakdown during the first meiotic cell cycle. We have found that cyclin B-cdc2 kinase activity drops earlier in emetine-treated than in control starfish oocytes, although the protein synthesis inhibitor does not activate the cyclin degradation pathway prematurely. Moreover, protein synthesis is required to prevent meiotic cleavage to occur prematurely, sometimes before chromosomes have segregated on the metaphase plate. In normal conditions, increased synthesis of cyclin B after germinal vesicle breakdown (GVBD) balances cyclin degradation and increases the time required for cyclin B-cdc2 kinase to drop below the level that inhibits cleavage. Taken together, these results point to cyclin B as a possible candidate that could explain the need for increased protein synthesis during meiosis I. Although direct experimental evidence was not provided in the present work, cyclin B synthesis after GVBD may be important for correct segregation of homologous chromosomes at the end of first meiotic metaphase, as shown by a variety of cytological disorders that accompany premature cleavage. Although the overall stimulation of protein synthesis because of cdc2 kinase activation is still observed in oocytes from which the germinal vesicle has been removed before hormonal stimulation, the main increase of cyclin B synthesis normally observed after germinal vesicle breakdown is suppressed. The nuclear factor required for specific translation of cyclin B after GVBD is not cyclin B mRNA, as shown by using a highly sensitive reverse transcription followed by polymerase chain reaction procedure that failed to detect any cyclin B mRNA in isolated germinal vesicles. Images PMID:7513215

  6. Cyclin-dependent protein kinase inhibitors including palbociclib as anticancer drugs.

    PubMed

    Roskoski, Robert

    2016-05-01

    Cyclins and cyclin-dependent protein kinases (CDKs) are important regulatory components that are required for cell cycle progression. The levels of the cell cycle CDKs are generally constant and their activities are controlled by cyclins, proteins whose levels oscillate during each cell cycle. Additional CDK family members were subsequently discovered that play significant roles in a wide range of activities including the control of gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1 phase of the cell cycle produce cyclins of the D type that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein. Then CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factors, which in turn lead to the generation of several proteins required for cell cycle progression. As a result, cells pass through the G1-restriction point and are committed to complete cell division. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S, G2, and M-phase progression. Increased cyclin or CDK expression or decreased levels of endogenous CDK inhibitors such as INK4 or CIP/KIP have been observed in various cancers. In contrast to the mutational activation of EGFR, Kit, or B-Raf in the pathogenesis of malignancies, mutations in the CDKs that cause cancers are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Abemaciclib (LY2835219), ribociclib (Lee011), and palbociclib (Ibrance(®) or PD0332991) target CDK4/6 with IC50 values in the low nanomolar range. Palbociclib and other CDK inhibitors bind in the cleft between the small and large lobes of the CDKs and inhibit the binding of ATP. Like ATP, palbociclib forms hydrogen bonds with residues in the hinge segment of the cleft. Like the adenine base of ATP, palbociclib interacts with catalytic spine residues CS6 and CS7

  7. PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

    PubMed

    Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

    2001-10-11

    Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth. PMID:11709714

  8. Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms.

    PubMed

    van Drogen, Frank; Sangfelt, Olle; Malyukova, Aljona; Matskova, Ludmila; Yeh, Elizabeth; Means, Anthony R; Reed, Steven I

    2006-07-01

    Cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), is targeted for proteasomal degradation by phosphorylation-dependent multiubiquitylation via the ubiquitin ligase SCF(hCdc4). SCF ubiquitin ligases are composed of a core of conserved subunits and one variable subunit (an F box protein) involved in substrate recognition. We show here that multiubiquitylation of cyclin E requires the sequential function of two distinct splice variant isoforms of the F box protein hCdc4 known as alpha and gamma. SCF(hCdc4alpha) binds a complex containing cyclin E, Cdk2, and the prolyl cis/trans isomerase Pin1 and promotes the activity of Pin1 without directly ubiquitylating cyclin E. However, due to the action of this SCF(hCdc4alpha)-Pin1 complex, cyclin E becomes an efficient ubiquitylation substrate of SCF(hCdc4gamma). Furthermore, in the context of Cdc4alpha and cyclin E, mutational data suggest that Pin1 isomerizes a noncanonical proline-proline bond, with the possibility that Cdc4alpha may serve as a cofactor for altering the specificity of Pin1. PMID:16818231

  9. Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs.

    PubMed

    Nabti, Ibtissem; Reis, Alexandra; Levasseur, Mark; Stemmann, Olaf; Jones, Keith T

    2008-09-15

    Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1. PMID:18639540

  10. Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

    SciTech Connect

    Yasmin, Tania; Takahashi-Yanaga, Fumi . E-mail: yanaga@clipharm.med.kyushu-u.ac.jp; Mori, Jun; Miwa, Yoshikazu; Hirata, Masato; Watanabe, Yutaka; Morimoto, Sachio; Sasaguri, Toshiyuki

    2005-12-16

    To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of {beta}-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3{beta} (GSK-3{beta}) and inhibition of GSK-3{beta} attenuated the DIF-1-induced {beta}-catenin degradation, indicating the involvement of GSK-3{beta} in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/{beta}-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.

  11. Regulation of Glioblastoma Progression by Cord Blood Stem Cells Is Mediated by Downregulation of Cyclin D1

    PubMed Central

    Velpula, Kiran Kumar; Dasari, Venkata Ramesh; Tsung, Andrew J.; Gondi, Christopher S.; Klopfenstein, Jeffrey D.; Mohanam, Sanjeeva; Rao, Jasti S.

    2011-01-01

    Background The normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, often is considered a rate-limiting step during cell cycle progression through the G1 phase. Methodology/Principal Findings In the present study, human umbilical cord blood stem cells (hUCBSC) in co-cultures with glioblastoma cells (U251 and 5310) not only induced G0-G1 phase arrest, but also reduced the number of cells at S and G2-M phases of cell cycle. Cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC as revealed by Western and FACS analyses. Inhibition of cyclin D1 activity by hUCBSC treatment is sufficient to abolish the expression levels of Cdk 4, Cdk 6, cyclin B1, β-Catenin levels. Our immuno precipitation experiments present evidence that, treatment of glioma cells with hUCBSC leads to the arrest of cell-cycle progression through inactivation of both cyclin D1/Cdk 4 and cyclin D1/Cdk 6 complexes. It is observed that hUCBSC, when co-cultured with glioma cells, caused an increased G0-G1 phase despite the reduction of G0-G1 regulatory proteins cyclin D1 and Cdk 4. We found that this reduction of G0-G1 regulatory proteins, cyclin D1 and Cdk 4 may be in part compensated by the expression of cyclin E1, when co-cultured with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated, decreased expression of cyclin D1 in the presence of hUCBSC. Conclusions/Significance This paper elucidates a model to regulate glioma cell cycle progression in which hUCBSC acts to control cyclin D1 induction and in concert its partner kinases, Cdk 4 and Cdk 6 by mediating cell cycle arrest at G0-G1 phase. PMID:21455311

  12. Centrosomal Localization of Cyclin E-Cdk2 is Required for Initiation of DNA Synthesis

    PubMed Central

    Ferguson, Rebecca L.; Maller, James L.

    2010-01-01

    Summary Cyclin E-Cdk2 is known to regulate both DNA replication and centrosome duplication during the G1-S transition in the cell cycle [1–4], and disruption of centrosomes results in a G1 arrest in some cell types [5–7]. Localization of cyclin E on centrosomes is mediated by a 20 amino acid domain termed the centrosomal localization sequence (CLS), and expression of the GFP-tagged CLS displaces both cyclin E and cyclin A from the centrosome [8]. In asynchronous cells CLS expression inhibits the incorporation of bromodeoxyuridine (BrdU) into DNA, an effect proposed to reflect a G1 arrest. Here we show in synchronized cells that the reduction in BrdU incorporation reflects not a G1 arrest but rather direct inhibition of the initiation of DNA replication in S phase. The loading of essential DNA replication factors such as Cdc45 and PCNA onto chromatin is blocked by CLS expression, but DNA synthesis can be rescued by retargeting active cyclin E-Cdk2 to the centrosome. These results suggest that initial steps of DNA replication require centrosomally localized Cdk activity and link the nuclear cycle with the centrosome cycle at the G1-S transition. PMID:20399658

  13. Arsenic trioxide suppressed mantle cell lymphoma by downregulation of cyclin D1.

    PubMed

    Lo, Rico K H; Kwong, Yok-Lam

    2014-02-01

    Mantle cell lymphoma (MCL) is aggressive with poor prognosis. Due to t(11;14)(q13;q32), cyclin D1 is overexpressed. The in vitro activities of arsenic trioxide (As2O3) in MCL were investigated. In MCL lines Jeko-1 and Granta-519, As2O3 induced dose-dependent and time-dependent increases in apoptosis accompanied by cyclin D1 suppression. Downregulation of cyclin D1 resulted in decreased retinoblastoma protein phosphorylation, which led to repressed G1 progression to S/G2 phases. As2O3 did not affect cyclin D1 gene transcription. Instead, As2O3 activated glycogen synthase kinase-3beta (by tyrosine-216 phosphorylation) and IkappaB kinase alpha/beta (by serine-176/180 phosphorylation), both of which phosphorylated cyclin D1 at threonine-286, leading to its poly-ubiquitination and degradation in the proteasome. These observations were recapitulated partly in primary MCL samples obtained from patients refractory to conventional treatment. Our findings suggested that As2O3 might be clinically useful in MCL. PMID:23949314

  14. Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.

    PubMed Central

    Sigrist, S; Jacobs, H; Stratmann, R; Lehner, C F

    1995-01-01

    While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition. Images PMID:7588612

  15. Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner.

    PubMed

    Casanovas, O; Miró, F; Estanyol, J M; Itarte, E; Agell, N; Bachs, O

    2000-11-10

    We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway. The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation. PMID:10952989

  16. Competitive Nuclear Export of Cyclin D1 and Hic-5 Regulates Anchorage Dependence of Cell Growth and Survival

    PubMed Central

    Mori, Kazunori; Hirao, Etsuko; Toya, Yosuke; Oshima, Yukiko; Ishikawa, Fumihiro; Nose, Kiyoshi

    2009-01-01

    Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival. PMID:18946086

  17. Fbw7α and Fbw7γ collaborate to shuttle cyclin E1 into the nucleolus for multiubiquitylation.

    PubMed

    Bhaskaran, Nimesh; van Drogen, Frank; Ng, Hwee-Fang; Kumar, Raman; Ekholm-Reed, Susanna; Peter, Matthias; Sangfelt, Olle; Reed, Steven I

    2013-01-01

    Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation. PMID:23109421

  18. Fbw7α and Fbw7γ Collaborate To Shuttle Cyclin E1 into the Nucleolus for Multiubiquitylation

    PubMed Central

    Bhaskaran, Nimesh; van Drogen, Frank; Ng, Hwee-Fang; Kumar, Raman; Ekholm-Reed, Susanna; Peter, Matthias

    2013-01-01

    Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G1-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G1 and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCFFbw7 (also known as SCFCdc4), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCFFbw7α in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCFFbw7γ. Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCFFbw7γ prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation. PMID:23109421

  19. Interphase APC/C–Cdc20 inhibition by cyclin A2–Cdk2 ensures efficient mitotic entry

    PubMed Central

    Hein, Jamin B.; Nilsson, Jakob

    2016-01-01

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C–Cdc20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2–Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C–Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C–Cdc20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2–Cdk2 inhibition of interphase APC/C–Cdc20 to allow further cyclin A2 accumulation and mitotic entry. PMID:26960431

  20. HIV-1 expression induces cyclin D1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

    PubMed Central

    Nelson, Peter J; Sunamoto, Masaaki; Husain, Mohammad; Gelman, Irwin H

    2002-01-01

    Background The aberrant cell-cycle progression of HIV-1-infected kidney cells plays a major role in the pathogenesis of HIV-associated nephropathy, however the mechanisms whereby HIV-1 induces infected glomerular podocytes or infected tubular epithelium to exit quiescence are largely unknown. Here, we ask whether the expression of HIV-1 genes in infected podocytes induces cyclin D1 and phospho-pRb (Ser780) expression, hallmarks of cyclin D1-mediated G1 → S phase progression. Results We assessed cyclin D1 and phospho-pRb (Ser780) expression in two well-characterized models of HIV-associated nephropathy pathogenesis: HIV-1 infection of cultured podocytes and HIV-1 transgenic mice (Tg26). Compared to controls, cultured podocytes expressing HIV-1 genes, and podocytes and tubular epithelium from hyperplastic nephrons in Tg26 kidneys, had increased levels of phospho-pRb (Ser780), a target of active cyclin D1/cyclin-dependent kinase-4/6 known to promote G1 → S phase progression. HIV-1-infected podocytes showed markedly elevated cyclin D1 mRNA and cyclin D1 protein, the latter of which did not down-regulate during cell-cell contact or differentiation, suggesting post-transcriptional stabilization of cyclin D1 protein levels by HIV-1. The selective suppression of HIV-1 transcription by the cyclin-dependent kinase inhibitor, flavopiridol, abrogated cyclin D1 expression, underlying the requirement for HIV-1 encoded products to induce cyclin D1. Indeed, HIV-1 virus deleted of nef failed to induce cyclin D1 mRNA to the level of other single gene mutant viruses. Conclusions HIV-1 expression induces cyclin D1 and phospho-pRb (Ser780) expression in infected podocytes, suggesting that HIV-1 activates cyclin D1-dependent cell-cycle mechanisms to promote proliferation of infected renal epithelium. PMID:12241561

  1. Cyclin D1 promotes BRCA2-Rad51 interaction by restricting cyclin A/B-dependent BRCA2 phosphorylation.

    PubMed

    Chalermrujinanant, C; Michowski, W; Sittithumcharee, G; Esashi, F; Jirawatnotai, S

    2016-06-01

    BRCA2 has an important role in the maintenance of genome stability by interacting with RAD51 recombinase through its C-terminal domain. This interaction is abrogated by cyclin A-CDK2-mediated phosphorylation of BRCA2 at serine 3291 (Ser3291). Recently, we showed that cyclin D1 facilitates RAD51 recruitment to BRCA2-containing DNA repair foci, and that downregulation of cyclin D1 leads to inefficient homologous-mediated DNA repair. Here, we demonstrate that cyclin D1, via amino acids 20-90, interacts with the C-terminal domain of BRCA2, and that this interaction is increased in response to DNA damage. Interestingly, CDK4-cyclin D1 does not phosphorylate Ser3291. Instead, cyclin D1 bars cyclin A from the C-terminus of BRCA2, prevents cyclin A-CDK2-dependent Ser3291 phosphorylation and facilitates RAD51 binding to the C-terminal domain of BRCA2. These findings indicate that the interplay between cyclin D1 and other cyclins such as cyclin A regulates DNA integrity through RAD51 interaction with the BRCA2 C-terminal domain. PMID:26387543

  2. A Presumptive Developmental Role for a Sea Urchin Cyclin B Splice Variant

    PubMed Central

    Lozano, Jean-Claude; Schatt, Philippe; Marquès, François; Peaucellier, Gérard; Fort, Philippe; Féral, Jean-Pierre; Genevière, Anne-Marie; Picard, André

    1998-01-01

    We show that a splice variant–derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation. PMID:9442104

  3. ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination

    PubMed Central

    Capecchi, Mario R.; Pozner, Amir

    2016-01-01

    We generate a mouse model for the human microcephaly syndrome by mutating the ASPM locus, and demonstrate a premature exhaustion of the neuronal progenitor pool due to dysfunctional self-renewal processes. Earlier studies have linked ASPM mutant progenitor excessive cell cycle exit to a mitotic orientation defect. Here, we demonstrate a mitotic orientation-independent effect of ASPM on cell cycle duration. We pinpoint the cell fate-determining factor to the length of time spent in early G1 before traversing the restriction point. Characterization of the molecular mechanism reveals an interaction between ASPM and the Cdk2/Cyclin E complex, regulating the Cyclin activity by modulating its ubiquitination, phosphorylation and localization into the nucleus, before the cell is fated to transverse the restriction point. Thus, we reveal a novel function of ASPM in mediating the tightly coordinated Ubiquitin- Cyclin E- Retinoblastoma- E2F bistable-signalling pathway controlling restriction point progression and stem cell maintenance. PMID:26581405

  4. ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination.

    PubMed

    Capecchi, Mario R; Pozner, Amir

    2015-01-01

    We generate a mouse model for the human microcephaly syndrome by mutating the ASPM locus, and demonstrate a premature exhaustion of the neuronal progenitor pool due to dysfunctional self-renewal processes. Earlier studies have linked ASPM mutant progenitor excessive cell cycle exit to a mitotic orientation defect. Here, we demonstrate a mitotic orientation-independent effect of ASPM on cell cycle duration. We pinpoint the cell fate-determining factor to the length of time spent in early G1 before traversing the restriction point. Characterization of the molecular mechanism reveals an interaction between ASPM and the Cdk2/Cyclin E complex, regulating the Cyclin activity by modulating its ubiquitination, phosphorylation and localization into the nucleus, before the cell is fated to transverse the restriction point. Thus, we reveal a novel function of ASPM in mediating the tightly coordinated Ubiquitin- Cyclin E- Retinoblastoma- E2F bistable-signalling pathway controlling restriction point progression and stem cell maintenance. PMID:26581405

  5. A presumptive developmental role for a sea urchin cyclin B splice variant.

    PubMed

    Lozano, J C; Schatt, P; Marquès, F; Peaucellier, G; Fort, P; Féral, J P; Genevière, A M; Picard, A

    1998-01-26

    We show that a splice variant-derived cyclin B is produced in sea urchin oocytes and embryos. This splice variant protein lacks highly conserved sequences in the COOH terminus of the protein. It is found strikingly abundant in growing oocytes and cells committed to differentiation during embryogenesis. Cyclin B splice variant (CBsv) protein associates weakly in the cell with Xenopus cdc2 and with budding yeast CDC28p. In contrast to classical cyclin B, CBsv very poorly complements a triple CLN deletion in budding yeast, and its microinjection prevents an initial step in MPF activation, leading to an important delay in oocyte meiosis reinitiation. CBsv microinjection in fertilized eggs induces cell cycle delay and abnormal development. We assume that CBsv is produced in growing oocytes to keep them in prophase, and during embryogenesis to slow down cell cycle in cells that will be committed to differentiation. PMID:9442104

  6. Cyclin D3-dependent control of the dNTP pool and HIV-1 replication in human macrophages.

    PubMed

    Ruiz, Alba; Pauls, Eduardo; Badia, Roger; Torres-Torronteras, Javier; Riveira-Muñoz, Eva; Clotet, Bonaventura; Martí, Ramon; Ballana, Ester; Esté, José A

    2015-01-01

    Cyclins control the activation of cyclin-dependent kinases (CDK), which in turn, control the cell cycle and cell division. Intracellular availability of deoxynucleotides (dNTP) plays a fundamental role in cell cycle progression. SAM domain and HD domain-containing protein 1 (SAMHD1) degrades nucleotide triphosphates and controls the size of the dNTP pool. SAMHD1 activity appears to be controlled by CDK. Here, we show that knockdown of cyclin D3 a partner of CDK6 and E2 a partner of CDK2 had a major impact in SAMHD1 phosphorylation and inactivation and led to decreased dNTP levels and inhibition of HIV-1 at the reverse transcription step in primary human macrophages. The effect of cyclin D3 RNA interference was lost after degradation of SAMHD1 by HIV-2 Vpx, demonstrating the specificity of the mechanism. Cyclin D3 inhibition correlated with decreased activation of CDK2. Our results confirm the fundamental role of the CDK6-cyclin D3 pair in controlling CDK2-dependent SAMHD1 phosphorylation and dNTP pool in primary macrophages. PMID:25927932

  7. Molecular dynamic behavior and binding affinity of flavonoid analogues to the cyclin dependent kinase 6/cyclin D complex.

    PubMed

    Khuntawee, Wasinee; Rungrotmongkol, Thanyada; Hannongbua, Supot

    2012-01-23

    The cyclin dependent kinases (CDKs), each with their respective regulatory partner cyclin that are involved in the regulation of the cell cycle, apoptosis, and transcription, are potentially interesting targets for cancer therapy. The CDK6 complex with cyclin D (CDK6/cycD) drives cellular proliferation by phosphorylation of specific key target proteins. To understand the flavonoids that inhibit the CDK6/cycD functions, molecular dynamics simulations (MDSs) were performed on three inhibitors, fisetin (FST), apigenin (AGN), and chrysin (CHS), complexed with CDK6/cycD, including the two different binding orientations of CHS: FST-like (CHS_A) and deschloro-flavopiridol-like (CHS_B). For all three inhibitors, including both CHS orientations, the conserved interaction between the 4-keto group of the flavonoid and the backbone V101 nitrogen of CDK6 was strongly detected. The 3'- and 4'-OH groups on the flavonoid phenyl ring and the 3-OH group on the benzopyranone ring of inhibitor were found to significantly increase the binding and inhibitory efficiency. Besides the electrostatic interactions, especially through hydrogen bond formation, the van der Waals (vdW) interactions with the I19, V27, F98, H100, and L152 residues of CDK6 are also important factors in the binding efficiency of flavonoids against the CDK6/cycD complex. On the basis of the docking calculation and MM-PBSA method, the order of the predicted inhibitory affinities of these three inhibitors toward the CDK6/cycD was FST > AGN > CHS, which is in good agreement with the experimental data. In addition, CHS preferentially binds to the active CDK6 in a different orientation to FST and AGN but similar to its related analog, deschloro-flavopiridol. The obtained results are useful as the basic information for the further design of potent anticancer drugs specifically targeting the CDK6 enzyme. PMID:22172011

  8. Sesamin, a lignan of sesame, down-regulates cyclin D1 protein expression in human tumor cells.

    PubMed

    Yokota, Tomoya; Matsuzaki, Youichirou; Koyama, Makoto; Hitomi, Toshiaki; Kawanaka, Mayumi; Enoki-Konishi, Masako; Okuyama, Yusuke; Takayasu, Junko; Nishino, Hoyoku; Nishikawa, Akiyoshi; Osawa, Toshihiko; Sakai, Toshiyuki

    2007-09-01

    Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF-7. Furthermore, sesamin dephosphorylates tumor-suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF-7 cell proliferation by sesamin is correlated with down-regulated cyclin D1 protein expression, a proto-oncogene that is overexpressed in many human cancer cells. It was found that sesamin-induced down-regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down-regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down-regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent. PMID:17640297

  9. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    PubMed

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers. PMID:17437483

  10. Cyclin B–Cdk1 inhibits protein phosphatase PP2A-B55 via a Greatwall kinase–independent mechanism

    PubMed Central

    Okumura, Eiichi; Morita, Atsushi; Wakai, Mizuho; Mochida, Satoru; Hara, Masatoshi

    2014-01-01

    Entry into M phase is governed by cyclin B–Cdk1, which undergoes both an initial activation and subsequent autoregulatory activation. A key part of the autoregulatory activation is the cyclin B–Cdk1–dependent inhibition of the protein phosphatase 2A (PP2A)–B55, which antagonizes cyclin B–Cdk1. Greatwall kinase (Gwl) is believed to be essential for the autoregulatory activation because Gwl is activated downstream of cyclin B–Cdk1 to phosphorylate and activate α-endosulfine (Ensa)/Arpp19, an inhibitor of PP2A-B55. However, cyclin B–Cdk1 becomes fully activated in some conditions lacking Gwl, yet how this is accomplished remains unclear. We show here that cyclin B–Cdk1 can directly phosphorylate Arpp19 on a different conserved site, resulting in inhibition of PP2A-B55. Importantly, this novel bypass is sufficient for cyclin B–Cdk1 autoregulatory activation. Gwl-dependent phosphorylation of Arpp19 is nonetheless necessary for downstream mitotic progression because chromosomes fail to segregate properly in the absence of Gwl. Such a biphasic regulation of Arpp19 results in different levels of PP2A-B55 inhibition and hence might govern its different cellular roles. PMID:24616226

  11. Isolation, characterization and expression of cyclin and cyclin-dependent kinase genes in Jerusalem artichoke (Helianthus tuberosus L.).

    PubMed

    Freeman, Donna; Riou-Khamlichi, Catherine; Oakenfull, E Ann; Murray, James A H

    2003-01-01

    Tuber explants of Jerusalem artichoke (Helianthus tuberosus L.) are a model system for cell-cycle re-entry from a quiescent state, involving the activation of division of tuber parenchyma cells in response to exogenous auxin. To enable molecular studies of this system, two cyclin (Heltu;CYCD1;1 and Heltu; CYCD3;1) and two cyclin-dependent kinase (Heltu; CDKA;1 and Heltu;CDKB1;1) genes have been isolated from a Jerusalem artichoke cDNA library and their expression demonstrated during the activation of cell division. It was found that CDKA;1 transcripts are present in quiescent tubers, whereas CYCD1;1, CYCD3;1 and CDKB1;1 transcripts are induced during cell-cycle re-entry as well as during bud growth of whole tubers. Both CYCD1;1 and CYCD3;1 transcripts appear shortly before, or coincident with, the onset of S phase. PMID:12493857

  12. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    SciTech Connect

    Lee, Juhyung; Yun, Nuri; Kim, Chiho; Song, Min-Young; Park, Kang-Sik; Oh, Young J.

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  13. AKAP95 promotes cell cycle progression via interactions with cyclin E and low molecular weight cyclin E

    PubMed Central

    Kong, Xiang-Yu; Zhang, Deng-Cheng; Zhuang, Wen-Xin; Hua, Su-Hang; Dai, Yue; Yuan, Yang-Yang; Feng, Li-Li; Huang, Qian; Teng, Bo-Gang; Yu, Xiu-Yi; Liu, Wen-Zhi; Zhang, Yong-Xing

    2016-01-01

    AKAP95 in lung cancer tissues showed higher expression than in paracancerous tissues. AKAP95 can bind with cyclin D and cyclin E during G1/S cell cycle transition, but its molecular mechanisms remain unclear. To identify the mechanism of AKAP95 in cell cycle progression, we performed AKAP95 transfection and silencing in A549 cells, examined AKAP95, cyclin E1 and cyclin E2 expression, and the interactions of AKAP95 with cyclins E1 and E2. Results showed that over-expression of AKAP95 promoted cell growth and AKAP95 bound cyclin E1 and E2, low molecular weight cyclin E1 (LWM-E1) and LWM-E2. Additionally AKAP95 bound cyclin E1 and LMW-E2 in the nucleus during G1/S transition, bound LMW-E1 during G1, S and G2/M, and bound cyclin E2 mainly on the nuclear membrane during interphase. Cyclin E2 and LMW-E2 were also detected. AKAP95 over-expression increased cyclin E1 and LMW-E2 expression but decreased cyclin E2 levels. Unlike cyclin E1 and LMW-E2 that were nuclear located during the G1, S and G1/S phases, cyclin E2 and LMW-E1 were expressed in all cell cycle phases, with cyclin E2 present in the cytoplasm and nuclear membrane, with traces in the nucleus. LMW-E1 was present in both the cytoplasm and nucleus. The 20 kDa form of LMW-E1 showed only cytoplasmic expression, while the 40 kDa form was nuclear expressed. The expression of AKAP95, cyclin E1, LMW-E1 and -E2, might be regulated by cAMP. We conclude that AKAP95 might promote cell cycle progression by interacting with cyclin E1 and LMW-E2. LMW-E2, but not cyclin E2, might be involved in G1/S transition. The binding of AKAP95 and LMW-E1 was found throughout cell cycle. PMID:27158371

  14. Participation of cyclin A in Myc-induced apoptosis.

    PubMed Central

    Hoang, A T; Cohen, K J; Barrett, J F; Bergstrom, D A; Dang, C V

    1994-01-01

    The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B, C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Rat1a fibroblasts. We further demonstrated that Zn(2+)-inducible cyclin A expression was sufficient to cause apoptosis. When Myc-induced apoptosis was blocked by coexpression of Bcl-2, the levels of cyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptosis by coexpressed Bcl-2 is associated with a complementary increase in cyclin C, D1, and E mRNAs. Images PMID:8041712

  15. Arsenic trioxide-mediated growth inhibition in gallbladder carcinoma cells via down-regulation of Cyclin D1 transcription mediated by Sp1 transcription factor

    SciTech Connect

    Ai, Zhilong; Lu, Weiqi; Ton, Saixiong; Liu, Houbao; Sou, Tao; Shen, Zhenbin; Qin, Xinyu . E-mail: smc_jjh@yahoo.com.cn

    2007-08-31

    Gallbladder carcinoma (GBC), an aggressive and mostly lethal malignancy, is known to be resistant to a number of drug stimuli. Here, we demonstrated that arsenic trioxide inhibited the proliferation of gallbladder carcinoma in vivo and in vitro as well as the transcription of cell cycle-related protein Cyclin D1. And, Cyclin D1 overexpression inhibited the negative role of arsenic trioxide in cell cycle progression. We further explored the mechanisms by which arsenic trioxide affected Cyclin D1 transcription and found that the Sp1 transcription factor was down-regulated by arsenic trioxide, with a corresponding decrease in Cyclin D1 promoter activity. Taken together, these results suggested that arsenic trioxide inhibited gallbladder carcinoma cell proliferation via down-regulation of Cyclin D1 transcription in a Sp1-dependent manner, which provided a new mechanism of arsenic trioxide-involved cell proliferation and may have important therapeutic implications in gallbladder carcinoma patients.

  16. A jekyll and hyde role of cyclin E in the genotoxic stress response: switching from cell cycle control to apoptosis regulation.

    PubMed

    Mazumder, Suparna; Plesca, Dragos; Almasan, Alexandru

    2007-06-15

    Cyclin E protein levels and associated kinase activity rise in late G(1) phase, reach a peak at the G(1)/S transition, and quickly decline during S phase. The cyclin E/Cdk2 complex has a well-established function in regulating two fundamental biological processes: cell cycle progression and DNA replication. However, cyclin E expression is deregulated in a wide range of tumors. Our recent reports have uncovered a critical role for cyclin E, independent of Cdk2, in the cell death of hematopoietic tumor cells exposed to genotoxic stress. An 18-kD C-terminal fragment of cyclin E, p18-cyclin E, which is generated by caspase-mediated cleavage in hematopoietic cells during genotoxic stress-induced apoptosis has a critical role in the amplification of the intrinsic apoptotic pathway. By interacting with Ku70, p18-cyclin E liberates Bax, which participates in the amplification of apoptosis by sustaining a positive feedback loop targeting mitochondria. This process is independent of p53 function and new RNA or protein synthesis. Therefore, cyclin E emerges as an arbiter of the genotoxic stress response by regulating a finite physiological balance between cell proliferation and death in hematopoietic cells. PMID:17581275

  17. Selective repression of the oncogene cyclin D1 by the tumor suppressor miR-206 in cancers.

    PubMed

    Elliman, S J; Howley, B V; Mehta, D S; Fearnhead, H O; Kemp, D M; Barkley, L R

    2014-01-01

    MicroRNAs (miRNAs) are deregulated in cancer and have been shown to exhibit both oncogenic and tumor suppressive functions. Although the functional effects of several miRNAs have been elucidated, those of many remain to be discovered. In silico analysis identified microRNA-206 (miR-206) binding sites in the 3'-untranslated regions (3'-UTR) of both the mouse and human CCND1 gene. Cyclin D1 is a recognized oncogene involved in direct phosphorylation of the retinoblastoma (Rb) protein and promoting cell cycle transition from G1 to S. miR-206 specifically binds to the CCND1 3'-UTR and mediates reduction of both cyclin D1 protein and mRNA. Expression of miR-206 induced a G1 arrest and a decrease in cell proliferation in breast cancer cells. Ectopic expression of miRNA-resistant cyclin D1 was able to reverse the miR-206-induced decrease in cell proliferation. Therefore, we identified miR-206 as an activator of cell cycle arrest resulting in a decrease in cell proliferation that is dependent on the inhibition of cyclin D1. Interestingly, prostatic cancer (PCa) cells express low levels of miR-206 resulting in deregulated cyclin D1 expression compared with non-transformed primary prostatic epithelial cells (PrEC). Finally, we demonstrate that cyclin D1 is regulated by miR-206 in PrEC but not in PCa cells and this is due to the absence of a CCND1 3'-UTR in these cells. This suggests that miR-206-based anti-cyclin D1 targeted therapy would be beneficial in cancers where cyclin D1 is overexpressed and contains a 3'-UTR. PMID:25111862

  18. Hog1 Targets Whi5 and Msa1 Transcription Factors To Downregulate Cyclin Expression upon Stress

    PubMed Central

    González-Novo, Alberto; Jiménez, Javier; Clotet, Josep; Nadal-Ribelles, Mariona; Cavero, Santiago

    2015-01-01

    Yeast cells have developed complex mechanisms to cope with extracellular insults. An increase in external osmolarity leads to activation of the stress-activated protein kinase Hog1, which is the main regulator of adaptive responses, such as gene expression and cell cycle progression, that are essential for cellular survival. Upon osmostress, the G1-to-S transition is regulated by Hog1 through stabilization of the cyclin-dependent kinase inhibitor Sic1 and the downregulation of G1 cyclin expression by an unclear mechanism. Here, we show that Hog1 interacts with and phosphorylates components of the core cell cycle transcriptional machinery such as Whi5 and the coregulator Msa1. Phosphorylation of these two transcriptional regulators by Hog1 is essential for inhibition of G1 cyclin expression, for control of cell morphogenesis, and for maximal cell survival upon stress. The control of both Whi5 and Msa1 by Hog1 also revealed the necessity for proper coordination of budding and DNA replication. Thus, Hog1 regulates G1 cyclin transcription upon osmostress to ensure coherent passage through Start. PMID:25733686

  19. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1–dependent cellular transformation

    PubMed Central

    Alt, Jodi R.; Cleveland, John L.; Hannink, Mark; Diehl, J. Alan

    2000-01-01

    GSK-3β-dependent phosphorylation of cyclin D1 at Thr-286 promotes the nuclear-to-cytoplasmic redistribution of cyclin D1 during S phase of the cell cycle, but how phosphorylation regulates redistribution has not been resolved. For example, phosphorylation of nuclear cyclin D1 could increase its rate of nuclear export relative to nuclear import; alternatively, phosphorylation of cytoplasmic cyclin D1 by GSK-3β could inhibit nuclear import. Here, we report that GSK-3β-dependent phosphorylation promotes cyclin D1 nuclear export by facilitating the association of cyclin D1 with the nuclear exportin CRM1. D1-T286A, a cyclin D1 mutant that cannot be phosphorylated by GSK-3β, remains nuclear throughout the cell cycle, a consequence of its reduced binding to CRM1. Constitutive overexpression of the nuclear cyclin D1-T286A in murine fibroblasts results in cellular transformation and promotes tumor growth in immune compromised mice. Thus, removal of cyclin D1 from the nucleus during S phase appears essential for regulated cell division. PMID:11124803

  20. Cyclin Dl expression in B-cell non Hodgkin lymphoma.

    PubMed

    Aref, Salah; Mossad, Y; El-Khodary, T; Awad, M; El-Shahat, E

    2006-10-01

    Disorders of the cell cycle regulatory machinery play a key role in the pathogenesis of cancer. Over-expression of cyclin D1 protein has been reported in several solid tumors and certain lymphoid malignancies, but little is known about the effect of its expression on clinical behavior and outcome in B-cell Non-Hodgkin lymphoma (NHL). In this study, we investigated the expression of cyclin Dl in group of patients with NHL and correlated the results with the clinical and laboratory data. The degree of expression of cyclin Dl protein was evaluated by flow cytometry in a group of NHL patients (n = 46) and in normal control group (n = 10). Cyclin Dl over expression was detected in 10 out of 46 (21.7%) patients; they were 5/5-mantle cell lymphoma (MCL) (100%) and 5/28 large B-cell lymphoma (17.8%). All other NHL subtypes showed normal cyclin D1 expression. The clinical signs (hepatomegaly, splenomegaly and B-symptoms, clinical staging) and laboratory data (hemoglobin, white cell count (WBCs), platelet count, and bone marrow infiltration) were not significantly different between NHL subgroup with cyclin Dl over expression and that with normal cyclin Dl expression. Serum lactic dehydrogenase (LDH) levels and lymphadenopathy were significantly higher in NHL group with cyclin D1 over expression as compared to those without. Also, cyclin D1 over expression is associated with poor outcome of NHL patients. Cyclin Dl over expression was evident among all cases of MCL and few cases of large B-cell lymphoma. Cyclin Dl over expression might be used as adjuvant tool for diagnosis of MCL; has role in NHL biology and is bad prognostic index in NHL. PMID:17607588

  1. Phylogenetic analysis of CDK and cyclin proteins in premetazoan lineages

    PubMed Central

    2014-01-01

    Background The molecular history of animal evolution from single-celled ancestors remains a major question in biology, and little is known regarding the evolution of cell cycle regulation during animal emergence. In this study, we conducted a comprehensive evolutionary analysis of CDK and cyclin proteins in metazoans and their unicellular relatives. Results Our analysis divided the CDK family into eight subfamilies. Seven subfamilies (CDK1/2/3, CDK5, CDK7, CDK 20, CDK8/19, CDK9, and CDK10/11) are conserved in metazoans and fungi, with the remaining subfamily, CDK4/6, found only in eumetazoans. With respect to cyclins, cyclin C, H, L, Y subfamilies, and cyclin K and T as a whole subfamily, are generally conserved in animal, fungi, and amoeba Dictyostelium discoideum. In contrast, cyclin subfamilies B, A, E, and D, which are cell cycle-related, have distinct evolutionary histories. The cyclin B subfamily is generally conserved in D. discoideum, fungi, and animals, whereas cyclin A and E subfamilies are both present in animals and their unicellular relatives such as choanoflagellate Monosiga brevicollis and filasterean Capsaspora owczarzaki, but are absent in fungi and D. discoideum. Although absent in fungi and D. discoideum, cyclin D subfamily orthologs can be found in the early-emerging, non-opisthokont apusozoan Thecamonas trahens. Within opisthokonta, the cyclin D subfamily is conserved only in eumetazoans, and is absent in fungi, choanoflagellates, and the basal metazoan Amphimedon queenslandica. Conclusions Our data indicate that the CDK4/6 subfamily and eumetazoans emerged simultaneously, with the evolutionary conservation of the cyclin D subfamily also tightly linked with eumetazoan appearance. Establishment of the CDK4/6-cyclin D complex may have been the key step in the evolution of cell cycle control during eumetazoan emergence. PMID:24433236

  2. Enhanced tumor formation in cyclin D1 x transforming growth factor beta1 double transgenic mice with characterization by magnetic resonance imaging.

    PubMed

    Deane, Natasha G; Lee, Haakil; Hamaamen, Jalal; Ruley, Anna; Washington, M Kay; LaFleur, Bonnie; Thorgeirsson, Snorri S; Price, Ronald; Beauchamp, R Daniel

    2004-02-15

    Transgenic mice that overexpress cyclin D1 protein in the liver develop liver carcinomas with high penetrance. Transforming growth factor beta (TGF-beta) serves as either an epithelial cell growth inhibitor or a tumor promoter, depending on the cellular context. We interbred LFABP-cyclin D1 and Alb-TGF-beta1 transgenic mice to produce cyclin D1/TGF-beta1 double transgenic mice and followed the development of liver tumors over time, characterizing cellular and molecular changes, tumor incidence, tumor burden, and tumor physiology noninvasively by magnetic resonance imaging. Compared with age-matched LFABP-cyclin D1 single transgenic littermates, cyclin D1/TGF-beta1 mice exhibited a significant increase in tumor incidence. Tumor multiplicity, tumor burden, and tumor heterogeneity were higher in cyclin D1/TGF-beta1 mice compared with single transgenic littermates. Characteristics of cyclin D1/TGF-beta1 livers correlated with a marked induction of the peripheral periductal oval cell/stem cell compartment of the liver. A number of cancerous lesions from cyclin D1/TGF-beta1 mice exhibited unique features such as ductal plate malformations and hemorrhagic nodules. Some lesions were contiguous with the severely diseased background liver and, in some cases, replaced the normal architecture of the entire organ. Cyclin D1/TGF-beta1 lesions, in particular, were associated with malignant features such as areas of vascular invasion by hepatocytes and heterogeneous hyperintensity of signal on T2-weighted magnetic resonance imaging. These findings demonstrate that TGF-beta1 promotes stem cell activation and tumor progression in the context of cyclin D1 overexpression in the liver. PMID:14973059

  3. Transgenic expression of walleye dermal sarcoma virus rv-cyclin gene in zebrafish and its suppressive effect on liver tumor development after carcinogen treatment.

    PubMed

    Zhan, Huiqing; Spitsbergen, Jan M; Qing, Wei; Wu, Yi Lian; Paul, Thomas A; Casey, James W; Her, Guor Muor; Gong, Zhiyuan

    2010-11-01

    A retrovirus homologue gene of cellular cyclin D₁, walleye dermal sarcoma virus rv-cyclin gene (orf A or rv-cyclin), was expressed in the livers of zebrafish under the control of liver fatty acid-binding protein (lfabp) promoter. To prevent possible fatality caused by overexpression of the oncogene, the GAL4/upstream activation sequence (GAL4/UAS) system was used to maintain the transgenic lines. Thus, both GAL4-activator [Tg(lfabp:GAL4)] and UAS-effector [Tg(UAS:rvcyclin)] lines were generated, and the rv-cyclin gene was activated in the liver after crossing these two lines. Since no obvious neoplasia phenotypes were observed in the double-transgenic line, cancer susceptibility of the transgenic fish expressing rv-cyclin was tested by carcinogen treatment. Unexpectedly, transgenic fish expressing rv-cyclin gene (rvcyclin+) were more resistant to the carcinogen than siblings not expressing this gene (rvcyclin-). Lower incidences of multiple and malignant liver tumors were observed in rvcyclin+ than in rvcyclin- fish, and the liver tumors in the rvcyclin+ group appeared later and were less malignant. These results suggest that expression of rv-cyclin protects the fish liver from carcinogen damage and delays onset of malignancy. These findings indicate that transgenic fish models are powerful systems for investigating mechanisms of inhibition and regression of liver tumors. PMID:20052603

  4. An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts.

    PubMed Central

    Lee, T H; Kirschner, M W

    1996-01-01

    The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition. Images Fig. 1 Fig. 3 Fig. 4 PMID:8552637

  5. NPAT links cyclin E-Cdk2 to the regulation of replication-dependent histone gene transcription.

    PubMed

    Zhao, J; Kennedy, B K; Lawrence, B D; Barbie, D A; Matera, A G; Fletcher, J A; Harlow, E

    2000-09-15

    In eukaryotic cells, histone gene expression is one of the major events that mark entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. Here we show that NPAT, a substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. We demonstrate that NPAT activates histone gene transcription and that this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, our results both show that NPAT is involved in a key S phase event and provide a link between the cell cycle machinery and activation of histone gene transcription. PMID:10995386

  6. Cannabinoids Regulate Bcl-2 and Cyclin D2 Expression in Pancreatic β Cells

    PubMed Central

    Kim, Jung Seok; Rho, Jun Gi; Shin, Jung Jae; Song, Woo Keun; Lee, Eun Kyung; Egan, Josephine M.; Kim, Wook

    2016-01-01

    Recent reports have shown that cannabinoid 1 receptors (CB1Rs) are expressed in pancreatic β cells, where they induce cell death and cell cycle arrest by directly inhibiting insulin receptor activation. Here, we report that CB1Rs regulate the expression of the anti-apoptotic protein Bcl-2 and cell cycle regulator cyclin D2 in pancreatic β cells. Treatment of MIN6 and βTC6 cells with a synthetic CB1R agonist, WIN55,212–2, led to a decrease in the expression of Bcl-2 and cyclin D2, in turn inducing cell cycle arrest in G0/G1 phase and caspase-3-dependent apoptosis. Additionally, genetic deletion and pharmacological blockade of CB1Rs after injury in mice led to increased levels of Bcl-2 and cyclin D2 in pancreatic β cells. These findings provide evidence for the involvement of Bcl-2 and cyclin D2 mediated by CB1Rs in the regulation of β-cell survival and growth, and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and growth in diabetes. PMID:26967640

  7. CyclinB1/Cdk1 Coordinates Mitochondrial Respiration for Cell Cycle G2/M Progression

    PubMed Central

    Wang, Zhaoqing; Fan, Ming; Candas, Demet; Zhang, Tie-Qiao; Eldridge, Angela; Wachsmann-Hogiu, Sebastian; Ahmed, Kazi M.; Chromy, Brett A.; Nantajit, Danupon; Duru, Nadire; He, Fuchu; Chen, Min; Finkel, Toren; Weinstein, Lee S.; Li, Jian Jian

    2014-01-01

    SUMMARY A substantial amount of mitochondrial energy is required for cell cycle progression. However, the mechanisms coordinating the mitochondrial respiration with G2/M transition, a critical step in cell division, remains to be elucidated. Here we show that a fraction of cell cycle CyclinB1/Cdk1 proteins localizes into the matrix of mitochondria and phosphorylates a cluster of mitochondrial proteins including the complex I (CI) subunits in the respiratory chain. The CyclinB1/Cdk1-mediated CI subunit phosphorylation enhances CI activity, whereas deficiency of such phosphorylation in each of the relevant CI subunits results in impairment of CI function. Mitochondria-targeted CyclinB1/Cdk1 increases mitochondrial respiration with enhanced oxygen consumption and ATP generation, which provides cells with efficient bioenergy for G2/M transition and shortens overall cycling time. Thus, CyclinB1/Cdk1-mediated phosphorylation of mitochondrial substrates allows cells to sense and respond to an increased energy demand for G2/M transition, and subsequently to up-regulate mitochondrial respiration for a successful cell cycle progression. PMID:24746669

  8. Paradoxical roles of cyclin D1 in DNA stability.

    PubMed

    Jirawatnotai, Siwanon; Sittithumcharee, Gunya

    2016-06-01

    Maintenance of DNA integrity is vital for all of the living organisms. Consequence of DNA damaging ranges from, introducing harmless synonymous mutations, to causing disease-associated mutations, genome instability, and cell death. A cell cycle protein cyclin D1 is an established cancer-driving protein. However, contribution of cyclin D1 to cancer formation and cancer survival is not entirely known. In cancer tissues, overexpression of cyclin D1 is associated with both cancer genome instability, and resistance to DNA-damaging cancer drugs. Emerging evidence indicated that cyclin D1 may play novel direct roles in regulating DNA repair. Here we provide an insight how cyclin D1 expression may contribute to DNA repair and chromosome instability, and how these functions may facilitate cancer formation, and drug resistance. PMID:27155130

  9. Cyclin D2 and the CDK substrate p220(NPAT) are required for self-renewal of human embryonic stem cells.

    PubMed

    Becker, Klaus A; Ghule, Prachi N; Lian, Jane B; Stein, Janet L; van Wijnen, Andre J; Stein, Gary S

    2010-02-01

    Self-renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB-dependent growth control. We investigated whether self-renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220(NPAT)/HiNF-P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220(NPAT) causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220(NPAT), decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220(NPAT) are principal cell cycle regulators that determine competency for self-renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220(NPAT)/HiNF-P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. PMID:19890848

  10. Essential Roles of Cyclin Y-Like 1 and Cyclin Y in Dividing Wnt-Responsive Mammary Stem/Progenitor Cells

    PubMed Central

    Li, Shan; Wang, Wenjuan; Li, Yaping; Chen, Jiangye; Zhu, Xueliang; Zeng, Yi Arial

    2016-01-01

    Cyclin Y family can enhance Wnt/β-catenin signaling in mitosis. Their physiological roles in mammalian development are yet unknown. Here we show that Cyclin Y-like 1 (Ccnyl1) and Cyclin Y (Ccny) have overlapping function and are crucial for mouse embryonic development and mammary stem/progenitor cell functions. Double knockout of Ccnys results in embryonic lethality at E16.5. In pubertal development, mammary terminal end buds robustly express Ccnyl1. Depletion of Ccnys leads to reduction of Lrp6 phosphorylation, hampering β-catenin activities and abolishing mammary stem/progenitor cell expansion in vitro. In lineage tracing experiments, Ccnys-deficient mammary cells lose their competitiveness and cease to contribute to mammary development. In transplantation assays, Ccnys-deficient mammary cells fail to reconstitute, whereas constitutively active β-catenin restores their regeneration abilities. Together, our results demonstrate the physiological significance of Ccnys-mediated mitotic Wnt signaling in embryonic development and mammary stem/progenitor cells, and reveal insights in the molecular mechanisms orchestrating cell cycle progression and maintenance of stem cell properties. PMID:27203244

  11. Essential Roles of Cyclin Y-Like 1 and Cyclin Y in Dividing Wnt-Responsive Mammary Stem/Progenitor Cells.

    PubMed

    Zeng, Liyong; Cai, Cheguo; Li, Shan; Wang, Wenjuan; Li, Yaping; Chen, Jiangye; Zhu, Xueliang; Zeng, Yi Arial

    2016-05-01

    Cyclin Y family can enhance Wnt/β-catenin signaling in mitosis. Their physiological roles in mammalian development are yet unknown. Here we show that Cyclin Y-like 1 (Ccnyl1) and Cyclin Y (Ccny) have overlapping function and are crucial for mouse embryonic development and mammary stem/progenitor cell functions. Double knockout of Ccnys results in embryonic lethality at E16.5. In pubertal development, mammary terminal end buds robustly express Ccnyl1. Depletion of Ccnys leads to reduction of Lrp6 phosphorylation, hampering β-catenin activities and abolishing mammary stem/progenitor cell expansion in vitro. In lineage tracing experiments, Ccnys-deficient mammary cells lose their competitiveness and cease to contribute to mammary development. In transplantation assays, Ccnys-deficient mammary cells fail to reconstitute, whereas constitutively active β-catenin restores their regeneration abilities. Together, our results demonstrate the physiological significance of Ccnys-mediated mitotic Wnt signaling in embryonic development and mammary stem/progenitor cells, and reveal insights in the molecular mechanisms orchestrating cell cycle progression and maintenance of stem cell properties. PMID:27203244

  12. Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization.

    PubMed

    Renaudin, J P; Doonan, J H; Freeman, D; Hashimoto, J; Hirt, H; Inzé, D; Jacobs, T; Kouchi, H; Rouzé, P; Sauter, M; Savouré, A; Sorrell, D A; Sundaresan, V; Murray, J A

    1996-12-01

    The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3. PMID:9002599

  13. Epigenetically altered miR-193b targets cyclin D1 in prostate cancer

    PubMed Central

    Kaukoniemi, Kirsi M; Rauhala, Hanna E; Scaravilli, Mauro; Latonen, Leena; Annala, Matti; Vessella, Robert L; Nykter, Matti; Tammela, Teuvo L J; Visakorpi, Tapio

    2015-01-01

    Micro-RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR-193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR-193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR-193b targets cyclin D1 in prostate cancer. Our data show that miR-193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR-193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR-193b expression, the overexpression of miR-193b reduced CCND1mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR-193b decreased the phosphorylation level of RB, a target of the cyclin D1-CDK4/6 pathway. Moreover, according to a reporter assay, miR-193b targeted the 3’UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3’UTR. Immunohistochemical analysis of cyclin D1 showed that castration-resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone-naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR-193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Taken together, our data demonstrate that miR-193b targets cyclin D1 in prostate cancer. PMID:26129688

  14. Epigenetically altered miR-193b targets cyclin D1 in prostate cancer.

    PubMed

    Kaukoniemi, Kirsi M; Rauhala, Hanna E; Scaravilli, Mauro; Latonen, Leena; Annala, Matti; Vessella, Robert L; Nykter, Matti; Tammela, Teuvo L J; Visakorpi, Tapio

    2015-09-01

    Micro-RNAs (miRNA) are important regulators of gene expression and often differentially expressed in cancer and other diseases. We have previously shown that miR-193b is hypermethylated in prostate cancer (PC) and suppresses cell growth. It has been suggested that miR-193b targets cyclin D1 in several malignancies. Here, our aim was to determine if miR-193b targets cyclin D1 in prostate cancer. Our data show that miR-193b is commonly methylated in PC samples compared to benign prostate hyperplasia. We found reduced miR-193b expression (P < 0.05) in stage pT3 tumors compared to pT2 tumors in a cohort of prostatectomy specimens. In 22Rv1 PC cells with low endogenous miR-193b expression, the overexpression of miR-193b reduced CCND1 mRNA levels and cyclin D1 protein levels. In addition, the exogenous expression of miR-193b decreased the phosphorylation level of RB, a target of the cyclin D1-CDK4/6 pathway. Moreover, according to a reporter assay, miR-193b targeted the 3'UTR of CCND1 in PC cells and the CCND1 activity was rescued by expressing CCND1 lacking its 3'UTR. Immunohistochemical analysis of cyclin D1 showed that castration-resistant prostate cancers have significantly (P = 0.0237) higher expression of cyclin D1 compared to hormone-naïve cases. Furthermore, the PC cell lines 22Rv1 and VCaP, which express low levels of miR-193b and high levels of CCND1, showed significant growth retardation when treated with a CDK4/6 inhibitor. In contrast, the inhibitor had no effect on the growth of PC-3 and DU145 cells with high miR-193b and low CCND1 expression. Taken together, our data demonstrate that miR-193b targets cyclin D1 in prostate cancer. PMID:26129688

  15. Pharmacological inhibitors of cyclin-dependent kinases.

    PubMed

    Knockaert, Marie; Greengard, Paul; Meijer, Laurent

    2002-09-01

    Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription and differentiation in addition to functions in the nervous system. Deregulation of CDKs in various diseases has stimulated an intensive search for selective pharmacological inhibitors of these kinases. More than 50 inhibitors have been identified, among which >20 have been co-crystallized with CDK2. These inhibitors all target the ATP-binding pocket of the catalytic site of the kinase. The actual selectivity of most known CDK inhibitors, and thus the underlying mechanism of their cellular effects, is poorly known. Pharmacological inhibitors of CDKs are currently being evaluated for therapeutic use against cancer, alopecia, neurodegenerative disorders (e.g. Alzheimer's disease, amyotrophic lateral sclerosis and stroke), cardiovascular disorders (e.g. atherosclerosis and restenosis), glomerulonephritis, viral infections (e.g. HCMV, HIV and HSV) and parasitic protozoa (Plasmodium sp. and Leishmania sp.). PMID:12237154

  16. Tylophorine Analog DCB-3503 Inhibited Cyclin D1 Translation through Allosteric Regulation of Heat Shock Cognate Protein 70

    PubMed Central

    Wang, Ying; Lam, Wing; Chen, Shao-Ru; Guan, Fu-Lan; Dutchman, Ginger E.; Francis, Samson; Baker, David C.; Cheng, Yung-Chi

    2016-01-01

    Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3′-untranslated region (3′ UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3′ UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1. PMID:27596272

  17. Tylophorine Analog DCB-3503 Inhibited Cyclin D1 Translation through Allosteric Regulation of Heat Shock Cognate Protein 70.

    PubMed

    Wang, Ying; Lam, Wing; Chen, Shao-Ru; Guan, Fu-Lan; Dutchman, Ginger E; Francis, Samson; Baker, David C; Cheng, Yung-Chi

    2016-01-01

    Tylophorine analog DCB-3503 is a potential anticancer and immunosuppressive agent that suppresses the translation of cellular regulatory proteins, including cyclin D1, at the elongation step. However, the molecular mechanism underlying this phenomenon remains unknown. This study demonstrates that DCB-3503 preferentially binds to heat shock cognate protein 70 (HSC70), which is a determinant for cyclin D1 translation by binding to the 3'-untranslated region (3' UTR) of its mRNA. DCB-3503 allosterically regulates the ATPase and chaperone activities of HSC70 by promoting ATP hydrolysis in the presence of specific RNA binding motifs (AUUUA) of cyclin D1 mRNA. The suppression of cyclin D1 translation by DCB-3503 is not solely caused by perturbation of the homeostasis of microRNAs, although the microRNA processing complex is dissociated with DCB-3503 treatment. This study highlights a novel regulatory mechanism of protein translation with AUUUA motifs in the 3' UTR of mRNA by HSC70, and its activity can be allosterically modulated by DCB-3503. DCB-3503 may be used to treat malignancies, such as hepatocellular carcinoma or breast cancer with elevated expression of cyclin D1. PMID:27596272

  18. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation.

    PubMed

    Baumli, Sonja; Lolli, Graziano; Lowe, Edward D; Troiani, Sonia; Rusconi, Luisa; Bullock, Alex N; Debreczeni, Judit E; Knapp, Stefan; Johnson, Louise N

    2008-07-01

    The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis. PMID:18566585

  19. Transcriptional analysis of an E2F gene signature as a biomarker of activity of the cyclin-dependent kinase inhibitor PHA-793887 in tumor and skin biopsies from a phase I clinical study.

    PubMed

    Locatelli, Giuseppe; Bosotti, Roberta; Ciomei, Marina; Brasca, Maria G; Calogero, Raffaele; Mercurio, Ciro; Fiorentini, Francesco; Bertolotti, Matteo; Scacheri, Emanuela; Scaburri, Angela; Galvani, Arturo; Pesenti, Enrico; De Baere, Thierry; Soria, Jean-Charles; Lazar, Vladimir; Isacchi, Antonella

    2010-05-01

    A transcriptional signature of the pan-cyclin-dependent kinase (Cdk) inhibitor PHA-793887 was evaluated as a potential pharmacodynamic and/or response biomarker in tumor and skin biopsies from patients treated in a phase I clinical study. We first analyzed the expression of a number of known E2F-dependent genes that were predicted to be modulated after Cdk2 and Cdk4 inhibition in xenograft tumor and skin samples of mice treated with the compound. This panel of 58 selected genes was then analyzed in biopsies from seven patients treated with PHA-793887 in a phase I dose escalation clinical trial in solid tumors. Quantitative real-time PCR or microarray analyses were done in paired skin and tumor biopsies obtained at baseline and at cycle 1. Analysis by quantitative real-time PCR of the signature in skin biopsies of patients treated at three different doses showed significant transcriptional downregulation with a dose-response correlation. These data show that PHA-793887 modulates genes involved in cell cycle regulation and proliferation in a clinical setting. The observed changes are consistent with its mechanism of action and correlate with target modulation in skin and with clinical benefit in tumors. PMID:20423997

  20. DNA damage signaling guards against perturbation of cyclin D1 expression triggered by low-dose long-term fractionated radiation

    PubMed Central

    Shimura, T; Kobayashi, J; Komatsu, K; Kunugita, N

    2014-01-01

    Cyclin D1 expression is precisely controlled during cell-cycle progression. However, repeated exposure to low-dose fractionated radiation (FR) abrogates cell cycle-dependent cyclin D1 degradation by constitutive activation of AKT survival signaling in normal human fibroblasts. The resulting abnormal nuclear cyclin D1 accumulation induces defects in DNA replication and resulting DNA double-strand breaks, and is associated with induction of genomic instability in low-dose irradiated cells. Here, we investigated the role of DNA damage signaling against such perturbed cell-cycle control of cyclin D1 expression. Nuclear cyclin D1 accumulation was induced within 7 days after low-dose FR (0.01 Gy or 0.05 Gy per fraction) in ATM-deficient cells (AT5BIVA), but appeared later in AT5BIVA cells harboring human ATM cDNA. Thus, ATM prevents abnormal nuclear cyclin D1 accumulation at early time points after low-dose FR. We further demonstrated that ATM-mediated downregulation of protein phosphatase 2A activity caused activation of the AKT/cyclin D1 pathway after long-term FR. Perturbation of cyclin D1 expression induced Rad51 foci that indicate homologous recombination repair (HRR) in control cells, while ATM- and NBS1-deficient cells (GM7166) failed to induce Rad51 foci after long-term low-dose FR. After 21 days of FR, NBS1- and ATM-deficient cells showed a decrease in nuclear cyclin D1-positive cells, and an increase in apoptotic cells. Similarly, inhibition of ATM with KU55933 abrogated nuclear cyclin D1 accumulation by induction of apoptosis in ATM-complemented cells exposed to low-dose FR. In conclusion, we here demonstrate that ATM is involved in controlling cyclin D1 levels after low-dose FR. DNA damage signaling mitigates the harmful effects of low-dose long-term FR by suppression of cell death induced by perturbation of cyclin D1 expression. PMID:25486524

  1. The coffee diterpene kahweol suppresses the cell proliferation by inducing cyclin D1 proteasomal degradation via ERK1/2, JNK and GKS3β-dependent threonine-286 phosphorylation in human colorectal cancer cells.

    PubMed

    Park, Gwang Hun; Song, Hun Min; Jeong, Jin Boo

    2016-09-01

    Kahweol as a coffee-specific diterpene has been reported to exert anti-cancer properties. However, the mechanism responsible for the anti-cancer effects of kahweol is not fully understood. The main aim of this investigation was to determine the effect of kahweol on cell proliferation and the possible mechanisms in human colorectal cancer cells. Kahweol inhibited markedly the proliferation of human colorectal cancer cell lines such as HCT116, SW480. Kahweol decreased cyclin D1 protein level in HCT116 and SW480 cells. Contrast to protein levels, cyclin D1 mRNA level and promoter activity did not be changed by kahweol treatment. MG132 treatment attenuated kahweol-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in kahweol-treated cells. Kahweol increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by kahweol. Inhibition of ERK1/2 by PD98059, JNK by SP600125 or GSK3β by LiCl suppressed cyclin D1 phosphorylation and downregulation by kahweol. Furthermore, the inhibition of nuclear export by LMB attenuated cyclin D1 degradation by kahweol. In conclusion, kahweol-mediated cyclin D1 degradation may contribute to the inhibition of the proliferation in human colorectal cancer cells. PMID:27424123

  2. Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells.

    PubMed

    Muñiz, Luis C; Yehia, Ghassan; Mémin, Elisabeth; Ratnakar, Pillarisetty V A L; Molina, Carlos A

    2006-08-01

    Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis. PMID:16625003

  3. Targeting the AKT/GSK3{beta}/Cyclin D1/Cdk4 Survival Signaling Pathway for Eradication of Tumor Radioresistance Acquired by Fractionated Radiotherapy

    SciTech Connect

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Purpose: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3{beta}-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3{beta}/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Methods and Materials: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Results: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Conclusion: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3{beta}/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor

  4. Transgenic Expression of Cyclin-Dependent Kinase 4 Results in Epidermal Hyperplasia, Hypertrophy, and Severe Dermal Fibrosis

    PubMed Central

    Miliani de Marval, Paula L.; Gimenez-Conti, Irma B.; LaCava, Margaret; Martinez, Luis A.; Conti, Claudio J.; Rodriguez-Puebla, Marcelo L.

    2001-01-01

    In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27Kip1 to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression. PMID:11438484

  5. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells

    PubMed Central

    Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M.; Ballana, Ester

    2016-01-01

    Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

  6. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-proliferating Cells.

    PubMed

    Badia, Roger; Pujantell, Maria; Riveira-Muñoz, Eva; Puig, Teresa; Torres-Torronteras, Javier; Martí, Ramón; Clotet, Bonaventura; Ampudia, Rosa M; Vives-Pi, Marta; Esté, José A; Ballana, Ester

    2016-08-01

    Macrophages are a heterogeneous cell population strongly influenced by differentiation stimuli that become susceptible to HIV-1 infection after inactivation of the restriction factor SAMHD1 by cyclin-dependent kinases (CDK). Here, we have used primary human monocyte-derived macrophages differentiated through different stimuli to evaluate macrophage heterogeneity on cell activation and proliferation and susceptibility to HIV-1 infection. Stimulation of monocytes with GM-CSF induces a non-proliferating macrophage population highly restrictive to HIV-1 infection, characterized by the upregulation of the G1/S-specific cyclin D2, known to control early steps of cell cycle progression. Knockdown of cyclin D2, enhances HIV-1 replication in GM-CSF macrophages through inactivation of SAMHD1 restriction factor by phosphorylation. Co-immunoprecipitation experiments show that cyclin D2 forms a complex with CDK4 and p21, a factor known to restrict HIV-1 replication by affecting the function of the downstream cascade that leads to SAMHD1 deactivation. Thus, we demonstrate that cyclin D2 acts as regulator of cell cycle proteins affecting SAMHD1-mediated HIV-1 restriction in non-proliferating macrophages. PMID:27541004

  7. Cyclin-dependent kinase 2 protects podocytes from apoptosis

    PubMed Central

    Saurus, Pauliina; Kuusela, Sara; Dumont, Vincent; Lehtonen, Eero; Fogarty, Christopher L.; Lassenius, Mariann I.; Forsblom, Carol; Lehto, Markku; Saleem, Moin A.; Groop, Per-Henrik; Lehtonen, Sanna

    2016-01-01

    Loss of podocytes is an early feature of diabetic nephropathy (DN) and predicts its progression. We found that treatment of podocytes with sera from normoalbuminuric type 1 diabetes patients with high lipopolysaccharide (LPS) activity, known to predict progression of DN, downregulated CDK2 (cyclin-dependent kinase 2). LPS-treatment of mice also reduced CDK2 expression. LPS-induced downregulation of CDK2 was prevented in vitro and in vivo by inhibiting the Toll-like receptor (TLR) pathway using immunomodulatory agent GIT27. We also observed that CDK2 is downregulated in the glomeruli of obese Zucker rats before the onset of proteinuria. Knockdown of CDK2, or inhibiting its activity with roscovitine in podocytes increased apoptosis. CDK2 knockdown also reduced expression of PDK1, an activator of the cell survival kinase Akt, and reduced Akt phosphorylation. This suggests that CDK2 regulates the activity of the cell survival pathway via PDK1. Furthermore, PDK1 knockdown reduced the expression of CDK2 suggesting a regulatory loop between CDK2 and PDK1. Collectively, our data show that CDK2 protects podocytes from apoptosis and that reduced expression of CDK2 associates with the development of DN. Preventing downregulation of CDK2 by blocking the TLR pathway with GIT27 may provide a means to prevent podocyte apoptosis and progression of DN. PMID:26876672

  8. Differential targeting of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1, by chelators with anti-proliferative activity in a range of tumor cell-types

    PubMed Central

    Moussa, Rayan S.; Kovacevic, Zaklina; Richardson, Des R.

    2015-01-01

    Chelators such as 2-hydroxy-1-napthylaldehyde isonicotinoyl hydrazone (311) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) target tumor cell iron pools and inhibit proliferation. These agents also modulate multiple targets, one of which is the cyclin-dependent kinase inhibitor, p21. Hence, this investigation examined the mechanism of action of these compounds in targeting p21. All the chelators up-regulated p21 mRNA in the five tumor cell-types assessed. In contrast, examining their effect on total p21 protein levels, these agents induced either: (1) down-regulation in MCF-7 cells; (2) up-regulation in SK-MEL-28 and CFPAC-1 cells; or (3) had no effect in LNCaP and SK-N-MC cells. The nuclear localization of p21 was also differentially affected by the ligands depending upon the cell-type, with it being decreased in MCF-7 cells, but increased in SK-MEL-28 and CFPAC-1 cells. Further studies assessing the mechanisms responsible for these effects demonstrated that p21 expression was not correlated with p53 status, suggesting a p53-independent mechanism. Considering this, we examined proteins that modulate p21 independently of p53, namely NDRG1, MDM2 and ΔNp63. These studies demonstrated that a dominant negative MDM2 isoform (p75MDM2) closely resembled p21 expression in response to chelation in three cell lines. These data suggest MDM2 may be involved in the regulation of p21 by chelators. PMID:26335183

  9. Dual Targeting of the Cyclin/Rb/E2F and Mitochondrial Pathways in Mantle Cell Lymphoma with the Translation Inhibitor Silvestrol

    PubMed Central

    Alinari, Lapo; Prince, Courtney J.; Edwards, Ryan B.; Towns, William; Mani, Rajeswaran; Lehman, Amy; Zhang, Xiaoli; Jarjoura, David; Pan, Li; Kinghorn, A. Douglas; Grever, Michael R.; Baiocchi, Robert A.; Lucas, David M.

    2013-01-01

    Purpose During cell cycle progression, D-cyclins activate cyclin-dependent kinases (CDKs) 4/6 to inactivate Rb, permitting E2F1-mediated S-phase gene transcription. This critical pathway is typically deregulated in cancer, and novel inhibitory strategies would be effective in a variety of tumors. The protein synthesis inhibitor silvestrol has potent activity in B-cell leukemias via the mitochondrial pathway of apoptosis, and also reduces cyclin D1 expression in breast cancer and lymphoma cell lines. We hypothesized that this dual activity of silvestrol would make it especially effective in malignancies driven by aberrant cyclin D1 expression. Experimental Design Mantle Cell Lymphoma (MCL), characterized by elevated cyclin D1, was used as a model to test this approach. The cyclin D/Rb/E2F1 pathway was investigated in vitro using MCL cell lines and primary tumor cells. Silvestrol was also evaluated in vivo using an aggressive model of MCL. Results Silvestrol showed low nanomolar potency both in MCL cell lines and primary MCL tumor cells. D-cyclins were depleted with just 10 nM silvestrol at 16 hr, with subsequent reductions of phosphorylated Rb, E2F1 protein, and E2F1 target transcription. As demonstrated in other leukemias, silvestrol caused Mcl-1 depletion followed by mitochondrial depolarization and caspase-dependent apoptosis, effects not related to inhibition of CDK4/6. Silvestrol significantly (P<0.0001) prolonged survival in a MCL xenograft model without detectable toxicity. Conclusions These data indicate that silvestrol effectively targets the cyclin/CDK/Rb pathway, and additionally induces cytotoxicity via intrinsic apoptosis. This dual activity may be an effective therapeutic strategy in MCL and other malignancies. PMID:22791882

  10. Downregulation of cyclin D1-CDK4 protein in human embryonic lung fibroblasts (HELF) induced by silica is mediated through the ERK and JNK pathway.

    PubMed

    Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Gao, Ai; Du, Hongju; Ye, Meng; You, Baorong; Huang, Chuanshu; Shi, Xianglin

    2008-10-01

    Silica is a factor in the induction of acute injury and chronic pulmonary fibrosis. In 1996, silica was also listed as a human carcinogen by the International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved in its pathologic effects are not well understood. We found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica for 2h decreased cyclin D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Extracellular signal-regulated protein kinase (ERKs), c-Jun NH2-terminal amino kinase (JNKs), and p38 kinase, as well as their downstream transcription factor, AP-1, had different effects on the regulation of expression levels of cyclin D1 and CDK4 alterations induced by silica. Silica activates multiple signal transduction pathways involved in coordinating cellular responses to stress. We established the requirements for ERK and JNK, members of the mitogen-activated protein kinase (MAPK) family, in mediating G1 phase arrest of HELF induced by silica. Silica treatment activated ERK in a dose-dependent manner. AG126 (a chemical inhibitor of the ERK signaling pathway) and the dominant negative mutant of ERK2 (a molecular inhibitor of ERK2) prevented decreases in cyclin D1 and CDK4 expression levels. A chemical inhibitor of JNK, SP600125, prevented the decreased expression of both cyclin D1 and CDK4, whereas SB203580, a chemical inhibitor of p38, did not. Interestingly, curcumin prevented the decrease in DK4 expression, but not in cyclin D1. These results demonstrate that ERKs and JNKs are responsible for the decrease of cyclin D1 and CDK4 expression levels in HELF induced by silica. Activator protein-1 (AP-1) was responsible for the decrease of CDK4 expression level, but not that of cyclin D1. The findings help to explain the mechanisms of diseases induced by silica. PMID:18703151

  11. Fluorescence energy transfer monitoring of protein-protein interaction in human cells: the Cyclin T1-HIV1 Tat case.

    NASA Astrophysics Data System (ADS)

    Ferrari, Aldo; Cinelli, Riccardo A. G.; Pellegrini, Vittorio; Beltram, Fabio; Marcello, Alessandro; Tyagi, Mudit; Giacca, Mauro

    2001-03-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein promotes transcriptional elongation of viral RNAs. Here we show that human Cyclin T1 directly binds Tat in cultured cells. By mapping fluorescence resonance energy transfer (FRET) in different cellular compartments we shall present a quantitative analysis of this interaction. The matched tagging pair consists of two optically matched variants of the green fluorescent protein: the enhanced GFP and the blue fluorescent protein. Strong energy transfer was observed between Cyclin T1 and Tat both in the cytoplasm and in specific subnuclear regions. We shall argue that such high-resolution optical studies can provide significant new insight in molecular processes and demonstrate that, for the specific case-study presented, they lead to a model by which Tat recruits Cyclin T1 out of the nuclear compartments where the protein resides to promote transcriptional activation.

  12. Cyclin D1 expression in colorectal cancer is a favorable prognostic factor in men but not in women in a prospective, population-based cohort study

    PubMed Central

    2011-01-01

    Background Although colorectal cancer (CRC) is generally not considered to be a hormone-dependent malignancy, several sex-related differences in incidence, molecular characteristics and survival have been reported. Epidemiological studies have consistently shown that increased exposure to female sex hormones is associated with a lower risk of CRC in women, and cyclin D1, an important downstream effector in estrogen-mediated signaling, is commonly activated in CRC. In this study, we analyzed the prognostic significance of cyclin D1 expression in CRC, with particular reference to sex-related differences, in tumors from a large, prospective, population-based cohort. Methods Using tissue microarrays and immunohistochemistry, the fraction and intensity of cyclin D1 expression was evaluated in 527 incident CRC cases from the Malmö Diet and Cancer Study. The χ2 and Spearman's rho (ρ) tests were used for comparison of cyclin D1 expression and relevant clinicopathological characteristics. Kaplan-Meier analysis and Cox proportional hazards modeling were used to assess the effect of cyclin D1 expression on cancer-specific survival (CSS) in univariate and multivariate analysis, adjusted for established prognostic factors. Results Cyclin D1 intensity was significantly lower in male compared with female CRC (P = 0.018). In the full cohort, cyclin D1 expression was associated with a significantly prolonged CSS (hazard ratio (HR) = 0.69; 95% CI 0.49 to 0.96, P = 0.026) but subgroup analysis according to gender revealed a strongly accentuated prognostic effect of cyclin D1 in male CRC (HR = 0.48; 95% CI 0.31 to 0.74, P < 0.001), which was in contrast to female CRC, where cyclin D1 was not prognostic (HR = 1.05; 95% CI 0.62 to 1.78, P = 0.864) (Pinteraction = 0.024). The prognostic value of cyclin D1 was not retained in multivariate analysis, either in the full cohort or in male CRC. Conclusions Cyclin D1 expression is strongly associated with prolonged survival in male CRC

  13. Meiotic induction by Xenopus cyclin B is accelerated by coexpression with mosXe.

    PubMed Central

    Freeman, R S; Ballantyne, S M; Donoghue, D J

    1991-01-01

    We have investigated the relationship between Xenopus laevis c-mos (mosXe) and the cyclin B component of maturation-promoting factor. Microinjection of Xenopus oocytes with in vitro-synthesized RNAs encoding Xenopus cyclin B1 or cyclin B2 induces the progression of meiosis, characterized by germinal vesicle breakdown (GVBD). By preinjecting oocytes with a mosXe-specific antisense oligonucleotide, we show that GVBD induced by cyclin B does not require expression of the mosXe protein. GVBD induced by cyclin B proceeds significantly faster than GVBD induced by progesterone or MosXe. However, coinjection of RNAs encoding cyclin B1 or cyclin B2 with mosXe RNA results in a 2.5- to 3-fold acceleration in GVBD relative to that induced by cyclin B alone. This acceleration of GVBD does not correlate with changes in the level of cyclin B1 and cyclin B2 phosphorylation. Images PMID:1825350

  14. Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects.

    PubMed

    Levine, K; Oehlen, L J; Cross, F R

    1998-01-01

    The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits. PMID:9418876

  15. Cancer-testis antigen MAGE-C2 binds Rbx1 and inhibits ubiquitin ligase-mediated turnover of cyclin E

    PubMed Central

    Wang, Jingjing; Guo, Chengli; Li, Yan; Li, Bing; Zhang, Yu; Yin, Yanhui

    2015-01-01

    Cancer-testis antigen MAGE-C2 is normally expressed in testis but aberrantly expressed in various kinds of tumors. Its functions in tumor cells are mostly unknown. Here, we show that MAGE-C2 binds directly to the RING domain protein Rbx1, and participates in Skp1-Cullin1-F box protein (SCF) complex. Furthermore, MAGE-C2 can inhibit the E3 ubiquitin ligase activity of SCF complex. Ablation of endogenous MAGE-C2 decreases the level of cyclin E and accelerates cyclin E turnover by inhibiting ubiquitin-mediated proteasome degradation. Overexpression of MAGE-C2 increases the level of cyclin E and promotes G1-S transition and cell proliferation, and the results are further confirmed by knockdown of MAGE-C2. Overall, the study indicates that MAGE-C2 is involved in SCF complex and increases the stability of cyclin E in tumor cells. PMID:26540345

  16. Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly

    PubMed Central

    Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell

    2013-01-01

    A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds. PMID:23323521

  17. Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

    PubMed Central

    Dahmann, C.; Futcher, B.

    1995-01-01

    The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be much more important for meiosis than for mitosis and may play some special role in meiosis II. In contrast, CLB2 is important for mitosis but not meiosis. The level of Cdc28-Clb activity may be important in determining whether meiosis II will occur. PMID:7672594

  18. Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression

    SciTech Connect

    Boehm, Johann; Kaiser, Frank J.; Borozdin, Wiktor; Depping, Reinhard; Kohlhase, Juergen . E-mail: jkohlhase@humangenetik-freiburg.de

    2007-05-11

    Loss of function mutations in SALL4 cause Okihiro syndrome, an autosomal dominant disorder characterised by radial ray malformations associated with Duane anomaly. In zebrafish and mouse Sall4 interacts with TBX5 during limb and heart development and plays a crucial role for embryonic stem (ES) cell pluripotency. Here we report the nuclear interaction of murine Sall4 with Cyclin D1, one of the main regulators of G{sub 1} to S phase transition in cell cycle, verified by yeast two-hybrid assay, co-immunoprecipitation and intracellular co-localisation. Furthermore, using luciferase reporter gene assays we demonstrate that Sall4 operates as a transcriptional repressor located to heterochromatin and that this activity is modulated by Cyclin D1.

  19. Genome-Wide Analysis of the Cyclin Gene Family in Tomato

    PubMed Central

    Zhang, Tingyan; Wang, Xin; Lu, Yongen; Cai, Xiaofeng; Ye, Zhibiao; Zhang, Junhong

    2014-01-01

    Cyclins play important roles in cell division and cell expansion. They also interact with cyclin-dependent kinases to control cell cycle progression in plants. Our genome-wide analysis identified 52 expressed cyclin genes in tomato. Phylogenetic analysis of the deduced amino sequences of tomato and Arabidopsis cyclin genes divided them into 10 types, A-, B-, C-, D-, H-, L-, T-, U-, SDS- and J18. Pfam analysis indicated that most tomato cyclins contain a cyclin-N domain. C-, H- and J18 types only contain a cyclin-C domain, and U-type cyclins contain another potential cyclin domain. All of the cyclin genes are distributed throughout the tomato genome except for chromosome 8, and 30 of them were found to be segmentally duplicated; they are found on the duplicate segments of chromosome 1, 2, 3, 4, 5, 6, 10, 11 and 12, suggesting that tomato cyclin genes experienced a mass of segmental duplication. Quantitative real-time polymerase chain reaction analysis indicates that the expression patterns of tomato cyclin genes were significantly different in vegetative and reproductive stages. Transcription of most cyclin genes can be enhanced or repressed by exogenous application of gibberellin, which implies that gibberellin maybe a direct regulator of cyclin genes. The study presented here may be useful as a guide for further functional research on tomato cyclins. PMID:24366066

  20. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    SciTech Connect

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.

  1. Nuclear envelope breakdown may deliver an inhibitor of protein phosphatase 1 which triggers cyclin B translation in starfish oocytes.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Lozano, Jean-Claude; Peaucellier, Gérard; Picard, André

    2005-09-01

    In vertebrates, enhanced translation of mRNAs in oocytes and early embryos entering M-phase is thought to occur through polyadenylation, involving binding, hyperphosphorylation and proteolytic degradation of Aurora-activated CPEB. In starfish, an unknown component of the oocyte nucleus is required for cyclin B synthesis following the release of G2/prophase block by hormonal stimulation. We have found that CPEB cannot be hyperphosphorylated following hormonal stimulation in starfish oocytes from which the nucleus has been removed. Activation of Aurora kinase, known to interact with protein phosphatase 1 and its specific inhibitor Inh-2, is also prevented. The microinjection of Inh-2 restores Aurora activation, CPEB hyperphosphorylation and cyclin B translation in enucleated oocytes. Nevertheless, we provide evidence that CPEB is in fact hyperphosphorylated by cdc2, without apparent involvement of Aurora or MAP kinase, and that cyclin B synthesis can be stimulated without previous degradation of phosphorylated CPEB. Thus, the regulation of cyclin B synthesis necessary for progression through meiosis can be explained by an equilibrium between CPEB phosphorylation and dephosphorylation, and both aspects of this control may rely on the sole activation of Cdc2 and subsequent nuclear breakdown. PMID:16081061

  2. G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.

    PubMed Central

    Kudoh, T; Ishidate, T; Moriyama, M; Toyoshima, K; Akiyama, T

    1995-01-01

    WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block. Images Fig. 1 Fig. 2 PMID:7753836

  3. Prognostic value of MET, cyclin D1 and MET gene copy number in non-small cell lung cancer

    PubMed Central

    Sun, Wenze; Song, Liping; Ai, Ting; Zhang, Yingbing; Gao, Ying; Cui, Jie

    2013-01-01

    The aim of this study was to analyze the correlation of the expression of MET and cyclin D1 and MET gene copy number in non-small cell lung cancer (NSCLC) tissues and patient clinicopathologic characteristics and survival. Sixty-one NSCLC tissue specimens were included in the study. The expression of MET and cyclin D1 was evaluated by immunohistochemistry and MET gene copy number was assessed by quantitative real-time polymerase chain reaction (Q-PCR). Positive expression of MET and cyclin D1 protein and increased MET gene copy number occurred in 59.0%, 59.0% and 18.0% of 61 NSCLC tissues, respectively. MET-positivity correlated with poor differentiation (P = 0.009). Increased MET gene copy number was significantly associated with lymph node metastasis (P = 0.004) and advanced tumor stage (P = 0.048), while the expression of cyclin D1 was not associated with any clinicopathologic parameters. There was a significant correlation between the expression of MET and MET gene copy number (P = 0.002). Additionally, the expression of cyclin D1 had a significant association with the expression of MET as well as MET gene copy number (P = 0.002 and P = 0.017, respectively). MET-positivity and increased MET gene copy number were significantly associated with poor overall survival (P = 0.003 and P < 0.001, respectively) in univariate analysis. Multivariate Cox proportional hazard analysis confirmed that the expression of MET and MET gene copy number were prognostic indicators of NSCLC (P = 0.003 and P = 0.001, respectively). The overexpression of MET and the increased MET gene copy number might be adverse prognostic factors for NSCLC patients. The activation of the MET/cyclin D1 signaling pathway may contribute to carcinogenesis and the development of NSCLC, and may represent a target for therapy. PMID:23720678

  4. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    PubMed

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. PMID:25346443

  5. Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection

    PubMed Central

    Chen, Xuefeng; Niu, Hengyao; Yu, Yang; Wang, Jingjing; Zhu, Shuangyi; Zhou, Jianjie; Papusha, Alma; Cui, Dandan; Pan, Xuewen; Kwon, Youngho; Sung, Patrick; Ira, Grzegorz

    2016-01-01

    DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins. PMID:26801641

  6. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    PubMed Central

    Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.

    2015-01-01

    Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291

  7. Unexpected reduction of skin tumorigenesis on expression of cyclin-dependent kinase 6 in mouse epidermis.

    PubMed

    Wang, Xian; Sistrunk, Christopher; Rodriguez-Puebla, Marcelo L

    2011-01-01

    Cyclin-dependent kinases (CDKs) 4 and 6 are important regulators of the G(1) phase of the cell cycle, share 71% amino acid identity, and are expressed ubiquitously. As a result, it was assumed that each of these kinases plays a redundant role regulating normal and neoplastic proliferation. In previous reports, we have described the effects of CDK4 expression in transgenic mice, including the development of epidermal hyperplasia and increased malignant progression to squamous cell carcinoma. To study the role of CDK6 in epithelial growth and tumorigenesis, we generated transgenic mice carrying the CDK6 gene under the keratin 5 promoter (K5CDK6). Similar to K5CDK4 mice, epidermal proliferation increased substantially in K5CDK6 mice; however, no hyperplasia was observed. CDK6 overexpression also triggered keratinocyte apoptosis in interfollicular and follicular epidermis as a compensatory mechanism to override aberrant proliferation. Unexpectedly, CDK6 overexpression results in decreased skin tumor development compared with wild-type siblings. The inhibition in skin tumorigenesis was similar to that previously reported in K5-cyclin D3 mice. Furthermore, biochemical analysis of the K5CDK6 epidermis showed preferential complex formation between CDK6 and cyclin D3, suggesting that this particular complex plays an important role in tumor restraint. These studies provide in vivo evidence that CDK4 and CDK6 play a similar role as a mediator of keratinocyte proliferation but differ in apoptosis activation and skin tumor development. PMID:21224071

  8. Disruption of the G1/S Transition in Human Papillomavirus Type 16 E7-Expressing Human Cells Is Associated with Altered Regulation of Cyclin E

    PubMed Central

    Martin, Larry G.; Demers, G. William; Galloway, Denise A.

    1998-01-01

    The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F. PMID:9444990

  9. Cyclin D1 Is Transcriptionally Down-Regulated by ZO-2 via an E Box and the Transcription Factor c-Myc

    PubMed Central

    Huerta, Miriam; Muñoz, Rodrigo; Tapia, Rocío; Soto-Reyes, Ernesto; Ramírez, Leticia; Recillas-Targa, Félix; González-Mariscal, Lorenza

    2007-01-01

    Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2–repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1. PMID:17881732

  10. Cloning and sequence analysis of two cDNAs encoding cyclin A and cyclin B in the zebra mussel Dreissena polymorpha.

    PubMed

    Lamers, A E; Heiney, J P; Ram, J L

    1999-01-11

    Cyclins are key components in the progression of both mitotic and meiotic cell cycle control. Full-length cDNA clones encoding cyclin A and cyclin B were isolated from a zebra mussel testis cDNA library. The clones contained open reading frames of 419 and 434 amino acids, had similarity to cyclins A and B from other species, but also some unique features in their sequences. Cyclin A and B mRNA was expressed in testis, ovary, gill, mantle, muscle, and eggs, as shown by specific polymerase chain reaction. PMID:9990304

  11. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription.

    PubMed

    Hossain, Manzar; Stillman, Bruce

    2016-01-01

    Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment. PMID:27458800

  12. Two distinct mechanisms control the accumulation of cyclin B1 and Mos in Xenopus oocytes in response to progesterone.

    PubMed

    Frank-Vaillant, M; Jessus, C; Ozon, R; Maller, J L; Haccard, O

    1999-10-01

    Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA. PMID:10512866

  13. Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

    PubMed Central

    Frank-Vaillant, Marie; Jessus, Catherine; Ozon, René; Maller, James L.; Haccard, Olivier

    1999-01-01

    Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA. PMID:10512866

  14. Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

    PubMed Central

    Hossain, Manzar; Stillman, Bruce

    2016-01-01

    Newly born cells either continue to proliferate or exit the cell division cycle. This decision involves delaying expression of Cyclin E that promotes DNA replication. ORC1, the Origin Recognition Complex (ORC) large subunit, is inherited into newly born cells after it binds to condensing chromosomes during the preceding mitosis. We demonstrate that ORC1 represses Cyclin E gene (CCNE1) transcription, an E2F1 activated gene that is also repressed by the Retinoblastoma (RB) protein. ORC1 binds to RB, the histone methyltransferase SUV39H1 and to its repressive histone H3K9me3 mark. ORC1 cooperates with SUV39H1 and RB protein to repress E2F1-dependent CCNE1 transcription. In contrast, the ORC1-related replication protein CDC6 binds Cyclin E-CDK2 kinase and in a feedback loop removes RB from ORC1, thereby hyper-activating CCNE1 transcription. The opposing effects of ORC1 and CDC6 in controlling the level of Cyclin E ensures genome stability and a mechanism for linking directly DNA replication and cell division commitment. DOI: http://dx.doi.org/10.7554/eLife.12785.001 PMID:27458800

  15. Consequence of the tumor-associated conversion to cyclin D1b

    PubMed Central

    Augello, Michael A; Berman-Booty, Lisa D; Carr, Richard; Yoshida, Akihiro; Dean, Jeffry L; Schiewer, Matthew J; Feng, Felix Y; Tomlins, Scott A; Gao, Erhe; Koch, Walter J; Benovic, Jeffrey L; Diehl, John Alan; Knudsen, Karen E

    2015-01-01

    Clinical evidence suggests that cyclin D1b, a variant of cyclin D1, is associated with tumor progression and poor outcome. However, the underlying molecular basis was unknown. Here, novel models were created to generate a genetic switch from cyclin D1 to cyclin D1b. Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo. Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo. Further molecular interrogation uncovered unexpected links between cyclin D1b and the DNA damage/PARP1 regulatory networks, which could be exploited to suppress cyclin D1b-driven tumors. Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant. PMID:25787974

  16. Cyclin Y regulates the proliferation, migration, and invasion of ovarian cancer cells via Wnt signaling pathway.

    PubMed

    Liu, Haiyuan; Shi, Honghui; Fan, Qingbo; Sun, Xiangxiu

    2016-08-01

    This study is designated to investigate the roles of cyclin Y (CCNY) and Wnt signaling pathway in regulating ovarian cancer (OC) cell proliferation, migration, and invasion. Quantitative real-time PCR (qRT-PCR), Western blot, MTT assay, cell scratch, and transwell test were used in our study, and transplanted tumor model was constructed on nude mice. C-Myc, cyclin D1, PFTK1, ki67, OGT, and β-catenin protein expressions in tumor tissues were detected. CCNY was significantly upregulated in OC cell lines and tissues (both P < 0.05); significant association was observed between CCNY expression and clinicopathological stage, lymph node metastasis (LNM) (P < 0.05); and the CCNY expression in stages III to IV was higher than that in stages I to II, and patients with LNM had higher CCNY expression when compared with those in patients without LNM (P < 0.05); expressions of c-Myc, cyclin D, PFTK1, ki67, and OGT were upregulated in OC tissues compared with ovarian benign tissues, suggesting that these expressions were significantly different between the two groups (P < 0.05); CCNY significantly exacerbated proliferation, migration, and invasion of A2780 cells; c-Myc and cyclin D1 protein expressions increased as the expression of CCNY increased (P < 0.001); β-catenin expressions in A2780 cells with over-expression of CCNY were significantly increased in the nucleus, but significantly decreased in the cytoplasm (both P < 0.05); high expressions of CCNY exacerbated the proliferation of A2780 cells in nude mice and significantly increased c-Myc, cyclin D1, PFTK1, ki67, and OGT protein expressions in tumor tissues which were transplanted into nude mice (P < 0.01). CCNY might exacerbate the proliferation, migration, and invasion of OC cells via activating the Wnt signaling pathway. Thus, this study provides a theoretical foundation for the development of therapeutic drugs that are able to cure OC by targeting CCNY. PMID:26831658

  17. Expression, Purification, and Identification of Associated Proteins of the Full-length hCDK12/CyclinK Complex*

    PubMed Central

    Bartkowiak, Bartlomiej; Greenleaf, Arno L.

    2015-01-01

    The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3′-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a “2A” peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors. PMID:25429106

  18. The Role of EGFR/PI3K/Akt/cyclinD1 Signaling Pathway in Acquired Middle Ear Cholesteatoma

    PubMed Central

    Liu, Wei; Ren, Hongmiao; Ren, Jihao; Yin, Tuanfang; Hu, Bing; Xie, Shumin; Dai, Yinghuan; Wu, Weijing; Xiao, Zian; Yang, Xinming; Xie, Dinghua

    2013-01-01

    Cholesteatoma is a benign keratinizing and hyper proliferative squamous epithelial lesion of the temporal bone. Epidermal growth factor (EGF) is one of the most important cytokines which has been shown to play a critical role in cholesteatoma. In this investigation, we studied the effects of EGF on the proliferation of keratinocytes and EGF-mediated signaling pathways underlying the pathogenesis of cholesteatoma. We examined the expressions of phosphorylated EGF receptor (p-EGFR), phosphorylated Akt (p-Akt), cyclinD1, and proliferating cell nuclear antigen (PCNA) in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium by immunohistochemical method. Furthermore, in vitro studies were performed to investigate EGF-induced downstream signaling pathways in primary external auditory canal keratinocytes (EACKs). The expressions of p-EGFR, p-Akt, cyclinD1, and PCNA in cholesteatoma epithelium were significantly increased when compared with those of control subjects. We also demonstrated that EGF led to the activation of the EGFR/PI3K/Akt/cyclinD1 signaling pathway, which played a critical role in EGF-induced cell proliferation and cell cycle progression of EACKs. Both EGFR inhibitor AG1478 and PI3K inhibitor wortmannin inhibited the EGF-induced EGFR/PI3K/Akt/cyclinD1 signaling pathway concomitantly with inhibition of cell proliferation and cell cycle progression of EACKs. Taken together, our data suggest that the EGFR/PI3K/Akt/cyclinD1 signaling pathway is active in cholesteatoma and may play a crucial role in cholesteatoma epithelial hyper-proliferation. This study will facilitate the development of potential therapeutic targets for intratympanic drug therapy for cholesteatoma. PMID:24311896

  19. Cyclin D1 silencing suppresses tumorigenicity, impairs DNA double strand break repair and thus radiosensitizes androgen-independent prostate cancer cells to DNA damage

    PubMed Central

    Ju, Xiaoming; Vetuschi, Antonella; Sferra, Roberta; Casimiro, Mathew C.; Pompili, Simona; Festuccia, Claudio; Colapietro, Alessandro; Gaudio, Eugenio; Di Cesare, Ernesto; Tombolini, Vincenzo; Pestell, Richard G.

    2016-01-01

    Patients with hormone-resistant prostate cancer (PCa) have higher biochemical failure rates following radiation therapy (RT). Cyclin D1 deregulated expression in PCa is associated with a more aggressive disease: however its role in radioresistance has not been determined. Cyclin D1 levels in the androgen-independent PC3 and 22Rv1 PCa cells were stably inhibited by infecting with cyclin D1-shRNA. Tumorigenicity and radiosensitivity were investigated using in vitro and in vivo experimental assays. Cyclin D1 silencing interfered with PCa oncogenic phenotype by inducing growth arrest in the G1 phase of cell cycle and reducing soft agar colony formation, migration, invasion in vitro and tumor formation and neo-angiogenesis in vivo. Depletion of cyclin D1 significantly radiosensitizes PCa cells by increasing the RT-induced DNA damages by affecting the NHEJ and HR pathways responsible of the DNA double-strand break repair. Following treatment of cells with RT the abundance of a biomarker of DNA damage, γ-H2AX, was dramatically increased in sh-cyclin D1 treated cells compared to shRNA control. Concordant with these observations DNA-PKcs-activation and RAD51-accumulation, part of the DNA double-strand break repair machinery, were reduced in shRNA-cyclin D1 treated cells compared to shRNA control. We further demonstrate the physical interaction between CCND1 with activated-ATM, -DNA-PKcs and RAD51 is enhanced by RT. Finally, siRNA-mediated silencing experiments indicated DNA-PKcs and RAD51 are downstream targets of CCND1-mediated PCa cells radioresistance. In summary, these observations suggest that CCND1 is a key mediator of PCa radioresistance and could represent a potential target for radioresistant hormone-resistant PCa. PMID:26689991

  20. MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK.

    PubMed Central

    Devault, A; Martinez, A M; Fesquet, D; Labbé, J C; Morin, N; Tassan, J P; Nigg, E A; Cavadore, J C; Dorée, M

    1995-01-01

    The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage à trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain. Images PMID:7588631

  1. V3 loop of human immunodeficiency virus type 1 reduces cyclin E expression and induces G1 arrest in interleukin 2-dependent T cells.

    PubMed

    Sakaida, H; Kawamata, S; Hattori, T; Uchiyama, T

    1998-01-01

    We previously described that V3 loop derived from the HTLV-III BH10 clone V3-BH10 markedly suppressed IL-2-driven T cell proliferation and produced G1 arrest of the cells. Here, we tested the effect of V3-BH10 on the molecules that are involved in transition from the G1 to S phase of the cell cycle. The effect of V3-BH10 on the IL-2-induced expression of G1 cyclins, Cdk inhibitors, and phosphorylation of retinoblastoma protein (pRb) was tested by immunoblotting, using the IL-2-dependent CD4-positive cell line Kit 225. Furthermore, IL-2-dependent kinase activity of the cyclin E-Cdk2 complex was investigated with histone H1 as a substrate. V3-BH10 reduced the IL-2-dependent expression of cyclin E, but not that of cyclin D and Cdk inhibitors such as p21 and p27. As the result of reduction of cyclin E, histone H1 kinase activity of the cyclin E-Cdk2 complex was markedly reduced even in the presence of rIL-2, followed by incomplete phosphorylation of pRb. The reduction in hyperphosphorylation of pRb by V3-BH10 led to G1 arrest of the cell cycle. Thus, V3-BH10 induced G1 arrest in IL-2-dependent cell cycle progression by reducing cyclin E expression, which may be one of the mechanisms underlying the dysfunction of T cells in HIV-1-infected people. PMID:9453249

  2. Cyclin D2 Overexpression in Transgenic Mice Induces Thymic and Epidermal Hyperplasia whereas Cyclin D3 Expression Results Only in Epidermal Hyperplasia

    PubMed Central

    Rodriguez-Puebla, Marcelo L.; LaCava, Margaret; Miliani de Marval, Paula L.; Jorcano, Jose L.; Richie, Ellen R.; Conti, Claudio J.

    2000-01-01

    In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells. PMID:10980142

  3. Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

    PubMed Central

    Bauer, Andrea; Mylroie, Hayley; Thornton, C. Clare; Calay, Damien; Birdsey, Graeme M.; Kiprianos, Allan P.; Wilson, Garrick K.; Soares, Miguel P.; Yin, Xiaoke; Mayr, Manuel; Randi, Anna M.; Mason, Justin C.

    2016-01-01

    Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1−/− mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis. PMID:27388959

  4. Expression of δ-cyclins of Brassica rapa L. embryos by clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, O. A.

    Cyclins is one of the important regulators of cell cycle. There are several types of cyclins exists. They are responding for different phases of cycle and have high homology in plant's and mammalian's cells. δ -cyclins are specific for plants and controlling the presynthetic phase events. These cyclins likes to mammalian D-cyclins and have similar functions. This class consist three types of cyclins -- δ 1, δ 2 and δ 3. Cyclin δ 1 is responding for events in cell, which take place before exiting from stage of quiet (G0). Cyclin δ 1 is responding for entering and outputting from G0, and cyclin δ 3 -- for events, which happen in cell after stage of quiet, by entering to S-phase (phase of DNA's synthesis). In present research was used δ 1- and δ 3-cyclins. For determination of δ -cyclins gene's expression level was excreted RNA from embryos: 3-days (spherical stage), 6-days (heart-shaped stage) and 9-days (generated stage) seedlings of Brassica rapa L. in control and under clinorotation. For definition the cyclins gene's expression level applied Northern Blot Analysis. Obtained data testify about difference in level of gene's expression of cyclin δ 1 between control and clinorotation variants. After three days by pollination the expression of this gene in embryos was observed in control only. By clinorotation the gene's expression was detected on 6 days later, but it level was lower than in control variant. On 9 days it was gently expressed by clinorotation, where as by control it was not detected absolutely. Cyclin δ 3 gene's expression was observed during all time of the experiment. These data also confirm known one about expression δ 1- cyclin, which expressed on beginning of cell cycle only. And δ 3 --cyclin that express during whole presinthetic phase of cell cycle (Sony et al., 1995, Murray, 1994, Inze et al, 1999, Umeda, 2000).

  5. Expression of the retinoblastoma protein in low-grade B-cell lymphoma: relationship to cyclin D1.

    PubMed

    Zukerberg, L R; Benedict, W F; Arnold, A; Dyson, N; Harlow, E; Harris, N L

    1996-07-01

    The product of the retinoblastoma tumor-suppressor gene (pRB), a nuclear phosphoprotein that regulates transcription factors such as E2F, is involved in cell cycle control and differentiation. Its activity is regulated by phosphorylation; the underphosphorylated form inhibits transcription whereas the highly phosphorylated form is inactive. Cyclin D1 and its associated kinase (CDK 4/6) phosphorylate pRB in vitro, and therefore are thought to contribute to the regulation of pRB function. To examine the effect of cyclin D1 overexpression on pRB in primary tumor tissue, we studied pRB expression in low-grade B-cell neoplasms, with particular regard to mantle cell lymphoma, which is characterized by cyclin D1 (bcl-1) overexpression. pRB expression was studied by immunostaining with a well-characterized anti-pRB antibody; the phosphorylation status of pRB was examined by immunoblots; and the functional binding capacity of pRB was examined by in vitro binding to adenovirus E1A protein. We studied 3 reactive lymph nodes, 28 low grade B-cell lymphomas, 4 cases of hairy cell leukemia (HCL) and 3 plasmacytomas. Reactive lymph nodes showed intense pRB staining of germinal centers, with strongest (2+) staining in the large cells (centroblasts) of the proliferating (dark) zone and weak or no staining of small lymphocytes, including those of the mantle zone. In B-chronic lymphocytic leukemia (B-CLL) (4 cases), follicular lymphoma (3 cases) and mucosa-associated (MALT) lymphoma (3 cases) strong (2+) pRB staining was limited to centroblasts in reactive and neoplastic follicles and occasional proliferation centers, with only faint staining of small lymphoid cells. In contrast, 15 of 16 cases of mantle cell lymphoma showed strong (1-2+) staining of most cells; one blastoid mantle cell lymphoma showed only faint pRB staining. All cases of (HCL) and plasmacytoma showed strong pRB staining. Although most lymphomas with strong pRB expression were cyclin D1(+), three cyclin D1(+) cases

  6. p18Ink4c and p53 Act as tumor suppressors in cyclin D1-driven primitive neuroectodermal tumor.

    PubMed

    Saab, Raya; Rodriguez-Galindo, Carlos; Matmati, Kelly; Rehg, Jerold E; Baumer, Shannon H; Khoury, Joseph D; Billups, Catherine; Neale, Geoffrey; Helton, Kathleen J; Skapek, Stephen X

    2009-01-15

    The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption. PMID:19147556

  7. Role of cyclin D1 immunoreactivity and AgNOR staining in the evaluation of benign and malignant lesions of the prostate

    PubMed Central

    Gupta, Veena; Garg, Monika; Chaudhry, Manish; Singh, Sunita; Sen, Rajeev; Gill, Meenu; Sangwaiya, Ashok

    2014-01-01

    Purpose: Prostatic carcinoma is a common and growing public health problem. Histological evaluation is fairly adequate for assessing tumor differentiation, but tumor proliferative activity is difficult to measure. Increasing evidence suggests that the factors controlling cell cycle progression also modulate the rate of ribosome biogenesis. Despite the influence of cyclin D1 and argyrophilic nuclear organizer region (AgNOR) on prostate cancer proliferation, few studies have evaluated the diagnostic importance of these markers. Therefore, the present study was carried out to analyze the diagnostic value of the proliferative markers cyclin D1 and AgNOR in various prostatic lesions and to determine whether any association or relation between these markers and different Gleason grades exists. Methods: A total 50 cases of various prostatic lesions were studied. Tumor grade, AgNOR staining, and cyclin D1 expression were evaluated in all cases. Correlations between the intensity and differential localization of these markers and Gleason grades were evaluated. Results: The mean AgNOR count in cases of prostatic intraepithelial neoplasia was high compared with cases of benign prostatic hyperplasia (BPH) but lower than that of carcinoma cases. The intensity of cyclin D1 expression was high in carcinoma. A total of 14 cases (46.67%) showed strong positivity. No significant correlation was found between the intensity of cyclin D1 expression, AgNOR count, and histologic grades of prostatic carcinoma, whereas a significant correlation was observed between intensity and percentage expression of cyclin D1 in BPH and carcinoma (P<0.01). Nuclear as well as cytoplasmic positivity was seen among various grades of carcinoma. Conclusions: AgNOR count and cyclin D1 may be helpful in distinguishing between BPH and carcinoma of the prostate but may not be used as reliable indicators of the grade of prostatic adenocarcinoma because of overlapping values in various grades. However, further

  8. Apoptosis in male germ cells in response to cyclin A1-deficiency and cell cycle arrest.

    PubMed

    Salazar, Glicella; Liu, Dong; Liao, Ching; Batkiewicz, Leah; Arbing, Rachel; Chung, Sanny S W; Lele, Karen; Wolgemuth, Debra J

    2003-10-15

    Male mice homozygous for a mutated allele of the cyclin A1 gene (Ccna1) are sterile due to a block in cell cycle progression before the first meiotic division. Meiosis arrest in Ccna1(-/-) spermatocytes is associated with desynapsis abnormalities, lowered MPF activity, and apoptosis as evidenced by TUNEL-positive staining. With time, adult testicular tubules exhibit severe degeneration: some tubules in the older animals are almost devoid of germ cells at various stages of spermatogenesis. The mechanisms by which the cells sense the cell cycle arrest and the regulation of the decision to undergo cell death are under investigation. PMID:14555236

  9. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2

    SciTech Connect

    Bischoff, J.R.; Marshak, D.R.; Beach, D. ); Friedman, P.N.; Prives, C. )

    1990-06-01

    The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G{sub 1} phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.

  10. Cyclin-Dependent Kinase Co-Ordinates Carbohydrate Metabolism and Cell Cycle in S. cerevisiae.

    PubMed

    Zhao, Gang; Chen, Yuping; Carey, Lucas; Futcher, Bruce

    2016-05-19

    Cyclin-dependent kinases (CDKs) control cell division in eukaryotes by phosphorylating proteins involved in division. But successful proliferation requires co-ordination between division and cellular growth in mass. Previous proteomic studies suggested that metabolic proteins, as well as cell division proteins, could potentially be substrates of cyclin-dependent kinases. Here we focus on two metabolic enzymes of the yeast S. cerevisiae, neutral trehalase (Nth1) and glycogen phosphorylase (Gph1), and show that their activities are likely directly controlled by CDK activity, thus allowing co-ordinate regulation of carbohydrate metabolism with cell division processes. In this case, co-ordinate regulation may optimize the decision to undertake a final cell division as nutrients are being exhausted. Co-regulation of cell division processes and metabolic processes by CDK activity may be a general phenomenon important for co-ordinating the cell cycle with growth. PMID:27203179

  11. Cyclin E/Cdk2, P/CAF, and E1A regulate the transactivation of the c-myc promoter by FOXM1

    SciTech Connect

    Wierstra, Inken Alves, Juergen

    2008-03-28

    FOXM1c transactivates the c-myc promoter by binding directly to its TATA-boxes. The present study demonstrates that the transactivation of the c-myc promoter by FOXM1c is enhanced by the key proliferation signal cyclin E/Cdk2, but repressed by P/CAF and the adenoviral oncoprotein E1A. Furthermore, FOXM1c interacts with the coactivator and histone acetyltransferase P/CAF. This study shows that, on the c-myc-P1 TATA-box, FOXM1c does not function simply as normal transcription factor just binding to an unusual site. Moreover, the inhibitory N-terminus of FOXM1c does not inhibit its transrepression domain or its EDA. Others reported that a cyclin/Cdk-binding LXL-motif of the splice variant FoxM1b is required for its interaction with Cdk2, Cdk1, and p27, its phosphorylation by Cdk1 and its activation by Cdc25B. In contrast, we now demonstrate that this LXL-motif is not required for the activation of FOXM1c by cyclin D1/Cdk4, cyclin E/Cdk and cyclin A/Cdk2 or for the repression of FOXM1c by p27.

  12. 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 NF-kB transcriptional signaling

    PubMed Central

    Tran, Kalvin Q.; Tin, Antony S.; Firestone, Gary L.

    2014-01-01

    Relatively little is known about the anti-proliferative effects of Artemisinin, a naturally occurring anti-malarial 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 down regulated 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 kappa-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 via 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 untreated cells, reversed the artemisinin down-regulation 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 anti-proliferative effects in endometrial cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter. PMID:24296733

  13. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

    SciTech Connect

    Aulia, Selina; Tang, Bor Luen . E-mail: bchtbl@nus.edu.sg

    2006-01-06

    The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons-a function that could become impaired in Alzheimer's and other neurodegenerative diseases.

  14. Dominant-negative cyclin-selective ubiquitin carrier protein E2-C/UbcH10 blocks cells in metaphase

    PubMed Central

    Townsley, Fiona M.; Aristarkhov, Alexander; Beck, Sharon; Hershko, Avram; Ruderman, Joan V.

    1997-01-01

    Destruction of mitotic cyclins by ubiquitin-dependent proteolysis is required for cells to complete mitosis and enter interphase of the next cell cycle. In clam eggs, this process is catalyzed by a cyclin-selective ubiquitin carrier protein, E2-C, and the cyclosome/anaphase promoting complex (APC), a 20S particle containing cyclin-selective ubiquitin ligase activity. Here we report cloning a human homolog of E2-C, UbcH10, which shares 61% amino acid identity with clam E2-C and can substitute for clam E2-C in vitro. Dominant-negative clam E2-C and human UbcH10 proteins, created by altering the catalytic cysteine to serine, inhibit the in vitro ubiquitination and destruction of cyclin B in clam oocyte extracts. When transfected into mammalian cells, mutant UbcH10 inhibits the destruction of both cyclin A and B, arrests cells in M phase, and inhibits the onset of anaphase, presumably by blocking the ubiquitin-dependent proteolysis of proteins responsible for sister chromatid separation. Thus, E2-C/UbcH10-mediated ubiquitination is involved in both cdc2 inactivation and sister chromatid separation, processes that are normally coordinated during exit from mitosis. PMID:9122200

  15. Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes.

    PubMed Central

    Picard, A; Galas, S; Peaucellier, G; Dorée, M

    1996-01-01

    Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body. Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins. We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body. MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation. However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage. We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. Images PMID:8670862

  16. Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis.

    PubMed

    Hoffmann, Steffen; Tsurumi, Chizuko; Kubiak, Jacek Z; Polanski, Zbigniew

    2006-04-01

    We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material. PMID:16490186

  17. Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad*

    PubMed Central

    Park, Youngkyu; Maizels, Evelyn T.; Feiger, Zachary J.; Alam, Hena; Peters, Carl A.; Woodruff, Teresa K.; Unterman, Terry G.; Lee, Eun Jig; Jameson, J. Larry; Hunzicker-Dunn, Mary

    2006-01-01

    Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-α, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphati-dylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-α, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-α, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-α, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3. PMID:15613482

  18. Atypical Regulation of a Green Lineage-Specific B-Type Cyclin-Dependent Kinase1

    PubMed Central

    Corellou, Florence; Camasses, Alain; Ligat, Laetitia; Peaucellier, Gérard; Bouget, François-Yves

    2005-01-01

    Cyclin-dependent kinases (CDKs) are the main regulators of cell cycle progression in eukaryotes. The role and regulation of canonical CDKs, such as the yeast (Saccharomyces cerevisiae) Cdc2 or plant CDKA, have been extensively characterized. However, the function of the plant-specific CDKB is not as well understood. Besides being involved in cell cycle control, Arabidopsis (Arabidopsis thaliana) CDKB would integrate developmental processes to cell cycle progression. We investigated the role of CDKB in Ostreococcus (Ostreococcus tauri), a unicellular green algae with a minimal set of cell cycle genes. In this primitive alga, at the basis of the green lineage, CDKB has integrated two levels of regulations: It is regulated by Tyr phosphorylation like cdc2/CDKA and at the level of synthesis-like B-type CDKs. Furthermore, Ostreococcus CDKB/cyclin B accounts for the main peak of mitotic activity, and CDKB is able to rescue a yeast cdc28ts mutant. By contrast, Ostreococcus CDKA is not regulated by Tyr phosphorylation, and it exhibits a low and steady-state activity from DNA replication to exit of mitosis. This suggests that from a major role in the control of mitosis in green algae, CDKB has evolved in higher plants to assume other functions outside the cell cycle. PMID:15965018

  19. A novel DAG-dependent mechanism links PKCa and Cyclin B1 regulating cell cycle progression

    PubMed Central

    Poli, Alessandro; Ramazzotti, Giulia; Matteucci, Alessandro; Manzoli, Lucia; Lonetti, Annalisa; Suh, Pann-Ghill; McCubrey, James A.; Cocco, Lucio

    2014-01-01

    Through the years, different studies showed the involvement of Protein Kinase C (PKC) in cell cycle control, in particular during G1/S transition. Little is known about their role at G2/M checkpoint. In this study, using K562 human erythroleukemia cell line, we found a novel and specific mechanism through which the conventional isoform PKC⍺ positively affects Cyclin B1 modulating G2/M progression of cell cycle. Since the kinase activity of this PKC isoform was not necessary in this process, we demonstrated that PKC⍺, physically interacting with Cyclin B1, avoided its degradation and stimulated its nuclear import at mitosis. Moreover, the process resulted to be strictly connected with the increase in nuclear diacylglycerol levels (DAG) at G2/M checkpoint, due to the activity of nuclear Phospholipase C β1 (PLCβ1), the only PLC isoform mainly localized in the nucleus of K562 cells. Taken together, our findings indicated a novel DAG dependent mechanism able to regulate the G2/M progression of the cell cycle. PMID:25362646

  20. Foxp3 Protein Stability Is Regulated by Cyclin-dependent Kinase 2*

    PubMed Central

    Morawski, Peter A.; Mehra, Parul; Chen, Chunxia; Bhatti, Tricia; Wells, Andrew D.

    2013-01-01

    Foxp3 is a transcription factor required for the development of regulatory T cells (Treg). Mice and humans with a loss of Foxp3 function suffer from uncontrolled autoimmunity and inflammatory disease. Expression of Foxp3 is necessary for the anti-inflammatory capacity of Treg, but whether Foxp3 activity is further subject to regulation by extracellular signals is unclear. The primary structure of Foxp3 contains four cyclin-dependent kinase (CDK) motifs (Ser/Thr-Pro) within the N-terminal repressor domain, and we show that CDK2 can partner with cyclin E to phosphorylate Foxp3 at these sites. Consistent with our previous demonstration that CDK2 negatively regulates Treg function, we find that mutation of the serine or threonine at each CDK motif to alanine (S/T→A) results in enhanced Foxp3 protein stability in CD4+ T cells. T cells expressing the S/T→A mutant of Foxp3 showed enhanced induction (e.g. CD25) and repression (e.g. IL2) of canonical Foxp3-responsive genes, exhibited an increased capacity to suppress conventional T cell proliferation in vitro, and were highly effective at ameliorating colitis in an in vivo model of inflammatory bowel disease. These results indicate that CDK2 negatively regulates the stability and activity of Foxp3 and implicate CDK-coupled receptor signal transduction in the control of regulatory T cell function and stability. PMID:23853094

  1. The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention

    PubMed Central

    Alao, John P

    2007-01-01

    Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptionl co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. A number of therapeutic agents have been shown to induce cyclin D1 degradation. The therapeutic ablation of cyclin D1 may be useful for the prevention and treatment of cancer. In this review, current knowledge on the regulation of cyclin D1 degradation is discussed. Novel insights into cyclin D1 degradation are also discussed in the context of ablative therapy. A number of unresolved questions regarding the regulation of cellular cyclin D1 levels are also addressed. PMID:17407548

  2. Pharmacological cyclin dependent kinase inhibitors: Implications for colorectal cancer

    PubMed Central

    Balakrishnan, Archana; Vyas, Arpita; Deshpande, Kaivalya; Vyas, Dinesh

    2016-01-01

    Colorectal cancer accounts for a significant proportion of cancer deaths worldwide. The need to develop more chemotherapeutic agents to combat this disease is critical. Cyclin dependent kinases (CDKs), along with its binding partner cyclins, serve to control the growth of cells through the cell cycle. A new class of drugs, termed CDK inhibitors, has been studied in preclinical and now clinical trials. These inhibitors are believed to act as an anti-cancer drug by blocking CDKs to block the uncontrolled cellular proliferation that is hallmark of cancers like colorectal cancer. CDK article provides overview of the emerging drug class of CDK inhibitors and provides a list of ones that are currently in clinical trials. PMID:26900281

  3. A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

    PubMed Central

    2014-01-01

    Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

  4. Drosophila Cyclin J is a mitotically stable Cdk1 partner without essential functions.

    PubMed

    Althoff, Friederike; Viktorinová, Ivana; Kastl, Johanna; Lehner, Christian F

    2009-09-15

    Cyclin J is a cyclin family member that appears to have evolved before the metazoan radiation. Its evolutionary conservation argues for an important role but functional characterizations of Cyclin J have remained very limited. In Drosophila, Cyclin J is expressed only in females. Using transgenic Drosophila lines expressing Cyclin J versions with N- or C-terminal GFP extensions, we demonstrate that it is expressed exclusively in the germline. After low level expression in all nuclei within the germarium, it gets highly enriched in the germinal vesicle within the oocyte until stage 12 of oogenesis, followed by disappearance after germinal vesicle breakdown before the first meiotic division. Surprisingly, Cyclin J is not required for female fertility. Chromosome segregation during female meiosis, as well as the rapid early embryonic cell cycles after fertilization, occurs normally in the complete absence of Cyclin J. Cyclin J with EGFP fused at either N- or C-terminus binds to Cdk1 and not to Cdk2. However, in contrast to the other known Cdk1 partners, the A- and B-type cyclins, Cyclin J is not degraded during mitosis. PMID:19591820

  5. Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D.

    PubMed

    Pauklin, Siim; Madrigal, Pedro; Bertero, Alessandro; Vallier, Ludovic

    2016-02-15

    Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1-3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions. PMID:26883361

  6. Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D

    PubMed Central

    Pauklin, Siim; Madrigal, Pedro; Bertero, Alessandro; Vallier, Ludovic

    2016-01-01

    Coordination of differentiation and cell cycle progression represents an essential process for embryonic development and adult tissue homeostasis. These mechanisms ultimately determine the quantities of specific cell types that are generated. Despite their importance, the precise molecular interplays between cell cycle machinery and master regulators of cell fate choice remain to be fully uncovered. Here, we demonstrate that cell cycle regulators Cyclin D1–3 control cell fate decisions in human pluripotent stem cells by recruiting transcriptional corepressors and coactivator complexes onto neuroectoderm, mesoderm, and endoderm genes. This activity results in blocking the core transcriptional network necessary for endoderm specification while promoting neuroectoderm factors. The genomic location of Cyclin Ds is determined by their interactions with the transcription factors SP1 and E2Fs, which result in the assembly of cell cycle-controlled transcriptional complexes. These results reveal how the cell cycle orchestrates transcriptional networks and epigenetic modifiers to instruct cell fate decisions. PMID:26883361

  7. Combinatorial control of cyclin B1 nuclear trafficking through phosphorylation at multiple sites.

    PubMed

    Yang, J; Song, H; Walsh, S; Bardes, E S; Kornbluth, S

    2001-02-01

    Entry into mitosis is regulated by the Cdc2 kinase complexed to B-type cyclins. We and others recently reported that cyclin B1/Cdc2 complexes, which appear to be constitutively cytoplasmic during interphase, actually shuttle continually into and out of the nucleus, with the rate of nuclear export exceeding the import rate (). At the time of entry into mitosis, the import rate is increased, whereas the export rate is decreased, leading to rapid nuclear accumulation of Cdc2/cyclin B1. Although it has recently been reported that phosphorylation of 4 serines within cyclin B1 promotes the rapid nuclear translocation of Cdc2/cyclin B1 at G(2)/M, the role that individual phosphorylation sites play in this process has not been examined (, ). We report here that phosphorylation of a single serine residue (Ser(113) of Xenopus cyclin B1) abrogates nuclear export of cyclin B1. This serine lies directly within the cyclin B1 nuclear export sequence and, when phosphorylated, prevents binding of the nuclear export factor, CRM1. In contrast, analysis of phosphorylation site mutants suggests that coordinate phosphorylation of all 4 serines (94, 96, 101, and 113) is required for the accelerated nuclear import of cyclin B1/Cdc2 characteristic of G(2)/M. Additionally, binding of cyclin B1 to importin-beta, the factor known to be responsible for the slow interphase nuclear entry of cyclin B1, appears to be unaffected by the phosphorylation state of cyclin B. These data suggest that a distinct import factor must be recruited to enhance nuclear entry of Cdc2/cyclin B1 at the G(2)/M transition. PMID:11060306

  8. Down-regulation of SOSTDC1 promotes thyroid cancer cell proliferation via regulating cyclin A2 and cyclin E2

    PubMed Central

    He, Xiaoying; Ke, Weijian; Xu, Lijuan; Liu, Liehua; Xiao, Haipeng; Li, Yanbing

    2015-01-01

    Sclerostin domain containing protein 1 (SOSTDC1) is down-regulated and acts as a tumor suppressor in some kinds of cancers. However, the expression pattern and biological significance of SOSTDC1 in thyroid cancer are largely unknown. We demonstrated that SOSTDC1 was significantly down-regulated in thyroid cancer. Ectopic over-expression of SOSTDC1 inhibited proliferation and induced G1/S arrest in thyroid cancer cells. Moreover, SOSTDC1 over-expression suppressed the growth of tumor xenografts in nude mice. We also found that elevated SOSTDC1 led to inhibition of cyclin A2 and cyclin E2. Together, our results demonstrate that SOSTDC1 is down-regulated in thyroid cancer and might be a potential therapeutic target in the treatment of thyroid cancer. PMID:26378658

  9. The prognostic significance of altered cyclin-dependent kinase inhibitors in human cancer.

    PubMed

    Tsihlias, J; Kapusta, L; Slingerland, J

    1999-01-01

    Progression through the cell cycle is governed by cyclin-dependent kinases (cdks), whose activity is inhibited by the cdk inhibitors. Cyclins, cdks, and cdk inhibitors are frequently deregulated in cancers. This chapter reviews the prognostic significance of alterations in cdk inhibitors. Loss of p27 protein provides independent prognostic information in breast, prostate, colon, and gastric carcinomas, and immunohistochemical (IHC) staining for p27 may eventually become part of routine histopathologic processing of cancers. Loss of IHC staining for p21 may be prognostic in certain cancers but conflicting results are reported in breast cancer. Reports on homozygous deletion of p16 and p15 genes suggest the value of larger, prospective studies with standardized treatment protocols to definitively establish the prognostic utility of p15/p16 deletions in acute leukemias. Larger trials and the development of a consensus on methods for deletion analysis, IHC staining, and tumor scoring will be needed to move these molecular assays from bench to bedside. PMID:10073286

  10. Cyclin D2 rescues size and function of GATA4 haplo-insufficient hearts

    PubMed Central

    Yamak, Abir; Temsah, Rana; Maharsy, Wael; Caron, Sophie; Paradis, Pierre; Aries, Anne

    2012-01-01

    Transcription factor GATA4 is a key regulator of cardiomyocyte growth, and differentiation and 50% reduction in GATA4 levels results in hypoplastic hearts. Search for GATA4 targets/effectors revealed cyclin D2 (CD2), a member of the D-type cyclins (D1, D2, and D3) that play a vital role in cell growth and differentiation as a direct transcriptional target and a mediator of GATA4 growth in postnatal cardiomyocytes. GATA4 associates with the CD2 promoter in cardiomyocytes and is sufficient to induce endogenous CD2 transcription and to dose-dependently activate the CD2 promoter in heterologous cells. Cardiomyocyte-specific overexpression of CD2 results in enhanced postnatal cardiac growth because of increased cardiomyocyte proliferation. When these transgenic mice are crossed with Gata4 heterozygote mice, they rescue the hypoplastic cardiac phenotype of Gata4+/− mice and enhance cardiomyocyte survival and heart function. The data uncover a role for CD2 in the postnatal heart as an effector of GATA4 in myocyte growth and survival. The finding that postnatal upregulation of a cell-cycle gene in GATA4 haplo-insufficient hearts may be protective opens new avenues for maintaining or restoring cardiac function in GATA4-dependent cardiac disease. PMID:22923619

  11. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate

    PubMed Central

    Lee, Young-Sam; Huang, Kexin; Quiocho, Florante A; O’Shea, Erin K

    2008-01-01

    When Saccharomyces cerevisiae cells are starved of inorganic phosphate, the Pho80-Pho85 cyclin–cyclin-dependent kinase (CDK) is inactivated by the Pho81 CDK inhibitor (CKI). The regulation of Pho80-Pho85 is distinct from previously characterized mechanisms of CDK regulation: the Pho81 CKI is constitutively associated with Pho80-Pho85, and a small-molecule ligand, inositol heptakisphosphate (IP7), is required for kinase inactivation. We investigated the molecular basis of the IP7- and Pho81-dependent Pho80-Pho85 inactivation using electrophoretic mobility shift assays, enzyme kinetics and fluorescence spectroscopy. We found that IP7 interacts noncovalently with Pho80-Pho85-Pho81 and induces additional interactions between Pho81 and Pho80-Pho85 that prevent substrates from accessing the kinase active site. Using synthetic peptides corresponding to Pho81, we define regions of Pho81 responsible for constitutive Pho80-Pho85 binding and IP7-regulated interaction and inhibition. These findings expand our understanding of the mechanisms of cyclin-CDK regulation and of the biochemical mechanisms of IP7 action. PMID:18059263

  12. Differentiation-inducing factor-1 induces cyclin D1 degradation through the phosphorylation of Thr{sup 286} in squamous cell carcinoma

    SciTech Connect

    Mori, Jun; Takahashi-Yanaga, Fumi . E-mail: yanaga@clipharm.med.kyushu-u.ac.jp; Miwa, Yoshikazu; Watanabe, Yutaka; Hirata, Masato; Morimoto, Sachio; Shirasuna, Kanemitsu; Sasaguri, Toshiyuki

    2005-11-01

    Differentiation-inducing factors (DIFs) are morphogens which induce cell differentiation in Dictyostelium. We reported that DIF-1 and DIF-3 inhibit proliferation and induce differentiation in mammalian cells. In this study, we investigated the effect of DIF-1 on oral squamous cell carcinoma cell lines NA and SAS, well differentiated and poorly differentiated cell lines, respectively. Although DIF-1 did not induce the expression of cell differentiation makers in these cell lines, it inhibited the proliferation of NA and SAS in a dose-dependent manner by restricting the cell cycle in the G{sub 0}/G{sub 1} phase. DIF-1 induced cyclin D1 degradation, but this effect was prevented by treatment with lithium chloride and SB216763, the inhibitors of glycogen synthase kinase-3{beta} (GSK-3{beta}). Depletion of endogenous GSK-3{beta} by RNA interference also attenuated the effect of DIF-1 on cyclin D1 degradation. Therefore, we investigated the effect of DIF-1 on GSK-3{beta} and found that DIF-1 dephosphorylated GSK-3{beta} on Ser{sup 9} and induced the nuclear translocation of GSK-3{beta}, suggesting that DIF-1 activated GSK-3{beta}. Then, we examined the effect of DIF-1 on cyclin D1 mutants (Thr286Ala, Thr288Ala, and Thr286/288Ala). We revealed that Thr286Ala and Thr286/288Ala mutants were highly resistant to DIF-1-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr{sup 286} was critical for cyclin D1 degradation induced by DIF-1. These results suggest that DIF-1 induces degradation of cyclin D1 through the GSK-3{beta}-mediated phosphorylation of Thr{sup 286}.

  13. Immunohistological recognition of cyclin D1 expression by non-lymphoid cells among lymphoid neoplastic cells.

    PubMed

    Abdulla, Zainalabideen; Turley, Helen; Gatter, Kevin; Pezzella, Francesco

    2014-03-01

    Cyclin D1 immunostaining of non-neoplastic cells has been a source of diagnostic confusion especially in lymphoproliferative lesions. This study has reviewed these in two hundred and thirty-one haematopathological samples stained for cyclin D1. Most cases were formalin-fixed except for a few bone marrow trephines, which were B-5 fixed, and EDTA decalcified. Overall, 94% (216/231) of cases showed one or more types of non-neoplastic cells expressing Cyclin D1 of variable intensity. Endothelial cells and histiocytes were the most commonly identified Cyclin D1 positive cells being positive in 92% (214/231) of cases. Other normal cell types identified included fat cells, stromal fibroblasts, glial cells, spermatocytes, smooth muscle cells, osteoblasts and where present epithelial cells. Many normal cell types can express cyclinD1. Knowledge of these is useful to prevent misinterpretation of cyclin D1 positive tumours. PMID:23758159

  14. Cyclin D1 and Ewing's sarcoma/PNET: A microarray analysis.

    PubMed

    Fagone, Paolo; Nicoletti, Ferdinando; Salvatorelli, Lucia; Musumeci, Giuseppe; Magro, Gaetano

    2015-10-01

    Recent immunohistochemical analyses have showed that cyclin D1 is expressed in soft tissue Ewing's sarcoma/peripheral neuroectodermal tumor (PNET) of childhood and adolescents, while it is undetectable in both embryonal and alveolar rhabdomyosarcoma. In the present paper, microarray analysis provided evidence of a significant upregulation of cyclin D1 in Ewing's sarcoma as compared to normal tissues. In addition, we confirmed our previous findings of a significant over-expression of cyclin D1 in Ewing sarcoma as compared to rhabdomyosarcoma. Bioinformatic analysis also allowed to identify some other genes, strongly correlated to cyclin D1, which, although not previously studied in pediatric tumors, could represent novel markers for the diagnosis and prognosis of Ewing's sarcoma/PNET. The data herein provided support not only the use of cyclin D1 as a diagnostic marker of Ewing sarcoma/PNET but also the possibility of using drugs targeting cyclin D1 as potential therapeutic strategies. PMID:26363896

  15. ARTD1 regulates cyclin E expression and consequently cell-cycle re-entry and G1/S progression in T24 bladder carcinoma cells.

    PubMed

    Léger, Karolin; Hopp, Ann-Katrin; Fey, Monika; Hottiger, Michael O

    2016-08-01

    ADP-ribosylation is involved in a variety of biological processes, many of which are chromatin-dependent and linked to important functions during the cell cycle. However, any study on ADP-ribosylation and the cell cycle faces the problem that synchronization with chemical agents or by serum starvation and subsequent growth factor addition already activates ADP-ribosylation by itself. Here, we investigated the functional contribution of ARTD1 in cell cycle re-entry and G1/S cell cycle progression using T24 urinary bladder carcinoma cells, which synchronously re-enter the cell cycle after splitting without any additional stimuli. In synchronized cells, ARTD1 knockdown, but not inhibition of its enzymatic activity, caused specific down-regulation of cyclin E during cell cycle re-entry and G1/S progression through alterations of the chromatin composition and histone acetylation, but not of other E2F-1 target genes. Although Cdk2 formed a functional complex with the residual cyclin E, p27(Kip 1) protein levels increased in G1 upon ARTD1 knockdown most likely due to inappropriate cyclin E-Cdk2-induced phosphorylation-dependent degradation, leading to decelerated G1/S progression. These results provide evidence that ARTD1 regulates cell cycle re-entry and G1/S progression via cyclin E expression and p27(Kip 1) stability independently of its enzymatic activity, uncovering a novel cell cycle regulatory mechanism. PMID:27295004

  16. The BH3-mimetic gossypol and noncytotoxic doses of valproic acid induce apoptosis by suppressing cyclin-A2/Akt/FOXO3a signaling

    PubMed Central

    Pan, Hao; Lin, Qiu-Ru; Huang, Mei-Yun; Cai, Ji-Ye; Ouyang, Dong-Yun; He, Xian-Hui

    2015-01-01

    Previously we reported that valproic acid (VPA) acts in synergy with GOS to enhance cell death in human DU145 cells. However, the underlying mechanism remains elusive. In this study, we observed that such synergistic cytotoxicity of GOS and VPA could be extended to human A375, HeLa, and PC-3 cancer cells. GOS and VPA co-treatment induced robust apoptosis as evidenced by caspase-8/-9/-3 activation, PARP cleavage, and nuclear fragmentation. GOS and VPA also markedly decreased cyclin A2 protein expression. Owing to the reduction of cyclin A2, Akt signaling was suppressed, leading to dephosphorylation of FOXO3a. Consequently, FOXO3a was activated and the expression of its target genes, including pro-apoptotic FasL and Bim, was upregulated. Supporting this, FOXO3a knockdown attenuated FasL and Bim upregulation and apoptosis induction in GOS+VPA-treated cells. Furthermore, blocking proteasome activity by MG132 prevented the downregulation of cyclin A2, dephosphorylation of Akt and FOXO3a, and induction of apoptosis in cells co-treated with GOS and VPA. In mouse model, GOS and VPA combination significantly inhibited the growth of A375 melanoma xenografts. Our findings indicate that GOS and VPA co-treatment induces apoptosis in human cancer cells by suppressing the cyclin-A2/Akt/FOXO3a pathway. PMID:26517515

  17. Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents

    PubMed Central

    He, G; Kuang, J; Koomen, J; Kobayashi, R; Khokhar, A R; Siddik, Z H

    2013-01-01

    Background: In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated. Methods: Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry. Results: The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ∼140 kDa, which increased to ∼290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ∼150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21. Conclusion: G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs activity of p21 within the complex. PMID:24104967

  18. O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.

    PubMed

    Ding, Fang; Yu, Lu; Wang, Meihui; Xu, Shengjie; Xia, Qiang; Fu, Guosheng

    2013-08-01

    Continuous hyperglycemia is considered to be the most significant pathogenesis of diabetic cardiomyopathy, which manifests as cardiac hypertrophy and subsequent heart failure. O-GlcNAcylation has attracted attention as a post-translational protein modification in the past decade. The role of O-GlcNAcylation in high glucose-induced cardiomyocyte hypertrophy remains unclear. We studied the effect of O-GlcNAcylation on neonatal rat cardiomyocytes that were exposed to high glucose and myocardium in diabetic rats induced by streptozocin. High glucose (30 mM) incubation induced a greater than twofold increase in cell size and increased hypertrophy marker gene expression accompanied by elevated O-GlcNAcylation protein levels. High glucose increased ERK1/2 but not p38 MAPK or JNK activity, and cyclin D2 expression was also increased. PUGNAc, an inhibitor of β-N-acetylglucosaminidase, enhanced O-GlcNAcylation and imitated the effects of high glucose. OGT siRNA and ERK1/2 inhibition with PD98059 treatment blunted the hypertrophic response and cyclin D2 upregulation. OGT inhibition also prevented ERK1/2 activation. We also observed concentric hypertrophy and similar changes of O-GlcNAcylation level, ERK1/2 activation and cyclin D2 expression in myocardium of diabetic rats induced by streptozocin. In conclusion, O-GlcNAcylation plays a role in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2. PMID:23665912

  19. Schizophrenia susceptibility gene product dysbindin-1 regulates the homeostasis of cyclin D1.

    PubMed

    Ito, Hidenori; Morishita, Rika; Nagata, Koh-Ichi

    2016-08-01

    Dysbindin-1 (dystrobrevin binding protein-1, DTNBP1) is now widely accepted as a potential schizophrenia susceptibility gene and accumulating evidence indicates its functions in the neural development. In this study, we tried to identify new binding partners for dysbindin-1 to clarify the novel function of this molecule. When consulted with BioGRID protein interaction database, cyclin D3 was found to be a possible binding partner for dysbindin-1. We then examined the interaction between various dysbindin-1 isoforms (dysbindin-1A, -1B and -1C) and all three D-type cyclins (cyclin D1, D2, and D3) by immunoprecipitation with the COS7 cell expression system, and found that dysbindin-1A preferentially interacts with cyclin D1. The mode of interaction between these molecules was considered as direct binding since recombinant dysbindin-1A and cyclin D1 formed a complex in vitro. Mapping analyses revealed that the C-terminal region of dysbindin-1A binds to the C-terminal of cyclin D1. Consistent with the results of the biochemical analyses, endogenous dysbindin-1was partially colocalized with cyclin D1 in NIH3T3 fibroblast cells and in neuronal stem and/or progenitor cells in embryonic mouse brain. While co-expression of dysbindin-1A with cyclin D1 changed the localization of the latter from the nucleus to cytosol, cyclin D1-binding partner CDK4 inhibited the dysbindin-cyclin D1 interaction. Meanwhile, depletion of endogenous dysbindin-1A increased cyclin D1 expression. These results indicate that dysbindin-1A may control the cyclin D1 function spatiotemporally and might contribute to better understanding of the pathophysiology of dysbindin-1-associated disorders. PMID:27130439

  20. Positive cyclin T expression as a favorable prognostic factor in treating gastric gastrointestinal stromal tumors

    PubMed Central

    LIN, LIEN-FU; JIN, JONG-SHIAW; CHEN, JUI-CHANG; HUANG, CHIA-CHI; SHEU, JENG-HORNG; CHEN, WENLUNG; TSAO, TANG-YI; HSU, CHIH-WEI

    2016-01-01

    Positive transcriptional elongation factor b (P-TEFb) contains the catalytic subunit cyclin-dependent kinase 9 (Cdk9) and the regulatory subunit cyclin T. Cyclin T1 and Cdk9 are the key factors of the PTEFb pathways and are overexpressed in the human head and neck carcinoma cell line. However, there have been limited studies regarding the role of cyclin T1 and Cdk9 in gastric gastrointestinal stromal tumors (GISTs). The aim of the present study was to assess the association between cyclin T1 and Cdk9 and their clinical significance in gastric GISTs. A total of 30 gastric GIST patients who underwent either laparoscopic or laparotomic partial gastrectomy were enrolled in the study. The surgical tissue slides were stained with Cdk9 and cyclin T1 antibodies, and the immunohistochemistry scores and disease-free survival (DFS) were analyzed. Ten patients were cyclin T1-positive, and 20 were negative. All 11 patients with recurrent tumors or distant metastases were cyclin T1-negative patients. Old age, large tumor size, a high Ki67 IHC staining score, high mitotic count and negative cyclin T1 staining revealed a worse clinical outcome in univariate analysis. By contrast, the Cdk9 score was not associated with clinical parameters. The Kaplan-Meier survival curve illustrated that the DFS rate of the patients with negative cyclin T1 staining was significantly lower than that of the patients with positive cyclin T1 staining. Positive expression of cyclin T1 was a good prognostic factor in patients with gastric GISTs. PMID:27284431

  1. Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells

    PubMed Central

    Ouellet, Stéphane; Vigneault, François; Lessard, Maryse; Leclerc, Steeve; Drouin, Régen; Guérin, Sylvain L.

    2006-01-01

    The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via specific protein–protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considerably increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Over-expression of each of the four NFI isoforms in HCT116 cells established that each of them contribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby establishing a role for NFI in the cell cycle dependent expression of p21. PMID:17130157

  2. Glucose Enhances Basal or Melanocortin-Induced cAMP-Response Element Activity in Hypothalamic Cells.

    PubMed

    Breit, Andreas; Wicht, Kristina; Boekhoff, Ingrid; Glas, Evi; Lauffer, Lisa; Mückter, Harald; Gudermann, Thomas

    2016-07-01

    Melanocyte-stimulating hormone (MSH)-induced activation of the cAMP-response element (CRE) via the CRE-binding protein in hypothalamic cells promotes expression of TRH and thereby restricts food intake and increases energy expenditure. Glucose also induces central anorexigenic effects by acting on hypothalamic neurons, but the underlying mechanisms are not completely understood. It has been proposed that glucose activates the CRE-binding protein-regulated transcriptional coactivator 2 (CRTC-2) in hypothalamic neurons by inhibition of AMP-activated protein kinases (AMPKs), but whether glucose directly affects hypothalamic CRE activity has not yet been shown. Hence, we dissected effects of glucose on basal and MSH-induced CRE activation in terms of kinetics, affinity, and desensitization in murine, hypothalamic mHypoA-2/10-CRE cells that stably express a CRE-dependent reporter gene construct. Physiologically relevant increases in extracellular glucose enhanced basal or MSH-induced CRE-dependent gene transcription, whereas prolonged elevated glucose concentrations reduced the sensitivity of mHypoA-2/10-CRE cells towards glucose. Glucose also induced CRCT-2 translocation into the nucleus and the AMPK activator metformin decreased basal and glucose-induced CRE activity, suggesting a role for AMPK/CRTC-2 in glucose-induced CRE activation. Accordingly, small interfering RNA-induced down-regulation of CRTC-2 expression decreased glucose-induced CRE-dependent reporter activation. Of note, glucose also induced expression of TRH, suggesting that glucose might affect the hypothalamic-pituitary-thyroid axis via the regulation of hypothalamic CRE activity. These findings significantly advance our knowledge about the impact of glucose on hypothalamic signaling and suggest that TRH release might account for the central anorexigenic effects of glucose and could represent a new molecular link between hyperglycaemia and thyroid dysfunction. PMID:27144291

  3. Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas. Functional studies in a cell line (Granta 519).

    PubMed

    Jadayel, D M; Lukas, J; Nacheva, E; Bartkova, J; Stranks, G; De Schouwer, P J; Lens, D; Bartek, J; Dyer, M J; Kruger, A R; Catovsky, D

    1997-01-01

    Abnormalities of several cell-cycle regulatory genes including cyclin D1, p16CDKN2 and p15CDKN2B have been described in B cell non-Hodgkin's lymphoma (B-NHL). We describe a new B-NHL cell line (Granta 519), with concurrent abnormalities of the cyclin D1, pl6CDKN2 and pl5CDKN2B genes. An independent clinical case of mantle cell NHL (Mc-NHL) with concomitant overexpression of cyclin D1, and deletion of the p16CDKN2 gene was also identified, suggesting that this combination of oncogenic aberration is a pathophysiologic contribution to a subset of NHL cases. More in-depth functional studies of this concept were facilitated by the availability of the cell line Granta 519 which was derived from a case of high-grade NHL and has a mature B cell immunophenotype. Cytogenetic analysis identified translocation t(11;14)(q13;q32) and complex rearrangements involving chromosomes 9p22, 13p21, 17pl1, and 18q21. Molecular analysis identified overexpression of cyclin D1 mRNA and biallelic deletion of the p16CDKN2 and p15CDKN2B genes. To elucidate the effect of these genetic abnormalities on the G1 control of Granta 519 cells, the level and function of the major components of the cyclinD/retinoblastoma (RB) pathway were investigated. Cyclin D1 was dominant among the D-type cyclins, formed abundant complexes with cyclin-dependent kinase (Cdk) Cdk4 rather than Cdk6, and the immunoprecipitated cyclin D1/Cdk4 holoenzyme was active as a pRB kinase. Electroporation of wild-type pl6CDKN2 arrested the Granta 519 cells in G1, consistent with the p16CDKN2 loss as a biologically relevant event during multistep evolution of the tumor, and with the expression of functional pRB. Direct cooperation of these distinct abnormalities to cell-cycle, deregulation in NHL cells was suggested by G1 acceleration upon inducible overexpression of cyclin D1 in a control breast cancer cell line lacking p16CDKN2, an effect which could be prevented by ectopic expression of p16CDKN2. Taken together, these data

  4. [Pronostic value of the immunohistochemical expression of cyclin D1 (DCS6) in epidermoid larynx carcinoma].

    PubMed

    García Lozano, M C; Orradre Romero, J L; Sánchez Carrión, S; Caro Garcia, M; Lasso Luis, O; Piris Pinilla, M A

    2006-01-01

    In this paper we carried out an immunohistochemical study of cyclin D1 (DCS6 ) expression in a series of 195 patients with laryngeal carcinoma that were diagnosticated, treated and followed at the Department of Otolaryngology at "Virgen de la Salud" Hospital (Toledo, Spain) for a time of 5 years. In the cases with lymph node metastasis we also studied cyclin D1 expression at this level. Furthermore we have analysed the value of cyclin D1 expression as a prognostic factor (tumor recurrence, deads due to cancer and survival) and we evaluate the relationship between cyclin D1 expression and other clinic and pathologic parameters. PMID:16881553

  5. 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. PMID:27203178

  6. Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes.

    PubMed

    Varadarajan, Ramya; Ayeni, Joseph; Jin, Zhigang; Homola, Ellen; Campbell, Shelagh D

    2016-07-01

    Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing. PMID:27170181

  7. Gankyrin facilitates follicle-stimulating hormone-driven ovarian cancer cell proliferation through the PI3K/AKT/HIF-1α/cyclin D1 pathway.

    PubMed

    Chen, J; Bai, M; Ning, C; Xie, B; Zhang, J; Liao, H; Xiong, J; Tao, X; Yan, D; Xi, X; Chen, X; Yu, Y; Bast, R C; Zhang, Z; Feng, Y; Zheng, W

    2016-05-12

    Gankyrin is a regulatory subunit of the 26kD proteasome complex. As a novel oncoprotein, gankyrin is expressed aberrantly in cancers from several different sites and has been shown to contribute to oncogenesis in endometrial and cervical carcinomas. Neither gankyrin's contribution to the development of epithelial ovarian cancer nor its interaction with follicle-stimulating hormone (FSH)-driven proliferation in ovarian cancer has been studied. Here we have found that gankyrin is overexpressed in ovarian cancers compared with benign ovarian cystadenomas and that gankyrin regulates FSH upregulation of cyclin D1. Importantly, gankyrin regulates PI3K/AKT signaling by downregulating PTEN. Prolonged AKT activation by FSH stimulation of the FSH receptor (FSHR) promotes gankyrin expression, which, in turn, enhances AKT activation by inhibiting PTEN. Overexpression of gankyrin decreases hypoxia inducible factor-1α (HIF-1α) protein levels, but has little effect on HIF-1α mRNA levels, which could be attributed to gankyrin mediating HIF-1α protein stability via the ubiquitin-proteasome pathway. Reduction in HIF-1α protein stability led to attenuation of the binding with cyclin D1 promoter, resulted in abolishment of the negative regulation of cyclin D1 by HIF-1α, which promotes proliferation of ovarian cancer cells. Our results document that gankyrin regulates HIF-1α protein stability and cyclin D1 expression, ultimately mediating FSH-driven ovarian cancer cell proliferation. PMID:26364616

  8. AUREOCHROME1a-Mediated Induction of the Diatom-Specific Cyclin dsCYC2 Controls the Onset of Cell Division in Diatoms (Phaeodactylum tricornutum)[W

    PubMed Central

    Huysman, Marie J.J.; Fortunato, Antonio E.; Matthijs, Michiel; Costa, Benjamin Schellenberger; Vanderhaeghen, Rudy; Van den Daele, Hilde; Sachse, Matthias; Inzé, Dirk; Bowler, Chris; Kroth, Peter G.; Wilhelm, Christian; Falciatore, Angela; Vyverman, Wim; De Veylder, Lieven

    2013-01-01

    Cell division in photosynthetic organisms is tightly regulated by light. Although the light dependency of the onset of the cell cycle has been well characterized in various phototrophs, little is known about the cellular signaling cascades connecting light perception to cell cycle activation and progression. Here, we demonstrate that diatom-specific cyclin 2 (dsCYC2) in Phaeodactylum tricornutum displays a transcriptional peak within 15 min after light exposure, long before the onset of cell division. The product of dsCYC2 binds to the cyclin-dependent kinase CDKA1 and can complement G1 cyclin-deficient yeast. Consistent with the role of dsCYC2 in controlling a G1-to-S light-dependent cell cycle checkpoint, dsCYC2 silencing decreases the rate of cell division in diatoms exposed to light-dark cycles but not to constant light. Transcriptional induction of dsCYC2 is triggered by blue light in a fluence rate-dependent manner. Consistent with this, dsCYC2 is a transcriptional target of the blue light sensor AUREOCHROME1a, which functions synergistically with the basic leucine zipper (bZIP) transcription factor bZIP10 to induce dsCYC2 transcription. The functional characterization of a cyclin whose transcription is controlled by light and whose activity connects light signaling to cell cycle progression contributes significantly to our understanding of the molecular mechanisms underlying light-dependent cell cycle onset in diatoms. PMID:23292736

  9. Cyclin D1 interacts and collaborates with Ral GTPases enhancing cell detachment and motility.

    PubMed

    Fernández, R M H; Ruiz-Miró, M; Dolcet, X; Aldea, M; Garí, E

    2011-04-21

    Alterations in the levels of adhesion and motility of cells are critical events in the development of metastasis. Cyclin D1 (CycD1) is one of the most frequently amplified oncogenes in many types of cancers and it is also associated with the development of metastasis. Despite this, we still do not know which are all the relevant pathways by which CycD1 induces oncogenic processes. CycD1 functions can be either dependent or independent of the cyclin-dependent kinase Cdk4, and they affect several cellular aspects such as proliferation, cell attachment and migration. In this work, we reveal a novel function of CycD1 that fosters our understanding of the oncogenic potential of CycD1. We show that CycD1 binds to the small GTPases Ral A and B, which are involved, through exocyst regulation, in the progression of metastatic cancers, inducing anchorage-independent growth and cell survival of transformed cells. We show that CycD1 binds active Ral complexes and the exocyst protein Sec6, and co-localizes with Ral GTPases in trans-Golgi and exocyst-rich regions. We have also observed that CycD1-Cdk4 phosphorylates the Ral GEF Rgl2 'in vitro' and that CycD1-Cdk4 activity stimulates accumulation of the Ral GTP active forms. In accordance with this, our data suggest that CycD1-Cdk4 enhances cell detachment and motility in collaboration with Ral GTPases. This new function may help explain the contribution of CycD1 to tumor spreading. PMID:21242975

  10. Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1

    SciTech Connect

    Kang, Du-Seock; Hong, Kyeong-Man; Park, Joobae; Bae, Chang-Dae

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer We identified a GC box and a CHR element in human CKAP2 minimal promoter. Black-Right-Pointing-Pointer The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. Black-Right-Pointing-Pointer The GC box was essential for the basic promoter activity of human CKAP2. Black-Right-Pointing-Pointer The GC box was also essential for the cyclic expression of human CKAP2. Black-Right-Pointing-Pointer The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (-110 to -104 bp) and a GC box (-41 to -32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

  11. Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation.

    PubMed

    Gomes, Helga; Moraes, Jorge; Githaka, Naftaly; Martins, Renato; Isezaki, Masayoshi; Vaz, Itabajara da Silva; Logullo, Carlos; Konnai, Satoru; Ohashi, Kazuhiko

    2015-07-30

    Among arthropods, ticks lead as vectors of animal diseases and rank second to mosquitoes in transmitting human pathogens. Cyclin-dependent kinases (CDK) participate in cell cycle control in eukaryotes. CDKs are serine/threonine protein kinases and these catalytic subunits are activated or inactivated at specific stages of the cell cycle. To determine the potential of using CDKs as anti-tick vaccine antigens, hamsters were immunized with recombinant Ixodes persulcatus CDK10, followed by a homologous tick challenge. Though it was not exactly unexpected, IpCDK10 vaccination significantly impaired tick blood feeding and fecundity, which manifested as low engorgement weights, poor oviposition, and a reduction in 80% of hatching rates. These findings may underpin the development of more efficacious anti-tick vaccines based on the targeting of cell cycle control proteins. PMID:26073111

  12. Selective inhibitors of Cyclin-G associated kinase (GAK) as anti-HCV agents

    PubMed Central

    Kovackova, Sona; Chang, Lei; Bekerman, Elena; Neveu, Gregory; Barouch-Bentov, Rina; Chaikuad, Apirat; Heroven, Christina; Šála, Michal; De Jonghe, Steven; Knapp, Stefan; Einav, Shirit; Herdewijn, Piet

    2015-01-01

    Cyclin-G associated kinase (GAK) emerged as a promising drug target for the treatment of viral infections. However, no potent and selective GAK inhibitors have been reported in the literature to date. This paper describes the discovery of isothiazolo[5,4-b]pyridines as selective GAK inhibitors, with the most potent congeners displaying low nanomolar binding affinity for GAK. Co-crystallization experiments revealed that these compounds behaved as classic type I ATP-competitive kinase inhibitors. In addition, we have demonstrated that these compounds exhibit a potent activity against hepatitis C virus (HCV) by inhibiting two temporally distinct steps in the HCV lifecycle (i.e. viral entry and assembly). Hence, these GAK inhibitors represent chemical probes to study GAK function in different disease areas where GAK has been implicated (including viral infection, cancer and Parkinson's disease). PMID:25822739

  13. The effect of cyclin-dependent kinases inhibitor treatment on experimental herpes simplex encephalitis mice.

    PubMed

    Zhou, Yu; Zeng, Yan-Ping; Zhou, Qin; Guan, Jing-Xia; Lu, Zu-Neng

    2016-08-01

    Herpes simplex encephalitis(HSE) is the most common and serious viral encephalitis in humans. There is a lack of effective medication to date for HSE. A better understanding of the mediators of tissue damage is essential for finding new targets for therapeutic intervention. In this project, we explored the effect of cyclin-dependent kinases inhibitor olomoucine treatment on experimental HSE mice. The following results were obtained: (1) olomoucine increased survival in HSE mice; (2) olomoucine inhibited microglial activation and reduced HSV-1-induced cytokines release; (3) olomoucine prevented neural cells apoptosis and attenuated brain tissue pathological changes following HSV-1 infection; (4) olomoucine reduced brain edema and improved neurological function in HSE. Overall, olomoucine can induce a blunted inflammatory response, maintain the blood vessel wall intact, improve neurological function and increase survival in HSE mice. PMID:27241721

  14. E-type cyclins modulate telomere integrity in mammalian male meiosis.

    PubMed

    Manterola, Marcia; Sicinski, Piotr; Wolgemuth, Debra J

    2016-06-01

    We have shown that E-type cyclins are key regulators of mammalian male meiosis. Depletion of cyclin E2 reduced fertility in male mice due to meiotic defects, involving abnormal pairing and synapsis, unrepaired DNA, and loss of telomere structure. These defects were exacerbated by additional loss of cyclin E1, and complete absence of both E-type cyclins produces a meiotic catastrophe. Here, we investigated the involvement of E-type cyclins in maintaining telomere integrity in male meiosis. Spermatocytes lacking cyclin E2 and one E1 allele (E1+/-E2-/-) displayed a high rate of telomere abnormalities but can progress to pachytene and diplotene stages. We show that their telomeres exhibited an aberrant DNA damage repair response during pachynema and that the shelterin complex proteins TRF2 and RAP2 were significantly decreased in the proximal telomeres. Moreover, the insufficient level of these proteins correlated with an increase of γ-H2AX foci in the affected telomeres and resulted in telomere associations involving TRF1 and telomere detachment in later prophase-I stages. These results suggest that E-type cyclins are key modulators of telomere integrity during meiosis by, at least in part, maintaining the balance of shelterin complex proteins, and uncover a novel role of E-type cyclins in regulating chromosome structure during male meiosis. PMID:26712234

  15. Analysis of a Drosophila cyclin E hypomorphic mutation suggests a novel role for cyclin E in cell proliferation control during eye imaginal disc development.

    PubMed Central

    Secombe, J; Pispa, J; Saint, R; Richardson, H

    1998-01-01

    We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycEJP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycEJP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development. PMID:9691043

  16. A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1.

    PubMed

    Scaglione-Sewell, B A; Bissonnette, M; Skarosi, S; Abraham, C; Brasitus, T A

    2000-11-01

    Previous studies by our laboratory have shown that a noncalcemic fluorinated analog of 1alpha,25-dihydroxyvitamin D3, 1alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholcal ciferol (F6-D3), significantly reduced the frequency of colonic adenomas and completely abolished the development of colonic adenocarcinomas in rats treated with azoxymethane. The mechanisms involved in this analog's chemopreventive actions, however, remain unclear. In the present study, we now show that although both 1alpha,25-dihydroxyvitamin D3 and F6-D3 inhibited the proliferation of CaCo-2 cells, a human colonic adenocarcinoma cell line, by increasing their doubling times, only F6-D3 caused an arrest of these cells in the G1 phase of their cell cycle. This arrest was accompanied by an increase in the expression of the cyclin-dependent kinase (cdk) inhibitor proteins, p2Waf1 and p27Kip1, which served to decrease the activity of cyclin-dependent kinase 2 and cyclin-dependent kinase 6, whereas the expression and phosphorylation of pRB were unchanged. In contrast to the increased expression of these cdk inhibitors, the expression of cyclin E was decreased, which further inhibited the activity of cyclin-dependent kinase 2. Collectively, the inhibition of these cyclin-dependent kinases served to arrest the CaCo-2 cells, independent of changes in pRB. Furthermore, antibody neutralization studies suggest that transforming growth factor-beta may mediate the coassociations between cdk2 and p27Kip1 and cyclin E induced by F6-D3. These data indicate that cell cycle arrest may, at least in part, underlie the chemopreventive actions of F6-D3 observed in the azoxymethane model of colon cancer. Furthermore, if the antiproliferative action observed in CaCo-2 cells also occurs in human colonic epithelium, F6-D3 may have chemopreventive potential against human colon cancer, as well. PMID:11089522

  17. Developmental cis-regulatory analysis of the cyclin D gene in the sea urchin Strongylocentrotus purpuratus

    PubMed Central

    McCarty, Christopher M.

    2013-01-01

    Cyclin D genes regulate the cell cycle, growth and differentiation in response to intercellular signaling. While the promoters of vertebrate cyclin D genes have been analyzed, the cis-regulatory sequences across an entire cyclin D locus have not. Doing so would increase understanding of how cyclin D genes respond to the regulatory states established by developmental gene regulatory networks, linking cell cycle and growth control to the ontogenetic program. Therefore, we conducted a cis-regulatory analysis on the cyclin D gene, SpcycD, of the sea urchin, Strongylocentrotus purpuratus, during embryogenesis, identifying upstream and intronic sequences, located within six defined regions bearing one or more cis-regulatory modules each. PMID:24090975

  18. Cyclin-dependent kinase 2 is an ideal target for ovary tumors with elevated cyclin E1 expression.

    PubMed

    Yang, Liu; Fang, Dongdong; Chen, Huijun; Lu, Yiyu; Dong, Zheng; Ding, Han-Fei; Jing, Qing; Su, Shi-Bing; Huang, Shuang

    2015-08-28

    CCNE1 gene amplification is present in 15-20% ovary tumor specimens. Here, we showed that Cyclin E1 (CCNE1) was overexpressed in 30% of established ovarian cancer cell lines. We also showed that CCNE1 was stained positive in over 40% of primary ovary tumor specimens regardless of their histological types while CCNE1 staining was either negative or low in normal ovary and benign ovary tumor tissues. However, the status of CCNE1 overexpression was not associated with the tumorigenic potential of ovarian cancer cell lines and also did not correlate with pathological grades of ovary tumor specimens. Subsequent experiments with CCNE1 siRNAs showed that knockdown of CCNE1 reduced cell growth only in cells with inherent CCNE1 overexpression, indicating that these cells may have developed an addiction to CCNE1 for growth/survival. As CCNE1 is a regulatory factor of cyclin-dependent kinase 2 (Cdk2), we investigated the effect of Cdk2 inhibitor on ovary tumorigenecity. Ovarian cancer cells with elevated CCNE1 expression were 40 times more sensitive to Cdk2 inhibitorSNS-032 than those without inherent CCNE1 overexpression. Moreover, SNS-032 greatly prolonged the survival of mice bearing ovary tumors with inherent CCNE1 overexpression. This study suggests that ovary tumors with elevated CCNE1 expression may be staged for Cdk2-targeted therapy. PMID:26204491

  19. Expression of D-type cyclins in mantle cell and diffuse large B-cell lymphomas.

    PubMed

    Zlamalikova, Lenka; Moulis, Mojmir; Salek, David; Jarkovsky, Jiri; Smarda, Jan; Smardova, Jana

    2016-05-01

    D-type cyclins are involved in cell cycle regulation and play an important role in the pathogenesis of lymphomas. Aberrant expression of cyclin D1 is associated with mantle cell lymphoma (MCL) and serves as a diagnostic marker of MCL. Analysis of cyclin D expression in tumor tissues of patients with diffuse large B-cell lymphoma (DLBCL) which comprises a heterogeneous group of tumors may contribute to their stratification. We analyzed expression of cyclin D1, D2, and D3 mRNAs in 30 MCL and 104 DLBCL patients using qRT-PCR and addressed their significance for disease outcome. We confirmed a high level of cyclin D1 mRNA in 29 MCL cases (97%). One case (3%) was identified as positive for cyclin D2. Expression of cyclin D1 was limited to MCL and did not occur in DLBCL. Overexpression of cyclin D2, which is rare in MCL, occurred more frequently in DLBCL (11 cases, 10.6%). We showed that high expression of cyclin D2 in DLBCL cases de novo decreased the overall survival rate (P=0.016) and progression-free survival (P=0.009). The expression pattern of cyclin D3 was similar in both types of studied lymphomas and it did not affect the disease outcome. PMID:26985765

  20. Cyclin D1 splice variants: polymorphism, risk, and isoform specific regulation in prostate cancer

    PubMed Central

    Comstock, Clay E.S.; Augello, Michael A.; Benito, Ruth Pe; Karch, Jason; Tran, Thai H.; Utama, Fransiscus E.; Tindall, Elizabeth A.; Wang, Ying; Burd, Craig J.; Groh, Eric M.; Hoang, Hoa N.; Giles, Graham G.; Severi, Gianluca; Hayes, Vanessa M.; Henderson, Brian E.; Marchand, Loic Le; Kolonel, Laurence N.; Haiman, Christopher A.; Baffa, Raffaele; Gomella, Leonard G.; Knudsen, Erik S.; Rui, Hallgeir; Henshall, Susan M.; Sutherland, Robert L.; Knudsen, Karen E.

    2010-01-01

    Purpose Alternative CCND1 splicing results in cyclin D1b, which has specialized, pro-tumorigenic functions in prostate not shared by the cyclin D1a (full-length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged. Experimental Design First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings validated in primary tissue. Fourth, impact of G/A870 on cancer risk was assessed in two large case-control studies. Results Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed non-correlated expression patterns, and hormone status did not alter splicing. While G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism. Conclusions These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. While A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate, and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. PMID:19706803

  1. Mechanisms Controlling Subcellular Localization of the G1 Cyclins Cln2p and Cln3p in Budding Yeast

    PubMed Central

    Miller, Mary E.; Cross, Frederick R.

    2001-01-01

    Different G1 cyclins confer functional specificity to the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G1 cyclin is localized primarily to the nucleus, while Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p, suggesting that this process may be physiologically regulated. The accumulation of hypophosphorylated Cln2 in the nucleus is an energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained by elutriation, and this lowered phosphorylation correlates with reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have a brief period of nuclear residence early in the cell cycle. In contrast, the nuclear localization pattern of Cln3p is not influenced by Cdk activity. Cln3p localization requires a bipartite nuclear localization signal (NLS) located at the C terminus of the protein. This sequence is required for nuclear localization of Cln3p and is sufficient to confer nuclear localization to green fluorescent protein in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much less active in genetic assays specific for Cln3p, but more active in assays normally specific for Cln2p, consistent with the idea that Cln3p localization explains a significant part of Clnp functional specificity. PMID:11509671

  2. A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death*

    PubMed Central

    de Araujo, Christiane B.; Russo, Lilian C.; Castro, Leandro M.; Forti, Fábio L.; do Monte, Elisabete R.; Rioli, Vanessa; Gozzo, Fabio C.; Colquhoun, Alison; Ferro, Emer S.

    2014-01-01

    Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides. PMID:24764300

  3. De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome.

    PubMed

    Mirzaa, Ghayda M; Parry, David A; Fry, Andrew E; Giamanco, Kristin A; Schwartzentruber, Jeremy; Vanstone, Megan; Logan, Clare V; Roberts, Nicola; Johnson, Colin A; Singh, Shawn; Kholmanskikh, Stanislav S; Adams, Carissa; Hodge, Rebecca D; Hevner, Robert F; Bonthron, David T; Braun, Kees P J; Faivre, Laurence; Rivière, Jean-Baptiste; St-Onge, Judith; Gripp, Karen W; Mancini, Grazia M S; Pang, Ki; Sweeney, Elizabeth; van Esch, Hilde; Verbeek, Nienke; Wieczorek, Dagmar; Steinraths, Michelle; Majewski, Jacek; Boycott, Kym M; Pilz, Daniela T; Ross, M Elizabeth; Dobyns, William B; Sheridan, Eamonn G

    2014-05-01

    Activating mutations in genes encoding phosphatidylinositol 3-kinase (PI3K)-AKT pathway components cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH, OMIM 603387). Here we report that individuals with MPPH lacking upstream PI3K-AKT pathway mutations carry de novo mutations in CCND2 (encoding cyclin D2) that are clustered around a residue that can be phosphorylated by glycogen synthase kinase 3β (GSK-3β). Mutant CCND2 was resistant to proteasomal degradation in vitro compared to wild-type CCND2. The PI3K-AKT pathway modulates GSK-3β activity, and cells from individuals with PIK3CA, PIK3R2 or AKT3 mutations showed similar CCND2 accumulation. CCND2 was expressed at higher levels in brains of mouse embryos expressing activated AKT3. In utero electroporation of mutant CCND2 into embryonic mouse brains produced more proliferating transfected progenitors and a smaller fraction of progenitors exiting the cell cycle compared to cells electroporated with wild-type CCND2. These observations suggest that cyclin D2 stabilization, caused by CCND2 mutation or PI3K-AKT activation, is a unifying mechanism in PI3K-AKT-related megalencephaly syndromes. PMID:24705253

  4. Cyclin E Associates with the Lipogenic Enzyme ATP-Citrate Lyase to Enable Malignant Growth of Breast Cancer Cells.

    PubMed

    Lucenay, Kimberly S; Doostan, Iman; Karakas, Cansu; Bui, Tuyen; Ding, Zhiyong; Mills, Gordon B; Hunt, Kelly K; Keyomarsi, Khandan

    2016-04-15

    Cyclin E is altered in nearly a third of invasive breast cancers where it is a powerful independent predictor of survival in women with stage I-III disease. Full-length cyclin E is posttranslationally cleaved into low molecular weight (LMW-E) isoforms, which are tumor-specific and accumulate in the cytoplasm because they lack a nuclear localization sequence. We hypothesized that aberrant localization of cytosolic LMW-E isoforms alters target binding and activation ultimately contributing to LMW-E-induced tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay to find LMW-E binding proteins in breast cancer, identifying ATP-citrate lyase (ACLY), an enzyme in the de novo lipogenesis pathway, as a novel LMW-E-interacting protein in the cytoplasm. LMW-E upregulated ACLY enzymatic activity, subsequently increasing lipid droplet formation, thereby providing cells with essential building blocks to support growth. ACLY was also required for LMW-E-mediated transformation, migration, and invasion of breast cancer cells in vitro along with tumor growth in vivo In clinical specimens of breast cancer, the absence of LMW-E and low expression of adipophilin (PLIN2), a marker of lipid droplet formation, associated with favorable prognosis, whereas overexpression of both proteins correlated with a markedly worse prognosis. Taken together, our findings establish a novel relationship between LMW-E isoforms of cyclin E and aberrant lipid metabolism pathways in breast cancer tumorigenesis, warranting further investigation in additional malignancies exhibiting their expression. Cancer Res; 76(8); 2406-18. ©2016 AACR. PMID:26928812

  5. The role of cyclins in the maturation of Patella vulgata oocytes.

    PubMed Central

    van Loon, A E; Colas, P; Goedemans, H J; Néant, I; Dalbon, P; Guerrier, P

    1991-01-01

    We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using p13suc1-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect. Images PMID:1655419

  6. The loss of PIN1 deregulates cyclin E and sensitizes mouse embryo fibroblasts to genomic instability.

    PubMed

    Yeh, Elizabeth S; Lew, Brian O; Means, Anthony R

    2006-01-01

    During the G0/G1-S phase transition, the timely synthesis and degradation of key regulatory proteins is required for normal cell cycle progression. Two of these proteins, c-Myc and cyclin E, are recognized by the Cdc4 E3 ligase of the Skp1/Cul1/Rbx1 (SCF) complex. SCF(Cdc4) binds to a similar phosphodegron sequence in c-Myc and cyclin E proteins resulting in ubiquitylation and degradation of both proteins via the 26 S proteosome. Since the prolyl isomerase Pin1 binds the c-Myc phosphodegron and participates in regulation of c-Myc turnover, we hypothesized that Pin1 would bind to and regulate cyclin E turnover in a similar manner. Here we show that Pin1 regulates the turnover of cyclin E in mouse embryo fibroblasts. Pin1 binds to the cyclin E-Cdk2 complex in a manner that depends on Ser384 of cyclin E, which is phosphorylated by Cdk2. The absence of Pin1 results in an increased steady-state level of cyclin E and stalling of the cells in the G1/S phase of the cell cycle. The cellular changes that result from the loss of Pin1 predispose Pin1 null mouse embryo fibroblasts to undergo more rapid genomic instability when immortalized by conditional inactivation of p53 and sensitizes these cells to more aggressive Ras-dependent transformation and tumorigenesis. PMID:16223725

  7. Mutation of hCDC4 leads to cell cycle deregulation of cyclin E in cancer.

    PubMed

    Ekholm-Reed, Susanna; Spruck, Charles H; Sangfelt, Olle; van Drogen, Frank; Mueller-Holzner, Elisabeth; Widschwendter, Martin; Zetterberg, Anders; Reed, Steven I; Reed, Susanna Ekholm

    2004-02-01

    hCDC4, the gene that encodes the F-box protein responsible for targeting cyclin E for ubiquitin-mediated proteolysis, has been found to be mutated in a number of primary cancers and cancer-derived cell lines. We have observed that functional inactivation of hCDC4 does not necessarily correlate with elevated levels of cyclin E in tumors. Here we show, however, that hCDC4 mutation in primary tumors correlates strongly with loss of cell cycle regulation of cyclin E. Similarly, a breast carcinoma-derived cell line mutated for hCDC4 exhibits cell cycle deregulation of cyclin E, but periodic expression is restored by reintroducing hCDC4 via retroviral transduction. Conversely, small interfering RNA-mediated silencing of hCdc4 deregulates cyclin E with respect to the cell cycle. These results indicate that hCdc4 function is an absolute prerequisite for cell cycle regulation of cyclin E levels, and loss of hCdc4 function is sufficient to deregulate cyclin E. PMID:14871801

  8. Cyclin A2 promotes DNA repair in the brain during both development and aging.

    PubMed

    Gygli, Patrick E; Chang, Joshua C; Gokozan, Hamza N; Catacutan, Fay P; Schmidt, Theresa A; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J; Czeisler, Catherine; Otero, José J

    2016-07-01

    Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation inCCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice. PMID:27425845

  9. Cyclin D1 as a universally expressed mantle cell lymphoma-associated tumor antigen for immunotherapy.

    PubMed

    Wang, M; Sun, L; Qian, J; Han, X; Zhang, L; Lin, P; Cai, Z; Yi, Q

    2009-07-01

    Mantle cell lymphoma (MCL) accounts for 5-10% of all non-Hodgkin lymphomas and has the worst prognosis among all lymphomas. The hallmark of MCL is a t(11;14) translocation that results in overexpression of cyclin D1 by tumor cells of virtually all patients. In this study, we examined whether cyclin D1 could be an effective tumor-associated antigen for immunotherapy. We identified cyclin D1 peptides for HLA-A(*)0201 and generated peptide-specific CD8(+) T-cell lines from HLA-A(*)0201(+) blood donors and MCL patients. These cell lines proliferated in response to cyclin D1 peptide-pulsed stimulatory cells. Moreover, the T cells efficiently lysed peptide-pulsed but not unpulsed T2 cells and autologous dendritic cells; cyclin D1(+) and HLA-A(*)0201(+) human MCL lines MINO, SP53, Jeko-1 and Granta 519; and more importantly, HLA-A(*)0201(+) primary lymphoma cells from MCL patients. No killing was observed with HLA-A(*)0201(-) primary lymphoma cells or HLA-A(*)0201(+) normal blood cells, including B cells. These results indicate that these T cells are potent cytotoxic T cells and recognize cyclin D1 peptides naturally presented by patient lymphoma cells in the context of HLA-A(*)0201 molecules. Taken together, our work identifies cyclin D1 as a potentially important antigen for immunotherapy of MCL. PMID:19225534

  10. Cyclin A2 promotes DNA repair in the brain during both development and aging

    PubMed Central

    Gygli, Patrick E.; Chang, Joshua C.; Gokozan, Hamza N.; Catacutan, Fay P.; Schmidt, Theresa A.; Kaya, Behiye; Goksel, Mustafa; Baig, Faisal S.; Chen, Shannon; Griveau, Amelie; Michowski, Wojciech; Wong, Michael; Palanichamy, Kamalakannan; Sicinski, Piotr; Nelson, Randy J.; Czeisler, Catherine; Otero, José J.

    2016-01-01

    Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation in CCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice. PMID:27425845

  11. Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2

    PubMed Central

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A.

    2014-01-01

    Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug. PMID:25136960

  12. Cell growth of BG-1 ovarian cancer cells is promoted by di-n-butyl phthalate and hexabromocyclododecane via upregulation of the cyclin D and cyclin-dependent kinase-4 genes.

    PubMed

    Park, Min-Ah; Hwang, Kyung-A; Lee, Hye-Rim; Yi, Bo-Rim; Jeung, Eui-Bae; Choi, Kyung-Chul

    2012-03-01

    Endocrine-disrupting chemicals (EDCs) are environmentally persistent exogenous compounds released from various industrial products such as plastics, pesticides, drugs, detergents and cosmetics. They can cause a variety of adverse effects to the reproductive, developmental, immune and nervous systems in humans and wildlife. Di-n-butyl phthalate (DBP) is the main compound of phthalates and is reported to inhibit estrogen receptor (ER)-mediated gene expression and to interfere with normal fetal development of the male reproductive system. Hexabromocyclododecane (HBCD or HBCDD) is one of the brominated flame retardants (BFRs) which have been widely used in plastic, electronic and textile applications and are known to cause endocrine disruption with toxicity of the nervous system. In the present study, the estrogenic effects of DBP and HBCD were examined in an ovarian cancer cell line, BG-1, expressing high levels of ER via MTT assay and semi-quantitative reverse-transcription PCR. Treatment with DBP (10(-8)-10(-5) M) or HBCD (2 x 10(-8) -2 x 10(-6) M) resulted in increased cell proliferation of BG-1 cells as observed with 17-β estradiol (E2). In addition, both DBP and HBCD upregulated the expression levels of cell cycle-regulatory genes, such as cyclin D and cyclin-dependent kinase-4 (cdk-4), which are downstream target genes of ER, at 6 h after treatment. However, the expression of the p21 gene was not altered by DBP or HBCD at any time as with E2. Taken together, these results suggest that DBP and HBCD are EDCs which have apparent estrogenic activities by stimulating the cell proliferation of BG-1 cells and by inducing the expression of cyclin D and cdk-4. Our results suggest that DBP and HBCD have sufficient potency to disrupt the endocrine system and to stimulate cell growth in ER-positive cancer cells. PMID:22179484

  13. Targeting the cyclin D-cyclin-dependent kinase (CDK) 4/6-retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions.

    PubMed

    Spring, Laura; Bardia, Aditya; Modi, Shanu

    2016-01-01

    Dysregulation of the cyclin D-cyclin-dependent kinase (CDK) 4/6-INK4-retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively non-selective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicated promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II-III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D-CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combination strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer. PMID:26896604

  14. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.

    PubMed

    Deverman, Benjamin E; Pravdo, Piers L; Simpson, Bryan P; Kumar, Sripriya Ravindra; Chan, Ken Y; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P; Gradinaru, Viviana

    2016-02-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  15. A novel cyclin gene (CCNF) in the region of the polycystic kidney disease gene (PKD1)

    SciTech Connect

    Kraus, B.; Pohlschmidt, M.; Leung, L.S.

    1994-11-01

    The major locus for autosomal dominant polycystic kidney disease (PKD1) is located in a gene-rich region on chromosome 16p13.3. Recently the identification of the gene responsible for PKD1 has been described. While searching for candidate genes in this region, the authors isolated a new member of the cyclin family. They have characterized the transcript by sequencing, determination of the exon intron boundaries, and Northern blot analysis. Cyclin F is related to A- and B-type cyclins by sequence, but its function is unknown.

  16. PIK3CA mutation uncouples tumor growth and Cyclin D1 regulation from MEK/ERK and mutant KRAS signaling

    PubMed Central

    Halilovic, Ensar; She, Qing-Bai; Ye, Qing; Pagliarini, Raymond; Sellers, William R.; Solit, David B.; Rosen, Neal

    2010-01-01

    Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small molecule MEK inhibitor, decreases MEK/ERK pathway signaling, and destabilizes Cyclin D1, resulting in significant anti-cancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores Cyclin D1 expression and G1/S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and PI3K mutations are unlikely to respond to inhibition of the MEK pathway alone but will require effective inhibition of both MEK and PI3K/AKT pathway signaling. PMID:20699365

  17. Cell division cycle 6, a mitotic substrate of polo-like kinase 1, regulates chromosomal segregation mediated by cyclin-dependent kinase 1 and separase

    PubMed Central

    Yim, Hyungshin; Erikson, Raymond L.

    2010-01-01

    Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression. PMID:21041660

  18. Phosphorylation of HOX11/TLX1 on Threonine-247 during mitosis modulates expression of cyclin B1

    PubMed Central

    2010-01-01

    Background The HOX11/TLX1 (hereafter referred to as HOX11) homeobox gene was originally identified at a t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T cell acute lymphoblastic leukemias (T-ALLs). We previously reported a predisposition to aberrant spindle assembly checkpoint arrest and heightened incidences of chromosome missegregation in HOX11-overexpressing B lymphocytes following exposure to spindle poisons. The purpose of the current study was to evaluate cell cycle specific expression of HOX11. Results Cell cycle specific expression studies revealed a phosphorylated form of HOX11 detectable only in the mitotic fraction of cells after treatment with inhibitors to arrest cells at different stages of the cell cycle. Mutational analyses revealed phosphorylation on threonine-247 (Thr247), a conserved amino acid that defines the HOX11 gene family and is integral for the association with DNA binding elements. The effect of HOX11 phosphorylation on its ability to modulate expression of the downstream target, cyclin B1, was tested. A HOX11 mutant in which Thr247 was substituted with glutamic acid (HOX11 T247E), thereby mimicking a constitutively phosphorylated HOX11 isoform, was unable to bind the cyclin B1 promoter or enhance levels of the cyclin B1 protein. Expression of the wildtype HOX11 was associated with accelerated progression through the G2/M phase of the cell cycle, impaired synchronization in prometaphase and reduced apoptosis whereas expression of the HOX11 T247E mutant restored cell cycle kinetics, the spindle checkpoint and apoptosis. Conclusions Our results demonstrate that the transcriptional activity of HOX11 is regulated by phosphorylation of Thr247 in a cell cycle-specific manner and that this phosphorylation modulates the expression of the target gene, cyclin B1. Since it is likely that Thr247 phosphorylation regulates DNA binding activity to multiple HOX11 target sequences, it is conceivable that

  19. Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies

    PubMed Central

    Chaikuad, Apirat; Keates, Tracy; Vincke, Cécile; Kaufholz, Melanie; Zenn, Michael; Zimmermann, Bastian; Gutiérrez, Carlos; Zhang, Rong-guang; Hatzos-Skintges, Catherine; Joachimiak, Andrzej; Muyldermans, Serge; Herberg, Friedrich W.; Knapp, Stefan; Müller, Susanne

    2014-01-01

    GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent ‘off-target’ of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK. PMID:24438162

  20. Combinatorial PX-866 and Raloxifene Decrease Rb Phosphorylation, Cyclin E2 Transcription, and Proliferation of MCF-7 Breast Cancer Cells.

    PubMed

    Peek, Gregory W; Tollefsbol, Trygve O

    2016-07-01

    As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-β pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc. PMID:26660119

  1. Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

    SciTech Connect

    Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan; Lee, Nam Y.

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previously identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.

  2. Cyclin partners determine Pho85 protein kinase substrate specificity in vitro and in vivo: control of glycogen biosynthesis by Pcl8 and Pcl10.

    PubMed

    Huang, D; Moffat, J; Wilson, W A; Moore, L; Cheng, C; Roach, P J; Andrews, B

    1998-06-01

    In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlike pho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation of PHO85 suppressed the glycogen storage deficiency of snf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8 and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1 mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10. PMID:9584169

  3. Methanol extract of wheatgrass induces G1 cell cycle arrest in a p53-dependent manner and down regulates the expression of cyclin D1 in human laryngeal cancer cells-an in vitro and in silico approach

    PubMed Central

    Shakya, Garima; Balasubramanian, Sangeetha; Rajagopalan, Rukkumani

    2015-01-01

    Background: Deregs been implicated in the malignancy of cancer. Since many years investigation on the traditional herbs has been the focus to develop novel and effective drug for cancer remedies. Wheatgrass is a medicinal plant, used in folk medicine to cure various diseases. The present study was undertaken to gain insights into antiproliferative effect of methanol extract of wheatgrass. Materials Methods: Cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Lactate Dehydrogenase assays. Cell cycle was analyzed by flow cytometry. Western blot was performed to determine the p53 and cyclin D1 levels. In silico docking interaction of the 14 active components (identified by high-performance liquid chromatography/gas chromatography-mass spectroscopy) of the methanol extract was tested with cyclin D1 (Protein Data Bank ID: 2W96) and compared with the reference cyclin D1/Cdk4 inhibitor. Results: Methanol extract of wheatgrass effectively reduced the cell viability. The cell cycle analysis showed that the extract treatment caused G1 arrest. The level of cyclin D1 was decreased, whereas p53 level was increased. Molecular docking studies revealed interaction of seven active compounds of the extract with the vital residues (Lys112/Glu141) of cyclin D1. Conclusion: These findings indicate that the methanol extract of wheatgrass inhibits human laryngeal cancer cell proliferation via cell cycle G1 arrest and p53 induction. The seven active compounds of the extract were also found to be directly involved in the inhibition of cyclin D1/Cdk4 binding, thus inhibiting the cell proliferation. PMID:26109759

  4. Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression.

    PubMed

    Chowdhury, Rajdeep; Chatterjee, Raghunath; Giri, Ashok K; Mandal, Chitra; Chaudhuri, Keya

    2010-10-01

    Arsenic is a well-established human carcinogen; however molecular mechanisms to arsenic-induced carcinogenesis are complex and elusive. The present study identifies a potential biomarker of arsenic exposure, and redefines arsenic-induced signaling in stimulation of cell proliferation. The effect of arsenic exposure on gene expression was evaluated in PBMC of arsenic-exposed individuals selected from a severely affected district of West Bengal, India. A novel, un-documented biomarker of arsenic exposure, CyclinA was identified by microarray analysis from the study. Non-transformed cell lines HaCat and Int407 when exposed to clinically achievable arsenic concentration showed significant increase of CyclinA substantiating the clinical data. An associated increase in S phase population of cells in cell cycle, indicative of enhanced proliferation was also noticed. On further investigation of the pathway to arsenic-induced proliferation, we observed that arsenic resulted: ROS generation; activated Erk signaling; stimulated AP-1 activity, including immediate early genes, c-Jun and c-Fos. N-Acetyl-l-cysteine, a ROS quencher, blocked the arsenic-induced effects. Our study underlines a previously undefined mechanism by which arsenic imparts its toxicity and results in uncontrolled cell proliferation. PMID:20654705

  5. RNA Inhibition Highlights Cyclin D1 as a Potential Therapeutic Target for Mantle Cell Lymphoma

    PubMed Central

    Weinstein, Shiri; Emmanuel, Rafi; Jacobi, Ashley M.; Abraham, Avigdor; Behlke, Mark A.; Sprague, Andrew G.; Novobrantseva, Tatiana I.; Nagler, Arnon; Peer, Dan

    2012-01-01

    Mantle cell lymphoma is characterized by a genetic translocation results in aberrant overexpression of the CCND1 gene, which encodes cyclin D1. This protein functions as a regulator of the cell cycle progression, hence is considered to play an important role in the pathogenesis of the disease. In this study, we used RNA interference strategies to examine whether cyclin D1 might serve as a therapeutic target for mantle cell lymphoma. Knocking down cyclin D1 resulted in significant growth retardation, cell cycle arrest, and most importantly, induction of apoptosis. These results mark cyclin D1 as a target for mantle cell lymphoma and emphasize the therapeutic potential hidden in its silencing. PMID:22905260

  6. Cybip, a starfish cyclin B-binding protein, is involved in meiotic M-phase exit.

    PubMed

    Offner, Nicolas; Derancourt, Jean; Lozano, Jean Claude; Schatt, Philippe; Picard, André; Peaucellier, Gérard

    2003-01-01

    We designed a screen to identify starfish oocyte proteins able to bind monomeric cyclin B by affinity chromatography on a cyclin B splice variant displaying low affinity for cdc2. We identified a 15kDa protein previously described as a cdk-binding protein [Biochim. Biophys. Acta Mol. Cell Res. 1589 (2002) 219-231]. Cybip is encoded by a single polymorphic gene and the native protein is matured by cleaving a signal peptide. We firmly establish the fact that it is a true cyclin B-binding protein, since the recombinant protein binds recombinant cyclin B in absence of any cdk. Finally, we show that the microinjection of GST-cybip, and of anti-cybip antibody, in maturing starfish oocytes, inhibits H1 kinase and MPF inactivation, and first polar body emission. PMID:12480530

  7. The δ-cyclin expression at early stages of embryogenesis of Brassica rapa L. under clinorotation

    NASA Astrophysics Data System (ADS)

    Artemenko, O. A.; Popova, A. F.

    We present some results of comparison studying of Brassica embryo development and the δ-cyclin genes expression under slow horizontal clinorotation and in the laboratory control. Some backlog of the δ1-cyclin genes expression at early stages of embryogenesis under clinorotation was revealed in comparison with the laboratory control. The similar level of the δ3-cyclin expression at all stages of embryo formation (from one to nine days) in both variants is shown. Some delays in the rate of Brassica rapa embryo development under clinorotation in comparison with the laboratory control can be a result of decrease of a level and some backlog of the δ1-cyclin expression at early stages of embryogenesis.

  8. Sulforaphane induced cell cycle arrest in the G2/M phase via the blockade of cyclin B1/CDC2 in human ovarian cancer cells

    PubMed Central

    2013-01-01

    Background Malignant tumors are the single most common cause of death and the mortality rate of ovarian cancer is the highest among gynecological disorders. The excision of benign tumors is generally followed by complete recovery; however, the activity of cancer cells often results in rapid proliferation even after the tumor has been excised completely. Thus, clinical treatment must be supplemented by auxiliary chemotherapy or radiotherapy. Sulforaphane (SFN) is an extract from the mustard family recognized for its anti-oxidation abilities, phase 2 enzyme induction, and anti-tumor activity. Methods This study investigated the cell cycle arrest in G2/M by SFN and the expression of cyclin B1, Cdc2, and the cyclin B1/CDC2 complex in PA-1 cells using western blotting and co-IP western blotting. Results This study investigated the anticancer effects of dietary isothiocyanate SFN on ovarian cancer, using cancer cells line PA-1. SFN-treated cells accumulated in metaphase by CDC2 down-regulation and dissociation of the cyclin B1/CDC2 complex. Conclusion Our findings suggest that, in addition to the known effects on cancer prevention, SFN may also provide antitumor activity in established ovarian cancer. PMID:23799914

  9. Cyclin-Dependent Kinase 1-Mediated Bcl-xL/Bcl-2 Phosphorylation Acts as a Functional Link Coupling Mitotic Arrest and Apoptosis ▿

    PubMed Central

    Terrano, David T.; Upreti, Meenakshi; Chambers, Timothy C.

    2010-01-01

    Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-xL and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-xL/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-xL and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-xL mutant but not by a phosphomimetic Bcl-xL mutant, confirming Bcl-xL as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-xL/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis. PMID:19917720

  10. Flavopiridol: the first cyclin-dependent kinase inhibitor in human clinical trials.

    PubMed

    Senderowicz, A M

    1999-01-01

    The discovery and cloning of the cyclin-dependent kinases (cdks), main regulators of cell cycle progression, allowed several investigators to design novel modulators of cdk activity. Flavopiridol (HMR 1275, L86-8275), a flavonoid derived from an indigenous plant from India, demonstrated potent and specific in vitro inhibition of all cdks tested (cdks 1, 2, 4 and 7) with clear block in cell cycle progression at the G1/S and G2/M boundaries. Moreover, preclinical studies demonstrated the capacity of flavopiridol to induce programmed cell death, promote differentiation, inhibit angiogenic processes and modulate transcriptional events. The relationship between the latter effects and cdk inhibition is still unclear. Initial testing in early clinical human trials with infusional flavopiridol showed activity in some patients with non-Hodgkin's lymphoma, renal, prostate, colon and gastric carcinomas. Main side effects were secretory diarrhea and a pro-inflammatory syndrome associated with hypotension. Biologically active plasma concentrations of flavopiridol (approximately 300-500 nM) are easily achievable in patients receiving infusional flavopiridol. Phase 2 trials with infusional flavopiridol in several tumor types, other schedules and combination with standard chemotherapies are being assessed. In conclusion, flavopiridol is the first cdk inhibitor to be tested in clinical trials. Although important questions remain to be answered, this positive experience will stimulate the development of novel cdk modulators for cancer therapy. PMID:10665481

  11. C-reactive protein promotes acute kidney injury via Smad3-dependent inhibition of CDK2/cyclin E.

    PubMed

    Lai, Weiyan; Tang, Ying; Huang, Xiao R; Ming-Kuen Tang, Patrick; Xu, Anping; Szalai, Alexander J; Lou, Tan-Qi; Lan, Hui Y

    2016-09-01

    Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-β/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition of tubular epithelial cells. In vitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-β-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. In vivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach for AKI. PMID:27470679

  12. Synthesis and evaluation of indenopyrazoles as cyclin-dependent kinase inhibitors. 2. Probing the indeno ring substituent pattern.

    PubMed

    Nugiel, David A; Vidwans, Anup; Etzkorn, Anna-Marie; Rossi, Karen A; Benfield, Pamela A; Burton, Catherine R; Cox, Sarah; Doleniak, Deborah; Seitz, Steven P

    2002-11-21

    We disclose a novel series of indenopyrazole-based cyclin-dependent kinase (CDK) inhibitors. Kinetic experiments confirmed our initial molecular modeling studies that the compounds are competitive with respect to adenosine 5'-triphosphate (ATP) and bind in the kinase ATP pocket. A unique combination of active pharmacophores led us to a series of semicarbazide-based inhibitors that are highly potent against CDK2 and CDK4 while maintaining selectivity against other relevant serine/threonine kinases. These compounds were active against a transformed human colon cancer cell line (HCT116) while maintaining an acceptable margin of activity against a normal fibroblast cell line. The compounds were found to be highly protein bound in our cell-based assay with the exception of 11k, which maintained a reasonable level of activity in the presence of human plasma proteins. PMID:12431050

  13. Expression of Cyclin D1 and P16 in Esophageal Squamous Cell Carcinoma

    PubMed Central

    Dey, Biswajit; Raphael, Vandana; Khonglah, Yookarin; GiriLynrah, Kyrshanlang

    2015-01-01

    BACKGROUND Esophageal squamous cell carcinoma (ESCC) is one of the lethal cancers with a high incidence rate in Asia. Many genes including cyclin D1 and p16 play important role in its carcinogenesis. We aimed to analyze the expressions of cyclin D1 and p16 with the various clinicopathological characteristics of ESCC. METHODS We examined 30 biopsy samples of ESCC for cyclin D1 and p16 protein expressions using immunohistochemistry. Immunointensity was classified as no immunostaining (-), weakly immunostaining (+), weak immunostaining (++) and strongly positive immunostaining (+++). RESULTS Out of the 30 cases, positive expression of cyclin D1 was detected in 26 cases (86.7%). The percentage of tumors with invasion to the adventitia (88.2%), lymph node metastasis (87.5%), and tumors which were poorly differentiated (92.9%) were higher in cyclin D1 positive tumors than in the cyclin D1 negative tumors. However no significant association was found between cyclin D1 expression and the different clinicopathological parameters.There were 22 cases of ESCC (73.3 %) which showed negativity for p16. The percentage of tumors with invasion to the adventitia (82.4%) and poorly differentiated tumors (92.9%) were higher in the p16 negative tumors than in the p16 positive tumors. There was significant association between the histological grade and p16 expression (p=0.012). However, there were no significant association with regard to site, size and lymph node status of the tumors and p16 expression. CONCLUSION The study shows that alterations of cyclin D1 and p16 play an important role in ESCC. Loss of p16 expression was associated with poor differentiation. PMID:26609350

  14. Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

    PubMed

    Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

    2016-08-01

    Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. PMID:27302109

  15. Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2.

    PubMed Central

    Huang, K N; Odinsky, S A; Cross, F R

    1997-01-01

    We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function. PMID:9234722

  16. Coexpression of cyclin D1 and alpha-internexin in oligodendroglial tumors.

    PubMed

    Matsumura, Nozomi; Nobusawa, Sumihito; Ikota, Hayato; Hirato, Junko; Hirose, Takanori; Yokoo, Hideaki; Nakazato, Yoichi

    2015-10-01

    Oligodendroglial tumors with neuronal differentiation cases have been reported in recent studies. Oligodendrocyte precursor cells (OPCs) give rise to both oligodendrocytes and neurons; however, little is known about the association between OPCs and oligodendroglial tumors with neuronal differentiation. Previously, we observed the coexpression of cyclin D1, one of the OPC markers, and alpha-internexin (INA) in oligodendroglial tumor cells. INA is a neuronal marker, and has been indicated as an immunohistochemical surrogate of chromosome 1p/19q co-deletion in oligodendroglial tumors. In this study, we investigated the expression status in 83 gliomas immunohistochemically, and found that cyclin D1-positive cells were commonly detected in gliomas. There was no correlation between the cyclin D1 and Ki-67 labeling indices, suggesting an unrecognized role of cyclin D1 other than a cell cycle regulator in gliomas. Cyclin D1/INA double-positive cells were consistently observed in oligodendroglial tumors regardless of histological grade. In 2 cases of oligodendroglioma with neuronal differentiation, the tumor cells of neuronal morphology showed higher expression of INA, suggesting INA expression may be associated with a bona fide neuronal phenotype. The prevalence of cyclin D1/INA double-positive cells is a distinct feature of oligodendroglial tumors. This new characteristic finding may have practical utility in glioma classification. PMID:26233522

  17. Analysis of the role of phosphorylation in fission yeast Cdc13p/cyclinB function.

    PubMed

    Ren, Liping; Feoktistova, Anna; McDonald, W Hayes; Haese, Greg Den; Morrell, Jennifer L; Gould, Kathleen L

    2005-04-15

    The Cdk1p-cyclin B complex drives entry into mitosis in all eukaryotes. Cdc13p is the single essential cyclin in Schizosaccharomyces pombe and a member of the cyclin B family. Cdc13p abundance rises during G(2)-phase and falls as cells progress through mitosis and G(1). Cdc13p degradation, mediated by the anaphase-promoting complex, is an important mechanism of Cdk1p inhibition and mitotic exit. Cdk1p-cyclin B1 complexes shuttle between the nucleus and cytoplasm, and preventing nuclear accumulation of Cdk1p-cyclin B1 in mammalian cells appears to be one mechanism of preventing entry into mitosis during a DNA damage-induced checkpoint delay. In vertebrates, phosphorylation plays a key role in regulating the intracellular distribution of cyclins. Previous mass spectrometric analysis identified sites of Cdc13p phosphorylation. Here, we have confirmed that these sites are the sole in vivo Cdc13p phosphorylation sites and have studied the role that phosphorylation plays in Cdc13p localization and function. Our data indicate that Cdc13p accumulates in the nucleolus in response to G(2) checkpoint delays, rather than in the cytoplasm, and that phosphorylation plays no role in Cdc13p localization or function. PMID:15705571

  18. Involvement of cyclin-dependent kinase 5 in 2,5-hexanedione-induced neuropathy.

    PubMed

    Wang, Qing-Shan; Zhang, Cui-Li; Hou, Li-Yan; Zhao, Xiu-Lan; Yang, Xi-Wei; Xie, Ke-Qin

    2008-06-01

    Occupational exposure to n-hexane produces a neuropathy characterized as a central-peripheral distal axonopathy, which is mediated by 2,5-hexanedione (HD). To investigate the mechanisms of the neuropathy induced by HD, the contents and activities of cyclin-dependent kinase 5 (CDK5) and activators (p35 precursor, p35 and p25) in rats' cerebrum cortex (CC), spinal cord (SC) and sciatic nerve (SN) were determined. The results showed that the levels and activities of CDK5 in CC of 200 or 400mg/kg HD-treated rats were significantly decreased in both the cytosolic and membrane fractions and negatively correlated with gait abnormality in the cytosolic fraction. However, CDK5 contents and activities in SN of rats treated with 200 or 400mg/kg HD were significantly increased and positively correlated with gait abnormality in both the cytosolic and membrane fractions. Although increases of CDK5 contents in both the cytosolic and membrane fractions of SC in 200 and 400mg/kg HD-treated rats were also observed, CDK5 activities were significantly decreased in the cytosolic fraction and negatively correlated with gait abnormality. The changes of p35 precursor, p35 and p25 contents in CC, SC and SN showed the same pattern with that of CDK5 activities. Thus, HD intoxication was associated with deregulation of CDK5 and its activator p35 or p25 in nerve tissues. The inconsistent changes of CDK5 activities in CNS and PNS might delegate the different mechanisms of HD-induced peripheral neuropathy. PMID:18423835

  19. Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes.

    PubMed

    Lu, Gang; Seta, Karen A; Millhorn, David E

    2005-06-10

    Cyclin-dependent kinases (CDKs) are a family of evolutionarily conserved serine/threonine kinases. CDK2 acts as a checkpoint for the G(1)/S transition in the cell cycle. Despite a down-regulation of CDK2 activity in postmitotic cells, many cell types, including muscle cells, maintain abundant levels of CDK2 protein. This led us to hypothesize that CDK2 may have a function in postmitotic cells. We show here for the first time that CDK2 can be activated by neuregulin (NRG) in differentiated C2C12 myotubes. In addition, this activity is required for expression of the acetylcholine receptor (AChR) epsilon subunit. The switch from the fetal AChRgamma subunit to the adult-type AChRepsilon is required for synapse maturation and the neuromuscular junction. Inhibition of CDK2 activity with either the specific CDK2 inhibitory peptide Tat-LFG or by RNA interference abolished neuregulin-induced AChRepsilon expression. Neuregulin-induced activation of CDK2 also depended on the ErbB receptor, MAPK, and PI3K, all of which have previously been shown to be required for AChRepsilon expression. Neuregulin regulated CDK2 activity through coordinating phosphorylation of CDK2 on Thr-160, accumulation of CDK2 in the nucleus, and down-regulation of the CDK2 inhibitory protein p27 in the nucleus. In addition, we also observed a novel mechanism of regulation of CDK2 activity by a low molecular weight variant of cyclin E in response to NRG. These findings establish CDK2 as an intermediate molecule that integrates NRG-activated signals from both the MAPK and PI3K pathways to AChRepsilon expression and reveal an undiscovered physiological role for CDK2 in postmitotic cells. PMID:15824106

  20. The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

    PubMed

    Fong, Clifford W

    2016-08-01

    Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. PMID:27317642

  1. After portal branch ligation in the rat, cellular proliferation in associated with selective induction of c-Ha-ras, p53, cyclin E, and Cdk2

    PubMed Central

    Starkel, P; Lambotte, L; Sempoux, C; De Saeger, C; Saliez, A; Maiter, D; Horsmans, Y

    2001-01-01

    BACKGROUND—In liver regeneration after portal branch ligation we previously showed that early cellular changes are observed in both the proliferating and atrophying liver lobes. They are therefore not indicative of future proliferative response. In this study we attempted to define precisely, in the same model, the time at which the cellular processes diverge between the lobes by measuring various parameters associated with cellular proliferation. We also investigated the possible role of inhibitors of cell proliferation in the absence of progression towards the S phase in the atrophying lobes.
AIMS—Expression of p53, c-Ha-ras, cyclin E, cyclin dependent kinase (Cdk2), transforming growth factor (TGF)-β, and interleukin (IL)-1α and IL-1β were assessed in relation to their potential role in proliferating and atrophying cellular phenomenons.
METHODS—Immunohistochemistry, northern blotting, western blotting, and reverse transcription-polymerase chain reaction were performed, mainly at time points corresponding to mid-G1/S phase progression (8-24 hours after surgery).
RESULTS—The common and thus most likely non-specific response was still evident 5-8 hours after surgery and included an increase in IL-1 mRNA as well as p53 and cyclin E proteins. From 12 hours onwards, p53, c-Ha-ras, cyclin E, and Cdk2 were selectively induced in proliferating lobes whereas IL-1β was predominantly activated in atrophying lobes. No changes in TGF-β or IL-1α expression were observed at the same time points in any of the liver lobes.
CONCLUSIONS—The initial response to portal branch ligation and thus probably to partial hepatectomy seems to be non-specific for at least eight hours. Thereafter, p53, c-Ha-ras, cyclin E, and Cdk2 seem to drive cellular proliferation while IL-1β is associated with cellular atrophy. In contrast, TGF-β and IL-1α do not seem to play a role in determining the commitment of cells towards atrophy or proliferation.


Keywords: portal

  2. c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

    SciTech Connect

    Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu

    2009-02-15

    Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.

  3. MiR-34a Promotes Osteogenic Differentiation of Human Adipose-Derived Stem Cells via the RBP2/NOTCH1/CYCLIN D1 Coregulatory Network.

    PubMed

    Fan, Cong; Jia, Lingfei; Zheng, Yunfei; Jin, Chanyuan; Liu, Yunsong; Liu, Hao; Zhou, Yongsheng

    2016-08-01

    MiR-34a was demonstrated to be upregulated during the osteogenic differentiation of human adipose-derived stem cells (hASCs). Overexpression of miR-34a significantly increased alkaline phosphatase activity, mineralization capacity, and the expression of osteogenesis-associated genes in hASCs in vitro. Enhanced heterotopic bone formation in vivo was also observed upon overexpression of miR-34a in hASCs. Mechanistic investigations revealed that miR-34a inhibited the expression of retinoblastoma binding protein 2 (RBP2) and reduced the luciferase activity of reporter gene construct comprising putative miR-34a binding sites in the 3' UTR of RBP2. Moreover, miR-34a downregulated the expression of NOTCH1 and CYCLIN D1 and upregulated the expression of RUNX2 by targeting RBP2, NOTCH1, and CYCLIN D1. Taken together, our results suggested that miR-34a promotes the osteogenic differentiation of hASCs via the RBP2/NOTCH1/CYCLIN D1 coregulatory network, indicating that miR-34a-targeted therapy could be a valuable approach to promote bone regeneration. PMID:27453008

  4. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis

    PubMed Central

    Collins, Carl; Maruthi, N. M.; Jahn, Courtney E.

    2015-01-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants. PMID:26022252

  5. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin.

    PubMed

    Fusté, Noel P; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  6. Fragment-Based De Novo Design of Cyclin-Dependent Kinase 2 Inhibitors.

    PubMed

    Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    Cyclin-dependent kinases (CDKs) are core components of the cell cycle machinery that govern the transition between phases during cell cycle progression. Abnormalities in CDKs activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. One of the main bottlenecks hampering the development of drugs for kinase is the difficulty to attain selectivity. A huge variety of small molecules have been reported as CDK inhibitors, as potential anticancer agents, but none of these has been approved for commercial use. Computer-based molecular design supports drug discovery by suggesting novel new chemotypes and compound modifications for lead candidate optimization. One of the methods known as de novo ligand design technique has emerged as a complementary approach to high-throughput screening. Several automated de novo software programs have been written, which automatically design novel structures to perfectly fit in known binding site. The de novo design supports drug discovery assignments by generating novel pharmaceutically active agents with desired properties in a cost as well as time efficient approach. This chapter describes procedure and an overview of computer-based molecular de novo design methods on a conceptual level with successful examples of CDKs inhibitors. PMID:26231707

  7. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin

    PubMed Central

    Fusté, Noel P.; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  8. Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1▿ †

    PubMed Central

    Sanhaji, Mourad; Friel, Claire Therese; Kreis, Nina-Naomi; Krämer, Andrea; Martin, Claudia; Howard, Jonathon; Strebhardt, Klaus; Yuan, Juping

    2010-01-01

    Mitotic centromere-associated kinesin (MCAK) plays an essential role in spindle formation and in correction of improper microtubule-kinetochore attachments. The localization and activity of MCAK at the centromere/kinetochore are controlled by Aurora B kinase. However, MCAK is also abundant in the cytosol and at centrosomes during mitosis, and its regulatory mechanism at these sites is unknown. We show here that cyclin-dependent kinase 1 (Cdk1) phosphorylates T537 in the core domain of MCAK and attenuates its microtubule-destabilizing activity in vitro and in vivo. Phosphorylation of MCAK by Cdk1 promotes the release of MCAK from centrosomes and is required for proper spindle formation. Interfering with the regulation of MCAK by Cdk1 causes dramatic defects in spindle formation and in chromosome positioning. This is the first study demonstrating that Cdk1 regulates the localization and activity of MCAK in mitosis by directly phosphorylating the catalytic core domain of MCAK. PMID:20368358

  9. A new mitochondrial pool of cyclin E, regulated by Drp1, is linked to cell-density-dependent cell proliferation.

    PubMed

    Parker, Danitra J; Iyer, Archana; Shah, Shikha; Moran, Aida; Hjelmeland, Anita B; Basu, Malay Kumar; Liu, Runhua; Mitra, Kasturi

    2015-11-15

    The regulation and function of the crucial cell cycle regulator cyclin E (CycE) remains elusive. Unlike other cyclins, CycE can be uniquely controlled by mitochondrial energetics, the exact mechanism being unclear. Using mammalian cells (in vitro) and Drosophila (in vivo) model systems in parallel, we show that CycE can be directly regulated by mitochondria through its recruitment to the organelle. Active mitochondrial bioenergetics maintains a distinct mitochondrial pool of CycE (mtCycE) lacking a key phosphorylation required for its degradation. Loss of the mitochondrial fission protein dynamin-related protein 1 (Drp1, SwissProt O00429 in humans) augments mitochondrial respiration and elevates the mtCycE pool allowing CycE deregulation, cell cycle alterations and enrichment of stem cell markers. Such CycE deregulation after Drp1 loss attenuates cell proliferation in low-cell-density environments. However, in high-cell-density environments, elevated MEK-ERK signaling in the absence of Drp1 releases mtCycE to support escape of contact inhibition and maintain aberrant cell proliferation. Such Drp1-driven regulation of CycE recruitment to mitochondria might be a mechanism to modulate CycE degradation during normal developmental processes as well as in tumorigenic events. PMID:26446260

  10. Introns, alternative splicing, spliced leader trans-splicing and differential expression of pcna and cyclin in Perkinsus marinus.

    PubMed

    Zhang, Huan; Dungan, Christopher F; Lin, Senjie

    2011-01-01

    To gain understanding on the structure and regulation of growth-related genes of the parasitic alveolatePerkinsus marinus, we analyzed genes encoding proliferating cell nuclear antigen (pcna) and cyclins (cyclin). Comparison of the full-length cDNAs with the corresponding genomic sequences revealedtrans-splicing of the mRNAs of these genes with a conserved 21-22 nt spliced leader. Over 10 copies ofpcnawere detected, with identical gene structures and similar nucleotide (nt) sequences (88-99%), encoding largely identical amino acid sequences (aa). Two distinct types ofcyclin(Pmacyclin1 andPmacyclin2) were identified, with 66-69% nt and 81-85% aa similarities.Pmacyclin2 was organized in tandem repeats, and was alternatively spliced, giving rise to five subtypes of transcripts. For bothpcnaandcyclingenes, 6-10 introns were found. Quantitative RT-PCR assays showed thatpcnaandPmacyclin2 expression levels were low with small variations during a 28-h time course, whereasPmacyclin1 transcript abundance was 10-100 times higher, and increased markedly during active cell division, suggesting that it is a mitoticcyclinand can be a useful growth marker for this species. The gene structure and expression features along with phylogenetic results position this organism between dinoflagellates and apicomplexans, but its definitive affiliation among alveolates requires further studies. PMID:20650682

  11. Monoterpenes inhibit cell growth, cell cycle progression, and cyclin D1 gene expression in human breast cancer cell lines.

    PubMed

    Bardon, S; Picard, K; Martel, P

    1998-01-01

    Monoterpenes are found in the essential oils of many commonly consumed fruits and vegetables. These compounds have been shown to exert chemopreventive and chemotherapeutic activities in mammary tumor models and represent a new class of breast cancer therapeutic agents. In this study, we investigated the effects of limonene and limonene-related monoterpenes, perillyl alcohol and perillic acid, on cell growth, cell cycle progression, and expression of cyclin D1 cell cycle-regulatory gene in T-47D, MCF-7, and MDA-MB-231 breast cancer cell lines. Our results revealed that limonene-related monoterpenes caused a dose-dependent inhibition of cell proliferation. Of the three monoterpenes tested, perillyl alcohol was the most potent and limonene was the least potent inhibitor of cell growth. The enantiomeric composition of limonene and perillyl alcohol did not interfere with their effect on cell growth. Sensitivity of breast cancer cell lines to monoterpenes was in the following order: T-47D > MCF-7 > MDA-MB-231. Growth inhibition induced by perillyl alcohol and perillic acid was associated with a fall in the proportion of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Finally, we showed that the effects of limonene-related monoterpenes on cell proliferation and cell cycle progression were preceded by a decrease in cyclin D1 mRNA levels. PMID:9824849

  12. Cyc17, a meiosis-specific cyclin, is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila.

    PubMed

    Yan, Guan-Xiong; Dang, Huai; Tian, Miao; Zhang, Jing; Shodhan, Anura; Ning, Ying-Zhi; Xiong, Jie; Miao, Wei

    2016-07-17

    Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17. PMID:27192402

  13. Characteristics of Cyclin B and its potential role in regulating oogenesis in the red claw crayfish (Cherax quadricarinatus).

    PubMed

    Wang, L M; Lv, W W; Zuo, D; Dong, Z J; Zhao, Y L

    2015-01-01

    Cyclin B is a regulatory subunit of maturation-promoting factor (MPF), which has a key role in the induction of meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, the complete cDNA sequence of Cyclin B was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. Cyclin B cDNA (1779 bp) encoded a 401 amino acid protein with a calculated molecular weight of 45.1 kDa. Quantitative real-time PCR demonstrated that Cyclin B mRNA was expressed mainly in the ovarian tissue and that the expression decreased as the ovaries developed. Immunofluorescence analysis revealed that the Cyclin B protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cyclin B plays an important role in gametogenesis and gonad development in C. quadricarinatus. PMID:26400307

  14. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

    PubMed Central

    Ferguson, David J. P.; Kaindama, Mbinda L.; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S.; Wheatley, Sally P.; Yamano, Hiroyuki; Holder, Anthony A.; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-01-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei. PMID:26565797

  15. Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry

    PubMed Central

    Jain, P; Giustolisi, G M; Atkinson, S; Elnenaei, M O; Morilla, R; Owusu-Ankomah, K; Rafiq-Mohammed, F; Matutes, E; Wotherspoon, A; Catovsky, D

    2002-01-01

    Aims: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). Methods: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1–120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. Results: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. Conclusion: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay. PMID:12461064

  16. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer.

    PubMed

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

    2014-04-25

    Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168-245 nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers. PMID:24704453

  17. Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes.

    PubMed

    Roques, Magali; Wall, Richard J; Douglass, Alexander P; Ramaprasad, Abhinay; Ferguson, David J P; Kaindama, Mbinda L; Brusini, Lorenzo; Joshi, Nimitray; Rchiad, Zineb; Brady, Declan; Guttery, David S; Wheatley, Sally P; Yamano, Hiroyuki; Holder, Anthony A; Pain, Arnab; Wickstead, Bill; Tewari, Rita

    2015-11-01

    Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei. PMID:26565797

  18. Cdk5-mediated inhibition of APC/C-Cdh1 switches on the cyclin D1-Cdk4-pRb pathway causing aberrant S-phase entry of postmitotic neurons

    PubMed Central

    Veas-Pérez de Tudela, Miguel; Maestre, Carolina; Delgado-Esteban, María; Bolaños, Juan P.; Almeida, Angeles

    2015-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells. To enter the S-phase, APC/C must be inactivated by phosphorylation of its cofactor, Cdh1. In post-mitotic cells such as neurons APC/C-Cdh1 complex is highly active and responsible for the continuous degradation of mitotic cyclins. However, the specific molecular pathway that determines neuronal cell cycle blockade in post-mitotic neurons is unknown. Here, we show that activation of glutamatergic receptors in rat cortical primary neurons endogenously triggers cyclin-dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1 leading to its cytoplasmic accumulation and disassembly from the APC3 core protein, causing APC/C inactivation. Conversely, pharmacological or genetic inhibition of Cdk5 promotes Cdh1 ubiquitination and proteasomal degradation. Furthermore, we show that Cdk5-mediated phosphorylation and inactivation of Cdh1 leads to p27 depletion, which switches on the cyclin D1-cyclin-dependent kinase-4 (Cdk4)-retinoblastoma protein (pRb) pathway to allow the S-phase entry of neurons. However, neurons do not proceed through the cell cycle and die by apoptosis. These results indicate that APC/C-Cdh1 actively suppresses an aberrant cell cycle entry and death of neurons, highlighting its critical function in neuroprotection. PMID:26658992

  19. Cyclin-dependent kinase 4 phosphorylates and positively regulates PAX3-FOXO1 in human alveolar rhabdomyosarcoma cells.

    PubMed

    Liu, Lingling; Wu, Jing; Ong, Su Sien; Chen, Taosheng

    2013-01-01

    Alveolar rhabdomyosarcoma (ARMS) is an aggressive childhood muscle sarcoma with a 5-year survival rate of less than 30%. More than 80% of ARMSs harbor a PAX3-FOXO1 fusion transcription factor. However, expression of PAX3-FOXO1 in muscle cells alone is not sufficient and requires the loss of function of Ink4a/ARF to promote malignant proliferation of muscle cells in vitro or initiate ARMS tumor formation in vivo. This prompted us to examine the signaling pathways required to activate the function of PAX3-FOXO1 and to explore the functional interaction between the Ink4a/ARF and PAX3-FOXO1 signaling pathways. Here we report that inhibition of cyclin-dependent kinase 4 (Cdk4) by fascaplysin (a small molecule selective inhibitor of Cdk4/cyclin D1 that we identified in a screen for compounds that inhibit PAX3-FOXO1) led to inhibition of the transcriptional activity of PAX3-FOXO1 in ARMS cell line Rh30. Consistent with this finding, activation of Cdk4 enhanced the activity of PAX3-FOXO1. In vitro kinase assays revealed that Cdk4 directly phosphorylated PAX3-FOXO1 at Ser(430). Whereas fascaplysin did not affect the protein level of PAX3-FOXO1, it did increase the cytoplasmic level of PAX3-FOXO1 in a portion of cells. Our findings indicate that Cdk4 phosphorylates and positively regulates PAX3-FOXO1 and suggest that inhibition of Cdk4 activity should be explored as a promising avenue for developing therapy for ARMS. PMID:23469153

  20. Downregulation of cyclin-dependent kinase inhibitor; p57{sup kip2}, is involved in the cell cycle progression of vascular smooth muscle cells

    SciTech Connect

    Nakano, Noritsugu . E-mail: norida@med.hokudai.ac.jp; Urasawa, Kazushi; Takagi, Yasushi; Saito, Takahiko; Kaneta, Satoshi; Ishikawa, Susumu; Higashi, Hideaki; Tsutsui, Hiroyuki; Hatakeyama, Masanori; Kitabatake, Akira

    2005-12-23

    Immature vascular smooth muscle cells (VSMCs) proliferate responding to extrinsic mitogens and accumulate in neointima after arterial injuries. Cell proliferation is positively regulated by cyclin/cyclin-dependent kinase (CDK) complex and negatively controlled by CDK inhibitors; CKIs such as p27{sup kip1} and p57{sup kip2}. In this study, embryonic rat thoracic aorta VSMCs; A10 were G0/G1 arrested by serum starvation, re-stimulated with serum, and harvested every four hours. Both CKIs co-expressed in quiescent VSMCs and rapidly diminished by stimulation. Protein level of p27{sup kip1} was regulated by both transcription and post-transcription, but that of p57{sup kip2} was mainly by post-transcription. Supplemental overexpression of p57{sup kip2} inhibited the activations of G1 cyclin/CDKs and subsequent hyperphosphorylations of all three retinoblastoma pocket proteins as well as G1/S transition of cell cycle. Our findings suggest that the downregulations of not only p27{sup kip1}, but also p57{sup kip2} responding to mitogenic stimulation, play key roles in the cell cycle progression of VSMCs.

  1. Sulforaphane, a Dietary Isothiocyanate, Induces G₂/M Arrest in Cervical Cancer Cells through CyclinB1 Downregulation and GADD45β/CDC2 Association.

    PubMed

    Cheng, Ya-Min; Tsai, Ching-Chou; Hsu, Yi-Chiang

    2016-01-01

    Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G₂/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G₂/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G₂/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins. PMID:27626412

  2. Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

    PubMed Central

    Zuber, M; Tan, E M; Ryoji, M

    1989-01-01

    Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication. Images PMID:2564636

  3. Cyclin-dependent kinase inhibitor dinaciclib potently synergizes with cisplatin in preclinical models of ovarian cancer

    PubMed Central

    Zhu, Xiu-Jie; Lin, Feng; Pan, Shi-Shi; Gong, Li-Hua; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Mei, Xiao-Long; Xue, You-Qiu; Qin, Wu-Ming; Shi, Zhi; Yan, Xiao-Jian

    2015-01-01

    Ovarian cancer is one of the most lethal of woman cancers, and its clinical therapeutic outcome currently is unsatisfied. Dinaciclib, a novel small molecule inhibitor of CDK1, CDK2, CDK5 and CDK9, is assessed in clinical trials for the treatment of several types of cancers. In this study, we investigated the anticancer effects and mechanisms of dinaciclib alone or combined with cisplatin in ovarian cancer. Dinaciclib alone actively induced cell growth inhibition, cell cycle arrest and apoptosis with the increased intracellular ROS levels, which were accompanied by obvious alterations of related proteins such as CDKs, Cyclins, Mcl-1, XIAP and survivin. Pretreatment with N-acety-L-cysteine significantly blocked ROS generation but only partially rescued apoptosis triggered by dinaciclib. Moreover, the combination of dinaciclib with cisplatin synergistically promoted cell cycle arrest and apoptosis, and inhibited the subcutaneous xenograft growth of ovarian cancer in nude mice. Altogether, dinaciclib potently synergizes with cisplatin in preclinical models of ovarian cancer, indicating this beneficial combinational therapy may be a promising strategy for treatment of ovarian cancer. PMID:25962959

  4. The history and future of targeting cyclin-dependent kinases in cancer therapy

    PubMed Central

    Asghar, Uzma; Witkiewicz, Agnieszka K.; Turner, Nicholas C.; Knudsen, Erik S.

    2015-01-01

    Cancer represents a pathological manifestation of uncontrolled cell division; therefore, it has long been anticipated that our understanding of the basic principles of cell cycle control would result in effective cancer therapies. In particular, cyclin-dependent kinases (CDKs) that promote transition through the cell cycle were expected to be key therapeutic targets because many tumorigenic events ultimately drive proliferation by impinging on CDK4 or CDK6 complexes in the G1 phase of the cell cycle. Moreover, perturbations in chromosomal stability and aspects of S phase and G2/M control mediated by CDK2 and CDK1 are pivotal tumorigenic events. Translating this knowledge into successful clinical development of CDK inhibitors has historically been challenging, and numerous CDK inhibitors have demonstrated disappointing results in clinical trials. Here, we review the biology of CDKs, the rationale for therapeutically targeting discrete kinase complexes and historical clinical results of CDK inhibitors. We also discuss how CDK inhibitors with high selectivity (particularly for both CDK4 and CDK6), in combination with patient stratification, have resulted in more substantial clinical activity. PMID:25633797

  5. Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases

    PubMed Central

    Schonbrunn, Ernst; Betzi, Stephane; Alam, Riazul; Martin, Mathew P.; Becker, Andreas; Han, Huijong; Francis, Rawle; Chakrasali, Ramappa; Jakkaraj, Sudhakar; Kazi, Aslamuzzaman; Sebti, Said M.; Cubitt, Christopher L.; Gebhard, Anthony W.; Hazlehurst, Lori A.; Tash, Joseph S.; Georg, Gunda I.

    2013-01-01

    Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009 – 0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 co-crystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4 and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics. PMID:23600925

  6. Drosophila Cyclin G Is a Regulator of the Notch Signalling Pathway during Wing Development

    PubMed Central

    Nagel, Anja C.; Szawinski, Jutta; Zimmermann, Mirjam; Preiss, Anette

    2016-01-01

    Notch signalling regulates a multitude of differentiation processes during Drosophila development. For example, Notch activity is required for proper wing vein differentiation which is hampered in mutants of either the receptor Notch, the ligand Delta or the antagonist Hairless. Moreover, the Notch pathway is involved in several aspects of Drosophila oogenesis as well. We have identified Drosophila Cyclin G (CycG) as a molecular interaction partner of Hairless, the major antagonist in the Notch signalling pathway, in vitro and in vivo. Loss of CycG was shown before to cause female sterility and to disturb the architecture of the egg shell. Nevertheless, Notch dependent processes during oogenesis appeared largely unaffected in cycG mutant egg chambers. Loss of CycG modified the dominant wing phenotypes of Notch, Delta and Hairless mutants. Whereas the Notch loss of function phenotype was ameliorated by a loss of CycG, the phenotypes of either Notch gain of function or of Delta or Hairless loss of function were enhanced. In contrast, loss of CycG had only a minor effect on the wing vein phenotype of mutants affecting the EGFR signalling pathway emphasizing the specificity of the interaction of CycG and Notch pathway members. PMID:26963612

  7. Protein-Protein Interaction for the De Novo Design of Cyclin-Dependent Kinase Peptide Inhibitors.

    PubMed

    Arumugasamy, Karthiga; Tripathi, Sunil Kumar; Singh, Poonam; Singh, Sanjeev Kumar

    2016-01-01

    The homology of the inhibitor binding site regions on the surface of cyclin-dependent kinases (CDKs) makes actual CDK inhibitors unable to bind specifically to their molecular targets. Most of them are ATP competitive inhibitors with low specificity that also affect the phosphorylation mechanisms of other nontarget kinases giving rise to harmful side effects. So, the search of specific and potent inhibitors able to bind to the desired CDK target is still a pending issue. Structure based drug design minimized the erroneous binding and increased the affinity of the inhibitor interaction. In the case of CDKs their activation and regulation mechanisms mainly depend on protein-protein interactions (PPIs). The design of drugs targeting these PPIs makes feasible and promising towards the discovery of new and specific CDK inhibitors. Development of peptide inhibitors for a target protein is an emerging approach in computer aided drug designing. This chapter describes in detail methodology for use of the VitAL-Viterbi algorithm for de novo peptide design of CDK2 inhibitors. PMID:26231708

  8. The Indispensable Role of Cyclin-Dependent Kinase 1 in Skeletal Development

    PubMed Central

    Saito, Masanori; Mulati, Mieradili; Talib, S. Zakiah A.; Kaldis, Philipp; Takeda, Shu; Okawa, Atsushi; Inose, Hiroyuki

    2016-01-01

    Skeletal development is tightly regulated through the processes of chondrocyte proliferation and differentiation. Although the involvement of transcription and growth factors on the regulation of skeletal development has been extensively studied, the role of cell cycle regulatory proteins in this process remains elusive. To date, through cell-specific loss-of-function experiments in vivo, no cell cycle regulatory proteins have yet been conclusively shown to regulate skeletal development. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates skeletal development based on chondrocyte-specific loss-of-function experiments performed in a mouse model. Cdk1 is highly expressed in columnar proliferative chondrocytes and is greatly downregulated upon differentiation into hypertrophic chondrocytes. Cdk1 is essential for proper chondrocyte proliferation and deletion of Cdk1 resulted in accelerated differentiation of chondrocytes. In vitro and ex vivo analyses revealed that Cdk1 is an essential cell cycle regulatory protein for parathyroid hormone-related peptide (PTHrP) signaling pathway, which is critical to chondrocyte proliferation and differentiation. These results demonstrate that Cdk1 functions as a molecular switch from proliferation to hypertrophic differentiation of chondrocytes and thus is indispensable for skeletal development. Given the availability of inhibitors of Cdk1 activity, our results could provide insight for the treatment of diseases involving abnormal chondrocyte proliferation, such as osteoarthritis. PMID:26860366

  9. A detailed analysis of cyclin A accumulation at the G(1)/S border in normal and transformed cells.

    PubMed

    Erlandsson, F; Linnman, C; Ekholm, S; Bengtsson, E; Zetterberg, A

    2000-08-25

    The temporal relationship between cyclin A accumulation and the onset of DNA replication was analyzed in detail. Five untransformed and nine transformed asynchronously growing cell cultures were investigated using a triple immunofluorescence staining protocol combined with computerized evaluation of staining intensities in individual cells. The simultaneous staining of BrdU, cyclin A, and cyclin E made it possible to determine the cell cycle position of each cell investigated. Cells at the G(1)/S border were identified on the basis of cyclin E content and were further analyzed with respect to cyclin A and BrdU content. A method was developed to calculate objective thresholds defining the highest staining intensity found in the negative cells in the population. Using the thresholds we could distinguish cells with minute amounts of cyclin A and BrdU from truly negative cells. We show that the onset of cyclin A accumulation and the start of DNA replication occurs at the same time, or deviating by a few minutes at the most. We also show that cyclin A accumulates continuously during S. This study clearly demonstrates that nuclear cyclin A can be used as a reliable marker for the S and G(2) phases in both normal and transformed interphase cells. PMID:10942581

  10. Identification and characterization of 2 testicular germ cell markers, Glut3 and CyclinA2.

    PubMed

    Howitt, Brooke E; Brooks, James D; Jones, Sunita; Higgins, John P T

    2013-10-01

    Testicular germ cell tumors (TGCT) are the most common type of testicular tumor and encompass different histologic types that greatly influence treatment and prognosis. Immunohistochemical studies may be required for accurate classification, particularly when these tumors present at extragonadal sites, and to aid in distinguishing histologic types. Traditional markers for identifying and distinguishing TGCT include PLAP, CD117, AFP, and CD30. More recently, the addition of OCT3/4 and SALL4 has increased sensitivity for immunohistochemical detection of germ cell tumors. We examined gene expression data from a previously published microarray study that compared normal testis mRNA expression to various TGCT. We also performed a search of the literature to identify less well-characterized markers. Glut3 and cyclinA2 showed promise as TGCT markers. Therefore, we evaluated expression of glut3 and cyclinA2 by immunohistochemistry using tissue microarrays (TMAs). Of 66 seminomas included in the TMA, 64 (97%) showed positive nuclear staining for cyclinA2 and 58 (88%) were strongly positive. Strong positive staining for cyclinA2 was also seen in the spermatocytic seminoma. All 20 of the embryonal carcinomas stained positively with cyclinA2, and 19 (95%) displayed strong nuclear staining for cyclinA2. Twenty of the 20 embryonal carcinomas stained for glut3 in a strong membranous pattern. Of 8 yolk sac tumors, 100% stained with glut3. We also evaluated glut3 and cyclinA2 staining on a general TMA containing 486 samples representing 156 different tumors. CyclinA2 stained a number of other tumor types, but the majority of these were weak or focal staining. Glut3 was rarely positive in other tumors; interestingly, most of these were of ovarian origin. We conclude that glut3 is a sensitive (96%) and specific (92%) marker for embryonal carcinomas and yolk sac tumors. Although cyclinA2 is a sensitive marker of seminomas and embryonal carcinomas (98%), its specificity is lower if

  11. Roscovitine blocks leukocyte extravasation by inhibition of cyclin-dependent kinases 5 and 9

    PubMed Central

    Berberich, Nina; Uhl, Bernd; Joore, Jos; Schmerwitz, Ulrike K; Mayer, Bettina A; Reichel, Christoph A; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert

    2011-01-01

    BACKGROUND AND PURPOSE Roscovitine, a cyclin-dependent kinase (CDK) inhibitor that induces tumour cell death, is under evaluation as an anti-cancer drug. By triggering leukocyte apoptosis, roscovitine can also enhance the resolution of inflammation. Beyond death-inducing properties, we tested whether roscovitine affects leukocyte-endothelial cell interaction, a vital step in the onset of inflammation. EXPERIMENTAL APPROACH Leukocyte-endothelial cell interactions were evaluated in venules of mouse cremaster muscle, using intravital microscopy. In primary human endothelial cells, we studied the influence of roscovitine on adhesion molecules and on the nuclear factor-κB (NF-κB) pathway. A cellular kinome array, in vitro CDK profiling and RNAi methods were used to identify targets of roscovitine. KEY RESULTS In vivo, roscovitine attenuated the tumour necrosis factor-α (TNF-α)-induced leukocyte adherence to and transmigration through, the endothelium. In vitro, roscovitine strongly inhibited TNF-α-evoked expression of endothelial adhesion molecules (E-selectin, intercellular cell adhesion molecule, vascular cell adhesion molecule). Roscovitine blocked NF-κB-dependent gene transcription, but not the NF-κB activation cascade [inhibitor of κB (IκB) kinase activity, IκB-α degradation, p65 translocation]. Using a cellular kinome array and an in vitro CDK panel, we found that roscovitine inhibited protein kinase A, ribosomal S6 kinase and CDKs 2, 5, 7 and 9. Experiments using kinase inhibitors and siRNA showed that the decreased endothelial activation was due solely to blockade of CDK5 and CDK9 by roscovitine. CONCLUSIONS AND IMPLICATIONS Our study highlights a novel mode of action for roscovitine, preventing endothelial activation and leukocyte-endothelial cell interaction by inhibition of CDK5 and 9. This might expand its usage as a promising anti-inflammatory compound. PMID:21391976

  12. Regulation of hippocampal and behavioral excitability by cyclin-dependent kinase 5.

    PubMed

    Hawasli, Ammar H; Koovakkattu, Della; Hayashi, Kanehiro; Anderson, Anne E; Powell, Craig M; Sinton, Christopher M; Bibb, James A; Cooper, Donald C

    2009-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that has been implicated in learning, synaptic plasticity, neurotransmission, and numerous neurological disorders. We previously showed that conditional loss of Cdk5 in adult mice enhanced hippocampal learning and plasticity via modulation of calpain-mediated N-methyl-D-aspartic acid receptor (NMDAR) degradation. In the present study, we characterize the enhanced synaptic plasticity and examine the effects of long-term Cdk5 loss on hippocampal excitability in adult mice. Field excitatory post-synaptic potentials (fEPSPs) from the Schaffer collateral CA1 subregion of the hippocampus (SC/CA1) reveal that loss of Cdk5 altered theta burst topography and enhanced post-tetanic potentiation. Since Cdk5 governs NMDAR NR2B subunit levels, we investigated the effects of long-term Cdk5 knockout on hippocampal neuronal excitability by measuring NMDAR-mediated fEPSP magnitudes and population-spike thresholds. Long-term loss of Cdk5 led to increased Mg(2+)-sensitive potentials and a lower threshold for epileptiform activity and seizures. Biochemical analyses were performed to better understand the role of Cdk5 in seizures. Induced-seizures in wild-type animals led to elevated amounts of p25, the Cdk5-activating cofactor. Long-term, but not acute, loss of Cdk5 led to decreased p25 levels, suggesting that Cdk5/p25 may be activated as a homeostatic mechanism to attenuate epileptiform activity. These findings indicate that Cdk5 regulates synaptic plasticity, controls neuronal and behavioral stimulus-induced excitability and may be a novel pharmacological target for cognitive and anticonvulsant therapies. PMID:19529798

  13. Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin.

    PubMed

    Drapkin, Benjamin J; Lu, Ying; Procko, Andrea L; Timney, Benjamin L; Cross, Frederick R

    2009-01-01

    Cyclin-dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose-response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose-dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2-Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions. PMID:19920813

  14. Arctiin induces cell growth inhibition through the down-regulation of cyclin D1 expression.

    PubMed

    Matsuzaki, Youichirou; Koyama, Makoto; Hitomi, Toshiaki; Yokota, Tomoya; Kawanaka, Mayumi; Nishikawa, Akiyoshi; Germain, Doris; Sakai, Toshiyuki

    2008-03-01

    Arctiin is a major lignan constituent of Arctium lappa and has anti-cancer properties in animal models. It was recently reported that arctiin induces growth inhibition in human prostate cancer PC-3 cells. However, the growth inhibitory mechanism of arctiin remains unknown. Herein we report that arctiin induces growth inhibition and dephosphorylates the tumor-suppressor retinoblastoma protein in human immortalized keratinocyte HaCaT cells. We also show that the growth inhibition caused by arctiin is associated with the down-regulation of cyclin D1 protein expression. Furthermore, the arctiin-induced suppression of cyclin D1 protein expression occurs in various types of human tumor cells, including osteosarcoma, lung, colorectal, cervical and breast cancer, melanoma, transformed renal cells and prostate cancer. Depletion of the cyclin D1 protein using small interfering RNA-rendered human breast cancer MCF-7 cells insensitive to the growth inhibitory effects of arctiin, implicates cyclin D1 as an important target of arctiin. Taken together, these results suggest that arctiin down-regulates cyclin D1 protein expression and that this at least partially contributes to the anti-proliferative effect of arctiin. PMID:18288407

  15. Cyclin D1 and Ki-67 expression in normal, hyperplastic and neoplastic endometrium

    PubMed Central

    Shevra, CR; Ghosh, A; Kumar, M

    2015-01-01

    Background: Proliferation and differentiation of cancer cells are regulated by various cell cycle promoting and inhibiting factors. Our knowledge about these proteins and mechanisms regulating cell cycle progression has increased dramatically in recent years. Aim: The present study was undertaken to examine the expression profile of cell cycle regulatory proteins in normal proliferative endometrium, hyperplasias (simple, complex and atypical) and endometrial carcinoma in a quantitative approach as also to assess correlations of Cyclin D1 expression with Ki-67 a proliferation marker. Settings and Design: A retrospective case control study in a tertiary referral centre. Materials and Methods: We evaluated and compared the expression profile of Cyclin D1 and Ki-67 expressions in 61 endometrial samples submitted as either endometrial curetting or hysterectomy specimens, which were diagnosed as simple hyperplasia (n =11), complex hyperplasia (n = 13), atypical hyperplasia (n = 7), and endometrial carcinoma (n = 20). Results: There was increased expression of Cyclin D1 and Ki-67 in patients with endometrial carcinoma relative to proliferative endometrium and simple hyperplasia, but there was no such difference between cases of atypical hyperplasia and endometrial carcinoma. Cyclin D1 expression had a positive correlation with Ki-67 expression. Cyclin D1 together with Ki-67 may be a marker for endometrial carcinogenesis and tumor cell proliferation. PMID:25511212

  16. Correlation between Cyclin Dependent Kinases and Artemisinin-Induced Dormancy in Plasmodium falciparum In Vitro

    PubMed Central

    Gray, Karen-Ann; Gresty, Karryn J.; Chen, Nanhua; Zhang, Veronica; Gutteridge, Clare E.; Peatey, Christopher L.; Chavchich, Marina; Waters, Norman C.; Cheng, Qin

    2016-01-01

    Background Artemisinin-induced dormancy provides a plausible explanation for recrudescence following artemisinin monotherapy. This phenomenon shares similarities with cell cycle arrest where cyclin dependent kinases (CDKs) and cyclins play an important role. Methods Transcription profiles of Plasmodium falciparum CDKs and cyclins before and after dihydroartemisinin (DHA) treatment in three parasite lines, and the effect of CDK inhibitors on parasite recovery from DHA-induced dormancy were investigated. Results After DHA treatment, parasites enter a dormancy phase followed by a recovery phase. During the dormancy phase parasites up-regulate pfcrk1, pfcrk4, pfcyc2 and pfcyc4, and down-regulate pfmrk, pfpk5, pfpk6, pfcrk3, pfcyc1 and pfcyc3. When entering the recovery phase parasites immediately up-regulate all CDK and cyclin genes. Three CDK inhibitors, olomoucine, WR636638 and roscovitine, produced distinct effects on different phases of DHA-induced dormancy, blocking parasites recovery. Conclusions The up-regulation of PfCRK1 and PfCRK4, and down regulation of other CDKs and cyclins correlate with parasite survival in the dormant state. Changes in CDK expression are likely to negatively regulate parasite progression from G1 to S phase. These findings provide new insights into the mechanism of artemisinin-induced dormancy and cell cycle regulation of P. falciparum, opening new opportunities for preventing recrudescence following artemisinin treatment. PMID:27326764

  17. The E2F functional analogue SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1, and the FACT chromatin reorganizer, to yeast G1 cyclin promoters.

    PubMed

    Takahata, Shinya; Yu, Yaxin; Stillman, David J

    2009-11-01

    Regulation of the CLN1 and CLN2 G1 cyclin genes controls cell cycle progression. The SBF activator binds to these promoters but is kept inactive by the Whi5 and Stb1 inhibitors. The Cdc28 cyclin-dependent kinase phosphorylates Whi5, ending the inhibition. Our chromatin immunoprecipitation (ChIP) experiments show that SBF, Whi5 and Stb1 recruit both Cdc28 and the Rpd3(L) histone deacetylase to CLN promoters, extending the analogy with mammalian G1 cyclin promoters in which Rb recruits histone deacetylases. Finally, we show that the SBF subunit Swi6 recruits the FACT chromatin reorganizer to SBF- and MBF-regulated genes. Mutations affecting FACT reduce the transient nucleosome eviction seen at these promoters during a normal cell cycle and also reduce expression. Temperature-sensitive mutations affecting FACT and Cdc28 can be suppressed by disruption of STB1 and WHI5, suggesting that one critical function of FACT and Cdc28 is overcoming chromatin repression at G1 cyclin promoters. Thus, SBF recruits complexes to promoters that either enhance (FACT) or repress (Rpd3L) accessibility to chromatin, and also recruits the kinase that activates START. PMID:19745812

  18. Down-regulation of cyclin G2 by insulin, IGF-I (insulin-like growth factor 1) and X10 (AspB10 insulin): role in mitogenesis.

    PubMed

    Svendsen, Angela M; Winge, Sofia B; Zimmermann, Maike; Lindvig, Anne B; Warzecha, Caroline B; Sajid, Waseem; Horne, Mary C; De Meyts, Pierre

    2014-01-01

    The mechanisms whereby insulin analogues may cause enhanced mitogenicity through activation of either the IR (insulin receptor) or the IGF-IR (insulin-like growth factor 1 receptor) are incompletely understood. We demonstrate that in L6 myoblasts expressing only IGF-IRs as well as in the same cells overexpressing the IR, IGF-I (insulin-like growth factor 1), insulin and X10 (AspB10 insulin) down-regulate the mRNA expression level of the cell cycle inhibitor cyclin G2, as measured by qRT-PCR (quantitative reverse transcription-PCR), and induce cell growth measured by [6-(3)H]thymidine incorporation into DNA. Western blotting showed a marked down-regulation of cyclin G2 at the protein level in both cell lines. Overexpression of cyclin G2 in the two cell lines diminished the mitogenic effect of all three ligands. The use of specific inhibitors indicated that both the MAPK (mitogen-activated protein kinase) and the PI3K (phosphoinositide 3-kinase) pathways mediate the down-regulation of Ccng2. The down-regulation of CCNG2 by the three ligands was also observed in other cell lines: MCF-7, HMEC, Saos-2, R(-)/IR and INS-1. These results indicate that regulation of cyclin G2 is a key mechanism whereby insulin, insulin analogues and IGF-I stimulate cell proliferation. PMID:24059861

  19. Regulation of μ and δ opioid receptor functions: involvement of cyclin-dependent kinase 5

    PubMed Central

    Beaudry, H; Mercier-Blais, A-A; Delaygue, C; Lavoie, C; Parent, J-L; Neugebauer, W; Gendron, L

    2015-01-01

    Background and Purpose Phosphorylation of δ opioid receptors (DOP receptors) by cyclin-dependent kinase 5 (CDK5) was shown to regulate the trafficking of this receptor. Therefore, we aimed to determine the role of CDK5 in regulating DOP receptors in rats treated with morphine or with complete Freund's adjuvant (CFA). As μ (MOP) and DOP receptors are known to be co-regulated, we also sought to determine if CDK5-mediated regulation of DOP receptors also affects MOP receptor functions. Experimental Approach The role of CDK5 in regulating opioid receptors in CFA- and morphine-treated rats was studied using roscovitine as a CDK inhibitor and a cell-penetrant peptide mimicking the second intracellular loop of DOP receptors (C11-DOPri2). Opioid receptor functions were assessed in vivo in a series of behavioural experiments and correlated by measuring ERK1/2 activity in dorsal root ganglia homogenates. Key Results Chronic roscovitine treatment reduced the antinociceptive and antihyperalgesic effects of deltorphin II (Dlt II) in morphine- and CFA-treated rats respectively. Repeated administrations of C11-DOPri2 also robustly decreased Dlt II-induced analgesia. Interestingly, DAMGO-induced analgesia was significantly increased by roscovitine and C11-DOPri2. Concomitantly, in roscovitine-treated rats the Dlt II-induced ERK1/2 activation was decreased, whereas the DAMGO-induced ERK1/2 activation was increased. An acute roscovitine treatment had no effect on Dlt II- or DAMGO-induced analgesia. Conclusions and Implications Together, our results demonstrate that CDK5 is a key player in the regulation of DOP receptors in morphine- and CFA-treated rats and that the regulation of DOP receptors by CDK5 is sufficient to modulate MOP receptor functions through an indirect process. PMID:25598508

  20. Small molecule modulators of cyclin-dependent kinases for cancer therapy.

    PubMed

    Senderowicz, A M

    2000-12-27

    The majority of human malignancies have aberrancies in the Retinoblastoma (Rb) pathway. Loss in Rb function results from the phosphorylation and inactivation of Rb by the cyclin-dependent kinases (cdks), main regulators of cell cycle progression. Thus, modulators of cdks may have a role in the treatment of human malignancies. Flavopiridol, the first cdk modulator tested in clinical trials, demonstrates interesting preclinical features: cell cycle block, induction of apoptosis, promotion of differentiation, inhibition of angiogenic processes and modulation of transcriptional events. Initial clinical trials with infusional flavopiridol demonstrated activity in some patients with lymphomas and renal, colon gastric carcinomas. Main side effects were diarrhea and hypotension. Phase 2 trials with infusional flavopiridol, other schedules and combination with standard chemotherapies are ongoing. The second cdk modulator tested in clinical trials, UCN-01, is a PKC inhibitor that can also modulate cdk activity. Similar to flavopiridol, UCN-01 blocks cell cycle progression and promotes apoptosis. Moreover, UCN-01 may abrogate checkpoints induced by genotoxic stress due to inhibition of chk1 kinase. The first clinical trial of UCN-01 demonstrated very prolonged half-life (approximately 600 h), due to high binding affinity of UCN-01 to the human alpha-1-acid glycoprotein. Main side effects were headaches, vomiting, hypoxemia and hyperglycemia. Clinical activity was observed in some patients with melanoma and lymphoma. Trials of shorter infusions of UCN-01 or in combination with standard chemotherapeutic agents are ongoing. Although several important basic and clinical questions remain unanswered, development of cdk modulators is a reasonable strategy for cancer therapy. PMID:11426645

  1. A chrysin derivative suppresses skin cancer growth by inhibiting cyclin-dependent kinases.

    PubMed

    Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N R; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon

    2013-09-01

    Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052

  2. A Cyclin D2-Rb Pathway Regulates Cardiac Myocyte Size and RNA Polymerase III After Biomechanical Stress in Adult Myocardium

    PubMed Central

    Angelis, Ekaterini; Garcia, Alejandro; Chan, Shing S.; Schenke-Layland, Katja; Ren, Shuxen; Goodfellow, Sarah J.; Jordan, Maria C.; Roos, Kenneth P.; White, Robert J.; MacLellan, W. Robb

    2008-01-01

    Normally, cell cycle progression is tightly coupled to the accumulation of cell mass; however, the mechanisms whereby proliferation and cell growth are linked are poorly understood. We have identified Cyclin D2 (CycD2), a G1 cyclin implicated in mediating S phase entry, as a potential regulator of hypertrophic growth in adult post mitotic myocardium. To examine the role of CycD2 and its downstream targets, we subjected CycD2-null mice to mechanical stress. Hypertrophic growth in response to transverse aortic constriction (TAC) was attenuated in CycD2 null compared to wildtype mice. Blocking the increase in CycD2 in response to hypertrophic agonists prevented phosphorylation of CycD2-target Rb in vitro and mice deficient for Rb had potentiated hypertrophic growth. Hypertrophic growth requires new protein synthesis and transcription of tRNA genes by RNA pol III, which increases with hypertrophic signals. This load-induced increase in RNA pol III activity is augmented in Rb-deficient hearts. Rb binds and represses Brf-1 and TBP, subunits of RNA pol III-specific transcription factor B, in adult myocardium under basal conditions. However this association is disrupted in response to TAC. RNA pol III activity is unchanged in CycD2-/- myocardium after TAC, and there is no dissociation of TBP from Rb. These investigations identify an essential role for the CycD2-Rb pathway as a governor of cardiac myocyte enlargement in response to biomechanical stress and, more fundamentally, as a regulator of the load-induced activation of RNA pol III. PMID:18420946

  3. Placental Estrogen Suppresses Cyclin D1 Expression in the Nonhuman Primate Fetal Adrenal Cortex*

    PubMed Central

    Dumitrescu, Adina; Aberdeen, Graham W.; Pepe, Gerald J.

    2014-01-01

    We have previously shown that estrogen selectively suppresses growth of the fetal zone of the baboon fetal adrenal cortex, which produces the C19-steroid precursors, eg, dehydroepiandrosterone sulfate, which are aromatized to estrogen within the placenta. In the present study, we determined whether fetal adrenal expression of cell cycle regulators are altered by estrogen and thus provide a mechanism by which estrogen regulates fetal adrenocortical development. Cyclin D1 mRNA levels in the whole fetal adrenal were increased 50% (P < .05), and the number of cells in the fetal adrenal definitive zone expressing cyclin D1 protein was increased 2.5-fold (P < .05), whereas the total number of cells in the fetal zone and fetal serum dehydroepiandrosterone sulfate levels were elevated 2-fold (P < .05) near term in baboons in which fetal serum estradiol levels were decreased by 95% (P < .05) after maternal administration of the aromatase inhibitor letrozole and restored to normal by concomitant administration of letrozole plus estradiol throughout second half of gestation. However, fetal adrenocortical expression of cyclin D2, the cyclin-dependent kinase (Cdk)-2, Cdk4, and Cdk6, and Cdk regulatory proteins p27Kip1 and p57Kip2 were not changed by letrozole or letrozole plus estradiol administration. We suggest that estrogen controls the growth of the fetal zone of the fetal adrenal by down-regulating cyclin D1 expression and thus proliferation of progenitor cells within the definitive zone that migrate to the fetal zone. We propose that estrogen restrains growth and function of the fetal zone via cyclin D1 to maintain estrogen levels in a physiological range during primate pregnancy. PMID:25247468

  4. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  5. AINTEGUMENTA and the D-type cyclin CYCD3;1 regulate root secondary growth and respond to cytokinins.

    PubMed

    Randall, Ricardo S; Miyashima, Shunsuke; Blomster, Tiina; Zhang, Jing; Elo, Annakaisa; Karlberg, Anna; Immanen, Juha; Nieminen, Kaisa; Lee, Ji-Young; Kakimoto, Tatsuo; Blajecka, Karolina; Melnyk, Charles W; Alcasabas, Annette; Forzani, Celine; Matsumoto-Kitano, Miho; Mähönen, Ari Pekka; Bhalerao, Rishikesh; Dewitte, Walter; Helariutta, Ykä; Murray, James A H

    2015-01-01

    Higher plant vasculature is characterized by two distinct developmental phases. Initially, a well-defined radial primary pattern is established. In eudicots, this is followed by secondary growth, which involves development of the cambium and is required for efficient water and nutrient transport and wood formation. Regulation of secondary growth involves several phytohormones, and cytokinins have been implicated as key players, particularly in the activation of cell proliferation, but the molecular mechanisms mediating this hormonal control remain unknown. Here we show that the genes encoding the transcription factor AINTEGUMENTA (ANT) and the D-type cyclin CYCD3;1 are expressed in the vascular cambium of Arabidopsis roots, respond to cytokinins and are both required for proper root secondary thickening. Cytokinin regulation of ANT and CYCD3 also occurs during secondary thickening of poplar stems, suggesting this represents a conserved regulatory mechanism. PMID:26340943

  6. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

    SciTech Connect

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

    2014-04-25

    Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

  7. Inotodiol inhabits proliferation and induces apoptosis through modulating expression of cyclinE, p27, bcl-2, and bax in human cervical cancer HeLa cells.

    PubMed

    Zhao, Li-Wei; Zhong, Xiu-Hong; Yang, Shu-Yan; Zhang, Yi-Zhong; Yang, Ning-Jiang

    2014-01-01

    Inonotus obliquus is a medicinal mushroom that has been used as an effective agent to treat various diseases such as diabetes, tuberculosis and cancer. Inotodiol, an included triterpenoid shows significant anti-tumor effect. However, the mechanisms have not been well documented. In this study, we aimed to explore the effect of inotodiol on proliferation and apoptosis in human cervical cancer HeLa cells and investigated the underlying molecular mechanisms. HeLa cells were treated with different concentrations of inotodiol. The MTT assay was used to evaluate cell proliferating ability, flow cytometry (FCM) was employed for cell cycle analysis and cell apoptosis, while expression of cyclinE, p27, bcl-2 and bax was detected by immunocytochemistry. Proliferation of HeLa cells was inhibited by inotodiolin a dose-dependent manner at 24h (r=0.9999, p<0.01). A sub-G1 peak (apoptotic cells) of HeLa cells was detected after treatment and the apoptosis rate with the concentration and longer incubation time (r=1.0, p<0.01), while the percentage of cells in S phase and G2/M phase decreased significantly. Immunocytochemistry assay showed that the expression of cyclin E and bcl-2 in the treated cells significantly decreased, while the expression of p27 and bax obviously increased, compared with the control group (p<0.05). The results of our research indicate that inotodiol isolated from Inonotus obliquus inhibited the proliferation of HeLa cells and induced apoptosis in vitro. The mechanisms may be related to promoting apoptosis through increasing the expression of bax and cutting bcl-2 and affecting the cell cycle by down-regulation the expression of cyclin E and up-regulation of p27. The results further indicate the potential value of inotodiol for treatment of human cervical cancer. PMID:24815470

  8. Cyclin B1 Expression and p53 Status in Squamous Cell Carcinomas of the Head and Neck

    PubMed Central

    Hoffmann, Thomas K.; Trellakis, Sokratis; Okulicz, Kornelia; Schuler, Patrick; Greve, Jens; Arnolds, Judith; Bergmann, Christoph; Bas, Murat; Lang, Stephan; Lehnerdt, Götz; Brandau, Sven; Mattheis, Stefan; Scheckenbach, Kathrin; Finn, Oliviera J.; Whiteside, Theresa L.; Sonkoly, Enikö

    2013-01-01

    Background The cyclin B1/CDC2 complex governs entry into mitosis by regulating the G2/M checkpoint, and it can be repressed by the tumor suppressor p53. We aimed to determine cyclin B1 expression in squamous cell carcinomas of the head and neck (SCCHN) and correlate it with p53 status and clinicopathological parameters. Patients and Methods Cyclin B1 and p53 protein expression was analyzed by immunohistochemistry, and p53 mutation analyses were performed. Results Cytoplasmic expression of cyclin B1 was found in all 26 SCCHN studied. In contrast, nuclear staining was seen in the basal layers of normal mucosa. A total of 46% of tumors showed high cyclin B1 expression. p53 was overexpressed in 53.8% of cases, and of these 79% carried a p53 gene mutation. High cyclin B1 expression significantly correlated with the high tumor grade, but not with gender, tumor size, nodal status, local tumor recurrence or p53 expression. Conclusion Cyclin B1 is frequently overexpressed in SCCHN, and its high expression is significantly associated with a high tumor grade. These data suggest that cyclin B1 may serve as a potential prognostic biomarker in SCCHN. PMID:21965721

  9. Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila.

    PubMed

    Fischer, Patrick; La Rosa, Martina K; Schulz, Adriana; Preiss, Anette; Nagel, Anja C

    2015-08-01

    In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B'-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila. PMID:26274446