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Sample records for cdk inhibition revealed

  1. Novel structural features of CDK inhibition revealed by an ab initio computational method combined with dynamic simulations

    CERN Document Server

    Heady, Lucy; Mancera, Ricardo L; Joyce, Sian; Venkitaraman, Ashok R; Artacho, Emilio; Skylaris, Chris-Kriton; Ciacchi, Lucio Colombi; Payne, Mike C

    2008-01-01

    The rational development of specific inhibitors for the ~500 protein kinases encoded in the human genome is impeded by a poor understanding of the structural basis for the activity and selectivity of small molecules that compete for ATP binding. Combining classical dynamic simulations with a novel ab initio computational approach linear-scalable to molecular interactions involving thousands of atoms, we have investigated the binding of five distinct inhibitors to the cyclin-dependent kinase CDK2. We report here that polarization and dynamic hydrogen bonding effects, so far undetected by crystallography, affect both their activity and selectivity. The effects arise from the specific solvation patterns of water molecules in the ATP binding pocket or the intermittent formation of hydrogen bonds during the dynamics of CDK/inhibitor interactions and explain the unexpectedly high potency of certain inhibitors such as 3-(3H-imidazol-4-ylmethylene)-5-methoxy-1,3-dihydro-indol-2-one (SU9516). The Lys89 residue in the ...

  2. Inhibition of human immunodeficiency virus type-1 by cdk inhibitors

    Directory of Open Access Journals (Sweden)

    Kehn-Hall Kylene

    2010-03-01

    Full Text Available Abstract Current therapy for human immunodeficiency virus (HIV-1 infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202 in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs.

  3. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

  4. Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities

    OpenAIRE

    Jorge Franco; Uthra Balaji; Elizaveta Freinkman; Agnieszka K. Witkiewicz; Erik S. Knudsen

    2016-01-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was ass...

  5. Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

    Science.gov (United States)

    Kumari, Namita; Iordanskiy, Sergey; Kovalskyy, Dmytro; Breuer, Denitra; Niu, Xiaomei; Lin, Xionghao; Xu, Min; Gavrilenko, Konstantin; Kashanchi, Fatah; Dhawan, Subhash

    2014-01-01

    HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection. PMID:25155598

  6. Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities.

    Science.gov (United States)

    Franco, Jorge; Balaji, Uthra; Freinkman, Elizaveta; Witkiewicz, Agnieszka K; Knudsen, Erik S

    2016-02-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was associated with an increase in mTORC1 activity. MTOR and MEK inhibitors potently cooperate with CDK4/6 inhibition in eliciting cell-cycle exit. However, MTOR inhibition fully suppressed metabolism and yielded apoptosis and suppression of tumor growth in xenograft models. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and reactive oxygen species (ROS). Concordantly, the suppression of ROS scavenging or BCL2 antagonists cooperated with CDK4/6 inhibition. Together, these data define the impact of therapeutics on PDA metabolism and provide strategies for converting cytostatic response to tumor cell killing. PMID:26804906

  7. Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

    Directory of Open Access Journals (Sweden)

    Lara Wohlbold

    2012-08-01

    Full Text Available The cyclin-dependent kinases (CDKs that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS Cdk2 after exposure to ionizing radiation (IR enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

  8. Inhibition of CDK9 as a therapeutic strategy for inflammatory arthritis.

    Science.gov (United States)

    Hellvard, Annelie; Zeitlmann, Lutz; Heiser, Ulrich; Kehlen, Astrid; Niestroj, André; Demuth, Hans-Ulrich; Koziel, Joanna; Delaleu, Nicolas; Jan Potempa; Mydel, Piotr

    2016-01-01

    Rheumatoid arthritis is characterised by synovial inflammation and proliferation of fibroblast-like synoviocytes. The induction of apoptosis has long been proposed as a target for proliferative autoimmune diseases, and has further been shown to act as a successful treatment of experimental models of arthritis, such as collagen-induced arthritis. Here we examined the effects of specific oral small-molecule inhibitors of the transcription regulating cyclin-dependent kinase 9 on the development and progression of collagen-induced arthritis. DBA/1 mice were immunised with bovine collagen type II and treated orally with specific CDK9 inhibitors. The effects of CDK9 inhibition on RNA levels and protein expression, apoptosis induction, caspase activation and lymphocyte phenotype were further analysed. Mice showed a significant delay in disease onset and a reduction in disease severity following treatment with CDK9 inhibitors. Inhibiting CDK9 activity in peripheral blood mononuclear cells resulted in the loss of Mcl-1 expression at both the protein and RNA levels, along with a subsequent increase in apoptosis. CDK9 specific inhibitors may be a potential alternative treatment not only of cancer, but also for autoimmune- and inflammatory diseases. Taken together, these results show that transient inhibition of CDK9 induces apoptosis in leukocyte subsets and modulates the immune response. PMID:27511630

  9. MicroRNA-206 induces G1 arrest in melanoma by inhibition of CDK4 and Cyclin D.

    Science.gov (United States)

    Georgantas, Robert W; Streicher, Katie; Luo, Xiaobing; Greenlees, Lydia; Zhu, Wei; Liu, Zheng; Brohawn, Philip; Morehouse, Christopher; Higgs, Brandon W; Richman, Laura; Jallal, Bahija; Yao, Yihong; Ranade, Koustubh

    2014-03-01

    Expression profiling of microRNAs in melanoma lesional skin biopsies compared with normal donor skin biopsies, as well as melanoma cell lines compared with normal melanocytes, revealed that hsa-miR-206 was down-regulated in melanoma (-75.4-fold, P = 1.7 × 10(-4)). MiR-206 has been implicated in a large number of cancers, including breast, lung, colorectal, ovarian, and prostate cancers; however, its role in tumor development remains largely unknown, its biologic function is poorly characterized, and its targets affecting cancer cells are largely unknown. MiR-206 reduced growth and migration/invasion of multiple melanoma cell lines. Bioinformatics identified cell cycle genes CDK2, CDK4, Cyclin C, and Cyclin D1 as strong candidate targets. Western blots and 3'UTR reporter gene assays revealed that miR-206 inhibited translation of CDK4, Cyclin D1, and Cyclin C. Additionally, hsa-miR-206 transfection induced G1 arrest in multiple melanoma cell lines. These observations support hsa-miR-206 as a tumor suppressor in melanoma and identify Cyclin C, Cyclin D1, and CDK4 as miR-206 targets. PMID:24289491

  10. INHIBITION STUDIES OF TERPENE BASED NATURAL PRODUCTS WITH CYCLIN-DEPENDENT KINASE 4 (CDK4 MIMIC CDK2)

    OpenAIRE

    Dr. Sunil H. Ganatra et al

    2012-01-01

    Cyclin dependent kinases (CDKs) are known as cell cycle regulators in eukaryotic cell cycle. Different CDKs (CDK2, CDK4 etc.) are having structure homology among them. Using computer based molecular modeling tools, interactions between naturally occurring terpene based compounds with crystal structure of CDK4 mimic CDK2 enzyme having PDB ID : 1GII. Using In-silico techniques, the binding energies between terpene based compounds and receptor enzymes are calculated in the form of ΔG in kcal/mol...

  11. Metabolic re-programming of pancreatic cancer mediated by CDK4/6 inhibition elicits unique vulnerabilities

    OpenAIRE

    Franco, Jorge; Balaji, Uthra; Freinkman, Elizaveta; Witkiewicz, Agnieszka K.; Knudsen, Erik S.

    2016-01-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models CDK4/6 inhibition had variable effect on cell cycle, but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was assoc...

  12. INHIBITION STUDIES OF TERPENE BASED NATURAL PRODUCTS WITH CYCLIN-DEPENDENT KINASE 4 (CDK4 MIMIC CDK2

    Directory of Open Access Journals (Sweden)

    Dr. Sunil H. Ganatra et al

    2012-09-01

    Full Text Available Cyclin dependent kinases (CDKs are known as cell cycle regulators in eukaryotic cell cycle. Different CDKs (CDK2, CDK4 etc. are having structure homology among them. Using computer based molecular modeling tools, interactions between naturally occurring terpene based compounds with crystal structure of CDK4 mimic CDK2 enzyme having PDB ID : 1GII. Using In-silico techniques, the binding energies between terpene based compounds and receptor enzymes are calculated in the form of ΔG in kcal/mol. The reported binding energies for series of molecules are ranging from –5.35 to –13.20 kcal/mol. The negative docking energies and a few hydrogen bonds between selected ligands and receptor enzyme support the affinity of Terpene based compounds with CDK4 mimic CDK2 enzymes. It is also found out that those compounds having carbon atoms 30-31 interacts better with enzyme, whereas larger size compounds having carbon atoms higher than 40 show weak interactions. It is concluded that Tri-terpene class of compounds are the best CDK4 mimic CDK2 inhibitors.

  13. Pharmacologic inhibition of cdk4/6 arrests the growth of glioblastoma multiforme intracranial xenografts

    OpenAIRE

    Michaud, Karine; Solomon, David A.; Oermann, Eric; Kim, Jung-Sik; Zhong, Wei-Zhu; Prados, Michael D.; Ozawa, Tomoko; James, C. David; Waldman, Todd

    2010-01-01

    Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6 specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G1 cell cycle arrest and induc...

  14. Iron Chelators of the Di-2-pyridylketone Thiosemicarbazone and 2-Benzoylpyridine Thiosemicarbazone Series Inhibit HIV-1 Transcription: Identification of Novel Cellular Targets—Iron, Cyclin-Dependent Kinase (CDK) 2, and CDK9S⃞

    OpenAIRE

    Debebe, Zufan; Ammosova, Tatyana; Breuer, Denitra; Lovejoy, David B.; Kalinowski, Danuta S.; Karla, Pradeep K.; Kumar, Krishna; Jerebtsova, Marina; Ray, Patricio; KASHANCHI, FATAH; Gordeuk, Victor R; Richardson, Des R.; Nekhai, Sergei

    2011-01-01

    HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 tran...

  15. Complete inhibition of Cdk/cyclin by one molecule of p21Cip1

    OpenAIRE

    Hengst, L; Göpfert, U.; Lashuel, H. A.; Reed, S I

    1998-01-01

    Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase activities are Cdk inhibitors, proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states, in that Cdk complexes associated with one p21 molecule re...

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xuesong; Gong, Xuhai [Department of Neurology, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163001 (China); Chen, Jing [Department of Neurology, Daqing Longnan Hospital, Daqing, Heilongjiang, 163001 China (China); Zhang, Jinghui [Department of Cardiology, The Fourth Hospital of Harbin City, Harbin, Heilongjiang 150026 (China); Sun, Jiahang [Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086 (China); Guo, Mian, E-mail: guomian_hyd@163.com [Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150086 (China)

    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.

  17. Iron Chelators of the Di-2-pyridylketone Thiosemicarbazone and 2-Benzoylpyridine Thiosemicarbazone Series Inhibit HIV-1 Transcription: Identification of Novel Cellular Targets—Iron, Cyclin-Dependent Kinase (CDK) 2, and CDK9S⃞

    Science.gov (United States)

    Debebe, Zufan; Ammosova, Tatyana; Breuer, Denitra; Lovejoy, David B.; Kalinowski, Danuta S.; Karla, Pradeep K.; Kumar, Krishna; Jerebtsova, Marina; Ray, Patricio; Kashanchi, Fatah; Gordeuk, Victor R.; Richardson, Des R.

    2011-01-01

    HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 transcription. Here we have analyzed a group of novel di-2-pyridylketone thiosemicarbazone- and 2-benzoylpyridine thiosemicarbazone-based iron chelators that exhibit marked anticancer activity in vitro and in vivo (Proc Natl Acad Sci USA 103:7670–7675, 2006; J Med Chem 50:3716–3729, 2007). Several of these iron chelators, in particular 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), inhibited HIV-1 transcription and replication at much lower concentrations than did 311 and ICL670. Neither Bp4aT nor Bp4eT were toxic after a 24-h incubation. However, longer incubations for 48 h or 72 h resulted in cytotoxicity. Analysis of the molecular mechanism of HIV-1 inhibition showed that the novel iron chelators inhibited basal HIV-1 transcription, but not the nuclear factor-κB-dependent transcription or transcription from an HIV-1 promoter with inactivated SP1 sites. The chelators inhibited the activities of CDK2 and CDK9/cyclin T1, suggesting that inhibition of CDK9 may contribute to the inhibition of HIV-1 transcription. Our study suggests the potential usefulness of Bp4aT or Bp4eT in antiretroviral regimens, particularly where resistance to standard treatment occurs. PMID:20956357

  18. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1.

    Science.gov (United States)

    Voets, Erik; Marsman, Judith; Demmers, Jeroen; Beijersbergen, Roderick; Wolthuis, Rob

    2015-01-01

    Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase. PMID:26423135

  19. Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yanjun [Shanghai Celstar Bio-Pharmaceutical Co. Ltd. (China). Cancer Research Center; Takebe, Hiraku; Yagi, Takashi

    2000-12-01

    Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21 {sup wafl/Cipl/Sdil} prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21 +/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21 -/-) and DLD1 (p21 +/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. (author)

  20. Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells

    International Nuclear Information System (INIS)

    Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21 wafl/Cipl/Sdil prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21 +/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21 -/-) and DLD1 (p21 +/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. (author)

  1. Interphase APC/C-Cdc20 inhibition by cyclin A2-Cdk2 ensures efficient mitotic entry

    DEFF Research Database (Denmark)

    Hein, Jamin B; Nilsson, Jakob

    2016-01-01

    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...

  2. Established and New Mouse Models Reveal E2f1 and Cdk2 Dependency of Retinoblastoma and Expose Strategies to Block Tumor Initiation

    Science.gov (United States)

    Sangwan, Monika; McCurdy, Sean R.; Livne-bar, Izzy; Ahmad, Mohammad; Wrana, Jeffery L.; Chen, Danian; Bremner, Rod

    2016-01-01

    RB +/− individuals develop retinoblastoma and, subsequently, many other tumors. The Rb relatives p107 and p130 protect the tumor-resistant Rb−/− mouse retina. Determining the mechanism underlying this tumor suppressor function may expose novel strategies to block Rb-pathway cancers. p107/p130 are best known as E2f inhibitors, but here we implicate E2f-independent Cdk2 inhibition as the critical p107 tumor suppressor function in vivo. Like p107 loss, deleting p27 or inactivating its Cdk inhibitor (CKI) function (p27CK−) cooperated with Rb loss to induce retinoblastoma. Genetically, p107 behaved like a CKI because inactivating Rb and one allele each of p27 and p107 was tumorigenic. While Rb loss induced canonical E2f targets, unexpectedly p107 loss did not further induce these genes but instead caused post-transcriptional Skp2-induction and Cdk2 activation. Strikingly, Cdk2 activity correlated with tumor penetrance across all the retinoblastoma models. Therefore, Rb restrains E2f, but p107 inhibits cross-talk to Cdk. While removing either E2f2 or E2f3 genes had little effect, removing only one E2f1 allele blocked tumorigenesis. More importantly, exposing retinoblastoma-prone fetuses to small molecule E2f or Cdk inhibitors for merely one week dramatically inhibited subsequent tumorigenesis in adult mice. Protection was achieved without disrupting normal proliferation. Thus, exquisite sensitivity of the cell-of-origin to E2f and Cdk activity can be exploited to prevent Rb pathway-induced cancer in vivo without perturbing normal cell division. These data suggest that E2f inhibitors, never before tested in vivo, or Cdk inhibitors, largely disappointing as therapeutics, may be effective preventive agents. PMID:22286767

  3. Evaluation and comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibition by paullones

    DEFF Research Database (Denmark)

    Kunick, Conrad; Lauenroth, Kathrin; Wieking, Karen; Xie, Xu; Schultz, Christiane; Gussio, Rick; Zaharevitz, Daniel; Leost, Maryse; Meijer, Laurent; Weber, Alexander; Jørgensen, Flemming Steen; Lemcke, Thomas

    2004-01-01

    data of 52 paullone entities, which were aligned by a docking routine into the ATP-binding cleft of a CDK1/cyclin B homology model. Variation of grid spacing and column filtering were used during the optimization of the models. The predictive ability of the models was shown by a leave-one-out cross...

  4. The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition.

    Directory of Open Access Journals (Sweden)

    Thomas K Albert

    Full Text Available CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb that controls the transition of RNA polymerase II (RNAPII into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21 and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription.

  5. CDK1 Inhibition Targets the p53-NOXA-MCL1 Axis, Selectively Kills Embryonic Stem Cells, and Prevents Teratoma Formation

    Directory of Open Access Journals (Sweden)

    Noelle E. Huskey

    2015-03-01

    Full Text Available Embryonic stem cells (ESCs have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs. We examined the effect of CDK inhibition on the pathways regulating proliferation and survival of ESCs. We found that inhibiting cyclin-dependent kinase 1 (CDK1 leads to activation of the DNA damage response, nuclear p53 stabilization, activation of a subset of p53 target genes including NOXA, and negative regulation of the anti-apoptotic protein MCL1 in human and mouse ESCs, but not differentiated cells. We demonstrate that MCL1 is highly expressed in ESCs and loss of MCL1 leads to ESC death. Finally, we show that clinically relevant CDK1 inhibitors prevent formation of ESC-derived tumors and induce necrosis in established ESC-derived tumors. Our data demonstrate that ES cells are uniquely sensitive to CDK1 inhibition via a p53/NOXA/MCL1 pathway.

  6. A chromatography-free isolation of rohitukine from leaves of Dysoxylum binectariferum: Evaluation for in vitro cytotoxicity, Cdk inhibition and physicochemical properties.

    Science.gov (United States)

    Kumar, Vikas; Guru, Santosh K; Jain, Shreyans K; Joshi, Prashant; Gandhi, Sumit G; Bharate, Sandip B; Bhushan, Shashi; Bharate, Sonali S; Vishwakarma, Ram A

    2016-08-01

    Rohitukine is a chromone alkaloid isolated from an Indian medicinal plant Dysoxylum binectariferum. This natural product has led to the discovery of two clinical candidates (flavopiridol and P276-00) for the treatment of cancer. Herein, for the first time we report an efficient protocol for isolation and purification of this precious natural product in a bulk-quantity from leaves (a renewable source) of D. binectariferum (>98% purity) without use of chromatography or any acid-base treatment. Despite of the fact that this scaffold has reached up to clinical stage, particularly for leukemia; however the antileukemic activity of a parent natural product has never been investigated. Furthermore, rohitukine has never been studied for cyclin-dependent kinase (Cdk) inhibition, kinase profiling and for its experimental physicochemical properties. Thus, herein, we report in vitro cytotoxicity of rohitukine in a panel of 20 cancer cell lines (including leukemia, pancreatic, prostate, breast and CNS) and 2 normal cell lines; kinase profiling, Cdk2/9 inhibition, and physicochemical properties (solubility and stability in biological medias, pKa, LogP, LogD). In cytotoxicity screening, rohitukine displayed promising activity in HL-60 and Molt-4 (leukemia) cell lines with GI50 of 10 and 12μM, respectively. It showed inhibition of Cdk2/A and Cdk9/T1 with IC50 values of 7.3 and 0.3μM, respectively. The key interactions of rohitukine with Cdk9 was also studied by molecular modeling. Rohitukine was found to be highly water soluble (Swater=10.3mg/mL) and its LogP value was -0.55. The ionization constant of rohitukine was found to be 5.83. Rohitukine was stable in various biological media's including rat plasma. The data presented herein will help in designing better anticancer agents in future. PMID:27363938

  7. Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells.

    Science.gov (United States)

    Koehler, Michael F T; Bergeron, Philippe; Blackwood, Elizabeth M; Bowman, Krista; Clark, Kevin R; Firestein, Ron; Kiefer, James R; Maskos, Klaus; McCleland, Mark L; Orren, Linda; Salphati, Laurent; Schmidt, Steve; Schneider, Elisabeth V; Wu, Jiansheng; Beresini, Maureen H

    2016-03-10

    Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-π interaction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells. PMID:26985305

  8. INHIBITION OF BREAST CANCER CELL PROLIFERATION AND TUMORIGENECITY BY cdk2 ANTISENSE RNA%cdk2反义RNA对乳腺癌细胞增殖及致瘤性的抑制作用

    Institute of Scientific and Technical Information of China (English)

    桑建利; 边昕; 王永潮

    2001-01-01

    为了研究cdk2对乳腺癌细胞生长及cyclinA, cyclinB1和cdk1(cdc2) mRNA表达水平的影响,利用真核表达载体pXJ41-neo构建了表达cdk2反义RNA的重组载体,并用此载体转染了人乳腺癌细胞系Bcap37,获得了cdk2表达受到抑制的细胞模型Bcap37-CDK2AS,然后将Bcap37-CDK2AS细胞的生长能力及cyclinA, cyclinB1和cdk1 mRNA的水平与转入空载体的对照细胞进行了对比分析.结果显示cdk2表达受到抑制时,细胞生长速率下降,根据测定出的细胞生长曲线,细胞培养至第7天时,细胞生长抑制率为64%.在流式细胞术的分析结果中,G1期细胞占的百分比从39%增加到47%,S期细胞由51%下降到39%.裸鼠接种的实验表明,Bcap37-CDK2AS的致瘤性明显减弱.在对cyclinA, cyclinB1和cdk1 mRNA的分析中发现,Bcap37-CDK2AS中这3种基因的mRNA水平均有不同程度的下降,依据这些结果可以推测,cdk2反义RNA可使乳腺癌细胞生长及致瘤性受到抑制,并且cdk2表达的抑制将影响cyclinA, cyclinB和cdk1的表达水平.

  9. Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    Full Text Available Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1 is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a no increase in senescence associated beta-galactosidase activity, (b decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1

  10. Effects of GAPSGF on Inhibiting Hepatoma Cell Line SMMC7721 Proliferation and CDK2 Expression%树舌多糖GF抑制肝癌细胞SMMC7721增殖及对CDK2基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    刘佳维; 宋高臣; 于水澜; 于英君

    2012-01-01

    Objective: To study the effects of Ganoderma appanatum polysaccharides GF(GAPSGF)on proliferation and CDK2 expression in hepatoma cell line SMMC7721. Methods: MTT assay was used to examine the growth and proliferation of SMMC7721 cells after treatment with different concentrations of GAPSGF. Real - time PCR and Western blot were performed to measure the expression of CDK2 at mRNA and protein level. Results: GAPSGF could effectively inhibit proliferation of hepatoma cell line SMMC7721, and GAPSGF at concentration of 2. 5μg · mL-1 and 10μg · mL-1 had a significant effect on inhibiting proliferation. The expression of mRNA and protein of CDK2 effectively reduced. Conclusion: GAPSGF restrains proliferation of hepatoma cell line SMMC7721 by reducing CDK2 expression.%目的:探明树舌多糖GF(Ganoderma appanatum polysaccharides GF,GAPSGF)对肝癌细胞SMMC7721增殖及CDK2表达的影响.方法:不同浓度树舌多糖GF处理肝癌细胞SMMC7721,采用MTT法检测树舌多糖GF对肝癌细胞SMMC7721增殖抑制作用 ;实时定量聚合酶链反应(Real-Time PCR)检测CDK2 mRNA表达 ;蛋白印迹法(Western blot)检测CDK2蛋白表达情况.结果:树舌多糖GF对肝癌细胞SMMC7721具有明显的生长抑制作用.其中树舌多糖GF2.5 μg/mL组与树舌多糖GF10μg/mL组作用显著,且CDK2的mRNA和蛋白表达下降.结论:树舌多糖GF可能通过降低CDK2表达,抑制肝癌细胞SMMC7721增殖.

  11. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

    NARCIS (Netherlands)

    E. Voets (Erik); J. Marsman (Judith); J.A.A. Demmers (Jeroen); R.L. Beijersbergen (Roderick); R. Wolthuis (Rob)

    2015-01-01

    textabstractCyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cd

  12. A dual role of Cdk2 in DNA damage response

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2009-05-01

    Full Text Available Abstract Once it was believed that Cdk2 was the master regulator of S phase entry. Gene knockout mouse studies of cell cycle regulators revealed that Cdk2 is dispensable for S phase initiation and progression whereby Cdk1 can compensate for the loss of Cdk2. Nevertheless, recent evidence indicates that Cdk2 is involved in cell cycle independent functions such as DNA damage repair. Whether these properties are unique to Cdk2 or also being compensated by other Cdks in the absence of Cdk2 is under extensive investigation. Here we review the emerging new role of Cdk2 in DNA damage repair and also discuss how the loss of Cdk2 impacts the G1/S phase DNA damage checkpoint.

  13. The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

    Directory of Open Access Journals (Sweden)

    Grassmann Ralph

    2005-09-01

    Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

  14. Dual inhibition of CDK4/Rb and PI3K/AKT/mTOR pathways by ON123300 induces synthetic lethality in mantle cell lymphomas.

    Science.gov (United States)

    Divakar, S K A; Ramana Reddy, M V; Cosenza, S C; Baker, S J; Perumal, D; Antonelli, A C; Brody, J; Akula, B; Parekh, S; Reddy, E Premkumar

    2016-01-01

    This study describes the characterization of a novel kinase inhibitor, ON123300, which inhibits CDK4/6 (cyclin-dependent kinases 4 and 6) and phosphatidylinositol 3 kinase-δ (PI3K-δ) and exhibits potent activity against mantle cell lymphomas (MCLs) both in vitro and in vivo. We examined the effects of PD0332991 and ON123300 on cell cycle progression, modulation of the retinoblastoma (Rb) and PI3K/AKT pathways, and the induction of apoptosis in MCL cell lines and patient-derived samples. When Granta 519 and Z138C cells were incubated with PD0332991 and ON123300, both compounds were equally efficient in their ability to inhibit the phosphorylation of Rb family proteins. However, only ON123300 inhibited the phosphorylation of proteins associated with the PI3K/AKT pathway. Cells treated with PD0332991 rapidly accumulated in the G0/G1 phase of cell cycle as a function of increasing concentration. Although ON123300-treated cells arrested similarly at lower concentrations, higher concentrations resulted in the induction of apoptosis, which was not observed in PD0332991-treated samples. Mouse xenograft assays also showed a strong inhibition of MCL tumor growth in ON123300-treated animals. Finally, treatment of ibrutinib-sensitive and -resistant patient-derived MCLs with ON123300 also triggered apoptosis and inhibition of the Rb and PI3K/AKT pathways, suggesting that this compound might be an effective agent in MCL, including ibrutinib-resistant forms of the disease. PMID:26174628

  15. Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells

    International Nuclear Information System (INIS)

    Although MYC is an attractive therapeutic target for breast cancer treatment, it has proven challenging to inhibit MYC directly, and clinically effective pharmaceutical agents targeting MYC are not yet available. An alternative approach is to identify genes that are synthetically lethal in MYC-dependent cancer. Recent studies have identified several cell cycle kinases as MYC synthetic-lethal genes. We therefore investigated the therapeutic potential of specific cyclin-dependent kinase (CDK) inhibition in MYC-driven breast cancer. Using small interfering RNA (siRNA), MYC expression was depleted in 26 human breast cancer cell lines and cell proliferation evaluated by BrdU incorporation. MYC-dependent and MYC-independent cell lines were classified based on their sensitivity to siRNA-mediated MYC knockdown. We then inhibited CDKs including CDK4/6, CDK2 and CDK1 individually using either RNAi or small molecule inhibitors, and compared sensitivity to CDK inhibition with MYC dependence in breast cancer cells. Breast cancer cells displayed a wide range of sensitivity to siRNA-mediated MYC knockdown. The sensitivity was correlated with MYC protein expression and MYC phosphorylation level. Sensitivity to siRNA-mediated MYC knockdown did not parallel sensitivity to the CDK4/6 inhibitor PD0332991; instead MYC-independent cell lines were generally sensitive to PD0332991. Cell cycle arrest induced by MYC knockdown was accompanied by a decrease in CDK2 activity, but inactivation of CDK2 did not selectively affect the viability of MYC-dependent breast cancer cells. In contrast, CDK1 inactivation significantly induced apoptosis and reduced viability of MYC-dependent cells but not MYC- independent cells. This selective induction of apoptosis by CDK1 inhibitors was associated with up-regulation of the pro-apoptotic molecule BIM and was p53-independent. Overall, these results suggest that further investigation of CDK1 inhibition as a potential therapy for MYC-dependent breast cancer

  16. CDK5 activator protein p25 preferentially binds and activates GSK3β.

    Science.gov (United States)

    Chow, Hei-Man; Guo, Dong; Zhou, Jie-Chao; Zhang, Guan-Yun; Li, Hui-Fang; Herrup, Karl; Zhang, Jie

    2014-11-11

    Glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins--the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3β does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3β outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3β:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3β substrate specificity. The GSK3β:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of β-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3β is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3β/AXIN/APC destruction complex, thus preventing GSK3β from binding to and phosphorylating β-catenin. Coexpression of GSK3β and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3β than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3β activity. PMID:25331900

  17. Influence of CDK1 and CDK2 siRNA interference on tumor cell cycle and cell apoptosis%CDK1、CDK2 siRNA干扰对肿瘤细胞凋亡和细胞周期的影响

    Institute of Scientific and Technical Information of China (English)

    Hui Xiao; Wanjun Gong; Jingpeng Cao; Xiaolan Li; Deding Tao; Junbo Hu; Jianping Gong

    2009-01-01

    Objective: We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods: The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results: CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotrensfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate cells among cotransfected cells were observed under the microscope. Conclu- sion: The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.

  18. Functional specialization of chordate CDK1 paralogs during oogenic meiosis.

    Science.gov (United States)

    Øvrebø, Jan Inge; Campsteijn, Coen; Kourtesis, Ioannis; Hausen, Harald; Raasholm, Martina; Thompson, Eric M

    2015-01-01

    Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program. PMID:25714331

  19. Bufalin induces G0/G1 phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells.

    Science.gov (United States)

    Huang, Wen-Wen; Yang, Jai-Sing; Pai, Shu-Jen; Wu, Ping-Ping; Chang, Shu-Jen; Chueh, Fu-Shin; Fan, Ming-Jen; Chiou, Shang-Ming; Kuo, Hsiu-Maan; Yeh, Chin-Chung; Chen, Po-Yuan; Tsuzuki, Minoru; Chung, Jing-Gung

    2012-04-01

    Most of the chemotherapy treatments for bladder cancer aim to kill the cancer cells, but a high recurrence rate after medical treatments is still occurred. Bufalin from the skin and parotid venom glands of toad has been shown to induce apoptotic cell death in many types of cancer cell lines. However, there is no report addressing that bufalin induced cell death in human bladder cancer cells. The purpose of this study was investigated the mechanisms of bufalin-induced apoptosis in a human bladder cancer cell line (T24). We demonstrated the effects of bufalin on the cell growth and apoptosis in T24 cells by using DAPI/TUNEL double staining, a PI exclusion and flow cytometric analysis. The effects of bufalin on the production of reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔΨ(m)), and DNA content including sub-G1 (apoptosis) in T24 cells were also determined by flow cytometry. Western blot analysis was used to examine the expression of G(0)/G(1) phase-regulated and apoptosis-associated protein levels in bufalin-treated T24 cells. The results indicated that bufalin significantly decreased the percentage of viability, induced the G(0)/G(1) phase arrest and triggered apoptosis in T24 cells. The down-regulation of the protein levels for cyclin D, CDK4, cyclin E, CDK2, phospho-Rb, phospho-AKT and Bcl-2 with the simultaneous up-regulation of the cytochrome c, Apaf-1, AIF, caspase-3, -7 and -9 and Bax protein expressions and caspase activities were observed in T24 cells after bufalin treatment. Based on our results, bufalin induces apoptotic cell death in T24 cells through suppressing AKT activity and anti-apoptotic Bcl-2 protein as well as inducing pro-apoptotic Bax protein. The levels of caspase-3, -7 and -9 are also mediated apoptosis in bufalin-treated T24 cells. Therefore, bufalin might be used as a therapeutic agent for the treatment of human bladder cancer in the future. PMID:22285700

  20. Targets downstream of Cdk8 in Dictyostelium development

    Directory of Open Access Journals (Sweden)

    Skelton Jason

    2011-01-01

    Full Text Available Abstract Background Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. Results Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2 with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD and one transcriptionally (short chain dehydrogenase/reductase (SDR1. Conclusions This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

  1. Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1.

    Science.gov (United States)

    Candas, Demet; Qin, Lili; Fan, Ming; Li, Jian-Jian

    2016-01-01

    Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2'-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches

  2. CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

    Science.gov (United States)

    Wang, HaiYang; Kim, Nam-Hyung

    2016-08-01

    Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression. PMID:27307074

  3. Water extract of Hedyotis Diffusa Willd suppresses proliferation of human HepG2 cells and potentiates the anticancer efficacy of low-dose 5-fluorouracil by inhibiting the CDK2-E2F1 pathway.

    Science.gov (United States)

    Chen, Xu-Zheng; Cao, Zhi-Yun; Chen, Tuan-Sheng; Zhang, You-Quan; Liu, Zhi-Zhen; Su, Yin-Tao; Liao, Lian-Ming; Du, Jian

    2012-08-01

    Hedyotis Diffusa Willd (HDW), a Chinese herbal medicine, has been widely used as an adjuvant therapy against various cancers, including hepatocellular carcinoma (HCC). However, the underlying anticancer mechanisms are yet to be elucidated. In the present study, the anticancer effects of HDW were evaluated and the efficacy and safety of HDW combined with low-dose 5-fluorouracil (5-FU) were investigated. HepG2 cells were cultured in vitro and nude mouse xenografts were established in vivo. The proliferation of HepG2 cells was measured using the MTT method and flow cytometry. The mRNA and protein expression levels of cyclin-dependent kinase 2 (CDK2), cyclin E and E2F1 were examined using relative quantitative real-time PCR and western blot analysis, respectively. The results showed that water extract of HDW remarkably inhibited HepG2 cell proliferation in a dose-dependent manner via arrest of HepG2 cells at the G0/G1 phase and induction of S phase delay. This suppression was accompanied by a great decrease of E2F1 and CDK2 mRNA expression. In addition, HDW remarkably potentiated the anticancer effect of low-dose 5-FU in the absence of overt toxicity by downregulating the mRNA and protein levels of CDK2, cyclin E and E2F1. Our findings support the use of HDW as adjuvant therapy of chemotherapy and suggest that HDW may potentiate the efficiency of low-dose 5-FU in treating HCC. PMID:22641337

  4. The Role of Cdk5 in Alzheimer's Disease.

    Science.gov (United States)

    Liu, Shu-Lei; Wang, Chong; Jiang, Teng; Tan, Lan; Xing, Ang; Yu, Jin-Tai

    2016-09-01

    Alzheimer's disease (AD) is known as the most fatal chronic neurodegenerative disease in adults along with progressive loss of memory and other cognitive function disorders. Cyclin-dependent kinase 5 (Cdk5), a unique member of the cyclin-dependent kinases (Cdks), is reported to intimately associate with the process of the pathogenesis of AD. Cdk5 is of vital importance in the development of CNS and neuron movements such as neuronal migration and differentiation, synaptic functions, and memory consolidation. However, when neurons suffer from pathological stimuli, Cdk5 activity becomes hyperactive and causes aberrant hyperphosphorylation of various substrates of Cdk5 like amyloid precursor protein (APP), tau and neurofilament, resulting in neurodegenerative diseases like AD. Deregulation of Cdk5 contributes to an array of pathological events in AD, ranging from formation of senile plaques and neurofibrillary tangles, synaptic damage, mitochondrial dysfunction to cell cycle reactivation as well as neuronal cell apoptosis. More importantly, an inhibition of Cdk5 activity with inhibitors such as RNA inference (RNAi) could protect from memory decline and neuronal cell loss through suppressing β-amyloid (Aβ)-induced neurotoxicity and tauopathies. This review will briefly describe the above-mentioned possible roles of Cdk5 in the physiological and pathological mechanisms of AD, further discussing recent advances and challenges in Cdk5 as a therapeutic target. PMID:26227906

  5. Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

    Science.gov (United States)

    Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B.; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

    2013-01-01

    Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition. PMID:24098701

  6. Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

    Directory of Open Access Journals (Sweden)

    Alessandra Bosutti

    Full Text Available Cyclin-dependent kinase-5 (Cdk5 is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

  7. Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle Progression

    OpenAIRE

    Silva, Mariana C.C.; Bodor, Dani L.; Stellfox, Madison E.; Martins, Nuno M.C.; Hochegger, Helfrid; Foltz, Daniel R.; Jansen, Lars E.T.

    2012-01-01

    Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We fur...

  8. The proline-histidine-rich CDK2/CDK4 interaction region of C/EBPalpha is dispensable for C/EBPalpha-mediated growth regulation in vivo

    DEFF Research Database (Denmark)

    Porse, Bo Torben; Pedersen, Thomas Askov; Hasemann, Marie Sigurd;

    2006-01-01

    a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice...

  9. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    Science.gov (United States)

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. PMID:27134283

  10. CDK4 is an essential insulin effector in adipocytes

    Science.gov (United States)

    Lagarrigue, Sylviane; Lopez-Mejia, Isabel C.; Denechaud, Pierre-Damien; Escoté, Xavier; Castillo-Armengol, Judit; Jimenez, Veronica; Chavey, Carine; Giralt, Albert; Lai, Qiuwen; Zhang, Lianjun; Martinez-Carreres, Laia; Delacuisine, Brigitte; Annicotte, Jean-Sébastien; Blanchet, Emilie; Huré, Sébastien; Abella, Anna; Tinahones, Francisco J.; Vendrell, Joan; Dubus, Pierre; Bosch, Fatima; Kahn, C. Ronald; Fajas, Lluis

    2015-01-01

    Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT. PMID:26657864

  11. Structural basis for CDK6 activation by a virus-encoded cyclin

    Energy Technology Data Exchange (ETDEWEB)

    Schulze-Gahmen, Ursula; Kim, Sung-Hou

    2002-01-17

    Cyclin from herpesvirus saimiri (Vcyclin) preferentially forms complexes with cyclin-dependent kinase 6 (CDK6) from primate host cells. These complexes show higher kinase activity than host cell CDK complexes with cellular cyclins and are resistant to cyclin-dependent inhibitory proteins (CDKIs). The crystal structure of human CDK6-Vcyclin in an active state was determined to 3.1 Angstrom resolution to get a better understanding of the structural basis of CDK6 activation by viral cyclins. The unphosphorylated CDK6 complexed to Vcyclin has many features characteristic of cyclinA-activated, phosphorylated CDK2. There are, however, differences in the conformation at the tip of the T-loop and its interactions with Vcyclin. Residues in the N-terminal extension of Vcyclin wrap around the tip of the CDK6 T-loop and form a short b-sheet with the T-loop backbone. These interactions lead to a 20 percent larger buried surface in the CDK6-Vcyclin interface than in the CDK2-cyclinA complex and are probably largely responsible for Vcyclin specificity for CDK6 and resistance of the complex to inhibition by INK-typeCDKIs.

  12. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    DEFF Research Database (Denmark)

    Groth, Anja; Willumsen, Berthe Marie

    2005-01-01

    and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent......-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal...

  13. Iterative Conversion of Cyclin Binding Groove Peptides into Druglike CDK Inhibitors with Antitumor Activity

    OpenAIRE

    Premnath, Padmavathy Nandha; Craig, Sandra N.; Liu, Shu; Anderson, Erin L.; Grigoroudis, Asterios I.; Kontopidis, George; Perkins, Tracy L.; Wyatt, Michael D.; Pittman, Douglas L.; McInnes, Campbell

    2014-01-01

    The cyclin groove is an important recognition site for substrates of the cell cycle cyclin dependent kinases and provides an opportunity for highly selective inhibition of kinase activity through a non-ATP competitive mechanism. The key peptide residues of the cyclin binding motif have been studied in order to precisely define the structure–activity relationship for CDK kinase inhibition. Through this information, new insights into the interactions of peptide CDK inhibitors with key subsites ...

  14. Cdk12 is essential for embryonic development and the maintenance of genomic stability.

    Science.gov (United States)

    Juan, H-C; Lin, Y; Chen, H-R; Fann, M-J

    2016-06-01

    The maintenance of genomic integrity during early embryonic development is important in order to ensure the proper development of the embryo. Studies from cultured cells have demonstrated that cyclin-dependent kinase 12 (Cdk12) is a multifunctional protein that maintains genomic stability and the pluripotency of embryonic stem cells. Perturbation of its functions is also known to be associated with pathogenesis and drug resistance in human cancers. However, the biological significance of Cdk12 in vivo is unclear. Here we bred mice that are deficient in Cdk12 and demonstrated that Cdk12 depletion leads to embryonic lethality shortly after implantation. We also used an in vitro culture system of blastocysts to examine the molecular mechanisms associated with the embryonic lethality of Cdk12-deficient embryos. Cdk12(-/-) blastocysts fail to undergo outgrowth of the inner cell mass because of an increase in the apoptosis of these cells. Spontaneous DNA damage was revealed by an increase in 53BP1 foci among cells cultured from Cdk12(-/-) embryos. Furthermore, the expression levels of various DNA damage response genes, namely Atr, Brca1, Fanci and Fancd2, are reduced in Cdk12(-/-) embryos. These findings indicate that Cdk12 is important for the correct expression of some DNA damage response genes and indirectly has an influence on the efficiency of DNA repair. Our report also highlights that DNA breaks occurring during DNA replication are frequent in mouse embryonic cells and repair of such damage is critical to the successful development of mouse embryos. PMID:26658019

  15. CDK2 accelerates early erythroid differentiation of K562 cells%CDK2促进K562细胞早期红系分化

    Institute of Scientific and Technical Information of China (English)

    李均; 岳瑞华; 沈钧乐; 肖俊

    2011-01-01

    目的 探讨细胞周期调节蛋白CDK2对K562细胞红系分化的影响.方法 分别用CDK2表达质粒和干扰RNA分子转染K562细胞,用Western blot法检测过表达或干扰效率,使用real-time PCR和联苯胺染色法检测K562细胞分化.结果 CDK2在K562细胞红系分化早期呈现表达上升趋势;在K562细胞中过表达CDK2可促进hemin诱导的红系分化;反之,干扰K562内源的CDK2表达会对K562红系分化产生抑制作用.结论 CDK2在K562细胞早期红系分化过程中发挥促进作用.%Objective To study the roles of a cell cycle regulator cyclin-dependent kinase 2 (CDK2) in erythroid differentiation of K562 cells. Methods K562 cells were transfected with the construct expressing CDK2 and siRNAs specifically targeting at CDK2. The effects of over-expression or knocking-down of CDK2 were examined by Western blot. Quantitative RT-PCR was performed to detect the level of γ-globin mRNA expression. The benzidine staining assay was used to identify the differentiation state of K562 cells. Results CDK2 was up-regulated at the early stage of K562 erythroid differentiation. Over-expression of CDK2 in K562 cells accelerated erythroid differentiation. Inhibition of CDK2 attenuates globin accumulation in K562 cells. Conclusion CDK2 is necessary for early erythroid differentiation of K562 cells.

  16. Benzamide capped peptidomimetics as non-ATP competitive inhibitors of CDK2 using the REPLACE strategy.

    Science.gov (United States)

    Premnath, Padmavathy Nandha; Craig, Sandra N; Liu, Shu; McInnes, Campbell

    2016-08-01

    Inhibition of cyclin dependent kinase 2 (CDK2) in complex with cyclin A in G1/S phase of the cell cycle has been shown to promote selective apoptosis of cancer cells through the E2F1 pathway. An alternative approach to catalytic inhibition is to target the substrate recruitment site also known as the cyclin binding groove (CBG) to generate selective non-ATP competitive inhibitors. The REPLACE strategy has been applied to identify fragment alternatives and substituted benzoic acid derivatives were evaluated as a promising scaffold to present appropriate functionality to mimic key peptide determinants. Fragment Ligated Inhibitory Peptides (FLIPs) are described which potently inhibit both CDK2/cyclin A and CDK4/cyclin D1 and have preliminary anti-tumor activity. A structural rationale for binding was obtained through molecular modeling further demonstrating their potential for further development as next generation non ATP competitive CDK inhibitors. PMID:27297568

  17. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.

    Science.gov (United States)

    Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L; Salih, Helmut R; Heidel, Florian H; Krämer, Alwin; Root, David E; Barbie, David A; Krivtsov, Andrei V; Armstrong, Scott A; Hahn, William C; Huntly, Brian J; Sykes, Stephen M; Milsom, Michael D; Scholl, Claudia; Fröhling, Stefan

    2014-07-01

    Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. PMID:24764564

  18. CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression.

    Science.gov (United States)

    Liang, Qian; Li, Lili; Zhang, Jianchao; Lei, Yang; Wang, Liping; Liu, Dong-Xu; Feng, Jingxin; Hou, Pingfu; Yao, Ruosi; Zhang, Yu; Huang, Baiqu; Lu, Jun

    2013-01-01

    Epithelial-mesenchymal transition is a change of cellular plasticity critical for embryonic development and tumor metastasis. CDK5 is a proline-directed serine/threonine kinase playing important roles in cancer progression. Here we show that CDK5 is commonly overexpressed and significantly correlated with several poor prognostic parameters of breast cancer. We found that CDK5 participated in TGF-β1-induced EMT. In MCF10A, TGF-β1 upregulated the CDK5 and p35 expression, and CDK5 knockdown inhibited TGF-β1-induced EMT. CDK5 overexpression also exhibited a potential synergy in promoting TGF-β1-induced EMT. In mesenchymal breast cancer cells MDA-MB-231 and BT549, CDK5 knockdown suppressed cell motility and tumorigenesis. We further demonstrated that CDK5 modulated cancer cell migration and tumor formation by regulating the phosphorylation of FAK at Ser-732. Therefore, CDK5-FAK pathway, as a downstream step of TGF-β1 signaling, is essential for EMT and motility in breast cancer cells. This study implicates the potential value of CDK5 as a molecular marker for breast cancer. PMID:24121667

  19. Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells

    OpenAIRE

    Gulappa, Thippeswamy; Reddy, Ramadevi Subramani; Suman, Suman; Nyakeriga, Alice M; Damodaran, Chendil

    2013-01-01

    This study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan (pso), a furanocoumarin, on PC-3 and C4-2B castration-resistant prostate cancer (CRPC) cell lines. Pso caused significant G0/G1 cell cycle arrest and inhibition of cell growth. Molecular analysis of cyclin (D1, D2, D3, and E), cyclin-dependent kinase (cdk) (cdks 2, 4, and 6), and cdk inhibitor (p21 and p27) expression suggested transcriptional regulation of the cdk inhibitors and more significant downregulation of cdk4 than of...

  20. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

    Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

  1. High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yue [Department of Diagnostics, Hebei Medical University, Shijiazhuang 050017 (China); Li, Hongbo; Hao, Jun [Department of Pathology, Hebei Medical University, Shijiazhuang 050017 (China); Zhou, Yi [Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000 (China); Liu, Wei, E-mail: lwei929@126.com [Department of Pathology, Hebei Medical University, Shijiazhuang 050017 (China)

    2014-08-15

    Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1

  2. Automated quantification reveals hyperglycemia inhibits endothelial angiogenic function.

    Directory of Open Access Journals (Sweden)

    Anthony R Prisco

    Full Text Available Diabetes Mellitus (DM has reached epidemic levels globally. A contributing factor to the development of DM is high blood glucose (hyperglycemia. One complication associated with DM is a decreased angiogenesis. The Matrigel tube formation assay (TFA is the most widely utilized in vitro assay designed to assess angiogenic factors and conditions. In spite of the widespread use of Matrigel TFAs, quantification is labor-intensive and subjective, often limiting experiential design and interpretation of results. This study describes the development and validation of an open source software tool for high throughput, morphometric analysis of TFA images and the validation of an in vitro hyperglycemic model of DM.Endothelial cells mimic angiogenesis when placed onto a Matrigel coated surface by forming tube-like structures. The goal of this study was to develop an open-source software algorithm requiring minimal user input (Pipeline v1.3 to automatically quantify tubular metrics from TFA images. Using Pipeline, the ability of endothelial cells to form tubes was assessed after culture in normal or high glucose for 1 or 2 weeks. A significant decrease in the total tube length and number of branch points was found when comparing groups treated with high glucose for 2 weeks versus normal glucose or 1 week of high glucose.Using Pipeline, it was determined that hyperglycemia inhibits formation of endothelial tubes in vitro. Analysis using Pipeline was more accurate and significantly faster than manual analysis. The Pipeline algorithm was shown to have additional applications, such as detection of retinal vasculature.

  3. Radiosensitivity modulating factors: Role of PARP-1, PARP-2 and Cdk5 proteins and chromatin implication

    International Nuclear Information System (INIS)

    The post-translational modifications of DNA repair proteins and histone remodeling factors by poly(ADP-ribose)ylation and phosphorylation are essential for the maintenance of DNA integrity and chromatin structure, and in particular in response to DNA damaging produced by ionizing radiation (IR). Amongst the proteins implicated in these two processes are the poly(ADP-ribose) polymerase -1 (PARP-1) and PARP-2, and the cyclin-dependent kinase Cdk5: PARP-1 and 2 are involved in DNA single strand break (SSB) repair (SSBR) and Cdk5 depletion has been linked with increased cell sensitivity to PARP inhibition. We have shown by using HeLa cells stably depleted for either CdK5 or PARP-2, that the recruitment profile of PARP-1 and XRCC-1, two proteins involved in the short-patch (SP) SSBR sub-pathway, to DNA damage sites is sub-maximal and that of PCNA, a protein involved in the long-patch (LP) repair pathway, is increased in the absence of Cdk5 and decreased in the absence of PARP-2 suggesting that both Cdk5 and PARP-2 are involved in both SSBR sub-pathways. PARP-2 and Cdk5 also impact on the poly(ADP-ribose) levels in cells as in the absence of Cdk5 a hyper-activation of PARP-1 was found and in the absence of PARP-2 a reduction in poly(ADP-ribose) glyco-hydrolase (PARG) activity was seen. However, in spite of these changes no impact on the repair of SSBs induced by IR was seen in either the Cdk5 or PARP-2 depleted cells (Cdk5KD or PARP-2KD cells) but, interestingly, increased radiation sensitivity in terms of cell killing was noted in the Cdk5 depleted cells. We also found that Cdk5, PARP-2 and PARG were all implicated in the regulation of the recruitment and the dissociation of the chromatin-remodeling factor ALC1 from DNA damage sites suggesting a role for these three proteins in changes in chromatin structure after DNA photo-damage. These results, taken together with the observation that PARP-1 recruitment is sub-optimal in both Cdk5KD and PARP-2KD cells, show that an

  4. CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

    Directory of Open Access Journals (Sweden)

    Sanghoon Lee

    Full Text Available The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD and dedifferentiated (DD liposarcomas.From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR.There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05. Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7% and MDM2 amplification in 46 cases (95.8%. WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041. High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54 was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012 and multivariate analyses (P = 0.020.Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

  5. 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

    Science.gov (United States)

    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

  6. Proteomics of CDK inhibition in cancer cells

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Hana; Skalníková, Helena; Halada, Petr; Strnad, M.; Hajdúch, M.

    Olomouc: -, 2007, s. 1-1. [Symposium and Workshop on Molecular Pathology /3./. Olomouc (CZ), 04.05.2007-05.05.2007] R&D Projects: GA ČR GA301/05/0418; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : cyclin-dependent kinase inhibitors * cancer * proteomics Subject RIV: EB - Genetics ; Molecular Biology

  7. Cdk5-mediated mitochondrial fission: A key player in dopaminergic toxicity in Huntington's disease.

    Science.gov (United States)

    Cherubini, Marta; Puigdellívol, Mar; Alberch, Jordi; Ginés, Silvia

    2015-10-01

    The molecular mechanisms underlying striatal vulnerability in Huntington's disease (HD) are still unknown. However, growing evidence suggest that mitochondrial dysfunction could play a major role. In searching for a potential link between striatal neurodegeneration and mitochondrial defects we focused on cyclin-dependent kinase 5 (Cdk5). Here, we demonstrate that increased mitochondrial fission in mutant huntingtin striatal cells can be a consequence of Cdk5-mediated alterations in Drp1 subcellular distribution and activity since pharmacological or genetic inhibition of Cdk5 normalizes Drp1 function ameliorating mitochondrial fragmentation. Interestingly, mitochondrial defects in mutant huntingtin striatal cells can be worsened by D1 receptor activation a process also mediated by Cdk5 as down-regulation of Cdk5 activity abrogates the increase in mitochondrial fission, the translocation of Drp1 to the mitochondria and the raise of Drp1 activity induced by dopaminergic stimulation. In sum, we have demonstrated a new role for Cdk5 in HD pathology by mediating dopaminergic neurotoxicity through modulation of Drp1-induced mitochondrial fragmentation, which underscores the relevance for pharmacologic interference of Cdk5 signaling to prevent or ameliorate striatal neurodegeneration in HD. PMID:26143143

  8. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    OpenAIRE

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M.

    2012-01-01

    Rubin and colleagues describe the first structures of full-length and phosphorylated Retinoblastoma (Rb) protein. These structures reveal the mechanism of Rb inactivation and provide valuable insight into this critical tumor suppressor protein's allosteric inhibition via multisite Cdk phosphorylation and its E2F and cell cycle regulation.

  9. Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2

    Science.gov (United States)

    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

  10. Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

    Directory of Open Access Journals (Sweden)

    Jasmina Makarević

    Full Text Available 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.

  11. Knockdown of cyclin-dependent kinase 10 (cdk10) gene impairs neural progenitor survival via modulation of raf1a gene expression.

    Science.gov (United States)

    Yeh, Chi-Wei; Kao, Shoa-Hsuan; Cheng, Yi-Chuan; Hsu, Li-Sung

    2013-09-27

    In this study, we used zebrafish as an animal model to elucidate the developmental function of cdk10 in vertebrates. In situ hybridization analyses demonstrated that cdk10 is expressed throughout development with a relative enrichment in the brain in the late stages. Similar to its mammalian ortholog, cdk10 can interact with the transcription factor ETS2 and exhibit kinase activity by phosphorylating histone H1. Morpholino-based loss of cdk10 expression caused apoptosis in sox2-positive cells and decreased the expression of subsequent neuronal markers. Acetylated tubulin staining revealed a significant reduction in the number of Rohon-Beard sensory neurons in cdk10 morphants. This result is similar to that demonstrated by decreased islet2 expression in the dorsal regions. Moreover, cdk10 morphants exhibited a marked loss of huC-positive neurons in the telencephalon and throughout the spinal cord axis. The population of retinal ganglion cells was also diminished in cdk10 morphants. These phenotypes were rescued by co-injection of cdk10 mRNA. Interestingly, the knockdown of cdk10 significantly elevated raf1a mRNA expression. Meanwhile, an MEK inhibitor (U0126) recovered sox2 and ngn1 transcript levels in cdk10 morphants. Our findings provide the first functional characterization of cdk10 in vertebrate development and reveal its critical function in neurogenesis by modulation of raf1a expression. PMID:23902762

  12. Inhibitor of CDK interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

    DEFF Research Database (Denmark)

    Baumer, Nicole; Tickenbrock, Lara; Tschanter, Petra;

    2011-01-01

    The cell cycle is driven by the kinase activity of cyclin/CDK complexes which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as interaction partner and substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin binding site in...... the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inihibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, while it was induced by cell cycle arrest. We established a deletional mouse model that showed...... increased CDK2 activity in spleen with altered spleen architecture in Inca1-/- mice. Inca1-/- embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from ALL and AML patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control in...

  13. Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling*

    OpenAIRE

    Bäumer, Nicole; Tickenbrock, Lara; Tschanter, Petra; Lohmeyer, Lisa; Diederichs, Sven; Bäumer, Sebastian; Skryabin, Boris V.; Zhang, Feng; Agrawal-Singh, Shuchi; Köhler, Gabriele; Berdel, Wolfgang E.; Serve, Hubert; Koschmieder, Steffen; Müller-Tidow, Carsten

    2011-01-01

    The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin-binding site in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic sig...

  14. ING5 is phosphorylated by CDK2 and controls cell proliferation independently of p53.

    Directory of Open Access Journals (Sweden)

    Ulrike Linzen

    Full Text Available Inhibitor of growth (ING proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2 in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53.

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

    Science.gov (United States)

    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

  16. 1α,25 dihydroxi-vitamin D{sub 3} modulates CDK4 and CDK6 expression and localization

    Energy Technology Data Exchange (ETDEWEB)

    Irazoqui, Ana P.; Heim, Nadia B.; Boland, Ricardo L.; Buitrago, Claudia G., E-mail: cbuitrag@criba.edu.ar

    2015-03-27

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH){sub 2}-vitamin D{sub 3} [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21{sup Waf1/Cip1} and p27{sup Kip1} expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D –induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs –dependent mechanism in hormone modulation of myogenesis. - Highlights: • 1,25D modulates CDKs 4 and 6 expression in skeletal muscle cells. • CDK4 co

  17. 1α,25 dihydroxi-vitamin D3 modulates CDK4 and CDK6 expression and localization

    International Nuclear Information System (INIS)

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21Waf1/Cip1 and p27Kip1 expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D –induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs –dependent mechanism in hormone modulation of myogenesis. - Highlights: • 1,25D modulates CDKs 4 and 6 expression in skeletal muscle cells. • CDK4 co-localizates with VDR after 1,25D

  18. Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

    Science.gov (United States)

    Mikolcevic, Petra; Isoda, Michitaka; Shibuya, Hiroki; del Barco Barrantes, Ivan; Igea, Ana; Suja, José A.; Shackleton, Sue; Watanabe, Yoshinori; Nebreda, Angel R.

    2016-01-01

    Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins. PMID:27025256

  19. The amylase inhibitor montbretin A reveals a new glycosidase inhibition motif.

    Science.gov (United States)

    Williams, Leslie K; Zhang, Xiaohua; Caner, Sami; Tysoe, Christina; Nguyen, Nham T; Wicki, Jacqueline; Williams, David E; Coleman, John; McNeill, John H; Yuen, Violet; Andersen, Raymond J; Withers, Stephen G; Brayer, Gary D

    2015-09-01

    The complex plant flavonol glycoside montbretin A is a potent (Ki = 8 nM) and specific inhibitor of human pancreatic α-amylase with potential as a therapeutic for diabetes and obesity. Controlled degradation studies on montbretin A, coupled with inhibition analyses, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide. X-ray structural analyses of the montbretin A-human α-amylase complex confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal π-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the α-amylase catalytic residues D197 and E233. This novel inhibitory motif can be reproduced in a greatly simplified analog, offering potential for new strategies for glycosidase inhibition and therapeutic development. PMID:26214255

  20. U12, a UDCA derivative, acts as an anti-hepatoma drug lead and inhibits the mTOR/S6K1 and cyclin/CDK complex pathways.

    Directory of Open Access Journals (Sweden)

    Yang Xu

    Full Text Available U12, one of 20 derivatives synthesized from ursodeoxycholic acid (UDCA, has been found to have anticancer effects in liver cancer cell lines (SMMC-7721 and HepG2 and to protect normal liver cells from deoxycholic acid (DCA damage (QSG-7701. Its anticancer mechanism was investigated using computer-aided network pharmacology and comparative proteomics. Results showed that its anti-malignancy activities were activated by mTOR/S6K1, cyclinD1/CDK2/4 and caspase-dependent apoptotic signaling pathways in hepatocellular carcinoma cells (HCC. The action of U12 may be similar to that of rapamycin. Animal testing confirmed that U12 exerted better anti-tumor activity than UDCA and had less severe side effects than fluorouracil (5-Fu. These observations indicate that U12 differs from UDCA and other derivatives and may be a suitable lead for the development of compounds useful in the treatment of HCC.

  1. Functional ablation of pRb activates Cdk2 and causes antiestrogen resistance in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Hemant Varma

    Full Text Available Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT, and cdk activity was inhibited using the cdk inhibitors p16(INK4A and p21(Waf1/Cip1. Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.

  2. CDK2-AP1通过调控细胞周期抑制乳腺癌生长

    Institute of Scientific and Technical Information of China (English)

    何向明; 黄润; 俞洋; 向华; 杨红健; 宗祥云

    2015-01-01

    目的:探讨CDK2-AP1在乳腺癌的作用及其机制。方法分别在正常乳腺组织及不同分期乳腺癌组织中检测CDK2-AP1的表达情况;进行CDK2-AP1的LOF & GOF细胞功能实验;接种CDK2-AP1干扰或过表达的乳腺癌细胞及对照细胞在裸鼠观察成瘤及相应指标。结果在乳腺癌存在CDK2-AP1表达降低/缺失而CDK2/CyclinD1表达升高的情况,且CDK2-AP1的表达在正常乳腺组织细胞、乳腺导管原位癌、侵袭性乳腺癌、复发转移性乳腺癌渐次降低(P<0.001),与CDK2/CyclinD1相反。体内、外实验均发现抑制CDK2-AP1表达后乳腺癌细胞周期后移、增殖加快;过表达CDK2-AP1的乳腺癌细胞周期阻滞在G0/G1和G2/M期,生长受抑制、裸鼠成瘤速度及大小均受抑制。结论 CDK2-AP1的表达降低以至缺失促进乳腺细胞进入恶性增殖形成肿瘤,缺乏细胞周期负性调控的乳腺癌细胞增殖能力增强。%Objective To observe the role of CDK2-AP1 in breast cancer.Methods Expressions of CDK2-AP1,CDK2 and CyclinD1 were examined in 209 cases of pathological specimens using IHC staining. Lost-of-function and Gain-of-function assays were performed in vivo and in vitro to assess the specific role of CDK2-AP1 in breast cancer. ResultsThe positive ratio of CDK2-AP1 expression was reduced successively in normal breast tissue,DCIS,invasive breast cancer and relapsed breast cancer,suggesting that CDK2-AP1 was correlated closely with the tumor’s genesis and progress and might work as a tumor suppressor. After down-regulating CDK2-AP1 in breast cancer cells,the cell cycle was accelerated and the cell proliferation was promoted. The cell cycle was arrested in G0/G1 phase and G2/M phase after up-regulating CDK2-AP1 in breast cancer cells,resulting in inhibited cell proliferation. The same results were obtained by animal assays.Conclusions CDK2-AP1 affects tumor genesis and tumor growth by cell cycle regulation,which has the potential to be

  3. CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Jena, N; Sheng, J; Hu, J K; Li, W; Zhou, W; Lee, G; Tsichlis, N; Pathak, A; Brown, N; Deshpande, A; Luo, C; Hu, G F; Hinds, P W; Van Etten, R A; Hu, M G

    2016-05-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy. PMID:26707936

  4. Brands and Inhibition: A Go/No-Go Task Reveals the Power of Brand Influence

    Science.gov (United States)

    Peatfield, Nicholas; Caulfield, Joanne; Parkinson, John; Intriligator, James

    2015-01-01

    Whether selecting a candy in a shop or picking a digital camera online, there are usually many options from which consumers may choose. With such abundance, consumers must use a variety of cognitive, emotional, and heuristic means to filter out and inhibit some of their responses. Here we use brand logos within a Go/No-Go task to probe inhibitory control during the presentation of familiar and unfamiliar logos. The results showed no differences in response times or in commission errors (CE) between familiar and unfamiliar logos. However, participants demonstrated a generally more cautious attitude of responding to the familiar brands: they were significantly slower and less accurate at responding to these brands in the Go trials. These findings suggest that inhibitory control can be exercised quite effectively for familiar brands, but that when such inhibition fails, the potent appetitive nature of brands is revealed. PMID:26544606

  5. Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

    International Nuclear Information System (INIS)

    Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma

  6. Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

    Energy Technology Data Exchange (ETDEWEB)

    Natoni, Alessandro [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Coyne, Mark R. E. [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Department of Medicine, National University of Ireland Galway, Galway (Ireland); Department of Haematology, Galway University Hospital, Galway (Ireland); Jacobsen, Alan; Rainey, Michael D.; O’Brien, Gemma; Healy, Sandra [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Montagnoli, Alessia; Moll, Jürgen [Nerviano Medical Sciences S.r.l., Via Pasteur 10, Nerviano 20014 (Italy); O’Dwyer, Michael, E-mail: michael.odwyer@nuigalway.ie [Department of Medicine, National University of Ireland Galway, Galway (Ireland); Department of Haematology, Galway University Hospital, Galway (Ireland); Santocanale, Corrado, E-mail: michael.odwyer@nuigalway.ie [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland)

    2013-07-24

    Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

  7. Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4(R24C/R24C)/Tyr-Nras(Q61K) mice following neonatal UVR.

    Science.gov (United States)

    Hacker, Elke; Muller, H Konrad; Hayward, Nicholas; Fahey, Paul; Walker, Graeme

    2010-02-01

    To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4(R24C/R24C)/Nras(Q61K) mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes. PMID:19788533

  8. Gene expression profiling in equine polysaccharide storage myopathy revealed inflammation, glycogenesis inhibition, hypoxia and mitochondrial dysfunctions

    Directory of Open Access Journals (Sweden)

    Benech Philippe

    2009-08-01

    Full Text Available Abstract Background Several cases of myopathies have been observed in the horse Norman Cob breed. Muscle histology examinations revealed that some families suffer from a polysaccharide storage myopathy (PSSM. It is assumed that a gene expression signature related to PSSM should be observed at the transcriptional level because the glycogen storage disease could also be linked to other dysfunctions in gene regulation. Thus, the functional genomic approach could be conducted in order to provide new knowledge about the metabolic disorders related to PSSM. We propose exploring the PSSM muscle fiber metabolic disorders by measuring gene expression in relationship with the histological phenotype. Results Genotypying analysis of GYS1 mutation revealed 2 homozygous (AA and 5 heterozygous (GA PSSM horses. In the PSSM muscles, histological data revealed PAS positive amylase resistant abnormal polysaccharides, inflammation, necrosis, and lipomatosis and active regeneration of fibers. Ultrastructural evaluation revealed a decrease of mitochondrial number and structural disorders. Extensive accumulation of an abnormal polysaccharide displaced and partially replaced mitochondria and myofibrils. The severity of the disease was higher in the two homozygous PSSM horses. Gene expression analysis revealed 129 genes significantly modulated (p Conclusion The main disorders observed in PSSM muscles could be related to mitochondrial dysfunctions, glycogenesis inhibition and the chronic hypoxia of the PSSM muscles.

  9. The functions of the cdk-cyclin kinase inhibitor p21WAF1

    International Nuclear Information System (INIS)

    p21WAF1 plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21WAF1 (hereafter referred to as p21) are the cdk-cyclins which regulate the progression of cukayotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase δ. p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit synthesis. These distinct properties map to the N- terminal and the C- terminal regions of p21, respectively. Cell cycle arrest in G-1 (G-1 checkpoint) following DNA damage is mediated by p53 and is deficient in p21 null cells. p53 thus up-regulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity. Retinoblastoma protein is regulated by cdk-cyclin kinases, and acts as a downstream target of p21 in DNA damage-induced G-1 arrest. Furthermore, accumulating evidence indicates that p21 may play a role in maintaining G-2 arrest after DNA damage. Transcriptional control of p21 by factors other than p53 is critical for growth arrest and for cell differentiation in many instances. (authors)

  10. CDK inhibitors, p21Cip1 and p27Kip1, participate in cell cycle exit of mammalian cardiomyocytes

    International Nuclear Information System (INIS)

    Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21Cip1 and p27Kip1 but not p57Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21Cip1 and p27Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21Cip1 and p27Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21Cip1 and p27Kip play important roles in the cell cycle exit of postnatal cardiomyocytes

  11. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    Energy Technology Data Exchange (ETDEWEB)

    Zaja, Ivan [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bai, Xiaowen, E-mail: xibai@mcw.edu [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bosnjak, Zeljko J. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States)

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  12. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    International Nuclear Information System (INIS)

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  13. Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

    International Nuclear Information System (INIS)

    The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML)

  14. Analysis list: Cdk7 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk7 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cd...k7.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk7.5.tsv http://dbarchive.bioscienced...bc.jp/kyushu-u/mm9/target/Cdk7.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk7.Pluripotent_s

  15. Analysis list: Cdk8 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk8 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cd...k8.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk8.5.tsv http://dbarchive.bioscienced...bc.jp/kyushu-u/mm9/target/Cdk8.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk8.Pluripotent_s

  16. Biochemical characterization of Cdk2-Speedy/Ringo A2

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2005-09-01

    Full Text Available Abstract Background Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs, which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. Results We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/TPX(K/R that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only ~0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of ~80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate

  17. Cell Cycle Regulating Kinase Cdk4 as a Potential Target for Tumor Cell Treatment and Tumor Imaging

    Directory of Open Access Journals (Sweden)

    Franziska Graf

    2009-01-01

    Full Text Available The cyclin-dependent kinase (Cdk-cyclin D/retinoblastoma (pRb/E2F cascade, which controls the G1/S transition of cell cycle, has been found to be altered in many neoplasias. Inhibition of this pathway by using, for example, selective Cdk4 inhibitors has been suggested to be a promising approach for cancer therapy. We hypothesized that appropriately radiolabeled Cdk4 inhibitors are suitable probes for tumor imaging and may be helpful studying cell proliferation processes in vivo by positron emission tomography. Herein, we report the synthesis and biological, biochemical, and radiopharmacological characterizations of two I124-labeled small molecule Cdk4 inhibitors (8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl-phenylamino-8H-pyrido[2,3-d]-pyrimidin-7-one (CKIA and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl-pyridin-2-yl-amino-8H-pyrido[2,3-d]pyrimidin-7-one (CKIB. Our data demonstrate a defined and specific inhibition of tumor cell proliferation through CKIA and CKIB by inhibition of the Cdk4/pRb/E2F pathway emphasizing potential therapeutic benefit of CKIA and CKIB. Furthermore, radiopharmacological properties of [I124]CKIA and [I124]CKIB observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies.

  18. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    International Nuclear Information System (INIS)

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics

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

    Science.gov (United States)

    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

  20. Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells

    OpenAIRE

    Nakayama, Satoshi; Torikoshi, Yasuhiro; Takahashi, Takeshi; Yoshida, Tomokazu; Sudo, Tamotsu; Matsushima, Tomoko; Kawasaki, Yuko; Katayama, Aya; Gohda, Keigo; Hortobagyi, Gabriel N.; Noguchi, Shinzaburo; Sakai, Toshiyuki; Ishihara, Hideki; Ueno, Naoto T.

    2009-01-01

    Introduction Paclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth ...

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

    Science.gov (United States)

    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

  2. Molecular basis for viral selective replication in cancer cells: activation of CDK2 by adenovirus-induced cyclin E.

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    Full Text Available Adenoviruses (Ads with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL, promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

  3. Retinoic Acid Induces Apoptosis of Prostate Cancer DU145 Cells through Cdk5 Overactivation

    Directory of Open Access Journals (Sweden)

    Mei-Chih Chen

    2012-01-01

    Full Text Available Retinoic acid (RA has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP. Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.

  4. Study of expression of CDK2 and CDK4 in Hamster Buckle Pouch Carcinogesis%CDK2、CDK4在金黄地鼠颊囊癌变过程中表达的研究

    Institute of Scientific and Technical Information of China (English)

    孙淑芬; 高文信; 罗兰; 顾彦成

    2006-01-01

    目的探讨CDK2 、CDK4在金黄地鼠颊囊黏膜从正常黏膜到单纯增生、异常增生及鳞状细胞癌的表达变化及相关性.方法采用DMBA诱导48只金黄地鼠颊囊癌变动物模型,SABC免疫组化法检测CDK2 、CDK4蛋白的表达.结果 CDK2 、CDK4均在异常增生上皮及鳞状细胞癌的表达与正常和单纯增生组相比明显提高(P<0.05),阳性染色等级随病理等级改变提高(P<0.05).CDK2与CDK4呈高度正相关.结论 CDK2 、CDK4参与了口腔黏膜癌前病变和鳞状细胞癌的发生与发展.

  5. Cross-class metallo-β-lactamase inhibition by bisthiazolidines reveals multiple binding modes.

    Science.gov (United States)

    Hinchliffe, Philip; González, Mariano M; Mojica, Maria F; González, Javier M; Castillo, Valerie; Saiz, Cecilia; Kosmopoulou, Magda; Tooke, Catherine L; Llarrull, Leticia I; Mahler, Graciela; Bonomo, Robert A; Vila, Alejandro J; Spencer, James

    2016-06-28

    Metallo-β-lactamases (MBLs) hydrolyze almost all β-lactam antibiotics and are unaffected by clinically available β-lactamase inhibitors (βLIs). Active-site architecture divides MBLs into three classes (B1, B2, and B3), complicating development of βLIs effective against all enzymes. Bisthiazolidines (BTZs) are carboxylate-containing, bicyclic compounds, considered as penicillin analogs with an additional free thiol. Here, we show both l- and d-BTZ enantiomers are micromolar competitive βLIs of all MBL classes in vitro, with Kis of 6-15 µM or 36-84 µM for subclass B1 MBLs (IMP-1 and BcII, respectively), and 10-12 µM for the B3 enzyme L1. Against the B2 MBL Sfh-I, the l-BTZ enantiomers exhibit 100-fold lower Kis (0.26-0.36 µM) than d-BTZs (26-29 µM). Importantly, cell-based time-kill assays show BTZs restore β-lactam susceptibility of Escherichia coli-producing MBLs (IMP-1, Sfh-1, BcII, and GOB-18) and, significantly, an extensively drug-resistant Stenotrophomonas maltophilia clinical isolate expressing L1. BTZs therefore inhibit the full range of MBLs and potentiate β-lactam activity against producer pathogens. X-ray crystal structures reveal insights into diverse BTZ binding modes, varying with orientation of the carboxylate and thiol moieties. BTZs bind the di-zinc centers of B1 (IMP-1; BcII) and B3 (L1) MBLs via the free thiol, but orient differently depending upon stereochemistry. In contrast, the l-BTZ carboxylate dominates interactions with the monozinc B2 MBL Sfh-I, with the thiol uninvolved. d-BTZ complexes most closely resemble β-lactam binding to B1 MBLs, but feature an unprecedented disruption of the D120-zinc interaction. Cross-class MBL inhibition therefore arises from the unexpected versatility of BTZ binding. PMID:27303030

  6. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair.

    Directory of Open Access Journals (Sweden)

    Marco Saponaro

    2010-02-01

    Full Text Available Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.

  7. Cdk1 targets Srs2 to complete synthesis-dependent strand annealing and to promote recombinational repair.

    Science.gov (United States)

    Saponaro, Marco; Callahan, Devon; Zheng, Xiuzhong; Krejci, Lumir; Haber, James E; Klein, Hannah L; Liberi, Giordano

    2010-02-01

    Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA-independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair. PMID:20195513

  8. Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor

    OpenAIRE

    Miguel A. Mata; Satterly, Neal; Versteeg, Gijs A.; Frantz, Doug; Wei, Shuguang; Williams, Noelle; Schmolke, Mirco; Peña-Llopis, Samuel; Brugarolas, James; Forst, Christian V.; White, Michael A.; García-Sastre, Adolfo; Roth, Michael G.; Fontoura, Beatriz M.A.

    2011-01-01

    A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occu...

  9. Chemical Inhibition of RNA Viruses Reveals REDD1 as Host Defense Factor

    OpenAIRE

    Miguel A. Mata; Satterly, Neal; Versteeg, Gijs A.; Frantz, Doug; Wei, Shuguang; Williams, Noelle; Schmolke, Mirco; Pena-Llopis, Samuel; Brugarolas, James; Forst, Christian; White, Michael A.; Garcia-Sastre, Adolfo; Roth, Michael G.; Fontoura, Beatriz M.A.

    2011-01-01

    A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reverted NS1-mediated inhibition of host gene expression. A counter-screen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus. The mechanism of action was throu...

  10. Expressions of CDK2 and CDK4 in intimal cell proliferation in autologous vein grafts%CDK2、CDK4基因与自体移植静脉内膜增殖的关系

    Institute of Scientific and Technical Information of China (English)

    亓明; 王新文; 罗英伟; 秦岭峰; 马文锋; 张强; 辛世杰; 段志泉

    2012-01-01

    [ Objecttive ] To observe CDK2, CDK4 expressions of proliferative intima in autologous grafted vein of rat. [Methods] Rat autologous vein graft model was established. Fifty rats were divided into five groups randomly, and the graft veins were respectively collectived at the 1st, 2nd, 3rd, 7th, and 14th day after the operation. Then the protein expressions of CDK2 and CDK4 were detected by immunohistochemistry and the expressions of their mRNA were detected by RT - PCR in intima of the graft vein. Normal veins were used for control. [ Results] At 7d after operation, the intima proliferation of autologous grafted vein reached nearly to the high point, and it was more obvious than those of control group and groups at 1 d, 2 d and 3 d after operation (P < 0.05) . The numbers of CDK2 and CDK4 positive cells in graft vein increased from 2 d after operation and reached to the peak at 7 d after operation. The expressions of CDK2, CDK4 mRNA reached to the peak during 7 d to 14 d after operation. [ Conclusions ] The expressions of CDK2 and CDK4 increased from early phrase after vein graft, and reached the peak during 7 d to 14 d after operation. The CDK2 and CDK4 played a role in intima proliferation of autologous graft vein.%[目的]了解CDK2、CDK4在大鼠移植血管的表达及对平滑肌细胞增殖的影响.[方法]Wistar大鼠50只,随机分为5组,建立自体静脉移植模型,分别于术后1、2、3、7及14 d取组织形态学观察,并用免疫组织化学和RT-PCR方法检测血管移植后不同时期CDK2、CDK4的表达情况,取正常静脉为对照组.[结果]移植后7d,内膜厚度与管壁厚度接近高峰,与对照组及移植后1、2、3d比较,差异有显著性意义(P<0.05).免疫组织化学显示,移植静脉CDK2、CDK4阳性细胞在移植后2d明显增加,7d达到高峰,与移植后1d比较,差异有显著性意义(P<0.05).RT-PCR检测结果显示,CDK2、CDK4基因mRNA表达产量7~14 d达到高峰,与移植后1、2、3d比

  11. Analysis list: Cdk9 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk9 Blood,Embryonic fibroblast,Pluripotent stem cell + mm9 http://dbarchive.biosci...encedbc.jp/kyushu-u/mm9/target/Cdk9.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk9.5.tsv h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk9.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cd...k9.Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk9....Embryonic_fibroblast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk9.Pluripotent_stem_cell.tsv

  12. Analysis list: CDK9 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available CDK9 Blood,Liver + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CDK9.1.tsv http://dba...rchive.biosciencedbc.jp/kyushu-u/hg19/target/CDK9.5.tsv http://dbarchive.biosciencedbc.jp/k...yushu-u/hg19/target/CDK9.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/CDK9.Blood.tsv,http://dba...rchive.biosciencedbc.jp/kyushu-u/hg19/colo/CDK9.Liver.tsv http://dbarchive.bios...ciencedbc.jp/kyushu-u/hg19/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Liver.gml ...

  13. Effects of new targets of CDK2 RNA interference on proliferation of SHG44 cells%新靶点CDK2干扰RNA对人脑胶质瘤增殖影响

    Institute of Scientific and Technical Information of China (English)

    呼格吉乐; 张军力; 段美庆; 王俊瑞; 高乃康

    2013-01-01

    Objective To construct four new eukaryotic expression vectors of RNA interference specific for cyclindependent kinase-2 (CDK2) and transfect the vectors into SHG44 cells for the detection of vectors with strong interferential effect.Methods Four new eukaryotic expression vectors of RNA interference specific for CDK2 were constructed.The human glioma SHG44 cell line was transfected with the four new vectors.The mRNA contents of CDK2 were detected using reverse transcriptase-PCR (RT-PCR).The change in proliferation of SHG44 cells was assayed.Results The new vectors ith new targets of eukaryotic expression of RNA interference specific for CDK2 were constructed (PCDK2-1,PCDK2-2,PCDK2-3,PCDK2-4).CDK2 small interfering RNA (siRNA) could suppress expression of mRNA and pCDK2-1.siRNA could inhibit the proliferation of SHG44 cell line.Conclusion The proliferation of human SHG44 cell line could be significantly inhibited after the transfection with new eukaryotic expression vectors of CDK2 siRNA.%目的 构建4个新靶点的人细胞周期蛋白依赖性激酶2(CDK2)干扰RNA真核表达载体,转染人脑胶质瘤细胞后,检测出干扰效果最好的载体及细胞增殖能力的变化.方法 构建4个新靶点CDK2干扰RNA真核表达载体并用双酶切和测序鉴定;分别转染上述4个载体到人脑胶质瘤细胞株SHG44;通过逆转录聚合酶链反应(RT-PCR)比较转染后CDK2 mRNA的表达量,选出干扰效果最好的一个,检测细胞增殖能力的变化.结果 成功构建4个新靶点的CDK2干扰RNA真核表达载体PCDK2-1、PCDK2-2、PCDK2-3、PCDK2-4;CDK2 mRNA表达和细胞增殖明显受到抑制,PCDK2-1的干扰效果为56%;PCDK2-1-SHG44细胞与对照组相比增殖能力减弱.结论 成功构建并筛选出效果最好的新靶点CDK2干扰RNA真核表达载体,并使SHG44细胞的增殖水平降低.

  14. TPPII, MYBBP1A and CDK2 form a protein-protein interaction network.

    Science.gov (United States)

    Nahálková, Jarmila; Tomkinson, Birgitta

    2014-12-15

    Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins. PMID:25303791

  15. Diverse models for the prediction of CDK4 inhibitory activity of substituted 4-aminomethylene isoquinoline-1, 3-diones

    Indian Academy of Sciences (India)

    Monika Gupta; A K Madan

    2013-05-01

    In the present study, both classification and correlation approaches have been successfully employed for development of models for the prediction of CDK4 inhibitory activity using a dataset comprising of 52 analogues of 4-aminomethylene isoquinoline-1,3-(2,4)-dione. Decision tree, random forest, moving average analysis (MAA), multiple linear regression (MLR), partial least square regression (PLSR) and principal component regression (PCR) were used to develop models for prediction of CDK4 inhibitory activity. The statistical significance of models was assessed through specificity, sensitivity, overall accuracy, Mathew’s correlation coefficient (MCC), cross validated correlation coefficient, test, 2 for external test set (pred_r2), coefficient of correlation of predicted dataset (pred_r2Se) and intercorrelation analysis. High accuracy of prediction offers proposed models a vast potential for providing lead structures for the development of potent therapeutic agents for CDK4 inhibition.

  16. Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization.

    Science.gov (United States)

    Mahale, S; Bharate, S B; Manda, S; Joshi, P; Jenkins, P R; Vishwakarma, R A; Chaudhuri, B

    2015-01-01

    The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development. PMID:25950473

  17. CDK inhibitors, p21{sup Cip1} and p27{sup Kip1}, participate in cell cycle exit of mammalian cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi; Iwamoto, Noriko [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan); Nakayama, Keiichi I. [Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Takeuchi, Takashi, E-mail: takeuchi@med.tottori-u.ac.jp [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan)

    2014-01-17

    Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21{sup Cip1} and p27{sup Kip1} but not p57{sup Kip2} showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21{sup Cip1} and p27{sup Kip1} bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21{sup Cip1} and p27{sup Kip1} knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21{sup Cip1} and p27{sup Kip} play important roles in the cell cycle exit of postnatal cardiomyocytes.

  18. Fast silencing reveals a lost role for reciprocal inhibition in locomotion

    OpenAIRE

    Moult, Peter Robert; Cottrell, Glen Alfred; Li, Wenchang

    2013-01-01

    Summary Alternating contractions of antagonistic muscle groups during locomotion are generated by spinal “half-center” networks coupled in antiphase by reciprocal inhibition. It is widely thought that reciprocal inhibition only coordinates the activity of these muscles. We have devised two methods to rapidly and selectively silence neurons on just one side of Xenopus tadpole spinal cord and hindbrain, which generate swimming rhythms. Silencing activity on one side led to rapid cessation of ac...

  19. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    Science.gov (United States)

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  20. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    Directory of Open Access Journals (Sweden)

    Eva Maria Putz

    2013-08-01

    Full Text Available The transcription factor STAT1 is important in natural killer (NK cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8. Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.

  1. CDK2干扰RNA对人脑胶质瘤细胞质蛋白质组的影响%Effects of CDK2 RNA interference on the cytoplasm proteome of SHG44 cells

    Institute of Scientific and Technical Information of China (English)

    呼格吉乐; 张军力; 段美庆; 王俊瑞; 高乃康

    2012-01-01

    Objective Eukaryotic expression vector of RNA interference specific for CDK2 that was stable transfectioned to inhibit the expression of CDK2 in SHG44 cell was constructed via the technique of RNA interference. The differentially expression of subcellular structure of the protein in SHG 44 cell line was investigated by 2D4mage master-MS and to offer valuable theoretical evidence for the occurrence and progress in glioma . Methods New eukaryotic expression vector of RNA interference specific for CDK 2 were constructed and verified via dual enzyme cleavage and sequencing. Stable transfection cell line was obtained by G418 selection and to cultivate transfected SHG44 cell line. The mRNA contents of CDK2 was contrasted by RT-PCR. Differential expression proteins of cytoplasm were compared via MALDI -TOF-MS and searching of database. Results New targets of eukaryotic expression vector of RNA interference specific for CDK 2 was constructed. Stable transfected cell line was constructed and named as P -SHG44. Five differential expression proteins in cytoplasm was identified by 2D4mage master -MS. Conclusions SHG44 cell line was transfected by CDK2 siRNA. Differentially expressed proteins related to cell proliferation and apoptosis in regulation of signal transduction , malignant transformation of normal cells and tumor development, as well as the occurrence of regulation , development and the formation of drug resistance by mass spectrometry identification.%目的 构建人CDK2的干扰RNA真核表达载体,稳定转染人脑胶质细胞瘤SHG44细胞,经RT-PCR检测后,运用双向电泳-图像分析-质谱技术研究人脑胶质瘤细胞质蛋白质组的改变,探讨CDK2在SHG44细胞中的作用,为人脑胶质瘤的发生、发展的研究及诊断与治疗提供有价值的资料.方法 (1)构建CDK2干扰RNA真核表达载体并用双酶切和测序鉴定.(2)G418筛选阳性转染细胞克隆,制备稳定转染的SHG44细胞系.(3)通过RT-PCR比较转染后CDK2 m

  2. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    Directory of Open Access Journals (Sweden)

    Erick Contreras-Vallejos

    Full Text Available Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5 is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC, which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate and Grin1 (G protein regulated inducer of neurite outgrowth 1. MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

  3. Phosphorylation of Ubc9 by Cdk1 enhances SUMOylation activity.

    Directory of Open Access Journals (Sweden)

    Yee-Fun Su

    Full Text Available Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9.

  4. 皮肤瘢痕癌中CDK4、CDK6蛋白的表达及意义%The expression and its significance of CDK4 and CDK6 in skin scar cancer

    Institute of Scientific and Technical Information of China (English)

    林宇静; 郭瑞珍

    2013-01-01

    目的 探讨细胞周期素依赖激酶CDK4、CDK6蛋白在皮肤瘢痕癌组织中的表达及意义.方法 采用免疫组织化学S-P法检测正常皮肤表皮、皮肤病理性瘢痕被覆上皮和瘢痕癌组织中CDK4、CDK6蛋白的表达.结果 CDK4、CDK6蛋白在皮肤瘢痕癌组中呈阳性或强阳性表达,在皮肤病理性瘢痕组中呈弱阳性表达,在正常皮肤组中呈阴性或弱阳性表达.瘢痕癌组分别与正常皮肤组和皮肤病理性瘢痕组比较,差别有统计学意义(P<0.01).结论 CDK4、CDK6蛋白的过表达可能与瘢痕癌的发生具有相关性.

  5. A caspase active site probe reveals high fractional inhibition needed to block DNA fragmentation.

    Science.gov (United States)

    Méthot, Nathalie; Vaillancourt, John P; Huang, JingQi; Colucci, John; Han, Yongxin; Ménard, Stéphane; Zamboni, Robert; Toulmond, Sylvie; Nicholson, Donald W; Roy, Sophie

    2004-07-01

    Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies. PMID:15067000

  6. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.

    Science.gov (United States)

    Nabti, Ibtissem; Marangos, Petros; Bormann, Jenny; Kudo, Nobuaki R; Carroll, John

    2014-03-17

    Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes. PMID:24637322

  7. Constitutive CCND1/CDK2 activity substitutes for p53 loss, or MYC or oncogenic RAS expression in the transformation of human mammary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Damian J Junk

    Full Text Available Cancer develops following the accumulation of genetic and epigenetic alterations that inactivate tumor suppressor genes and activate proto-oncogenes. Dysregulated cyclin-dependent kinase (CDK activity has oncogenic potential in breast cancer due to its ability to inactivate key tumor suppressor networks and drive aberrant proliferation. Accumulation or over-expression of cyclin D1 (CCND1 occurs in a majority of breast cancers and over-expression of CCND1 leads to accumulation of activated CCND1/CDK2 complexes in breast cancer cells. We describe here the role of constitutively active CCND1/CDK2 complexes in human mammary epithelial cell (HMEC transformation. A genetically-defined, stepwise HMEC transformation model was generated by inhibiting p16 and p53 with shRNA, and expressing exogenous MYC and mutant RAS. By replacing components of this model, we demonstrate that constitutive CCND1/CDK2 activity effectively confers anchorage independent growth by inhibiting p53 or replacing MYC or oncogenic RAS expression. These findings are consistent with several clinical observations of luminal breast cancer sub-types that show elevated CCND1 typically occurs in specimens that retain wild-type p53, do not amplify MYC, and contain no RAS mutations. Taken together, these data suggest that targeted inhibition of constitutive CCND1/CDK2 activity may enhance the effectiveness of current treatments for luminal breast cancer.

  8. Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson's disease.

    Science.gov (United States)

    Binukumar, B K; Shukla, Varsha; Amin, Niranjana D; Grant, Philip; Bhaskar, M; Skuntz, Susan; Steiner, Joseph; Pant, Harish C

    2015-12-01

    Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. Recent evidence indicates that cyclin-dependent kinase 5 (Cdk5) is inappropriately activated in several neurodegenerative conditions, including PD. To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity. Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease. Here we show that TFP5/TP5 selective inhibition of Cdk5/p25 hyperactivation in vivo and in vitro rescues nigrostriatal dopaminergic neurodegeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) in a mouse model of PD. TP5 peptide treatment also blocked dopamine depletion in the striatum and improved gait dysfunction after MPTP administration. The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis. Here we show selective inhibition of Cdk5/p25 -hyperactivation by TFP5/TP5 peptide, which identifies the kinase as a potential therapeutic target to reduce neurodegeneration in Parkinson's disease. PMID:26399293

  9. CDK2-AP1基因过表达对乳腺癌MCF-7细胞增殖及周期的影响%Effect of CDK2-AP1 gene over-expression on proliferation and cell cycle regulation of breast cancer cell line MCF-7

    Institute of Scientific and Technical Information of China (English)

    关晓燕; 周卫兵; 黄隽; 王龙云; 廖遇平

    2012-01-01

    Objective: To over-express cyclin-dependent kinase 2-associated protein 1 (CDK2-AP1) gene, and investigate its effect on the proliferation and cell cycle regulation in breast cancer cell line MCF-7. Methods: CDK2-AP1 gene coding region was cloned into lentivirus vector. Lentivirus particles were infected into MCF-7 cells to upregulate the expression of CDK2-AP1 gene. The expression level of CDK2-AP1 was detected at both mRNA and protein levels by real-time PCR and Western blot. MTT assay, colony formatting assay, and flow cytometry were performed to detect the change of proliferation and cell cycle in MCF-7 cells. We examined the expression of cell cycle associated genes (CDK2, CDK4, P16Ink4A, and P2lCiP1/Wafl) followed by CDK2-AP1 over-expression by Western blot.Results: CDK2-AP1 gene was up-regulated significantly at both mRNA (6.94 folds) and protein level. MTT based growth curve, colony formatting assay and flow cytometry showed that CDK2- API over-expression lentivirus inhibited the proliferation of MCF-7 cells with statistical difference (P<0.05). In addition, with CDK2-AP1 over-expression, MCF-7 cells were arrested in G1 phase accompanied by apoptosis. Western blot showed that the expression level of P21Clpl/wafl and P16Int4A was upregulated, while the expression level of CDK2 and CDK4, members of the CDK family, was downregulated.Conclusion: CDK2-AP1 gene plays a cancer suppressor role in breast cancer. Its function includes inhibiting the proliferation of MCF-7 cells and arresting the cell cycle in G, phase.%目的:通过过表达手段上调细胞周期调节蛋白依赖性激酶2-关联蛋白1(CDK2-AP1)基因在乳腺癌细胞MCF-7中的表达,并观察其对MCF-7细胞生长和细胞周期调控的作用.方法:将CDK2-AP1基因的编码框构建于慢病毒表达载体,导入MCF-7细胞,应用实时定量PCR和Western印迹验证CDK2-AP1基因mRNA和蛋白的表达效率.利用MTT法绘制生长曲线、克隆形成实验观察CDK2-AP1

  10. An in vitro screening with emerging contaminants reveals inhibition of carboxylesterase activity in aquatic organisms.

    Science.gov (United States)

    Solé, Montserrat; Sanchez-Hernandez, Juan C

    2015-12-01

    Pharmaceuticals and personal care products (PPCPs) form part of the new generation of pollutants present in many freshwater and marine ecosystems. Although environmental concentrations of these bioactive substances are low, they cause sublethal effects (e.g., enzyme inhibition) in non-target organisms. However, little is known on metabolism of PPCPs by non-mammal species. Herein, an in vitro enzyme trial was performed to explore sensitivity of carboxylesterase (CE) activity of aquatic organisms to fourteen PPCPs. The esterase activity was determined in the liver of Mediterranean freshwater fish (Barbus meridionalis and Squalius laietanus), coastal marine fish (Dicentrarchus labrax and Solea solea), middle-slope fish (Trachyrhynchus scabrus), deep-sea fish (Alepocephalus rostratus and Cataetix laticeps), and in the digestive gland of a decapod crustacean (Aristeus antennatus). Results showed that 100μM of the lipid regulators simvastatin and fenofibrate significantly inhibited (30-80% of controls) the CE activity of all target species. Among the personal care products, nonylphenol and triclosan were strong esterase inhibitors in most species (36-68% of controls). Comparison with literature data suggests that fish CE activity is as sensitive to inhibition by some PPCPs as that of mammals, although their basal activity levels are lower than in mammals. Pending further studies on the interaction between PPCPs and CE activity, we postulate that this enzyme may act as a molecular sink for certain PPCPs in a comparable way than that described for the organophosphorus pesticides. PMID:26562051

  11. Signal Inhibition Reveals JAK/STAT3 Pathway as Critical for Bovine Inner Cell Mass Development.

    Science.gov (United States)

    Meng, Fanli; Forrester-Gauntlett, Blaise; Turner, Pavla; Henderson, Harold; Oback, Björn

    2015-12-01

    The inner cell mass (ICM) of mammalian blastocysts consists of pluripotent epiblast and hypoblast lineages, which develop into embryonic and extraembryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine Day 8 blastocysts. From the morula stage onward, in vitro fertilized embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signaling pathway components, including TGFbeta1, BMP, EGF, VEGF, PDGF, FGF, cAMP, PI3K, and JAK signals. Using 1) blastocyst quality (by morphological grading), 2) cell numbers (by differential stain), and 3) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR), we identified positive and negative regulators of ICM development and pluripotency. TGFbeta1, BMP, and cAMP and combined VEGF/PDGF/FGF signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF/PDGF/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signaling with AZD1480. This treatment interfered with ICM formation, but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts as well as naive pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3(Y705)) was restricted to ICM nuclei, where it colocalized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3(Y705) signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naive pluripotency markers. PMID:26510863

  12. Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.

    Directory of Open Access Journals (Sweden)

    Yanying Miao

    Full Text Available We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5 signaling pathway in modifying NMDA receptors (NMDARs in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15 protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs, which are both Ca(2+-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15, calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232 and NR2A (p-NR2A(S1232/NR2A, and caused a translocation of p-NR2A(S1232 from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+ concentrations ([Ca(2+](i of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232 expression.

  13. NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair

    OpenAIRE

    Proto, J D; Tang, Y.; Lu, A.; Chen, W C W; Stahl, E; Poddar, M.; Beckman, S A; Robbins, P. D.; Nidernhofer, L J; Imbrogno, K; Hannigan, T; Mars, W M; Wang, B; Huard, J

    2015-01-01

    The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found tha...

  14. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    International Nuclear Information System (INIS)

    Highlights: ► miR-191 expression is upregulated in senescencent human epidermal keratinocytes. ► miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. ► SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. ► SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-β-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3′UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  15. Structural requirements of pyrido[2,3-d]pyrimidin-7-one as CDK4/D inhibitors: 2D autocorrelation, CoMFA and CoMSIA analyses.

    Science.gov (United States)

    Caballero, Julio; Fernández, Michael; González-Nilo, Fernando D

    2008-06-01

    2D autocorrelation, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were undertaken for a series of pyrido[2,3-d]pyrimidin-7-ones to correlate cyclin-dependent kinase (CDK) cyclin D/CDK4 inhibition with 2D and 3D structural properties of 60 known compounds. QSAR models with considerable internal as well as external predictive ability were obtained. The relevant 2D autocorrelation descriptors for modeling CDK4/D inhibitory activity were selected by linear and nonlinear genetic algorithms (GAs) using multiple linear regression (MLR) and Bayesian-regularized genetic neural network (BRGNN) approaches, respectively. Both models showed good predictive statistics; but BRGNN model enables better external predictions. A weight-based input ranking scheme and Kohonen self-organized maps (SOMs) were carried out to interpret the final net weights. The 2D autocorrelation space brings different descriptors for CDK4/D inhibition, and suggests the atomic properties relevant for the inhibitors to interact with CDK4/D active site. CoMFA and CoMSIA analyses were developed with a focus on interpretative ability using coefficient contour maps. CoMSIA produced significantly better results. The results indicate a strong correlation between the inhibitory activity of the modeled compounds and the electrostatic and hydrophobic fields around them. PMID:18468903

  16. Inhibition pathways of the potent organophosphate CBDP with cholinesterases revealed by X-ray crystallographic snapshots and mass spectrometry

    International Nuclear Information System (INIS)

    Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aero-toxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bio-scavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 Angstroms snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H2 16O or H2 18O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP. (authors)

  17. NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

    Science.gov (United States)

    Proto, J D; Tang, Y; Lu, A; Chen, W C W; Stahl, E; Poddar, M; Beckman, S A; Robbins, P D; Nidernhofer, L J; Imbrogno, K; Hannigan, T; Mars, W M; Wang, B; Huard, J

    2015-01-01

    The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD. PMID:25906153

  18. Shikonin Suppresses Skin Carcinogenesis via Inhibiting Cell Proliferation.

    Science.gov (United States)

    Li, Wenjuan; Zhang, Chunjing; Ren, Amy; Li, Teena; Jin, Rong; Li, Guohong; Gu, Xin; Shi, Runhua; Zhao, Yunfeng

    2015-01-01

    The M2 isoform of pyruvate kinase M2 (PKM2) has been shown to be up-regulated in human skin cancers. To test whether PKM2 may be a target for chemoprevention, shikonin, a natural product from the root of Lithospermum erythrorhizon and a specific inhibitor of PKM2, was used in a chemically-induced mouse skin carcinogenesis study. The results revealed that shikonin treatment suppressed skin tumor formation. Morphological examinations and immunohistochemical staining of the skin epidermal tissues suggested that shikonin inhibited cell proliferation without inducing apoptosis. Although shikonin alone suppressed PKM2 activity, it did not suppress tumor promoter-induced PKM2 activation in the skin epidermal tissues at the end of the skin carcinogenesis study. To reveal the potential chemopreventive mechanism of shikonin, an antibody microarray analysis was performed, and the results showed that the transcription factor ATF2 and its downstream target Cdk4 were up-regulated by chemical carcinogen treatment; whereas these up-regulations were suppressed by shikonin. In a promotable skin cell model, the nuclear levels of ATF2 were increased during tumor promotion, whereas this increase was inhibited by shikonin. Furthermore, knockdown of ATF2 decreased the expression levels of Cdk4 and Fra-1 (a key subunit of the activator protein 1. In summary, these results suggest that shikonin, rather than inhibiting PKM2 in vivo, suppresses the ATF2 pathway in skin carcinogenesis. PMID:25961580

  19. Structure-based prediction reveals capping motifs that inhibit β-helix aggregation

    OpenAIRE

    Bryan, Allen W.; Starner-Kreinbrink, Jennifer L.; Hosur, Raghavendra; Clark, Patricia L.; Berger, Bonnie

    2011-01-01

    The parallel β-helix is a geometrically regular fold commonly found in the proteomes of bacteria, viruses, fungi, archaea, and some vertebrates. β-helix structure has been observed in monomeric units of some aggregated amyloid fibers. In contrast, soluble β-helices, both right- and left-handed, are usually “capped” on each end by one or more secondary structures. Here, an in-depth classification of the diverse range of β-helix cap structures reveals subtle commonalities in structural componen...

  20. Mutations in GDF5 reveal a key residue mediating BMP inhibition by NOGGIN.

    Directory of Open Access Journals (Sweden)

    Petra Seemann

    2009-11-01

    Full Text Available Signaling output of bone morphogenetic proteins (BMPs is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T in patients with multiple synostosis syndrome (SYM1 in the BMP-related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP-inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling.

  1. Hsf1 activation inhibits rapamycin resistance and TOR signaling in yeast revealed by combined proteomic and genetic analysis.

    Directory of Open Access Journals (Sweden)

    Sricharan Bandhakavi

    Full Text Available TOR kinases integrate environmental and nutritional signals to regulate cell growth in eukaryotic organisms. Here, we describe results from a study combining quantitative proteomics and comparative expression analysis in the budding yeast, S. cerevisiae, to gain insights into TOR function and regulation. We profiled protein abundance changes under conditions of TOR inhibition by rapamycin treatment, and compared this data to existing expression information for corresponding gene products measured under a variety of conditions in yeast. Among proteins showing abundance changes upon rapamycin treatment, almost 90% of them demonstrated homodirectional (i.e., in similar direction transcriptomic changes under conditions of heat/oxidative stress. Because the known downstream responses regulated by Tor1/2 did not fully explain the extent of overlap between these two conditions, we tested for novel connections between the major regulators of heat/oxidative stress response and the TOR pathway. Specifically, we hypothesized that activation of regulator(s of heat/oxidative stress responses phenocopied TOR inhibition and sought to identify these putative TOR inhibitor(s. Among the stress regulators tested, we found that cells (hsf1-R206S, F256S and ssa1-3 ssa2-2 constitutively activated for heat shock transcription factor 1, Hsf1, inhibited rapamycin resistance. Further analysis of the hsf1-R206S, F256S allele revealed that these cells also displayed multiple phenotypes consistent with reduced TOR signaling. Among the multiple Hsf1 targets elevated in hsf1-R206S, F256S cells, deletion of PIR3 and YRO2 suppressed the TOR-regulated phenotypes. In contrast to our observations in cells activated for Hsf1, constitutive activation of other regulators of heat/oxidative stress responses, such as Msn2/4 and Hyr1, did not inhibit TOR signaling. Thus, we propose that activated Hsf1 inhibits rapamycin resistance and TOR signaling via elevated expression of specific

  2. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    Science.gov (United States)

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-01

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  3. Resistance to Apoptosis Conferred by Cdk Inhibitors During Myocyte Differentiation

    OpenAIRE

    Jian WANG; Walsh, Kenneth

    1996-01-01

    Proliferating murine C2C12 myoblasts can undergo either terminal differentiation or programmed cell death under conditions of mitogen deprivation. Unlike myoblasts, differentiated myotubes were resistant to apoptosis. During myogenesis the appearance of the apoptosis-resistant phenotype was correlated with the induction of the cyclin-dependent kinase (Cdk) inhibitor p21CIP1 but not with the appearance of myogenin, a marker expressed earlier in differentiation. Forced expression of the Cdk inh...

  4. Quantitative Phosphoproteomics Reveals Wee1 Kinase as a Therapeutic Target in a Model of Proneural Glioblastoma.

    Science.gov (United States)

    Lescarbeau, Rebecca S; Lei, Liang; Bakken, Katrina K; Sims, Peter A; Sarkaria, Jann N; Canoll, Peter; White, Forest M

    2016-06-01

    Glioblastoma (GBM) is the most common malignant primary brain cancer. With a median survival of about a year, new approaches to treating this disease are necessary. To identify signaling molecules regulating GBM progression in a genetically engineered murine model of proneural GBM, we quantified phosphotyrosine-mediated signaling using mass spectrometry. Oncogenic signals, including phosphorylated ERK MAPK, PI3K, and PDGFR, were found to be increased in the murine tumors relative to brain. Phosphorylation of CDK1 pY15, associated with the G2 arrest checkpoint, was identified as the most differentially phosphorylated site, with a 14-fold increase in phosphorylation in the tumors. To assess the role of this checkpoint as a potential therapeutic target, syngeneic primary cell lines derived from these tumors were treated with MK-1775, an inhibitor of Wee1, the kinase responsible for CDK1 Y15 phosphorylation. MK-1775 treatment led to mitotic catastrophe, as defined by increased DNA damage and cell death by apoptosis. To assess the extensibility of targeting Wee1/CDK1 in GBM, patient-derived xenograft (PDX) cell lines were also treated with MK-1775. Although the response was more heterogeneous, on-target Wee1 inhibition led to decreased CDK1 Y15 phosphorylation and increased DNA damage and apoptosis in each line. These results were also validated in vivo, where single-agent MK-1775 demonstrated an antitumor effect on a flank PDX tumor model, increasing mouse survival by 1.74-fold. This study highlights the ability of unbiased quantitative phosphoproteomics to reveal therapeutic targets in tumor models, and the potential for Wee1 inhibition as a treatment approach in preclinical models of GBM. Mol Cancer Ther; 15(6); 1332-43. ©2016 AACR. PMID:27196784

  5. The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Warren; Shahinas, Dea; Bonvin, Julie; Hou, Wenjuan; Kimber, Matthew S.; Turnbull, Joanne; Christendat, Dinesh; (Guelph); (Toronto); (ConU)

    2009-08-14

    TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD(+) plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.

  6. Specific Antileukemic Activity of PD0332991, a CDK4/6 Inhibitor, against Philadelphia Chromosome-Positive Lymphoid Leukemia.

    Science.gov (United States)

    Nemoto, Atsushi; Saida, Satoshi; Kato, Itaru; Kikuchi, Jiro; Furukawa, Yusuke; Maeda, Yasuhiro; Akahane, Koshi; Honna-Oshiro, Hiroko; Goi, Kumiko; Kagami, Keiko; Kimura, Shinya; Sato, Yuko; Okabe, Seiichi; Niwa, Akira; Watanabe, Kenichiro; Nakahata, Tatsutoshi; Heike, Toshio; Sugita, Kanji; Inukai, Takeshi

    2016-01-01

    S-phase progression of the cell cycle is accelerated in tumors through various genetic abnormalities, and, thus, pharmacologic inhibition of altered cell-cycle progression would be an effective strategy to control tumors. In the current study, we analyzed the antileukemic activity of three available small molecules targeting CDK4/CDK6 against lymphoid crisis of chronic myeloid leukemia (CML-LC) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL), and found that all three molecules showed specific activities against leukemic cell lines derived from CML-LC and Ph(+) ALL. In particular, PD0332991 exhibited extremely high antileukemic activity against CML-LC and Ph(+) ALL cell lines in the nanomolar range by the induction of G0-G1 arrest and partially cell death through dephosphorylation of pRb and downregulation of the genes that are involved in S-phase transition. As an underlying mechanism for favorable sensitivity to the small molecules targeting CDK4/CDK6, cell-cycle progression of Ph(+) lymphoid leukemia cells was regulated by transcriptional and posttranscriptional modulation of CDK4 as well as Cyclin D2 gene expression under the control of BCR-ABL probably through the PI3K pathway. Consistently, the gene expression level of Cyclin D2 in Ph(+) lymphoid leukemia cells was significantly higher than that in Ph(-) lymphoid leukemia cells. Of note, three Ph(+) ALL cell lines having the T315I mutation also showed sensitivity to PD0332991. In a xenograft model, PD0332991, but not imatinib, suppressed dissemination of Ph(+) ALL having the T315I mutation and prolonged survival, demonstrating that this reagent would be a new therapeutic modality for relapsed CML-LC and Ph(+) ALL patients after treatment with tyrosine kinase inhibitors. PMID:26637365

  7. Dysregulation of CDK8 and Cyclin C in tumorigenesis %Dysregulation of CDK8 and Cyclin C in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Wu Xu; Jun-Yuan Ji

    2011-01-01

    Appropriately controlled gene expression is fundamental for normal growth and survival of all living organisms.In eukaryotes,the transcription of protein-coding mRNAs is dependent on RNA polymerase Ⅱ (Pol Ⅱ).The multi-subunit transcription cofactor Mediator complex is proposed to regulate most,if not all,of the Pol Ⅱ-dependent transcription.Here we focus our discussion on two subunits of the Mediator complex,cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC),because they are either mutated or amplified in a variety of human cancers.CDK8 functions as an oncoprotein in melanoma and colorectal cancers,thus there are considerable interests in developing drugs specifically targeting the CDK8 kinase activity.However,to evaluate the feasibility of targeting CDK8 for cancer therapy and to understand how their dysregulation contributes to tumorigenesis,it is essential to elucidate the in vivo function and regulation of CDK8-CycC,which are still poorly understood in multi-cellular organisms.We summarize the evidence linking their dysregulation to various cancers and present our bioinformatics and computational analyses on the structure and evolution of CDK8.We also discuss the implications of these observations in tumorigenesis.Because most of the Mediator subunits,including CDK8 and CycC,are highly conserved during eukaryotic evolution,we expect that investigations using model organisms such as Drosophila will provide important insights into the function and regulation of CDK8 and CycC in different cellular and developmental contexts.

  8. Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

    Science.gov (United States)

    Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

    2015-01-01

    The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target. PMID:26355431

  9. Genetic Evidence for Functional Dependency of p18Ink4c on Cdk4

    OpenAIRE

    Pei, Xin-Hai; Bai, Feng; Tsutsui, Tateki; Kiyokawa, Hiroaki; Xiong, Yue

    2004-01-01

    The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, wh...

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

    Energy Technology Data Exchange (ETDEWEB)

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U. [Lawrence Berkeley National Lab., CA (United States)] [and others

    1996-04-02

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

  11. Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) Model, Which Is Associated with the Induction of Cell Cycle G1 Arrest.

    Science.gov (United States)

    Cho, Han Jin; Lim, Do Young; Kwon, Gyoo Taik; Kim, Ji Hee; Huang, Zunnan; Song, Hyerim; Oh, Yoon Sin; Kang, Young-Hee; Lee, Ki Won; Dong, Zigang; Park, Jung Han Yoon

    2016-01-01

    Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC. PMID:26907265

  12. Benzyl Isothiocyanate Inhibits Prostate Cancer Development in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP Model, Which Is Associated with the Induction of Cell Cycle G1 Arrest

    Directory of Open Access Journals (Sweden)

    Han Jin Cho

    2016-02-01

    Full Text Available Benzyl isothiocyanate (BITC is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker, cyclin A, cyclin D1, and cyclin-dependent kinase (CDK2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.

  13. A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression.

    Science.gov (United States)

    Zeineldin, M; Cunningham, J; McGuinness, W; Alltizer, P; Cowley, B; Blanchat, B; Xu, W; Pinson, D; Neufeld, K L

    2012-05-10

    Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt-signaling pathway by targeting the proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription cofactor for genes required for cell proliferation such as cyclin-D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and the cytoplasm, with nuclear APC implicated in the inhibition of Wnt target gene expression. Adopting a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals (NLSs) of Apc (Apc(mNLS)). Apc(mNLS/mNLS) mice are viable and fractionation of mouse embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc as compared with Apc(+/+) MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from Apc(mNLS/mNLS) mice. Compared with Apc(+/+) mice, Apc(mNLS/mNLS) mice showed increased proliferation in epithelial cells from the jejunum, ileum and colon. These same tissues from Apc(mNLS/mNLS) mice showed more mRNA from three genes upregulated in response to canonical Wnt signal, c-Myc, axin-2 and cyclin-D1, and less mRNA from Hath-1, which is downregulated in response to Wnt. These observations suggest a role for nuclear Apc in the inhibition of canonical Wnt signaling and the control of epithelial proliferation in intestinal tissue. Furthermore, we found Apc(Min/+) mice, which harbor a mutation that truncates Apc, to have an increased polyp size and multiplicity if they also carry the Apc(mNLS) allele. Taken together, this analysis of the novel Apc(mNLS) mouse model supports a role for nuclear Apc in the control of Wnt target genes

  14. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

    Science.gov (United States)

    Toledo, Chad M; Ding, Yu; Hoellerbauer, Pia; Davis, Ryan J; Basom, Ryan; Girard, Emily J; Lee, Eunjee; Corrin, Philip; Hart, Traver; Bolouri, Hamid; Davison, Jerry; Zhang, Qing; Hardcastle, Justin; Aronow, Bruce J; Plaisier, Christopher L; Baliga, Nitin S; Moffat, Jason; Lin, Qi; Li, Xiao-Nan; Nam, Do-Hyun; Lee, Jeongwu; Pollard, Steven M; Zhu, Jun; Delrow, Jeffery J; Clurman, Bruce E; Olson, James M; Paddison, Patrick J

    2015-12-22

    To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality. PMID:26673326

  15. Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells

    Directory of Open Access Journals (Sweden)

    Chad M. Toledo

    2015-12-01

    Full Text Available To identify therapeutic targets for glioblastoma (GBM, we performed genome-wide CRISPR-Cas9 knockout (KO screens in patient-derived GBM stem-like cells (GSCs and human neural stem/progenitors (NSCs, non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers. In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

  16. Molecular Mechanism of Inhibition of the Mitochondrial Carnitine/Acylcarnitine Transporter by Omeprazole Revealed by Proteoliposome Assay, Mutagenesis and Bioinformatics

    OpenAIRE

    Annamaria Tonazzi; Ivano Eberini; Cesare Indiveri

    2013-01-01

    The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC50 of the inhibition...

  17. Non-CDK-bound p27 (p27{sup NCDK}) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerklund, Mia A. [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Vaahtomeri, Kari [Genome-Scale Biology Program and Institute of Biotechnology, 00014 University of Helsinki, Helsinki (Finland); Peltonen, Karita [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Viollet, Benoit [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), 75014 Paris (France); INSERM U567, 75014 Paris (France); Maekelae, Tomi P. [Genome-Scale Biology Program and Institute of Biotechnology, 00014 University of Helsinki, Helsinki (Finland); Band, Arja M. [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Laiho, Marikki, E-mail: mlaiho1@jhmi.edu [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD (United States)

    2010-03-10

    p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27{sup NCDK}) but the nature of p27{sup NCDK} has remained unresolved. Here we demonstrate that the epitope recognized by the p27{sup NCDK}-specific antibody resides in the p27 CDK-interaction domain and that p27{sup NCDK} is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27{sup NCDK} levels whereas total p27 levels are unaffected. p27{sup NCDK}, but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27{sup NCDK} responses to metabolic stress and PI3K inhibition. These results indicate that p27{sup NCDK} is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.

  18. Mutations in CDK5RAP2 cause Seckel syndrome

    OpenAIRE

    Karabey Kayserili, Hülya; Yiğit, G.; Brown, KE.; Pohl, E.; Caliebe, A.; Zahnleiter, D.; Rosser, E.; Bögershausen, N.; Uyguner, ZO.; Altunoğlu, U.; Nürnberg, G.; Nürnberg, P.; Rauch, A.; Li, Y.; Thiel, CT.; Wollnik, B.

    2015-01-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been sho...

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

    Directory of Open Access Journals (Sweden)

    Jon H Chung

    2010-02-01

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

  20. File list: Oth.EmF.05.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.05.Cdk9.AllCell mm9 TFs and others Cdk9 Embryonic fibroblast SRX620288,SRX6...20289,SRX255482,SRX620286,SRX620287 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.05.Cdk9.AllCell.bed ...

  1. File list: Oth.Bld.20.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.20.Cdk9.AllCell mm9 TFs and others Cdk9 Blood SRX277329,SRX020973,SRX020972...,SRX020974,SRX020971 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.20.Cdk9.AllCell.bed ...

  2. File list: Oth.PSC.05.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX236482,SRX...668247 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Cdk8.AllCell.bed ...

  3. File list: Oth.Bld.05.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.Cdk9.AllCell mm9 TFs and others Cdk9 Blood SRX277329,SRX020971,SRX020972...,SRX020973,SRX020974 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.05.Cdk9.AllCell.bed ...

  4. File list: Oth.PSC.10.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.10.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX668247,SRX...236482 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.10.Cdk8.AllCell.bed ...

  5. File list: Oth.PSC.50.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX236482,SRX...668247 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Cdk8.AllCell.bed ...

  6. File list: Oth.EmF.50.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Cdk9.AllCell mm9 TFs and others Cdk9 Embryonic fibroblast SRX620288,SRX6...20289,SRX620287,SRX620286,SRX255482 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Cdk9.AllCell.bed ...

  7. Biosynthesis of Antibiotic Leucinostatins in Bio-control Fungus Purpureocillium lilacinum and Their Inhibition on Phytophthora Revealed by Genome Mining

    Science.gov (United States)

    Li, Erfeng; Mao, Zhenchuan; Ling, Jian; Yang, Yuhong; Yin, Wen-Bing; Xie, Bingyan

    2016-01-01

    Purpureocillium lilacinum of Ophiocordycipitaceae is one of the most promising and commercialized agents for controlling plant parasitic nematodes, as well as other insects and plant pathogens. However, how the fungus functions at the molecular level remains unknown. Here, we sequenced two isolates (PLBJ-1 and PLFJ-1) of P. lilacinum from different places Beijing and Fujian. Genomic analysis showed high synteny of the two isolates, and the phylogenetic analysis indicated they were most related to the insect pathogen Tolypocladium inflatum. A comparison with other species revealed that this fungus was enriched in carbohydrate-active enzymes (CAZymes), proteases and pathogenesis related genes. Whole genome search revealed a rich repertoire of secondary metabolites (SMs) encoding genes. The non-ribosomal peptide synthetase LcsA, which is comprised of ten C-A-PCP modules, was identified as the core biosynthetic gene of lipopeptide leucinostatins, which was specific to P. lilacinum and T. ophioglossoides, as confirmed by phylogenetic analysis. Furthermore, gene expression level was analyzed when PLBJ-1 was grown in leucinostatin-inducing and non-inducing medium, and 20 genes involved in the biosynthesis of leucionostatins were identified. Disruption mutants allowed us to propose a putative biosynthetic pathway of leucinostatin A. Moreover, overexpression of the transcription factor lcsF increased the production (1.5-fold) of leucinostatins A and B compared to wild type. Bioassays explored a new bioactivity of leucinostatins and P. lilacinum: inhibiting the growth of Phytophthora infestans and P. capsici. These results contribute to our understanding of the biosynthetic mechanism of leucinostatins and may allow us to utilize P. lilacinum better as bio-control agent. PMID:27416025

  8. CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

    International Nuclear Information System (INIS)

    Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27kip1. The elevated p27kip1 expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. - Highlights: • Isoangustone A suppresses growth of PC3 and LNCaP prostate cancer cells. • Administration of isoangustone A inhibits tumor growth in mice. • Treatment of isoangustone A induces cell cycle arrest and accumulation of p27kip1. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells

  9. CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Liu, Kangdong [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Kim, Jiyoung [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Lim, Soon Sung; Park, Jung Han Yoon [Department of Food Science and Nutrition, College of Natural Science, Hallym University, Chuncheon, 200-702 (Korea, Republic of); Dong, Zigang [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Lee, Ki Won, E-mail: kiwon@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of); Lee, Hyong Joo, E-mail: leehyjo@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of)

    2013-10-01

    Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27{sup kip1}. The elevated p27{sup kip1} expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. - Highlights: • Isoangustone A suppresses growth of PC3 and LNCaP prostate cancer cells. • Administration of isoangustone A inhibits tumor growth in mice. • Treatment of isoangustone A induces cell cycle arrest and accumulation of p27{sup kip1}. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells.

  10. Identification of new targets of human glioma CDK2 siRNA%人脑胶质瘤CDK2干扰RNA新靶点的检验

    Institute of Scientific and Technical Information of China (English)

    呼格吉乐; 张军力; 段美庆; 王俊瑞; 高乃康

    2012-01-01

    Objective To construct four new eukaryotic expression vectors of small interference RNA(siRNA) specific for CDK2 and confirm the interferential efficiency of siRNA on the expression of CDK2. Methods (l)Four new eukaryotic expression vectors of siRNA specific for CDK2 were constructed and identified by double enzymic digestion. (2)SHG44 cell line of human brain gliocytoma was transiently transfected with the four new vectors via oligofectamine. (3) Vector, with the strongest interferential efficiency, was confirmed by detecting the expression level of CDK2 Mrna using reverse transcription-polymerase chain reactionCRT-PCR). Results (l)Four eukaryotic expression vectors of siRNA specific for new targets of CDK2 was constructed and denominated as Pgpu6/GFP/Neo-CDK2-l,Pgpu6/GFP/Neo-CDK2-2,Pgpu6/GFP/Neo-CDK2-3 and Pgpu6/GFP/Neo-CDK2-4. (2)The expression of CDK2 Mrna was obviously suppressed and the vector with the strongest interferential efficiency was obtained. Conclusion The eukaryotic expression vectors of siRNA,specific for new target of CDK2 and with the strongest interferential efficiency, was successfully constructed and indentified,which could obviously suppress the expression of CDK2 Mrna in SHG44 cell line.%目的 构建4个新靶点CDK2干扰RNA真核表达载体,转染人脑胶质细胞瘤SHG44细胞,经逆转录-聚合酶链反应(RT-PCR)检测mRNA表达,获得干扰效果最好的真核表达载体,为CDK2成为人脑肿瘤标志物提供有价值的资料.方法 (1)构建4个新靶点CDK2干扰RNA真核表达载体并用双酶切和测序鉴定;(2)用脂质体法瞬时转染上述4个载体到SHG44细胞株;(3)通过 RT-PCR 比较转染后CDK2 mRNA表达量,选出干扰效果最好的一个载体.结果 (1)成功构建了4个新靶点CDK2干扰RNA真核表达载体即pGPU6/GFP/Neo-CDK2-1、pGPU6/GFP/Neo-CDK2-2、pGPU6/GFP/Neo-CDK2-3、pGPU6/GFP/Neo-CDK2-4;(2)CDK2 mRNA表达明显受抑制,并获得效果最好的CDK2干扰RNA真核表达载体.结论 成

  11. Effect of CDK1/CDK2 interference on cell cycle by lentivirus vector in cancer cells%慢病毒介导 CDK1/CDK2干扰对肿瘤细胞周期的影响❉

    Institute of Scientific and Technical Information of China (English)

    江文娇; 李慧萍; 齐庆远

    2015-01-01

    In order to investigate the influence of CDK1 and CDK2 interference on cell cycle in CBRH-7919 cell, the CDK1, CDK2 specific shRNA lentiviral expression vectors were structured, then three plasmids were contransfected into 293 FT cells to produce viral particles, which infected the CBRH-7919 cells after collecting and concentrating the virals.The morphological changes of cells were observed by fluorescence microscope, Real-time PCR and Western Blotting demonstrated the level changes of CDK1 , CDK2 mRNA and protein ex-pression in CBRH-7919 cells.It was analyzed the changes of cell proliferation and cycle effect by MTT and flow cytometry.The results showed that it was successful to construct the CDK1 and CDK2 specific shRNA lentiviral expression vector;silencing of CDK1 led to arrest of cells in G2/M phase, cell proliferation rate de-creased obviously, and increased cell debris, while silencing CDK2 cells remained growth as normal.%为了检测细胞周期性蛋白激酶CDK1与CDK2干扰对CBRH-7919细胞周期的影响,构建了CDK1和CDK2特异性shRNA慢病毒沉默表达载体,三质粒共转染293 FT细胞产生病毒颗粒,收集浓缩后感染CBRH-7919细胞,荧光显微镜下观察了细胞形态,实时定量荧光PCR和聚丙烯酰胺凝胶电泳检测了细胞中CDK1和CDK2 mRNA和蛋白质表达水平的变化,MTT法和流式细胞仪分别检测了细胞增殖和细胞周期的变化情况。结果表明:成功构建了CDK1与CDK2特异性shRNA慢病毒表达载体,干扰CDK1导致细胞G2/M期的阻滞,细胞增殖明显降低,细胞碎片增多;而干扰CDK2后细胞仍正常生长。

  12. Cdk5 is essential for synaptic vesicle endocytosis

    DEFF Research Database (Denmark)

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S;

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynami...

  13. Micro-electrode array recordings reveal reductions in both excitation and inhibition in cultured cortical neuron networks lacking Shank3.

    Science.gov (United States)

    Lu, C; Chen, Q; Zhou, T; Bozic, D; Fu, Z; Pan, J Q; Feng, G

    2016-02-01

    Numerous risk genes have recently been implicated in susceptibility to autism and schizophrenia. Translating such genetic findings into disease-relevant neurobiological mechanisms is challenging due to the lack of throughput assays that can be used to assess their functions on an appropriate scale. To address this issue, we explored the feasibility of using a micro-electrode array (MEA) as a potentially scalable assay to identify the electrical network phenotypes associated with risk genes. We first characterized local and global network firing in cortical neurons with MEAs, and then developed methods to analyze the alternation between the network active period (NAP) and the network inactive period (NIP), each of which lasts tens of seconds. We then evaluated the electric phenotypes of neurons derived from Shank3 knockout (KO) mice. Cortical neurons cultured on MEAs displayed a rich repertoire of spontaneous firing, and Shank3 deletion led to reduced firing activity. Enhancing excitation with CX546 rescued the deficit in the spike rate in the Shank3 KO network. In addition, the Shank3 KO network produced a shorter NIP, and this altered network firing pattern was normalized by clonazepam, a positive modulator of the GABAA receptor. MEA recordings revealed electric phenotypes that displayed altered excitation and inhibition in the network lacking Shank3. Thus, our study highlights MEAs as an experimental framework for measuring multiple robust neurobiological end points in dynamic networks and as an assay system that could be used to identify electric phenotypes in cultured neuronal networks and to analyze additional risk genes identified in psychiatric genetics. PMID:26598066

  14. Preventing DNA over-replication: a Cdk perspective

    Directory of Open Access Journals (Sweden)

    Porter Andrew CG

    2008-01-01

    Full Text Available Abstract The cell cycle is tightly controlled to ensure that replication origins fire only once per cycle and that consecutive S-phases are separated by mitosis. When controls fail, DNA over-replication ensues: individual origins fire more than once per S-phase (re-replication or consecutive S-phases occur without intervening mitoses (endoreduplication. In yeast the cell cycle is controlled by a single cyclin dependent kinase (Cdk that prevents origin licensing at times when it promotes origin firing, and that is inactivated, via proteolysis of its partner cyclin, as cells undergo mitosis. A quantitative model describes three levels of Cdk activity: low activity allows licensing, intermediate activity allows firing but prevents licensing, and high activity promotes mitosis. In higher eukaryotes the situation is complicated by the existence of additional proteins (geminin, Cul4-Ddb1Cdt2, and Emi1 that control licensing. A current challenge is to understand how these various control mechanisms are co-ordinated and why the degree of redundancy between them is so variable. Here the experimental induction of DNA over-replication is reviewed in the context of the quantitative model of Cdk action. Endoreduplication is viewed as a consequence of procedures that cause Cdk activity to fall below the threshold required to prevent licensing, and re-replication as the result of procedures that increase that threshold value. This may help to explain why over-replication does not necessarily require reduced Cdk activity and how different mechanisms conspire to prevent over-replication. Further work is nevertheless required to determine exactly how losing just one licensing control mechanism often causes over-replication, and why this varies between cell systems.

  15. (S)-Lacosamide Binding to Collapsin Response Mediator Protein 2 (CRMP2) Regulates CaV2.2 Activity by Subverting Its Phosphorylation by Cdk5.

    Science.gov (United States)

    Moutal, Aubin; François-Moutal, Liberty; Perez-Miller, Samantha; Cottier, Karissa; Chew, Lindsey Anne; Yeon, Seul Ki; Dai, Jixun; Park, Ki Duk; Khanna, May; Khanna, Rajesh

    2016-04-01

    The neuronal circuit remodels during development as well as in human neuropathologies such as epilepsy. Neurite outgrowth is an obligatory step in these events. We recently reported that alterations in the phosphorylation state of an axon specification/guidance protein, the collapsin response mediator protein 2 (CRMP2), play a major role in the activity-dependent regulation of neurite outgrowth. We also identified (S)-LCM, an inactive stereoisomer of the clinically used antiepileptic drug (R)-LCM (Vimpat®), as a novel tool for preferentially targeting CRMP2-mediated neurite outgrowth. Here, we investigated the mechanism by which (S)-LCM affects CRMP2 phosphorylation by two key kinases, cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β (GSK-3β). (S)-LCM application to embryonic cortical neurons resulted in reduced levels of Cdk5- and GSK-3β-phosphorylated CRMP2. Mechanistically, (S)-LCM increased CRMP2 binding to both Cdk5- and GSK-3β without affecting binding of CRMP2 to its canonical partner tubulin. Saturation transfer difference nuclear magnetic resonance (STD NMR) and differential scanning fluorimetry (DSF) experiments demonstrated direct binding of (S)-LCM to CRMP2. Using an in vitro luminescent kinase assay, we observed that (S)-LCM specifically inhibited Cdk5-mediated phosphorylation of CRMP2. Cross-linking experiments and analytical ultracentrifugation showed no effect of (S)-LCM on the oligomerization state of CRMP2. The increased association between Cdk5-phosphorylated CRMP2 and CaV2.2 was reduced by (S)-LCM in vitro and in vivo. This reduction translated into a decrease of calcium influx via CaV2.2 in (S)-LCM-treated neurons compared to controls. (S)-LCM, to our knowledge, is the first molecule described to directly inhibit CRMP2 phosphorylation and may be useful for delineating CRMP2-facilitated functions. PMID:25846820

  16. Pin1-mediated Sp1 phosphorylation by CDK1 increases Sp1 stability and decreases its DNA-binding activity during mitosis.

    Science.gov (United States)

    Yang, Hang-Che; Chuang, Jian-Ying; Jeng, Wen-Yih; Liu, Chia-I; Wang, Andrew H-J; Lu, Pei-Jung; Chang, Wen-Chang; Hung, Jan-Jong

    2014-12-16

    We have shown that Sp1 phosphorylation at Thr739 decreases its DNA-binding activity. In this study, we found that phosphorylation of Sp1 at Thr739 alone is necessary, but not sufficient for the inhibition of its DNA-binding activity during mitosis. We demonstrated that Pin1 could be recruited to the Thr739(p)-Pro motif of Sp1 to modulate the interaction between phospho-Sp1 and CDK1, thereby facilitating CDK1-mediated phosphorylation of Sp1 at Ser720, Thr723 and Thr737 during mitosis. Loss of the C-terminal end of Sp1 (amino acids 741-785) significantly increased Sp1 phosphorylation, implying that the C-terminus inhibits CDK1-mediated Sp1 phosphorylation. Binding analysis of Sp1 peptides to Pin1 by isothermal titration calorimetry indicated that Pin1 interacts with Thr739(p)-Sp1 peptide but not with Thr739-Sp1 peptide. X-ray crystallography data showed that the Thr739(p)-Sp1 peptide occupies the active site of Pin1. Increased Sp1 phosphorylation by CDK1 during mitosis not only stabilized Sp1 levels by decreasing interaction with ubiquitin E3-ligase RNF4 but also caused Sp1 to move out of the chromosomes completely by decreasing its DNA-binding activity, thereby facilitating cell cycle progression. Thus, Pin1-mediated conformational changes in the C-terminal region of Sp1 are critical for increased CDK1-mediated Sp1 phosphorylation to facilitate cell cycle progression during mitosis. PMID:25398907

  17. Suppressing the truth as a mechanism of deception: Delta plots reveal the role of response inhibition in lying

    NARCIS (Netherlands)

    E. Debey; K.R. Ridderinkhof; J. de Houwer; M. Schryver; B. Verschuere

    2015-01-01

    Lying takes more time than telling the truth. Because lying involves withholding the truth, this "lie effect" has been related to response inhibition. We investigated the response inhibition hypothesis of lying using the delta-plot method: A leveling-off of the standard increase of the lie effect wi

  18. Sequencing analysis of mutant allele cdc28-srm of protein kinase CDC28 and molecular dynamics study of glycine-rich loop in wild-type and mutant allele G16S of CDK2 as model

    International Nuclear Information System (INIS)

    The central role that cyclin-dependent kinases play in the timing of cell division and the high incidence of genetic alteration of CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of the yeast Saccharomyces cerevisiae a very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including cdc28-srm affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A cdc28-srm mutation is not a temperature-sensitive mutation and differs from the known cdc28-ts mutations because it has the evident phenotypic manifestations at 30 deg C. Sequencing analysis of cdc28-srm revealed a single nucleotide substitution G20S. This is a third glycine in a conserved sequence GxGxxG in the G-rich loop positioned opposite the activation T-loop. Despite its demonstrated importance, the role of the G-loop has remained unclear. The crystal structure of the human CDK2 has served as a model for the catalytic core of other CDKs, including CDC28. Nanoseconds long molecular dynamics (MD) trajectories of the CDK2/ATP complex were analyzed. The MD simulations of CDK2-G16S (CDC28-G20S) substitution show conformational changes of CDK2 structure resulting in the moving of the G-loop away from ATP and a new rearrangement of amino acids in the T-loop

  19. Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer

    Science.gov (United States)

    Thorenoor, Nithyananda; Faltejskova-Vychytilova, Petra; Hombach, Sonja; Mlcochova, Jitka; Kretz, Markus; Svoboda, Marek; Slaby, Ondrej

    2016-01-01

    We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P HCT116+/+, HCT116−/− and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified. PMID:26506418

  20. 2,8-Disubstituted-1,6-Naphthyridines and 4,6-Disubstituted-Isoquinolines with Potent, Selective Affinity for CDK8/19.

    Science.gov (United States)

    Mallinger, Aurélie; Schiemann, Kai; Rink, Christian; Sejberg, Jimmy; Honey, Mark A; Czodrowski, Paul; Stubbs, Mark; Poeschke, Oliver; Busch, Michael; Schneider, Richard; Schwarz, Daniel; Musil, Djordje; Burke, Rosemary; Urbahns, Klaus; Workman, Paul; Wienke, Dirk; Clarke, Paul A; Raynaud, Florence I; Eccles, Suzanne A; Esdar, Christina; Rohdich, Felix; Blagg, Julian

    2016-06-01

    We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing. PMID:27326329

  1. Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements

    Science.gov (United States)

    Liu, Shu; Bolger, Joshua K.; Kirkland, Lindsay O.; Premnath, Padmavathy N.; McInnes, Campbell

    2012-01-01

    An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized

  2. Development of mice without Cip/Kip CDK inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582 (Japan); CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Hara, Eiji [Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Nakayama, Keiko [Department of Developmental Genetics, Center for Translational and Advanced Animal Research, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 (Japan); Nakayama, Keiichi I., E-mail: nakayak1@bioreg.kyushu-u.ac.jp [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582 (Japan); CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. Black-Right-Pointing-Pointer Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. Black-Right-Pointing-Pointer CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G{sub 0} to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in

  3. Development of mice without Cip/Kip CDK inhibitors

    International Nuclear Information System (INIS)

    Highlights: ► Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. ► Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. ► CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G0 to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to

  4. Cdk2-Null Mice Are Resistant to ErbB-2-Induced Mammary Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Dipankar Ray

    2011-05-01

    Full Text Available The concept of targeting G1 cyclin-dependent kinases (CDKs in breast cancer treatments is supported by the fact that the genetic ablation of Cdk4 had minimal impacts on normal cell proliferation in majority of cell types, resulting in near-normal mouse development, whereas such loss of Cdk4 completely abrogated ErbB-2/neu-induced mammary tumorigenesis in mice. In most human breast cancer tissues, another G1-regulatory CDK, CDK2, is also hyperactivated by various mechanisms and is believed to be an important therapeutic target. In this report, we provide genetic evidence that CDK2 is essential for proliferation and oncogenesis of murine mammary epithelial cells. We observed that 87% of Cdk2-null mice were protected from ErbB-2-induced mammary tumorigenesis. Mouse embryonic fibroblasts isolated from Cdk2-null mouse showed resistance to various oncogene-induced transformation. Previously, we have reported that hemizygous loss of Cdc25A, the major activator of CDK2, can also protect mice from ErbB-2-induced mammary tumorigenesis [Cancer Res (2007 67(14: 6605–11]. Thus, we propose that CDC25A-CDK2 pathway is critical for the oncogenic action of ErbB-2 in mammary epithelial cells, in a manner similar to Cyclin D1/CDK4 pathway.

  5. ALDH maintains the stemness of lung adenoma stem cells by suppressing the Notch/CDK2/CCNE pathway.

    Directory of Open Access Journals (Sweden)

    Zhongjun Li

    Full Text Available To evaluate the expression of ALDH1A1 in lung adenoma stem cells (LASCs and maintenance of their stemness through the Notch pathway.LASCs (A549s were isolated from lung adenoma cells (A549 and identified by their coexpression of CD133 and CD326 and their capacity formulti-directional differentiation. Expression of ALDH1A1 in A549 and A549s cells were evaluated by Real-time PCR. Effects of ALDH1A1 upregulation in A549 cells and its downregulation in A549s cells on the clonogenicity and cell cycle were assessed by colony-forming unit assay. Moreover, the effects of ALDH1A1 on the Notch pathway, and thus on the cell cycle, were studied.A549s cells were successfully isolated and identified.ALDH1A1expression was significantly higher in A549s than in A549 cells. Clonogenicity was significantly decreased in A549s cells treated with ALDH1A1 siRNA. Duration of the G1 stage of the cell cycle increased after ALDH1A1 was overexpressed, or decreased with ALDH1A1 siRNA. ALDH1A1, Notch1, -2, and -3, CDK2, and CCNE1 expression levels were higher in A549s cells than in A549 cells. Expression of Notch1, -2, and -3, CDK2, and CCNE1 was significantly decreased by upregulation of ALDH1A1 in A549 cells, but increased by its interruption in A549s cells. When Notch3 or CDK2 expression was downregulated, the expression levels of ALDH1A1, Notch1, -2, and -3, CDK2, and CCNE1 were reduced in all cell types.ALDH1A1 expression improved clonogenicity and inhibited the cell cycle, maintaining the stemness of the A549s cells; this may involve suppression of the Notch/CDK2/Cyclin pathway.

  6. Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

    Science.gov (United States)

    Kaldis, Philipp; Aleem, Eiman

    2005-11-01

    It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings. PMID:16258277

  7. Rising cyclin-CDK levels order cell cycle events.

    Directory of Open Access Journals (Sweden)

    Catherine Oikonomou

    Full Text Available BACKGROUND: Diverse mitotic events can be triggered in the correct order and time by a single cyclin-CDK. A single regulator could confer order and timing on multiple events if later events require higher cyclin-CDK than earlier events, so that gradually rising cyclin-CDK levels can sequentially trigger responsive events: the "quantitative model" of ordering. METHODOLOGY/PRINCIPAL FINDINGS: This 'quantitative model' makes predictions for the effect of locking cyclin at fixed levels for a protracted period: at low cyclin levels, early events should occur rapidly, while late events should be slow, defective, or highly variable (depending on threshold mechanism. We titrated the budding yeast mitotic cyclin Clb2 within its endogenous expression range to a stable, fixed level and measured time to occurrence of three mitotic events: growth depolarization, spindle formation, and spindle elongation, as a function of fixed Clb2 level. These events require increasingly more Clb2 according to their normal order of occurrence. Events occur efficiently and with low variability at fixed Clb2 levels similar to those observed when the events normally occur. A second prediction of the model is that increasing the rate of cyclin accumulation should globally advance timing of all events. Moderate (<2-fold overexpression of Clb2 accelerates all events of mitosis, resulting in consistently rapid sequential cell cycles. However, this moderate overexpression also causes a significant frequency of premature mitoses leading to inviability, suggesting that Clb2 expression level is optimized to balance the fitness costs of variability and catastrophe. CONCLUSIONS/SIGNIFICANCE: We conclude that mitotic events are regulated by discrete cyclin-CDK thresholds. These thresholds are sequentially triggered as cyclin increases, yielding reliable order and timing. In many biological processes a graded input must be translated into discrete outputs. In such systems, expression of

  8. Cyclin-dependent kinase CDK1/CDC28 and checkpoints

    International Nuclear Information System (INIS)

    The genetic instability induced by defects in the cell cycle progression contributes to different human diseases, particularly neoplastic transformation. The control mechanisms of correct cell cycle progression are the most studied in the yeast Saccharomyces cerevisiae, in which checkpoint was first discovered. Many components of these processes have been identified by now. Here, the role of the central kinase of cell cycle CDK1/CDC28 is considered in checkpoint in different phases.

  9. Quantitative Proteomics Reveals That the Inhibition of Na(+)/K(+)-ATPase Activity Affects S-Phase Progression Leading to a Chromosome Segregation Disorder by Attenuating the Aurora A Function in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Xu, Zhongwei; Wang, Fengmei; Fan, Fengxu; Gu, Yanjun; Shan, Nana; Meng, Xiangyan; Cheng, Shixiang; Liu, Yingfu; Wang, Chengyan; Song, Yueying; Xu, Ruicheng

    2015-11-01

    Many studies have shown the Na(+)/K(+)-ATPase (NKA) might be a potential target for anticancer therapy. Cardiac glycosides (CGs), as a family of naturally compounds, inhibited the NKA activity. The present study investigates the antitumor effect of ouabain and elucidates the pharmacological mechanisms of CG activity in liver cancer HepG2 cell using SILAC coupled to LC-MS/MS method. Bioinformatics analysis of 330 proteins that were changed in cells under treatment with 0.5 μmol/L ouabain showed that the biological processes are associated with an acute inflammatory response, cell cycle, oxidation reduction, chromosome segregation, and DNA metabolism. We confirmed that ouabain induced chromosome segregation disorder and S-cell cycle block by decreasing the expression of AURKA, SMC2, Cyclin D, and p-CDK1 as well as increasing the expression of p53. We found that the overexpression or inhibition of AURKA significantly reduced or enhanced the ouabain-mediated the anticancer effects. Our findings suggest that AURKA is involved in the anticancer mechanisms of ouabain in HepG2 cells. PMID:26491887

  10. Structures of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control

    Energy Technology Data Exchange (ETDEWEB)

    Burke, Jason R.; Hura, Greg L.; Rubin, Seth M. (UCSC); (LBNL)

    2012-07-18

    Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible 'pocket' domain loop such that it mimics and directly blocks E2F transactivation domain (E2F{sup TD}) binding. T373 phosphorylation induces a global conformational change that associates the pocket and N-terminal domains (RbN). This first multidomain Rb structure demonstrates a novel role for RbN in allosterically inhibiting the E2F{sup TD}-pocket association and protein binding to the pocket 'LxCxE' site. Together, these structures detail the regulatory mechanism for a canonical growth-repressive complex and provide a novel example of how multisite Cdk phosphorylation induces diverse structural changes to influence cell cycle signaling.

  11. Germ Line Transmission of the Cdk4R24C Mutation Facilitates Tumorigenesis and Escape from Cellular Senescence

    OpenAIRE

    Rane, Sushil G; Cosenza, Stephen C.; Mettus, Richard V.; Reddy, E. Premkumar

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16INK4a have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4a. To determine the role of the Cdk4R24C germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by usi...

  12. Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

    OpenAIRE

    Honey, Sangeet; Futcher, Bruce

    2007-01-01

    The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of the...

  13. Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    HE Kan; CHEN Yue; LI Jing-hua; ZHAN Zhuo; WU Yong-ge; KONG Wei; JIN Ying-hua

    2007-01-01

    Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli. To determine a specific substrate of Cyclin A-Cdk2 for apoptosis, in this study, we carried out anin vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source, and recombinant protein GST-Bad as a substrate. Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro. To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo, we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn, a dominant negative version of Cdk2, in Hela cells and determined the phosphorylation status of protein Bad. The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent. Moreover, etoposide also caused the phosphorylation of endogenetic Bad. In conclusion, here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

  14. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    Science.gov (United States)

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. PMID:27554859

  15. Arsenic-induced promoter hypomethylation and over-expression of ERCC2 reduces DNA repair capacity in humans by non-disjunction of the ERCC2-Cdk7 complex.

    Science.gov (United States)

    Paul, Somnath; Banerjee, Nilanjana; Chatterjee, Aditi; Sau, Tanmoy J; Das, Jayanta K; Mishra, Prafulla K; Chakrabarti, Partha; Bandyopadhyay, Arun; Giri, Ashok K

    2014-04-01

    Arsenic in drinking water is of critical concern in West Bengal, India, as it results in several physiological symptoms including dermatological lesions and cancers. Impairment of the DNA repair mechanism has been associated with arsenic-induced genetic damage as well as with several cancers. ERCC2 (Excision Repair Cross-Complementing rodent repair, complementation group 2), mediates DNA-repair by interacting with Cdk-activating kinase (CAK) complex, which helps in DNA proof-reading during transcription. Arsenic metabolism alters epigenetic regulation; we tried to elucidate the regulation of ERCC2 in arsenic-exposed humans. Water, urine, nails, hair and blood samples from one hundred and fifty seven exposed and eighty eight unexposed individuals were collected. Dose dependent validation was done in vitro using HepG2 and HEK-293. Arsenic content in the biological samples was higher in the exposed individuals compared with the content in unexposed individuals (p < 0.001). Bisulfite-modified methylation specific PCR showed a significant (p < 0.0001) hypomethylation of the ERCC2 promoter in the arsenic-exposed individuals. Densitometric analysis of immunoblots showed a nearly two-fold increase in expression of ERCC2 in exposed individuals, but there was an enhanced genotoxic insult as measured by micronuclei frequency. Immuno-precipitation and western blotting revealed an increased (p < 0.001) association of Cdk7 with ERCC2 in highly arsenic exposed individuals. The decrease in CAK activity was determined by observing the intensity of Ser(392) phosphorylation in p53, in vitro, which decreased with an increase in arsenic dose. Thus we infer that arsenic biotransformation leads to promoter hypomethylation of ERCC2, which in turn inhibits the normal functioning of the CAK-complex, thus affecting DNA-repair; this effect was highest among the arsenic exposed individuals with dermatological lesions. PMID:24473091

  16. Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling.

    Science.gov (United States)

    Jarvis, Gavin E; Barbosa, Roseli; Thompson, Andrew J

    2016-03-01

    Citral, eucalyptol, and linalool are widely used as flavorings, fragrances, and cosmetics. Here, we examined their effects on electrophysiological and binding properties of human 5-HT3 receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. Data were analyzed using nonlinear mixed-effects modeling to account for random variance in the peak current response between oocytes. The oils caused an insurmountable inhibition of 5-HT-evoked currents (citral IC50 = 120 µM; eucalyptol = 258 µM; linalool = 141 µM) and did not compete with fluorescently labeled granisetron, suggesting a noncompetitive mechanism of action. Inhibition was not use-dependent but required a 30-second preapplication. Compound washout caused a slow (∼180 seconds) but complete recovery. Coapplication of the oils with bilobalide or diltiazem indicated they did not bind at the same locations as these channel blockers. Homology modeling and ligand docking predicted binding to a transmembrane cavity at the interface of adjacent subunits. Liquid chromatography coupled to mass spectrometry showed that an essential oil extracted from Lippia alba contained 75.9% citral. This inhibited expressed 5-HT3 receptors (IC50 = 45 µg ml(-1)) and smooth muscle contractions in rat trachea (IC50 = 200 µg ml(-1)) and guinea pig ileum (IC50 = 20 µg ml(-1)), providing a possible mechanistic explanation for why this oil has been used to treat gastrointestinal and respiratory ailments. These results demonstrate that citral, eucalyptol, and linalool inhibit 5-HT3 receptors, and their binding to a conserved cavity suggests a valuable target for novel allosteric modulators. PMID:26669427

  17. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis

    OpenAIRE

    Yu, Tao; Tao, Yonghui; Yang, Meiqiang; Chen, Peng; Gao, XiaoBo; Zhang, Yanbo; Zhang,Tao; Chen, Zi; Hou, Jian; Zhang, Yan; Ruan, Kangcheng; Wang, Hongyan; Hu, Ronggui

    2014-01-01

    Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be a...

  18. A chrysin derivative suppresses skin cancer growth by inhibiting cyclin-dependent kinases.

    Science.gov (United States)

    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

  19. Free Energy Analysis of CDK2-Inhibitor Interaction%CDK2-抑制剂结合自由能计算

    Institute of Scientific and Technical Information of China (English)

    蒋勇军; 曾敏; 周先波; 邹建卫; 俞庆森

    2004-01-01

    细胞周期蛋白依赖性激酶Ⅱ(cyclin-dependent kinase 2,CDK2)是一种重要的治疗癌症的靶标.本文中采用分子动力学取样,运用MM-PBSA/GBSA两种方法计算了CDK2-NU6102复合物的绝对结合自由能.通过能量分解的方法考察了CDK2大分子主要残基与配体NU6102之间的相互作用和识别.

  20. AC1MMYR2 impairs high dose paclitaxel-induced tumor metastasis by targeting miR-21/CDK5 axis.

    Science.gov (United States)

    Ren, Yu; Zhou, Xuan; Yang, Juan-Juan; Liu, Xia; Zhao, Xiao-hui; Wang, Qi-xue; Han, Lei; Song, Xin; Zhu, Zhi-yan; Tian, Wei-ping; Zhang, Lun; Mei, Mei; Kang, Chun-sheng

    2015-07-01

    Paclitaxel (taxol) is a widely used chemo-drug for many solid tumors, while continual taxol treatment is revealed to stimulate tumor dissemination. We previously found that a small molecule inhibitor of miR-21, termed AC1MMYR2, had the potential to impair tumorigenesis and metastasis. The aim of this study was to investigate whether combining AC1MMYR2 with taxol could be explored as a means to limit tumor metastasis. Here we showed that abnormal activation of miR-21/CDK5 axis was associated with breast cancer lymph node metastasis, which was also contribute to high dose taxol-induced invasion and epithelial mesenchymal transition (EMT) in both breast cancer cell line MDA-MB-231 and glioblastoma cell line U87VIII. AC1MMYR2 attenuated CDK5 activity by functional targeting CDK5RAP1, CDK5 activator p39 and target p-FAK(ser732). A series of in vitro assays indicated that treatment of AC1MMYR2 combined with taxol suppressed tumor migration and invasion ability in both MDA-MB-231 and U87VIII cell. More importantly, combination therapy impaired high-dose taxol induced invadopodia, and EMT markers including β-catenin, E-cadherin and vimentin. Strikingly, a significant reduction of lung metastasis in mice was observed in the AC1MMYR2 plus taxol treatment. Taken together, our work demonstrated that AC1MMYR2 appeared to be a promising strategy in combating taxol induced cancer metastasis by targeting miR-21/CDK5 axis, which highlighted the potential for development of therapeutic modalities for better clinic taxol application. PMID:25827073

  1. Influence of esculentoside A on proliferation and CDK2 of IL-1βinduced glomerular mesangial cell *%商陆皂苷甲对IL-1β诱导的肾小球系膜细胞增殖及CDK2、P27的影响

    Institute of Scientific and Technical Information of China (English)

    张祥贵; 汤杰印

    2013-01-01

    Objective To observe the effect of esculemoside A (EsA)on the proliferation and expression of CDK2 and P27 of rats glomerular mesangial cell .Methods rGMC was cultured in vitro ,The cell growth and cell toxicity were detected by MTT assay ;rGMC proliferation was observed by Rnase/PI-Flous ;The expression of CDK2 and P27 were measured by Western blotting .Results EsA at observed dose has not apparent cytotoxicity effect on rGMC .EsA(2 .5-5 .0 mg/L) inhibited the proliferation of rGMC after 48h .EsA increased the number of the G1 phase and reduced the number of the S phase of IL-1βinduced rGMC .At the same time ,EsA inhibited the expression of CDK2 and promoted the expression of P27 of IL-1βinduced rGMC .Conclusion The GMC is one of the mainly target cell which EsA bing therapeu tical action .EsA inhibited proliferation of IL-1βinduced the GMC ,inhibited the expression of CDK2 and activated the expression of P27 may be its mechanism .%目的观察商陆皂苷甲(EsA )对白细胞介素(IL )-1β诱导的大鼠肾小球系膜细胞(rGM C )增殖和细胞周期依赖性蛋白激酶(CDK2)及其抑制蛋白(P27)表达的影响。方法噻唑蓝(MTT)法检测EsA对 rGMC增殖与毒性的影响;碘化丙啶(PI)染色法,流式细胞仪检测细胞周期;蛋白免疫印迹法(Western blotting )检测CDK2、P27的表达。结果观察剂量中EsA对rG-MC没有细胞毒作用,EsA(2.5~5.0 mg/L)作用rGMC 48 h后明显抑制其增殖;IL-1β减少了rGMC的G1期细胞数并增加了S期的细胞数,促进了rGMC的CDK2的表达,抑制了P27的表达;EsA增加了IL-1β诱导的rGMC的G1期的细胞数并减少了S期的细胞数,抑制了IL-1β诱导的rGMC的CDK2的表达,并促进了P27的表达。结论 rGMC可能是EsA的作用靶细胞,EsA通过抑制CDK2及激活P27的表达抑制了IL-1β诱导的rGMC的增殖,阻滞了细胞周期的进程。

  2. Proteomic profiling reveals that resveratrol inhibits HSP27 expression and sensitizes breast cancer cells to doxorubicin therapy.

    Directory of Open Access Journals (Sweden)

    José Díaz-Chávez

    Full Text Available The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4',5-trans-trihydroxystilbilene is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05 in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS as heat shock protein 27 (HSP27, translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5'-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27

  3. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    Directory of Open Access Journals (Sweden)

    Anna Lundin

    2014-05-01

    Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  4. Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

    Directory of Open Access Journals (Sweden)

    Gabriele Möller

    Full Text Available Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1 for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

  5. A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression

    OpenAIRE

    Zeineldin, Maged; Cunningham, Jamie; McGuinness, William; Alltizer, Preston; Cowley, Brett; Blanchat, Bryan; Xu, Wenhao; Pinson, David; Neufeld, Kristi L.

    2011-01-01

    Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt signaling pathway by targeting proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription co-factor for genes required for cell proliferation such as cyclin D1 and c-Myc. In cultured cells, APC shutt...

  6. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Raghavan, Pavithra; Tumati, Vasu; Yu Lan [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Chan, Norman [Departments of Medical Biophysics and Radiation Oncology, Princess Margaret Hospital, University Health Network, University of Toronto, Ontario (Canada); Tomimatsu, Nozomi [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Burma, Sandeep [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Simmons Comprehensive Cancer Center, Dallas, Texas (United States); Bristow, Robert G. [Departments of Medical Biophysics and Radiation Oncology, Princess Margaret Hospital, University Health Network, University of Toronto, Ontario (Canada); Saha, Debabrata, E-mail: debabrata.saha@utsouthwestern.edu [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Simmons Comprehensive Cancer Center, Dallas, Texas (United States)

    2012-11-15

    Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G{sub 2}-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

  7. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

    International Nuclear Information System (INIS)

    Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G2-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

  8. Wogonin induced G1 cell cycle arrest by regulating Wnt/β-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells

    International Nuclear Information System (INIS)

    Highlights: • Wogonin inhibited HCT116 cells growth and arrested at G1 phase of the cell cycle. • Wogonin down-regulated the canonical Wnt/β-catenin signaling pathway. • Wogonin interfered in the combination of β-catenin and TCF/Lef. • Wogonin limited the kinase activity of CDK8. - Abstract: Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/β-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted β-catenin for proteasomal degradation. Wogonin inhibited β-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/β-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer

  9. Simulation of Different Truncated p16INK4a Forms and In Silico Study of Interaction with Cdk4

    Directory of Open Access Journals (Sweden)

    Najmeh Fahham

    2009-01-01

    Full Text Available Protein-protein interactions studies can greatly increase the amount of structural and functional information pertaining to biologically active molecules and processes. The information obtained from such studies can lead to design and application of new modification in order to obtain a desired bioactivity. Many application packages and servers performing docking, such as HEX, DOT, AUTODOCK, and ZDOCK are now available for predicting the lowest free energy state of a protein complex. In this study, we have focused on cyclin-dependent kinase 4 (Cdk4, a key molecule in the regulation of cell cycle progression at the G1-S phase restriction point and p16INK4a, a tumor suppressor which inhibits Cdk4 activity. Truncated structures were created to find the more critical regions of p16 for interaction. The tertiary structures were determined by ProSAL, GENO3D Web Server. We evaluated their interactions with Cdk4 using two docking systems, HEX 4.5 and DOT 1. Calculations were performed on a high-speed computer. Minimizations and visualizations were carried out by PdbViewer 3.7. Considering shape and shape/electrostatic total energy, structures containing ANK II, III and IV motifs that lack the N-terminal region of the full length p16 molecule showed the best fi t complexes among the p16 truncated forms. The free energies were compatible with that of p16 full length original form, the full length. It seems that the N-terminal of the molecule is not crucial for the interaction since the truncated structure containing only this region did not show a good total energy.

  10. Novel Insights Into The Mode of Inhibition of Class A SHV-1 Beta-Lactamases Revealed by Boronic Acid Transition State Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    W Ke; J Sampson; C Ori; F Prati; S Drawz; C Bethel; R Bonomo; F van den Akker

    2011-12-31

    Boronic acid transition state inhibitors (BATSIs) are potent class A and C {beta}-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 {beta}-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 {beta}-lactamase with micromolar affinity that is considerably weaker than their inhibition of other {beta}-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other {beta}-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

  11. CDK5RAP2 function during Zebrafish neurogenesis

    OpenAIRE

    Martins, Tiago Filipe Mendes

    2014-01-01

    Microcefalia de origem primária é uma doença caracterizada por afectar o desenvolvimento cerebral. Cdk5rap2, Aspm e Wdr62 são algumas das proteínas centrossomáis que têm sido descritas como sendo associadas a microcefalias. As proteínas associadas aos centrossomas são evidenciadas como reguladoras da divisão celular e tem sido sugerido que a saída prematura do ciclo celular e a interferência com o tipo de divisão de células progenitoras pode causar microcefalias. O objetivo des...

  12. Versatile templates for the development of novel kinase inhibitors: Discovery of novel CDK inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Dwyer, Michael P.; Paruch, Kamil; Alvarez, Carmen; Doll, Ronald J.; Keertikar, Kerry; Duca, Jose; Fischmann, Thierry O.; Hruza, Alan; Madison, Vincent; Lees, Emma; Parry, David; Seghezzi, Wolfgang; Sgambellone, Nicole; Shanahan, Frances; Wiswell, Derek; Guzi, Timothy J. (SPRI)

    2008-06-30

    A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors.

  13. Upregulation of CDK7 in gastric cancer cell promotes tumor cell proliferation and predicts poor prognosis.

    Science.gov (United States)

    Wang, Qiuhong; Li, Manhua; Zhang, Xunlei; Huang, Hua; Huang, Jianfei; Ke, Jing; Ding, Haifang; Xiao, Jinzhang; Shan, Xiaohang; Liu, Qingqing; Bao, Bojun; Yang, Lei

    2016-06-01

    CDK7 has been known as a component of CDK activating kinase (CAK) complex, the complex was composed of CDK7, Cyclin H and RING finger protein Mat1 that contribute to cell cycle progression by phosphorylating other CDKs. In addition, the complex is also an essential component of general transcription factor TFIIH which controls transcription via activating RNA polymerase II by serines 5 and 7 phosphorylation of the carboxyl-terminal domain (CTD) of its largest subunit. However, the role of CDK7 in the pathogenesis of gastric cancer has not been identified. Our study showed that CDK7 was significantly upregulated and positively correlated with tumor grade, infiltration depth, lymph node, Ki-67, and predicted poor prognosis in 173 gastric cancer specimens by immunohistochemistrical analyses. Furthermore, in vitro results indicated that CDK7 promoted proliferation of gastric cancer cells by CCK8, clone formation analyses and flow cytometric analyses, while CDK7 knockdown led to decreased cell proliferation. Our study will provide a theoretical basis for the study of CDK7 in gastric cancer. PMID:27155449

  14. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    International Nuclear Information System (INIS)

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  15. Inhibition of CDC25B Phosphatase Through Disruption of Protein-Protein Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Lund, George; Dudkin, Sergii; Borkin, Dmitry; Ni, Wendi; Grembecka, Jolanta; Cierpicki, Tomasz [Michigan

    2015-04-29

    CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

  16. 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.

    Science.gov (United States)

    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

  17. CDK2在非小细胞肺癌组织中的表达%Expression of CDK2 in Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    林炜明; 罗茂春; 陈彤; 尹会方

    2014-01-01

    探讨CDK2在非小细胞肺癌组织中的表达与肺癌转移关系.将50例非小细胞肺癌组织分为转移组和非转移组,采用免疫组织化学和Western blot检测癌组织中CDK2蛋白的表达.结果表明:CDK2蛋白在肺癌细胞中主要位于细胞核.CDK2蛋白在肺癌组织中的表达水平显著高于癌旁组织(P<0.05).CDK2蛋白高水平表达与肺癌淋巴结转移呈正相关(P<0.05),但与肿瘤类型无关(P>0.05).CDK2的过表达可能与肺癌的形成有关,并与淋巴转移有关.

  18. Expression of miR-124 inhibits growth of medulloblastoma cells

    OpenAIRE

    Silber, Joachim; Hashizume, Rintaro; Felix, Tristan; Hariono, Sujatmi; Yu, Mamie; Berger, Mitchel S.; Huse, Jason T.; VandenBerg, Scott R.; James, C. David; Hodgson, J Graeme; Gupta, Nalin

    2012-01-01

    Medulloblastoma is the most common malignant brain tumor in children, and a substantial number of patients die as a result of tumor progression. Overexpression of CDK6 is present in approximately one-third of medulloblastomas and is an independent poor prognostic marker for this disease. MicroRNA (miR)-124 inhibits expression of CDK6 and prevents proliferation of glioblastoma and medulloblastoma cells in vitro. We examined the effects of miR-124 overexpression on medulloblastoma cells both in...

  19. Inhibition of HMG CoA reductase reveals an unexpected role for cholesterol during PGC migration in the mouse

    Directory of Open Access Journals (Sweden)

    Ewing Andrew G

    2008-12-01

    Full Text Available Abstract Background Primordial germ cells (PGCs are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. Results We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. Conclusion In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.

  20. CRISPR-Mediated Drug-Target Validation Reveals Selective Pharmacological Inhibition of the RNA Helicase, eIF4A

    Directory of Open Access Journals (Sweden)

    Jennifer Chu

    2016-06-01

    Full Text Available Targeting translation initiation is an emerging anti-neoplastic strategy that capitalizes on de-regulated upstream MAPK and PI3K-mTOR signaling pathways in cancers. A key regulator of translation that controls ribosome recruitment flux is eukaryotic initiation factor (eIF 4F, a hetero-trimeric complex composed of the cap binding protein eIF4E, the scaffolding protein eIF4G, and the RNA helicase eIF4A. Small molecule inhibitors targeting eIF4F display promising anti-neoplastic activity in preclinical settings. Among these are some rocaglate family members that are well tolerated in vivo, deplete eIF4F of its eIF4A helicase subunit, have shown activity as single agents in several xenograft models, and can reverse acquired resistance to MAPK and PI3K-mTOR targeted therapies. Herein, we highlight the power of using genetic complementation approaches and CRISPR/Cas9-mediated editing for drug-target validation ex vivo and in vivo, linking the anti-tumor properties of rocaglates to eIF4A inhibition.

  1. Fluorine Substituted 1,2,4-Triazinones as Potential Anti-HIV-1 and CDK2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Mohammed S. I. Makki

    2014-01-01

    Full Text Available Fluorine substituted 1,2,4-triazinones have been synthesized via alkylation, amination, and/or oxidation of 6-(2-amino-5-fluorophenyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H-one 1 and 4-fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-ylphenylbenzamide 5 as possible anti-HIV-1 and CDK2 inhibitors. Alkylation on positions 2 and 4 in 1,2,4-triazinone gave compounds 6–8. Further modification was performed by selective alkylation and amination on position 3 to form compounds 9–15. However oxidation of 5 yielded compounds 16–18. Structures of the target compounds have been established by spectral analysis data. Five compounds (5, 11, 14, 16, and 17 have shown very good anti-HIV activity in MT-4 cells. Similarly, five compounds (1, 3, and 14–16 have exhibited very significant CDK2 inhibition activity. Compounds 14 and 16 were found to have dual anti-HIV and anticancer activities.

  2. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production

    Directory of Open Access Journals (Sweden)

    Emanuel Joseph Paul Nazareth

    2016-05-01

    Full Text Available Human pluripotent stem cells (hPSCs exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4 and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives.

  3. A Multi-Lineage Screen Reveals mTORC1 Inhibition Enhances Human Pluripotent Stem Cell Mesendoderm and Blood Progenitor Production.

    Science.gov (United States)

    Nazareth, Emanuel Joseph Paul; Rahman, Nafees; Yin, Ting; Zandstra, Peter William

    2016-05-10

    Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations, convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors, measuring yield and purity outputs of undifferentiated, neuroectoderm, mesendoderm, and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically, small interfering RNA knockdown of RAPTOR, a component of mTOR complex 1, phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold, with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives. PMID:27132889

  4. A closer look at cognitive control: Differences in resource allocation during updating, inhibition and switching as revealed by pupillometry

    Directory of Open Access Journals (Sweden)

    Eefje Rondeel

    2015-09-01

    Full Text Available The present study investigated resource allocation, as measured by pupil dilation, in tasks measuring updating (2-Back task, inhibition (Stroop task and switching (Number Switch task. Because each cognitive control component has unique characteristics, differences in patterns of resource allocation were expected. Pupil and behavioral data from 35 participants were analysed. In the 2-Back task (requiring correct matching of current stimulus identity at trial p with the stimulus two trials back, p-2 we found that better performance (low total of errors made in the task was positively correlated to the mean pupil dilation during correctly responding to targets. In the Stroop task, pupil dilation on incongruent trials was higher than those on congruent trials. Incongruent versus congruent trial pupil dilation differences were positively related to reaction time differences between incongruent and congruent trials. Furthermore, on congruent Stroop trials, pupil dilation was negatively related to reaction times, presumably because more effort allocation paid off in terms of faster responses. In addition, pupil dilation on correctly-responded-to congruent trials predicted a weaker Stroop interference effect in terms of errors, probably because pupil dilation on congruent trials were diagnostic of task motivation, resulting in better performance. In the Number Switch task we found higher pupil dilation in switch as compared to non-switch trials. On the Number Switch task, pupil dilation was not related to performance. We also explored error-related pupil dilation in all tasks. The results provide new insights in the diversity of the cognitive control components in terms of resource allocation as a function of individual differences, task difficulty and error processing.

  5. A closer look at cognitive control: differences in resource allocation during updating, inhibition and switching as revealed by pupillometry.

    Science.gov (United States)

    Rondeel, Eefje W M; van Steenbergen, Henk; Holland, Rob W; van Knippenberg, Ad

    2015-01-01

    The present study investigated resource allocation, as measured by pupil dilation, in tasks measuring updating (2-Back task), inhibition (Stroop task) and switching (Number Switch task). Because each cognitive control component has unique characteristics, differences in patterns of resource allocation were expected. Pupil and behavioral data from 35 participants were analyzed. In the 2-Back task (requiring correct matching of current stimulus identity at trial p with the stimulus two trials back, p -2) we found that better performance (low total of errors made in the task) was positively correlated to the mean pupil dilation during correctly responding to targets. In the Stroop task, pupil dilation on incongruent trials was higher than those on congruent trials. Incongruent vs. congruent trial pupil dilation differences were positively related to reaction time differences between incongruent and congruent trials. Furthermore, on congruent Stroop trials, pupil dilation was negatively related to reaction times, presumably because more effort allocation paid off in terms of faster responses. In addition, pupil dilation on correctly-responded-to congruent trials predicted a weaker Stroop interference effect in terms of errors, probably because pupil dilation on congruent trials were diagnostic of task motivation, resulting in better performance. In the Number Switch task we found higher pupil dilation in switch as compared to non-switch trials. On the Number Switch task, pupil dilation was not related to performance. We also explored error-related pupil dilation in all tasks. The results provide new insights in the diversity of the cognitive control components in terms of resource allocation as a function of individual differences, task difficulty and error processing. PMID:26441594

  6. Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition.

    Directory of Open Access Journals (Sweden)

    Christopher J Vavricka

    2011-10-01

    Full Text Available The 2009 H1N1 influenza pandemic (pH1N1 led to record sales of neuraminidase (NA inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958 and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1 is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity. Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1, p09N1 (atypical group 1 and N2 from the 1957 pandemic H2N2 (p57N2 (typical group 2 to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of

  7. FLT3 and CDK4/6 inhibitors: signaling mechanisms and tumor burden in subcutaneous and orthotopic mouse models of acute myeloid leukemia.

    Science.gov (United States)

    Zhang, Yaping; Hsu, Cheng-Pang; Lu, Jian-Feng; Kuchimanchi, Mita; Sun, Yu-Nien; Ma, Ji; Xu, Guifen; Zhang, Yilong; Xu, Yang; Weidner, Margaret; Huard, Justin; D'Argenio, David Z

    2014-12-01

    FLT3(ITD) subtype acute myeloid leukemia (AML) has a poor prognosis with currently available therapies. A number of small molecule inhibitors of FLT3 and/or CDK4/6 are currently under development. A more complete and quantitative understanding of the mechanisms of action of FLT3 and CDK4/6 inhibitors may better inform the development of current and future compounds that act on one or both of the molecular targets, and thus may lead to improved treatments for AML. In this study, we investigated in both subcutaneous and orthotopic AML mouse models, the mechanisms of action of three FLT3 and/or CDK4/6 inhibitors: AMG925 (Amgen), sorafenib (Bayer and Onyx), and quizartinib (Ambit Biosciences). A composite model was developed to integrate the plasma pharmacokinetics of these three compounds on their respective molecular targets, the coupling between the target pathways, as well as the resulting effects on tumor burden reduction in the subcutaneous xenograft model. A sequential modeling approach was used, wherein model structures and estimated parameters from upstream processes (e.g. PK, cellular signaling) were fixed for modeling subsequent downstream processes (cellular signaling, tumor burden). Pooled data analysis was employed for the plasma PK and cellular signaling modeling, while population modeling was applied to the tumor burden modeling. The resulting model allows the decomposition of the relative contributions of FLT3(ITD) and CDK4/6 inhibition on downstream signaling and tumor burden. In addition, the action of AMG925 on cellular signaling and tumor burden was further studied in an orthotopic tumor mouse model more closely representing the physiologically relevant environment for AML. PMID:25326874

  8. Cloning and Functional Analysis of Porcine Cdk2 Gene%猪Cdk2基因的克隆及其功能研究

    Institute of Scientific and Technical Information of China (English)

    唐青海; 张辉; 危艳武; 刘长明

    2013-01-01

    本研究旨在克隆猪Cdk2基因,并研究其编码蛋白CDK2的生物学功能.采用RT-PCR扩增猪Cdk2基因,运用生物信息学软件分析其核苷酸和编码氨基酸特征,并预测编码蛋白的生物学功能;利用半定量RT-PCR方法分析该基因在猪各个脏器和组织中的表达情况;共聚焦显微镜观察CDK2的亚细胞定位,采用过表达和shRNA干扰技术研究CDK2在细胞周期和细胞增殖中的调控作用.结果表明,猪Cdk2基因开放阅读框(ORF)为897 bp(GenBank:JX967576),该基因与绵羊、牛、山羊、人、金仓鼠、小鼠、仓鼠和沟鼠Cdk2的核苷酸相似性依次为94.2%、94.0%、93.8%、93.4%、91.8%、91.0%、90.6%和89.9%,与牛、山羊和绵羊的亲缘关系最近;Cdk2编码298 aa,CDK2分子质量为34 ku.Cdk2 mRNA在猪10个不同脏器和组织中均有表达.CDK2定位于细胞质和细胞核中,并通过蛋白酶体途径降解.猪CDK2在PK-15细胞中过表达引起S期细胞比例显著减少及G2/M期细胞比例显著增加(P<0.05),而G0/G1期无显著变化;相反,CDK2表达量降低引起S期细胞比例显著减少及G0/G1期细胞比例显著增加,而G2/M期无显著变化.本研究成功克隆了猪Cdk2基因并对其编码蛋白生物学功能进行了初步研究.

  9. CDK2在鼻咽癌中的表达及意义%The Expression and Significance of CDK-2 in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    官树雄; 蒋月荷; 王继群; 山艳春

    2011-01-01

    目的 探讨细胞周期蛋白依赖性激酶2(CDK2)在鼻咽癌和慢性鼻咽炎黏膜组织中的表达水平,及其与鼻咽癌的临床病理关系.方法 免疫组化SP法检测CDK2蛋白在鼻咽癌和慢性鼻咽炎黏膜组织中的表达情况.结果 CDK2蛋白阳性表达主要定位于细胞核,少数有胞浆着色,呈棕黄色或棕褐色不同强度的染色.62例鼻咽癌组织中阳性表达率为69.4%(43/62),慢性鼻咽炎黏膜组织中阳性率为32.0%(9/28),两组间差异有统计学意义(P<0.05).CDK2蛋白表达与鼻咽癌分化程度,淋巴结转移范围,TNM分期有关(P<0.05).结论 CDK2在鼻咽癌组织中的表达明显高于慢性鼻咽炎黏膜组织中的表达,提示CDK2与鼻咽癌的发生、发展有关.CDK2与鼻咽癌分化程度,淋巴结转移范围,TNM分期有关.%Objective The propose of the research was focused on the expression levels of CDK2 in the tissues of nasopharyngeal carcinoma and chronic inflammation nasopharyngeal membrane. And it may relate to the pathogenesis and clinical significance of nasopharyngeal carcinoma which had been observed in the field of cell cycle. Methods Immunohistochemistry.(sp) was used to examine the expression levels of CDK2 protein in the tissues of nasopharyngeal carcinoma and chronic inflammation nasopharyngeal membrane. Results Most of the CDK2 protein positive expression was found in cell nucleus,but some was found in the kytoplasm and its color was brown yellow or dark brown. The positive expression rate of nasopharyngeal carcinoma from 62 patients was 69.40% (43/62), and that of the chronic inflammation nasopharyngeal membrane was 32.0% (9/28). The difference of the two groups was found statistical significance( P < 0.05 ) ;the expression levels of CDK2 were related to the differential degree, the range of lymph node metastases and clinical staging of nasopharyngeal carcinoma, the difference was been found statistical significance( P < 0.05 ). Conclusion The expression

  10. A novel role for peptidylarginine deiminases in microvesicle release reveals therapeutic potential of PAD inhibition in sensitizing prostate cancer cells to chemotherapy

    Directory of Open Access Journals (Sweden)

    Sharad Kholia

    2015-06-01

    Full Text Available Introduction: Protein deimination, defined as the post-translational conversion of protein-bound arginine to citrulline, is carried out by a family of 5 calcium-dependent enzymes, the peptidylarginine deiminases (PADs and has been linked to various cancers. Cellular microvesicle (MV release, which is involved in cancer progression, and deimination have not been associated before. We hypothesize that elevated PAD expression, observed in cancers, causes increased MV release in cancer cells and contributes to cancer progression. Background: We have previously reported that inhibition of MV release sensitizes cancer cells to chemotherapeutic drugs. PAD2 and PAD4, the isozymes expressed in patients with malignant tumours, can be inhibited with the pan-PAD-inhibitor chloramidine (Cl-am. We sought to investigate whether Cl-am can inhibit MV release and whether this pathway could be utilized to further increase the sensitivity of cancer cells to drug-directed treatment. Methods: Prostate cancer cells (PC3 were induced to release high levels of MVs upon BzATP stimulation of P2X7 receptors. Western blotting with the pan-protein deimination antibody F95 was used to detect a range of deiminated proteins in cells stimulated to microvesiculate. Changes in deiminated proteins during microvesiculation were revealed by immunoprecipitation and immunoblotting, and mass spectrometry identified deiminated target proteins with putative roles in microvesiculation. Conclusion: We report for the first time a novel function of PADs in the biogenesis of MVs in cancer cells. Our results reveal that during the stimulation of prostate cancer cells (PC3 to microvesiculate, PAD2 and PAD4 expression levels and the deimination of cytoskeletal actin are increased. Pharmacological inhibition of PAD enzyme activity using Cl-am significantly reduced MV release and abrogated the deimination of cytoskeletal actin. We demonstrated that combined Cl-am and methotrexate (MTX treatment of

  11. The Effect of cdk- 5 Overexpression and Overactivation on Tau Hyperphosphorylation in Cultured N2a Cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Juan; LI Hong-lian; FENG You-mei; WANG Jian-zhi

    2005-01-01

    Neurofibrillary tangles (NFTs) are one of the neuropathological hallmarks of Alzheimer' s disease (AD) and abnormally hyperphosphorylated tau is the major protein of NFTs. It was reported that cyclin-dependent kinase5 (Cdk-5) could phosphorylate tau at most AD-related epitopes in vivo. In this study, we investigated the effect of cdk-5 overexpression on tau hyperphosphorylation in neuroblastoma N2a cells. We demonstrated that overexpression of cdk-5 which resulted in a 3.5-fold Cdk5 activation in the transfected cells induced a dramatic increase in phosphorylation of tau at several phosphorylation sites. Overexpression of cdk-5 led to a reduced staining with antibody Tau-1 and an enhanced staining with antibody PHF-1, suggesting hy perphosphorylation of tau at Ser199/202 and Ser396/404 sites. It implies that in vitro overexpression of cdk-5 leads to Cdk5 overactivation and tau hyperphosphorylation may be the underline mechanism.

  12. Honokiol, a phytochemical from the Magnolia plant, inhibits photocarcinogenesis by targeting UVB-induced inflammatory mediators and cell cycle regulators: development of topical formulation.

    Science.gov (United States)

    Vaid, Mudit; Sharma, Som D; Katiyar, Santosh K

    2010-11-01

    To develop newer and more effective chemopreventive agents for skin cancer, we assessed the effect of honokiol, a phytochemical from the Magnolia plant, on ultraviolet (UV) radiation-induced skin tumorigenesis using the SKH-1 hairless mouse model. Topical treatment of mice with honokiol in a hydrophilic cream-based topical formulation before or after UVB (180 mJ/cm(2)) irradiation resulted in a significant protection against photocarcinogenesis in terms of tumor multiplicity (28-60%, P skin samples from the tumor-bearing mice were analyzed for inflammatory mediators, cell cycle regulators and survival signals using immunostaining, western blotting and enzyme-linked immunosorbent assay. Treatment with honokiol significantly inhibited UVB-induced expression of cyclooxygenase-2, prostaglandin E(2) (P skin as well as in skin tumors. Western blot analysis revealed that honokiol: (i) inhibited the levels of cyclins D1, D2 and E and associated cyclin-dependent kinases (CDKs)2, CDK4 and CDK6, (ii) upregulated Cip/p21 and Kip/p27 and (iii) inhibited the levels of phosphatidylinositol 3-kinase and the phosphorylation of Akt at Ser(473) in UVB-induced skin tumors. Together, our results indicate that honokiol holds promise for the prevention of UVB-induced skin cancer by targeting inflammatory mediators, cell cycle regulators and cell survival signals in UVB-exposed skin. PMID:20823108

  13. Mutations in CDK5RAP2 cause Seckel syndrome

    OpenAIRE

    Karabey Kayserili, Hülya; Yiğit, G.; Brown, KE.; Pohl, E.; Caliebe, A.; Zahnleiter, D.; Rosser, E.; Bögershausen, N.; Uyguner, ZO.; Altunoğlu, U.; Nürnberg, G.; Nürnberg, P.; Rauch, A.; Li, Y.; Thiel, CT.; Wollnik, B.

    2015-01-01

    ORIGINAL ARTICLE Mutations in CDK5RAP2 cause Seckel syndrome Go¨ khan Yigit1,2,3,a, Karen E. Brown4,a, Hu¨ lya Kayserili5, Esther Pohl1,2,3, Almuth Caliebe6, Diana Zahnleiter7, Elisabeth Rosser8, Nina Bo¨ gershausen1,2,3, Zehra Oya Uyguner5, Umut Altunoglu5, Gudrun Nu¨ rnberg2,3,9, Peter Nu¨ rnberg2,3,9, Anita Rauch10, Yun Li1,2,3, Christian Thomas Thiel7 & Bernd Wollnik1,2,3 1Institute of Human Genetics, University of Cologne, Cologne, Germany 2Center for Molecular Medic...

  14. Maintenance of leukemia-initiating cells is regulated by the CDK inhibitor Inca1.

    Directory of Open Access Journals (Sweden)

    Nicole Bäumer

    Full Text Available Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1-/- bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1-/- in MLL-AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.

  15. Effects of CDK2 on DNA ploidy in laryngeal squamous cell carcinoma%喉鳞癌中CDK2表达对DNA倍体的作用

    Institute of Scientific and Technical Information of China (English)

    刘荣; 皇甫辉

    2008-01-01

    目的 研究喉鳞癌组织中CDK2表达在引起DNA异倍体发生过程中的作用. 方法 取手术中获得的50例喉鳞癌组织和30例声带息肉组织,用γ-微管蛋白抗体标记中心体,用免疫组织化学的方法检测CDK2激酶和γ-微管蛋白的表达;用流式细胞术检测喉鳞癌组织DNA倍体. 结果 在喉鳞癌组织中CDK2激酶和γ-微管蛋白阳性率表达[分别为68.0%(34/50)和78.0%(39/50)]都显著高于在声带息肉组织中(P<0.05)[分别为20.0%(6/30)和33.3%(10/30)],而且CDK2激酶的表达与γ-微管蛋白的表达具有相关性.21例CDK2表达阳性的喉鳞癌组织其DI为1.76±0.36;9例CDK2表达阴性的喉鳞癌组织其DI为1.05±0.07,CDK2阳性的喉鳞癌组织较阴性表达的组织DI增高(P<0.05). 结论 喉鳞癌中CDK2过度表达导致肿瘤细胞DNA异倍体发生.在诊断和治疗喉鳞癌中,CDK2可能是一个有重要作用的指标.

  16. Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44.

    Science.gov (United States)

    Yi, Jun-Mei; Huan, Xia-Juan; Song, Shan-Shan; Zhou, Hu; Wang, Ying-Qing; Miao, Ze-Hong

    2016-07-01

    Multidrug resistance (MDR) is a major cause of tumor treatment failure; therefore, drugs that can avoid this outcome are urgently needed. We studied triptolide, which directly kills MDR tumor cells with a high potency and a broad spectrum of cell death. Triptolide did not inhibit P-glycoprotein (P-gp) drug efflux and reduced P-gp and MDR1 mRNA resulting from transcription inhibition. Transcription factors including c-MYC, SOX-2, OCT-4, and NANOG were not correlated with triptolide-induced cell killing, but RPB1, the largest subunit of RNA polymerase II, was critical in mediating triptolide's inhibition of MDR cells. Triptolide elicited antitumor and anti-MDR activity through a universal mechanism: by activating CDK7 by phosphorylating Thr170 in both parental and MDR cell lines and in SK-OV-3 cells. The CDK7-selective inhibitor BS-181 partially rescued cell killing induced by 72-hour treatment of triptolide, which may be due to partial rescue of RPB1 degradation. We suggest that a precise phosphorylation site on RPB1 (Ser1878) was phosphorylated by CDK7 in response to triptolide. In addition, XPB and p44, two transcription factor TFIIH subunits, did not contribute to triptolide-driven RPB1 degradation and cell killing, although XPB was reported to covalently bind to triptolide. Several clinical trials are underway to test triptolide and its analogues for treating cancer and other diseases, so our data may help expand potential clinical uses of triptolide, as well as offer a compound that overcomes tumor MDR. Future investigations into the primary molecular target(s) of triptolide responsible for RPB1 degradation may suggest novel anti-MDR target(s) for therapeutic development. Mol Cancer Ther; 15(7); 1495-503. ©2016 AACR. PMID:27197304

  17. Cdk5-mediated phosphorylation of RapGEF2 controls neuronal migration in the developing cerebral cortex.

    Science.gov (United States)

    Ye, Tao; Ip, Jacque P K; Fu, Amy K Y; Ip, Nancy Y

    2014-01-01

    During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar-bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition. In utero electroporation and live-imaging studies reveal that RapGEF2 acts on the multipolar-bipolar transition during neuronal migration via a Rap1/N-cadherin pathway. Importantly, activation of RapGEF2 is controlled via phosphorylation by a serine/threonine kinase Cdk5, whose activity is largely restricted to the radial migration zone. Thus, the specific expression and Cdk5-dependent phosphorylation of RapGEF2 during multipolar-bipolar transition within the intermediate zone are essential for proper neuronal migration and wiring of the cerebral cortex. PMID:25189171

  18. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Petersen, B O; Lukas, J; Sørensen, Claus Storgaard;

    1999-01-01

    CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo...... relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we...... suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2....

  19. An opposite effect of the CDK inhibitor, p18(INK4c on embryonic stem cells compared with tumor and adult stem cells.

    Directory of Open Access Journals (Sweden)

    Yanxin Li

    Full Text Available Self-renewal is a feature common to both adult and embryonic stem (ES cells, as well as tumor stem cells (TSCs. The cyclin-dependent kinase inhibitor, p18(INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB; on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1 were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.

  20. Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

    Directory of Open Access Journals (Sweden)

    Yan Li

    2015-04-01

    Full Text Available Cyclin-dependent kinase 2 (CDK2 is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP binding site (Site I and two non-competitive binding sites (Site II and III. In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV. All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate. In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.

  1. CDK4 Amplification Predicts Recurrence of Well-Differentiated Liposarcoma of the Abdomen

    OpenAIRE

    Lee, Sanghoon; Park, Hyojun; Ha, Sang Yun; Paik, Kwang Yeol; Lee, Seung Eun; Kim, Jong Man; Park, Jae Berm; Kwon, Choon Hyuck David; Joh, Jae-Won; Choi, Yoon-La; Kim, Sung Joo

    2014-01-01

    Background The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas. Methods From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantit...

  2. PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

    Science.gov (United States)

    Hanafusa, Hiroshi; Kedashiro, Shin; Tezuka, Motohiro; Funatsu, Motoki; Usami, Satoshi; Toyoshima, Fumiko; Matsumoto, Kunihiro

    2015-08-01

    Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation. PMID:26192437

  3. Sumoylation of p35 modulates p35/cyclin-dependent kinase (Cdk) 5 complex activity.

    Science.gov (United States)

    Büchner, Anja; Krumova, Petranka; Ganesan, Sundar; Bähr, Mathias; Eckermann, Katrin; Weishaupt, Jochen H

    2015-03-01

    Cyclin-dependent kinase (Cdk) 5 is critical for central nervous system development and neuron-specific functions including neurite outgrowth as well as synaptic function and plasticity. Cdk5 activity requires association with one of the two regulatory subunits, called p35 and p39. p35 redistribution as well as misregulation of Cdk5 activity is followed by cell death in several models of neurodegeneration. Posttranslational protein modification by small ubiquitin-related modifier (SUMO) proteins (sumoylation) has emerged as key regulator of protein targeting and protein/protein interaction. Under cell-free in vitro conditions, we found p35 covalently modified by SUMO1. Using both biochemical and FRET-/FLIM-based approaches, we demonstrated that SUMO2 is robustly conjugated to p35 in cells and identified the two major SUMO acceptor lysines in p35, K246 and K290. Furthermore, different degrees of oxidative stress resulted in differential p35 sumoylation, linking oxidative stress that is encountered in neurodegenerative diseases to the altered activity of Cdk5. Functionally, sumoylation of p35 increased the activity of the p35/Cdk5 complex. We thus identified a novel neuronal SUMO target and show that sumoylation is a likely candidate mechanism for the rapid modulation of p35/Cdk5 activity in physiological situations as well as in disease. PMID:25391294

  4. Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.

    Science.gov (United States)

    Shirali, Saeid; Aghaei, Mahmoud; Shabani, Mahdi; Fathi, Mojtaba; Sohrabi, Majid; Moeinifard, Marzieh

    2013-04-01

    Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer. PMID:23345014

  5. The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks

    Directory of Open Access Journals (Sweden)

    Sang Hoon Ha

    2016-02-01

    Full Text Available Previous work has shown that Suc1/Cks proteins can promote the hyperphosphorylation of primed Cdk1 substrates through the formation of ternary Cdk1-Cks-phosphosubstrate complexes. This raises the possibility that Cks proteins might be able to both facilitate and interfere with hyperphosphorylation through a mechanism analogous to the prozone effect in antigen-antibody interactions, with substoichiometric Cks promoting the formation of Cdk1-Cks-phosphosubstrate complexes and suprastoichiometric Cks instead promoting the formation of Cdk1-Cks and Cks-phosphosubstrate complexes. We tested this hypothesis through a combination of theory, proof-of-principle experiments with oligonucleotide annealing, and experiments on the interaction of Xenopus cyclin B1-Cdk1-Cks2 with Wee1A in vitro and in Xenopus extracts. Our findings help explain why both Cks under-expression and overexpression interfere with cell-cycle progression and provide insight into the regulation of the Cdk1 system.

  6. Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

    Science.gov (United States)

    Perumal, Deepak; Kuo, Pei-Yu; Leshchenko, Violetta V; Jiang, Zewei; Divakar, Sai Krishna Athaluri; Cho, Hearn Jay; Chari, Ajai; Brody, Joshua; Reddy, M V Ramana; Zhang, Weijia; Reddy, E Premkumar; Jagannath, Sundar; Parekh, Samir

    2016-03-01

    Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma. PMID:26873845

  7. The RNA binding motif protein 15B (RBM15B/OTT3) is a functional competitor of serine-arginine (SR) proteins and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing.

    Science.gov (United States)

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J; Lahti, Jill M

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  8. The RNA Binding Motif Protein 15B (RBM15B/OTT3) Is a Functional Competitor of Serine-Arginine (SR) Proteins and Antagonizes the Positive Effect of the CDK11p110-Cyclin L2α Complex on Splicing*

    Science.gov (United States)

    Loyer, Pascal; Busson, Adeline; Trembley, Janeen H.; Hyle, Judith; Grenet, Jose; Zhao, Wei; Ribault, Catherine; Montier, Tristan; Kidd, Vincent J.; Lahti, Jill M.

    2011-01-01

    Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11p110 binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11p110, cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of ∼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11p110, cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11p110-cyclin L2α complex on splicing both in vitro and in vivo. PMID:21044963

  9. The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

    Science.gov (United States)

    Frontini, Mattia; Kukalev, Alexander; Leo, Elisabetta; Ng, Yiu-Ming; Cervantes, Marcella; Cheng, Chi-Wai; Holic, Roman; Dormann, Dirk; Tse, Eric; Pommier, Yves; Yu, Veronica

    2012-01-01

    Summary CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCFSKP2 to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2−/− knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2−/− cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In vivo, Cks2−/− cortical progenitor cells are limited in their capacity to differentiate into mature neurons, a phenotype akin to animals lacking p27. We propose that the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation. PMID:22898779

  10. Charged Propargyl-Linked Antifolates Reveal Mechanisms of Antifolate Resistance and Inhibit Trimethoprim-Resistant MRSA Strains Possessing Clinically Relevant Mutations.

    Science.gov (United States)

    Reeve, Stephanie M; Scocchera, Eric; Ferreira, Jacob J; G-Dayanandan, Narendran; Keshipeddy, Santosh; Wright, Dennis L; Anderson, Amy C

    2016-07-14

    Drug-resistant enzymes must balance catalytic function with inhibitor destabilization to provide a fitness advantage. This sensitive balance, often involving very subtle structural changes, must be achieved through a selection process involving a minimal number of eligible point mutations. As part of a program to design propargyl-linked antifolates (PLAs) against trimethoprim-resistant dihydrofolate reductase (DHFR) from Staphylococcus aureus, we have conducted a thorough study of several clinically observed chromosomal mutations in the enzyme at the cellular, biochemical, and structural levels. Through this work, we have identified a promising lead series that displays significantly greater activity against these mutant enzymes and strains than TMP. The best inhibitors have enzyme inhibition and MIC values near or below that of trimethoprim against wild-type S. aureus. Moreover, these studies employ a series of crystal structures of several mutant enzymes bound to the same inhibitor; analysis of the structures reveals a more detailed molecular understanding of drug resistance in this important enzyme. PMID:27308944

  11. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    International Nuclear Information System (INIS)

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24−/CD44+) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer

  12. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Lee, Janet [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Lee, Chang-Woo [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Suwon, Gyeonggi 440-746 (Korea, Republic of); Yang, Kwangmo [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Department of Radiation Oncology, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Department of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, Seoul 139-709 (Korea, Republic of); Lee, Chang Geun, E-mail: cglee@dirams.re.kr [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of)

    2013-01-25

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

  13. Expression and signification of cell cycle regulation protein Cyclin D1-CDK4-p21 in scar cancer%细胞周期调控系统相关因子 Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    林宇静; 郭瑞珍; 王海青

    2014-01-01

    Objective Dysfunction of cell cycle regulation is one of the key factors for cellular carcinogenesis .This paper aimed to study the expression and significance of cell cycle regulation protein Cyclin D 1-CDK4-p21 in scar cancer . Methods The expressions of Cyclin D1, CDK4 and p21 protains were detected in scar cancer group , pathological scar group and normal skin group respectively by using immunohistochemical staining (SP).The mRNA expression levels of Cyclin D1, CDK4 and p21 were detected by the use of nucleic acid-mediated in-situ hybridization .Correlation analysis was made on the indexes , and the average optical density and positive area were analyzed using image analysis . Results The expressions of Cyclin D1, CDK4 and p21 protains and the mRNA ex-pression levels of cyclin D1, CDK4 and p21 were high in scar cancer group, low in pathological scar group , and negative in normal skin group.The mean optical density and positive area in scar cancer group were significantly different from pathological scar group and normal skin group (P0.05).In terms of correlation analysis , the expressions of Cyclin D 1 and CDK4 as well as p21 and CDK4 in scar cancer tissue were both in posi-tive correlations. Conclusion The occurrence of scar cancer is related to the abnormal expression of Cyclin D 1 and CDK4.The complex formed by Cyclin D1 and CDK4 may promote the G1/S transition, proliferation and tumorigenesis of scar cancer .In scar canc-er, the inhibition of Cyclin D1-CDK4 complex might be caused by other members of CKI family or even inbibitors of other families apart from CDK family.%目的:细胞周期调控机制失调是细胞增生肿瘤发生的重要因素。文中探讨细胞周期调控系统相关因子Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义。方法选取遵义医学院病理教研室和中山大学附属第五医院病理科2005-2011年石蜡包埋标本,分为瘢痕癌组、病理性瘢痕组和正常皮肤组。应

  14. Synthesis and Biological Evaluation of Scutellaria Flavone Cyclaneaminol Mannich Base Derivatives as Novel CDK1 Inhibitors.

    Science.gov (United States)

    Ha, Lisha; Qian, Yuan; Zhang, Shixuan; Ju, Xiulan; Sun, Shiyou; Guo, Hongmin; Wang, Qianru; Li, Kangjian; Fan, Qingyu; Zheng, Yang; Li, Hailiang

    2016-01-01

    Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in the cell proliferation process and a new target in the research and development of anti-cancer drugs. Natural flavones are selective CDK1 inhibitors which can suppress the proliferation of cancer cells. However, their bioavailability is poor. To solve these problems, 6 Scutellaria flavones were isolated from hydrolyzed products of Scutellaria baicalensis and used as lead compounds, 18 Scutellaria flavones cyclane-aminol Mannich base derivatives were semi-synthesized and their biological activity as novel CDK1 inhibitors was evaluated. Results indicated that the biological activity of 8-Hydroxypiperidinemethyl-baicalein (BA-j) is the highest among these compounds. BA-j is a selective CDK1 inhibitor, and has broad-spectrum anti-proliferative activity in human cancer cells (IC50 12.3μM). BA-j can capture oxygen free radicals (.O2(-)) and selectively increase intracellular H2O2 level in cancer cells and activated lymphocytes, thus inducing their apoptosis rather than in normal cells. These findings suggest that BA-j selectively induces apoptosis in cancer and activated lymphocyte by controlling intracellular H2O2 level, and can be developed into a novel anti-proliferative agent for the treatment of cancer, AIDS, and some immune diseases. PMID:26411959

  15. CDK5 knockdown prevents hippocampal degeneration and cognitive dysfunction produced by cerebral ischemia.

    Science.gov (United States)

    Gutiérrez-Vargas, Johana A; Múnera, Alejandro; Cardona-Gómez, Gloria P

    2015-12-01

    Acute ischemic stroke is a cerebrovascular accident and it is the most common cause of physical disabilities around the globe. Patients may present with repeated ictuses, experiencing mental consequences, such as depression and cognitive disorders. Cyclin-dependent kinase 5 (CDK5) is a kinase that is involved in neurotransmission and plasticity, but its dysregulation contributes to cognitive disorders and dementia. Gene therapy targeting CDK5 was administered to the right hippocampus of ischemic rats during transient cerebral middle artery occlusion. Physiologic parameters (blood pressure, pH, pO2, and pCO2) were measured. The CDK5 downregulation resulted in neurologic and motor improvement during the first week after ischemia. Cyclin-dependent kinase 5 RNA interference (RNAi) prevented dysfunctions in learning, memory, and reversal learning at 1 month after ischemia. These observations were supported by the prevention of neuronal loss, the reduction of microtubule-associated protein 2 (MAP2) immunoreactivity, and a decrease in astroglial and microglia hyperreactivities and tauopathy. Additionally, CDK5 silencing led to an increase in the expression of brain-derived neurotrophic factor (BDNF), its Tropomyosin Receptor kinase B (TRKB) receptor, and activation of cyclic AMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), which are important targets in neuronal plasticity. Together, our findings suggest that gene therapy based on CDK5 silencing prevents cerebral ischemia-induced neurodegeneration and motor and cognitive deficits. PMID:26104286

  16. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  17. Effect of berberine on Cdk9 and cyclin T1 expressions in myocardium of diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Zhou Jiyin; Zhou Shiwen; Tang Jianlin; Xu Ying; Ying Yi

    2008-01-01

    Objective: To investigate the effect of berberine, one of the main alkaloids of Rhizoma coptidis, on myocardial orphology and the expressions of cyclin-dependent kinase 9 (Cdk9) and cyclin T1 protein in the myocardium of type diabetic rats. Methods: Type 2 diabetes mellitus rats were induced by an injection of 35 mg/kg streptozotocin (STZ) nd a high-carbohydrate/high-fat diet for 16 weeks. Diabetic rats were given low-, middle-, high-dose berberine (75,150, 300 mg/kg), fenofibrate (100 mg/kg) and rosiglitazone (4 mg/kg) for another 16 weeks, respectively. The myocardium structure was observed with hematoxylin & eosin (H&E) staining and Cdk9 and cyclin T1 protein expressions were detected by immunohistochemistry. Results: Middle-dose, high-dose berberine improved myocardial hypertrophy and interstitial fibrosis of diabetic rats. Cdk9 and cyclin T1 protein were significantly lower in diabetic myocardium than in control one (P<0.01), and middle-dose, high-dose berberine and fenofibrate obviously increased oth Cdk9 and cyclin T1 expression to near control level (P<0.01). Conclusion: Berberine modulates Cdk9 and cyclin I protein expression in diabetic myocardium which may contribute to ameliorate myocardium damage.

  18. Flavonoids activate pregnane × receptor-mediated CYP3A4 gene expression by inhibiting cyclin-dependent kinases in HepG2 liver carcinoma cells

    Directory of Open Access Journals (Sweden)

    Wu Jing

    2010-06-01

    Full Text Available Abstract Background The expression of the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4 is regulated by the pregnane × receptor (PXR, which is modulated by numerous signaling pathways, including the cyclin-dependent kinase (Cdk pathway. Flavonoids, commonly consumed by humans as dietary constituents, have been shown to modulate various signaling pathways (e.g., inhibiting Cdks. Flavonoids have also been shown to induce CYPs expression, but the underlying mechanism of action is unknown. Here, we report the mechanism responsible for flavonoid-mediated PXR activation and CYP expression. Results In a cell-based screen designed to identify compounds that activate PXR-mediated CYP3A4 gene expression in HepG2 human carcinoma cells, we identified several flavonoids, such as luteolin and apigenin, as PXR activators. The flavonoids did not directly bind to PXR, suggesting that an alternative mechanism may be responsible for flavonoid-mediated PXR activation. Consistent with the Cdk5-inhibitory effect of flavonoids, Cdk5 and p35 (a non-cyclin regulatory subunit required to activate Cdk5 were expressed in HepG2. The activation of Cdk5 attenuated PXR-mediated CYP3A4 expression whereas its downregulation enhanced it. The Cdk5-mediated downregulation of CYP3A4 promoter activity was restored by flavonoids, suggesting that flavonoids activate PXR by inactivating Cdk5. In vitro kinase assays showed that Cdk5 directly phosphorylates PXR. The Cdk kinase profiling assay showed that apigenin inhibits multiple Cdks, suggesting that several Cdks may be involved in activation of PXR by flavonoids. Conclusions Our results for the first time link the stimulatory effect of flavonoids on CYP expression to their inhibitory effect on Cdks, through a PXR-mediated mechanism. These results may have important implications on the pharmacokinetics of drugs co-administered with herbal remedy and herbal-drug interactions.

  19. CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Mariela C Marazita

    Full Text Available DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

  20. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    Science.gov (United States)

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage. PMID:18832014

  1. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

    Directory of Open Access Journals (Sweden)

    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  2. Revealing the Anti-Tumor Effect of Artificial miRNA p-27-5p on Human Breast Carcinoma Cell Line T-47D

    Directory of Open Access Journals (Sweden)

    Hsueh-Fen Juan

    2012-05-01

    Full Text Available microRNAs (miRNAs cause mRNA degradation or translation suppression of their target genes. Previous studies have found direct involvement of miRNAs in cancer initiation and progression. Artificial miRNAs, designed to target single or multiple genes of interest, provide a new therapeutic strategy for cancer. This study investigates the anti-tumor effect of a novel artificial miRNA, miR P-27-5p, on breast cancer. In this study, we reveal that miR P-27-5p downregulates the differential gene expressions associated with the protein modification process and regulation of cell cycle in T-47D cells. Introduction of this novel artificial miRNA, miR P-27-5p, into breast cell lines inhibits cell proliferation and induces the first “gap” phase (G1 cell cycle arrest in cancer cell lines but does not affect normal breast cells. We further show that miR P-27-5p targets the 3′-untranslated mRNA region (3′-UTR of cyclin-dependent kinase 4 (CDK4 and reduces both the mRNA and protein level of CDK4, which in turn, interferes with phosphorylation of the retinoblastoma protein (RB1. Overall, our data suggest that the effects of miR p-27-5p on cell proliferation and G1 cell cycle arrest are through the downregulation of CDK4 and the suppression of RB1 phosphorylation. This study opens avenues for future therapies targeting breast cancer.

  3. 哺乳动物细胞CDK2系列表达载体的构建与表达%Construction and expression of the CDK2 mammalian cell expression vectors

    Institute of Scientific and Technical Information of China (English)

    吴健谊; 蒋太峰; 谢剑君; 张锴; 杜则澎; 许丽艳

    2011-01-01

    目的:构建一系列细胞周期蛋白依赖激酶2(cyclin-dependent kinase 2,CDK2)在哺乳动物细胞的表达载体,为研究CDK2的功能和修饰提供实验材料.方法:从食管癌细胞中提取总RNA,逆转录PCR扩增CDK2编码区,然后将PCR产物克隆到T载体;扩增后的CDK2片段分别亚克隆入pcDNA3、pcDNA4、pNTAP和pEGFP等4种哺乳动物表达载体;最后,将获得的表达载体PEGFP/CDK2转染小鼠成纤维细胞NIH3T3进行初步的CDK2表达分析.结果:RT-PCR扩增获得约900 bp的目的片段,经T载体克隆和DNA序列分析,显示重组片段是人CDK2基因序列;CDK2片段分别亚克隆入上述4种载体后获得相应表达载体;运用构建的PEGFP/CDK2表达载体,在NIH3T3细胞中表达出CDK2蛋白.结论:成功构建了CDK2的哺乳动物细胞系列表达载体,并在NIH3T3细胞中成功表达目的蛋白.%OBJECTIVE: To construct a series of cyclin-dependent kinase 2 (CDK2) mammalian cell expression vectors and to assess CDK2 expression in NIH3T3 cells. METHODS: RNA was isolated from esophageal cancer cells and the full coding sequence of CDK2 gene was obtained by RT-PCR. The PCR product was then cloned into T vector and subsequently subcloned into four eukaryotic expression vectors (pcDNA3, pcDNA4, pNTAP and pEGFP). The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK2 was detected by western blot. RESULTS: The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequence of CDK2 gene. The product was subcloned into the eukaryotic expression vectors and four CDK2 expression vectors were constructed. Western blot showed that CDK2 could be expressed in the expression vector-transfected cells.CONCLUSION: Four CDK2 eukaryotic expression vectors were successfully constructed and CDK2 was effectively expressed in NIH3T3 cells.

  4. Inhibition of CDK4/6 as a novel therapeutic option for neuroblastoma

    OpenAIRE

    Rihani, Ali; Vandesompele, Jo; Speleman, Franki; VAN MAERKEN, TOM

    2015-01-01

    Background Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than 1 year of age. We hypothesized that aberrations in genes that control the cell cycle could play an important role in the pathogenesis of neuroblastoma and could provide a tractable therapeutic target. Methods In this study, we screened 131 genes involved in cell cycle regulation at different levels by analyzing the effect of siRNA-mediated gene silencing on the proliferation of neurobl...

  5. Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus.

    Directory of Open Access Journals (Sweden)

    Sebastian Jessberger

    2008-11-01

    Full Text Available Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5 activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.

  6. Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion

    OpenAIRE

    Ganuza, Miguel; Sáiz-Ladera, Cristina; Cañamero, Marta; Gómez, Gonzalo; Schneider, Ralph; Blasco, María A.; Pisano, David; Paramio, Jesús M.; Santamaría, David; Barbacid, Mariano

    2012-01-01

    Employing a conditionally inactive gene trap allele, Cdk7's function in regulating cellular proliferation by Cdk1/2-phosphorylation is convincingly dissected from alternative notions on CTD-phosphorylation of RNA Pol II. Premature aging phenotypes caused by stem cell depletion lend the necessary functional support.

  7. Aberrant cytological localization of p16 and CDK4 in colorectal epithelia in the normal adenoma carcinoma sequence

    Institute of Scientific and Technical Information of China (English)

    Po Zhao; Xin Mao; Ian C Talbot

    2006-01-01

    AIM: To study the correlation between the patterns of subcellular expression of p16 and CDK4 in colorectal epithelia in the normal-adenoma-carcinoma sequence.METHODS: Paraffin sections of 43 cases of normal colorectal epithelia and corresponding adenomas as well as carcinomas were analysed immunocytochemically for subcellular expression of p16 and CDK4 proteins.RESULTS: Most carcinomas showed more cytoplasmic overexpression for p16 and CDK4 than the adenomas from which they arised or the adjacent normal mucosa.Most normal or non-neoplastic epithelia showed more p16 and CDK4 expression in the nucleus than their adjacent adenomas and carcinomas. There was a significant difference between the subcellular expression pattern of p16 and CDK4 in normal-adenoma-carcinoma sequence epithelia (P < 0.001). Neither p16 nor CDK4 subcellular patterns correlated with histological grade or Dukes' stage.CONCLUSION: Interaction of expression of p16 and CDK4 plays an important role in the Rb/p16 pathway.Overexpression of p16 and CDK4 in the cytoplasm, as well as loss expression of p16 in the nucleus might be important in the evolution of colorectal carcinoma from adenoma and, of adenoma from normal epithelia.

  8. 非小细胞肺癌组织中CDK2及β-catenin的表达%Expression of CDK2 and3-Catenin in Non Small-Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    杨海燕; 李晓虹; 张映红

    2011-01-01

    Objective: To observe the expression of CDK2 and b-catenin in non small-cell lung cancer tissue and to investigate the relationship of CDK2 and b-catenin with metastatic lung cancer. Methods: 48 non small-cell lung cancer patient were divided into metachoresis and non metachoresis groups. Real-time FQ-PCR and western blot were applied respectively to detect the protein and mRNA expression of CDK2 and b-catenin in carcinoma tissue. Results: The protein and mRNA expression of CDK2 and b-catenin was obviously higher in metachoresis group than in non metachoresis group. Conclusion: The expression of CDK2 and b-catenin may be correlated with lung cancer metastasis.%目的:观察非小细胞肺癌组织中CDK2及β-catenin的表达,探讨CDK2及β-catenin与肺癌转移的关系.方法:48例非小细胞肺癌患者分为转移组和未转移组.手术取肺癌组织,分别采用实时荧光定量PCR法和western blot法检测脑组织中CDK2及β-catenin蛋白和mRNA的表达.结果:转移组肺癌组织中CDK2及β-catenin蛋白和mRNA的表达明显高于未转移组(P<0.01).结论:CDK2及β-catenin与肺癌转移有关.

  9. Role of CDK5/cyclin complexes in ischemia-induced death and survival of renal tubular cells

    Science.gov (United States)

    Guevara, Tatiana; Sancho, Mónica; Pérez-Payá, Enrique; Orzáez, Mar

    2014-01-01

    Ischemia reperfusion processes induce damage in renal tubules and compromise the viability of kidney transplants. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. CDK5 has been traditionally considered a neuronal kinase with dual roles in cell death and survival. Here, we demonstrate that CDK5 and their regulators p35/p25 and cyclin I are also expressed in renal tubular cells. We show that treatment with CDK inhibitors promotes the formation of pro-survival CDK5/cyclin I complexes and enhances cell survival upon an ischemia reperfusion pro-apoptotic insult. These findings support the benefit of treating with CDK inhibitors for renal preservation, assisting renal tubule protection. PMID:24675881

  10. CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    International Nuclear Information System (INIS)

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11p58 as a novel protein involved in the regulation of VDR. CDK11p58, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11p58 interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11p58 decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11p58 is involved in the negative regulation of VDR.

  11. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  12. Expression of cdk4 and p16 in Oral Lichen Planus

    OpenAIRE

    Sinny Goel; Nita Khurana; Akanksha Marvah; Sunita Gupta

    2015-01-01

    ABSTRACT Objectives The purpose of this study was to evaluate the expression of cdk4 and p16, the proteins implicated in hyperproliferation and arrest in oral lichen planus and to compare their expression in erosive and non-erosive oral lichen planus and with normal mucosa and oral squamous cell carcinoma. Material and Methods Analysis of cdk4 and p16 expression was done in 43 erosive oral lichen planus (EOLP) and 17 non-erosive oral lichen planus (NOLP) cases, 10 normal mucosa and 10 oral sq...

  13. Synthesis and CDK2 kinase inhibitory activity of 7/7′-azaindirubin derivatives

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A series of novel 7'-azaindirubin(1a-g) and 7-azaindirubin(2a,2c,2e and 2f) derivatives were designed and synthesized.Their structures were characterized by ~1H NMR and MS spectroscopy as well as by elemental analysis.Their inhibitory properties against CDK2/cylinA were evaluated in vitro.In contrast to indirubin,some of the described azaindirubins emerged as potent inhibitors of CDK2/cylinA and compound 2b had more potent activity.Biological tests also showed that nitrogen atom at 7-position of azaindir...

  14. In Silico Identification and In Vitro and In Vivo Validation of Anti-Psychotic Drug Fluspirilene as a Potential CDK2 Inhibitor and a Candidate Anti-Cancer Drug.

    Directory of Open Access Journals (Sweden)

    Xi-Nan Shi

    Full Text Available Hepatocellular carcinoma (HCC is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC's resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg. Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history

  15. Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies.

    Directory of Open Access Journals (Sweden)

    Nagasundaram N

    Full Text Available The cyclin-dependent kinase 4 (CDK4-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1 and protein-ligand (CDK4-flavopiridol interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment.

  16. PET32a-CDK2重组质粒的构建与表达%The construction and expression of recombinant plasmid PET32a-CDK2 in E. Coli

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 张战锋; 陈炜烨; 何敏; 庄俊华

    2011-01-01

    Objective To construct recomhinant plasmid PET32a - CDK2 in E. Coli, expressing human CDK2 protein. Methods Total RNA of human white blood cell was extracted for RT - PCR. CDK2 gene fragment was amplified by PCR, recombined into recombinant plasmid, and transformed into E. coli DH5α for cloning. Subsequently, recombinant plasmid was transformed into the competent cells BL21. The CDK2 proteins were induced with isopropy - β -D - thiogalactoside (IPTG) and detected with SDS - PAGE and Western - Blot. Results CDK2 gene recombinant plasmid PET32a was successfully constructed and expressed in E. coli according to DNA sequencing. IPTG - induced prokaryotic protein of 52 KD was detected in Western - blot. Conclusion Recombinant plasmid is constructed successfully.with expression of full - length protein of CDK2 in E. coli.%目的 构建含有细胞周期依赖性激酶2(CDK2)的PET32a-CDK2重组质粒,利用原核表达体系表达CDK2蛋白.方法 从人白细胞中提取总RNA, 采用聚合酶链反应从总RNA中扩增出CDK2基因,并将其插入PET32a质粒,构建重组质粒,化学法转化大肠杆菌DH5α进行克隆.将克隆得到的PET32a-CDK2重组质粒转化入表达菌株BL21,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导其蛋白表达,SDS-PAGE和Western-Blot鉴定蛋白表达情况.结果 菌落PCR及DNA测序证实CDK2基因已正确克隆到载体中;重组质粒成功转入表达菌株BL21(DE3),SDS-PAGE和Western-Blot结果显示表达菌经IPTG诱导后表达出52 kD左右的蛋白.结论 成功构建重组质粒,并且CDK2全长蛋白在原核表达菌BL21中成功表达.

  17. CCNG2和CDK2在结肠癌中的表达及意义%The Significance and the Expression of CCNG2 and CDK2 in colon cancer

    Institute of Scientific and Technical Information of China (English)

    王赛; 曾亚

    2015-01-01

    ObjectiveTo investigate the roles of Cyclin G2(CCNG2) and cycle protein dependent activating enzyme2(CDK2)in pathogenesis o f colon cancer and their relationships with tumor biological behavior.Methods Immunohis-tochemical methods were adopted to examine expressions of cyclin G2 and CDK2.Results In 89 cases CCNG2 in colonl cancer was significantly lower than that in normal colon tissues, while the expression of CDK2 was just in opposite. The expression of both CCNG2 and CDK2 were significantly related with tumor differention degree, lymph node metastasis and tumor TNM stage. The CCNG2 level was negatively related to the CDK2 level in human colon cancer tissues.Conclusion Abnormal expressions of CCNG2 and CDK2 play important roles in pathogenesis of colon cancer. Inverse correlation between CCNG2 and CDK2 ex-pression in human colon cancer tissues. The deficiency of CCNG2 could promote the invasion and metastasis of colon cancer by disinhibiting the expression of CDK2 protein.%目的:研究CCNG2及CDK2在结肠癌组织中的表达及其与结肠癌生物学行为的关系。方法:应用免疫组化法检测89例结肠癌组织中CCNG2及CDK2的表达。结果:89例结肠癌中,CCNG2蛋白阳性表达31例34.8%(31/89),CDK2蛋白阳性表达55例61.8%(55/89),两者与结肠癌的分化程度、淋巴结转移、临床分期分别呈负相关和正相关。且CCNG2与CDK2蛋白在结肠癌组织中的表达呈负相关性。结论:CCNG2的低表达可能导致CDK2表达的增强促进了结肠癌的发生发展。

  18. EIF3D silencing suppresses renal cell carcinoma tumorigenesis via inducing G2/M arrest through downregulation of Cyclin B1/CDK1 signaling.

    Science.gov (United States)

    Pan, Xiu-Wu; Chen, Lu; Hong, Yi; Xu, Dan-Feng; Liu, Xi; Li, Lin; Huang, Yi; Cui, Li-Ming; Gan, Si-Shun; Yang, Qi-Wei; Huang, Hai; Qu, Fa-Jun; Ye, Jian-Qing; Wang, Lin-Hui; Cui, Xin-Gang

    2016-06-01

    There are no effective therapies for advanced renal cell carcinoma (RCC), except for VEGFR inhibitors with only ~50% response rate. To identify novel targets and biomarkers for RCC is of great importance in treating RCC. In this study, we observed that eukaryotic initiation factor 3d (EIF3D) expression was significantly increased in RCC compared with paracarcinoma tissue using immunohistochemistry staining and western blot analysis. Furthermore, bioinformatics meta-analysis using ONCOMINE microarray datasets showed that EIF3D mRNA expressions in CCRCC tissue specimens were significantly higher than that in normal tissue specimens. In addition, RCC tissue microarray demonstrated that elevated EIF3D expression was positively correlated with TNM stage and tumor size. EIF3D silencing in human 786-O and ACHN CCRCC cell lines by RNA interference demonstrated that EIF3D knockdown obviously inhibited cell proliferation and colony formation, caused G2/M arrest through downregulation of Cyclin B1 and Cdk1 and upregulation of p21, and induced apoptosis shown by sub-G1 accumulation and RARP cleavage. Moreover, correlation analysis using ONCOMINE microarray datasets indicated that increased EIF3D mRNA expression was positively correlated to PCNA, Cyclin B1 and CDK1 mRNA expression in RCC. Collectively, these results provide reasonable evidences that EIF3D may function as a potential proto-oncogene that participates in the occurrence and progression of RCC. PMID:27035563

  19. Combined Cytological and Transcriptomic Analysis Reveals a Nitric Oxide Signaling Pathway Involved in Cold-Inhibited Camellia sinensis Pollen Tube Growth

    OpenAIRE

    Wang, Weidong; Sheng, Xianyong; Shu, Zaifa; Li, Dongqin; Pan, Junting; Ye, Xiaoli; Chang, Pinpin; Li, Xinghui; Wang, Yuhua

    2016-01-01

    Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca2+ gradient, increase in ROS content, acidification of cytoplas...

  20. Preliminary Analysis of CDK2 Sequence and Its Nuclear Import%CDK2蛋白质分子结构与其入核转运过程关系的初步分析

    Institute of Scientific and Technical Information of China (English)

    刘琦; 罗阳; 姜莉; 周伟强; 满晓辉; 张学

    2004-01-01

    应用重组技术构建野生型及缺失型CDK2基因的真核表达载体,分别使野生型及缺失型CDK2蛋白与增强型绿色荧光蛋白(EnhRnced-green Fluorescent Protein,EGFP)形成融合蛋白.通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO,经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达.结果表明,野生型CDK2基因的表达产物定位于细胞核,而两种缺失型CDK2基因分别编码的CDK2蛋白N-端1~201及98~298多肽均主要定位于细胞质.以上结果提示,CDK2蛋白序列中不含有与核定位直接相关的信号,其入核过程可能是由其N-端1~97及202~298多肽范围内的部分氨基酸共同形成高级结构,并依赖此高级结构与其他含有入核信号的蛋白形成复合物,从而被带动进入细胞核的.%We constructed the plasmids encoding enhanced green fluorescent protein (EGFP)-tagged wild type cyclin-de pendent kinase 2 (CDK2) (pEGFP-CDK2) and CDK2 deletion mutants ( pEGFP-CDK2N and pEGFP-CDK2C, lacking the last C-terminal and the first N-terminal 97 amino acids of CDK2 ,respectively) and transfected them into HeLa cell line and CHO cell line. After synchronization,green fluorescent signals were detected mainly in nucleus of the cells transfected with pEGFP-CDK2 and predominantly in cytoplasm of the cells transfected with the two mutant CDK2 constructs. Our results sug gested that there were no nuclear-import signals in CDK2 and that CDK2 nuclear import might be mediated by association with other proteins through the three-dimensional structure formed by amino acids including those from the N- and C-termi nal regions of CDK2.

  1. CDK2蛋白在胆管细胞癌中的表达及临床意义%Expression and Clinical Significance of CDK2 Protein in Cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    胡逸林; 张端莲

    2010-01-01

    目的 探讨细胞周期蛋白依赖性激酶2(cycle protein dependent activating enzyme 2, CDK2)在胆管细胞癌中的表达及临床意义.方法 收集武汉大学人民医院病理科2002/2008年胆管细胞癌存档蜡块40例,其中男性20例,女性20例.另取胆管细胞癌周围正常组织5例作对照.采用免疫组织化学方法检测各组中CDK2蛋白的表达,利用HPIAS-1000图像分析系统测定各组中CDK2蛋白表达的平均光密度和平均阳性面积率.结果 胆管细胞癌中CDK2蛋白呈高表达,对照组中CDK2蛋白呈低表达,两组CDK2表达的平均光密度及阳性面积率有显著性差异(P<0.05).结论 CDK2蛋白在胆管细胞癌的发生和发展过程中起了重要作用.

  2. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  3. NBM-T-BBX-OS01, Semisynthesized from Osthole, Induced G1 Growth Arrest through HDAC6 Inhibition in Lung Cancer Cells.

    Science.gov (United States)

    Pai, Jih-Tung; Hsu, Chia-Yun; Hua, Kuo-Tai; Yu, Sheng-Yung; Huang, Chung-Yang; Chen, Chia-Nan; Liao, Chiung-Ho; Weng, Meng-Shih

    2015-01-01

    Disrupting lung tumor growth via histone deacetylases (HDACs) inhibition is a strategy for cancer therapy or prevention. Targeting HDAC6 may disturb the maturation of heat shock protein 90 (Hsp90) mediated cell cycle regulation. In this study, we demonstrated the effects of semisynthesized NBM-T-BBX-OS01 (TBBX) from osthole on HDAC6-mediated growth arrest in lung cancer cells. The results exhibited that the anti-proliferative activity of TBBX in numerous lung cancer cells was more potent than suberoylanilide hydroxamic acid (SAHA), a clinically approved pan-HDAC inhibitor, and the growth inhibitory effect has been mediated through G1 growth arrest. Furthermore, the protein levels of cyclin D1, CDK2 and CDK4 were reduced while cyclin E and CDK inhibitor, p21Waf1/Cip1, were up-regulated in TBBX-treated H1299 cells. The results also displayed that TBBX inhibited HDAC6 activity via down-regulation HDAC6 protein expression. TBBX induced Hsp90 hyper-acetylation and led to the disruption of cyclin D1/Hsp90 and CDK4/Hsp90 association following the degradation of cyclin D1 and CDK4 proteins through proteasome. Ectopic expression of HDAC6 rescued TBBX-induced G1 arrest in H1299 cells. Conclusively, the data suggested that TBBX induced G1 growth arrest may mediate HDAC6-caused Hsp90 hyper-acetylation and consequently increased the degradation of cyclin D1 and CDK4. PMID:25946558

  4. Effects on CDK2 Gene Expression in H22 Cells with Ganoderma Appanatum Polysacharides GF%树舌多糖GF对小鼠H22瘤CDK2基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    于英君; 李雪松; 李和伟

    2010-01-01

    目的:探明树舌多糖GF对H22瘤细胞CDK2基因表达的影响.方法:运用Elisa法测定H22瘤细胞中CDK2蛋白的表达量.结果:树舌多糖组中CDK2蛋白的表达量均显著低于荷瘤对照组(P0.05).结论:树舌多糖GF可通过降低CDK2蛋白的表达,抑制H22瘤细胞的增殖.

  5. Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications.

    Science.gov (United States)

    Popova, Tatiana; Manié, Elodie; Boeva, Valentina; Battistella, Aude; Goundiam, Oumou; Smith, Nicholas K; Mueller, Christopher R; Raynal, Virginie; Mariani, Odette; Sastre-Garau, Xavier; Stern, Marc-Henri

    2016-04-01

    CDK12 is a recurrently mutated gene in serous ovarian carcinoma, whose downregulation is associated with impaired expression of DNA damage repair genes and subsequent hypersensitivity to DNA-damaging agents and PARP1/2 inhibitors. In this study, we investigated the genomic landscape associated with CDK12 inactivation in patients with serous ovarian carcinoma. We show that CDK12 loss was consistently associated with a particular genomic instability pattern characterized by hundreds of tandem duplications of up to 10 megabases (Mb) in size. Tandem duplications were characterized by a bimodal (∼0.3 and ∼3 Mb) size distribution and overlapping microhomology at the breakpoints. This genomic instability, denoted as the CDK12 TD-plus phenotype, is remarkably distinct from other alteration patterns described in breast and ovarian cancers. The CDK12 TD-plus phenotype was associated with a greater than 10% gain in genomic content and occurred at a 3% to 4% rate in The Cancer Genome Atlas-derived and in-house cohorts of patients with serous ovarian carcinoma. Moreover, CDK12-inactivating mutations together with the TD-plus phenotype were also observed in prostate cancers. Our finding provides new insight toward deciphering the function of CDK12 in genome maintenance and oncogenesis. Cancer Res; 76(7); 1882-91. ©2016 AACR. PMID:26787835

  6. Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b. Comparison with ligand binding to pCDK2-cyclin A complex.

    Science.gov (United States)

    Kosmopoulou, Magda N; Leonidas, Demetres D; Chrysina, Evangelia D; Bischler, Nicolas; Eisenbrand, Gerhard; Sakarellos, Constantinos E; Pauptit, Richard; Oikonomakos, Nikos G

    2004-06-01

    The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC(50) = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (K(i) = 13.8 +/- 0.2 micro m) and GPa (K(i) = 57.8 +/- 7.1 micro m) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 A resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566-34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb-E226, GPb-caffeine and GPb-flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb-E226 complex structure with the active pCDK2-cyclin A-E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme. PMID:15153119

  7. Impact of roscovitine, a selective CDK inhibitor, on cancer cells: bi-functionality increases its therapeutic potential.

    Science.gov (United States)

    Wesierska-Gadek, Józefa; Borza, Andreea; Komina, Oxana; Maurer, Margarita

    2009-01-01

    Increased expression and activity of proteins driving cell cycle progression as well as inactivation of endogenous inhibitors of cyclin-dependent kinases (CDKs) enhance the proliferative potential of cells. Escape of cells during malignant transformation from the proper cell cycle control rendering them independent from growth factors provides rationale for therapeutic targeting of CDKs. Exposure of rapidly growing human MCF-7 breast cancer and HeLa cervix cancer cells to roscovitine (ROSC), a selective inhibitor of CDKs, inhibits their proliferation by induction of cell cycle arrest and/or apoptosis. The outcome strongly depends on the intrinsic traits of the tumor cells, on their cell cycle status prior to the onset of treatment and also on ROSC concentration. At lower dose ROSC primarily inhibits the cell cycle-related CDKs resulting in a strong cell cycle arrest. Interestingly, ROSC arrests asynchronously growing cells at the G(2)/M transition irrespective of the status of their restriction checkpoint. However, the exposure of cancer cells synchronized after serum starvation in the late G(1) phase results in a transient G(1) arrest only in cells displaying the intact G(1)/S checkpoint. At higher dosage ROSC triggers apoptosis. In HeLa cells inhibition of the activity of CDK7 and, in consequence, that of RNA polymerase II is a major event that facilitates the initiation of caspase-dependent apoptosis. In contrast, in the caspase-3-deficient MCF-7 breast cancer cells ROSC induces apoptosis by a p53-dependent pathway. HIPK2-mediated activation of the p53 transcription factor by phosphorylation at Ser46 results in upregulation of p53AIP1 protein. This protein after de novo synthesis and translocation into the mitochondria promotes depolarization of the mitochondrial membrane. PMID:19724778

  8. Frequent amplification of CENPF, GMNN and CDK13 genes in hepatocellular carcinomas.

    Directory of Open Access Journals (Sweden)

    Hye-Eun Kim

    Full Text Available Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP chip to screen for gene copy number changes in primary hepatocellular carcinomas (HCCs, we initially detected amplification of 35 genes from four genomic regions (1q21-41, 6p21.2-24.1, 7p13 and 8q13-23. By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected CENPF (centromere protein F/mitosin, GMNN (geminin, DNA replication inhibitor, CDK13 (cyclin-dependent kinase 13, and FAM82B (family with sequence similarity 82, member B as common cancer genes. Each gene exhibited an amplification frequency of ~30% (range, 20-50% in primary HCC (n = 57 and colorectal cancer (n = 12, as well as in a panel of human cancer cell lines (n = 70. Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that CENPF, GMNN, and CDK13 were highly oncogenic whereas FAM82B was not. Interestingly, the oncogenic activity of these genes (excluding FAM82B was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423, indicating that amplifications of CENPF, GMNN, and CDK13 genes are tightly linked and coincident in tumors. Furthermore, we confirmed that CDK13 gene copy number was significantly associated with clinical onset age in patients with HCC (P = 0.0037. Taken together, our results suggest that coincidently amplified CDK13, GMNN, and CENPF genes can play a role as common cancer-driver genes in human cancers.

  9. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

    OpenAIRE

    Nabti, Ibtissem; Marangos, Petros; Bormann, Jenny; Kudo, Nobuaki R; Carroll, John

    2014-01-01

    Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates...

  10. Cdk-dependent phosphorylation regulates TRF1 recruitment to PML bodies and promotes C-circle production in ALT cells.

    Science.gov (United States)

    Wilson, Florence R; Ho, Angus; Walker, John R; Zhu, Xu-Dong

    2016-07-01

    TRF1, a duplex telomeric DNA binding protein, is implicated in homologous-recombination-based alternative lengthening of telomeres, known as ALT. However, how TRF1 promotes ALT activity has yet to be fully characterized. Here we report that Cdk-dependent TRF1 phosphorylation on T371 acts as a switch to create a pool of TRF1, referred to as (pT371)TRF1, which is recruited to ALT-associated PML bodies (APBs) in S and G2 phases independently of its binding to telomeric DNA. We find that phosphorylation of T371 is essential for APB formation and C-circle production, both of which are hallmarks of ALT. We show that the interaction of (pT371)TRF1 with APBs is dependent upon ATM and homologous-recombination-promoting factors Mre11 and BRCA1. In addition, (pT371)TRF1 interaction with APBs is sensitive to transcription inhibition, which also reduces DNA damage at telomeres. Furthermore, overexpression of RNaseH1 impairs (pT371)TRF1 recruitment to APBs in the presence of campothecin, an inhibitor that prevents topoisomerase I from resolving RNA-DNA hybrids. These results suggest that transcription-associated DNA damage, perhaps arising from processing RNA-DNA hybrids at telomeres, triggers (pT371)TRF1 recruitment to APBs to facilitate ALT activity. PMID:27185864

  11. Deletion of the MED13 and CDK8 subunits of the Mediator improves the phenotype of a long-lived respiratory deficient mutant of Podospora anserina.

    Science.gov (United States)

    Humbert, Adeline; Bovier, Elodie; Sellem, Carole H; Sainsard-Chanet, Annie

    2015-09-01

    In Podospora anserina, the loss of function of the cytochrome segment of the mitochondrial respiratory chain is viable. This is due to the presence in this organism, as in most filamentous fungi, of an alternative respiratory oxidase (AOX) that provides a bypass to the cytochrome pathway. However mutants lacking a functional cytochrome pathway present multiple phenotypes including poorly colored thin mycelium and slow growth. In a large genetic screen based on the improvement of these phenotypes, we isolated a large number of independent suppressor mutations. Most of them led to the constitutive overexpression of the aox gene. In this study, we characterize a new suppressor mutation that does not affect the production of AOX. It is a loss-of-function mutation in the gene encoding the MED13 subunit of the kinase module of the Mediator complex. Inactivation of the cdk8 gene encoding another subunit of the same module also results in partial suppression of a cytochrome-deficient mutant. Analysis of strains lacking the MED13 or CDK8 subunits points to the importance of these subunits as regulators involved in diverse physiological processes such as growth, longevity and sexual development. Interestingly, transcriptional analyses indicate that in P. anserina, loss of the respiratory cytochrome pathway results in the up-regulation of glycolysis-related genes revealing a new type of retrograde regulation. The loss of MED13 augments the up-regulation of some of these genes. PMID:26231682

  12. Study of Expression of CDK2 in Hamster Buckle Pouch Carcinogenesis%CDK2在金黄地鼠颊囊癌变过程中的表达的研究

    Institute of Scientific and Technical Information of China (English)

    孙淑芬; 高文信; 刘岩; 刘敏; 李晓丽; 刘树泰

    2004-01-01

    目的:探讨CDK2在金黄地鼠颊囊黏膜从正常黏膜到单纯增生、异常增生及鳞癌的表达变化.方法:采用DMBA诱导48只金黄地鼠颊囊癌变动物模型,SABC免疫组化法检测CDK2蛋白的表达.结果:CDK2在异常增生上皮及鳞癌的表达与正常和单纯增生组相比明显提高(P<0.05),阳性染色等级随病理等级改变提高(P<0.05).结论:CDK2参与了口腔黏膜癌前病变和鳞癌的发生与发展.

  13. Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development

    Science.gov (United States)

    Li, Shujun; Yang, Cuili; Xi, Yongyong; Wang, Liang; Zhang, Feng; Fu, Yunfeng; Li, Dejia

    2016-01-01

    Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1. PMID:26840018

  14. Virtual screening of and in vitro activity study on allosteric small-molecule CDK2 inhibitors%CDK2别构小分子抑制剂的虚拟筛选和体外活性研究

    Institute of Scientific and Technical Information of China (English)

    邵媛媛; 张璐; 沈瑛; 张健

    2016-01-01

    目的 根据CDK2的晶体结构(PDB ID:3PXF),在已验证的别构口袋处,拟筛选出CDK2新型别构小分子抑制剂.方法 通过计算机辅助药物设计方法,基于CDK2蛋白晶体别构位点进行虚拟筛选,综合分析化合物与CDK2的作用模式;构建CDK2体外激酶活性检测体系,对化合物进行初步的体外生物活性研究.结果 虚拟筛选得到打分前1 000名的化合物,最终挑选并购买10个候选化合物.其中,化合物S2和S5表现出较好的抑制效果,在100 μmol/L的浓度下对CDK2活性的抑制率分别为57.59%和41.64%.结论 综合利用虚拟筛选、结构分析以及生物活性测试,筛选出具有明显的CDK2抑制活性的先导化合物S2和S5,为设计开发新型的CDK2别构小分子抑制剂奠定了基础.

  15. Indole-3-carbinol inhibits nasopharyngeal carcinoma growth through cell cycle arrest in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Zhe Chen

    Full Text Available Nasopharyngeal carcinoma is a common malignant tumor in the head and neck. Because of frequent recurrence and distant metastasis which are the main causes of death, better treatment is needed. Indole-3-carbinol (I3C, a natural phytochemical found in the vegetables of the cruciferous family, shows anticancer effect through various signal pathways. I3C induces G1 arrest in NPC cell line with downregulation of cell cycle-related proteins, such as CDK4, CDK6, cyclin D1 and pRb. In vivo, nude mice receiving I3C protectively or therapeutically exhibited smaller tumors than control group after they were inoculated with nasopharyngeal carcinoma cells. The expression of CDK4, CDK6, cyclin D1 and pRb in preventive treatment group and drug treatment group both decreased compared with the control group. We conclude that I3C can inhibit the growth of NPC in vitro and in vivo by suppressing the expression of CDK and cyclin families. The drug was safe and had no toxic effects on normal tissues and organs.

  16. New developments on the cheminformatics open workflow environment CDK-Taverna

    Directory of Open Access Journals (Sweden)

    Truszkowski Andreas

    2011-12-01

    Full Text Available Abstract Background The computational processing and analysis of small molecules is at heart of cheminformatics and structural bioinformatics and their application in e.g. metabolomics or drug discovery. Pipelining or workflow tools allow for the Lego™-like, graphical assembly of I/O modules and algorithms into a complex workflow which can be easily deployed, modified and tested without the hassle of implementing it into a monolithic application. The CDK-Taverna project aims at building a free open-source cheminformatics pipelining solution through combination of different open-source projects such as Taverna, the Chemistry Development Kit (CDK or the Waikato Environment for Knowledge Analysis (WEKA. A first integrated version 1.0 of CDK-Taverna was recently released to the public. Results The CDK-Taverna project was migrated to the most up-to-date versions of its foundational software libraries with a complete re-engineering of its worker's architecture (version 2.0. 64-bit computing and multi-core usage by paralleled threads are now supported to allow for fast in-memory processing and analysis of large sets of molecules. Earlier deficiencies like workarounds for iterative data reading are removed. The combinatorial chemistry related reaction enumeration features are considerably enhanced. Additional functionality for calculating a natural product likeness score for small molecules is implemented to identify possible drug candidates. Finally the data analysis capabilities are extended with new workers that provide access to the open-source WEKA library for clustering and machine learning as well as training and test set partitioning. The new features are outlined with usage scenarios. Conclusions CDK-Taverna 2.0 as an open-source cheminformatics workflow solution matured to become a freely available and increasingly powerful tool for the biosciences. The combination of the new CDK-Taverna worker family with the already available workflows

  17. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.;

    2007-01-01

    Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV......) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may...

  18. Effects of CDK2 on cell cycle in laryngeal squamous cell carcinoma and its clinical significance%喉鳞癌中CDK2对细胞周期的影响及其意义

    Institute of Scientific and Technical Information of China (English)

    刘荣; 皇甫辉

    2008-01-01

    目的 探讨喉鳞癌组织CDK2激酶表达与肿瘤细胞增殖之间的关系. 方法 取手术中获得的50例喉鳞癌组织,12例非典型增生组织和30例声带息肉组织,用免疫组化的方法检测CDK2与PCNA的表达;用流式细胞术检测喉鳞癌组织细胞周期比率. 结果 在喉鳞癌组织中CDK2与PCNA的阳性率表达分别为68.0%和86.0%,显著高于声带息肉组织(P<0.05);并且CDK2的表达与临床分期、病理分级、淋巴转移密切相关,与患者的年龄、性别和原发部位无关. 结论 喉鳞癌中CDK2过度表达可能与肿瘤细胞增殖异常密切相关.在诊断和治疗喉鳞癌中,CDK2可能是一个有重要作用的指标.

  19. pPICZαA-CDK2重组质粒的构建及表达

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 张战锋; 谢诗园; 李朝霞; 李林; 陈炜烨; 何敏; 庄俊华

    2011-01-01

    目的:构建含有细胞周期依赖性激酶2(CDK2)的胞外分泌型pPICZαA-CDK2重组质粒,利用毕赤酵母表达体系表达CDK2蛋白.方法:从人白细胞中提取总RNA,逆转录后采用聚合酶链反应扩增出CDK2基因,并将其插入pPICZαA质粒,构建重组质粒,化学法转化大肠杆菌JM109进行克隆.重组质粒pPICZαA-CDK2转化毕赤酵母菌株GS115,甲醇诱导酵母细胞进行蛋白表达,SDS-PAGE和Western-Blot鉴定蛋白表达情况及抗原性.结果:PCR电泳及DNA测序证实CDK2基因已正确克隆到表达载体中;重组质粒转入酵母菌GS115,酵母经甲醇诱导表达后经SDS-PAGE检测发现在34 000左右有条带,Western-Blot检测发现有与CDK2单抗结合蛋白.结论:成功构建重组质粒,初步判断CDK2全长蛋白在毕赤酵母中表达成功且抗原性良好.

  20. Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis

    OpenAIRE

    Voit, Renate; Seiler, Jeanette; Grummt, Ingrid

    2015-01-01

    Author Summary In metazoans, transcription is arrested during mitosis. Previous studies have established that mitotic repression of cellular transcription is mediated by Cdk1/cyclin B-dependent phosphorylation of basal transcription factors that nucleate transcription complex formation. Repression of rDNA transcription at the onset of mitosis is brought about by inactivation of the TBP-containing transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of the TAFI110 subunit, which...

  1. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.; Chytil, A.; Forrester, E.; Nørgaard, Peter Henrik; Teoh, N.; Law, B.

    2007-01-01

    Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV...... development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors...

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

    OpenAIRE

    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

    2014-01-01

    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 E2...

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

    Science.gov (United States)

    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

  4. Heterologous expression of the transcriptional regulator escargot inhibits megakaryocytic endomitosis.

    Science.gov (United States)

    Ballester, A; Frampton, J; Vilaboa, N; Calés, C

    2001-11-16

    Certain cell types escape the strict mechanisms imposed on the majority of somatic cells to ensure the faithful inheritance of parental DNA content. This is the case in many embryonic tissues and certain adult cells such as mammalian hepatocytes and megakaryocytes. Megakaryocytic endomitosis is characterized by repeated S phases followed by abortive mitoses, resulting in mononucleated polyploid cells. Several cell cycle regulators have been proposed to play an active role in megakaryocytic polyploidization; however, little is known about upstream factors that could control endomitosis. Here we show that ectopic expression of the transcriptional repressor escargot interferes with the establishment of megakaryocytic endomitosis. Phorbol ester-induced polyploidization was inhibited in stably transfected megakaryoblastic HEL cells constitutively expressing escargot. Analysis of the expression and activity of different cell cycle factors revealed that Escargot affects the G(1)/S transition by influencing Cdk2 activity and cyclin A transcription. Nuclear proteins that specifically bind the Escargot-binding element were detected in endomitotic and non-endomitotic megakaryoblastic cells, but down-regulation occurred only during differentiation of cells that become polyploid. As Escargot was originally implicated in ploidy maintenance of Drosophila embryonic and larval cells, our results suggest that polyploidization in megakaryocytes might respond to mechanisms conserved from early development to adult cells that need to escape normal control of the diploid state. PMID:11498537

  5. Cdk5 Is Required for Memory Function and Hippocampal Plasticity via the cAMP Signaling Pathway

    Science.gov (United States)

    Gao, Jun; Joseph, Nadine; Xie, Zhigang; Zhou, Ying; Durak, Omer; Zhang, Lei; Zhu, J. Julius; Clauser, Karl R.; Carr, Steven A.; Tsai, Li-Huei

    2011-01-01

    Memory formation is modulated by pre- and post-synaptic signaling events in neurons. The neuronal protein kinase Cyclin-Dependent Kinase 5 (Cdk5) phosphorylates a variety of synaptic substrates and is implicated in memory formation. It has also been shown to play a role in homeostatic regulation of synaptic plasticity in cultured neurons. Surprisingly, we found that Cdk5 loss of function in hippocampal circuits results in severe impairments in memory formation and retrieval. Moreover, Cdk5 loss of function in the hippocampus disrupts cAMP signaling due to an aberrant increase in phosphodiesterase (PDE) proteins. Dysregulation of cAMP is associated with defective CREB phosphorylation and disrupted composition of synaptic proteins in Cdk5-deficient mice. Rolipram, a PDE4 inhibitor that prevents cAMP depletion, restores synaptic plasticity and memory formation in Cdk5-deficient mice. Collectively, our results demonstrate a critical role for Cdk5 in the regulation of cAMP-mediated hippocampal functions essential for synaptic plasticity and memory formation. PMID:21984943

  6. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase.

    Science.gov (United States)

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-06-23

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  7. Activation of GPR30 inhibits cardiac fibroblast proliferation.

    Science.gov (United States)

    Wang, Hao; Zhao, Zhuo; Lin, Marina; Groban, Leanne

    2015-07-01

    The incidence of left ventricular diastolic dysfunction significantly increases in postmenopausal women suggesting the association between estrogen loss and diastolic dysfunction. The in vivo activation of G protein-coupled estrogen receptor (GPR30) attenuates the adverse effects of estrogen loss on cardiac fibrosis and diastolic dysfunction in mRen2.Lewis rats. This study was designed to address the effects of GPR30 on cardiac fibroblast proliferation in rats. The expression of GPR30 in cardiac fibroblasts isolated from adult Sprague-Dawley rats was confirmed by RT-PCR, Western blot analysis, and immunofluorescence staining. Results from BrdU incorporation assays, cell counting, carboxyfluorescein diacetate succinimidyl ester labeling in conjunction with flow cytometry, and Ki-67 staining showed that treatment with G1, a specific agonist of GPR30, inhibited cardiac fibroblast proliferation in a dose-dependent manner, which was associated with decreases in CDK1 and cyclin B1 protein expressions. In the GPR30-KO cells, BrdU incorporation, and CDK1 and cyclin B1 expressions significantly increased when compared to GPR30-intact cells. G1 had no effect on BrdU incorporation, CDK1 and cyclin B1 mRNA levels in GPR30-KO cells. In vivo studies showed increases in CDK1 and cyclin B1 mRNA levels, Ki-67-positive cells, and the immunohistochemistry staining of vimentin, a fibroblast marker, in the left ventricles from ovariectomized mRen2.Lewis rats versus hearts from ovary-intact littermates; 2 weeks of G1 treatment attenuated these adverse effects of estrogen loss. This study demonstrates that GPR30 is expressed in rat cardiac fibroblasts, and activation of GPR30 limits proliferation of these cells likely via suppression of the cell cycle proteins, cyclin B1, and CDK1. PMID:25893735

  8. Combined cytological and transcriptomic analysis reveals a nitric oxide signaling pathway involved in cold-inhibited Camellia sinensis pollen tube growth

    Directory of Open Access Journals (Sweden)

    Weidong eWang

    2016-04-01

    Full Text Available Nitric oxide (NO as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca2+ gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs-related to signaling pathway, such as NO synthesis, cGMP, Ca2+, ROS, pH, actin, cell wall and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca2+, ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension.

  9. Combined Cytological and Transcriptomic Analysis Reveals a Nitric Oxide Signaling Pathway Involved in Cold-Inhibited Camellia sinensis Pollen Tube Growth.

    Science.gov (United States)

    Wang, Weidong; Sheng, Xianyong; Shu, Zaifa; Li, Dongqin; Pan, Junting; Ye, Xiaoli; Chang, Pinpin; Li, Xinghui; Wang, Yuhua

    2016-01-01

    Nitric oxide (NO) as a signaling molecule plays crucial roles in many abiotic stresses in plant development processes, including pollen tube growth. Here, the signaling networks dominated by NO during cold stress that inhibited Camellia sinensis pollen tube growth are investigated in vitro. Cytological analysis show that cold-induced NO is involved in the inhibition of pollen tube growth along with disruption of the cytoplasmic Ca(2+) gradient, increase in ROS content, acidification of cytoplasmic pH and abnormalities in organelle ultrastructure and cell wall component distribution in the pollen tube tip. Furthermore, differentially expressed genes (DEGs)-related to signaling pathway, such as NO synthesis, cGMP, Ca(2+), ROS, pH, actin, cell wall, and MAPK cascade signal pathways, are identified and quantified using transcriptomic analyses and qRT-PCR, which indicate a potential molecular mechanism for the above cytological results. Taken together, these findings suggest that a complex signaling network dominated by NO, including Ca(2+), ROS, pH, RACs signaling and the crosstalk among them, is stimulated in the C. sinensis pollen tube in response to cold stress, which further causes secondary and tertiary alterations, such as ultrastructural abnormalities in organelles and cell wall construction, ultimately resulting in perturbed pollen tube extension. PMID:27148289

  10. Exclusion of mRNPs and ribosomal particles from a thin zone beneath the nuclear envelope revealed upon inhibition of transport

    International Nuclear Information System (INIS)

    We have studied the nucleocytoplasmic transport of a specific messenger RNP (mRNP) particle, named Balbiani ring (BR) granule, and ribosomal RNP (rRNP) particles in the salivary glands of the dipteran Chironomus tentans. The passage of the RNPs through the nuclear pore complex (NPC) was inhibited with the nucleoporin-binding wheat germ agglutinin, and the effects were examined by electron microscopy. BR mRNPs bound to the nuclear basket increased in number, while BR mRNPs translocating through the central channel decreased, suggesting that the initiation of translocation proper had been inhibited. The rRNPs accumulated heavily in nucleoplasm, while no or very few rRNPs were recorded within nuclear baskets. Thus, the transport of rRNPs had been blocked prior to the entry into the baskets. Remarkably, the rRNPs had been excluded both from baskets and the space in between the baskets. We propose that normally basket fibrils move freely and repel RNPs from the exclusion zone unless the particles have affinity for and bind to nucleoporins within the baskets.

  11. CDK5RAP2 Regulates Centriole Engagement and Cohesion in Mice

    OpenAIRE

    Barrera, Jose A.; Kao, Ling-Rong; Robert E Hammer; Seemann, Joachim; Fuchs, Jannon L.; Megraw, Timothy L.

    2010-01-01

    Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microc...

  12. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

    OpenAIRE

    Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2014-01-01

    Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, w...

  13. Perspective of Cyclin-dependent kinase 9 (CDK9) as a Drug Target

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Baumli, S.; Fürst, R.

    2012-01-01

    Roč. 18, č. 20 (2012), s. 2883-2890. ISSN 1381-6128 R&D Projects: GA ČR GAP305/12/0783 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cancer * inflammation * kinase Subject RIV: ED - Physiology Impact factor: 3.311, year: 2012 http://www.benthamdirect.org/pages/article/1/3177374/perspective-of-cyclin-dependent-kinase-9-cdk9-as-a-drug-target.html

  14. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    OpenAIRE

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating b...

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

    OpenAIRE

    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 ...

  16. Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

    OpenAIRE

    Bäumer, Nicole; Bäumer, Sebastian; Berkenfeld, Frank; Stehling, Martin; Köhler, Gabriele; Berdel, Wolfgang E.; Müller-Tidow, Carsten; Tschanter, Petra

    2014-01-01

    Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling...

  17. Stereospecific phosphorylation by the central mitotic kinase Cdk1-cyclin B.

    Science.gov (United States)

    Etzkorn, Felicia A; Zhao, Song

    2015-04-17

    The cis vs trans conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the trans conformation, not cis, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation. PMID:25603287

  18. Targeting Transcriptional Addictions In Small Cell Lung Cancer With a Covalent CDK7 Inhibitor

    OpenAIRE

    Christensen, Camilla L.; Kwiatkowski, Nicholas; Abraham, Brian J; Carretero, Julian; Al-Shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S.; Akbay, Esra A; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B.; Cavanaugh, Jillian

    2014-01-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality. The identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library we observe that SCLC is sensitive to transcription-targeting drugs, and in particular to THZ1, a recent identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription fact...

  19. Effect of mimetic CDK9 inhibitors on HIV-1 activated transcription

    OpenAIRE

    Van Duyne, Rachel; Guendel, Irene; Jaworski, Elizabeth; Sampey, Gavin; Klase, Zachary; Chen, Hao; Zeng, Chen; Kovalskyy, Dmytro; el Kouni, Mahmoud H.; Lepene, Benjamin; Patanarut, Alexis; Nekhai, Sergei; Price, David H; Kashanchi, Fatah

    2012-01-01

    Potent antiretroviral therapy (ART) has transformed HIV-1 infection into a chronic manageable disease; however drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study we utilized a combination of structure based analysis of Cyclin/CDK complexes with our...

  20. New developments on the cheminformatics open workflow environment CDK-Taverna

    OpenAIRE

    Truszkowski Andreas; Jayaseelan Kalai; Neumann Stefan; Willighagen Egon L; Zielesny Achim; Steinbeck Christoph

    2011-01-01

    Abstract Background The computational processing and analysis of small molecules is at heart of cheminformatics and structural bioinformatics and their application in e.g. metabolomics or drug discovery. Pipelining or workflow tools allow for the Lego™-like, graphical assembly of I/O modules and algorithms into a complex workflow which can be easily deployed, modified and tested without the hassle of implementing it into a monolithic application. The CDK-Taverna project aims at building a fre...

  1. Methoxychlor inhibits growth of antral follicles by altering cell cycle regulators

    International Nuclear Information System (INIS)

    Methoxychlor (MXC) reduces fertility in female rodents, decreases antral follicle numbers, and increases atresia through oxidative stress pathways. MXC also inhibits antral follicle growth in vitro. The mechanism by which MXC inhibits growth of follicles is unknown. The growth of follicles is controlled, in part, by cell cycle regulators. Thus, we tested the hypothesis that MXC inhibits follicle growth by reducing the levels of selected cell cycle regulators. Further, we tested whether co-treatment with an antioxidant, N-acetyl cysteine (NAC), prevents the MXC-induced reduction in cell cycle regulators. For in vivo studies, adult cycling CD-1 mice were dosed with MXC or vehicle for 20 days. Treated ovaries were subjected to immunohistochemistry for proliferating cell nuclear antigen (PCNA) staining. For in vitro studies, antral follicles isolated from adult cycling CD-1 mouse ovaries were cultured with vehicle, MXC, and/or NAC for 48, 72 and 96 h. Levels of cyclin D2 (Ccnd2) and cyclin dependent kinase 4 (Cdk4) were measured using in vivo and in vitro samples. The results indicate that MXC decreased PCNA staining, and Ccnd2 and Cdk4 levels compared to controls. NAC co-treatment restored follicle growth and expression of Ccnd2 and Cdk4. Collectively, these data indicate that MXC exposure reduces the levels of Ccnd2 and Cdk4 in follicles, and that protection from oxidative stress restores Ccnd2 and Cdk4 levels. Therefore, MXC-induced oxidative stress may decrease the levels of cell cycle regulators, which in turn, results in inhibition of the growth of antral follicles.

  2. Dynamic macrophage polarization-specific miRNA patterns reveal increased soluble VEGF receptor 1 by miR-125a-5p inhibition.

    Science.gov (United States)

    Melton, David W; Lei, XiuFen; Gelfond, Jonathan A L; Shireman, Paula K

    2016-05-01

    Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of microRNAs (miRNAs or miRs) across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 h) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 h; however, robust responses for most markers occurred within 3 h. In parallel, many polarization-specific miRNAs were also changed by 3 h and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns: increased at 12-24 h in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared with M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media-only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media, partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p. PMID:26884460

  3. Cyclin E,CDK2,Ki-67在白血病中的表达及其临床意义%Expressions of cyclin E,CDK2,and Ki-67 and the clinical significance in leukemia

    Institute of Scientific and Technical Information of China (English)

    郝杰; 侯科佐; 刘云鹏; 于萍

    2004-01-01

    目的:探讨白血病细胞周期素E(cyclin E)、细胞周期蛋白依赖性激酶2(CDK2)、细胞增殖相关抗原(Ki-67)在白血病中的表达及其临床意义.方法:应用免疫组织化学S-P法检测50例白血病病人骨髓(急性40例,慢性10例)cyclin E、CDK2和Ki-67的表达情况.结果:cyclin E阳性表达率38.0%,CDK2阳性表达率64.0%,均明显高于正常对照组(P<0.05);Ki-67阳性表达率16.0%,与正常对照组比较无显著差异(P>0.05).cyclin E表达与CDK2表达呈密切正相关(P<0.05);CDK2表达与Ki-67表达密切正相关(P<0.05).cyclin E和CDK2共同阳性表达者复发率明显增高.结论:cyciin E和CDK2的异常表达在白血病发生发展和复发过程中起重要作用,其表达水平对判断疾病的预后和疗效有一定价值.

  4. The Expression and Clinic Significance of Cdk2 and CyclinE in Cholangiocarcinoma%Cdk2蛋白和CyclinE在胆管细胞癌中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    胡逸林; 张端莲; 曹廷加; 高友兵

    2010-01-01

    目的:探讨细胞周期蛋白依赖性激酶2(Cdk2)和细胞周期蛋白E(CyclinE)在胆管细胞癌中的表达及临床意义.方法:收集武汉大学人民医院病理科2002-2008年胆管细胞癌存档蜡块40例,其中男性20例,女性20例.另取胆管细胞癌周围正常组织5例作对照.采用免疫组织化学方法检测各组中Cdk2蛋白和CyclinE的表达,利用HPIAS-2000图像分析系统测定各组中Cdk2蛋白和CyclinE表达的平均光密度和平均阳性面积率.结果:胆管细胞癌中Cdk2蛋白和CyclinE呈高表达,对照组中Cdk2蛋白和CyclinE呈低表达,胆管细胞癌组与对照组之间Cdk2蛋白和CyclinE表达的平均光密度及阳性面积率有显著性差异(P<0.05).结论:Cdk2与CyclinE在胆管细胞癌的发生发展中发挥正性调节因子的作用.

  5. p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

    OpenAIRE

    Cen, Ling; Carlson, Brett L.; Schroeder, Mark A.; Ostrem, Jamie L.; Kitange, Gaspar J.; Mladek, Ann C.; Fink, Stephanie R.; Decker, Paul A.; Wu, Wenting; Kim, Jung-Sik; Waldman, Todd; Jenkins, Robert B.; Sarkaria, Jann N.

    2012-01-01

    Deregulation of the p16INK4a-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16INK4a-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16INK4a-Cdk4/6-Rb pathway. In vitro evalua...

  6. Cdk2在皮肤淋巴瘤中的表达及统计分析

    Institute of Scientific and Technical Information of China (English)

    冉启杰

    2010-01-01

    目的:探讨Cdk2在皮肤淋巴瘤中的表达及其在肿瘤的发生和发展中的作用.方法: 收集武汉大学人民医院病理科2006~2009 年手术切除及活检经病理明确诊断的皮肤淋巴瘤蜡块40 例, 其中22 例MF、7 例皮肤间变性大细胞淋巴瘤、6 例其他类型的T 细胞淋巴瘤、5 例B 细胞淋巴瘤.另外20例皮肤炎症病变,包括10例银屑病、10例副银屑病.采用免疫组织化学方法观察各组组织内Cdk2的表达.利用HPIAS-2000图像分析系统测定Cdk2在以上各组中表达的平均光密度和平均阳性面积率.结果:皮肤淋巴瘤中Cdk2呈高表达;癌旁组织中Cdk2呈低表达.图像分析结果显示:皮肤淋巴瘤与皮肤炎症病变之间Cdk2的平均光密度及阳性面积率的差异有显著性意义(P<0.05).结论:Cdk2异常高表达促进细胞增殖,从而在皮肤淋巴瘤发病过程中起重要作用.

  7. CDK2基因RNAi慢病毒载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 王前; 郑磊; 陈晓; 熊石龙; 包杰; 丁海明; 黄妩姣; 庄俊华

    2008-01-01

    目的:构建人细胞周期素依赖蛋白激酶2(CDK2)基因RNA干扰慢病毒载体.方法:利用Invitrogen公司在线软件设计人CDK2 (NM001798) shRNA序列,退火形成ds oligo后克隆到pENTRTM/U6载体的黏性末端,测序,再与慢病毒载体重组,测序鉴定,在脂质体的介导下将慢病毒的包装混合物和CDK2基因重组慢病毒载体转染293FT细胞,包装成病毒后,收集细胞培养上清液,测定病毒滴度.结果:测序证实pENTRTM/U6-CDK2-shRNA为阳性克隆,与慢病毒载体重组后测序结果显示也为阳性克隆,CDK2基因重组慢病毒载体传染293FT细胞后48h,细胞培养上清液,病毒的滴度为6×108TU/L.结论:成功构建人CDK2基因RNAi慢病毒载体,为研究CDK2在自身免疫病中的应用提供了稳定的转染细胞载体.

  8. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Clément Achille

    Full Text Available RNA interference (RNAi is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL alone (Control and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i and EGFP (EGFPi, also served as a control shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing and 0.2-3 µm (Control. While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

  9. Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    Directory of Open Access Journals (Sweden)

    Ade Kallas

    2014-01-01

    Full Text Available As cyclin-dependent kinases (CDKs regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction on human embryonic stem (hES cells and embryonal carcinoma-derived (hEC cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

  10. Pharmacologic inhibition of small-conductance calcium-activated potassium (SK) channels by NS8593 reveals atrial antiarrhythmic potential in horses

    DEFF Research Database (Denmark)

    Haugaard, Maria Mathilde; Hesselkilde, Eva Zander; Pehrson, Steen Michael;

    2015-01-01

    . METHODS: Cardiac biopsies were analyzed to investigate the expression level of the most prominent cardiac ion channels, with special focus on SK channels, in the equine heart. Subcellular distribution of SK isoform 2 (SK2) was assessed by immunohistochemistry and confocal microscopy. The...... ventricular depolarization and repolarization times. RESULTS: Analysis revealed equivalent mRNA transcript levels of the 3 SK channel isoforms in atria compared to ventricles. Immunohistochemistry and confocal microscopy displayed a widespread distribution of SK2 in both atrial and ventricular cardiomyocytes...

  11. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    Directory of Open Access Journals (Sweden)

    Laura Ordovás

    2015-11-01

    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  12. (99mTc-DTPA renal dynamic imaging method may be unsuitable to be used as the reference method in investigating the validity of CDK-EPI equation for determining glomerular filtration rate.

    Directory of Open Access Journals (Sweden)

    Peng Xie

    Full Text Available OBJECTIVE: To compare the measurements of glomerular filtration rate (GFR determined by (99mTc-diethylene triamine pentaacetic acid ((99mTc-DTPA renal dynamic imaging with those estimated by Chronic Kidney Disease Epidemiology Collaboration (CDK-EPI equation and to identify a more accurate measurement of GFR of chronic kidney disease (CKD patients in clinical practice. METHODS: The GFR was determined simultaneously by 3 methods: (a dual plasma sample clearance method (tGFR; (b renal dynamic imaging method (dGFR; (c CDK-EPI equation (eGFR. The tGFR was employed as the reference method. The correlation, regression, and limit of agreement of dGFR and eGFR were used to demonstrate the validity of the two methods. The comparison of bias, precision, and accuracy between dGFR and eGFR was analyzed to identify the most suitable method. The analysis of bias, precision and accuracy was repeated after stratifying patients by a measured tGFR cutpoint of 60 ml·min(-1·(1.73 m(2(-1. RESULTS: A total of 149 patients were enrolled. Both dGFR and eGFR correlated well with tGFR and the regression equation of dGFR and eGFR against tGFR was respectively Y = -4.289+0.962X (r = 0.919; RMSE = 14.323 ml.min(-1. (1.73 m(2(-1; P<0.001 and Y = 2.462+0.914X (r = 0.909; RMSE = 15.123 ml.min(-1. (1.73 m(2(-1; P<0.001. In addition, Bland-Altman analysis showed preferable agreement between the two methods and the reference method. The comparison revealed that eGFR, compared with dGFR, showed better performance on bias and 50% accuracy and similar performance on other indexes in the whole cohort and the lower-GFR subgroup, whereas in the higher-GFR subgroup the difference of the two methods was not significant in all parameters. CONCLUSIONS: Although both CDK-EPI equation and renal dynamic imaging can be used to determine the GFR of CKD patients, CDK-EPI equation is more accurate than renal dynamic imaging. As a result, (99mTc-DTPA renal dynamic imaging

  13. CyclinE和cdk2在眼睑基底细胞癌组织中的表达及其意义%Expression and significance of Cyclin E and Cdk2 in eyelid basal cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    陈志雄; 黄琼

    2012-01-01

    Objective To research the expression and significance of CyclinE and cdk2 in basal cell carcinoma of eyelids. Methods Twenty samples of eyelid basal cell carcinoma (BCC) from surgical excision and biopsy were collected from the departments of pathology of Wuhan Central Hospital and Wuhan University Zhongnan Hospital in 2002-2009, and 5 normal tissues around the cancer acted as controls. Immu-nohistochemical staining was performed to detect the expression of Cyclin E and Cdk2. The average optical density and the rate of positive area of Cyclin E and Cdk2 expression were analyzed using the HPIAS-2000 Image Analysis System. Results Cyclin E and Cdk2 showed high expression in eyelid basal cell carcinoma, but low expression in paracancerous tissue. Image analysis showed that the expression of CyclinE and cdk2 in eyelid basal cell carcinoma were significantly higher than that in paracancerous tissue (P<0. 05). Conclusion High expression of Cyclin E and Cdk2 plays an important role in the occurrence and development of eyelid basal cell carcinoma, and their co-expression is of guiding significance and for formulating therapeutic plans and for monitoring patients after treatment.%目的 探讨CyclinE和cdk2在眼睑基底细胞癌组织中的表达及其意义.方法 收集武汉市中心医院和武汉大学人民医院病理科2002-2009年手术切除及活检的眼睑基底细胞癌(basal cell carcinoma,BCC)标本其20例,另取癌周围组织5例作对照,采用免疫组织化学方法观察各组细胞内CyclinE和cdk2表达.利用HPIAS-2000图像分析系统测定CyclinE和cdk2在以上各组中表达的平均光密度和平均阳性面积率.结果 眼睑基底细胞癌组织中CyclinE和cdk2呈高表达;癌旁组织中CyclinE和cdk2呈低表达.图像分析结果显示:眼睑基底细胞癌组织与癌旁组织之间CyclinE和cdk2的平均光密度及阳性面积率的差异有显著性意义(P<0.05).结论 CyclinE和cdk2的高表达,在眼睑基底细胞癌的

  14. Expression and clinical significance of cyclin A and CDK2 in colorectal carcinoma%周期素A和CDK2在结直肠癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    潘长海; 彭洪云

    2011-01-01

    目的 研究周期素A(cyclin A)和CDK2在结直肠癌中的表达及其临床意义.方法 手术切除并经病理学确诊的结直肠癌及相应的癌周正常组织石蜡标本30例,采用免疫组化SP法检测周期素A及CDK2的表达.结果 结直肠癌中周期素A阳性表达率为67%,明显高于癌周正常组织的10%;结直肠癌中CDK2阳性表达率为50%,亦明显高于癌周正常组织的7%.两者的表达水平与直肠癌的分化、浸润深度及淋巴结转移有相关性.结论 周期素A和CDK2的高表达为结直肠癌发生的早期现象,两者参与了结直肠癌的发生过程.%Objective To study the expression and clinical significance of cyclin A and CDK2 in colorectal carcinoma. Methods With immunohistochemical SP method,the expressions of cyclin A and CDK2 were detected in 30 surgically resected clocrectal carcinomas(group A) verified by pathology and the tumor-adjacent normal colorectal tissues(group B). Results The positive rate of cyclin A was higher in group A than that in group B(67% vs. 10%),so did that of CDK2(50% vs. 7%). The expressions of cyclin A and CDK2 were closely related to histological grade, lymph node metastasis and invasion. Conclusion The overexpression of cyclin A and CDK2 was an early phenomena in the progression of colorectal carcinoma, both of which participate the colorectal carcinogenesis.

  15. Changes of protein expression in HepG2 cells with CDK2 RNA interference%CDK2 RNA干扰后HepG2细胞蛋白表达的变化

    Institute of Scientific and Technical Information of China (English)

    商进; 王震宇; 李君枣

    2013-01-01

    目的 探讨稳定转染CDK2干扰RNA对人肝癌细胞株HepG2细胞生物活性及细胞核蛋白质的改变.方法 构建稳定转染pGenesil-1-CDK2的HepG2细胞系,MTT法检测细胞增殖、流式细胞术检测细胞周期的改变.通过RT-PCR和双向凝胶电泳-质谱技术-数据库搜索,比较转染前后CDK2 mRNA的表达和细胞核蛋白质的变化.并通过Western blot法对显著差异蛋白进行验证.结果 与空质粒组PHK-siRNA-HepG2细胞和未转染组HepG2细胞相比,pCDK2-siRNA-HepG2组细胞的生长速度减慢(P<0.01),稳定转染CDK2 RNAi组细胞的CDK2 mRNA表达水平显著下降.通过双向电泳-质谱技术得到4个稳定转染CDK2 siRNA的HepG2细胞不表达的蛋白质,Westem blot法证实双向电泳结果的可信性.结论 CDK2干扰RNA可明显降低HepG2细胞CDK2 mRNA的表达,抑制HepG2细胞的增殖,干扰后的HepG2细胞不表达的蛋白质分别是类核糖体蛋白S12、β-肌动蛋白、锌指蛋白276和伴侣蛋白10相关蛋白.

  16. The Antidiabetic Drug Metformin Inhibits the Proliferation of Bladder Cancer Cells in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Tao Zhang

    2013-12-01

    Full Text Available Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4, E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK and suppressed mammalian target of rapamycin (mTOR, the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA, cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

  17. Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation

    Directory of Open Access Journals (Sweden)

    Dubrovsky Larisa

    2008-03-01

    Full Text Available Abstract It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

  18. The role of CDK2 in the meiosis of spermatocyte and oocyte%CDK2在精母细胞和卵母细胞减数分裂中的作用

    Institute of Scientific and Technical Information of China (English)

    关泽红; 旭日干

    2008-01-01

    细胞周期蛋白依赖性蛋白激酶(Cyclin-dependent kinase,CDK)2是驱动细胞通过G1/S期检验点进入S期完成DNA合成的关键性调控蛋白.过去一度认为CDK2在减数分裂中的作用不像在有丝分裂中那么重要.直至2003年在敲除小鼠CDK2基因后出乎意料地发现小鼠生长发育正常,只是不育:生殖细胞减数分裂受到影响.这一发现引起人们重新审视CDK2在细胞增殖中的作用,对CDK2在减数分裂中的作用研究受到关注,本文就此作一综述.

  19. The Significance and the Expression of p27kip1 and cdk2 in Gastric Carcinoma%p27kip1和cdk2在胃癌中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    张世同; 徐志林

    2002-01-01

    目的研究p27kip1及cdk2在胃癌组织中的表达及其与胃癌生物学行为的关系.方法应用免疫组化SABC法检测63例胃癌组织中p27kip1及cdk2的表达.结果本组63例胃癌中,p27kip1蛋白阳性表达30例(47.6%).p27kip1与胃癌的浸润深度、淋巴结转移、组织学分级均呈负相关(P<0.05).cdk2蛋白阳性表达33例(50.8%),cdk2与胃癌的组织学分级呈正相关(P<0.05).结论提示p27kip1表达减少及cdk2表达增加可能促进了胃癌的发生发展.

  20. Establishment of a HeLa Cell Line Stably Expressing Human Mutated CDK2(F80A)%人突变型CDK2(F80A)稳定细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    王佳思; 金英花

    2010-01-01

    CDK2激酶第80位的Phe突变成Ala, 使该激酶特异地利用ATP类似物N6-(2-苯乙基)-ATP(PE-ATP)筛选CDK2激酶的体内特异性底物. 将pCMV-CDK2(F80A)-myc载体转染人宫颈癌细胞(HeLa), 经持续G418选择和克隆化获得6株抗G418细胞系. Immunoblotting分析发现, 挑选的6株细胞系中有4株表达带有myc标签的人突变CDK2蛋白质, 其中2株表达量较高, 可以作为筛选CDK2底物的细胞系.

  1. 含氮查尔酮类细胞周期蛋白依赖性激酶抑制剂的合成及抗癌活性研究Ⅳ%Synthesis and anticancer activity study of nitrogen-containing chalcones as CDK inhibitors IV

    Institute of Scientific and Technical Information of China (English)

    李艳玲; 方浩; 徐文方

    2011-01-01

    The key role of CDKs in tumorigenesis have raised great interest for the development of CDK inhibitors as potential anticancer agents. Flavopiridol,a synthetic flavone,is the fist CDK inhibitors that entered clinical trial. And chalcones,as a branch of flavone,having important effects on cancer cell growth and proliferation. Herein, based on the structure of flavopiridol, eight novel chalcones analogs were synthesized by aldol condensation of a substituted acetophenone with various benzaldehyde, acetophenone was obtained by Hoesch reaction and methylation,chalcones was then converted target compounds by Mannich reaction with morpholine. The structures of target compounds were confirmed by IR,'H-NMR and ESI-MS,and their CDK1 inhibition as well as cytotoxicity activity against HCT116 were determined with flavopiridol as a positive control. The results showed that compounds 5a,5b,5c,5d,5f,5g and 5h exhibited higher CDK1 inhibition than flavopiridol,with the IC50 values of 63. 83,46.45,59. 70,48. 97,51. 40,52. 84,45. 70 nrnol·L-1 respectively, while flavopiridol was 64. 05 nmol·L-1 ,and compounds 5g and 5h showed higher cytotoxicity against HCT116 with the Icj,, values 2. 17,2. 80 μmol·L-1 respectively. The results showed that piperidine ring of flavopiridol can be replaced by Mannich base with morpholine ring.%目的 寻找活性更好的类黄酮细胞周期蛋白依赖激酶(CDKs)抑制剂.方法 利用Mannich反应制得8个查尔酮类黄酮.结果与结论 目标化合物的结构经1R、1 H-NMR、质谱确证,并测定了化合物对CDK1的抑制活性以及对HCT116肿瘤细胞的体外抗肿瘤活性,其中有7个化合物对CDK1抑制活性高于阳性对照flavopiridol,所有化合物对HCT116肿瘤细胞均显示出较强的抑制活性.

  2. Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

    Science.gov (United States)

    Kozasa, Takashi; Abe, Yuri; Mitsuhashi, Kazuya; Tamura, Tomokazu; Aoki, Hiroshi; Ishimaru, Masatoshi; Nakamura, Shigeyuki; Okamatsu, Masatoshi; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-05-01

    The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV. PMID:25648277

  3. 斑节对虾CDK2基因全长cDNA克隆及表达分析

    Institute of Scientific and Technical Information of China (English)

    戴文婷; 傅明骏; 赵超; 周发林; 杨其彬; 王艳; 史进选; 邱丽华

    2015-01-01

    该研究以斑节对虾(Penaeus monodon)c DNA文库中的CDK2片段为基础,利用RACE技术获得Pm CDK2的c DNA全长,并利用生物信息学对其进行了分析。Pm CDK2基因全长1 679 bp,包括编码306个氨基酸的921 bp的开放阅读框(ORF),258 bp的5'UTR和500 bp的3'UTR。同源性分析显示,Pm CDK2与罗氏沼虾(Macrobrachium rosenbergii)等物种的CDK2具有较高的同源性。使用荧光定量PCR技术研究了Pm CDK2的mRNA在不同组织和卵巢发育各时期的相对表达量和变化模式,结果显示,Pm CDK2在斑节对虾的脑、心、淋巴、肝胰腺、性腺等组织中均有表达,其中精巢中表达量显著高于其他组织;卵巢发育阶段表达分析则显示Pm CDK2在Ⅲ期表达量最高,其次是Ⅳ期。通过原核表达技术成功获得了含有6个His标签的融合蛋白,并对融合蛋白进行Western Blot试验,初步探讨了重组蛋白表达情况。结果表明,Pm CDK2基因在斑节对虾卵巢发育过程中可能发挥了重要作用,该结果对进一步探究斑节对虾卵巢发育机理提供了一定的理论依据。

  4. Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

    Energy Technology Data Exchange (ETDEWEB)

    Sávio, André Luiz Ventura, E-mail: savio.alv@gmail.com [UNESP – Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, Departamento de Patologia, Botucatu, SP (Brazil); Nicioli da Silva, Glenda [UFOP – Universidade Federal de Ouro Preto, Escola de Farmácia, Departamento de Análises Clínicas, Ouro Preto, MG (Brazil); Salvadori, Daisy Maria Fávero [UNESP – Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, Departamento de Patologia, Botucatu, SP (Brazil)

    2015-01-15

    Highlights: • AITC inhibits mutant and wild-type TP53 cell proliferation. • Morphological changes and cells debris were observed after AITC treatment in both cells. • BAX and BCL2 expression modulation was observed in wild-type TP53 cells. • BCL2, BAX and ANLN increased and S100P decreased expression was detected in mutated TP53 cells. • AITC effects in gene modulation are dependent TP53 gene status. - Abstract: Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5 μM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm

  5. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Zhuo [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Department of Cardiology, Jinan Central Hospital, Affiliated with Shandong University, 105 Jiefang Road, Jinan, 250013 (China); Wang, Hao; Lin, Marina [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Groban, Leanne, E-mail: lgroban@wakehealth.edu [Department of Anesthesiology, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27159-1009 (United States); Hypertension and Vascular Disease Center, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States); Office of Women in Medicine and Science, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157 (United States)

    2015-03-27

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression.

  6. Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

    International Nuclear Information System (INIS)

    Highlights: • AITC inhibits mutant and wild-type TP53 cell proliferation. • Morphological changes and cells debris were observed after AITC treatment in both cells. • BAX and BCL2 expression modulation was observed in wild-type TP53 cells. • BCL2, BAX and ANLN increased and S100P decreased expression was detected in mutated TP53 cells. • AITC effects in gene modulation are dependent TP53 gene status. - Abstract: Natural compounds hold great promise for combating antibiotic resistance, the failure to control some diseases, the emergence of new diseases and the toxicity of some contemporary medical products. Allyl isothiocyanate (AITC), which is abundant in cruciferous vegetables and mustard seeds and is commonly referred to as mustard essential oil, exhibits promising antineoplastic activity against bladder cancer, although its mechanism of action is not fully understood. Therefore, the aim of this study was to investigate the effects of AITC activity on bladder cancer cell lines carrying a wild type (wt; RT4) or mutated (T24) TP53 gene. Morphological changes, cell cycle kinetics and CDK1, SMAD4, BAX, BCL2, ANLN and S100P gene expression were evaluated. In both cell lines, treatment with AITC inhibited cell proliferation (at 62.5, 72.5, 82.5 and 92.5 μM AITC) and induced morphological changes, including scattered and elongated cells and cellular debris. Gene expression profiles revealed increased S100P and BAX and decreased BCL2 expression in RT4 cells following AITC treatment. T24 cells displayed increased BCL2, BAX and ANLN and decreased S100P expression. No changes in SMAD4 and CDK1 expression were observed in either cell line. In conclusion, AITC inhibits cell proliferation independent of TP53 status. However, the mechanism of action of AITC differed in the two cell lines; in RT4 cells, it mainly acted via the classical BAX/BCL2 pathway, while in T24 cells, AITC modulated the activities of ANLN (related to cytokinesis) and S100P. These data confirm

  7. GPR30 decreases cardiac chymase/angiotensin II by inhibiting local mast cell number

    International Nuclear Information System (INIS)

    Chronic activation of the novel estrogen receptor GPR30 by its agonist G1 mitigates the adverse effects of estrogen (E2) loss on cardiac structure and function. Using the ovariectomized (OVX) mRen2.Lewis rat, an E2-sensitive model of diastolic dysfunction, we found that E2 status is inversely correlated with local cardiac angiotensin II (Ang II) levels, likely via Ang I/chymase-mediated production. Since chymase is released from cardiac mast cells during stress (e.g., volume/pressure overload, inflammation), we hypothesized that GPR30-related cardioprotection after E2 loss might occur through its opposing actions on cardiac mast cell proliferation and chymase production. Using real-time quantitative PCR, immunohistochemistry, and immunoblot analysis, we found mast cell number, chymase expression, and cardiac Ang II levels were significantly increased in the hearts of OVX-compared to ovary-intact mRen2.Lewis rats and the GPR30 agonist G1 (50 mg/kg/day, s.c.) administered for 2 weeks limited the adverse effects of estrogen loss. In vitro studies revealed that GPR30 receptors are expressed in the RBL-2H3 mast cell line and G1 inhibits serum-induced cell proliferation in a dose-dependent manner, as determined by cell counting, BrdU incorporation assay, and Ki-67 staining. Using specific antagonists to estrogen receptors, blockage of GPR30, but not ERα or ERβ, attenuated the inhibitory effects of estrogen on BrdU incorporation in RBL-2H3 cells. Further study of the mechanism underlying the effect on cell proliferation showed that G1 inhibits cyclin-dependent kinase 1 (CDK1) mRNA and protein expression in RBL-2H3 cells in a dose-dependent manner. - Highlights: • GPR30 activation limits mast cell number in hearts from OVX mRen2.Lewis rats. • GPR30 activation decreases cardiac chymase/angiotensin II after estrogen loss. • GPR30 activation inhibits RBL-2H3 mast cell proliferation and CDK1 expression

  8. 喉鳞状细胞癌中CDK2对细胞增殖的作用%Effect of CDK2 on Proliferation in laryngeal squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    张海利; 王斌全; 刘荣; 温树信; 皇甫辉

    2008-01-01

    目的:研究喉鳞状细胞癌组织中CDK2激酶在引起DNA异倍体发生过程中的作用.方法:取手术中获得的50例喉鳞状细胞癌组织,12例非典型增生组织和30例声带息肉组织,用γ-微管蛋白抗体标记中心体,用免疫组织化学的方法检测CDK2激酶、γ-微管蛋白的表达.结果:在喉鳞状细胞癌组织中CDK2激酶,γ-微管蛋白阳性率表达分别为68%(34/50),78%(10/15),二者的表达都显著高于声带息肉组织(P<0.05);在喉鳞状细胞癌组织中,CDK2激酶的表达与γ-微管蛋白的表达具有相关性.结论:喉鳞状细胞癌中CDK2过度表达导致肿瘤细胞增殖异常.在诊断和治疗喉鳞状细胞癌中,CDK2可能是一个有重要作用的指标.

  9. Quantitative phosphoproteomic analysis reveals γ-bisabolene inducing p53-mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation.

    Science.gov (United States)

    Jou, Yu-Jen; Chen, Chao-Jung; Liu, Yu-Ching; Way, Tzong-Der; Lai, Chih-Ho; Hua, Chun-Hung; Wang, Ching-Ying; Huang, Su-Hua; Kao, Jung-Yie; Lin, Cheng-Wen

    2015-10-01

    γ-Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti-proliferative activities against human oral squamous cell carcinoma (OSCC). γ-Bisabolene activated caspases-3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9-22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ-bisabolene was identified using TiO2-PDMS plate and LC-MS/MS, then confirmed using Western blotting and real-time RT-PCR assays. Phosphoproteome profiling revealed that γ-bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein-protein interaction network analysis proposed the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in γ-bisabolene-induced apoptosis. Subsequent assays indicated γ-bisabolene eliciting p53 acetylation that enhanced the expression of p53-regulated apoptotic genes. PP1 inhibitor-2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ-bisabolene-treated Ca9-22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ-bisabolene-induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1-HDAC2-p53 and ERK1/2-p53 pathways in mitochondria-mediated apoptosis of γ-bisabolene-treated cells. This study demonstrated γ-bisabolene displaying potent anti-proliferative and apoptosis-inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ-bisabolene-induced apoptosis. The novel insight could be useful for developing anti-cancer drugs. PMID:26194454

  10. Crystal Structure of Acivicin-Inhibited [gamma]-Glutamyltranspeptidase Reveals Critical Roles for Its C-Terminus in Autoprocessing and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Kristin; Cullati, Sierra; Sand, Aaron; Biterova, Ekaterina I.; Barycki, Joseph J.; (UNL)

    2009-03-27

    Helicobacter pylori {gamma}-glutamyltranspeptidase (HpGT) is a general {gamma}-glutamyl hydrolase and a demonstrated virulence factor. The enzyme confers a growth advantage to the bacterium, providing essential amino acid precursors by initiating the degradation of extracellular glutathione and glutamine. HpGT is a member of the N-terminal nucleophile (Ntn) hydrolase superfamily and undergoes autoprocessing to generate the active form of the enzyme. Acivicin is a widely used {gamma}-glutamyltranspeptidase inhibitor that covalently modifies the enzyme, but its precise mechanism of action remains unclear. The time-dependent inactivation of HpGT exhibits a hyperbolic dependence on acivicin concentration with k{sub max} = 0.033 {+-} 0.006 s{sup -1} and K{sub I} = 19.7 {+-} 7.2 {micro}M. Structure determination of acivicin-modified HpGT (1.7 {angstrom}; R{sub factor} = 17.9%; R{sub free} = 20.8%) demonstrates that acivicin is accommodated within the {gamma}-glutamyl binding pocket of the enzyme. The hydroxyl group of Thr 380, the catalytic nucleophile in the autoprocessing and enzymatic reactions, displaces chloride from the acivicin ring to form the covalently linked complex. Within the acivicin-modified HpGT structure, the C-terminus of the protein becomes ordered with Phe 567 positioned over the active site. Substitution or deletion of Phe 567 leads to a >10-fold reduction in enzymatic activity, underscoring its importance in catalysis. The mobile C-terminus is positioned by several electrostatic interactions within the C-terminal region, most notably a salt bridge between Arg 475 and Glu 566. Mutational analysis reveals that Arg 475 is critical for the proper placement of the C-terminal region, the Tyr 433 containing loop, and the proposed oxyanion hole.

  11. Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling.

    Science.gov (United States)

    Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M; Shaw, Patrick G; Huang, Tai-Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dong-Wen; Zhang, Kun; Manda, Srikanth S; Pandey, Akhilesh; Hayward, S Diane

    2015-12-01

    Epstein-Barr virus (EBV) is etiologically linked to infectious mononucleosis and several human cancers. EBV encodes a conserved protein kinase BGLF4 that plays a key role in the viral life cycle. To provide new insight into the host proteins regulated by BGLF4, we utilized stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to compare site-specific phosphorylation in BGLF4-expressing Akata B cells. Our analysis revealed BGLF4-mediated hyperphosphorylation of 3,046 unique sites corresponding to 1,328 proteins. Frequency analysis of these phosphosites revealed a proline-rich motif signature downstream of BGLF4, indicating a broader substrate recognition for BGLF4 than its cellular ortholog cyclin-dependent kinase 1 (CDK1). Further, motif analysis of the hyperphosphorylated sites revealed enrichment in ATM, ATR and Aurora kinase substrates while functional analyses revealed significant enrichment of pathways related to the DNA damage response (DDR), mitosis and cell cycle. Phosphorylation of proteins associated with the mitotic spindle assembly checkpoint (SAC) indicated checkpoint activation, an event that inactivates the anaphase promoting complex/cyclosome, APC/C. Furthermore, we demonstrated that BGLF4 binds to and directly phosphorylates the key cellular proteins PP1, MPS1 and CDC20 that lie upstream of SAC activation and APC/C inhibition. Consistent with APC/C inactivation, we found that BGLF4 stabilizes the expression of many known APC/C substrates. We also noted hyperphosphorylation of 22 proteins associated the nuclear pore complex, which may contribute to nuclear pore disassembly and SAC activation. A drug that inhibits mitotic checkpoint activation also suppressed the accumulation of extracellular EBV virus. Taken together, our data reveal that, in addition to the DDR, manipulation of mitotic kinase signaling and SAC activation are mechanisms associated with lytic EBV replication. All MS data have been deposited in

  12. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

    Energy Technology Data Exchange (ETDEWEB)

    Memin, Elisabeth, E-mail: molinac@mail.montclair.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Genzale, Megan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Crow, Marni; Molina, Carlos A. [Department of Biology and Molecular Biology, Montclair State University, Montclair, NJ, 07043 (United States)

    2011-10-15

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

  13. TGF-β1 sensitizes TRPV1 through Cdk5 signaling in odontoblast-like cells

    OpenAIRE

    Utreras, Elias; Prochazkova, Michaela; Terse, Anita; Gross, Jacklyn; Keller, Jason; Iadarola, Michael J.; Kulkarni, Ashok B.

    2013-01-01

    Background Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-β1 (TGF-β1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-β1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role ...

  14. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

    International Nuclear Information System (INIS)

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

  15. Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1.

    Science.gov (United States)

    Rodriguez-Rodriguez, Jose-Antonio; Moyano, Yolanda; Játiva, Soraya; Queralt, Ethel

    2016-05-31

    To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions. PMID:27210759

  16. IL12A, MPHOSPH9/CDK2AP1 and RGS1 are novel multiple sclerosis susceptibility loci

    DEFF Research Database (Denmark)

    Sørensen, Per Soelberg

    2010-01-01

    same direction of effect observed in the discovery phase. Three loci exceeded genome-wide significance in the joint analysis: RGS1 (P value=3.55 x 10(-9)), IL12A (P=3.08 x 10(-8)) and MPHOSPH9/CDK2AP1 (P=3.96 x 10(-8)). The RGS1 risk allele is shared with celiac disease (CD), and the IL12A risk allele...... seems to be protective for celiac disease. Within the MPHOSPH9/CDK2AP1 locus, the risk allele correlates with diminished RNA expression of the cell cycle regulator CDK2AP1; this effect is seen in both lymphoblastic cell lines (P=1.18 x 10(-5)) and in peripheral blood mononuclear cells from subjects with...

  17. 应用MM/PBSA方法研究CDK2活性口袋内溶剂水分子对CDK2-配体结合自由能的影响%Influence of the Solvent Water Molecules at the Active Site of CDK2 on the Binding Free Energy of CDK2-1igand Complexes: an MM/PBSA Study

    Institute of Scientific and Technical Information of China (English)

    杨丽君; 贾若; 杨胜勇

    2009-01-01

    应用MM/PBSA方法研究了CDK2活性口袋内溶剂水分子对CDK2-配体结合自由能的影响.结果表明,活性口袋内溶剂水分子对CDK2-配体相互作用自由能有一定的贡献,其贡献的大小随配体不同而有所差异,导致这种差异的主要原因是活性位点内溶剂水分子与蛋白残基和配体之间形成了不同的氢键相互作用网络.

  18. Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

    Directory of Open Access Journals (Sweden)

    Zhuo Zhan

    Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

  19. CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup [Laboratory of Cell Signaling, Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahangno, Yusong, Daejeon 305-806 (Korea, Republic of); Kwon, Ki-Sun, E-mail: kwonks@kribb.re.kr [Laboratory of Cell Signaling, Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahangno, Yusong, Daejeon 305-806 (Korea, Republic of)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} differently adjusted senescence and proliferation in normal and cancer cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently decreased PCNA levels in normal cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently increased CDK2 activity in cancer cells. Black-Right-Pointing-Pointer p21{sup Cip1} is likely dispensable when H{sub 2}O{sub 2} induces senescence in normal cells. Black-Right-Pointing-Pointer Suggestively, CDK2 and PCNA play critical roles in H{sub 2}O{sub 2}-induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H{sub 2}O{sub 2} decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H{sub 2}O{sub 2} increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H{sub 2}O{sub 2}-induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21{sup Cip1}/PCNA complex plays an important role as a regulator of cell fate decisions.

  20. Cdk4 regulates recruitment of quiescent beta-cells and ductal epithelial progenitors to reconstitute beta-cell mass.

    Directory of Open Access Journals (Sweden)

    Ji-Hyeon Lee

    Full Text Available Insulin-producing pancreatic islet beta cells (beta-cells are destroyed, severely depleted or functionally impaired in diabetes. Therefore, replacing functional beta-cell mass would advance clinical diabetes management. We have previously demonstrated the importance of Cdk4 in regulating beta-cell mass. Cdk4-deficient mice display beta-cell hypoplasia and develop diabetes, whereas beta-cell hyperplasia is observed in mice expressing an active Cdk4R24C kinase. While beta-cell replication appears to be the primary mechanism responsible for beta-cell mass increase, considerable evidence also supports a contribution from the pancreatic ductal epithelium in generation of new beta-cells. Further, while it is believed that majority of beta-cells are in a state of 'dormancy', it is unclear if and to what extent the quiescent cells can be coaxed to participate in the beta-cell regenerative response. Here, we address these queries using a model of partial pancreatectomy (PX in Cdk4 mutant mice. To investigate the kinetics of the regeneration process precisely, we performed DNA analog-based lineage-tracing studies followed by mathematical modeling. Within a week after PX, we observed considerable proliferation of islet beta-cells and ductal epithelial cells. Interestingly, the mathematical model showed that recruitment of quiescent cells into the active cell cycle promotes beta-cell mass reconstitution in the Cdk4R24C pancreas. Moreover, within 24-48 hours post-PX, ductal epithelial cells expressing the transcription factor Pdx-1 dramatically increased. We also detected insulin-positive cells in the ductal epithelium along with a significant increase of islet-like cell clusters in the Cdk4R24C pancreas. We conclude that Cdk4 not only promotes beta-cell replication, but also facilitates the activation of beta-cell progenitors in the ductal epithelium. In addition, we show that Cdk4 controls beta-cell mass by recruiting quiescent cells to enter the cell

  1. ESTABLISHMENT OF A SHG44 CELL LINE STABLY TRANSFECTED BY CDK2 -SIRNA CONSTRUCT%稳定转染CDK2干扰RNA真核表达载体的人脑胶质细胞瘤SHG44细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    呼格吉乐; 苏仁娜; 高乃康

    2011-01-01

    Objective:To explore the function of CDK2, a stable expression of CDK2 SiRNA astro-cytoma cell line was established. Method: The Eukaryotic expression vector pGenesil-1-CDK2, CKD2 specific RNA interference, was constructed based on the sequence from Cenbank. The plasmid was se-quenced and transfected to SHG44 cell line using oligofectamine. The stable transfectants were selected by G418. Results; The sequence of the construct was confirmed right. All stable transfectants had significant lower expression of CDK2 compared with the control clones. Conclusion: The pGenesil-1-CDK2-SHG44 stable transfectants were successfully established with the constructed Pgenesil-1-CDK2 vector.%目的:构建CDK2的干扰RNA真核表达载体,并且稳定转染人脑胶质细胞瘤SHG44细胞来抑制CDK2的表达,为人脑胶质细胞瘤的研究提供有价值的资料.方法:1.根据siRNA设计原则和GeneBank数据库中CDK2的cDNA序列,构建CDK2干扰RNA真核表达载体PGenesil - 1- CDK2,并测序鉴定.2.利用脂质体法转染CDK2的干扰RNA真核表达栽体,G418筛选阳性转染细胞克隆,制备稳定转染干扰RNA真核表达栽体的SHG44细胞系,用倒置荧光显微镜观察荧光蛋白表达量.结果:1.成功构建了CDK2干扰RNA真核表达载体.经鉴定证实,构建的siRNAs序列与基因库中序列完全相同,并且未发现有突变、缺失、插入等异常存在.2.获得稳定转染CDK2干扰RNA真核表达栽体的SHG44细胞系,命名为pGenesil-1 - CDK2一SHG44.结论:成功的建立稳定转染CDK2干扰RNA的SHG44细胞系.

  2. 慢病毒介导的CDK2-shRNA促进黑色素瘤细胞A375凋亡

    Institute of Scientific and Technical Information of China (English)

    刘厚广; 刘卓; 姜颖; 肖井仁; 李红影; 李峥; 霍姗姗; 于英君

    2015-01-01

    目的 探讨慢病毒介导的细胞周期蛋白依赖性激酶(CDK)2-shRNA对人黑色素瘤细胞A375凋亡的影响.方法 依据CDK2基因序列,设计3条靶向干扰CDK2的序列,构建CDK2-shRNA慢病毒载体,质粒转染人胚肾细胞株(HEK293)细胞进行慢病毒包装,滴度测定包装的重组pGMLV-CDK2-shRNA慢病毒,免疫印迹检测对CDK2的干扰效率及对细胞周期蛋白(cyclin)E、E2F1、RB蛋白表达的影响,四甲基偶氮唑蓝比色(MTT)检测细胞增殖活力,Annexin V-FITC/PI染色流式细胞仪检测A375凋亡的发生.结果 成功获得包装的重组pGMLV-CDK2-shRNA慢病毒,病毒滴度为5×108TU/ml.重组的pGMLV-CDK2-shRNA慢病毒能有效下调CDK2、cyclinE、E2F1蛋白表达,抑制A375细胞增殖和触发凋亡.结论 基于慢病毒介导的CDK2-shRNA能够促进黑色素瘤细胞A375凋亡发生,为黑色素瘤的基因治疗提供理论支撑.

  3. Jak3, STAT3, and STAT5 inhibit expression of miR-22, a novel tumor suppressor microRNA, in cutaneous T-Cell lymphoma

    DEFF Research Database (Denmark)

    Sibbesen, Nina A; Kopp, Katharina L; Litvinov, Ivan V;

    2015-01-01

    malignant T cells with recombinant miR-22 inhibits the expression of validated miR-22 targets including NCoA1, a transcriptional co-activator in others cancers, as well as HDAC6, MAX, MYCBP, PTEN, and CDK2, which have all been implicated in CTCL pathogenesis. In conclusion, we provide the first evidence...

  4. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    Science.gov (United States)

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics. PMID:24286762

  5. CDK2 shRNA慢病毒载体的构建及其基因沉默效应

    Institute of Scientific and Technical Information of China (English)

    晋佳路; 朱仁书; 谢育媛; 刘红春

    2015-01-01

    目的:构建CDK2 shRNA慢病毒载体,并在黑色素瘤细胞B16-F1中鉴定其基因沉默效应。方法体外构建3个慢病毒重组目的质粒pUL-CDK2-shRNAs和1个阴性对照慢病毒重组质粒pUL-NC-shRNA,转化感受态细胞,PCR鉴定后进一步测序验证;293T细胞中测定病毒滴度;用重组慢病毒感染B16-F1细胞测定其感染效率,RT-PCR和Western印迹检测其对 B16-F1细胞中CDK2的基因沉默效应。结果 PCR鉴定后进一步测序表明,成功构建了重组慢病毒质粒;病毒滴度为4.5×107~5.5×107 TU/ml;用重组慢病毒感染 B16-F1细胞,当感染复数(MOI)为10时,感染效率可达90%;RT-PCR和Western印迹结果表明,与未感染组和NC-shRNA感染组细胞相比,CDK2-shRNA1、CDK2-shRNA2、CDK2-shRNA3感染的细胞中CDK2 mRNA和蛋白表达均受到不同程度抑制(P<0.05),以 CDK2-shRNA3感染组的抑制率最高,RT-PCR和 Western Blot 检测其抑制率分别为78.5%±4.23%和70.5%±3.54%。结论利用RNAi技术成功构建了3种CDK2-shRNA重组慢病毒载体,均可有效感染B16-F1细胞并具有一定的基因沉默效应,其中以针对靶位点1012-1020的pUL-CDK2-shRNA3基因沉默效应最强,为进一步研究CDK2基因沉默对黑色素瘤化疗敏感性的影响奠定了基础。

  6. Effect of gax gene on the expression of PCNA and CDK2 in vascular smooth muscle cells%gax基因对血管平滑肌细胞中PCNA和CDK2表达的影响

    Institute of Scientific and Technical Information of China (English)

    王耿; 韩雅玲; 冉擘力; 张萍; 景涛

    2005-01-01

    目的研究增殖细胞核抗原(PCNA)和细胞周期蛋白依赖性激酶2(CDK2)在gax基因抑制血管平滑肌细胞(VSMC)增殖中的作用.方法以携带大鼠gax基因表达序列的重组腺病毒载体(AdCMV-gax)转染VSMC后,检测gax、PCNA和CDK2的表达及3H-TdR掺入量的变化.结果 AdCMV-gax转染后,SMC中Gax蛋白的表达比转染前显著增高; AdCMV-gax转染后VSMC的PCNA和CDK2表达较未转染组显著降低; AdCMV-gax转染使VSMC的3H-TdR掺入量显著降低.结论 gax基因抑制VSMC增殖的机制与其抑制PCNA和CDK2的表达有关.

  7. CDK2、P57在子宫内膜腺癌中的表达及意义%Expression and significance of CDK2 prote and P57 prote in endometrial adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    张苗; 王永红

    2011-01-01

    目的 研究CDK2、P57在子宫内膜组织中的分布和表达,探讨其在子宫内膜腺癌发病中的作用.方法 运用原位杂交方法 检测CDK2、P57在14例正常子宫内膜、19例非典型增生子宫内膜、45例子宫内膜腺癌中的表达情况.蛄果CDK2蛋白在正常子宫内膜、非典型增生子宫内膜、子宫内膜腺癌的阳性表达率分别为7.14%、36.84%、71.11%,各组之间的差异有显著性(P0.05).P57蛋白在正常子宫内膜、非典型增生子宫内膜、子宫内膜腺癌的阳性表达率分别为71.43%、52.63%和44.44%,各组之问的差异有显著性(P0.05);P57蛋白表达与CDK2呈负相关(P<0.05).结论子宫内膜腺癌中存在CDK2蛋白的异常表达3LP57蛋白表达下降或缺失,促进了细胞的生长和肿瘤的发展,是子宫内膜腺癌的发生、发展中的重要事件.

  8. Cdk5 is a New Rapid Synaptic Homeostasis Regulator Capable of Initiating the Early Alzheimer-Like Pathology.

    Science.gov (United States)

    Sheng, Yanghui; Zhang, Lei; Su, Susan C; Tsai, Li-Huei; Julius Zhu, J

    2016-07-01

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase implicated in synaptic plasticity, behavior, and cognition, yet its synaptic function remains poorly understood. Here, we report that physiological Cdk5 signaling in rat hippocampal CA1 neurons regulates homeostatic synaptic transmission using an unexpectedly rapid mechanism that is different from all known slow homeostatic regulators, such as beta amyloid (Aβ) and activity-regulated cytoskeleton-associated protein (Arc, aka Arg3.1). Interestingly, overproduction of the potent Cdk5 activator p25 reduces synapse density, and dynamically regulates synaptic size by suppressing or enhancing Aβ/Arc production. Moreover, chronic overproduction of p25, seen in Alzheimer's patients, induces initially concurrent reduction in synapse density and increase in synaptic size characteristic of the early Alzheimer-like pathology, and later persistent synapse elimination in intact brains. These results identify Cdk5 as the regulator of a novel rapid form of homeostasis at central synapses and p25 as the first molecule capable of initiating the early Alzheimer's synaptic pathology. PMID:26088971

  9. Effects of p35 Mutations Associated with Mental Retardation on the Cellular Function of p35-CDK5.

    Directory of Open Access Journals (Sweden)

    Shunsuke Takada

    Full Text Available p35 is an activation subunit of the cyclin-dependent kinase 5 (CDK5, which is a Ser/Thr kinase that is expressed predominantly in neurons. Disruption of the CDK5 or p35 (CDK5R1 genes induces abnormal neuronal layering in various regions of the mouse brain via impaired neuronal migration, which may be relevant for mental retardation in humans. Accordingly, mutations in the p35 gene were reported in patients with nonsyndromic mental retardation; however, their effect on the biochemical function of p35 has not been examined. Here, we studied the biochemical effect of mutant p35 on its known properties, i.e., stability, CDK5 activation, and cellular localization, using heterologous expression in cultured cells. We also examined the effect of the mutations on axon elongation in cultured primary neurons and migration of newborn neurons in embryonic brains. However, we did not detect any significant differences in the effects of the mutant forms of p35 compared with wild-type p35. Therefore, we conclude that these p35 mutations are unlikely to cause mental retardation.

  10. Preclinical Metabolism and Pharmacokinetics of SB1317 (TG02), a Potent CDK/JAK2/FLT3 Inhibitor

    NARCIS (Netherlands)

    Pasha, Mohammed Khalid; Jayaraman, Ramesh; Reddy, Venkatesh Pilla; Yeo, Pauline; Goh, Evelyn; Williams, Anthony; Goh, Kee Chuan; Kantharaj, Ethirajulu

    2012-01-01

    SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed >99% binding to

  11. The Involvement of p53-miR-34a-CDK4 Signaling During the Development of Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Huijun Zuo

    2015-01-01

    Conclusion: With the progression of cervical cancer lesions, the positive rate of p53 was greatly increased which may indicate its mutation, while miR-34a was down-regulated and CDK4 was up-regulated, which suggested that all these factors might be involved in the process of cervical oncogenesis.

  12. EFFECTS OF siRNA TARGED CDK2 AND cyclinE ON CELL CYCLE AND APOPTOSIS OF HepG2 CELLS%靶向CDK2、cyclinE的siRNA对HepG2细胞周期及凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    曹银芳; 关泽红; 刘新风

    2009-01-01

    目的:探讨细胞周期蛋白依赖性激酶(cyclin-dependent-kinase 2,CDK2)活性对肝癌细胞株HepG2细胞周期和细胞凋亡的影响.方法:根据基因库中登录的人和鼠CDK2、cyclinE序列,设计并构建CDK2、cyclinE干扰RNA真核表达载体;脂质体法转染肝癌细胞株HepG2细胞,流式细胞术分析CDK2及cyclinE对HepG2细胞增殖的影响;蛋白质印迹法检测CDK2、cyclinE活性的变化caspase-3活性的影响.结果:1.成功构建CDK2及cyclinE干扰RNA真核表达载体psiCDK2、psiCyclinE,用脂质体法导入肝癌细胞株HepG2细胞中,有效表达.2.转染48h后与空载体组相比:psiCDK2、psiCyclinE组G1期细胞增多,G2/M和S期细胞减少;蛋白质印迹法分析表明psiCDK2、psiCyclinE组caspase-3酶原被激活.结论:靶向CDK2、cyclinE的siRNA能抑制HepG2细胞的增殖;靶向CDK2、cyclinE的siRNA能激活caspase-3,诱导肝癌细胞HepG2凋亡.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  14. 甲状腺癌中CyclinE及CDK2的表达及意义

    Institute of Scientific and Technical Information of China (English)

    张安文; 史琳; 唐艳妮; 罗宇

    2013-01-01

      目的探讨CyclinE和CDK2蛋白的表达与甲状腺癌发生、发展的关系.方法采用免疫组织化学S-P法检测甲状腺癌49例、甲状腺腺瘤26例、结节性甲状腺肿29例、正常甲状腺组织10例中CyclinE和CDK2蛋白的表达,并结合临床资料进行分析.结果 CyclinE和CDK2蛋白在甲状腺癌组的阳性表达率分别为61.22%(30/49)、75.51%(37/49),明显高于甲状腺腺瘤组、结节性甲状腺肿组及正常甲状腺组的表达率(P 0.05).CyclinE与CDK2在甲状腺癌中的表达呈正相关(r=0.6524,P <0.01).结论CyclinE和CDK2在甲状腺癌中呈高表达状态,提示两者可能与甲状腺癌的发生发展有关.二者联合检测或许能作为临床诊断及判断甲状腺肿瘤细胞增殖活性的参考指标.

  15. Osthole inhibits proliferation of human breast cancer cells by inducing cell cycle arrest and apoptosis

    Institute of Scientific and Technical Information of China (English)

    Lintao Wang; Yanyan Peng; Kaikai Shi; Haixiao Wang; Jianlei Lu; Yanli Li; Changyan Ma

    2015-01-01

    Recent studies have revealed that osthole,an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson,a traditional Chinese medicine,possesses anticancer activity.However,its effect on breast cancer cells so far has not been elucidated clearly.In the present study,we evaluated the effects of osthole on the proliferation,cell cycle and apoptosis of human breast cancer cells MDA-MB 435.We demonstrated that osthole is effective in inhibiting the proliferation of MDA-MB 435 cells,The mitochondrion-mediated apoptotic pathway was involved in apoptosis induced by osthole,as indicated by activation of caspase-9 and caspase-3 followed by PARP degradation.The mechanism underlying its effect on the induction of G1 phase arrest was due to the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Were observed taken together,these findings suggest that the anticancer efficacy of osthole is mediated via induction of cell cycle arrest and apoptosis in human breast cancer cells and osthole may be a potential chemotherapeutic agent against human breast cancer.

  16. Significance of Cyclin A、CDK2 Expression in Non-Small-Cell Lung Cancer%Cyclin A、CDK2基因在非小细胞肺癌中的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    张胜名; 刘铭球; 李奇志; 毛永荣; 王敏

    2005-01-01

    目的研究细胞周期素A(Cyclin A)和细胞周期素依赖性激酶2(cyclin-dependent kinases,CDK2)基因在非小细胞肺癌(non-small-cell lung cancer,NSCLC)组织中的表达及其相互关系,探讨其对NSCLC发生、发展、淋巴结转移及预后的影响.方法采用免疫组织化学二步法检测40例NSCLC(伴淋巴结转移21例,不伴淋巴结转移19例),11例支气管黏膜上皮增生或不典型增生,9例淋巴结转移癌组织中Cyclin A、CDK2蛋白的表达,并随访40例NSCLC患者3年生存期.结果在支气管黏膜上皮增生或不典型增生,不伴淋巴结转移的NSCLC,伴淋巴结转移的NSCLC,淋巴结转移癌组织中,Cyclin A蛋白的阳性表达率分别为9.09%(1/11),31.58%(6/19),80.95%(17/21),66.67%(6/9);CDK2蛋白的阳性表达率分别为9.09%(1/11),36.84%(7/19),76.19%(16/21),77.78%(7/9);不伴淋巴结转移的NSCLC组织中的Cyclin A、CDK2蛋白阳性表达率分别与伴淋巴结转移NSCLC组织、淋巴结转移癌组织的Cyclin A、CDK2蛋白阳性表达率比较,差异均有显著性(P均<0.05).23例Cyclin A蛋白表达阳性患者3年生存率为21.74%(5/23),17例表达阴性患者3年生存率为58.82%(10/17),两者比较有显著性差异(P<0.05);23例CDK2蛋白表达阳性患者3年生存率为17.39%(4/23),17例表达阴性患者3年生存率为64.71%(11/17),两者比较有显著性差异(P<0.05).NSCLC中Cyclin A与CDK2蛋白表达呈正相关(x2=19.22,P<0.001,列联系数Pearson=0.570).结论在NSCLC发生、演进、浸润、淋巴结转移过程中CyclinA、CDK2起正调控作用,NSCLC组织中CyclinA、CDK2表达上调可作为判断NSCLC预后不良的参考指标.

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

    Science.gov (United States)

    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

  18. HBx-upregulated lncRNA UCA1 promotes cell growth and tumorigenesis by recruiting EZH2 and repressing p27Kip1/CDK2 signaling.

    Science.gov (United States)

    Hu, Jiao-Jiao; Song, Wei; Zhang, Shao-Dan; Shen, Xiao-Hui; Qiu, Xue-Mei; Wu, Hua-Zhang; Gong, Pi-Hai; Lu, Sen; Zhao, Zhu-Jiang; He, Ming-Liang; Fan, Hong

    2016-01-01

    It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC. PMID:27009634

  19. Inhibition of Post-Transcriptional RNA Processing by CDK Inhibitors and Its Implication in Anti-Viral Therapy

    Czech Academy of Sciences Publication Activity Database

    Holčáková, J.; Müller, P.; Tomasec, P.; Hrstka, R.; Nekulová, M.; Kryštof, Vladimír; Strnad, Miroslav; Wilkinson, G. W. G.; Vojtěšek, B.

    2014-01-01

    Roč. 9, č. 2 (2014). E-ISSN 1932-6203 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61389030 Keywords : IMMUNODEFICIENCY-VIRUS TYPE-1 * DEPENDENT KINASE INHIBITORS * LARGE T-ANTIGEN Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  20. Sequencing Analysis of Mutant Allele $cdc$28-$srm$ of Protein Kinase CDC28 and Molecular Dynamics Study of Glycine-Rich Loop in Wild-Type and Mutant Allele G16S of CDK2 as Model

    CERN Document Server

    Koltovaya, N A; Kholmurodov, Kh T; Kretov, D A

    2005-01-01

    The central role that cyclin-dependent kinases play in the timing of cell division and the high incidence of genetic alteration of CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of the yeast \\textit{Saccharomyces cerevisiae }very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including \\textit{cdc28-srm} affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A~\\textit{cdc28-srm} mutation is not temperature-sensitive mutation and differs from the known \\textit{cdc28-ts }mutations because it has the evident phenotypic manifestations at 30 $^{\\circ}$C. Sequencing analysis of \\textit{cdc28-srm} revealed a single nucleotide substitution G20S. This is a third glycine in a conserved sequence GxGxxG in the G-rich loop positioned opposite the activation T-loop. Despite its demonstrated importance, the role of the G-loop has remained unclear. The crystal stru...

  1. 甲状腺癌中CyclinE及CDK2的表达及意义

    Institute of Scientific and Technical Information of China (English)

    张安文; 史琳; 唐艳妮; 罗宇

    2013-01-01

    目的探讨CyclinE和CDK2蛋白的表达与甲状腺癌发生、发展的关系。方法采用免疫组织化学S-P法检测甲状腺癌49例、甲状腺腺瘤26例、结节性甲状腺肿29例、正常甲状腺组织10例中CyclinE和CDK2蛋白的表达,并结合临床资料进行分析。结果 CyclinE和CDK2蛋白在甲状腺癌组的阳性表达率分别为61.22%(30/49)、75.51%(37/49),明显高于甲状腺腺瘤组、结节性甲状腺肿组及正常甲状腺组的表达率(P〈0.01)。甲状腺癌组中的CyclinE、CDK2蛋白表达率与患者的性别、年龄、肿瘤大小及淋巴结转移情况无相关性(P〉0.05)。CyclinE与CDK2在甲状腺癌中的表达呈正相关(r=0.6524,P〈0.01)。结论CyclinE和CDK2在甲状腺癌中呈高表达状态,提示两者可能与甲状腺癌的发生发展有关。二者联合检测或许能作为临床诊断及判断甲状腺肿瘤细胞增殖活性的参考指标。

  2. A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M;

    2001-01-01

    how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a...... conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding......, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates....

  3. Phosphorylation of CDK9 at Ser175 Enhances HIV Transcription and Is a Marker of Activated P-TEFb in CD4+ T Lymphocytes

    Science.gov (United States)

    Mbonye, Uri R.; Gokulrangan, Giridharan; Datt, Manish; Dobrowolski, Curtis; Cooper, Maxwell; Chance, Mark R.; Karn, Jonathan

    2013-01-01

    The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for

  4. Phosphorylation of CDK9 at Ser175 enhances HIV transcription and is a marker of activated P-TEFb in CD4(+ T lymphocytes.

    Directory of Open Access Journals (Sweden)

    Uri R Mbonye

    Full Text Available The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification-tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative

  5. 腺相关病毒介导的CDK2-shRNA促进黑色素瘤细胞A375凋亡

    Institute of Scientific and Technical Information of China (English)

    刘厚广; 刘卓; 姜颖; 肖井仁; 李红影; 李峥; 霍姗姗; 于英君

    2015-01-01

    目的 探讨腺相关病毒介导的CDK2-shRNA对人黑色素瘤细胞A375凋亡,为黑色素瘤的防治提供技术支持.方法 体外合成CDK2及转录终止序列,构建CDK2-shRNA的腺相关病毒载体和包装相应的重组腺相关病毒,荧光显微镜检测转染和感染条件下CDK2-shRNA表达效果,MTT法检测细胞活力,Annexin V-FITC/PI细胞凋亡试剂盒检测细胞凋亡,免疫印迹检测cyclin E和E2F1表达.结果 转染和感染的腺相关病毒介导的CDK2-shRNA在A375细胞均有表达且具有较低的细胞毒性;转染和感染的CDK2-shRNA均促进A375细胞发生凋亡;干扰CDK2可降低cyclin E和E2F1表达.结论 相关病毒介导的CDK2-shRNA促进黑色素瘤细胞A375凋亡.

  6. CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

    DEFF Research Database (Denmark)

    Cepeda, Diana; Ng, Hwee-Fang; Sharifi, Hamid Reza;

    2013-01-01

    results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in...... transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer....

  7. The Expression of cyclin E and CDK2 in Human Uterine Smooth Muscle Tumors%cyclinE、CDK2在子宫平滑肌肿瘤中的表达

    Institute of Scientific and Technical Information of China (English)

    张素丽; 郑红兵

    2006-01-01

    @@ 0 引言 子宫平滑肌肿瘤(uterine smooth muscle tumors, USMTs)是妇女最常见的肿瘤.细胞周期素(cyclinE)是G1期的周期蛋白,与细胞周期素依赖性激酶2(CDK2)在G1期末结合而发挥作用,促进细胞进入S期.近年来,在多种肿瘤的研究中发现有cyclinE、CDK2表达的异常,但其在USMTs中的研究,国内文献尚未见报道.增殖细胞核抗原(PCNA)是反映细胞,特别是恶性肿瘤细胞增殖活性的一个指标.本研究拟通过免疫组织化学方法探讨cyclinE、CDK2及PCNA在USMTs中的表达及临床意义.

  8. @@%熊果酸通过抑制CDK2活性诱导HepG2细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    吴晓薇; 林锐珊

    2014-01-01

    目的:研究熊果酸抑制HepG2细胞增殖的分子机制.方法:应用CDK2试剂盒检测熊果酸对CDK2的抑制效果以及浓度和时间等影响因素,用流式细胞术验证CDK2抑制所造成的细胞周期阻滞,并用Annexin V/PI检测细胞凋亡.结果:CDK2试剂盒实验显示熊果酸对CDK2具有抑制作用,并呈时间和浓度依赖性;细胞周期检测结果表明熊果酸能将HepG2细胞周期阻滞在G0/G1期,Annexin V/PI细胞凋亡检测其早期凋亡率达24.12%.结论:熊果酸能抑制CDK2活性,将HepG2细胞周期阻滞于G0/G1期,从而抑制HepG2细胞增殖,并进一步诱导细胞凋亡.

  9. Molecular modelling on small molecular CDK2 inhibitors: an integrated approach using a combination of molecular docking, 3D-QSAR and pharmacophore modelling.

    Science.gov (United States)

    Yuan, H; Liu, H; Tai, W; Wang, F; Zhang, Y; Yao, S; Ran, T; Lu, S; Ke, Z; Xiong, X; Xu, J; Chen, Y; Lu, T

    2013-10-01

    Cyclin-dependent kinase 2 (CDK2) has been identified as an important target for developing novel anticancer agents. Molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) and pharmacophore modelling were combined with the ultimate goal of studying the structure-activity relationship of CDK2 inhibitors. The comparative molecular similarity indices analysis (CoMSIA) model constructed based on a set of 3-aminopyrazole derivatives as CDK2 inhibitors gave statistically significant results (q (2) = 0.700; r (2) = 0.982). A HypoGen pharmacophore model, constructed using diverse CDK2 inhibitors, also showed significant statistics ([Formula: see text]Cost = 61.483; RMSD = 0.53; Correlation coefficient = 0.98). The small residues and error values between the estimated and experimental activities of the training and test set compounds proved their strong capability of activity prediction. The structural insights obtained from these two models were consistent with each other. The pharmacophore model summarized the important pharmacophoric features required for protein-ligand binding. The 3D contour maps in combination with the comprehensive pharmacophoric features helped to better interpret the structure-activity relationship. The results will be beneficial for the discovery and design of novel CDK2 inhibitors. The simplicity of this approach provides expansion to its applicability in optimizing other classes of small molecular CDK2 inhibitors. PMID:23941641

  10. A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

    Directory of Open Access Journals (Sweden)

    Nan Hua

    Full Text Available Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs. In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC.

  11. A novel muscarinic antagonist R2HBJJ inhibits non-small cell lung cancer cell growth and arrests the cell cycle in G0/G1.

    Science.gov (United States)

    Hua, Nan; Wei, Xiaoli; Liu, Xiaoyan; Ma, Xiaoyun; He, Xinhua; Zhuo, Rengong; Zhao, Zhe; Wang, Liyun; Yan, Haitao; Zhong, Bohua; Zheng, Jianquan

    2012-01-01

    Lung cancers express the cholinergic autocrine loop, which facilitates the progression of cancer cells. The antagonists of mAChRs have been demonstrated to depress the growth of small cell lung cancers (SCLCs). In this study we intended to investigate the growth inhibitory effect of R2HBJJ, a novel muscarinic antagonist, on non-small cell lung cancer (NSCLC) cells and the possible mechanisms. The competitive binding assay revealed that R2HBJJ had a high affinity to M3 and M1 AChRs. R2HBJJ presented a strong anticholinergic activity on carbachol-induced contraction of guinea-pig trachea. R2HBJJ markedly suppressed the growth of NSCLC cells, such as H1299, H460 and H157. In H1299 cells, both R2HBJJ and its leading compound R2-PHC displayed significant anti-proliferative activity as M3 receptor antagonist darifenacin. Exogenous replenish of ACh could attenuate R2HBJJ-induced growth inhibition. Silencing M3 receptor or ChAT by specific-siRNAs resulted in a growth inhibition of 55.5% and 37.9% on H1299 cells 96 h post transfection, respectively. Further studies revealed that treatment with R2HBJJ arrested the cell cycle in G0/G1 by down-regulation of cyclin D1-CDK4/6-Rb. Therefore, the current study reveals that NSCLC cells express an autocrine and paracrine cholinergic system which stimulates the growth of NSCLC cells. R2HBJJ, as a novel mAChRs antagonist, can block the local cholinergic loop by antagonizing predominantly M3 receptors and inhibit NSCLC cell growth, which suggest that M3 receptor antagonist might be a potential chemotherapeutic regimen for NSCLC. PMID:23285263

  12. Cdk1 Phosphorylates Drosophila Sas-4 to Recruit Polo to Daughter Centrioles and Convert Them to Centrosomes.

    Science.gov (United States)

    Novak, Zsofia A; Wainman, Alan; Gartenmann, Lisa; Raff, Jordan W

    2016-06-20

    Centrosomes and cilia are organized by a centriole pair comprising an older mother and a younger daughter. Centriole numbers are tightly regulated, and daughter centrioles (which assemble in S phase) cannot themselves duplicate or organize centrosomes until they have passed through mitosis. It is unclear how this mitotic "centriole conversion" is regulated, but it requires Plk1/Polo kinase. Here we show that in flies, Cdk1 phosphorylates the conserved centriole protein Sas-4 during mitosis. This creates a Polo-docking site that helps recruit Polo to daughter centrioles and is required for the subsequent recruitment of Asterless (Asl), a protein essential for centriole duplication and mitotic centrosome assembly. Point mutations in Sas-4 that prevent Cdk1 phosphorylation or Polo docking do not block centriole disengagement during mitosis, but block efficient centriole conversion and lead to embryonic lethality. These observations can explain why daughter centrioles have to pass through mitosis before they can duplicate and organize a centrosome. PMID:27326932

  13. MicroRNA-34a Inhibits Osteoblast Differentiation and In Vivo Bone Formation of Human Stromal Stem Cells

    DEFF Research Database (Denmark)

    Chen, Li; Holmstrøm, Kim; Qiu, Weimin; Ditzel, Nicholas; Shi, Kaikai; Hokland, Lea; Kassem, Moustapha

    2014-01-01

    target of miR-34a. siRNA-mediated reduction of JAG1 expression inhibited OB differentiation. Moreover, a number of known cell cycle regulator and cell proliferation proteins, such as cyclin D1, cyclin-dependent kinase 4 and 6 (CDK4 and CDK6), E2F transcription factor three, and cell division cycle 25...... regulatory effects controlling both hMSC proliferation and OB differentiation. Tissue-specific inhibition of miR-34a might be a potential novel therapeutic strategy for enhancing in vivo bone formation. Stem Cells 2014;32:902-912......Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, microRNAs (miRNAs) were identified as novel key regulators of human stromal (skeletal, mesenchymal) stem cells (hMSC) differentiation. Here, we identified mi...

  14. Cdk2 Silencing via a DNA/PCL Electrospun Scaffold Suppresses Proliferation and Increases Death of Breast Cancer Cells

    OpenAIRE

    Achille, Clément; Sundaresh, Sowmya; Chu, Benjamin; Hadjiargyrou, Michael

    2012-01-01

    RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolac...

  15. CDK2-dependent phosphorylation of Suv39H1 is involved in control of heterochromatin replication during cell cycle progression

    OpenAIRE

    Park, Su Hyung; Yu, Seung Eun; Chai, Young Gyu; Jang, Yeun Kyu

    2014-01-01

    Although several studies have suggested that the functions of heterochromatin regulators may be regulated by post-translational modifications during cell cycle progression, regulation of the histone methyltransferase Suv39H1 is not fully understood. Here, we demonstrate a direct link between Suv39H1 phosphorylation and cell cycle progression. We show that CDK2 phosphorylates Suv39H1 at Ser391 and these phosphorylation levels oscillate during the cell cycle, peaking at S phase and maintained d...

  16. Evidence that two Pcl-like cyclins control Cdk9 activity during cell differentiation in Aspergillus nidulans asexual development.

    Science.gov (United States)

    Kempf, Claudia; Bathe, Friederike; Fischer, Reinhard

    2013-01-01

    Cyclin-dependent protein kinases (CDKs) are usually involved in cell cycle regulation. However, Cdk9 is an exception and promotes RNA synthesis through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). The CTD is comprised of repeating heptapeptides, in which serine residues at positions 2, 5, and 7 are of crucial importance. Ser5 phosphorylation causes transcription initiation and promoter escape. However, RNAPII pauses 20 to 50 bp downstream from the transcription start site, until Cdk9 phosphorylates Ser2. This event relieves the checkpoint and promotes the processivity of elongation. Here we present evidence that in the filamentous fungus Aspergillus nidulans, a Cdk9 homologue, PtkA, serves specific functions in conidiophore development. It was previously shown that PtkA interacts with two cyclins, PclA and the T cyclin PchA. Using yeast two-hybrid screens, we identified a third cyclin, PclB, and a kinase, PipA(Bud32). Both proteins were expressed in hyphae and in conidiophores, but interaction between each protein and PtkA was restricted to the conidiophores. Deletion of pchA caused a severe growth defect, and deletion of pipA was lethal, suggesting basic functions in PtkA-dependent gene transcription. In contrast, deletion of pclB in combination with deletion of pclA essentially caused a block in spore formation. We present evidence that the phosphorylation status of the CTD of RNA polymerase II in the conidiophore changes upon deletion of pclA or pclB. Our results suggest that tissue-specific modulation of Cdk9 activity by PclA and PclB is required for proper differentiation. PMID:23104571

  17. A PSTAIRE CDK-like protein localizes in nuclei and cytoplasm of Physarum polycephalum and functions in the mitosis

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    CDKs play key roles in controlling cell cycle progression in all eukaryotes. In plants, multiple CDKs are present,among which the best characterized CDKs are PSTAIRE CDKs. In this study, we carried out Western blot,immunoelectron microscopy and antibody treatment with an anti-PSTAIRE monoclonal antibody to explore the subcellular localization and functions of PSTAIRE CDKs in Physarum polycephalum. The results of Western blot and immunoelectron microscopy showed that in P. polycephalum, a PSTAIRE CDK-like protein was 34 kD in molecular weight and located in both nuclei and cytoplasm. In nuclei, the protein was mainly associated with chromosomes and nucleoli. The expression of the PSTAIRE CDK-like protein in both the plasmodia and nuclei showed little fluctuation through the whole cell cycle. When treated with an anti-PSTAIRE monoclonal antibody at early S phase, the cells were arrested in S phase, and the mitotic onset of P. polycephalum was blocked for about 1 h when treated at early G2 phase.Our data indicated that the PSTAIRE CDK- like protein has a direct bearing on the mitosis.

  18. TRPV1 function is modulated by Cdk5-mediated phosphorylation: insights into the molecular mechanism of nociception.

    Science.gov (United States)

    Jendryke, Thomas; Prochazkova, Michaela; Hall, Bradford E; Nordmann, Grégory C; Schladt, Moritz; Milenkovic, Vladimir M; Kulkarni, Ashok B; Wetzel, Christian H

    2016-01-01

    TRPV1 is a polymodally activated cation channel acting as key receptor in nociceptive neurons. Its function is strongly affected by kinase-mediated phosphorylation leading to hyperalgesia and allodynia. We present behavioral and molecular data indicating that TRPV1 is strongly modulated by Cdk5-mediated phosphorylation at position threonine-407(mouse)/T406(rat). Increasing or decreasing Cdk5 activity in genetically engineered mice has severe consequences on TRPV1-mediated pain perception leading to altered capsaicin consumption and sensitivity to heat. To understand the molecular and structural/functional consequences of TRPV1 phosphorylation, we generated various rTRPV1T406 receptor variants to mimic phosphorylated or dephosphorylated receptor protein. We performed detailed functional characterization by means of electrophysiological whole-cell and single-channel recordings as well as Ca(2+)-imaging and challenged recombinant rTRPV1 receptors with capsaicin, low pH, or heat. We found that position T406 is critical for the function of TRPV1 by modulating ligand-sensitivity, activation, and desensitization kinetics as well as voltage-dependence. Based on high resolution structures of TRPV1, we discuss T406 being involved in the molecular transition pathway, its phosphorylation leading to a conformational change and influencing the gating of the receptor. Cdk5-mediated phosphorylation of T406 can be regarded as an important molecular switch modulating TRPV1-related behavior and pain sensitivity. PMID:26902776

  19. Preparation and identification of the polyclonal antibodies against cyclin-dependent kinase 2%细胞周期蛋白质依赖性激酶CDK2多克隆抗体的制备和鉴定

    Institute of Scientific and Technical Information of China (English)

    李清; 陈越; 陈勇; 佟立全; 孔维; 金英花

    2007-01-01

    我们采用PCR技术合成编码CDK2肽段的基因,将其置于谷胱甘肽转移酶(GST)编码基因的下游,在IPTG诱导下,于E.coli中诱导表达了GST-CDK2肽融合蛋白质,以此融合蛋白质作为免疫原免疫家兔制备抗CDK2的多克隆抗体.经Western Blot检测证明:该抗体能够特异地识别CDK2蛋白质,可作为CDK2的特异性检测抗体,用于研究细胞周期和细胞凋亡进程中CDK2的作用.

  20. Influence of human cytomegalovirus infection on CDK2 in host cell%人巨细胞病毒感染对宿主细胞周期素依赖性蛋白激酶2的影响

    Institute of Scientific and Technical Information of China (English)

    刘楠; 余艳红; 赵杨; 张(龙夭); 闻良珍

    2006-01-01

    目的 观察HCMV感染细胞周期素依赖性蛋白激酶2(Cdk2)的亚细胞定位,研究HCMV感染对Cdk2蛋白水平及对细胞周期蛋白E(CyclinE)/Cdk2激酶活性的影响.方法 通过密度抑制使细胞同步化于G0/G1期,用HCMV AD169毒株感染人胚肺成纤维细胞(HEL),用免疫细胞化学技术分别测定HCMV感染前及感染后24 h Cdk2亚细胞定位;Western Blot法测定HCMV Cdk2蛋白丰度;用免疫沉淀,激酶活性分析法检测HCMV感染细胞内Cdk2的活性.结果 接触抑制阻止在G0期细胞Cdk2游离在细胞质,HCMV感染24 h内导致Cdk2从细胞质移位到细胞核.同时HCMV感染可引起cyclinE/Cdk2激酶的强烈激活,但HCMV感染并不诱导Cdk2蛋白丰度增加.结论 HCMV感染G0/G1细胞,在24 h内导致Cdk2从细胞质移位到细胞核,使之与细胞核内的调节亚单位CyclinE结合,激活CyclinE/Cdk2激酶,使细胞周期越过G1/S限制点,进展至晚G1期.

  1. β2-Syntrophin is a Cdk5 substrate that restrains the motility of insulin secretory granules.

    Directory of Open Access Journals (Sweden)

    Sandra Schubert

    Full Text Available The molecular basis for the interaction of insulin granules with the cortical cytoskeleton of pancreatic β-cells remains unknown. We have proposed that binding of the granule protein ICA512 to the PDZ domain of β2-syntrophin anchors granules to actin filaments and that the phosphorylation/dephosphorylation of β2-syntrophin regulates this association. Here we tested this hypothesis by analyzing INS-1 cells expressing GFP-β2-syntrophin through the combined use of biochemical approaches, imaging studies by confocal and total internal reflection fluorescence microscopy as well as electron microscopy. Our results support the notion that β2-syntrophin restrains the mobility of cortical granules in insulinoma INS-1 cells, thereby reducing insulin secretion and increasing insulin stores in resting cells, while increasing insulin release upon stimulation. Using mass spectrometry, in vitro phosphorylation assays and β2-syntrophin phosphomutants we found that phosphorylation of β2-syntrophin on S75 near the PDZ domain decreases its binding to ICA512 and correlates with increased granule motility, while phosphorylation of S90 has opposite effects. We further show that Cdk5, which regulates insulin secretion, phosphorylates S75. These findings provide mechanistic insight into how stimulation displaces insulin granules from cortical actin, thus promoting their motility and exocytosis.

  2. Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

    Directory of Open Access Journals (Sweden)

    Margaret A Schwarz

    Full Text Available BACKGROUND: Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

  3. Mannose-binding lectin inhibits monocyte proliferation through transforming growth factor-β1 and p38 signaling pathways.

    Directory of Open Access Journals (Sweden)

    Yan Wang

    Full Text Available Mannose-binding lectin (MBL, a plasma C-type lectin, plays an important role in innate immunity. However, the interaction, and the consequences of it, between MBL and the immune system remain ill defined. We have investigated the contributing mechanisms and effects of MBL on the proliferation of human monocytes. At lower concentrations (≤4 μg/ml MBL was shown to partially enhance monocyte proliferation. By contrast, at higher concentrations (8-20 μg/ml of MBL, cell proliferation was markedly attenuated. MBL-induced growth inhibition was associated with G0/G1 arrest, down-regulation of cyclin D1/D3, cyclin-dependent kinase (Cdk 2/Cdk4 and up-regulation of the Cdk inhibitory protein Cip1/p21. Additionally, MBL induced apoptosis, and did so through caspase-3 activation and poly ADP-ribose polymerase (PARP cleavage. Moreover, transforming growth factor (TGF-β1 levels increased in the supernatants of MBL-stimulated monocyte cultures. We also found that MBL-dependent inhibition of monocyte proliferation could be reversed by the TGF-β receptor antagonist SB-431542, or by anti-TGF-β1 antibody, or by the mitogen-activated protein kinase (MAPK inhibitors specific for p38 (SB203580, but not ERK (U0126 or JNK (SP600125. Thus, at high concentrations, MBL can affect the immune system by inhibiting monocyte proliferation, which suggests that MBL may exhibit anti-inflammatory effects.

  4. 氯沙坦对Thy1肾炎大鼠肾小球系膜细胞CDK2表达的影响

    Institute of Scientific and Technical Information of China (English)

    秦福芳; 孙倩; 邵凤民; 阎磊

    2013-01-01

    目的 观察氯沙坦对Thy1肾炎大鼠肾小球系膜细胞人细胞周期蛋白依赖性激酶2(CDK2)表达的影响.方法 实验鼠分为Thy1肾炎组、Thy1肾炎+氯沙坦治疗组和正常组.诱导肾脏疾病后,第1、3、5、7天检查病理.用免疫组化方法检测;肾小球内PCNA和CDK2蛋白的表达情况,采用Western印迹分析CDK2的表达情况.结果 对于正常大鼠,其系膜细胞的CDK2表达量很低,但具有系膜细胞增生现象的肾炎大鼠,CDK2表达会有增加趋势.比较而言,用氯沙坦治疗3~7d后,肾小球内的PCNA表达明显低于肾炎组(P<0.05).结论 CDK2可导致肾小球系膜细胞增生,氯沙坦对系膜细胞CDK2的高表达有明显的抑制作用,进而抑制系膜细胞的增生,表明氯沙坦可以治疗Thy1肾炎大鼠系膜细胞增殖.

  5. Selective inhibition of jasmonic acid accumulation by a small α, β-unsaturated carbonyl and phenidone reveals different modes of octadecanoid signalling activation in response to insect elicitors and green leaf volatiles in Zea mays

    Directory of Open Access Journals (Sweden)

    Engelberth Jurgen

    2011-10-01

    Full Text Available Abstract Background Plants often release a complex blend of volatile organic compounds (VOC in response to insect herbivore damage. Among those blends of VOC green leaf volatiles (GLV have been demonstrated to function as defence signals between plants, thereby providing protection against impending herbivory. A problem in understanding the mode of action of these 6-carbon aldehydes, alcohols, and esters is caused by their structural diversity. Besides different degrees of oxidation, E-2- as well as Z-3-configured isomers are often released. This study was therefore initiated to determine the structural requirement necessary to exhibit biological activity measured as jasmonic acid (JA accumulation in Zea mays seedlings. Findings The structure/function analysis of green leaf volatiles and related compounds revealed that an olefinic bond in position 2 or 3 and a size of 6-8 carbons is required for biological activity in maize. Also, it was found that the presence of an α, β-unsaturated carbonyl is not a prerequisite for activity. However, by treating plants first with volatile acrolein it was discovered that this smallest α, β-unsaturated carbonyl inhibits JA accumulation in response to insect elicitor treatment, but not after GLV exposure. This selective inhibitory effect was also found for phenidone, an inhibitor of lipoxygenases. These findings led to the discovery of a pool of protein-associated 12-oxo-phytodienoic acid, a biosynthetic precursor of JA, which appeared to be rapidly converted into JA upon exposure to GLV. Conclusions The structure/function analysis of GLV demonstrates a high degree of correlation between the compounds released by wounded plants in nature and their biological activity. The selective inhibitory effects of acrolein and phenidone on insect elicitor- and GLV-induced JA accumulation in maize led to the discovery of a pool of protein-associated precursor, which is rapidly activated and transformed to JA after

  6. KSHV G protein-coupled receptor inhibits lytic gene transcription in primary-effusion lymphoma cells via p21-mediated inhibition of Cdk2

    OpenAIRE

    Cannon, M; Cesarman, E; Boshoff, C

    2006-01-01

    Kaposi sarcoma (KS) remains the most common AIDS-associated malignancy worldwide. In sub-Saharan Africa especially, this aggressive endothelial-cell tumor is a cause of widespread morbidity and mortality. Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is now known to be an etiologic force behind KS and primary-effusion lymphoma (PEL). Over time, KSHV has pirated many human genes whose products regulate angiogenesis, inflammation, and the cell cycle. One of these, the KSHV vGPCR, ...

  7. 茵陈蒿对实验性食道肿瘤大鼠P53和cdk2表达的影响%Study on Effect of Herba Artemisiae Scoparia to p53 and cdK2 Express of Experimental Esophageal Tumor Rats

    Institute of Scientific and Technical Information of China (English)

    洪振丰; 高碧珍; 许碧玉; 王郑选

    2001-01-01

    为研究中药茵陈蒿的抗肿瘤作用机理,观察了茵陈蒿水煎剂对实验性食道肿瘤大鼠病变组织P43和cdk2表达的影响.结果表明,肿瘤大鼠食道组织P53、cdk2表达增高,而饮用菌陈蒿水煎剂各组P53、cdk2的表达均不同程度受到抑制.提示茵陈蒿水煎剂对P53、cdk2的表达有下调作用.

  8. Effect of Valsartan on CDK2 Expression in Mesangial Cells of Thy1 Glomerulonephritis Rats%缬沙坦对Thy1肾炎大鼠肾小球系膜细胞CDK2表达的影响

    Institute of Scientific and Technical Information of China (English)

    余荣杰; 杨惠标; 赵景宏; 赵洪雯; 刘宏; 干磊

    2007-01-01

    目的:观察Thy1肾炎大鼠系膜细胞增生及CDK2表达,以及血管紧张素Ⅱ受体拮抗剂缬沙坦对其干预作用.方法:设正常组、Thy1肾炎组及Thy1肾炎+缬沙坦治疗组.分别于各组疾病诱导后第1、3、5、7 d取肾脏行病理检查,免疫组化检测肾小球内PCNA、CDK2蛋白的表达,Westernblot分析肾小球内CDK2的表达.结果:在正常大鼠系膜细胞CDK2存在低表达,而在肾炎大鼠随系膜细胞增生,其CDK2表达增加.缬沙坦治疗组第3~7 d肾小球系膜细胞增生、系膜区扩张程度以及肾小球内PCNA表达低于肾炎组(P<0.05),肾小球内CDK2表达也低于肾炎组相应时间点(P<0.05).结论:肾小球系膜细胞的增生与其CDK2的高表达相关,缬沙坦可抑制系膜细胞CDK2的高表达,抑制系膜细胞增殖及系膜扩张.提示缬沙坦对Thy1肾炎大鼠有一定治疗作用.

  9. Ormeloxifene efficiently inhibits ovarian cancer growth

    Science.gov (United States)

    Maher, Diane M.; Khan, Sheema; Nordquist, Jordan; Ebeling, Mara C.; Bauer, Nichole A.; Kopel, Lucas; Singh, Man Mohan; Halaweish, Fathi; Bell, Maria C.; Jaggi, Meena; Chauhan, Subhash C.

    2014-01-01

    Ovarian cancer continues to be a leading cause of cancer related deaths for women. Anticancer agents effective against chemo-resistant cells are greatly needed for ovarian cancer treatment. Repurposing drugs currently in human use is an attractive strategy for developing novel cancer treatments with expedited translation into clinical trials. Therefore, we examined whether ormeloxifene (ORM), a non-steroidal Selective Estrogen Receptor Modulator (SERM) currently used for contraception, is therapeutically effective at inhibiting ovarian cancer growth. We report that ORM treatment inhibits cell growth and induces apoptosis in ovarian cancer cell lines, including cell lines resistant to cisplatin. Furthermore, ORM treatment decreases Akt phosphorylation, increases p53 phosphorylation, and modulates the expression and localization patterns of p27, cyclin E, cyclin D1, and CDK2. In a pre-clinical xenograft mouse ORM treatment significantly reduces tumorigenesis and metastasis. These results indicate that ORM effectively inhibits the growth of cisplatin resistant ovarian cancer cells. ORM is currently in human use and has an established record of patient safety. Our encouraging in vitro and pre-clinical in vivo findings indicate that ORM is a promising candidate for the treatment of ovarian cancer. PMID:25306892

  10. The influence of human cytomegalovirus infection on activity of cyclin E/Cdk2 in host cell%人巨细胞病毒感染对宿主细胞CyclinE/Cdk2激酶活性影响的实验研究

    Institute of Scientific and Technical Information of China (English)

    刘楠

    2008-01-01

    目的 研究HCMV感染对Cdk2蛋白水平及对CyclinE/Cdk2激酶活性的影响.方法 通过接触抑制使细胞同步化于GO/G1期,用MOI=5PFU/per cell HCMV AD169毒株感染人胚肺成纤维细胞(HEL);用免疫沉淀,激酶活性分析法检测HCMV感染细胞Cdk2的活性.结果 HCMV感染可引起CyclinE/Cdk2激酶的强烈激活,但HCMV感染并不诱导Cdk2蛋白丰度增加.结论 HCMV感染GO/G1细胞,激活CyelinE/Cdk2激酶.使细胞周期越过G1/S限制点,进展至晚G1期.

  11. Expressions and Significance of COX-2,CDK2 to Human Gastric Carcinoma%环氧化酶2、细胞周期调节蛋白依耐性激酶CDK2在胃癌中的表达

    Institute of Scientific and Technical Information of China (English)

    谢妮

    2009-01-01

    目的 探讨环氧化酶2(COX-2)、细胞周期调节蛋白依耐性激酶(CDK2)在胃癌中表达及与胃癌侵袭转移的相关性. 方法 采用SP免疫组化技术检测90例胃癌组织、20例正常胃黏膜COX-2、CDK2指标. 结果 胃癌组织中COX-2、CDK2的表达明显高于正常对照组(P<0.05), 高表达与胃癌淋巴结转移、浸润深度、TNM分期显著相关(PCDK2呈显著正相关性. 结论 COX-2、CDK2与胃癌的发生、侵袭、转移有关.在诊断和治疗胃癌中COX-2和CDK2 可能是两个有重要作用的指标.

  12. RNA interference targeting raptor inhibits proliferation of gastric cancer cells

    International Nuclear Information System (INIS)

    Mammalian target of rapamycin complex 1 (mTORC1) is dysregulated in gastric cancer. The biologic function of mTORC1 in gastric carcinogenesis is unclear. Here, we demonstrate that disruption of mTORC1 function by RNA interference-mediated downregulation of raptor substantially inhibited gastric cancer cell proliferation through induction of G0/G1-phase cell cycle arrest. The anti-proliferative effect was accompanied by concomitant downregulation of activator protein-1 and upregulation of Smad2/3 transcriptional activities. In addition, the expression of cyclin D3 and p21Waf1, which stabilizes cyclin D/cdk4 complex for G1-S transition, was reduced by raptor knockdown. In conclusion, disruption of mTORC1 inhibits gastric cancer cell proliferation through multiple pathways. This discovery may have an implication in the application of mTORC1-directed therapy for the treatment of gastric cancer.

  13. Inhibition of cell cycle progression by penta-acetyl geniposide in rat C6 glioma cells

    International Nuclear Information System (INIS)

    Penta-acetyl geniposide, (Ac)5-GP, the acetylated compound of geniposide, is able to inhibit the growth of rat C6 glioma cells in culture and in the bearing rats. Our recent data indicated that the induction of cell apoptosis and cell cycle arrest at G0/gap phase 1 (G1) by (Ac)5-GP might be associated with the induction of p53 and c-Myc, and mediated via the apoptosis-related bcl-2 family proteins. In this report, we further investigated the mechanism involved in the cell cycle arrest induced by (Ac)5-GP in C6 glioma cells. The inhibitory effect of (Ac)5-GP on the cell cycle progression of C6 glioma cells which arrested cells at the G0/G1 phase was associated with a marked decrease in the protein expression of cyclin D1, and an induction in the content of cyclin-dependent kinase (cdk) inhibitor p21 protein. This effect was correlated with the elevation in p53 levels. Further immunoprecipitation studies found that, in response to the treatment, the formation of cyclin D1/cdk 4 complex declined, preventing the phosphorylation of retinoblastoma (Rb) and the subsequent dissociation of Rb/E2F complex. These results illustrated that the apoptotic effect of (Ac)5-GP, arresting cells at the G0/G1 phase, was exerted by inducing the expression of p21 that, in turn, repressed the activity of cyclin D1/cdk 4 and the phosphorylation of Rb

  14. Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

    NARCIS (Netherlands)

    J.J. Molenaar; M.E. Ebus; D. Geerts; J. Koster; F. Lamers; L.J. Valentijn; E.M. Westerhout; R. Versteeg; H.N. Caron

    2009-01-01

    Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cel

  15. Transferable scoring function based on semiempirical quantum mechanical PM6-DH2 method: CDK2 with 15 structurally diverse inhibitors

    Czech Academy of Sciences Publication Activity Database

    Dobeš, Petr; Fanfrlík, Jindřich; Řezáč, Jan; Otyepka, M.; Hobza, Pavel

    2011-01-01

    Roč. 25, č. 3 (2011), s. 223-235. ISSN 0920-654X R&D Projects: GA MŠk LC512; GA ČR GAP208/11/0295 Grant ostatní: European Social Fund(XE) CZ.1.05/2.1.00/03.0058 Institutional research plan: CEZ:AV0Z40550506 Keywords : CDK2 * semiempirical quantum mechanical method PM6-DH2 * drug design Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.386, year: 2011

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

    OpenAIRE

    Zhao, Jiyong; Kennedy, Brian K.; Lawrence, Brandon D.; Barbie, David A; Matera, A. Gregory; Fletcher, Jonathan A; Harlow, Ed

    2000-01-01

    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 depende...

  17. Free-energy-based methods for binding profile determination in a congeneric series of CDK2 inhibitors.

    Science.gov (United States)

    Fidelak, Jérémy; Juraszek, Jarek; Branduardi, Davide; Bianciotto, Marc; Gervasio, Francesco Luigi

    2010-07-29

    Free-energy pathway methods show great promise in computing the mode of action and the free energy profile associated with the binding of small molecules with proteins, but are generally very computationally demanding. Here we apply a novel approach based on metadynamics and path collective variables. We show that this combination is able to find an optimal reaction coordinate and the free energy profile of binding with explicit solvent and full flexibility, while minimizing human intervention and computational costs. We apply it to predict the binding affinity of a congeneric series of 5 CDK2 inhibitors. The predicted binding free energy profiles are in accordance with experiment. PMID:20593892

  18. Clone and expression of the human cyclin dependent kinase 2 in E.coli%人细胞周期蛋白依赖性激酶2(CDK2)基因克隆和原核表达

    Institute of Scientific and Technical Information of China (English)

    郑永晨; 曹岩; 毓明涛

    2005-01-01

    目的从人胃癌组织中克隆出人CDK2基因和原核表达CDK2蛋白.方法从胃癌组织中提取RNA,逆转录PCR扩增CDK2 cDNA'PCR产物进行TA克隆和DNA序列分析;阳性TA克隆CDK2片段亚克隆入PET28a+载体,IPTG诱导表达人CDK2.结果逆转录PCR扩增出约900 bp的DNA片段,TA克隆和DNA序列分析显示重组片段是人CDK2基因序列,全长897bp;CDK2 cDNA亚克隆入PET28a+载体NcoI和XhoI位点之间;IPTG诱导出约34 kD的蛋白.结论从人胃癌组织中成功地扩增出人CDK2基因,并且在大肠杆菌中得到表达.

  19. Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

    International Nuclear Information System (INIS)

    Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel–Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers. - Highlights: • Wogonin is an all around inhibitor of VEGF signaling. • We firstly demonstrate that wogonin inhibits secretion of VEGF by decreasing HIF-1α. • Wogonin enhances PDH and VHL expression and inhibits Hsp90 function.

  20. 稳定转染CDK2干扰RNA的人肝癌HepG2细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    王震宇; 白瑞霞; 商进; 李丽梅; 姜丽丽; 王文礼

    2008-01-01

    目的建立稳定转染CDK2干扰RNA的人肝癌HepG2细胞系。方法用LipofectamineTM 2000法将已构建好的特异性的携带绿色荧光蛋白的CDK2干扰RNA的重组质粒PGenesil-1-CDK2转染入HepG2细胞,经G418筛选后获得稳定转染细胞株,并用倒置荧光显微镜观察转染细胞的绿色荧光蛋白,利用逆转录-聚合酶链反应(RT-PCR)检测转染后的HepG2细胞CDK2 mRNA的表达。结果倒置荧光显微镜观察与KT-PCR检测,结果显示获得重组质粒PGenesil-1-CDK2稳定转染的细胞克隆。结论建立了可稳定转染CDK2干扰RNA的人肝癌HepG2细胞系,为进一步研究细胞周期调控蛋白激酶对肝细胞癌的治疗与开发新的特异性药物提供了实验依据。

  1. Relationship between Expression of CDK2 and p27kip1 Protein and Non-small Cell Lung Cancer in Xuanwei%CDK2、p27kip1蛋白与宣威地区非小细胞肺癌的关系

    Institute of Scientific and Technical Information of China (English)

    李晓明; 黄云超; 殷君太; 叶联华; 杨凯云; 赵光强

    2011-01-01

    目的 探讨CDK2、p27kip1蛋白与宣威地区非小细胞肺癌(non-small cell lung Cancer,NSCLC)生物学行为的关系.方法 免疫组织化学法检测宣威和非宣威地区非小细胞肺癌患者的癌组织和正常组织(距癌组织边缘距离>5 cm)CDK2、p27kip1蛋白的表达.结果 CDK2蛋白在癌组织中的表达主要位于胞浆.CDK2在宣威腺癌组织中表达最高,明显高于CDK2在宣威鳞癌、非宣威鳞癌、非宣威腺癌以及正常肺组织中的表达(P<0.05);CDK2在宣威鳞癌中的表达也高于其在非宣威鳞癌中的表达(P<0.05);CDK2在非宣威腺癌中的表达高于其在非宣威鳞癌和非宣威正常肺组织中的表达(P<0.05);但CDK2在非宣威鳞癌中的表达与其在正常肺组织中的表达无统计学差异(P>0.05);CDK2蛋白在癌组织中的表达与临床分期及组织分化程度无关(P>0.05);与淋巴结转移有关;与实验组的病理类型有关,实验组腺癌组织的表达高于对照组腺癌组织,差异有统计学意义(P<0.05).p27kip1蛋白的表达主要位于胞浆;p27kip1在不同地区(宣威或非宣威),不同病理类型(腺癌、鳞癌)肿瘤组织中的表达均无统计学差异(P>0.05);P27kip1在正常肺组织中的表达明显高于其在肿瘤组织中的表达(P<0.05);p27kip1蛋白的表达与临床分期、组织分化程度及病理类型无关(P>0.05),与淋巴结转移有关(P<0.05).CDK2蛋白的表达与p27kip1蛋白的表达呈负相关(P<0.05).结论 CDK2蛋白在宣威癌组织的表达高于非宣威癌组织;CDK2在非宣威腺癌中的表达高于非宣威鳞癌和正常肺组织;CDK2蛋白和p27kip1蛋白在癌组织中的表达与淋巴结转移有关.CDK2蛋白在癌组织中的表达与p27kip1蛋白的表达呈负相关.%Objective To explore the relationship between the expressions of CDK2 and p27kip1 protein and biological behavior of non-small cell lung cancer in Xuanwei.Methods Immuneohistochemical method was used to

  2. CDKN2A and CDK4 mutation analysis in Italian melanoma-prone families: functional characterization of a novel CDKN2A germ line mutation

    OpenAIRE

    Torre, G Della; Pasini, B; S. Frigerio; Donghi, R; Rovini, D; Delia, D; Peters, G.; Huot, T J G; Bianchi-Scarra, G; Lantieri, F; Rodolfo, M.; Parmiani, G.; Pierotti, M A

    2001-01-01

    Physical interaction between CDKN2A/p16 and CDK4 proteins regulates the cell cycle progression through the G1 phase and dysfunction of these proteins by gene mutation is implicated in genetic predisposition to melanoma. We analysed 15 Italian melanoma families for germ line mutations in the coding region of the CDKN2A gene and exon 2 of the CDK4 gene. One novel disease-associated mutation (P48T), 3 known pathological mutations (R24P, G101W and N71S) and 2 common polymorphisms (A148T and Nt500...

  3. HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

    OpenAIRE

    Hall, Caitlin; Nelson, David M.; Ye, Xiaofen; Baker, Kayla; DeCaprio, James A.; Seeholzer, Steven; Lipinski, Marc; Adams, Peter D

    2001-01-01

    Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo s...

  4. Loss of Keratinocytic RXRα Combined with Activated CDK4 or oncogenic NRAS Generates UVB-induced Melanomas via Loss of p53 and PTEN in the Tumor Microenvironment

    OpenAIRE

    Coleman, Daniel J.; Chagani, Sharmeen; Hyter, Stephen; Sherman, Anna M.; Christiane V. Löhr; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K.

    2014-01-01

    Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout...

  5. Effect of siRNA-induced CDK2 Expression Suppression on Expression of RB, CyclinE and E2F1 in Hepatic Carcinoma Cells%小分子干扰RNA沉默肝癌细胞CDK2基因对RB、CyclinE、E2F1基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    刘佳维; 于水澜; 宋高臣; 于英君

    2012-01-01

    Objective To investigate the effects of cyclin-dependent kinase 2 ( CDK2 ) expression suppression induced by small interfering RNAs (siRNAs) on mRNA expression of cell cycle related genes RB, CyclinE and E2F1 in hepatic carcinoma cells SMMC7721. Methods The siRNA eukaryotic expression plasmids of CDK2 gene were constructed firstly and then were transfected into SMMC7721 cells with the Lipofectmine TM 2000 liposome. The transfected cells were divided into six groups; recombinant plasmid 190 group, recombinant plasmid 191 group, SMMC7721 group, CDK2-siRNA transfection group, negative control group, and blank vector group. The expression of CDK2 gene was detected with Western blot method. Real-time fluorescent quantitation polymerase chain reaction (PCR) method was utilized to detect the mRNA expression of RB, Cyclin E and E2F1 which were related to CDK2 gene, and then the effective siRNA sequence of CDK2 gene was screened. Results After the siRNA eukaryotic expression plasmids of CDK2 gene was transfected into SMMC7721 cells, mRNA expression of RB was up-regulated and the mRNA expression of CyclinE and E2F1 was down-regulated. Conclusion CDK2 gene expression suppression can up-regulate the mRNA expression of RB in SMMC7721, and down-regulate the mRNA expression of CyclinE and E2F1, indicating that the mRNA expression of RB, Cyclin E and E2F1 genes is correlated with CDK2 gene expression.%[目的]观察小分子干扰RNA (siRNA)沉默细胞周期素依赖性蛋白激酶(CDK2)基因后,细胞周期相关基因RB、CyclinE、E2F1在肝癌细胞SMMC7721中mRNA的表达. [方法]将前期研究中已构建成功并筛选出的最有效干扰抑制CDK2基因的siRNA序列片段,采用Lipofectamine TM2000脂质体转染法转染肝癌细胞株SMMC7721后分6组:重组质粒组190、重组质粒组191、SMMC7721肝癌组、转染试剂组、阴性对照组、空质粒组.采用实时荧光定量PCR法检测RB、CyclinE、E2F1 mRNA水平.[结果]CDK2的siRNA转染SMMC7721细

  6. Mathematics revealed

    CERN Document Server

    Berman, Elizabeth

    1979-01-01

    Mathematics Revealed focuses on the principles, processes, operations, and exercises in mathematics.The book first offers information on whole numbers, fractions, and decimals and percents. Discussions focus on measuring length, percent, decimals, numbers as products, addition and subtraction of fractions, mixed numbers and ratios, division of fractions, addition, subtraction, multiplication, and division. The text then examines positive and negative numbers and powers and computation. Topics include division and averages, multiplication, ratios, and measurements, scientific notation and estim

  7. Revealed Attention

    OpenAIRE

    Masatlioglu, Yusufcan; NAKAJIMA, Daisuke; Ozbay, Erkut Y

    2012-01-01

    The standard revealed preference argument relies on an implicit assumption that a decision maker considers all feasible alternatives. The marketing and psychology literatures, however, provide wellestablished evidence that consumers do not consider all brands in a given market before making a purchase (Limited Attention). In this paper, we illustrate how one can deduce both the decision maker's preference and the alternatives to which she pays attention and inattention from the observed behav...

  8. Revealed Attention

    OpenAIRE

    Yusufcan Masatlioglu; Daisuke Nakajima; Ozbay, Erkut Y

    2012-01-01

    The standard revealed preference argument relies on an implicit assumption that a decision maker considers all feasible alternatives. The marketing and psychology literatures, however, provide well-established evidence that consumers do not consider all brands in a given market before making a purchase (Limited Attention). In this paper, we illustrate how one can deduce both the decision maker's preference and the alternatives to which she pays attention and inattention from the observed beha...

  9. CRIF1 interacting with CDK2 regulates bone marrow microenvironment-induced G0/G1 arrest of leukemia cells.

    Directory of Open Access Journals (Sweden)

    Qian Ran

    Full Text Available BACKGROUND: To assess the level of CR6-interacting factor 1 (CRIF1, a cell cycle negative regulator, in patients with leukemia and investigate the role of CRIF1 in regulating leukemia cell cycle. METHODS: We compared the CRIF1 level in bone marrow (BM samples from healthy and acute myeloid leukemia (AML, iron deficiency anemia (IDA and AML-complete remission (AML-CR subjects. We also manipulated CRIF1 level in the Jurkat cells using lentivirus-mediated overexpression or siRNA-mediated depletion. Co-culture with the BM stromal cells (BMSCs was used to induce leukemia cell cycle arrest and mimic the BM microenvironment. RESULTS: We found significant decreases of CRIF1 mRNA and protein in the AML group. CRIF1 overexpression increased the proportion of Jurkat cells arrested in G0/G1, while depletion of endogenous CRIF1 decreased cell cycle arrest. Depletion of CRIF1 reversed BMSCs induced cell cycle arrest in leukemia cells. Co-immunoprecipitation showed a specific binding of CDK2 to CRIF1 in Jurkat cells during cell cycle arrest. Co-localization of two proteins in both nucleus and cytoplasm was also observed with immunofluorescent staining. CONCLUSION: CRIF1 may play a regulatory role in the BM microenvironment-induced leukemia cell cycle arrest possibly through interacting with CDK2 and acting as a cyclin-dependent kinase inhibitor.

  10. HPV16 E6、P21WAF1、CDK2、Livin在宫颈癌中的表达及其意义%Expression and significance of HPV16 E6, P21WAF1, CDK2 and livin in cervical cancer

    Institute of Scientific and Technical Information of China (English)

    孙峰; 邢传平; 刘斌; 哈小琴; 王芳; 柳红; 曹晓哲

    2009-01-01

    目的 探讨人乳头瘤病毒(HPV)16 E6、P21WAF1、CDK2、livin在宫颈癌中的表达及P21WAF1、CDK2、livin与E6的关系.方法 采用免疫组化SP法检测HPV16 E6、P21 P21WAF1、CDK2、livin在20例宫颈癌、40例宫颈上皮内瘤变(CIN)及20例正常对照组中的表达.应用图像分析软件分别对此四个因子的表达进行平均光密度分析.结果 E6、P21WAF1、CDK2、livin蛋白于宫颈癌组高于CIN1-2组及正常组(P0.05).CIN1-2组与正常组之间差异无统计学意义(P>0.05).E6分别与P21WAF1、CDK2、livin成正相关(相关系数r分别为0.706、0.713、0.711,P<0.05).结论 E6、CDK2与宫颈癌的发生、发展密切相关,E6、CDK2异常高表达可能是宫颈疾病恶变早期事件.livin可能成为宫颈癌潜在的治疗分子靶向.

  11. Correlation of CDK2-AP1 gene over-expression with proliferation and cell cycle regulation of breast cancer cell line MCF-7%乳腺癌MCF-7细胞的增殖及周期与CDK2-AP1基因的表达的相关性研究

    Institute of Scientific and Technical Information of China (English)

    高双全; 高双荣; 肖高芳; 丁宇; 王林辉; 黄伟; 李静

    2013-01-01

    目的 探讨乳腺癌MCF-7细胞的增殖及周期与CDK2-AP1基因的表达的相关性研究,为临床乳腺癌的分子治疗提供基础.方法 取我院研究所保存的人乳腺癌细胞MCF-7进行培养,并构建CDK2-AP1基因编码的病毒表达载体,应用实时定量PCR验证CDK2-AP1基因mRNA和蛋白的表达率.利用流式细胞仪检测MCF-7细胞周期的改变.结果 过表达CDK2-AP1基因的慢病毒感染MCF-7细胞可上调其mRNA表达6.87倍.MCF-7细胞过表达CDK2-AP1基因后,增殖能力显著降低,差异具有统计学意义(P<0.05).流式细胞仪检测证实MCF-7细胞过表达CDK2-AP1能够使细胞周期出现G1期阻滞.结论 CDK2-AP1基因具有抑癌基因的功能,在乳腺癌MCF-7细胞过表达该基因能够抑制细胞的生长和克隆形成能力,并且使细胞阻滞于G1期.

  12. A novel phosphorylation site of N-methyl-D-aspartate receptor GluN2B at S1284 is regulated by Cdk5 in neuronal ischemia.

    Science.gov (United States)

    Lu, Wen; Ai, Heng; Peng, Lin; Wang, Jie-jie; Zhang, Bin; Liu, Xiao; Luo, Jian-hong

    2015-09-01

    N-methyl-D-aspartate receptors (NMDARs) are a key player in synaptic and several neurological diseases, such as stroke. Phosphorylation of NMDAR subunits at their cytoplasmic carboxyl termini has been considered to be an important mechanism to regulate the receptor function. Cyclin-dependent kinase 5 (Cdk5) has been demonstrated to be responsible for regulating phosphorylation and function of NMDARs. Besides, it is also suggested that Cdk5 is involved in ischemic insult. In the present study, we showed that GluN2B subunit serine 1284 at its cytoplasmic carboxyl termini was regulated by Cdk5 in neuronal ischemia. Interestingly, both oxygen glucose deprivation (OGD) in cultured hippocampal neurons and transient global ischemia in mice induce dramatic changes in the phosphorylated level of GluN2B at S1284. However, no significant changes in the phosphorylation of this site are found neither in chemical LTP stimulation in cultured hippocampal neurons nor fear conditioning in adult mice. Taken together, our study identified NMDAR GluN2B S1284 as a novel phosphorylation site regulated by Cdk5 with implication in neuronal ischemia. PMID:26093036

  13. Synergic effects of the cyclin-dependent kinase (CDK) inhibitor olomoucine and androgen-antagonist bicalutamide on prostatic cancer cell lines

    Czech Academy of Sciences Publication Activity Database

    Knillová, J.; Bouchal, J.; Hlobilková, A.; Strnad, Miroslav; Kolář, Z.

    2004-01-01

    Roč. 51, č. 5 (2004), s. 358-367. ISSN 0028-2685 R&D Projects: GA ČR(CZ) GA301/02/0475 Institutional research plan: CEZ:AV0Z5038910 Keywords : olomoucine * bikalutamid * prostate cancer * CDK Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.822, year: 2004

  14. Regulation of the G1/S Transition in Hepatocytes: Involvement of the Cyclin-Dependent Kinase Cdk1 in the DNA Replication

    Directory of Open Access Journals (Sweden)

    Anne Corlu

    2012-01-01

    Full Text Available A singular feature of adult differentiated hepatocytes is their capacity to proliferate allowing liver regeneration. This review emphasizes the literature published over the last 20 years that established the most important pathways regulating the hepatocyte cell cycle. Our article also aimed at illustrating that many discoveries in this field benefited from the combined use of in vivo models of liver regeneration and in vitro models of primary cultures of human and rodent hepatocytes. Using these models, our laboratory has contributed to decipher the different steps of the progression into the G1 phase and the commitment to S phase of proliferating hepatocytes. We identified the mitogen dependent restriction point located at the two-thirds of the G1 phase and the concomitant expression and activation of both Cdk1 and Cdk2 at the G1/S transition. Furthermore, we demonstrated that these two Cdks contribute to the DNA replication. Finally, we provided strong evidences that Cdk1 expression and activation is correlated to extracellular matrix degradation upon stimulation by the pro-inflammatory cytokine TNFα leading to the identification of a new signaling pathway regulating Cdk1 expression at the G1/S transition. It also further confirms the well-orchestrated regulation of liver regeneration via multiple extracellular signals and pathways.

  15. Synapsin III Acts Downstream of Semaphorin 3A/CDK5 Signaling to Regulate Radial Migration and Orientation of Pyramidal Neurons In Vivo

    Directory of Open Access Journals (Sweden)

    Laura E. Perlini

    2015-04-01

    Full Text Available Synapsin III (SynIII is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5 phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation.

  16. Tetramethylpyrazine Inhibits Activation of Hepatic Stellate Cells through Hedgehog Signaling Pathways In Vitro

    Directory of Open Access Journals (Sweden)

    Jue Hu

    2015-01-01

    Full Text Available Background and Aim. Tetramethylpyrazine (TMP, a major alkaloid isolated from Ligusticum chuanxiong, has been reported in hepatic fibrosis models. However, the action mechanism remains unclear. In the present study, effects of tetramethylpyrazine (TMP against hepatic stellate cell (HSC activation as well as the possible mechanisms were evaluated. Methods. Western blot assay was used to detect TMP effects on protein expression of Smo, Patched, Hhip, and Gli and to investigate the effects of TMP on Cyclin D1, Cyclin E1, CDK2, Bcl-2, Bax, and caspase expression with cyclopamine supplementation. Results. Our results showed that TMP significantly inhibits the expression of Cyclin D1, Cyclin E1, and Cyclin-dependent kinase CDK2 and changes the HSC cycle by inhibiting the proliferation of HSC. Moreover, TMP has also been shown to decrease the expression of Bcl-2 and increase the expression of Bax in HSC-T6 cells. Furthermore, TMP can inhibit the expression of connective tissue growth factor (CTGF, and the inhibitory effect was intensified after the application of joint treatment with TMP and cyclopamine. Conclusion. TMP may be an effective Hh signaling pathway inhibitor for hepatic fibrosis treatment.

  17. Piperine from black pepper inhibits activation-induced proliferation and effector function of T lymphocytes.

    Science.gov (United States)

    Doucette, Carolyn D; Rodgers, Gemma; Liwski, Robert S; Hoskin, David W

    2015-11-01

    Piperine is a major alkaloid component of black pepper (Piper nigrum Linn), which is a widely consumed spice. Here, we investigated the effect of piperine on mouse T lymphocyte activation. Piperine inhibited polyclonal and antigen-specific T lymphocyte proliferation without affecting cell viability. Piperine also suppressed T lymphocyte entry into the S and G2 /M phases of the cell cycle, and decreased expression of G1 -associated cyclin D3, CDK4, and CDK6. In addition, piperine inhibited CD25 expression, synthesis of interferon-γ, interleukin (IL)-2, IL-4, and IL-17A, and the generation of cytotoxic effector cells. The inhibitory effect of piperine on T lymphocytes was associated with hypophosphorylation of Akt, extracellular signal-regulated kinase, and inhibitor of κBα, but not ZAP-70. The ability of piperine to inhibit several key signaling pathways involved in T lymphocyte activation and the acquisition of effector function suggests that piperine might be useful in the management of T lymphocyte-mediated autoimmune and chronic inflammatory disorders. PMID:25900378

  18. Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin

    Directory of Open Access Journals (Sweden)

    Jacquemont Céline

    2012-04-01

    Full Text Available Abstract Background Platinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin. Results Through a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407. Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor, CA-074-Me (cathepsin B inhibitor and 17-AAG (HSP90 inhibitor, synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF, but not in FA-deficient isogenic cells (2008. In addition, geldanamycin (HSP90 inhibitor and two CHK1 inhibitors (UCN-01 and SB218078 exhibited a significantly stronger synergism with cisplatin in FA

  19. The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

    Science.gov (United States)

    Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

    2015-01-15

    Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. PMID:25498792

  20. Revealing Rembrandt

    Directory of Open Access Journals (Sweden)

    Andrew J Parker

    2014-04-01

    Full Text Available The power and significance of artwork in shaping human cognition is self-evident. The starting point for our empirical investigations is the view that the task of neuroscience is to integrate itself with other forms of knowledge, rather than to seek to supplant them. In our recent work, we examined a particular aspect of the appreciation of artwork using present-day functional magnetic resonance imaging (fMRI. Our results emphasised the continuity between viewing artwork and other human cognitive activities. We also showed that appreciation of a particular aspect of artwork, namely authenticity, depends upon the co-ordinated activity between the brain regions involved in multiple decision making and those responsible for processing visual information. The findings about brain function probably have no specific consequences for understanding how people respond to the art of Rembrandt in comparison with their response to other artworks. However, the use of images of Rembrandt’s portraits, his most intimate and personal works, clearly had a significant impact upon our viewers, even though they have been spatially confined to the interior of an MRI scanner at the time of viewing. Neuroscientific studies of humans viewing artwork have the capacity to reveal the diversity of human cognitive responses that may be induced by external advice or context as people view artwork in a variety of frameworks and settings.

  1. Clinical Significance of the Protient Expression of MCM7、CDK2 and Ki-67 in Thyroid Cancer%MCM7、CDK2及 Ki-67蛋白在甲状腺癌中表达的临床意义

    Institute of Scientific and Technical Information of China (English)

    李琰; 张娟

    2015-01-01

    Objective To explore the clinical significance of the protient expression of MCM 7、CDK2 and Ki-67 in thy-roid cancer .Methods 30 cases of papillary thyroid cancer patients ,30 cases of thyroid adenoma patients ,30 cases of nodular goiter patients and 30 cases of normal thyroid patients were collected .Then protein expression of MCM 7、CDK2 and Ki-67 were detected by immunohistochemical SP method .Results The positive expression rates of MCM 7、CDK2 and Ki-67 were 100.00%(30/30)、80.00%(24/30)and 83.33%(25/30)in thyroid cancer patients.The positive expression rates of MCM7、CDK2 and Ki-67 in thyroid cancer patients were significantly higher than those of thyroid adenoma patients ,nodular goiter patients and nor-mal thyroid patients .The expression of MCM 7、CDK2 and Ki-67 was positively correlated in thyroid cancer patients .Conclusion Positive expression rate of MCM7、CDK2 and Ki-67 and thyroid cancer have certain correlation .The detection of MCM7、CDK2 and Ki-67 expression can provide important reference for early diagnosis and evaluation of prognosis of papillary thyroid carcinoma in clinical .%目的:探讨甲状腺癌中MCM7、CDK2及Ki-67蛋白表达的临床意义。方法收集甲状腺乳头状癌、甲状腺腺瘤、结节性甲状腺肿及正常甲状腺标本各30例,然后用免疫组织化学SP法来检测所选甲状腺组织标本中MCM7、CDK2及Ki-67蛋白的表达。结果 MCM7、CDK2及Ki-67蛋白在甲状腺癌中表达的阳性率分别为100.00%(30/30)、80.00%(24/30)及83.33%(25/30),三者在甲状腺乳头状癌中的阳性率均明显高于甲状腺腺瘤、结节性甲状腺肿及正常甲状腺。甲状腺癌中MCM7、CDK2及Ki-67蛋白的表达呈正相关。结论 MCM7、CDK2及Ki-67蛋白的高表达与甲状腺乳头状癌存在一定的关联,三者表达的检测可为临床上早期诊断及评价甲状腺乳头状癌的预后提供重要的参考依据。

  2. NANOG Is Multiply Phosphorylated and Directly Modified by ERK2 and CDK1 In Vitro

    Directory of Open Access Journals (Sweden)

    Justin Brumbaugh

    2014-01-01

    Full Text Available NANOG is a divergent homeobox protein and a core component of the transcriptional circuitry that sustains pluripotency and self-renewal. Although NANOG has been extensively studied on the transcriptional level, little is known regarding its posttranslational regulation, likely due to its low abundance and challenging physical properties. Here, we identify eleven phosphorylation sites on endogenous human NANOG, nine of which mapped to single amino acids. To screen for the signaling molecules that impart these modifications, we developed the multiplexed assay for kinase specificity (MAKS. MAKS simultaneously tests activity for up to ten kinases while directly identifying the substrate and exact site of phosphorylation. Using MAKS, we discovered site-specific phosphorylation by ERK2 and CDK1/CyclinA2, providing a putative link between key signaling pathways and NANOG.

  3. Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro

    International Nuclear Information System (INIS)

    Any insult that affects survival of ovarian antral follicles can cause abnormal estradiol production and fertility problems. Phthalate esters (PEs) are plasticizers used in a wide range of consumer and industrial products. Exposure to these chemicals has been linked to reduced fertility in humans and animal models. Di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) decrease serum estradiol levels and aromatase (Arom) expression, prolong estrous cycles, and cause anovulation in animal and culture models. These observations suggest PEs directly target antral follicles. We therefore tested the hypothesis that DEHP (1-100 μg/ml) and MEHP (0.1-10 μg/ml) directly inhibit antral follicular growth and estradiol production. Antral follicles from adult mice were cultured with DEHP or MEHP, and/or estradiol for 96 h. During culture, follicle size was measured every 24 h as a measurement of follicle growth. After culture, media were collected for measurement of estradiol levels and follicles were subjected to measurement of cylin-D-2 (Ccnd2), cyclin-dependant-kinase-4 (Cdk4), and Arom. We found that DEHP and MEHP inhibited growth of follicles and decreased estradiol production compared to controls at the highest doses. DEHP and MEHP also decreased mRNA expression of Ccnd2, Cdk4, and Arom at the highest dose. Addition of estradiol to the culture medium prevented the follicles from DEHP- and MEHP-induced inhibition of growth, reduction in estradiol levels, and decreased Ccnd2 and Cdk4 expression. Collectively, our results indicate that DEHP and MEHP may directly inhibit antral follicle growth via a mechanism that partially includes reduction in levels of estradiol production and decreased expression of cell cycle regulators.

  4. Expression of Cdk2 and p21 in gastric cancer and its relationship with clinicopathological features and prognosis%胃癌中Cdk2和p21的表达及其与临床病理和预后的关系

    Institute of Scientific and Technical Information of China (English)

    刘雷; 陈国昌; 宋振云; 郜恒骏; 陈卫昌

    2014-01-01

    目的 研究Cdk2和p21在胃癌中的表达及其与临床病理和预后的关系.方法 将130例胃癌组织和248例癌旁胃组织标本石蜡块制成组织芯片,采用免疫组化法检测Cdk2及p21的表达.结果 胃癌组织组Cdk2及p21阳性表达率分别为63.8%(83/130)和70.0%(91/130),明显高于癌旁组织的18.1%(45/248)和37.1%(92/248)(P<0.05).p21的阳性表达与胃癌的分化程度有关,Cdk2的阳性表达与胃癌的分化程度、淋巴结转移、TNM分期有关(P<0.05).p21阳性表达组5年生存率低于阴性表达组.多因素分析显示,Cdk2和p21的表达可作为胃癌独立的预后因素.结论 Cdk2及p21在胃癌的发生、发展中可能起重要作用,可作为判断胃癌预后的指标.

  5. Expressions of cell cycle associated proteins cyclin E,cdk2 and p27kip1 in pituitary adenomas%细胞周期相关蛋白cyclin E、cdk2和p27kip1在垂体腺瘤中的表达

    Institute of Scientific and Technical Information of China (English)

    郑安锡; 霍钢; 黄晓明; 唐文华; 袁晓东; 陈世平

    2009-01-01

    目的 观察细胞周期相关蛋白cyclin E、cdk2和p27kip1在垂体腺瘤中的表达情况,探讨其与垂体腺瘤临床生物学行为的关系.方法 采用免疫组织化学SP法检测37例非侵袭性垂体腺瘤和21例侵袭性垂体腺瘤组织中cyclin E、cdk2和p27 kip1蛋白的表达情况.结果 在侵袭性垂体腺瘤中cyclin E和cdk2的表达明显高于非侵袭性垂体腺瘤(均P<0.01),p27kip1的表达明显低于非侵袭性垂体腺瘤(P<0.01);cyclin E与cdk2在垂体腺瘤中的表达呈正相关(r=1,P<0.01),两者与p27kip1的表达呈负相关(均r=-1,P<0.01);cyclin E和cdk2的表达与垂体腺瘤的侵袭性呈正相关(r=0.685,r=0.667,均P<0.01),而p27kip1的表达与垂体腺瘤的侵袭性呈负相关(r=0.659,P<0.01).结论 cyclin E、cdk2及p27kip1的表达情况可以作为判断垂体腺瘤的侵袭性和预后的指标.

  6. Evaluation of E2F4 Regulate CDK2 and CyclinA in Livet Cancer Cells%E2F4对CDK2和CyclinA在肝癌细胞系中的转录调控特异性研究

    Institute of Scientific and Technical Information of China (English)

    张圆

    2010-01-01

    目的 研究E2F4对细胞周期素依赖性激酶2(CDK2)和细胞周期素A(Cyclin A)在肝癌细胞系中的转录调控活性.方法 扩增并克隆不同片段长度的CDK2和CyclinA启动子序列,E2F4表达质粒序列,转染肝癌细胞系(HepG2、QGY1007),双荧光报告系统检测荧光素酶表达活性,实时定量反转录.聚合酶链反应检测肝癌细胞系中CDK2和Cyclin A mRNA的表达水平.结果 双荧光报告系统、卖时定量反转录-聚合酶链反应显示,在肝癌细胞系中转染E2F4表达质粒组CDK2和CyclinA启动子活性明显低于未转染E2F4表达质粒组,CDK2和CyclinA mRNA的表达水平降低.结论 E2F4在肝癌细胞系中可以抑制CDK2和CyclinA的转录活性.

  7. Effect of CDK9 Inhibitor F200 on the Apoptosis of Human Breast Cancer MCF7 Cells%CDK9抑制剂F200对乳腺癌细胞MCF7凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    侯贝; 罗楹; 陈文峻

    2014-01-01

    Objective This study aimed to investigate the effects of cell cycle dependent kinase inhibitor F200 on ap-optosis of human breast cancer cell MCF7. Methods MCF7 cells were cultured in RPMI-1640 medium with 0.01 mg·mL-1 human recombinant insulin and 10%fetal bovine serum. Cells were subcultured at exponential growth phase. 24 h later, cells were adherent to the plate and treatments were given according to groups setup, negative control (0.5%DMSO), posi-tive control (R-Roscovitine 5.66μM), F200 groups (F200 0.1μM, 0.71μM). At 48 h after treatment, cell morphological change of apoptosis was measured by TUNEL methods and the apoptosis rate was detected by flow cytometry. The expres-sion of PRAP was measured by Western Bolt. Results The TUNEL results showed that as the F200 concentration increased, the cell apoptosis features like cell pyknosis and dense granule in nucleus were significantly clearer. More cells were stained with DAB after treated with F200. The flow cytometry results showed 0.71μM F200 could induce 32.6%cell apoptosis. Moreover, the Western Blot results showed cleaved PARP was increased along with the increase of F200 concentration. Conclusion CDK inhibitor F200 could inhibit MCF7 cell growth and induce the cell apoptosis.%目的:研究细胞周期依赖性激酶(cell cycle dependent kinase,CDK)CDK9抑制剂F200对乳腺癌细胞MCF7凋亡的影响。方法 MCF7细胞培养于含0.01 mg·mL-1人重组胰岛素及10%胎牛血清的RPMI 1640培养液中,待对数生长期时接种细胞进行实验,24 h贴壁后给药:分为阴性对照组(0.5% DMSO)、阳性对照组(R-Roscovitine 5.66μM)及药物组(F2000.1μM,0.71μM),给药48 h后利用TUNEL法染色观察细胞凋亡DNA断裂情况及细胞凋亡形态学变化、流式细胞技术检测细胞的凋亡比率、免疫印迹法检测凋亡标志蛋白PARP的表达情况。结果 TUNEL结果显示,随着F200浓度增加,细胞出现明显的固缩变圆,细

  8. EFFECTS OF CDK2 siRNA ON CELL CYCLE AND PROLIFERATION OF HUMAN HEPATOMA CELLS HepG2%CDK2干扰RNA对人肝癌细胞HepG2细胞周期和增殖的影响

    Institute of Scientific and Technical Information of China (English)

    王文礼; 李丽梅; 关则红; 王震宇; 郝兴霞; 姜丽丽

    2008-01-01

    目的:探讨CDK2 siRNA对人肝癌细胞株HepG2细胞CDK2 mRNA表达及其对HepG2细胞周期和增殖的影响.方法:利用脂质体转染法将特异性的携带绿色荧光蛋白的CDK2干扰RNA的重组质粒PGenesil-1-CDK2导入HepG2细胞,同时设立阴性对照空载体转染组PGenesil-1-HK.G418筛选稳定表达细胞株扩增培养.并用倒置荧光显微镜观察转染细胞的绿色荧光蛋白.反转录-聚合酶链反应(RT-PCR)和流式细胞术(FCM),分别检测转染后的HepG2细胞CDK2 mRNA和细胞周期的变化,噻唑蓝(MTT)法检测癌细胞生长增殖率.结果:经1mo的G418筛选得到稳定表达细胞株,与阴性对照组及空白对照组相比,转染组CDK2 mRNA 水平明显下降,细胞周期明显改变,细胞从G1期到S期发生阻滞.结论:RNA干扰技术可明显抑制CDK2基因的表达,引起 HepG2细胞周期阻滞及肿瘤细胞生长抑制作用,为CDK2介导的肿瘤基因沉默疗法提供实验依据.

  9. Dihydroaustrasulfone Alcohol Inhibits PDGF-Induced Proliferation and Migration of Human Aortic Smooth Muscle Cells through Inhibition of the Cell Cycle

    Directory of Open Access Journals (Sweden)

    Yao-Chang Chen

    2015-04-01

    Full Text Available Dihydroaustrasulfone alcohol is the synthetic precursor of austrasulfone, which is a marine natural product, isolated from the Taiwanese soft coral Cladiella australis. Dihydroaustrasulfone alcohol has anti-inflammatory, neuroprotective, antitumor and anti-atherogenic properties. Although dihydroaustrasulfone alcohol has been shown to inhibit neointima formation, its effect on human vascular smooth muscle cells (VSMCs has not been elucidated. We examined the effects and the mechanisms of action of dihydroaustrasulfone alcohol on proliferation, migration and phenotypic modulation of human aortic smooth muscle cells (HASMCs. Dihydroaustrasulfone alcohol significantly inhibited proliferation, DNA synthesis and migration of HASMCs, without inducing cell death. Dihydroaustrasulfone alcohol also inhibited platelet-derived growth factor (PDGF-induced expression of cyclin-dependent kinases (CDK 2, CDK4, cyclin D1 and cyclin E. In addition, dihydroaustrasulfone alcohol inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2, whereas it had no effect on the phosphorylation of phosphatidylinositol 3-kinase (PI3K/(Akt. Moreover, treatment with PD98059, a highly selective ERK inhibitor, blocked PDGF-induced upregulation of cyclin D1 and cyclin E and downregulation of p27kip1. Furthermore, dihydroaustrasulfone alcohol also inhibits VSMC synthetic phenotype formation induced by PDGF. For in vivo studies, dihydroaustrasulfone alcohol decreased smooth muscle cell proliferation in a rat model of restenosis induced by balloon injury. Immunohistochemical staining showed that dihydroaustrasulfone alcohol noticeably decreased the expression of proliferating cell nuclear antigen (PCNA and altered VSMC phenotype from a synthetic to contractile state. Our findings provide important insights into the mechanisms underlying the vasoprotective actions of dihydroaustrasulfone alcohol and suggest that it may be a useful therapeutic agent

  10. Molecular docking and molecular dynamics studies reveal structural basis of inhibition and selectivity of inhibitors EGCG and OSU-03012 toward glucose regulated protein-78 (GRP78) overexpressed in glioblastoma.

    Science.gov (United States)

    Bhattacharjee, Rituparna; Devi, Arpita; Mishra, Seema

    2015-10-01

    Glioblastoma (GBM), a malignant form of brain tumor, has a high mortality rate. GRP78, one of the HSP70 protein family members, is overexpressed in GBM. GRP78 is the key chaperone protein involved in the unfolded protein response. Upregulated GRP78 expression in cancer cells inhibits apoptosis and promotes chemoresistance. GRP78 has an ATPase domain, a substrate-binding domain, and a linker region. ATP-competitive inhibitors such as EGCG and OSU-03012 inhibit GRP78 activity and reduce its expression in GBM. However, there is a lack of structural data on the binding modes of these inhibitors to GRP78 ATPase domain. Further, the mode of selectivity of these inhibitors toward GRP78 also is unknown. Toward this end, molecular docking was performed with AutoDock Vina and confirmation obtained by docking using ROSIE. The stability and MM-PBSA binding energy of GRP78-inhibitor complexes as well as energetic contribution of individual residues was analyzed by 50 ns molecular dynamics run with GROMACS. MSA by ClustalW2 identified unique amino acid residues in the ATPase domain of GRP78 which were different from the residues present in other HSP70 proteins. Important and unique amino acid residues of GRP78 such as Ile61, Glu293, Arg297, and Arg367 played a major role in the intermolecular interactions with these inhibitors. The interactions with unique residues of GRP78 as compared with those of HSP70-1A provided the basis for selectivity. It was found that the binding affinity and specificity/selectivity of EGCG toward GRP78 was higher than that toward HSP70-1A, and selectivity was even better than OSU-03012. OSU-03012 was predicted to bind to GRP78. Analyses from MD runs showed tight binding and stability of complexes, and the highest number of hydrogen bonds during the trajectory runs were comparable to those found in the docking studies. Energetic contribution of individual inhibitor-interacting residues showed that energy values of Ile61 and Glu293 were among the most

  11. Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARγ signaling as potential mechanism for the negative relationships between immune response and lipid metabolism

    Directory of Open Access Journals (Sweden)

    Rodriguez-Zas Sandra L

    2009-11-01

    Full Text Available Abstract Background Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (IMI with S. uberis and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with S. uberis O140J. Results Streptococcus uberis IMI resulted in 2,102 (1,939 annotated differentially expressed genes (DEG. Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each, the majority of which were signaling pathways. Among the most inhibited were LXR/RXR Signaling and PPARα/RXRα Signaling. Pathways activated by IMI were IL-10 Signaling and IL-6 Signaling which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA3. Genes down-regulated (1,020 included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Network analysis of DEG indicated that TNF had positive relationships with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2 and negative relationships with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL and antioxidant activity (SOD1. Conclusion Results provided novel information into the early signaling and metabolic pathways in mammary tissue that are associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J elicited

  12. Hedyotis diffusa Willd inhibits colorectal cancer growth in vivo via inhibition of STAT3 signaling pathway.

    Science.gov (United States)

    Cai, Qiaoyan; Lin, Jiumao; Wei, Lihui; Zhang, Ling; Wang, Lili; Zhan, Youzhi; Zeng, Jianwei; Xu, Wei; Shen, Aling; Hong, Zhenfeng; Peng, Jun

    2012-01-01

    Signal Transducer and Activator of Transcription 3 (STAT3), a common oncogenic mediator, is constitutively activated in many types of human cancers; therefore it is a major focus in the development of novel anti-cancer agents. Hedyotis diffusa Willd has been used as a major component in several Chinese medicine formulas for the clinical treatment of colorectal cancer (CRC). However, the precise mechanism of its anti-tumor activity remains largely unclear. Using a CRC mouse xenograft model, in the present study we evaluated the effect of the ethanol extract of Hedyotis diffusa Willd (EEHDW) on tumor growth in vivo and investigated the underlying molecular mechanisms. We found that EEHDW reduced tumor volume and tumor weight, but had no effect on body weight gain in CRC mice, demonstrating that EEHDW can inhibit CRC growth in vivo without apparent adverse effect. In addition, EEHDW treatment suppressed STAT3 phosphorylation in tumor tissues, which in turn resulted in the promotion of cancer cell apoptosis and inhibition of proliferation. Moreover, EEHDW treatment altered the expression pattern of several important target genes of the STAT3 signaling pathway, i.e., decreased expression of Cyclin D1, CDK4 and Bcl-2 as well as up-regulated p21 and Bax. These results suggest that suppression of the STAT3 pathway might be one of the mechanisms by which EEHDW treats colorectal cancer. PMID:22754353

  13. Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion▿

    OpenAIRE

    Kirschner, Austin N.; Lowrey, Amanda S.; Longnecker, Richard; Theodore S Jardetzky

    2007-01-01

    Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N...

  14. Inhibition in Autism: Children with Autism Have Difficulty Inhibiting Irrelevant Distractors but Not Prepotent Responses

    Science.gov (United States)

    Adams, Nena C.; Jarrold, Christopher

    2012-01-01

    Resistance to distractor inhibition tasks have previously revealed impairments in children with autism. However, on the classic Stroop task and other prepotent response tasks, children with autism show intact inhibition. These data may reflect a distinction between prepotent response and resistance to distractor inhibition. The current study…

  15. A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

    Science.gov (United States)

    Arai, Kazuya; Eguchi, Takanori; Rahman, M Mamunur; Sakamoto, Ruriko; Masuda, Norio; Nakatsura, Tetsuya; Calderwood, Stuart K; Kozaki, Ken-Ichi; Itoh, Manabu

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor

  16. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    Science.gov (United States)

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  17. Genome-Wide Transcriptional Analysis Reveals the Protection against Hypoxia-Induced Oxidative Injury in the Intestine of Tibetans via the Inhibition of GRB2/EGFR/PTPN11 Pathways

    Science.gov (United States)

    Gesang, Luobu; Dan, Zeng; Gusang, Lamu

    2016-01-01

    The molecular mechanisms for hypoxic environment causing the injury of intestinal mucosal barrier (IMB) are widely unknown. To address the issue, Han Chinese from 100 m altitude and Tibetans from high altitude (more than 3650 m) were recruited. Histological and transcriptome analyses were performed. The results showed intestinal villi were reduced and appeared irregular, and glandular epithelium was destroyed in the IMB of Tibetans when compared with Han Chinese. Transcriptome analysis revealed 2573 genes with altered expression. The levels of 1137 genes increased and 1436 genes decreased in Tibetans when compared with Han Chinese. Gene ontology (GO) analysis indicated most immunological responses were reduced in the IMB of Tibetans when compared with Han Chinese. Gene microarray showed that there were 25-, 22-, and 18-fold downregulation for growth factor receptor-bound protein 2 (GRB2), epidermal growth factor receptor (EGFR), and tyrosine-protein phosphatase nonreceptor type 11 (PTPN11) in the IMB of Tibetans when compared with Han Chinese. The downregulation of EGFR, GRB2, and PTPN11 will reduce the production of reactive oxygen species and protect against oxidative stress-induced injury for intestine. Thus, the transcriptome analysis showed the protecting functions of IMB patients against hypoxia-induced oxidative injury in the intestine of Tibetans via affecting GRB2/EGFR/PTPN11 pathways.

  18. 中药胃康宁对胃癌细胞周期因子Cyclin E、CDK2和p27表达的影响%Effect of Weikangning Decoction on Expression of Cyclin E, CDK2, and p27

    Institute of Scientific and Technical Information of China (English)

    阚方巨; 李庆明; 钟娃; 梁蔚文

    2006-01-01

    目的:观察中药胃康宁含药血清对胃癌细胞Cyclin E、CDK2(Cyclin dependant kinase 2,CDK2)和p27mRNA及其蛋白表达的作用.方法:分别以高、中、低不同剂量的胃康宁制剂对Wistar雄性大鼠进行灌胃,制备含药血清,然后分别用不同剂量组含药血清培养胃癌细胞,采用免疫组织化学SABC法检测各组胃癌细胞中CyclinE、CDK2和p27蛋白的表达,RT-PCR法检测各组胃癌细胞中Cyclin E、CDK2和p27 mRNA的表达.结果:免疫组化法结果显示,胃康宁高、中、低剂量组Cyclin E和CDK2灰度值均有明显升高,与空白对照组比较,P<0.05~0.01,而p27灰度值则显著降低(P<0.05~0.01);RT-PCR结果显示,与空白对照组比较,胃康宁低、中、高剂量组均能明显降低Cyclin E和CDK2 OPTDI值(P<0.05~0.01).同时,还能升高p27 OPTDI值(P<0.05~0.01).结论:中药胃康宁可抑制胃癌细胞周期因子Cyclin E、CDK2及其mRNA的表达,促进细胞周期抑制因子p27及其mRNA的表达,这可能是胃康宁抑制胃癌细胞生长的机制之一.

  19. 鲍恩样丘疹病中p21和CDK2的表达与人乳头瘤病毒感染的关系%Relationship between the expressions of p21 and CDK2 and infection of human papillomavirus in Bowenoid papulosis

    Institute of Scientific and Technical Information of China (English)

    杨永生; 梁俊; 杜娟; 林尽染; 金晶; 朱小华; 徐金华

    2010-01-01

    目的: 评价鲍恩样丘疹病(Bowenoid populosis, BP)皮损中p21和CDK2的表达与HPV感染的相关性.方法: 石蜡包埋标本免疫组化Envision法和IMS细胞图像分析系统分析p21和CDK2蛋白表达.冰冻标本采用煮沸法提取DNA,用导流杂交基因芯片技术作HPV分型检测.结果: 在28例BP中p21和CDK2阳性细胞数百分比分别为(31.6±10.5)%和(49.9±13.8)%,显著高于包皮对照组的(22.5±5.4)%和(17.3±8.5)%,P<0.05.HPV阳性组p21和CDK2阳性细胞百分比分别为(30.56±11.78)%和(54.42±13.69)%;HPV阴性组p21和CDK2阳性细胞百分比为(33.3±8.36)%和(43.15±11.55)%.p21两组差别无统计学意义(P>0.05),而CDK2差别有统计学意义(P<0.05).p21、CDK2直线相关分析无相关性(P>0.05).结论: BP中存在细胞周期相关蛋白的高表达,HPV可能通过作用于细胞周期相关蛋白使细胞周期紊乱,继而导致非典型增生.

  20. ImageJ软件在重组质粒pET32a-CDK2中蛋白表达的应用%Application of Image J software in analyzing protein expression of recombinant plasmid pET32a-CDK2

    Institute of Scientific and Technical Information of China (English)

    陈炜烨; 刘冬冬; 徐建华; 陈丹娜; 何敏; 张战锋; 黄宪章

    2014-01-01

    目的 应用Image J软件探索细胞周期依赖性蛋白激酶2(CDK2)在重组质粒pET32a-CDK2中的蛋白表达条件.方法 将重组质粒pET32a-CDK2转入表达菌株BL21 (DE3),然后在不同时间点(0、1、2、3、4h)和不同浓度(0.5、1、2 mmol/L)异丙基-β-D-硫代半乳糖苷(IPTG)下诱导表达目的蛋白CDK2,对CDK2采用SDS-PAGE法进行蛋白电泳,并应用Image J软件对电泳条带进行灰度分析.结果 CDK2诱导表达量在不同时间点差异有统计学意义(P<0.001),其中0h与1h、2h、3h、4h诱导表达量的差异均有统计学意义(P=0.007,P<0.001,P<0.001,P<0.001);1h与3h和4h诱导表达量的差异均有统计学意义(P=0.001);而2h和1h、3h、4h诱导表达量的差异均无统计学意义(均有P>0.05);不同浓度IPTG下的CDK2诱导表达量差异无统计学意义(P=0.336,P=0.240,P=1.000).结论 根据Image J软件分析结果,采用0.5 mmol/L浓度IPTG 2h的条件,节约诱导时间和试剂用量.

  1. 全反式维甲酸对兔颈动脉粥样硬化病变中Cdk2表达及血管平滑肌细胞增生的影响%Influence of All-trans Retinoic Acid on the Expression of Cdk2 and Proliferation of Vascular Smooth Muscle Cells in Carotid Atherosclerosis in Rabbits

    Institute of Scientific and Technical Information of China (English)

    郭琳琳; 董果雄; 张社华

    2007-01-01

    目的 探讨全反式维甲酸(ATRA)对兔颈动脉粥样硬化病灶中血管内膜的增生、血管平滑肌细胞(VSMCs)增殖、增殖细胞核抗原(PCNA)及细胞周期素依赖性激酶Cdk2表达规律的影响.方法 36只新西兰雄性大白兔随机分为3组:正常饮食组、手术组、ARTA治疗组,每组12只.手术组和治疗组均给予高脂饮食,两周后用空气干燥法制作颈动脉内膜损伤模型,治疗组于术前3d开始给予ATRA灌胃.于术后1、4周处死动物,取病变血管应用HE染色、免疫组化和计算机图像分析法进行形态学、PCNA和Cdk2表达水平的检测.结果 ①正常动脉壁未见PCNA及Cdk2表达.②手术组在术后第1周时内膜开始增生,第4周增生明显,且出现泡沫细胞、脂质核心形成、管腔狭窄;增生内膜中Cdk2表达水平增高.③治疗组Cdk2的表达明显低于手术组(P<0.05),VSMCs的迁移、增殖、内膜增生和管腔狭窄显著减轻(P<0.05).④PCNA表达与Cdk2表达呈显著正相关(P<0.01).结论 ARTA可通过抑制Cdk2表达,抑制VSMCs的迁移和增殖,从而抑制兔颈动脉粥样硬化新生内膜过度增生和管腔狭窄.

  2. Phosphorylation by PKA potentiates retinoic acid receptor α activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7

    OpenAIRE

    Gaillard, Emilie; Bruck, Nathalie; Brelivet, Yann; Bour, Gaétan; Lalevée, Sébastien; Bauer, Annie; Poch, Olivier; Moras, Dino; Rochette-Egly, Cécile

    2006-01-01

    Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RARα is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylat...

  3. 血管内皮生长因子及其受体与Cyclin E-CDK2在肝癌发生发展过程中的表达变化%Changes in Expression of vascular endothelial growth factor and its receptor and cyclin E-cdk2 in Liver cancer progression

    Institute of Scientific and Technical Information of China (English)

    王东; 李笑岩; 李红星; 白咸勇

    2011-01-01

    Objective To study the expression of vascular endothelial growth factor (VEGF) and its receptor, Cyclin E and CDK2 in the liver of the rat liver cancer model, and to explore the function and significance of VEGF, VEGFR1, Cyclin E and CDK2 in the liver cancer progression. Methods Based on the DEN-induced hepatocarcinogenesis model, the expressions of VEGFR1, Cyclin E and CDK2 were detected with immunohistochemical staining, and the quantity of VEGF in the serum was detected with enzyme linked immunosorbent assay. Results The quantity of VEGF in the serum of rats is lowest in the control group, and increased gradually in the experimental groups. The average optic density values of VEGFR1,Cyclin E and CDK2 protein expression had increasing trends during the development of liver cancer. The quantity of VEGF in the rat serum and the average optic density values of VEGFR1, Cyclin E and CDK2 protein were positively correlated (r = 0. 834, F = 42. 1274 , P<0. 05). Conclusion Abnormal expression of VEGF and its receptor, promotes hepatocarcinogenesis and tumor development, which might be related with the abnormal expression of Cyclin E and CDK2 protein.%目的 通过观察血管内皮生长因子(VEGF)及其受体和细胞周期蛋白E (Cyclin E)在肝癌模型大鼠肝脏中表达情况,探讨VEGF与细胞周期相关蛋白在肝癌发生发展过程中的作用.方法 建立诱发性肝癌模型,采用酶联免疫吸附试验检测血清中VEGF量的变化,免疫组化技术检测VEGFR1、Cyclin E和细胞周期蛋白依赖性激酶(CDK2)的表达情况.结果 血清中的VEGF含量在对照组中最低,在实验组中逐渐增多,以癌变期含量最高.VEGFR1、Cyclin E和CDK2蛋白表达的平均光密度值均随着肝癌的发生发展有增高的趋势,大鼠血清中的VEGF量与肝脏组织中VEGFR1、Cyclin E和CDK2蛋白表达的平均光密度值随着肝癌的发生发展呈正相关(r=0.834,F=42.1274,P<0.05).结论 VEGF及其受体VEGFR1在肝癌发生发

  4. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    Directory of Open Access Journals (Sweden)

    Ortbahn Casey T

    2011-05-01

    Full Text Available Abstract Background Mifepristone (MF has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR. The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Methods Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. Results MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Conclusions Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and

  5. Arsenic trioxide inhibits tumor cell growth in malignant rhabdoid tumors in vitro and in vivo by targeting overexpressed Gli1.

    Science.gov (United States)

    Kerl, Kornelius; Moreno, Natalia; Holsten, Till; Ahlfeld, Julia; Mertins, Julius; Hotfilder, Marc; Kool, Marcel; Bartelheim, Kerstin; Schleicher, Sabine; Handgretinger, Rupert; Schüller, Ulrich; Meisterernst, Michael; Frühwald, Michael C

    2014-08-15

    Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6-Rb-, the WNT- and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients. PMID:24420698

  6. Antiprogestin mifepristone inhibits the growth of cancer cells of reproductive and non-reproductive origin regardless of progesterone receptor expression

    International Nuclear Information System (INIS)

    Mifepristone (MF) has been largely used in reproductive medicine due to its capacity to modulate the progesterone receptor (PR). The study of MF has been expanded to the field of oncology; yet it remains unclear whether the expression of PR is required for MF to act as an anti-cancer agent. Our laboratory has shown that MF is a potent inhibitor of ovarian cancer cell growth. In this study we questioned whether the growth inhibitory properties of MF observed in ovarian cancer cells would translate to other cancers of reproductive and non-reproductive origin and, importantly, whether its efficacy is related to the expression of cognate PR. Dose-response experiments were conducted with cancer cell lines of the nervous system, breast, prostate, ovary, and bone. Cultures were exposed to vehicle or increasing concentrations of MF for 72 h and analysed for cell number and cell cycle traverse, and hypodiploid DNA content characteristic of apoptotic cell death. For all cell lines, expression of steroid hormone receptors upon treatment with vehicle or cytostatic doses of MF for 24 h was studied by Western blot, whereas the activity of the G1/S regulatory protein Cdk2 in both treatment groups was monitored in vitro by the capacity of Cdk2 to phosphorylate histone H1. MF growth inhibited all cancer cell lines regardless of tissue of origin and hormone responsiveness, and reduced the activity of Cdk2. Cancer cells in which MF induced G1 growth arrest were less susceptible to lethality in the presence of high concentrations of MF, when compared to cancer cells that did not accumulate in G1. While all cancer cell lines were growth inhibited by MF, only the breast cancer MCF-7 cells expressed cognate PR. Antiprogestin MF inhibits the growth of different cancer cell lines with a cytostatic effect at lower concentrations in association with a decline in the activity of the cell cycle regulatory protein Cdk2, and apoptotic lethality at higher doses in association with increased

  7. Briefly Bound to Activate: Transient Binding of a Second Catalytic Magnesium Activates the Structure and Dynamics of CDK2 Kinase for Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Zhao Qin; Jacobsen, Douglas M.; Young, Matthew A. (Michigan-Med)

    2014-10-02

    We have determined high-resolution crystal structures of a CDK2/Cyclin A transition state complex bound to ADP, substrate peptide, and MgF{sub 3}{sup -}. Compared to previous structures of active CDK2, the catalytic subunit of the kinase adopts a more closed conformation around the active site and now allows observation of a second Mg{sup 2+} ion in the active site. Coupled with a strong [Mg{sup 2+}] effect on in vitro kinase activity, the structures suggest that the transient binding of the second Mg{sup 2+} ion is necessary to achieve maximum rate enhancement of the chemical reaction, and Mg{sup 2+} concentration could represent an important regulator of CDK2 activity in vivo. Molecular dynamics simulations illustrate how the simultaneous binding of substrate peptide, ATP, and two Mg{sup 2+} ions is able to induce a more rigid and closed organization of the active site that functions to orient the phosphates, stabilize the buildup of negative charge, and shield the subsequently activated {gamma}-phosphate from solvent.

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

    Science.gov (United States)

    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

  9. Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/{beta}-catenin signaling pathway in HCT116 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); Kim, Chul Young [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); College of Pharmacy, Hanyang University, Ansan 426-791 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of)

    2012-11-16

    Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

  10. Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes.

    Science.gov (United States)

    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

  11. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    Science.gov (United States)

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells. PMID:27212103

  12. Expression of P27kip1, CDK2, Cyclin E in variant tissue of human gallbladder and relationship to each other%不同病变胆囊组织中P27kip1、CDK2、Cyclin E的表达及相关性研究

    Institute of Scientific and Technical Information of China (English)

    龚学军; 汤辉焕

    2005-01-01

    目的通过对胆囊结石、胆囊腺瘤性息肉及胆囊癌组织中细胞周期蛋白激酶抑制蛋白P27kip1、细胞周期蛋白依赖激酶CDK2、细胞周期素Cyclin E的表达及相关性研究,分析在3种胆囊病变组织中的表达与病理的关系,以探讨其与胆囊癌的发生、发展有关的可能因素.方法应用免疫组织化学的方法分析人类3组胆囊组织中的P27kip1、CDK2、Cyclin E的表达.结果P27kip1在胆囊癌的表达与CDK2的表达呈显著负相关(r=-0.949,P<0.05);CDK2与Cyclin E的表达呈显著正相关(r=0.908,P<0.01).胆囊癌中P27kip1的表达与Nevin病理分期呈显著负相关(r=-0.558,P<0.01),而与患者的性别、年龄、分化及病理类型无明显关系(P>0.05)P27kip1在胆囊癌与胆囊结石之间的表达差异有显著性(P<0.01),胆囊结石与胆囊息肉之间的表达差异也有显著性(P<0.01);CDK2、Cyclin E在胆囊癌与胆囊结石的表达差异有显著性(P<0.01),胆囊结石与胆囊息肉之间的表达差异也有显著性(P<0.05).结论胆囊癌中P27kip1、CDK2、Cyclin E基因出现异常表达,P27kip1的表达抑制及CDK2、Cyclin E的过度表达在胆囊癌的发生、发展过程中可能起促进作用.P27kip1、CDK2、Cyclin E并不能作为预测肿瘤发生,诊断早期肿瘤理想的指标;P27kip1的低表达是胆囊癌预后不良的预测指标之一,CDK2、Cyclin E作为单独的判断胆囊癌预后的指标尚缺乏足够的依据.

  13. 甲状腺乳头状癌中MCM7、CDK2、p27蛋白的表达及意义%The expression of MCM7, CDK2 and p27 in papillary thyroid carcinoma and their significance

    Institute of Scientific and Technical Information of China (English)

    史琳; 张安文; 罗宇; 杨燕初

    2011-01-01

    目的 探讨MCM7、CDK2及p27蛋白与人甲状腺乳头状癌(papillary thyroid carcinoma,PTC)发生、发展的关系.方法 采用免疫组织化学SP法检测40例PTC、30例甲状腺腺瘤、30例结节性甲状腺肿及20例正常甲状腺组织中MCM7、CDK2、p27蛋白的表达.结果 PTC中MCM7、CDK2蛋白的阳性表达率分别为100.00%(40/40)、80.00%(32/40),两者均明显高于甲状腺腺瘤、结节性甲状腺肿及正常甲状腺组织(P<0.01,P<0.01).PTC中p27蛋白的阳性表达率为22.50%(9/40),明显低于甲状腺腺瘤、结节性甲状腺肿及正常甲状腺组织(P<0.01).PTC中MCM7与CDK2蛋白的表达呈正相关(r=0.550,P<0.01),MCM7、CDK2及p27蛋白的表达呈负相关(r=-0.334,P<0.05;r=-0.413,P<0.01).结论 MCM7、CDK2蛋白的高表达及p27蛋白的低表达变化与PTC可能存在关联,三者联合检测或许可以作为临床早期诊断和评价甲状腺肿瘤细胞增殖活性的潜在的参考指标.%Purpose To explore the expression of MCM7 , CDK2 and p27 in papillary thyroid carcinoma and the mechanisms involved in the carcinogenesis and development of papillary thyroid carcinoma.Methods Immunohistochemical SP method was used to detect the expression of MCM7 , CDK2 and p27 protein in 40 cases of papillary thyroid carcinoma, 30 cases of thyroid adenoma.30 cases of nodular goitre and 20 cases of normal thyroid gland tissue.Results The positive rates of MCM7 and CDK2 expressions in papillary thyroid carcinoma were 100.00%( 40/40 ) and 80.00% (32/40 ).The expression of MCM7 and CDK2 protein in the papillary thyroid carcinoma group was significantly higher than that in the thyroid adenoma.nodular goitre and normal thyroid tissue groups ( P < 0.01 ,P < 0.01 ).The positive rates of p27 expressions in papillary thyroid carcinoma were 22.50%( 9/40 ).The expression of p27 protein in the papillary thyroid carcinoma group was significantly lower than that in the thyroid adenoma.nodular goitre and normal thyroid tissue

  14. Direct inhibition of Retinoblastoma phosphorylation by Nimbolide causes cell cycle arrest and suppresses glioblastoma growth

    Science.gov (United States)

    Anderson, Jane; Liu, Xiaona; Henry, Heather; Gasilina, Anjelika; Nassar, Nicholas; Ghosh, Jayeeta; Clark, Jason P; Kumar, Ashish; Pauletti, Giovanni M.; Ghosh, Pradip K; Dasgupta, Biplab

    2013-01-01

    Purpose Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth. Experimental Design Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. Results We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth. Conclusions Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo. PMID:24170547

  15. Effects of Buyang Huanwu decoction on the expression of Cyclin D1 and Cdk2 in rats with post-stroke depression%补阳还五汤对卒中后抑郁大鼠海马区Cyclin D1与Cdk2的影响

    Institute of Scientific and Technical Information of China (English)

    侯菊花; 刘圆月; 李灿; 邓叔华; 刘柏炎

    2015-01-01

    Objective To observe the effects of Buyang Huanwu decoction(BYHW) on the expression of Cyclin D1 and Cdk2 in rats with post-stroke depression(PSD).Methods The rats model of focal cerebral ischemia was established by means of middle cerebral artery occlusion(MCAO).Then three weeks of salute-living and chronic unpredictable mild stress(CUMS) was given to the animal after cerebral stroke to induce the post-stoke depression animal model.The rats were divided into 5 groups:the sham operated group,the midge cerebral artery occlusion(MCAO) group,the PSD group,the fluoxetine group and the BYHWD group.The rats were subjected to left MCAO rebuilding in consistent focal cerebral ischemia,followed by an 21-day exposure to chronic mild stress (CMS)and single housing to induce PSD animal model.All rats were killed in 7,14 and 21 day after operation.The expressions of Cyclin D1 and Cdk2 were measured by immunohistochemical staining.Results Pathological changes such as hippocampal nerve cell regression,degeneration and necrosis were observed in the model rats compare with the sham operated group.The expression of Cyclin D1 in normal hippocampus in 7,14 or 21 day after operation was (1.16±0.34)%,(1.62±0.29)% and (1.60±0.24)% respectively,and Cdk2 was (1.52±0.26)%,(1.85±0.23)% and (1.97±0.10)%.After PSD the expression of Cyclin D1 was (49.69±5.68)%,(58.17± 2.89) % and (50.87 ± 2.48) % respectively,and Cdk2 was (50.63 ± 2.93) %,(70.34± 1.47) % and (61.35 ± 3.04) %.Compared with model group,Fluoxetine and BYHW significantly reduced the numbers of Cyclin D1 and Cdk2 positive cells,the expression of Cyclin D1 was (16.62±4.27)%,(29.66±5.24)% and (26.71±1.32)% at fluoxetine group,and Cdk2 was (18.05±2.26) %,(33.84±3.12) % and (24.51±2.66) %.The expression of Cvclin D1 was (14.62±2.06)%,(26.89±3.41)% and (23.68±2.01)% at BYHWD group,and Cdk2 was (16.60± 2.42) %,(20.09±3.45) % and (22.19± 1.70) %.Conclusion The abnormal

  16. The gene expressions of cyclin E, CDK2 and p21WAF1 and their significant in esophageal carsinogenesis%Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    李丽; 齐凤英; 左连富; 李萍; 王辉

    2005-01-01

    目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P<0.01或P<0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P<0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P<0.01或P<0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.

  17. Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

    International Nuclear Information System (INIS)

    We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus

  18. Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung-Mi [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of); Yun, Ji Ho [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Lee, Dong Hwa [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Park, Young Gyun [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Son, Kun Ho [Department of Food Science and Nutrition, Andong National University, Andong 760-749 (Korea, Republic of); Nho, Chu Won, E-mail: cwnho@kist.re.kr [Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 210-340 (Korea, Republic of); Kim, Yeong Shik, E-mail: kims@snu.ac.kr [Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul (Korea, Republic of)

    2015-04-17

    We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.

  19. 洛伐他汀通过抑制Calpain和CDK5的过度激活减轻NMDA的毒性损害%Lovastatin attenuates Calpain and CDK5 over-activation induced by NMDA

    Institute of Scientific and Technical Information of China (English)

    马涛; 许著一; 姚晴宇; 孔岳南

    2012-01-01

    Objective To observe the effect of lovastatin on the excitotoxicity induced by NMDA in cortical neurons in rats and to investigate the underlying mechanism. Methods Cortical neurons prepared from E17 rats were assigned into 4 groups:NMDA group (addition of 100 μmol/L of NMDA for 15 minutes),LOV group (pretreatment of the neurons for 3 days with 500 nmol/L of LOV),LOV+NMDA group (pretreatment of the neurons for 3 days with 500 nmol/L of LOV and addition of 100 μmol/L of NMDA for 15 minutes) and untreated group (addition of isodose solvent).Cell viability was evaluated with the trypan blue dye exclusion test,the morphology and number of neurons were assessed with MAP-2 immunofluorescence staining,and the level of protein was measured with Western blotting assay. Results Trypan blue staining demonstrated that the pretreatment with 500 nmol/L of lovastatin for 3 days significantly protected the neurons against the excitotoxicity induced by NMDA (P<0.05 vs NMDA).Immunofluorescence staining demonstrated the number of MAP-2 positive neurons decreased and the surviving neurons showed a loss of MAP-2 positive dendrites after NMDA treatment (P<0.05 vs untreated),which were not observed after lovastatin pretreatment (P<0.05 vs NMDA).Excitotoxicity was mediated in part by the Calpain over-activation and the subsequent protein truncation events on Calpain substrate, CDK5 co-activator P35 to P25 cleavage. Lovastatin pretreatment remarkably suppressed Calpain over-activation and the conversion from P35 to P25 in response to NMDA exposure as detected by Western blotting analysis (P<0.05 vs NMDA). Conclusions Lovastatin significantly attenuates the excitotoxicity induced by NMDA. The neuroprotection of lovastatin may be mediated by blocking the Calpain and CDK5 over-activation.%目的 探讨洛伐他汀(LOV)对N-甲基-D-天门冬氨酸(NMDA)诱导的大鼠皮质神经元兴奋性毒性损害的神经保护作用及可能机制. 方法 原代培养的大鼠皮质神经

  20. Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site.

    Science.gov (United States)

    Oikonomakos, N G; Schnier, J B; Zographos, S E; Skamnaki, V T; Tsitsanou, K E; Johnson, L N

    2000-11-01

    Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes. PMID:10924512

  1. Involvement of cdk5/p35 and tau protein in the hippocampal mossy fiber sprouting in the PTZ kindling model%Cdk5/p35和tau蛋白对戊四氮点燃模型海马苔藓纤维出芽的作用

    Institute of Scientific and Technical Information of China (English)

    田发发; 郭婷辉; 党静; 马云峰; 陈金梅; 陈莹; 蔡小凤; 宋明谕

    2011-01-01

    Objective To observe the expression of eyclin-dependent kinase 5(Cdk5),p35,tau protein and the activity of Cdk5 in rat hippoeampus during pentylenetetrazole(PTZ)kindling process and their correlation with mossy fiber sprouting(MFS)so as to investigate the role of Cdk5/p35 in epileptogenesis.Methods A total of 240 healthy male SD rats were divided randomly into normal controls and pentylenetetrazole(PTZ)treatment groups.The epileptic models were established by injection of PTZ intraperitoneally.At Day 3,Weeks 1,2,4 & 6 after a daily injection of PTZ,Timm staining was scored in the CA3 region and dentate gyrus.At the same time,the mRNA and protein of Cdk5 and p35,total tau protein and its phosphorylation at set202 and Cdk5 activity were analyzed in the hilus and stratum granulosum of dentate gyms and the CA 1,CA3 regions of hippocampus.The methods of in situ hybridization,immunohistochemistry,Western blot and immuno-precipitation and liquid scintillation counter were employed respectively.Results Prominent MFS was observed in area CA3 rather than the inner molecular layer in PTZ-treated rats.And the degree of MFS progressed with the development of behavioral kindled seizures.The expressions of Cdk5/p35 mRNA and protein,tau protein and its phosphorylation at Set202 significantly increased from Day 3 to Week 4 in the PTZ treatment group.It was in accordance with the progression of MFS in area CA3.Conclusion CdkS/p35 and its substrate tau protein may be involed in M FS.Undeerstanding the molecular mechanisms of MFS may lead to therapeutic interventions for limiting epileptogenesis.%目的 动态观察细胞周期素依赖性激酶5(Cdk5)及调节亚基p35、骨架蛋白在戊四氮致痫大鼠海马各区的表达变化和苔藓纤维出芽(MFS)的情况,以探讨cdk5/p35对MFS的作用及其机制.方法 SD雄性成年大鼠240只,随机分为PTZ组和对照组;PTZ组分为PTZ第一次注射后3 d、1周、2周、4周、6周共5个亚组,每亚组24

  2. IDH1 mutant structures reveal a mechanism of dominant inhibition

    Institute of Scientific and Technical Information of China (English)

    Shimin Zhao; Kun-Liang Guan

    2010-01-01

    @@ Pioneered by a major cancer genome sequencing project [1] and followed by numerous cancer genomic sequencing studies [2-4], the cytosolic isocitrate dehydrogenase (IDH1) gene and mitochondria isocitrate dehydrogenase (IDH2) gene are found to be frequently mutated in low grade gliomas and secondary glioblastoma multiforme (GBM), acute myeloid leukemia (AML), and to a much lower frequency in other types of tumors.

  3. Transcriptional profiling of C. elegans DAF-19 uncovers a ciliary base-associated protein and a CDK/CCRK/LF2p-related kinase required for intraflagellar transport.

    Science.gov (United States)

    Phirke, Prasad; Efimenko, Evgeni; Mohan, Swetha; Burghoorn, Jan; Crona, Filip; Bakhoum, Mathieu W; Trieb, Maria; Schuske, Kim; Jorgensen, Erik M; Piasecki, Brian P; Leroux, Michel R; Swoboda, Peter

    2011-09-01

    Cilia are ubiquitous cell surface projections that mediate various sensory- and motility-based processes and are implicated in a growing number of multi-organ genetic disorders termed ciliopathies. To identify new components required for cilium biogenesis and function, we sought to further define and validate the transcriptional targets of DAF-19, the ciliogenic C. elegans RFX transcription factor. Transcriptional profiling of daf-19 mutants (which do not form cilia) and wild-type animals was performed using embryos staged to when the cell types developing cilia in the worm, the ciliated sensory neurons (CSNs), still differentiate. Comparisons between the two populations revealed 881 differentially regulated genes with greater than a 1.5-fold increase or decrease in expression. A subset of these was confirmed by quantitative RT-PCR. Transgenic worms expressing transcriptional GFP fusions revealed CSN-specific expression patterns for 11 of 14 candidate genes. We show that two uncharacterized candidate genes, termed dyf-17 and dyf-18 because their corresponding mutants display dye-filling (Dyf) defects, are important for ciliogenesis. DYF-17 localizes at the base of cilia and is specifically required for building the distal segment of sensory cilia. DYF-18 is an evolutionarily conserved CDK7/CCRK/LF2p-related serine/threonine kinase that is necessary for the proper function of intraflagellar transport, a process critical for cilium biogenesis. Together, our microarray study identifies targets of the evolutionarily conserved RFX transcription factor, DAF-19, providing a rich dataset from which to uncover-in addition to DYF-17 and DYF-18-cellular components important for cilium formation and function. PMID:21740898

  4. Clinical Significance and Expressions of Cyclin E andcdk2 in Bladder Transitinal Cell Carcinoma%CyclinE和cdk2在膀胱癌中表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    谢庆祥; 林福地; 韩聪祥

    2001-01-01

    目的:探讨细胞周期蛋白E(cyclinE)和细胞周期蛋白依赖性激酶2(cdk2)与膀胱癌发病的关系及其临床意义.方法:应用免疫组化方法检测16例正常膀胱组织和88例膀胱移行细胞癌中cyclinE、cdk2和PCNA的表达.结果:膀胱癌中cyclinE和cdk2表达均显著高于正常膀胱组织.cyclinE表达与膀胱癌病理分级、分期、复发和大小之间密切相关;cdk2表达则仅与肿瘤大小和PCNA指数密切相关.cyclinE和cdk2共同阳性表达者的肿瘤复发率明显增高.结论:cyclinE和cdK2的异常表达在膀胱癌发生发展过程中起重要作用,两者共同表达对于术后病人监测及治疗方案的制定具有指导意义.

  5. Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes.

    Science.gov (United States)

    Ogushi, Sugako; Fulka, Josef; Miyano, Takashi

    2005-10-01

    In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation. PMID:16153631

  6. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

    Science.gov (United States)

    Young, Richard J; Waldeck, Kelly; Martin, Claire; Foo, Jung H; Cameron, Donald P; Kirby, Laura; Do, Hongdo; Mitchell, Catherine; Cullinane, Carleen; Liu, Wendy; Fox, Stephen B; Dutton-Regester, Ken; Hayward, Nicholas K; Jene, Nicholas; Dobrovic, Alexander; Pearson, Richard B; Christensen, James G; Randolph, Sophia; McArthur, Grant A; Sheppard, Karen E

    2014-07-01

    We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance. PMID:24495407

  7. The inhibition of cell proliferation and induction of apoptosis in pancreatic ductal adenocarcinoma cells by verrucarin A, a macrocyclic trichothecene, is associated with the inhibition of Akt/NF-кB/mTOR prosurvival signaling.

    Science.gov (United States)

    Deeb, Dorrah; Gao, Xiaohua; Liu, Yongbo; Zhang, Yiguan; Shaw, Jiajiu; Valeriote, Frederick A; Gautam, Subhash C

    2016-09-01

    Pancreatic ductal adenocarcinoma (PDA) remains one of the most difficult to treat of all malignancies. Multimodality regimens provide only short-term symptomatic improvement with minor impact on survival, underscoring the urgent need for novel therapeutics and treatment strategies for PDA. Trichothecenes are powerful mycotoxins that inhibit protein synthesis and induce ribotoxic stress response in mammalian cells. Verrucarin A (VC-A) is a Type D macrocyclic mycotoxin which inhibited cell proliferation and induced apoptosis in breast cancer cells. However, the antitumor activity of VC-A for PDA cells has not been investigated. Here we show potent antitumor activity and the mechanism of action of VC-A in PDA cell lines. VC-A strongly inhibited the proliferation and arrested cells in the S phase of the cell cycle. The blocking of cell cycle progression by VC-A was associated with the inhibition of cell cycle regulatory proteins cyclin D1, cyclin E, cyclin-dependent kinases (cdks) cdk2, cdk4 and cdk inhibitor WAF1/21. VC-A induced apoptosis in PDA cells as indicated by the increased Annexin V FITC-binding, cleavage of poly(ADP-ribose) polymerase‑1 (PARP-1) and procaspases-3, -8 and -9. VC-A also induced mitochondrial depolarization and release of cytochrome c and it inhibited Bcl-2 family proteins that regulate apoptosis (Bcl-2, Bcl-xL, Bax and Bad). In addition, VC-A reduced the levels of inhibitors of apoptosis survivin and c-IAP-2. Finally, VC-A downregulated the expression of prosurvival phospho-Akt (p-Akt), nuclear factor κB (NF-κB) (p65) and mammalian target of rapamycin (p-mTOR) signaling proteins and their downstream mediators. Together, these results demonstrated strong antiproliferative and apoptosis-inducing activity of verrucarin A for PDA cells through cell cycle arrest and inhibition of the prosurvival (antiapoptotic) AKT/NF-κB/mTOR signaling. PMID:27573873

  8. Antidiabetic drug metformin inhibits esophageal adenocarcinoma cell proliferation in vitro and in vivo.

    Science.gov (United States)

    Fujihara, Shintaro; Kato, Kiyohito; Morishita, Asahiro; Iwama, Hisakazu; Nishioka, Tomoko; Chiyo, Taiga; Nishiyama, Noriko; Miyoshi, Hisaaki; Kobayashi, Mitsuyoshi; Kobara, Hideki; Mori, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Masaki, Tsutomu

    2015-05-01

    Esophageal carcinoma is the eighth most common cancer worldwide and the sixth leading cause of cancer-related deaths, with one of the worst prognoses of any form of cancer. Treatment with the anti-diabetic drug metformin has been associated with reduced cancer incidence in patients with type 2 diabetes. This study therefore evaluated the effects of metformin on the proliferation, in vitro and in vivo, of human esophageal adenocarcinoma cells, as well as the microRNAs associated with the antitumor effects of metformin. Metformin inhibited the proliferation of the esophageal adenocarcinoma cell lines OE19, OE33, SK-GT4 and OACM 5.1C, blo