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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. Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression.

    Science.gov (United States)

    Willoughby, Jamin A; Sundar, Shyam N; Cheung, Mark; Tin, Antony S; Modiano, Jaime; Firestone, Gary L

    2009-01-23

    Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter. PMID:19017637

  4. cdk4 Deficiency Inhibits Skin Tumor Development but Does Not Affect Normal Keratinocyte Proliferation

    Science.gov (United States)

    Rodriguez-Puebla, Marcelo L.; Miliani de Marval, Paula L.; LaCava, Margaret; Moons, David S.; Kiyokawa, Hiroaki; Conti, Claudio J.

    2002-01-01

    Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue. PMID:12163365

  5. 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...

  6. Inhibition of endothelial Cdk5 reduces tumor growth by promoting non-productive angiogenesis.

    Science.gov (United States)

    Merk, Henriette; Zhang, Siwei; Lehr, Thorsten; Müller, Christoph; Ulrich, Melanie; Bibb, James A; Adams, Ralf H; Bracher, Franz; Zahler, Stefan; Vollmar, Angelika M; Liebl, Johanna

    2016-02-01

    Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels. Here we pin down the in vivo impact of endothelial Cdk5 inhibition in angiogenesis and elucidate the underlying mechanism in order to judge the potential of Cdk5 as a novel anti-angiogenic and anti-cancer target. By the use of endothelial-specific Cdk5 knockout mouse models and various endothelial and tumor cell based assays including human tumor xenograft models, we show that endothelial-specific knockdown of Cdk5 results in excessive but non-productive angiogenesis during development but also in tumors, which subsequently leads to inhibition of tumor growth. As Cdk5 inhibition disrupted Notch function by reducing the generation of the active Notch intracellular domain (NICD) and Cdk5 modulates Notch-dependent endothelial cell proliferation and sprouting, we propose that the Dll4/Notch driven angiogenic signaling hub is an important and promising mechanistic target of Cdk5. In fact, Cdk5 inhibition can sensitize tumors to conventional anti-angiogenic treatment as shown in tumor xenograft models. In summary our data set the stage for Cdk5 as a drugable target to inhibit Notch-driven angiogenesis condensing the view that Cdk5 is a promising target for cancer therapy. PMID:26755662

  7. 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;

    2004-01-01

    With a view to the rational design of selective GSK-3beta inhibitors, 3D-QSAR CoMSIA models were developed for the inhibition of the three serine/threonine kinases CDK1/cyclin B, CDK5/p25, and GSK-3beta by compounds from the paullone inhibitor family. The models are based on the kinase inhibition...

  8. 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

  9. 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

  10. 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.

  11. Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle

    Science.gov (United States)

    Chiu, Hung-Chuan; Huang, Wei-Ru; Liao, Tsai-Ling; Wu, Hung-Yi; Munir, Muhammad; Shih, Wing-Ling; Liu, Hung-Jen

    2016-01-01

    The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1–60) of p17 and central region (aa 27–118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication. PMID:27603133

  12. 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

  13. 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

  14. 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.

  15. 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...

  16. 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...

  17. 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.

  18. 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...

  19. Metabolic re-programming 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.

    2016-01-01

    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 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. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and 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

  20. 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...

  1. Anti-diabetic drugs inhibit obesity-linked phosphorylation of PPARgamma by Cdk5.

    Science.gov (United States)

    Choi, Jang Hyun; Banks, Alexander S; Estall, Jennifer L; Kajimura, Shingo; Boström, Pontus; Laznik, Dina; Ruas, Jorge L; Chalmers, Michael J; Kamenecka, Theodore M; Blüher, Matthias; Griffin, Patrick R; Spiegelman, Bruce M

    2010-07-22

    Obesity induced in mice by high-fat feeding activates the protein kinase Cdk5 (cyclin-dependent kinase 5) in adipose tissues. This results in phosphorylation of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor gamma), a dominant regulator of adipogenesis and fat cell gene expression, at serine 273. This modification of PPARgamma does not alter its adipogenic capacity, but leads to dysregulation of a large number of genes whose expression is altered in obesity, including a reduction in the expression of the insulin-sensitizing adipokine, adiponectin. The phosphorylation of PPARgamma by Cdk5 is blocked by anti-diabetic PPARgamma ligands, such as rosiglitazone and MRL24. This inhibition works both in vivo and in vitro, and is completely independent of classical receptor transcriptional agonism. Similarly, inhibition of PPARgamma phosphorylation in obese patients by rosiglitazone is very tightly associated with the anti-diabetic effects of this drug. All these findings strongly suggest that Cdk5-mediated phosphorylation of PPARgamma may be involved in the pathogenesis of insulin-resistance, and present an opportunity for development of an improved generation of anti-diabetic drugs through PPARgamma.

  2. Brain-derived neurotrophic factor-dependent cdk1 inhibition prevents G2/M progression in differentiating tetraploid neurons.

    Directory of Open Access Journals (Sweden)

    María C Ovejero-Benito

    Full Text Available Neurodegeneration is often associated with DNA synthesis in neurons, the latter usually remaining for a long time as tetraploid cells before dying by apoptosis. The molecular mechanism preventing G2/M transition in these neurons remains unknown, but it may be reminiscent of the mechanism that maintains tetraploid retinal ganglion cells (RGCs in a G2-like state during normal development, thus preventing their death. Here we show that this latter process, known to depend on brain-derived neurotrophic factor (BDNF, requires the inhibition of cdk1 by TrkB. We demonstrate that a subpopulation of chick RGCs previously shown to become tetraploid co-expresses TrkB and cdk1 in vivo. By using an in vitro system that recapitulates differentiation and cell cycle re-entry of chick retinal neurons we show that BDNF, employed at concentrations specific for the TrkB receptor, reduces the expression of cdk1 in TrkB-positive, differentiating neurons. In this system, BDNF also inhibits the activity of both endogenous cdk1 and exogenously-expressed cdk1/cyclin B1 complex. This inhibition correlates with the phosphorylation of cdk1 at Tyr15, an effect that can be prevented with K252a, a tyrosine kinase inhibitor commonly used to prevent the activity of neurotrophins through their Trk receptors. The effect of BDNF on cdk1 activity is Tyr15-specific since BDNF cannot prevent the activity of a constitutively active form of cdk1 (Tyr15Phe when expressed in differentiating retinal neurons. We also show that BDNF-dependent phosphorylation of cdk1 at Tyr15 could not be blocked with MK-1775, a Wee1-selective inhibitor, indicating that Tyr15 phosphorylation in cdk1 does not seem to occur through the canonical mechanism observed in proliferating cells. We conclude that the inhibition of both expression and activity of cdk1 through a BDNF-dependent mechanism contributes to the maintenance of tetraploid RGCs in a G2-like state.

  3. Inhibiting the cyclin-dependent kinase CDK5 blocks pancreatic cancer formation and progression through the suppression of Ras-Ral signaling.

    Science.gov (United States)

    Feldmann, Georg; Mishra, Anjali; Hong, Seung-Mo; Bisht, Savita; Strock, Christopher J; Ball, Douglas W; Goggins, Michael; Maitra, Anirban; Nelkin, Barry D

    2010-06-01

    Cyclin-dependent kinase 5 (CDK5), a neuronal kinase that functions in migration, has been found to be activated in some human cancers in which it has been implicated in promoting metastasis. In this study, we investigated the role of CDK5 in pancreatic cancers in which metastatic disease is most common at diagnosis. CDK5 was widely active in pancreatic cancer cells. Functional ablation significantly inhibited invasion, migration, and anchorage-independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in the profound inhibition of Ras signaling through its critical effectors RalA and RalB. Conversely, restoring Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Our findings identify CDK5 as a pharmacologically tractable target to degrade Ras signaling in pancreatic cancer.

  4. Inhibiting the cyclin-dependent kinase CDK5 blocks pancreatic cancer formation and progression via suppression of Ras-Ral signaling

    Science.gov (United States)

    Feldmann, Georg; Mishra, Anjali; Hong, Seung-Mo; Bisht, Savita; Strock, Christopher J.; Ball, Douglas W.; Goggins, Michael; Maitra, Anirban; Nelkin, Barry D.

    2011-01-01

    Cyclin-dependent kinase 5 (CDK5), a neuronal kinase that functions in migration, has been found to be activated in some human cancers where it has been implicated in promoting metastasis. In this study, we investigated the role of CDK5 in pancreatic cancers where metastatic disease is most common at diagnosis. CDK5 was widely active in pancreatic cancer cells. Functional ablation significantly inhibited invasion, migration and anchorage-independent growth in vitro, and orthotopic tumor formation and systemic metastases in vivo. CDK5 blockade resulted in profound inhibition of Ras signaling through its critical effectors RalA and RalB. Conversely, restoring Ral function rescued the effects of CDK5 inhibition in pancreatic cancer cells. Our findings identify CDK5 as a pharmacologically tractable target to degrade Ras signaling in pancreatic cancer. PMID:20484029

  5. 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...

  6. 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.

  7. 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

  8. 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

  9. 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)

  10. 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)

  11. Inhibition of endothelial Cdk5 reduces tumor growth by promoting non-productive angiogenesis

    OpenAIRE

    Merk, Henriette; Zhang, Siwei; Lehr, Thorsten; Müller, Christoph; Ulrich, Melanie; Bibb, James A.; Adams, Ralf H.; Bracher, Franz; Zahler, Stefan; Vollmar, Angelika M.; Liebl, Johanna

    2016-01-01

    Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels. Here we pin down the in vivo impact of endothelial ...

  12. 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

  13. Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination

    NARCIS (Netherlands)

    Krajewska, M.; Heijink, A. M.; Bisselink, Y. J. W. M.; Seinstra, R. I.; Sillje, H. H. W.; de Vries, E. G. E.; van Vugt, M. A. T. M.

    2013-01-01

    In response to DNA breaks, the 'DNA damage response' provokes a cell cycle arrest to facilitate DNA repair. Recent findings have indicated that cells can respond to DNA damage throughout the cell cycle, except during mitosis. Specifically, various mitotic kinases, including Cdk1, Aurora A and Plk1,

  14. Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes.

    Science.gov (United States)

    Varadarajan, Ramya; Ayeni, Joseph; Jin, Zhigang; Homola, Ellen; Campbell, Shelagh D

    2016-07-01

    Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing. PMID:27170181

  15. PKA and CDK5 can phosphorylate specific serines on the intracellular domain of podoplanin (PDPN) to inhibit cell motility.

    Science.gov (United States)

    Krishnan, Harini; Retzbach, Edward P; Ramirez, Maria I; Liu, Tong; Li, Hong; Miller, W Todd; Goldberg, Gary S

    2015-07-01

    Podoplanin (PDPN) is a transmembrane glycoprotein that promotes tumor cell migration, invasion, and cancer metastasis. In fact, PDPN expression is induced in many types of cancer. Thus, PDPN has emerged as a functionally relevant cancer biomarker and chemotherapeutic target. PDPN contains 2 intracellular serine residues that are conserved between species ranging from mouse to humans. Recent studies indicate that protein kinase A (PKA) can phosphorylate PDPN in order to inhibit cell migration. However, the number and identification of specific residues phosphorylated by PKA have not been defined. In addition, roles of other kinases that may phosphorylate PDPN to control cell migration have not been investigated. We report here that cyclin dependent kinase 5 (CDK5) can phosphorylate PDPN in addition to PKA. Moreover, results from this study indicate that PKA and CDK5 cooperate to phosphorylate PDPN on both intracellular serine residues to decrease cell motility. These results provide new insight into PDPN phosphorylation dynamics and the role of PDPN in cell motility. Understanding novel mechanisms of PDPN intracellular signaling could assist with designing novel targeted chemotherapeutic agents and procedures. PMID:25959509

  16. CDK2 Regulates HIV-1 Transcription by Phosphorylation of CDK9 on Serine 90

    Directory of Open Access Journals (Sweden)

    Breuer Denitra

    2012-11-01

    Full Text Available Abstract Background HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity. Results The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94 that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation. Conclusion Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for

  17. Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation.

    Science.gov (United States)

    Khan, Naim A; Nishimura, Kazuhiro; Aires, Virginie; Yamashita, Tomoko; Oaxaca-Castillo, David; Kashiwagi, Keiko; Igarashi, Kazuei

    2006-10-01

    Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

  18. 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.

  19. 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.

  20. C-reactive protein promotes acute kidney injury via Smad3-dependent inhibition of CDK2/cyclin E.

    Science.gov (United States)

    Lai, Weiyan; Tang, Ying; Huang, Xiao R; Ming-Kuen Tang, Patrick; Xu, Anping; Szalai, Alexander J; Lou, Tan-Qi; Lan, Hui Y

    2016-09-01

    Acute kidney injury (AKI) is exacerbated in C-reactive protein transgenic mice but alleviated in Smad3 knockout mice. Here we used C-reactive protein transgenic/Smad3 wild-type and C-reactive protein transgenic/Smad3 knockout mice to investigate the signaling mechanisms by which C-reactive protein promotes AKI. Serum creatinine was elevated, and the extent of tubular epithelial cell necrosis following ischemia/reperfusion-induced AKI was greater in C-reactive protein transgenics but was blunted when Smad3 was deleted. Exacerbation of AKI in C-reactive protein transgenics was associated with increased TGF-β/Smad3 signaling and expression of the cyclin kinase inhibitor p27, but decreased phosphorylated CDK2 and expression of cyclin E. Concomitantly, tubular epithelial cell proliferation was arrested at the G1 phase in C-reactive protein transgenics with fewer cells entering the S-phase cell cycle as evidenced by fewer bromodeoxyuridine-positive cells. In contrast, the protection from AKI in C-reactive protein transgenic/Smad3 knockout mice was associated with decreased expression of p27 and promotion of CDK2/cyclin E-dependent G1/S transition of tubular epithelial cells. In vitro studies using tubular epithelial cells showed that C-reactive protein activates Smad3 via both TGF-β-dependent and ERK/MAPK cross talk mechanisms, Smad3 bound directly to p27, and blockade of Smad3 or the Fc receptor CD32 prevented C-reactive protein-induced p27-dependent G1 cell cycle arrest. In vivo, treatment of C-reactive protein transgenics with a Smad3 inhibitor largely improved AKI outcomes. Thus, C-reactive protein may promote AKI by impairing tubular epithelial cell regeneration via the CD32-Smad3-p27-driven inhibition of the CDK2/cyclin E complex. Targeting Smad3 may offer a new treatment approach for AKI. PMID:27470679

  1. 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

  2. Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

    Directory of Open Access Journals (Sweden)

    Kumari Ratna

    2010-07-01

    Full Text Available Abstract Background p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5 is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. Results To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. Conclusion Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

  3. Androgen suppresses the proliferation of androgen receptor-positive castration-resistant prostate cancer cells via inhibition of Cdk2, CyclinA, and Skp2.

    Directory of Open Access Journals (Sweden)

    John M Kokontis

    Full Text Available The majority of prostate cancer (PCa patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC. We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27(Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27(Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.

  4. Androgen suppresses the proliferation of androgen receptor-positive castration-resistant prostate cancer cells via inhibition of Cdk2, CyclinA, and Skp2.

    Science.gov (United States)

    Kokontis, John M; Lin, Hui-Ping; Jiang, Shih Sheng; Lin, Ching-Yu; Fukuchi, Junichi; Hiipakka, Richard A; Chung, Chi-Jung; Chan, Tzu-Min; Liao, Shutsung; Chang, Chung-Ho; Chuu, Chih-Pin

    2014-01-01

    The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27(Kip1); and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27(Kip1) and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.

  5. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

    Science.gov (United States)

    Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2015-02-01

    Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma.

  6. 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

    The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...... ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27Kip1 levels rose and the inhibitor accumulated in both Cdk4......-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...

  7. 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.

  8. 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.

  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. Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.

    Science.gov (United States)

    Verba, Kliment A; Wang, Ray Yu-Ruei; Arakawa, Akihiko; Liu, Yanxin; Shirouzu, Mikako; Yokoyama, Shigeyuki; Agard, David A

    2016-06-24

    The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions. PMID:27339980

  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. Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death.

    Science.gov (United States)

    Kim, C; Yun, N; Lee, J; Youdim, M B H; Ju, C; Kim, W-K; Han, P-L; Oh, Y J

    2016-02-01

    Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP's E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIP(S20A), but not CHIP(WT), attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli.

  16. Protein phosphatase-1 activates CDK9 by dephosphorylating Ser175.

    Directory of Open Access Journals (Sweden)

    Tatiana Ammosova

    Full Text Available The cyclin-dependent kinase CDK9/cyclin T1 induces HIV-1 transcription by phosphorylating the carboxyterminal domain (CTD of RNA polymerase II (RNAPII. CDK9 activity is regulated by protein phosphatase-1 (PP1 which was previously shown to dephosphorylate CDK9 Thr186. Here, we analyzed the effect of PP1 on RNAPII phosphorylation and CDK9 activity. The selective inhibition of PP1 by okadaic acid and by NIPP1 inhibited phosphorylation of RNAPII CTD in vitro and in vivo. Expression of the central domain of NIPP1 in cultured cells inhibited the enzymatic activity of CDK9 suggesting its activation by PP1. Comparison of dephosphorylation of CDK9 phosphorylated by ((32P in vivo and dephosphorylation of CDK9's Thr186 analyzed by Thr186 phospho-specific antibodies, indicated that a residue other than Thr186 might be dephosphorylated by PP1. Analysis of dephosphorylation of phosphorylated peptides derived from CDK9's T-loop suggested that PP1 dephosphorylates CDK9 Ser175. In cultured cells, CDK9 was found to be phosphorylated on Ser175 as determined by combination of Hunter 2D peptide mapping and LC-MS analysis. CDK9 S175A mutant was active and S175D--inactive, and dephosphorylation of CDK9's Ser175 upregulated HIV-1 transcription in PP1-dependent manner. Collectively, our results point to CDK9 Ser175 as novel PP1-regulatory site which dephosphorylation upregulates CDK9 activity and contribute to the activation of HIV-1 transcription.

  17. Proteomic analysis of the human cyclin-dependent kinase family reveals a novel CDK5 complex involved in cell growth and migration.

    Science.gov (United States)

    Xu, Shuangbing; Li, Xu; Gong, Zihua; Wang, Wenqi; Li, Yujing; Nair, Binoj Chandrasekharan; Piao, Hailong; Yang, Kunyu; Wu, Gang; Chen, Junjie

    2014-11-01

    Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases. PMID:25096995

  18. FDCs-miR-548m-CDK6轴在套细胞淋巴瘤集落形成中的研究%Role of FDCs-miR-548m-CDK6 axis in clonogenicity of mantle cell lymphoma

    Institute of Scientific and Technical Information of China (English)

    王芳; 张新伟; 张翼鷟; 魏枫; 任秀宝

    2014-01-01

    目的:探讨FDCs-miR-548m-CDK6轴在套细胞淋巴瘤(mantle cell lymphoma,MCL)集落形成中的作用。方法:分别采用RT-qPCR和Western blot检测MCL细胞与滤泡树突状细胞(FDCs)共培养后miR-548m和CDK6的变化。以生物信息学软件预测miR-548m的靶点,Western Blot检测MCL细胞系分别转染pre-miR-548m和anti-miR-548后细胞周期蛋白依赖激酶6(CDK6)的变化。荧光素酶报告基因实验验证CDK6是否为miR-548m的直接作用靶点。MCL细胞系与/不与FDCs共培养,过表达miR-548m或者敲低CDK6后MCL的集落形成能力。结果:FDCs与MCL的粘附作用可以下调miR-548m并上调CDK6。生物信息学软件预测显示CDK6的3'UTR是miR-548m的潜在靶点,且过表达miR-548m能够减少CDK6的表达,抑制miR-548m表达能够增加CDK6。荧光素酶报告基因实验证实CDK6的3'UTR是miR-548m的一个直接作用靶点。集落形成实验显示过表达miR-548m或者敲低CDK6后,细胞的集落形成能力明显减弱。结论:FDCs通过抑制MCL中miR-548m表达上调CDK6,增强MCL的集落形成能力。%Objective:To study the role of FDCs-miR-548m-CDK6 axis on clonogenicity in mantle cell lymphoma. Methods:RT-qPCR and Western blot were used respectively to test the expression of miR-548m and CDK6. Bioinformatics assay was applied to predict the targets of miR-548m, and Western Blot was used to test the expression level of CDK6 after miR-548m overexpression or in-hibition. Luciferase report assay was performed to test whether CDK6 was a direct target of miR-548m. Colony forming assay was used to test the colony forming activity in MCL after overexpression of miR-548m or knockdown of CDK6. Results:Cell adhesion to FDCs induced downregulation of miR-548m and CDK6 expression in MCL. Bioinformatics assay revealed that miR-548m could target the 3'-UTR of CDK6 and that a negative correlation exists between the level of miR-548m and the CDK6 expression. Luciferase report

  19. Enhanced Malignant Tumorigenesis in Cdk4-Transgenic Mice

    Science.gov (United States)

    Miliani de Marval, Paula L.; Macias, Everardo; Conti, Claudio J.; Rodriguez-Puebla, Marcelo L.

    2010-01-01

    In a previous study, we reported that overexpression of CDK4 in mouse epidermis results in epidermal hyperplasia, hypertrophy and severe dermal fibrosis. In this study, we have investigated the susceptibility to skin tumor formation by forced expression of CDK4. Skin tumors from transgenic mice showed a dramatic increase in the rate of malignant progression to squamous cell carcinomas (SCC) in an initiation-promotion protocol. Histopathological analysis of papillomas from transgenic mice showed an elevated number of premalignant lesions characterized by dysplasia and marked atypia. Interestingly, transgenic mice also developed tumors in initiated but not promoted skin, demonstrating that CDK4 replaced the action of tumor promoters. These results suggest that expression of cyclin D1 upon ras activation synergizes with CDK4 overexpression. However, cyclin D1 transgenic mice and double transgenic mice for cyclin D1 and CDK4 did not show increased malignant progression in comparison to CDK4 transgenic mice. Biochemical analysis of tumors showed that CDK4 sequesters the CDK2 inhibitors p27Kip1 and p21Cip1 suggesting that indirect activation of CDK2 plays an important role in tumor development. These results indicate that, contrary to the general assumption, the catalytic subunit, CDK4, has higher oncogenic activity than cyclin D1, revealing a potential use of CDK4 as therapeutic target. PMID:14647432

  20. 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.

  1. p120-catenin is necessary for neuroprotection induced by CDK5 silencing in models of Alzheimer's disease.

    Science.gov (United States)

    Uribe-Arias, Alejandro; Posada-Duque, Rafael Andrés; González-Billault, Christian; Villegas, Andrés; Lopera, Francisco; Cardona-Gómez, Gloria Patricia

    2016-08-01

    Cyclin-dependent kinase 5 (CDK5) plays important roles in synaptic function. Its unregulated over-activation has been, however, associated with neurodegeneration in Alzheimer's disease. Our previous studies revealed that CDK5 silencing ameliorates tauopathy and spatial memory impairment in the 3xTgAD mouse model. However, how CDK5 targeting affects synaptic adhesion proteins, such as those involved in the cadherin/catenin system, during learning and memory processes is not completely understood. In this study, we detected reduced expression of p120 catenin (p120 ctn), N-cadherin, and β-catenin in the brain of human Alzheimer's disease patients, in addition to a reduced PSD95 and GluN2B protein levels in a 3xTgAD mouse model. Such decrease in synaptic proteins was recovered by CDK5 silencing in mice leading to a better learning and memory performance. Additionally, CDK5 inhibition or knockout increased p120 ctn levels. Moreover, in a glutamate-induced excitotoxicity model, CDK5 silencing-induced neuroprotection depended on p120 ctn. Together, those findings suggest that p120 ctn plays an important role in the neuronal dysfunction of Alzheimer's disease models and contributes to CDK5 silencing-induced neuroprotection and improvement of memory function. p120ctn is part of the synaptic adhesion molecular complex N-cadh/p120ctn/B-ctn/PSD95, and it has a pivotal role in cell adhesion stabilization and dendritic spine modulation. Our data show that synaptic adhesion complex is affected in AD human brains and in AD models. This complex is recovered by the silencing of CDK5, preventing memory dysfunction in an AD mice model and contributing to the neuroprotection in a depend-mode of p120ctn. PMID:27273428

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. CDK1-dependent inhibition of the E3 ubiquitin ligase CRL4CDT2 ensures robust transition from S Phase to Mitosis.

    Science.gov (United States)

    Rizzardi, Lindsay F; Coleman, Kate E; Varma, Dileep; Matson, Jacob P; Oh, Seeun; Cook, Jeanette Gowen

    2015-01-01

    Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4(CDT2) ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA(DNA)) triggers the interaction between CRL4(CDT2) and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA(DNA) is no longer sufficient to trigger CRL4(CDT2)-mediated degradation. A CDK1-dependent mechanism that blocks CRL4(CDT2) activity by interfering with CDT2 recruitment to chromatin actively protects CRL4(CDT2) substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4(CDT2) inactivation contributes to efficient transition from S phase to mitosis.

  9. 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

  10. 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.

  11. Expression of constitutively active CDK1 stabilizes APC-Cdh1 substrates and potentiates premature spindle assembly and checkpoint function in G1 cells.

    Directory of Open Access Journals (Sweden)

    Yan Ma

    Full Text Available Mitotic progression in eukaryotic cells depends upon the activation of cyclin-dependent kinase 1 (CDK1, followed by its inactivation through the anaphase-promoting complex (APC/cyclosome-mediated degradation of M-phase cyclins. Previous work revealed that expression of a constitutively active CDK1 (CDK1AF in HeLa cells permitted their division, but yielded G1 daughter cells that underwent premature S-phase and early mitotic events. While CDK1AF was found to impede the sustained activity of APC-Cdh1, it was unknown if this defect improperly stabilized mitotic substrates and contributed to the occurrence of these premature M phases. Here, we show that CDK1AF expression in HeLa cells improperly stabilized APC-Cdh1 substrates in G1-phase daughter cells, including mitotic kinases and the APC adaptor, Cdc20. Division of CDK1AF-expressing cells produced G1 daughters with an accelerated S-phase onset, interrupted by the formation of premature bipolar spindles capable of spindle assembly checkpoint function. Further characterization of these phenotypes induced by CDK1AF expression revealed that this early spindle formation depended upon premature CDK1 and Aurora B activities, and their inhibition induced rapid spindle disassembly. Following its normal M-phase degradation, we found that the absence of Wee1 in these prematurely cycling daughter cells permitted the endogenous CDK1 to contribute to these premature mitotic events, since expression of a non-degradable Wee1 reduced the number of cells that exhibited premature cyclin B1oscillations. Lastly, we discovered that Cdh1-ablated cells could not be forced into a premature M phase, despite cyclin B1 overexpression and proteasome inhibition. Together, these results demonstrate that expression of constitutively active CDK1AF hampers the destruction of critical APC-Cdh1 targets, and that this type of condition could prevent newly divided cells from properly maintaining a prolonged interphase state. We

  12. 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...

  13. Molecular Mechanism of Enhanced Anticancer Effect of Nanoparticle Formulated LY2835219 via p16-CDK4/6-pRb Pathway in Colorectal Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Xu Tang

    2016-01-01

    Full Text Available LY2835219 is a dual inhibitor to CDK4 and CDK6. This study was to prepare LY2835219-loaded chitosan nanoparticles (CNP/LY and LY2835219-loaded hyaluronic acid-conjugated chitosan nanoparticles (HACNP/LY and revealed their anticancer effect and influence on p16-CDK4/6-pRb pathway against colon cell line. The nanoparticle sizes of CNP/LY and HACNP/LY were approximately 195±39.6 nm and 217±31.1 nm, respectively. The zeta potentials of CNP/LY and HACNP/LY were 37.3±1.5 mV and 30.3±2.2 mV, respectively. And the preparation process showed considerable drug encapsulation efficiency and loading efficiency. LY2835219, CNP/LY, and HACNP/LY inhibited HT29 cell proliferation with 0.68, 0.54, and 0.30 μM of IC50, respectively. G1 phase was arrested by LY2835219 and its formulations. Furthermore, inhibition of CDK4/6 by LY2835219 formulations induced CDK4, CDK6, cyclin D1, and pRb decrease and p16 increase at both protein and mRNA levels. Overall, nanoparticle formulated LY2835219 could enhance the cytotoxicity and cell cycle arrest, and HACNP/LY strengthened the trend furtherly compared to CNP/LY. It is the first time to demonstrate the anticancer effect and mechanism against HT29 by LY2835219 and its nanoparticles. The drug and its nanoparticle formulations delay the cell growth and arrest cell cycle through p16-CDK4/6-pRb pathway, while the nanoparticle formulated LY2835219 could strengthen the process.

  14. 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...

  15. 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

  16. 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

  17. 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.

  18. Structure-based drug design to the discovery of new 2-aminothiazole CDK2 inhibitors.

    Science.gov (United States)

    Vulpetti, Anna; Casale, Elena; Roletto, Fulvia; Amici, Raffaella; Villa, Manuela; Pevarello, Paolo

    2006-03-01

    N-(5-Bromo-1,3-thiazol-2-yl)butanamide (compound 1) was found active (IC50=808 nM) in a high throughput screening (HTS) for CDK2 inhibitors. By exploiting crystal structures of several complexes between CDK2 and inhibitors and applying structure-based drug design (SBDD), we rapidly discovered a very potent and selective CDK2 inhibitor 4-[(5-isopropyl-1,3-thiazol-2-yl)amino] benzenesulfonamide (compound 4, IC50=20 nM). The syntheses, structure-based analog design, kinases inhibition data and X-ray crystallographic structures of CDK2/inhibitor complexes are reported.

  19. O(2/3) exposure inhibits cell progression affecting cyclin B1/cdk1 activity in SK-N-SH while induces apoptosis in SK-N-DZ neuroblastoma cells.

    Science.gov (United States)

    Cannizzaro, A; Verga Falzacappa, C Verga; Martinelli, M; Misiti, S; Brunetti, E; Bucci, B

    2007-10-01

    In search for innovative therapeutic agents for children neuroblastoma, the oxygen therapy could be considered an alternative anti-tumoral treatment. Given the physiochemical properties of O(2/3) gas mixture including fairly low aqueous solubility and spreading, and the interesting perspective of hyperoxia, we analyzed the inhibitory effect of O(2/3) treatment on two human neuroblastoma cell lines (SK-N-SH and SK-N-DZ). In this study, we demonstrated that O(2/3) treatment was able to induce cell growth inhibition and cell cycle perturbation in both cell lines. We observed an arrest at G(2) phase, accompanied by an alteration in the expression and localization of cyclin B1/cdk1 complex and a reduction in its activity in SK-N-SH cells. This reduction was consistent with the increase in both Wee1 and chk1 protein levels. On the contrary, O(2/3) induced apoptosis in SK-N-DZ cells via caspase 3 activation and Poly ADP-ribose polymerase-1 (PARP) cleavage, associated with an increase in the pro-apoptotic Bax protein. Consequently, we considered the possibility of improving the responsiveness to chemotherapeutic agents such as Cisplatin, Etoposide, and Gemcitabine in combination with O(2/3) treatment. The combined treatments produced a stronger cell inhibitory effect than Cisplatin and Etoposide used alone in SK-N-SH cells. On the contrary, the combination data were not significantly different from O(2/3) treatment alone in SK-N-DZ cells, thus suggesting that the obtained changes in cell growth inhibition were due to the effect of O(2/3) alone. PMID:17477375

  20. All-Trans Retinoic Acid Induces DU145 Cell Cycle Arrest through Cdk5 Activation

    Directory of Open Access Journals (Sweden)

    Eugene Lin

    2014-05-01

    Full Text Available Background/Aims: All-trans retinoic acid (ATRA, the active form of vitamin A, plays an important role in the growth arrest of numerous types of cancer cells. It has been indicated that cyclin-dependent kinase 5 (Cdk5 activity can be affected by ATRA treatment. Our previous results demonstrate the involvement of Cdk5 in the fate of prostate cancer cells. The purpose of this study is to examine whether Cdk5 is involved in ATRA-induced growth arrest of the castration-resistant cancer cell line DU145 through up-regulating Cdk inhibitor protein, p27. Methods: DU145 cells were treated with ATRA, and cell proliferation, protein expression, and protein localization of Cdk5/p27 were examined. Cell proliferation and cell cycle distribution were also determined under Cdk5 inhibition induced by inhibitor or knockdown. Results: ATRA treatment inhibited DU145 cell proliferation and significantly increased p27 expression through Cdk5 up-regulation. Immunocytochemical data showed that a Cdk5 inhibitor reduced ATRA-triggered nuclear distribution of p27 in DU145 cells. The proliferation inhibition and G1 phase accumulation of DU145 cells were significantly increased by ATRA treatment, whereas Cdk5 inhibitor and siRNA could reverse these effects. Conclusions: Our results demonstrate that ATRA induced growth inhibition in castration-resistant prostate cancer cells through activating Cdk5 and p27. We hope this finding will increase the knowledge of prostate cancer treatment and can be applied in patients' nutritional control in the future.

  1. CK1δ activity is modulated by CDK2/E- and CDK5/p35-mediated phosphorylation.

    Science.gov (United States)

    Ianes, Chiara; Xu, Pengfei; Werz, Natalie; Meng, Zhigang; Henne-Bruns, Doris; Bischof, Joachim; Knippschild, Uwe

    2016-02-01

    CK1 protein kinases form a family of serine/threonine kinases which are highly conserved through different species and ubiquitously expressed. CK1 family members can phosphorylate numerous substrates thereby regulating different biological processes including membrane trafficking, cell cycle regulation, circadian rhythm, apoptosis, and signal transduction. Deregulation of CK1 activity and/or expression contributes to the development of neurological diseases and cancer. Therefore, CK1 became an interesting target for drug development and it is relevant to further understand the mechanisms of its regulation. In the present study, Cyclin-dependent kinase 2/Cyclin E (CDK2/E) and Cyclin-dependent kinase 5/p35 (CDK5/p35) were identified as cellular kinases able to modulate CK1δ activity through site-specific phosphorylation of its C-terminal domain. Furthermore, pre-incubation of CK1δ with CDK2/E or CDK5/p35 reduces CK1δ activity in vitro, indicating a functional impact of the interaction between CK1δ and CDK/cyclin complexes. Interestingly, inhibition of Cyclin-dependent kinases by Dinaciclib increases CK1δ activity in pancreatic cancer cells. In summary, these results suggest that CK1δ activity can be modulated by the interplay between CK1δ and CDK2/E or CDK5/p35. These findings extend our knowledge about CK1δ regulation and may be of use for future development of CK1-related therapeutic strategies in the treatment of neurological diseases or cancer. PMID:26464264

  2. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  3. 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.

  4. 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.

  5. 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

  6. Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

    Science.gov (United States)

    Deshmukh, Abhijit S.; Mitra, Pallabi; Maruthi, Mulaka

    2016-01-01

    Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5′-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis. PMID:27759017

  7. 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

  8. Cdk8 deletion in the Apc(Min) murine tumour model represses EZH2 activity and accelerates tumourigenesis.

    Science.gov (United States)

    McCleland, Mark L; Soukup, Tim M; Liu, Scot D; Esensten, Jonathan H; de Sousa e Melo, Felipe; Yaylaoglu, Murat; Warming, Soren; Roose-Girma, Merone; Firestein, Ron

    2015-12-01

    CDK8 is a dissociable kinase module of the Mediator complex and has been shown to play an important role in transcriptional regulation in organisms as diverse as yeast and humans. Recent studies suggest that CDK8 functions as an oncoprotein in melanoma and colon cancer. Importantly, these studies were conducted using in vitro cell line models and the role of CDK8 in tumourigenesis in vivo has not been explored. We have generated a mouse with a Cdk8 conditional knockout allele and examined the consequences of Cdk8 loss on normal tissue homeostasis and tumour development in vivo. Cdk8 deletion in the young adult mouse did not induce any gross or histopathological abnormalities, implying that Cdk8 is largely dispensable for somatic cellular homeostasis. In contrast, Cdk8 deletion in the Apc(Min) intestinal tumour model shortened the animals' survival and increased tumour burden. Although Cdk8 deletion did not affect tumour initiation, intestinal tumour size and growth rate were significantly increased in Cdk8-null animals. Transcriptome analysis performed on Cdk8-null intestinal cells revealed up-regulation of genes that are governed by the Polycomb group (PcG) complex. In support of these findings, Cdk8-null intestinal cells and tumours displayed a reduction in histone H3K27 trimethylation, both globally and at the promoters of a number of PcG-regulated genes involved in oncogenic signalling. Together, our findings uncover a tumour suppressor function for CDK8 in vivo and suggest that the role of CDK8 activity in driving oncogenesis is context-specific. Sequencing data were deposited at GEO (Accession No. GSE71385).

  9. 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...

  10. 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.

  11. 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.

  12. Proteins regulating cyclin dependent kinases Cdk4 and Cdk5

    NARCIS (Netherlands)

    Moorthamer, M.J.M.W.

    1999-01-01

    The exact passage through the eukaryotic cell cycle is regulated by the progressive activation and inactivation of a family Cdk-s. Cancer cells evolve from normal cells when some essential processes in a dividing cell malfunction. This causes inappropriate replication, segregation and repair of the

  13. 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

  14. Molecular modifiers reveal a mechanism of pathological crystal growth inhibition

    Science.gov (United States)

    Chung, Jihae; Granja, Ignacio; Taylor, Michael G.; Mpourmpakis, Giannis; Asplin, John R.; Rimer, Jeffrey D.

    2016-08-01

    Crystalline materials are crucial to the function of living organisms, in the shells of molluscs, the matrix of bone, the teeth of sea urchins, and the exoskeletons of coccoliths. However, pathological biomineralization can be an undesirable crystallization process associated with human diseases. The crystal growth of biogenic, natural and synthetic materials may be regulated by the action of modifiers, most commonly inhibitors, which range from small ions and molecules to large macromolecules. Inhibitors adsorb on crystal surfaces and impede the addition of solute, thereby reducing the rate of growth. Complex inhibitor-crystal interactions in biomineralization are often not well elucidated. Here we show that two molecular inhibitors of calcium oxalate monohydrate crystallization—citrate and hydroxycitrate—exhibit a mechanism that differs from classical theory in that inhibitor adsorption on crystal surfaces induces dissolution of the crystal under specific conditions rather than a reduced rate of crystal growth. This phenomenon occurs even in supersaturated solutions where inhibitor concentration is three orders of magnitude less than that of the solute. The results of bulk crystallization, in situ atomic force microscopy, and density functional theory studies are qualitatively consistent with a hypothesis that inhibitor-crystal interactions impart localized strain to the crystal lattice and that oxalate and calcium ions are released into solution to alleviate this strain. Calcium oxalate monohydrate is the principal component of human kidney stones and citrate is an often-used therapy, but hydroxycitrate is not. For hydroxycitrate to function as a kidney stone treatment, it must be excreted in urine. We report that hydroxycitrate ingested by non-stone-forming humans at an often-recommended dose leads to substantial urinary excretion. In vitro assays using human urine reveal that the molecular modifier hydroxycitrate is as effective an inhibitor of nucleation

  15. Molecular modifiers reveal a mechanism of pathological crystal growth inhibition

    Science.gov (United States)

    Chung, Jihae; Granja, Ignacio; Taylor, Michael G.; Mpourmpakis, Giannis; Asplin, John R.; Rimer, Jeffrey D.

    2016-08-01

    Crystalline materials are crucial to the function of living organisms, in the shells of molluscs, the matrix of bone, the teeth of sea urchins, and the exoskeletons of coccoliths. However, pathological biomineralization can be an undesirable crystallization process associated with human diseases. The crystal growth of biogenic, natural and synthetic materials may be regulated by the action of modifiers, most commonly inhibitors, which range from small ions and molecules to large macromolecules. Inhibitors adsorb on crystal surfaces and impede the addition of solute, thereby reducing the rate of growth. Complex inhibitor–crystal interactions in biomineralization are often not well elucidated. Here we show that two molecular inhibitors of calcium oxalate monohydrate crystallization—citrate and hydroxycitrate—exhibit a mechanism that differs from classical theory in that inhibitor adsorption on crystal surfaces induces dissolution of the crystal under specific conditions rather than a reduced rate of crystal growth. This phenomenon occurs even in supersaturated solutions where inhibitor concentration is three orders of magnitude less than that of the solute. The results of bulk crystallization, in situ atomic force microscopy, and density functional theory studies are qualitatively consistent with a hypothesis that inhibitor–crystal interactions impart localized strain to the crystal lattice and that oxalate and calcium ions are released into solution to alleviate this strain. Calcium oxalate monohydrate is the principal component of human kidney stones and citrate is an often-used therapy, but hydroxycitrate is not. For hydroxycitrate to function as a kidney stone treatment, it must be excreted in urine. We report that hydroxycitrate ingested by non-stone-forming humans at an often-recommended dose leads to substantial urinary excretion. In vitro assays using human urine reveal that the molecular modifier hydroxycitrate is as effective an inhibitor of

  16. 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

  17. 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.

  18. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    Science.gov (United States)

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  19. Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

    Science.gov (United States)

    Tran, Kalvin Q; Tin, Antony S; Firestone, Gary L

    2014-03-01

    Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter. PMID:24296733

  20. Effect of sorafenib on inhibition of liver cancer cells and the expression levels of CDK5%索拉菲尼对肝癌细胞的抑制作用及其对CDK5表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    刘冲; 赵浩亮

    2012-01-01

    目的 探讨索拉菲尼对体外培养的人肝癌HepG2 细胞的增殖抑制作用及其对周期素依赖性蛋白激酶5 表达的影 响.方法 体外培养人肝癌HepG2 细胞株,采用MTT 法检测不同浓度索拉菲尼对HepG2 细胞增殖的影响;同时经细胞 爬片后,采用免疫细胞化学法检测肝癌细胞内CDK5 的表达情况.结果 索拉菲尼可使人肝癌细胞增殖率明显下降,且 呈明显的时间- 剂量依赖效应;药物作用48 h 后,给药组CDK5 的表达较对照组明显减少,其着色程度随药物浓度增高 而逐渐变淡.结论 索拉菲尼对人肝癌HepG2 细胞增殖有抑制作用,并能下调肝癌细胞中CDK5 的表达,从而起到抗肿 瘤的治疗作用.

  1. Cdk1 and SUMO regulate Swe1 stability.

    Directory of Open Access Journals (Sweden)

    Kobi J Simpson-Lavy

    Full Text Available The Swe1/Wee1 kinase phosphorylates and inhibits Cdk1-Clb2 and is a major mitotic switch. Swe1 levels are controlled by ubiquitin mediated degradation, which is regulated by interactions with various mitotic kinases. We have recently reported that Swe1 levels are capable of sensing the progress of the cell cycle by measuring the levels of Cdk1-Clb2, Cdc5 and Hsl1. We report here a novel mechanism that regulates the levels of Swe1. We show that S. cerevisiae Swe1 is modified by Smt3/SUMO on residue K594 in a Cdk1 dependant manner. A degradation of the swe1(K594R mutant that cannot be modified by Smt3 is considerably delayed in comparison to wild type Swe1. Swe1(K594R cells express elevated levels of Swe1 protein and demonstrate higher levels of Swe1 activity as manifested by Cdk1-Y19 phosphorylation. Interestingly this mutant is not targeted, like wild type Swe1, to the bud neck where Swe1 degradation takes place. We show that Swe1 is SUMOylated by the Siz1 SUMO ligase, and consequently siz1Δ cells express elevated levels of Swe1 protein and activity. Finally we show that swe1(K594R cells are sensitive to osmotic stress, which is in line with their compromised regulation of Swe1 degradation.

  2. CDK7-Dependent Transcriptional Addiction in Triple-Negative Breast Cancer

    Science.gov (United States)

    Wang, Yubao; Zhang, Tinghu; Kwiatkowski, Nicholas; Abraham, Brian J.; Lee, Tong Ihn; Xie, Shaozhen; Yuzugullu, Haluk; Von, Thanh; Li, Heyuan; Lin, Ziao; Stover, Daniel G.; Lim, Elgene; Wang, Zhigang C.; Iglehart, J. Dirk; Young, Richard A.; Gray, Nathanael S.; Zhao, Jean J.

    2015-01-01

    SUMMARY Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An “Achilles cluster” of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer. PMID:26406377

  3. 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

  4. 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.

  5. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    Science.gov (United States)

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  6. 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

  7. 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.

  8. NPM phosphorylation stimulates Cdk1, overrides G2/M checkpoint and increases leukemic blasts in mice.

    Science.gov (United States)

    Du, Wei; Zhou, Yun; Pike, Suzette; Pang, Qishen

    2010-02-01

    An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CDK) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Simultaneous inactivation of two CDK phosphorylation sites at Ser10 and Ser70 (NPM-AA) induced G(2)/M cell cycle arrest, phosphorylation of Cdk1 at Tyr15 (Cdc2(Tyr15)) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1(Tyr15) and Cdc25C sequestration was suppressed by expression of a phosphomimetic NPM mutant created on the same CDK sites (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 was required for proper interaction between Cdk1 and Cdc25C. Moreover, NPM-EE directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G(2)/M arrest and increased leukemia blasts in a NOD/SCID xenograft model. Thus, these findings reveal a novel function of NPM on regulation of cell cycle progression, in which phosphorylation of NPM controls cell cycle progression at G(2)/M transition through modulation of Cdk1 and Cdc25C activities.

  9. Dichotomy of cellular inhibition by small-molecule inhibitors revealed by single-cell analysis

    Science.gov (United States)

    Vogel, Robert M.; Erez, Amir; Altan-Bonnet, Grégoire

    2016-01-01

    Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings. PMID:27687249

  10. Gauchos and ochos: a Wee1-Cdk tango regulating mitotic entry

    Directory of Open Access Journals (Sweden)

    Enders Greg H

    2010-05-01

    Full Text Available Abstract The kinase Wee1 has been recognized for a quarter century as a key inhibitor of Cyclin dependent kinase 1 (Cdk1 and mitotic entry in eukaryotes. Nonetheless, Wee1 regulation is not well understood and its large amino-terminal regulatory domain (NRD has remained largely uncharted. Evidence has accumulated that cyclin B/Cdk1 complexes reciprocally inhibit Wee1 activity through NRD phosphorylation. Recent studies have identified the first functional NRD elements and suggested that vertebrate cyclin A/Cdk2 complexes also phosphorylate the NRD. A short NRD peptide, termed the Wee box, augments the activity of the Wee1 kinase domain. Cdk1/2-mediated phosphorylation of the Wee box (on T239 antagonizes kinase activity. A nearby region harbors a conserved RxL motif (RxL1 that promotes cyclin A/Cdk2 binding and T239 phosphorylation. Mutation of either T239 or RxL1 bolsters the ability of Wee1 to block mitotic entry, consistent with negative regulation of Wee1 through these sites. The region in human somatic Wee1 that encompasses RxL1 also binds Crm1, directing Wee1 export from the nucleus. These studies have illuminated important aspects of Wee1 regulation and defined a specific molecular pathway through which cyclin A/Cdk2 complexes foster mitotic entry. The complexity, speed, and importance of regulation of mitotic entry suggest that there is more to be learned.

  11. Cdk5 Modulates Long-Term Synaptic Plasticity and Motor Learning in Dorsolateral Striatum.

    Science.gov (United States)

    Hernandez, Adan; Tan, Chunfeng; Mettlach, Gabriel; Pozo, Karine; Plattner, Florian; Bibb, James A

    2016-01-01

    The striatum controls multiple cognitive aspects including motivation, reward perception, decision-making and motor planning. In particular, the dorsolateral striatum contributes to motor learning. Here we define an approach for investigating synaptic plasticity in mouse dorsolateral cortico-striatal circuitry and interrogate the relative contributions of neurotransmitter receptors and intracellular signaling components. Consistent with previous studies, we show that long-term potentiation (LTP) in cortico-striatal circuitry is facilitated by dopamine, and requires activation of D1-dopamine receptors, as well as NMDA receptors (NMDAR) and their calcium-dependent downstream effectors, including CaMKII. Moreover, we assessed the contribution of the protein kinase Cdk5, a key neuronal signaling molecule, in cortico-striatal LTP. Pharmacological Cdk5 inhibition, brain-wide Cdk5 conditional knockout, or viral-mediated dorsolateral striatal-specific loss of Cdk5 all impaired dopamine-facilitated LTP or D1-dopamine receptor-facilitated LTP. Selective loss of Cdk5 in dorsolateral striatum increased locomotor activity and attenuated motor learning. Taken together, we report an approach for studying synaptic plasticity in mouse dorsolateral striatum and critically implicate D1-dopamine receptor, NMDAR, Cdk5, and CaMKII in cortico-striatal plasticity. Furthermore, we associate striatal plasticity deficits with effects upon behaviors mediated by this circuitry. This approach should prove useful for the study of the molecular basis of plasticity in the dorsolateral striatum. PMID:27443506

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

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

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    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.

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

    Science.gov (United States)

    Varma, Hemant; Skildum, Andrew J; Conrad, Susan E

    2007-12-05

    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.

  19. 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

  20. Cyclin-Dependent Kinase 5 (CDK5 Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread—Identification of a Promising Novel therapeutic target

    Directory of Open Access Journals (Sweden)

    Savita Bisht

    2015-08-01

    Full Text Available Despite considerable progress in recent years, the overall prognosis of metastatic malignant melanoma remains poor, and curative therapeutic options are lacking. Therefore, better understanding of molecular mechanisms underlying melanoma progression and metastasis, as well as identification of novel therapeutic targets that allow inhibition of metastatic spread, are urgently required. The current study provides evidence for aberrant cyclin-dependent kinase 5 (CDK5 activation in primary and metastatic melanoma lesions by overexpression of its activator protein CDK5R1/p35. Moreover, using melanoma in vitro model systems, shRNA-mediated inducible knockdown of CDK5 was found to cause marked inhibition of cell motility, invasiveness, and anchorage-independent growth, while at the same time net cell growth was not affected. In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases. Inhibition of lung metastasis was further validated in a syngenic murine melanoma model. CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype. Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells. Thus, experimental data presented here provide strong evidence for a crucial role of aberrantly activated CDK5 in melanoma progression and metastasis and establish CDK5 as promising target for therapeutic intervention.

  1. 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

  2. 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.

  3. Cdk2 deficiency decreases ras/CDK4-dependent malignant progression, but not myc-induced tumorigenesis.

    Science.gov (United States)

    Macias, Everardo; Kim, Yongbaek; Miliani de Marval, Paula L; Klein-Szanto, Andres; Rodriguez-Puebla, Marcelo L

    2007-10-15

    We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinoma (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development, causing a selection of cells bearing activating Ha-ras mutations. We have also shown that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin, loss of CDK2 rescues CDK4-induced, but not myc-induced epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decreased incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathologic analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2-/- tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report of K5Myc/CDK4-/-, K5Myc/CDK2-/- mice developed oral tumors with the same frequency as K5Myc mice. Overall, we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved.

  4. Cdk2 deficiency decrease ras/cdk4-dependent malignant progression, but not myc-induced tumorigenesis

    Science.gov (United States)

    Macias, Everardo; Kim, Yongbaek; Miliani de Marval, Paula L.; Klein-Szanto, Andres; Rodriguez-Puebla, Marcelo L.

    2010-01-01

    We have previously shown that forced expression of CDK4 in mouse skin (K5CDK4 mice) results in increased susceptibility to squamous cell carcinomas (SCC) development in a chemical carcinogenesis protocol. This protocol induces skin papilloma development causing a selection of cells bearing activating Ha-ras mutations. We have also demonstrated that myc-induced epidermal proliferation and oral tumorigenesis (K5Myc mice) depends on CDK4 expression. Biochemical analysis of K5CDK4 and K5Myc epidermis as well as skin tumors showed that keratinocyte proliferation is mediated by CDK4 sequestration of p27Kip1 and p21Cip1, and activation of CDK2. Here, we studied the role of CDK2 in epithelial tumorigenesis. In normal skin loss of CDK2 rescues CDK4-induced, but not myc-induce epidermal hyperproliferation. Ablation of CDK2 in K5CDK4 mice results in decrease incidences and multiplicity of skin tumors as well as malignant progression to SCC. Histopathological analysis showed that K5CDK4 tumors are drastically more aggressive than K5CDK4/CDK2−/− tumors. On the other hand, we show that CDK2 is dispensable for myc-induced tumorigenesis. In contrast to our previous report K5Myc/CDK4−/− mice, K5Myc/CDK2−/− mice developed oral tumors with the same frequency as K5Myc mice. Overall we have established that ras-induced tumors are more susceptible to CDK2 ablation than myc-induced tumors, suggesting that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the oncogenic pathway involved. PMID:17942901

  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. Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity

    Science.gov (United States)

    Dorand, R. Dixon; Nthale, Joseph; Myers, Jay T.; Barkauskas, Deborah S.; Avril, Stefanie; Chirieleison, Steven M.; Pareek, Tej K.; Abbott, Derek W.; Stearns, Duncan S.; Letterio, John J.

    2016-01-01

    Cancers often evade immune surveillance by adopting peripheral tissue–tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4+ T cell–mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells. PMID:27463676

  8. Cdk5 disruption attenuates tumor PD-L1 expression and promotes antitumor immunity.

    Science.gov (United States)

    Dorand, R Dixon; Nthale, Joseph; Myers, Jay T; Barkauskas, Deborah S; Avril, Stefanie; Chirieleison, Steven M; Pareek, Tej K; Abbott, Derek W; Stearns, Duncan S; Letterio, John J; Huang, Alex Y; Petrosiute, Agne

    2016-07-22

    Cancers often evade immune surveillance by adopting peripheral tissue- tolerance mechanisms, such as the expression of programmed cell death ligand 1 (PD-L1), the inhibition of which results in potent antitumor immunity. Here, we show that cyclin-dependent kinase 5 (Cdk5), a serine-threonine kinase that is highly active in postmitotic neurons and in many cancers, allows medulloblastoma (MB) to evade immune elimination. Interferon-γ (IFN-γ)-induced PD-L1 up-regulation on MB requires Cdk5, and disruption of Cdk5 expression in a mouse model of MB results in potent CD4(+) T cell-mediated tumor rejection. Loss of Cdk5 results in persistent expression of the PD-L1 transcriptional repressors, the interferon regulatory factors IRF2 and IRF2BP2, which likely leads to reduced PD-L1 expression on tumors. Our finding highlights a central role for Cdk5 in immune checkpoint regulation by tumor cells. PMID:27463676

  9. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

    Directory of Open Access Journals (Sweden)

    Shu Zhang

    Full Text Available Cyclin-dependent kinase 5 (CDK5 is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1 as well as TP53-dependent transcriptional induction of p21(Cip1 in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells.

  10. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis.

    Science.gov (United States)

    Zhang, Shu; Lu, Zhen; Mao, Weiqun; Ahmed, Ahmed A; Yang, Hailing; Zhou, Jinhua; Jennings, Nicholas; Rodriguez-Aguayo, Cristian; Lopez-Berestein, Gabriel; Miranda, Roberto; Qiao, Wei; Baladandayuthapani, Veera; Li, Zongfang; Sood, Anil K; Liu, Jinsong; Le, Xiao-Feng; Bast, Robert C

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was measured by immunohistochemical staining of an ovarian cancer tissue microarray to correlate CDK5 expression with overall patient survival. Knockdown of CDK5 with siRNAs inhibits activation of AKT which significantly correlates with decreased cell growth and enhanced paclitaxel sensitivity in ovarian cancer cell lines. In addition, CDK5 knockdown alone and in combination with paclitaxel induced G1 cell cycle arrest and caspase 3 dependent apoptotic cell death associated with post-translational upregulation and nuclear translocation of TP53 and p27(Kip1) as well as TP53-dependent transcriptional induction of p21(Cip1) in wild type TP53 cancer cells. Treatment of HEYA8 and A2780 wild type TP53 xenografts in nu/nu mice with CDK5 siRNA and paclitaxel produced significantly greater growth inhibition than either treatment alone. Increased expression of CDK5 in human ovarian cancers correlates inversely with overall survival. CDK5 modulates paclitaxel sensitivity by regulating AKT activation, the cell cycle and caspase-dependent apoptosis. CDK5 inhibition can potentiate paclitaxel activity in human ovarian cancer cells. PMID:26146988

  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. 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)

  13. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic.

    Science.gov (United States)

    Zhang, Hai-Nan; Yang, Lina; Ling, Jian-Ya; Czajkowsky, Daniel M; Wang, Jing-Fang; Zhang, Xiao-Wei; Zhou, Yi-Ming; Ge, Feng; Yang, Ming-Kun; Xiong, Qian; Guo, Shu-Juan; Le, Huang-Ying; Wu, Song-Fang; Yan, Wei; Liu, Bingya; Zhu, Heng; Chen, Zhu; Tao, Sheng-Ce

    2015-12-01

    Arsenic is highly effective for treating acute promyelocytic leukemia (APL) and has shown significant promise against many other tumors. However, although its mechanistic effects in APL are established, its broader anticancer mode of action is not understood. In this study, using a human proteome microarray, we identified 360 proteins that specifically bind arsenic. Among the most highly enriched proteins in this set are those in the glycolysis pathway, including the rate-limiting enzyme in glycolysis, hexokinase-1. Detailed biochemical and metabolomics analyses of the highly homologous hexokinase-2 (HK2), which is overexpressed in many cancers, revealed significant inhibition by arsenic. Furthermore, overexpression of HK2 rescued cells from arsenic-induced apoptosis. Our results thus strongly implicate glycolysis, and HK2 in particular, as a key target of arsenic. Moreover, the arsenic-binding proteins identified in this work are expected to serve as a valuable resource for the development of synergistic antitumor therapeutic strategies.

  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. 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.

  17. 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 ...

  18. Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

    Science.gov (United States)

    Chaffee, Blake R; Shang, Fu; Chang, Min-Lee; Clement, Tracy M; Eddy, Edward M; Wagner, Brad D; Nakahara, Masaki; Nagata, Shigekazu; Robinson, Michael L; Taylor, Allen

    2014-09-01

    Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

  19. Crystal structure of MC159 reveals molecular mechanism of DISC assembly and FLIP inhibition.

    Science.gov (United States)

    Yang, Jin Kuk; Wang, Liwei; Zheng, Lixin; Wan, Fengyi; Ahmed, Misonara; Lenardo, Michael J; Wu, Hao

    2005-12-22

    The death-inducing signaling complex (DISC) comprising Fas, Fas-associated death domain (FADD), and caspase-8/10 is assembled via homotypic associations between death domains (DDs) of Fas and FADD and between death effector domains (DEDs) of FADD and caspase-8/10. Caspase-8/10 and FLICE/caspase-8 inhibitory proteins (FLIPs) that inhibit caspase activation at the DISC level contain tandem DEDs. Here, we report the crystal structure of a viral FLIP, MC159, at 1.2 Angstroms resolution. It reveals a noncanonical fold of DED1, a dumbbell-shaped structure with rigidly associated DEDs and a different mode of interaction in the DD superfamily. Whereas the conserved hydrophobic patch of DED1 interacts with DED2, the corresponding region of DED2 mediates caspase-8 recruitment and contributes to DISC assembly. In contrast, MC159 cooperatively assembles with Fas and FADD via an extensive surface that encompasses the conserved charge triad. This interaction apparently competes with FADD self-association and disrupts higher-order oligomerization required for caspase activation in the DISC. PMID:16364918

  20. Design, Synthesis and Biological Evaluation of Novel Pyrimido[4,5-d]pyrimidine CDK2 Inhibitors as Anti-Tumor Agents

    Science.gov (United States)

    El-Moghazy, Samir M.; Ibrahim, Diaa A.; Abdelgawad, Nagwa M.; Farag, Nahla A. H.; El-Khouly, Ahmad S.

    2011-01-01

    A series of 2,5,7-trisubstituted pyrimido[4,5-d]pyrimidine cyclin-dependent kinase (CDK2) inhibitors is designed and synthesized. 6-Amino-2-thiouracil is reacted with an aldehyde and thiourea to prepare the pyrimido[4,5-d]-pyrimidines. Alkylation and amination of the latter ones give different amino derivatives. These compounds show potent and selective CDK inhibitory activities and inhibit in vitro cellular proliferation in cultured human tumor cells. PMID:21886895

  1. 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.

  2. 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.

  3. 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

  4. 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...

  5. 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...

  6. Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

    Directory of Open Access Journals (Sweden)

    Hai-Xiao Jin

    Full Text Available The current paradigm of cyclin-dependent kinase (CDK regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1 Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+. The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

  7. miR-1 suppresses the growth of esophageal squamous cell carcinoma in vivo and in vitro through the downregulation of MET, cyclin D1 and CDK4 expression

    Science.gov (United States)

    JIANG, SEN; ZHAO, CHAO; YANG, XIAODI; LI, XIANGYANG; PAN, QING; HUANG, HAIJIN; WEN, XUYANG; SHAN, HUSHENG; LI, QIANWEN; DU, YUNXIANG; ZHAO, YAPING

    2016-01-01

    Several aberrant microRNAs (miRNAs or miRs) have been implicated in esophageal cancer (EC), which is widely prevalent in China. However, their role in EC tumorigenesis has not yet been fully elucidated. In the present study, we determined that miR-1 was downregulated in esophageal squamous cell carcinoma (ESCC) tissues compared with adjacent non-neoplastic tissues using RT-qPCR, and confirmed this using an ESCC cell line. Using a nude mouse xenograft model, we confirmed that the re-expression of miR-1 significantly inhibited ESCC tumor growth. A tetrazolium assay and a trypan blue exclusion assay revealed that miR-1 suppressed ESCC cell proliferation and increased apoptosis, whereas the silencing of miR-1 promoted cell proliferation and decreased apoptosis, suggesting that miR-1 is a novel tumor suppressor. To elucidate the molecular mechanisms of action of miR-1 in ESCC, we investigated putative targets using bioinformatics tools. MET, cyclin D1 and cyclin-dependent kinase 4 (CDK4), which are involved in the hepatocyte growth factor (HGF)/MET signaling pathway, were found to be targets of miR-1. miR-1 expression inversely correlated with MET, cyclin D1 and CDK4 expression in ESCC cells. miR-1 directly targeted MET, cyclin D1 and CDK4, suppressing ESCC cell growth. The newly identified miR-1/MET/cyclin D1/CDK4 axis provides new insight into the molecular mechanisms of ESCC pathogenesis and indicates a novel strategy for the diagnosis and treatment of ESCC. PMID:27247259

  8. 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.

  9. Redundant Regulation of Cdk1 Tyrosine Dephosphorylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kennedy, Erin K; Dysart, Michael; Lianga, Noel; Williams, Elizabeth C; Pilon, Sophie; Doré, Carole; Deneault, Jean-Sebastien; Rudner, Adam D

    2016-03-01

    Cdk1 activity drives both mitotic entry and the metaphase-to-anaphase transition in all eukaryotes. The kinase Wee1 and the phosphatase Cdc25 regulate the mitotic activity of Cdk1 by the reversible phosphorylation of a conserved tyrosine residue. Mutation of cdc25 in Schizosaccharomyces pombe blocks Cdk1 dephosphorylation and causes cell cycle arrest. In contrast, deletion of MIH1, the cdc25 homolog in Saccharomyces cerevisiae, is viable. Although Cdk1-Y19 phosphorylation is elevated during mitosis in mih1∆ cells, Cdk1 is dephosphorylated as cells progress into G1, suggesting that additional phosphatases regulate Cdk1 dephosphorylation. Here we show that the phosphatase Ptp1 also regulates Cdk1 dephosphorylation in vivo and can directly dephosphorylate Cdk1 in vitro. Using a novel in vivo phosphatase assay, we also show that PP2A bound to Rts1, the budding yeast B56-regulatory subunit, regulates dephosphorylation of Cdk1 independently of a function regulating Swe1, Mih1, or Ptp1, suggesting that PP2A(Rts1) either directly dephosphorylates Cdk1-Y19 or regulates an unidentified phosphatase. PMID:26715668

  10. Cyclin D1 represses gluconeogenesis via inhibition of the transcriptional coactivator PGC1α.

    Science.gov (United States)

    Bhalla, Kavita; Liu, Wan-Ju; Thompson, Keyata; Anders, Lars; Devarakonda, Srikripa; Dewi, Ruby; Buckley, Stephanie; Hwang, Bor-Jang; Polster, Brian; Dorsey, Susan G; Sun, Yezhou; Sicinski, Piotr; Girnun, Geoffrey D

    2014-10-01

    Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1-mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

  11. 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

    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...

  12. 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.

  13. 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

  14. The Cdk5 inhibitor Roscovitine increases LTP induction in corticostriatal synapses

    Directory of Open Access Journals (Sweden)

    Jorge Miranda‑Barrientos

    2014-03-01

    Full Text Available In corticostriatal synapses, LTD (long-term depression and LTP (long-term potentiation are modulated by the activation of DA (dopamine receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5 phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa at Thr75, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz. Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.

  15. 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

  16. 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 ...

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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...

  2. 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.

  3. A subset of cancer cell lines is acutely sensitive to the Chk1 inhibitor MK-8776 as monotherapy due to CDK2 activation in S phase.

    Science.gov (United States)

    Sakurikar, Nandini; Thompson, Ruth; Montano, Ryan; Eastman, Alan

    2016-01-12

    DNA damage activates Checkpoint kinase 1 (Chk1) to halt cell cycle progression thereby preventing further DNA replication and mitosis until the damage has been repaired. Consequently, Chk1 inhibitors have emerged as promising anticancer therapeutics in combination with DNA damaging drugs, but their single agent activity also provides a novel approach that may be particularly effective in a subset of patients. From analysis of a large panel of cell lines, we demonstrate that 15% are very sensitive to the Chk1 inhibitor MK-8776. Upon inhibition of Chk1, sensitive cells rapidly accumulate DNA double-strand breaks in S phase in a CDK2- and cyclin A-dependent manner. In contrast, resistant cells can continue to grow for at least 7 days despite continued inhibition of Chk1. Resistance can be circumvented by inhibiting Wee1 kinase and thereby directly activating CDK2. Hence, sensitivity to Chk1 inhibition is regulated upstream of CDK2 and correlates with accumulation of CDC25A. We conclude that cells poorly tolerate CDK2 activity in S phase and that a major function of Chk1 is to ensure it remains inactive. Indeed, inhibitors of CDK1 and CDK2 arrest cells in G1 or G2, respectively, but do not prevent progression through S phase demonstrating that neither kinase is required for S phase progression. Inappropriate activation of CDK2 in S phase underlies the sensitivity of a subset of cell lines to Chk1 inhibitors, and this may provide a novel therapeutic opportunity for appropriately stratified patients. PMID:26595527

  4. 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.

  5. 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

  6. TIGAR regulates DNA damage and repair through pentosephosphate pathway and Cdk5-ATM pathway.

    Science.gov (United States)

    Yu, Hong-Pei; Xie, Jia-Ming; Li, Bin; Sun, Yi-Hui; Gao, Quan-Geng; Ding, Zhi-Hui; Wu, Hao-Rong; Qin, Zheng-Hong

    2015-01-01

    Previous study revealed that the protective effect of TIGAR in cell survival is mediated through the increase in PPP (pentose phosphate pathway) flux. However, it remains unexplored if TIGAR plays an important role in DNA damage and repair. This study investigated the role of TIGAR in DNA damage response (DDR) induced by genotoxic drugs and hypoxia in tumor cells. Results showed that TIGAR was increased and relocated to the nucleus after epirubicin or hypoxia treatment in cancer cells. Knockdown of TIGAR exacerbated DNA damage and the effects were partly reversed by the supplementation of PPP products NADPH, ribose, or the ROS scavenger NAC. Further studies with pharmacological and genetic approaches revealed that TIGAR regulated the phosphorylation of ATM, a key protein in DDR, through Cdk5. The Cdk5-AMT signal pathway involved in regulation of DDR by TIGAR defines a new role of TIGAR in cancer cell survival and it suggests that TIGAR may be a therapeutic target for cancers. PMID:25928429

  7. CDK2 Activation in Mouse Epidermis Induces Keratinocyte Proliferation but Does Not Affect Skin Tumor Development

    Science.gov (United States)

    Macias, Everardo; Miliani de Marval, Paula L.; De Siervi, Adriana; Conti, Claudio J.; Senderowicz, Adrian M.; Rodriguez-Puebla, Marcelo L.

    2008-01-01

    It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21Cip1 and p27Kip1. Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. PMID:18599613

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. The role of Cdk5 in neuroendocrine thyroid cancer.

    Science.gov (United States)

    Pozo, Karine; Castro-Rivera, Emely; Tan, Chunfeng; Plattner, Florian; Schwach, Gert; Siegl, Veronika; Meyer, Douglas; Guo, Ailan; Gundara, Justin; Mettlach, Gabriel; Richer, Edmond; Guevara, Jonathan A; Ning, Li; Gupta, Anjali; Hao, Guiyang; Tsai, Li-Huei; Sun, Xiankai; Antich, Pietro; Sidhu, Stanley; Robinson, Bruce G; Chen, Herbert; Nwariaku, Fiemu E; Pfragner, Roswitha; Richardson, James A; Bibb, James A

    2013-10-14

    Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/Ser811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression. PMID:24135281

  15. 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.

  16. 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

  17. A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

    Directory of Open Access Journals (Sweden)

    Pinhero Reena

    2004-01-01

    Full Text Available We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14. Our protocol provides a novel systematic approach for the purification of these three (and possibly other recombinant CDKs.

  18. In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein

    Directory of Open Access Journals (Sweden)

    Arase Sachiko

    2012-03-01

    Full Text Available Abstract Background Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism. Results Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants. Conclusions Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

  19. 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...

  20. Cdk5及p35在NGF撤退诱导的已分化PC12细胞凋亡中的作用研究%The roles of Cdk5 and p35 in apoptosis of differentiated PC12 cells induced by NGF withdrawal

    Institute of Scientific and Technical Information of China (English)

    沈晗; 吴少波; 张百芳; 彭芳芳; 武栋成

    2012-01-01

    overexpression of Cdk5/ p35 leaded to the occurrence of apoptosis in PC 12 cells that were transfcted with pCMV-p35-IRES-Cdk5 plasmids. Conclusion-.There is a close relationship between Cdk5/ p35 activation and apoptosis of differentiated PC12 cell induced by NGF withdrawal. Inhibition of activation of Cdk5 may contribute to reducing the rate of apoptosis and protecting neurons.

  1. Functional interplay between MSL1 and CDK7 controls RNA polymerase II Ser5 phosphorylation.

    Science.gov (United States)

    Chlamydas, Sarantis; Holz, Herbert; Samata, Maria; Chelmicki, Tomasz; Georgiev, Plamen; Pelechano, Vicent; Dündar, Friederike; Dasmeh, Pouria; Mittler, Gerhard; Cadete, Filipe Tavares; Ramírez, Fidel; Conrad, Thomas; Wei, Wu; Raja, Sunil; Manke, Thomas; Luscombe, Nicholas M; Steinmetz, Lars M; Akhtar, Asifa

    2016-06-01

    Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies. PMID:27183194

  2. Temporal profiling of the chromatin proteome reveals system-wide responses to replication inhibition

    DEFF Research Database (Denmark)

    Khoudoli, Guennadi A; Gillespie, Peter J; Stewart, Graeme;

    2008-01-01

    Although the replication, expression, and maintenance of DNA are well-studied processes, the way that they are coordinated is poorly understood. Here, we report an analysis of the changing association of proteins with chromatin (the chromatin proteome) during progression through interphase...... of the cell cycle. Sperm nuclei were incubated in Xenopus egg extracts, and chromatin-associated proteins were analyzed by mass spectrometry at different times. Approximately 75% of the proteins varied in abundance on chromatin by more than 15%, suggesting that the chromatin proteome is highly dynamic....... Proteins were then assigned to one of 12 different clusters on the basis of their pattern of chromatin association. Each cluster contained functional groups of proteins involved in different nuclear processes related to progression through interphase. We also blocked DNA replication by inhibiting either...

  3. 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.

  4. 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

  5. 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)

  6. 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

  7. 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.

  8. 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...

  9. Prodigiosin inhibits motility and activates bacterial cell death revealing molecular biomarkers of programmed cell death.

    Science.gov (United States)

    Darshan, N; Manonmani, H K

    2016-12-01

    The antimicrobial activity of prodigiosin from Serratia nematodiphila darsh1, a bacterial pigment was tested against few food borne bacterial pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The mode of action of prodigiosin was studied. Prodigiosin induced bactericidal activity indicating a stereotypical set of biochemical and morphological feature of Programmed cell death (PCD). PCD involves DNA fragmentation, generation of ROS, and expression of a protein with caspase-like substrate specificity in bacterial cells. Prodigiosin was observed to be internalized into bacterial cells and was localized predominantly in the membrane and the nuclear fraction, thus, facilitating intracellular trafficking and then binding of prodigiosin to the bacterial DNA. Corresponding to an increasing concentration of prodigiosin, the level of certain proteases were observed to increase in bacteria studied, thus initiating the onset of PCD. Prodigiosin at a sub-inhibitory concentration inhibits motility of pathogens. Our observations indicated that prodigiosin could be a promising antibacterial agent and could be used in the prevention of bacterial infections. PMID:27460563

  10. The Role of Cdk5 in Neuroendocrine Thyroid Cancer

    OpenAIRE

    Pozo, Karine; Castro-Rivera, Emely; Tan, Chunfeng; Plattner, Florian; SCHWACH, GERT; Siegl, Veronika; Meyer, Douglas; Guo, Ailan; Gundara, Justin; Mettlach, Gabriel; RICHER, Edmond; Guevara, Jonathan A.; Ning, Li; Tsai, Li-Huei; Sun, Xiankai

    2013-01-01

    Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors p35 and p25 are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repre...

  11. Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

    Science.gov (United States)

    Shindo, Takayuki; Kaschani, Farnusch; Yang, Fan; Kovács, Judit; Tian, Fang; Kourelis, Jiorgos; Hong, Tram Ngoc; Colby, Tom; Shabab, Mohammed; Chawla, Rohini; Kumari, Selva; Ilyas, Muhammad; Hörger, Anja C; Alfano, James R; van der Hoorn, Renier A L

    2016-09-01

    Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes. PMID:27603016

  12. 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

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

    OpenAIRE

    Mei-Chih Chen; Chih-Yang Huang; Shih-Lan Hsu; Eugene Lin; Chien-Te Ku; Ho Lin; Chuan-Mu Chen

    2012-01-01

    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 ...

  14. Natural Aristolactams and Aporphine Alkaloids as Inhibitors of CDK1/Cyclin B and DYRK1A

    Directory of Open Access Journals (Sweden)

    Françoise Guéritte

    2013-03-01

    Full Text Available In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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...

  3. 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

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

    Directory of Open Access Journals (Sweden)

    Gang Wang

    2016-07-01

    Full Text Available 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.

  5. 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.

  6. 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

  7. MiR-506 suppresses proliferation and induces senescence by directly targeting the CDK4/6-FOXM1 axis in ovarian cancer.

    Science.gov (United States)

    Liu, Guoyan; Sun, Yan; Ji, Ping; Li, Xia; Cogdell, David; Yang, Da; Parker Kerrigan, Brittany C; Shmulevich, Ilya; Chen, Kexin; Sood, Anil K; Xue, Fengxia; Zhang, Wei

    2014-07-01

    Ovarian carcinoma is the most lethal gynaecological malignancy. Better understanding of the molecular pathogenesis of this disease and effective targeted therapies are needed to improve patient outcomes. MicroRNAs play important roles in cancer progression and have the potential for use as either therapeutic agents or targets. Studies in other cancers have suggested that miR-506 has anti-tumour activity, but its function has yet to be elucidated. We found that deregulation of miR-506 in ovarian carcinoma promotes an aggressive phenotype. Ectopic over-expression of miR-506 in ovarian cancer cells was sufficient to inhibit proliferation and to promote senescence. We also demonstrated that CDK4 and CDK6 are direct targets of miR-506, and that miR-506 can inhibit CDK4/6-FOXM1 signalling, which is activated in the majority of serous ovarian carcinomas. This newly recognized miR-506-CDK4/6-FOXM1 axis provides further insight into the pathogenesis of ovarian carcinoma and identifies a potential novel therapeutic agent.

  8. 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...

  9. 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.

  10. 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 ...

  11. 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 ...

  12. 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 ...

  13. 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 ...

  14. 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 ...

  15. 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 ...

  16. 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 ...

  17. 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.

  18. 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

  19. Progranulin Deficiency Reduces CDK4/6/pRb Activation and Survival of Human Neuroblastoma SH-SY5Y Cells.

    Science.gov (United States)

    de la Encarnación, Ana; Alquézar, Carolina; Esteras, Noemí; Martín-Requero, Ángeles

    2015-12-01

    Null mutations in GRN are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). However, the influence of progranulin (PGRN) deficiency in neurodegeneration is largely unknown. In neuroblastoma cells, silencing of GRN gene causes significantly reduced cell survival after serum withdrawal. The following observations suggest that alterations of the CDK4/6/retinoblastoma protein (pRb) pathway, secondary to changes in PI3K/Akt and ERK1/2 activation induced by PGRN deficiency, are involved in the control of serum deprivation-induced apoptosis: (i) inhibiting CDK4/6 levels or their associated kinase activity by sodium butyrate or PD332991 sensitized control SH-SY5Y cells to serum deprivation-induced apoptosis without affecting survival of PGRN-deficient cells; (ii) CDK4/6/pRb seems to be downstream of the PI3K/Akt and ERK1/2 signaling pathways since their specific inhibitors, LY294002 and PD98059, were able to decrease CDK6-associated kinase activity and induce death of control SH-SY5Y cells; (iii) PGRN-deficient cells show reduced stimulation of PI3K/Akt, ERK1/2, and CDK4/6 activities compared with control cells in the absence of serum; and (iv) supplementation of recombinant human PGRN was able to rescue survival of PGRN-deficient cells. These observations highlight the important role of PGRN-mediated stimulation of the PI3K/Akt-ERK1/2/CDK4/6/pRb pathway in determining the cell fate survival/death under serum deprivation.

  20. The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

    Science.gov (United States)

    Govindaraghavan, Meera; Anglin, Sarah Lea; Osmani, Aysha H; Osmani, Stephen A

    2014-08-01

    Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast.

  1. 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

  2. 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。

  3. 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

  4. 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 dynamin...

  5. 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.

  6. (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.

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

    Science.gov (United States)

    Debey, Evelyne; Ridderinkhof, Richard K; De Houwer, Jan; De Schryver, Maarten; Verschuere, Bruno

    2015-12-01

    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 with slower reaction times would be indicative of successful response inhibition. Participants performed a reaction-time task that required them to alternate between lying and truth telling in response to autobiographical questions. In two experiments, we found that the delta plot of the lie effect leveled off with longer response latencies, but only in a group of participants who had better inhibitory skills as indexed by relatively small lie effects. This finding supports the role of response inhibition in lying. We elaborate on repercussions for cognitive models of deception and the data analysis of reaction-time based lie tests.

  8. Regulation of Survivin and CDK4 by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Mi Dan AI; Li Li LI; Xiao Rong ZHAO; Yong WU; Jian Ping GONG; Ya CAO

    2005-01-01

    Latent membrane protein 1 (LMP1), an important protein encoded by Epstein Barr virus (EBV), has been implied to link with the pathogenesis of nasopharyngeal carcinoma (NPC). Its dual effects of increasing cell proliferation and inhibiting cell apoptosis have been confirmed. In this study, we showed that the expression of Survivin and CDK4 protein in CNE-LMP1, a LMP1 positive NPC epithelial cell line, is higher than in LMP1 negative NPC epithelial cell line CNE1, and the expression is LMP1 dosage-dependent. Although it was reported that Survivin specifically expressed in cell cycle G2/M phase, our studies suggested that LMP1 could promote the expression of Survivin in G0/G1, S and G2/M phase. It also showed that Survivin and CDK4 could be accumulated more in the nuclei triggered by LMP1. More interestingly, Survivin and CDK4 could form a protein complex in the nuclei of CNE-LMP1 rather than in that of CNE1, which demonstrated that the interaction between these two proteins could be promoted by LMP1. These results strongly suggested that the role of LMP1 in the regulation of Survivin and CDK4 may also shed some light on the mechanism research of LMP1 in NPC.

  9. Searching for Novel Cdk5 Substrates in Brain by Comparative Phosphoproteomics of Wild Type and Cdk5−/− Mice

    Science.gov (United States)

    Contreras-Vallejos, Erick; Utreras, Elías; Bórquez, Daniel A.; Prochazkova, Michaela; Terse, Anita; Jaffe, Howard; Toledo, Andrea; Arruti, Cristina; Pant, Harish C.; Kulkarni, Ashok B.; González-Billault, Christian

    2014-01-01

    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. PMID:24658276

  10. 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

  11. 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

  12. Small-molecule screening of PC3 prostate cancer cells identifies tilorone dihydrochloride to selectively inhibit cell growth based on cyclin-dependent kinase 5 expression.

    Science.gov (United States)

    Wissing, Michel D; Dadon, Tikva; Kim, Eunice; Piontek, Klaus B; Shim, Joong S; Kaelber, Nadine S; Liu, Jun O; Kachhap, Sushant K; Nelkin, Barry D

    2014-07-01

    Cyclin-dependent kinase 5 (CDK5) is a potential target for prostate cancer treatment, the enzyme being essential for prostate tumor growth and formation of metastases. In the present study, we identified agents that target prostate cancer cells based on CDK5 expression. CDK5 activity was suppressed by transfection of PC3 prostate cancer cells with a dominant-negative construct (PC3 CDK5dn). PC3 CDK5dn and PC3 control cells were screened for compounds that selectively target cells based on CDK5 expression, utilizing the Johns Hopkins Drug Library. MTS proliferation, clonogenic and 3D growth assays were performed to validate the selected hits. Screening of 3,360 compounds identified rutilantin, ethacridine lactate and cetalkonium chloride as compounds that selectively target PC3 control cells and a tilorone analog as a selective inhibitor of PC3 CDK5dn cells. A PubMed literature study indicated that tilorone may have clinical use in patients. Validation experiments confirmed that tilorone treatment resulted in decreased PC3 cell growth and invasion; PC3 cells with inactive CDK5 were inhibited more effectively. Future studies are needed to unravel the mechanism of action of tilorone in CDK5 deficient prostate cancer cells and to test combination therapies with tilorone and a CDK5 inhibitor for its potential use in clinical practice. PMID:24841903

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. Consequences of abnormal CDK activity in S phase.

    Science.gov (United States)

    Anda, Silje; Rothe, Christiane; Boye, Erik; Grallert, Beáta

    2016-01-01

    Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1(-) strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress. PMID:26918805

  18. Lack of Cyclin-Dependent Kinase 4 Inhibits c-myc Tumorigenic Activities in Epithelial Tissues

    Science.gov (United States)

    Miliani de Marval, Paula L.; Macias, Everardo; Rounbehler, Robert; Sicinski, Piotr; Kiyokawa, Hiroaki; Johnson, David G.; Conti, Claudio J.; Rodriguez-Puebla, Marcelo L.

    2004-01-01

    The proto-oncogene c-myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis and that has also been found to be deregulated in several forms of human and experimental tumors. We have shown that forced expression of c-myc in epithelial tissues of transgenic mice (K5-Myc) resulted in keratinocyte hyperproliferation and the development of spontaneous tumors in the skin and oral cavity. Although a number of genes involved in cancer development are regulated by c-myc, the actual mechanisms leading to Myc-induced neoplasia are not known. Among the genes regulated by Myc is the cyclin-dependent kinase 4 (CDK4) gene. Interestingly, previous studies from our laboratory showed that the overexpression of CDK4 led to keratinocyte hyperproliferation, although no spontaneous tumor development was observed. Thus, we tested the hypothesis that CDK4 may be one of the critical downstream genes involved in Myc carcinogenesis. Our results showed that CDK4 inhibition in K5-Myc transgenic mice resulted in the complete inhibition of tumor development, suggesting that CDK4 is a critical mediator of tumor formation induced by deregulated Myc. Furthermore, a lack of CDK4 expression resulted in marked decreases in epidermal thickness and keratinocyte proliferation compared to the results obtained for K5-Myc littermates. Biochemical analysis of the K5-Myc epidermis showed that CDK4 mediates the proliferative activities of Myc by sequestering p21Cip1 and p27Kip1 and thereby indirectly activating CDK2 kinase activity. These results show that CDK4 mediates the proliferative and oncogenic activities of Myc in vivo through a mechanism that involves the sequestration of specific CDK inhibitors. PMID:15314163

  19. 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.

  20. 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...

  1. 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

  2. Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

    OpenAIRE

    Ovejero, Sara; Ayala, Patricia; Bueno, Avelino; Sacristán, María P.

    2012-01-01

    The activity of Cdk1–cyclin B1 mitotic complexes is regulated by the balance between the counteracting activities of Wee1/Myt1 kinases and Cdc25 phosphatases. These kinases and phosphatases must be strictly regulated to ensure proper mitotic timing. One masterpiece of this regulatory network is Cdk1, which promotes Cdc25 activity and suppresses inhibitory Wee1/Myt1 kinases through direct phosphorylation. The Cdk1-dependent phosphorylation of Wee1 primes phosphorylation by additional kinases s...

  3. 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.

  4. 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...

  5. 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

  6. 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.

  7. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis

    Science.gov (United States)

    Kollmann, Karoline; Heller, Gerwin; Schneckenleithner, Christine; Warsch, Wolfgang; Scheicher, Ruth; Ott, Rene G.; Schäfer, Markus; Fajmann, Sabine; Schlederer, Michaela; Schiefer, Ana-Iris; Reichart, Ursula; Mayerhofer, Matthias; Hoeller, Christoph; Zöchbauer-Müller, Sabine; Kerjaschki, Dontscho; Bock, Christoph; Kenner, Lukas; Hoefler, Gerald; Freissmuth, Michael; Green, Anthony R.; Moriggl, Richard; Busslinger, Meinrad; Malumbres, Marcos; Sexl, Veronika

    2013-01-01

    Summary In contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a. PMID:23948297

  8. 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

  9. 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.

  10. 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.

  11. The crystal structure of an HSL-homolog EstE5 complex with PMSF reveals a unique configuration that inhibits the nucleophile Ser144 in catalytic triads.

    Science.gov (United States)

    Nam, Ki Hyun; Kim, Soo-Jin; Priyadarshi, Amit; Kim, Hyun Sook; Hwang, Kwang Yeon

    2009-11-13

    The esterase/lipase family (EC 3.1.1.3/EC 3.1.1.1) represents a diverse group of hydrolases that catalyze the cleavage of ester bonds and are widely distributed in animals, plants and microorganisms. Among these enzymes, hormone-sensitive lipases, play a critical role in the regulation of rodent fat cell lipolysis and are regarded as adipose tissue-specific enzymes. Recently, we reported the structural and biological characterization of EstE5 from the metagenome library [K.H. Nam, M.Y. Kim, S.J. Kim, A. Priyadarshi, W.H. Lee, K.Y. Hwang, Structural and functional analysis of a novel EstE5 belonging to the subfamily of hormone-sensitive lipase, Biochem. Biophys. Res. Commun. 379 (2009) 553-556]. The structure of this protein revealed that it belongs to the HSL-family. Here, we report the inhibition of the activity of the HSL-homolog EstE5 protein as determined by the use of esterase/lipase inhibitors. Our results revealed that the EstE5 protein is significantly inhibited by PMSF. In addition, this is the first study to identify the crystal structures of EstE5-PMSF at 2.4 and 2.5A among the HSL-homolog structures. This structural configuration is similar to that adopted when serine proteases are inhibited by PMSF. The results presented here provide valuable information regarding the properties of the HSL-family.

  12. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    2016-04-01

    Full Text Available One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  13. 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

  14. 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.

  15. 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.

  16. 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

    Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits...... the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism...... of 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...

  17. 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

  18. 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.

  19. 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。

  20. A functional connection between pRB and transforming growth factor beta in growth inhibition and mammary gland development.

    Science.gov (United States)

    Francis, Sarah M; Bergsied, Jacqueline; Isaac, Christian E; Coschi, Courtney H; Martens, Alison L; Hojilla, Carlo V; Chakrabarti, Subrata; Dimattia, Gabriel E; Khoka, Rama; Wang, Jean Y J; Dick, Frederick A

    2009-08-01

    Transforming growth factor beta (TGF-beta) is a crucial mediator of breast development, and loss of TGF-beta-induced growth arrest is a hallmark of breast cancer. TGF-beta has been shown to inhibit cyclin-dependent kinase (CDK) activity, which leads to the accumulation of hypophosphorylated pRB. However, unlike other components of TGF-beta cytostatic signaling, pRB is thought to be dispensable for mammary development. Using gene-targeted mice carrying subtle missense changes in pRB (Rb1(DeltaL) and Rb1(NF)), we have discovered that pRB plays a critical role in mammary gland development. In particular, Rb1 mutant female mice have hyperplastic mammary epithelium and defects in nursing due to insensitivity to TGF-beta growth inhibition. In contrast with previous studies that highlighted the inhibition of cyclin/CDK activity by TGF-beta signaling, our experiments revealed that active transcriptional repression of E2F target genes by pRB downstream of CDKs is also a key component of TGF-beta cytostatic signaling. Taken together, our work demonstrates a unique functional connection between pRB and TGF-beta in growth control and mammary gland development.

  1. Simvastatin induces cell cycle arrest and inhibits proliferation of bladder cancer cells via PPARγ signalling pathway

    Science.gov (United States)

    Wang, Gang; Cao, Rui; Wang, Yongzhi; Qian, Guofeng; Dan, Han C.; Jiang, Wei; Ju, Lingao; Wu, Min; Xiao, Yu; Wang, Xinghuan

    2016-01-01

    Simvastatin is currently one of the most common drugs for old patients with hyperlipidemia, hypercholesterolemia and atherosclerotic diseases by reducing cholesterol level and anti-lipid properties. Importantly, simvastatin has also been reported to have anti-tumor effect, but the underlying mechanism is largely unknown. We collected several human bladder samples and performed microarray. Data analysis suggested bladder cancer (BCa) was significantly associated with fatty acid/lipid metabolism via PPAR signalling pathway. We observed simvastatin did not trigger BCa cell apoptosis, but reduced cell proliferation in a dose- and time-dependent manner, accompanied by PPARγ-activation. Moreover, flow cytometry analysis indicated that simvastatin induced cell cycle arrest at G0/G1 phase, suggested by downregulation of CDK4/6 and Cyclin D1. Furthermore, simvastatin suppressed BCa cell metastasis by inhibiting EMT and affecting AKT/GSK3β. More importantly, we found that the cell cycle arrest at G0/G1 phase and the alterations of CDK4/6 and Cyclin D1 triggered by simvastatin could be recovered by PPARγ-antagonist (GW9662), whereas the treatment of PPARα-antagonist (GW6471) shown no significant effects on the BCa cells. Taken together, our study for the first time revealed that simvastatin inhibited bladder cancer cell proliferation and induced cell cycle arrest at G1/G0 phase via PPARγ signalling pathway. PMID:27779188

  2. 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.

  3. 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.

  4. 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.

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

    Science.gov (United States)

    Fahham, Najmeh; Ghahremani, Mohammad Hossein; Sardari, Soroush; Vaziri, Behrouz; Ostad, Seyed Nasser

    2008-01-01

    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 fit 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. PMID:19352455

  6. 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.

  7. 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...

  8. 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.

  9. Differential expression of cell cycle regulators in CDK5-dependent medullary thyroid carcinoma tumorigenesis.

    Science.gov (United States)

    Pozo, Karine; Hillmann, Antje; Augustyn, Alexander; Plattner, Florian; Hai, Tao; Singh, Tanvir; Ramezani, Saleh; Sun, Xiankai; Pfragner, Roswitha; Minna, John D; Cote, Gilbert J; Chen, Herbert; Bibb, James A; Nwariaku, Fiemu E

    2015-05-20

    Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer of thyroid C-cells, for which few treatment options are available. We have recently reported a role for cyclin-dependent kinase 5 (CDK5) in MTC pathogenesis. We have generated a mouse model, in which MTC proliferation is induced upon conditional overexpression of the CDK5 activator, p25, in C-cells, and arrested by interrupting p25 overexpression. Here, we identify genes and proteins that are differentially expressed in proliferating versus arrested benign mouse MTC. We find that downstream target genes of the tumor suppressor, retinoblastoma protein, including genes encoding cell cycle regulators such as CDKs, cyclins and CDK inhibitors, are significantly upregulated in malignant mouse tumors in a CDK5-dependent manner. Reducing CDK5 activity in human MTC cells down-regulated these cell cycle regulators suggesting that CDK5 activity is critical for cell cycle progression and MTC proliferation. Finally, the same set of cell cycle proteins was consistently overexpressed in human sporadic MTC but not in hereditary MTC. Together these findings suggest that aberrant CDK5 activity precedes cell cycle initiation and thus may function as a tumor-promoting factor facilitating cell cycle protein expression in MTC. Targeting aberrant CDK5 or its downstream effectors may be a strategy to halt MTC tumorigenesis. PMID:25900242

  10. Low Expression of CDK5 and p27 Are Associated with Poor Prognosis in Patients with Gastric Cancer

    OpenAIRE

    Sun, Yu-Qin; Xie, Jian-Wei; Chen, Peng-Chen; Zheng, Chao-Hui; Li, Ping; Wang, Jia-Bin; Lin, Jian-Xian; Lu, Jun; Chen, Qi-Yue; Cao, Long-Long; Lin, Mi; Tu, Ru-Hong; Lin, Yao; Huang, Chang-Ming

    2016-01-01

    Several previous studies have demonstrated that CDK5 or p27 expression in gastric cancer are associated with overall survival. We have previously reported that tumor suppressive function of CDK5 is related to p27. The aim of this study was to investigate correlation between the clinicopathological parameters and overall survival with different CDK5/p27 expression statuses in 244 gastric cancer patients using immunohistochemistry. Low CDK5 expression was detected in 93 cases (38.11%) and low p...

  11. 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.

  12. Crystal structures of the SAM-III/S[subscript MK] riboswitch reveal the SAM-dependent translation inhibition mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lu, C.; Smith, A.M.; Fuchs, R.T.; Ding, F.; Rajashankar, K.; Henkin, T.M.; Ke, A. (Cornell); (OSU)

    2010-01-07

    Three distinct classes of S-adenosyl-L-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-{angstrom} crystal structure of the Enterococcus faecalis SMK box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti-Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the SMK box riboswitch sampling the near-cognate ligand S-adenosyl-L-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

  13. 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.

  14. Antibacterial phage ORFans of Pseudomonas aeruginosa phage LUZ24 reveal a novel MvaT inhibiting protein

    Directory of Open Access Journals (Sweden)

    Jeroen eWagemans

    2015-11-01

    Full Text Available The functional elucidation of small unknown phage proteins (‘ORFans’ presents itself as one of the major challenges of bacteriophage molecular biology. In this work, we mined the Pseudomonas aeruginosa infecting phage LUZ24 proteome for antibacterial and antibiofilm proteins against its host. Subsequently, their putative host target was identified. In one example, we observed an interaction between LUZ24 gp4 and the host transcriptional regulator MvaT. The polymerization of MvaT across AT-rich DNA strands permits gene silencing of foreign DNA, thereby limiting any potentially adverse effects of such DNA. Gel shift assays proved the inhibitory effect of LUZ24 gp4 on MvaT DNA binding activity. Therefore, we termed this gene product as Mip, the MvaT inhibiting protein. We hypothesize Mip prevents the AT-rich LUZ24 DNA from being physically blocked by MvaT oligomers right after its injection in the host cell, thereby allowing phage transcription and thus completion of the phage infection cycle.

  15. A structure activity-relationship study of the bacterial signal molecule HHQ reveals swarming motility inhibition in Bacillus atrophaeus.

    Science.gov (United States)

    Reen, F Jerry; Shanahan, Rachel; Cano, Rafael; O'Gara, Fergal; McGlacken, Gerard P

    2015-05-21

    The sharp rise in antimicrobial resistance has been matched by a decline in the identification and clinical introduction of new classes of drugs to target microbial infections. Thus new approaches are being sought to counter the pending threat of a post-antibiotic era. In that context, the use of non-growth limiting small molecules, that target virulence behaviour in pathogens, has emerged as a solution with real clinical potential. We have previously shown that two signal molecules (HHQ and PQS) from the nosocomial pathogen Pseudomonas aeruginosa have modulatory activity towards other microorganisms. This current study involves the synthesis and evaluation of analogues of HHQ towards swarming and biofilm virulence behaviour in Bacillus atrophaeus, a soil bacterium and co-inhibitor with P. aeruginosa. Compounds with altered C6-C8 positions on the anthranilate-derived ring of HHQ, display a surprising degree of biological specificity, with certain candidates displaying complete motility inhibition. In contrast, anti-biofilm activity of the parent molecule was completely lost upon alteration at any position indicating a remarkable degree of specificity and delineation of phenotype. PMID:25880413

  16. Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism.

    Science.gov (United States)

    Murray, David S; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V; Fisher, Susan H; Schumacher, Maria A

    2013-12-13

    Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

  17. UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons.

    Science.gov (United States)

    Edwards, Stacey L; Morrison, Logan M; Yorks, Rosalina M; Hoover, Christopher M; Boominathan, Soorajnath; Miller, Kenneth G

    2015-09-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function.

  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. 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 eRondeel

    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.

  20. 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

  1. The cell cycle rallies the transcription cycle: Cdc28/Cdk1 is a cell cycle-regulated transcriptional CDK.

    Science.gov (United States)

    Chymkowitch, Pierre; Enserink, Jorrit M

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinases (CDKs) Kin28, Bur1 and Ctk1 regulate basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. However, very little is known about the involvement of the cell cycle CDK Cdc28 in the transcription process. We have recently shown that, upon cell cycle entry, Cdc28 kinase activity boosts transcription of a subset of genes by directly stimulating the basal transcription machinery. Here, we discuss the biological significance of this finding and give our view of the kinase-dependent role of Cdc28 in regulation of RNA polymerase II.

  2. 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.

  3. Expression of Cyclin E and CDK5 in lung cancer%Cyclin E 、CDK5在肺癌中的表达

    Institute of Scientific and Technical Information of China (English)

    孙延; 唐建武

    2003-01-01

    [目的] 研究Cyclin E、CDK5在不同组织类型肺癌中的表达情况及其与肺癌发生发展的关系.[方法] 采用免疫组织化学法检测91例原发性肺癌及10例正常支气管粘膜组织中的Cyclin E、CDK5表达水平.[结果] Cyclin E、CDK5在肺癌中总的阳性表达率分别为 46.15%、37.36%,而在正常支气管粘膜中几乎无表达. Cyclin E、CDK5表达水平在肺癌各组织类型、分化程度间无显著性差异(P>0.05).Cyclin E与CDK5存在交叉共存现象(91例中有65例表达相同),经相关分析发现两者呈显著正相关(P<0.01). [结论] Cyclin E、CDK5与肺癌发生有关,可作为肺癌的潜在的诊断指标.

  4. 氧化苦参碱对结肠癌LoVo细胞c-myc,PSMD9,CDK4mRNA表达的影响%Effect of Oxymatrine on Expression of c-myc, PSMD9 and CDK4 mRNA in Human Colon Carcinoma LoVo Cells

    Institute of Scientific and Technical Information of China (English)

    彭燕; 韩凌; 孙静; 危建安

    2012-01-01

    目的:探讨氧化苦参碱( oxymatrine,OM)抑制人结肠癌LoVo细胞增殖和诱导凋亡的分子作用机制.方法:采用流式细胞仪检测LoVo细胞凋亡率以及细胞周期分布;采用荧光定量PCR法检测0.25,0.5 g·L-1 OM对LoVo细胞增殖相关基因c -myc,蛋白酶调解因子9(PSMD9),CDK4的基因表达的影响.结果:0.5 g·L-1以下浓度的OM作用结肠癌LoVo细胞48 h,对细胞凋亡无明显影响.0.25 g·L-1 OM作用48 h时可明显抑制人结肠癌LoVo细胞c-myc基因表达(P<0.05).0.5g·L-1 OM作用48 h时可明显抑制LoVo细胞c-myc,CDK4的基因表达(P <0.01,P<0.01,).药物作用时间为96 h时,0.5g·L-1 OM可明显抑制c-myc,PSM D9,CDK4基因表达(P<0.05,或P<0.01).结论:较低剂量OM显著抑制人结肠癌LoVo细胞增殖的作用机制,可能与下调LoVo细胞c-myc,PSM D9,CDK4表达有关.%Objective: To explore the molecular mechanism of inhibiting colon cancer cell strein LoVo proliferation and inducing apoptosis by oxymatrine ( OM ) Method: Flow cytometry was used to detect the LoVo cells apoptosis and cell cycle distribution. Fluorescence quantitative PCR was used to detect cell proliferation-related genes like the c-myc, proteasome modulator 9 (PSMD9) , CDK4 gene expression when LoVo was treated with 0. 25, 0. 5 g · L-1OM. Result: OM had no significant effect on apoptosis in colon cancer LoVo cells when the treatment of OM lasted 48 h and the concentration was lower than 0.5, 0.25 g · L-1 OM can inhibit c-myc gene expression in LoVo when duration of action last 24 h ( P < 0. 05 ). When the dose increated to 0. 5 g · L-1 and duration of action was 48 h, OM could inhibit c-myc, CDK4 gene expression in LoVo cells (P <0. 01 , P < 0. 01). When duration of action was extended to 96 h, 0. 5 g · L-1 OM could inhibit the c-myc, PSMD9, CDK4 gene expression in LoVo cells ( P < 0. 05, P < 0. 01, P < 0. 01 ). Conclusion; OM at Lower dose could significantly inhibit the proliferation of human colon cancer Lo

  5. CDK1 phosphorylates WRN at collapsed replication forks

    Science.gov (United States)

    Palermo, Valentina; Rinalducci, Sara; Sanchez, Massimo; Grillini, Francesca; Sommers, Joshua A.; Brosh, Robert M.; Zolla, Lello; Franchitto, Annapaola; Pichierri, Pietro

    2016-01-01

    Regulation of end-processing is critical for accurate repair and to switch between homologous recombination (HR) and non-homologous end joining (NHEJ). End resection is a two-stage process but very little is known about regulation of the long-range resection, especially in humans. WRN participates in one of the two alternative long-range resection pathways mediated by DNA2 or EXO1. Here we demonstrate that phosphorylation of WRN by CDK1 is essential to perform DNA2-dependent end resection at replication-related DSBs, promoting HR, replication recovery and chromosome stability. Mechanistically, S1133 phosphorylation of WRN is dispensable for relocalization in foci but is involved in the interaction with the MRE11 complex. Loss of WRN phosphorylation negatively affects MRE11 foci formation and acts in a dominant negative manner to prevent long-range resection altogether, thereby licensing NHEJ at collapsed forks. Collectively, we unveil a CDK1-dependent regulation of the WRN-DNA2-mediated resection and identify an undescribed function of WRN as a DSB repair pathway switch. PMID:27634057

  6. Positron Emission Tomography with [18F]FLT Revealed Sevoflurane-induced Inhibition of Neural Progenitor Cell Expansion in vivo

    Directory of Open Access Journals (Sweden)

    Shuliang eLiu

    2014-11-01

    Full Text Available Neural progenitor cell expansion is critical for normal brain development and an appropriate response to injury. During the brain growth spurt, exposures to general anesthetics which either block the N-methyl D-aspartate receptor or enhance the γ-aminobutyric acid receptor type A can disturb neuronal transduction. This effect can be detrimental to brain development. Until now, the effects of anesthetic exposure on neural progenitor cell expansion in vivo had seldom been reported. Here, minimally invasive micro positron emission tomography (microPET coupled with 3'-deoxy-3' [18F] fluoro-L-thymidine ([18F]FLT was utilized to assess the effects of sevoflurane exposure on neural progenitor cell proliferation. FLT, a thymidine analogue, is taken up by proliferating cells and phosphorylated in the cytoplasm, leading to its intracellular trapping. Intracellular retention of [18F]FLT, thus, represents an observable in vivo marker of cell proliferation. Here, postnatal day (PND 7 rats (n = 11/ group were exposed to 2.5% sevoflurane or room air for 9 hr. For up to two weeks following the exposure, standard uptake values (SUVs for [18F]-FLT in the hippocampal formation were significantly attenuated in the sevoflurane-exposed rats (p <0.0001, suggesting decreased uptake and retention of [18F]FLT (decreased proliferation in these regions. Four weeks following exposure, SUVs for [18F]FLT were comparable in the sevoflurane-exposed rats and in controls. Co-administration of 7-nitroindazole (7-NI, 30 mg/kg, n = 5, a selective inhibitor of neuronal nitric oxide synthase, significantly attenuated the SUVs for [18F]FLT in both the air-exposed (p = 0.00006 and sevoflurane-exposed rats (p = 0.0427 in the first week following the exposure. These findings suggested that microPET in couple with [18F]FLT as cell proliferation marker could be used as a non-invasive modality to monitor the sevoflurane-induced inhibition of neural progenitor cell proliferation in vivo.

  7. 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.

  8. Expression of CDK1(Tyr15, pCDK1(Thr161, Cyclin B1 (total and pCyclin B1(Ser126 in vulvar squamous cell carcinoma and their relations with clinicopatological features and prognosis.

    Directory of Open Access Journals (Sweden)

    Zhihui Wang

    Full Text Available Cyclin B1-CDK1 complex plays an important role in the regulation of cell cycle. Activation of Cyclin B1 and CDK1 and the formation of the complex in G2/M are under multiple regulations involving many regulators such as isoforms of 14-3-3 and CDC25 and Wee1. Abnormal expression of Cyclin B1 and CDK1 has been detected in various tumors. However, to our knowledge no previous study has investigated Cyclin B1 and CDK1 in vulvar cancer. Therefore, we evaluated the statuses of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total and pCyclin B1Ser126 in 297 cases of vulvar squamous cell carcinomas by immunohistochemistry. Statistical analyses were performed to explore their clinicopathological and prognostic values. In at least 25% of tumor cases high expression of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total and pCyclin B1Ser126 was observed, compared to the low levels in normal vulvar squamous epithelium. Elevated levels of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total and pCyclin B1Ser126 were correlated with advanced tumor behaviors and aggressive features. Although CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total and pCyclin B1Ser126 could not be identified as prognostic factors, combinations of (pCDK1Thr161 C+N + 14-3-3σN, (pCDK1Thr161 C+N + 14-3-3ηC, (pCDK1Thr161 C+N + Wee1C and (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C were correlated with disease-specific survival (p = 0.036, p = 0.029, p = 0.042 and p = 0.007, respectively in univariate analysis. The independent prognostic significance of (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C was confirmed by multivariate analysis. In conclusion, CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total and pCyclin B1Ser126 may be involved in progression of vulvar squamous cell carcinoma. The combination of pCDK1Thr161, 14-3-3σ, 14-3-3η and Wee1 was a statistically independent prognostic factor.

  9. Structures of proline-rich peptides bound to the ribosome reveal a common mechanism of protein synthesis inhibition.

    Science.gov (United States)

    Gagnon, Matthieu G; Roy, Raktim N; Lomakin, Ivan B; Florin, Tanja; Mankin, Alexander S; Steitz, Thomas A

    2016-03-18

    With bacterial resistance becoming a serious threat to global public health, antimicrobial peptides (AMPs) have become a promising area of focus in antibiotic research. AMPs are derived from a diverse range of species, from prokaryotes to humans, with a mechanism of action that often involves disruption of the bacterial cell membrane. Proline-rich antimicrobial peptides (PrAMPs) are instead actively transported inside the bacterial cell where they bind and inactivate specific targets. Recently, it was reported that some PrAMPs, such as Bac71 -35, oncocins and apidaecins, bind and inactivate the bacterial ribosome. Here we report the crystal structures of Bac71 -35, Pyrrhocoricin, Metalnikowin and two oncocin derivatives, bound to the Thermus thermophilus 70S ribosome. Each of the PrAMPs blocks the peptide exit tunnel of the ribosome by simultaneously occupying three well characterized antibiotic-binding sites and interferes with the initiation step of translation, thereby revealing a common mechanism of action used by these PrAMPs to inactivate protein synthesis. Our study expands the repertoire of PrAMPs and provides a framework for designing new-generation therapeutics. PMID:26809677

  10. From quiescence to proliferation : Cdk oscillations drive the mammalian cell cycle

    Directory of Open Access Journals (Sweden)

    Claude eGérard

    2012-11-01

    Full Text Available We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks driving the mammalian cell cycle [Gérard, C. and Goldbeter, A. (2009. Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle. Proc. Natl. Acad. Sci. USA 106, 21643-21648]. The model contains four modules, each centered around one cyclin/Cdk complex. Cyclin D/Cdk4-6 and cyclin E/Cdk2 promote progression in G1 and elicit the G1/S transition, respectively; cyclin A/Cdk2 ensures progression in S and the transition S/G2, while the activity of cyclin B/Cdk1 brings about the G2/M transition. This model shows that in the presence of sufficient amounts of growth factor the Cdk network is capable of temporal self-organization in the form of sustained oscillations, which correspond to the ordered, sequential activation of the various cyclin/Cdk complexes that control the successive phases of the cell cycle. The results suggest that the switch from cellular quiescence to cell proliferation corresponds to the transition from a stable steady state to sustained oscillations in the Cdk network. The transition depends on a finely tuned balance between factors that promote or hinder progression in the cell cycle. We show that the transition from quiescence to proliferation can occur in multiple ways that alter this balance. By resorting to bifurcation diagrams, we analyze the mechanism of oscillations in the Cdk network. Finally, we show that the complexity of the detailed model can be greatly reduced, without losing its key dynamical properties, by considering a skeleton model for the Cdk network. Using such a skeleton model for the mammalian cell cycle we show that positive feedback loops enhance the amplitude and the robustness of Cdk oscillations with respect to molecular noise. We compare the relative merits of the detailed and skeleton versions of the model for the Cdk network driving the mammalian cell cycle.

  11. 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

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

    OpenAIRE

    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 inhi...

  13. 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.

  14. 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

  15. 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.

  16. Role of CyclinD1 and CDK4 in the Carcinogenesis Induced by Silica

    Institute of Scientific and Technical Information of China (English)

    KE-XIA YAN; BING-CI LIU; XIANG-LIN SHI; BAO-RONG YOU; MING XU

    2005-01-01

    Objective To study the role of cyclinD1 and CDK4 in malignant transformation of human fetal lung diploid fibroblast cell line(2BS) induced by silica. Methods Recombination vectors with sense and antisense pXJ41-cyclinD1 and pXJ41-CDK4 were constructed, and then transfected into the malignant transformed cells induced by silica, respectively. At the same time, pXJ41-neo was used as the control. Results During the progress of the malignant transformation of 2BS cells induced by silica, cyclinD1 and CDK4 were overexpressed. Antisense RNA suppressed cyclinD1 and CDK4 gene expression in the antisense pXJ41-cyclinD1 and pXJ41-CDK4 transfected cells. Antisense RNA led to cell cycle arrest, resulting in lengthened G1 phase (the percentages of cells in the G1 phase changed from 45.1% to 52.7% and 58.0% for cyclinD1 and CDK4 transfected cells, respectively), and eventually attenuated the increase of the proliferation of malignant transformed cells induced by silica. Compared with malignant transformed cells induced by silica, cells transfected with antisense pXJ41-cyclinD1 and pXJ41-CDK4 showed obviously reduced growth rates. On the 8th day, the suppression rates were 58.69 and 77.43% (the growth rate of malignant transformed cells induced by silica was 100%), doubling time changed from 21.0 h to 31.4 h and 21.0 h to 42.7 h, respectively, the growth capacities on soft agar of cells transfected by antisense pXJ41-cyclinD1 and pXJ41-CDK4 decreased obviously. Conclusion CyclinD1 and CDK4 play an important role in maintaining transformed phenotype of the cancer cells.

  17. 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

  18. Low Expression of CDK5 and p27 Are Associated with Poor Prognosis in Patients with Gastric Cancer.

    Science.gov (United States)

    Sun, Yu-Qin; Xie, Jian-Wei; Chen, Peng-Chen; Zheng, Chao-Hui; Li, Ping; Wang, Jia-Bin; Lin, Jian-Xian; Lu, Jun; Chen, Qi-Yue; Cao, Long-Long; Lin, Mi; Tu, Ru-Hong; Lin, Yao; Huang, Chang-Ming

    2016-01-01

    Several previous studies have demonstrated that CDK5 or p27 expression in gastric cancer are associated with overall survival. We have previously reported that tumor suppressive function of CDK5 is related to p27. The aim of this study was to investigate correlation between the clinicopathological parameters and overall survival with different CDK5/p27 expression statuses in 244 gastric cancer patients using immunohistochemistry. Low CDK5 expression was detected in 93 cases (38.11%) and low p27 in 157 cases (64.34%). The expression of CDK5 was significantly related to sex (P = 0.034) and Lauren's classification (P = 0.013). The expression of p27 was significantly related to sex (P = 0.012), differentiation (P = 0.003), TNM stage (P = 0.013) and lymph node metastasis (P = 0.001). Based on the combined expression of CDK5 and p27, we classified the patients into four subtypes: CDK5 Low/p27 Low (n = 69), CDK5 High/p27 Low (n = 88), CDK5 Low/p27 High (n = 24) and CDK5 High/p27 High (n = 63). The CDK5 Low/p27 Low expression was closely related to female (P = 0.026), diffuse type (P = 0.027) and lymph node metastasis (P = 0.010). The CDK5 Low/p27 Low patients displayed poorer survival in comparison with the rest of the patients in Kaplan-Meier analysis. No significant overall survival difference was observed among the patients with CDK5 High and/or p27 High expression. In the multivariate analysis, CDK5 and p27 co-expression status was identified as an independent prognostic factor for patients with gastric cancer. PMID:27326247

  19. Unique Cyclin-Dependent Kinase (CDK) Inhibitors at the ATP-site

    Institute of Scientific and Technical Information of China (English)

    LI Lin; LUNDGREN Karen; ESCOBAR Jorge; MINNICK Sharon price; HUBER Andrea; KOUDRIAKOVA Tatiana; ARRUDA Jeannie; SISSON Wes; AUST Robert M.; VERKHIVKER Gennady M.; SCHAFFER Lana; CHONG Wesley K. M.; ROSE Peter w.; LEWIS Cristrina T; DUVADIE Rohit K.; CHU Shao Song; YANG Y. Michelle; NONOMIYA Jim; TUCKER Kadthleen D.; KNIGHTON Daniel R.; FERRE RoseAnn

    2001-01-01

    @@ Control of the cell cycle could be applicable in new approaches for cancer chemotherapy. The cyclin-dependent kinases (CDK's) and their corresponding complexes with cyclins are regulatory enzymes for which we have discovered a novel small molecule series of inhibitors, with potencies in the nanomolar range and good selectivity for the CDK's versus other kinases. We will discuss structure-based drug design efforts with crystal structures of complexes with certain CDK's. Cellular effects and some preliminary examination of in vivo cancer efficacy by these inhibitors will also be discussed.

  20. 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...

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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....

  6. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    Science.gov (United States)

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  7. 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.

  8. 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.

  9. 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

  10. The effect of cdk-5 overexpression on tau phosphorylation and spatial memory of rat

    Institute of Scientific and Technical Information of China (English)

    LIAO Xiaomei; ZHANG Yingchun; WANG Yipeng; WANG Jianzhi

    2004-01-01

    In Alzheimer's disease (AD), hyperphosphorylation of tau may be the underlying mechanism for the cytoskeletal abnormalities and neuronal death. It was reported that cyclin-dependent kinase5 (cdk-5) could phosphorylate tau at most AD-related epitopes in vitro. In this study, we investigated the effect of cdk-5 overexpression on tau phosphorylation and spatial memory in rat. We demonstrated that 24 h after transfection into rat hippocampus, cdk-5 was overexpressed and induced a reduced staining with antibody tau-1 and an enhanced staining with antibodies 12e8 and PHF-1, suggesting hyperphosphorylation of tau at Ser199/202, Ser262/356 and Ser396/404 sites. Additionally, the cdk-5 transfected rats showed long latency to find the hidden platform in Morris water maze compared to the control rat. 48 h after transfection, the level of cdk-5 was decreased significantly, and the latency of rats to find the hidden platform was prolonged. It implies that in vivo overexpression of cdk-5 leads to impairment of spatial memory in rat and tau hyperphosphorylation may be the underlying mechanism.

  11. Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3β and CDK5 dependent manner: Relevance to neurological disorders.

    Science.gov (United States)

    Gąssowska, Magdalena; Baranowska-Bosiacka, Irena; Moczydłowska, Joanna; Tarnowski, Maciej; Pilutin, Anna; Gutowska, Izabela; Strużyńska, Lidia; Chlubek, Dariusz; Adamczyk, Agata

    2016-03-10

    Hyperphosphorylation of Tau is involved in the pathomechanism of neurological disorders such as Alzheimer's, Parkinson's diseases as well as Autism. Epidemiological data suggest the significance of early life exposure to lead (Pb) in etiology of disorders affecting brain function. However, the precise mechanisms by which Pb exerts neurotoxic effects are not fully elucidated. The purpose of this study was to evaluate the effect of perinatal exposure to low dose of Pb on the Tau pathology in the developing rat brain. Furthermore, the involvement of two major Tau-kinases: glycogen synthase kinase-3 beta (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in Pb-induced Tau modification was evaluated. Pregnant female rats were divided into control and Pb-treated group. The control animals were maintained on drinking water while females from the Pb-treated group received 0.1% lead acetate (PbAc) in drinking water, starting from the first day of gestation until weaning of the offspring. During the feeding of pups, mothers from the Pb-treated group were still receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and Tau mRNA and protein level as well as Tau phosphorylation were analyzed in forebrain cortex (FC), cerebellum (C) and hippocampus (H). Concomitantly, we examined the effect of Pb exposure on GSK-3β and CDK5 activation. Our data revealed that pre- and neonatal exposure to Pb (concentration of Pb in whole blood below 10μg/dL, considered safe for humans) caused significant increase in the phosphorylation of Tau at Ser396 and Ser199/202 with parallel rise in the level of total Tau protein in FC and C. Tau hyperphosphorylation in Pb-treated animals was accompanied by elevated activity of GSK-3β and CDK5. Western blot analysis revealed activation of GSK-3β in FC and C as well as CDK5 in C, via increased phosphorylation of Tyr-216 and calpain-dependent p25

  12. Perinatal exposure to lead (Pb) promotes Tau phosphorylation in the rat brain in a GSK-3β and CDK5 dependent manner: Relevance to neurological disorders.

    Science.gov (United States)

    Gąssowska, Magdalena; Baranowska-Bosiacka, Irena; Moczydłowska, Joanna; Tarnowski, Maciej; Pilutin, Anna; Gutowska, Izabela; Strużyńska, Lidia; Chlubek, Dariusz; Adamczyk, Agata

    2016-03-10

    Hyperphosphorylation of Tau is involved in the pathomechanism of neurological disorders such as Alzheimer's, Parkinson's diseases as well as Autism. Epidemiological data suggest the significance of early life exposure to lead (Pb) in etiology of disorders affecting brain function. However, the precise mechanisms by which Pb exerts neurotoxic effects are not fully elucidated. The purpose of this study was to evaluate the effect of perinatal exposure to low dose of Pb on the Tau pathology in the developing rat brain. Furthermore, the involvement of two major Tau-kinases: glycogen synthase kinase-3 beta (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in Pb-induced Tau modification was evaluated. Pregnant female rats were divided into control and Pb-treated group. The control animals were maintained on drinking water while females from the Pb-treated group received 0.1% lead acetate (PbAc) in drinking water, starting from the first day of gestation until weaning of the offspring. During the feeding of pups, mothers from the Pb-treated group were still receiving PbAc. Pups of both groups were weaned at postnatal day 21 and then until postnatal day 28 received only drinking water. 28-day old pups were sacrificed and Tau mRNA and protein level as well as Tau phosphorylation were analyzed in forebrain cortex (FC), cerebellum (C) and hippocampus (H). Concomitantly, we examined the effect of Pb exposure on GSK-3β and CDK5 activation. Our data revealed that pre- and neonatal exposure to Pb (concentration of Pb in whole blood below 10μg/dL, considered safe for humans) caused significant increase in the phosphorylation of Tau at Ser396 and Ser199/202 with parallel rise in the level of total Tau protein in FC and C. Tau hyperphosphorylation in Pb-treated animals was accompanied by elevated activity of GSK-3β and CDK5. Western blot analysis revealed activation of GSK-3β in FC and C as well as CDK5 in C, via increased phosphorylation of Tyr-216 and calpain-dependent p25

  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. 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.

  15. 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

  16. 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.

  17. Synthetic, enzyme kinetic, and protein crystallographic studies of C-β-d-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase.

    Science.gov (United States)

    Kantsadi, Anastassia L; Bokor, Éva; Kun, Sándor; Stravodimos, George A; Chatzileontiadou, Demetra S M; Leonidas, Demetres D; Juhász-Tóth, Éva; Szakács, Andrea; Batta, Gyula; Docsa, Tibor; Gergely, Pál; Somsák, László

    2016-11-10

    C-β-d-Glucopyranosyl pyrrole derivatives were prepared in the reactions of pyrrole, 2-, and 3-aryl-pyrroles with O-peracetylated β-d-glucopyranosyl trichloroacetimidate, while 2-(β-d-glucopyranosyl) indole was obtained by a cross coupling of O-perbenzylated β-d-glucopyranosyl acetylene with N-tosyl-2-iodoaniline followed by spontaneous ring closure. An improved synthesis of O-perbenzoylated 2-(β-d-glucopyranosyl) imidazoles was achieved by reacting C-glucopyranosyl formimidates with α-aminoketones. The deprotected compounds were assayed with isoforms of glycogen phosphorylase (GP) to show no activity of the pyrroles against rabbit muscle GPb. The imidazoles proved to be the best known glucose derived inhibitors of not only the muscle enzymes (both a and b) but also of the pharmacologically relevant human liver GPa (Ki = 156 and 26 nM for the 4(5)-phenyl and -(2-naphthyl) derivatives, respectively). An X-ray crystallographic study of the rmGPb-imidazole complexes revealed structural features of the strong binding, and also allowed to explain the absence of inhibition for the pyrrole derivatives. PMID:27522507

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. CDK5 is a major regulator of the tumor suppressor DLC1.

    Science.gov (United States)

    Tripathi, Brajendra K; Qian, Xiaolan; Mertins, Philipp; Wang, Dunrui; Papageorge, Alex G; Carr, Steven A; Lowy, Douglas R

    2014-12-01

    DLC1 is a tumor suppressor protein whose full activity depends on its presence at focal adhesions, its Rho-GTPase activating protein (Rho-GAP) function, and its ability to bind several ligands, including tensin and talin. However, the mechanisms that regulate and coordinate these activities remain poorly understood. Here we identify CDK5, a predominantly cytoplasmic serine/threonine kinase, as an important regulator of DLC1 functions. The CDK5 kinase phosphorylates four serines in DLC1 located N-terminal to the Rho-GAP domain. When not phosphorylated, this N-terminal region functions as an autoinhibitory domain that places DLC1 in a closed, inactive conformation by efficiently binding to the Rho-GAP domain. CDK5 phosphorylation reduces this binding and orchestrates the coordinate activation DLC1, including its localization to focal adhesions, its Rho-GAP activity, and its ability to bind tensin and talin. In cancer, these anti-oncogenic effects of CDK5 can provide selective pressure for the down-regulation of DLC1, which occurs frequently in tumors, and can contribute to the pro-oncogenic activity of CDK5 in lung adenocarcinoma. PMID:25452387

  7. 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.

  8. Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence.

    Science.gov (United States)

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

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16(INK4a) 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 p16(INK4a). To determine the role of the Cdk4(R24C) germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated "knock-in" technology. Cdk4(R24C/R24C) mouse embryo fibroblasts (MEFs) displayed increased Cdk4 kinase activity resulting in hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130. MEFs derived from Cdk4(R24C/R24C) mice displayed decreased doubling times, escape from replicative senescence, and escape sensitivity to contact-induced growth arrest. These MEFs also exhibited a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4(R24C) mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with the in vitro data, homozygous Cdk4(R24C/R24C) mice developed tumors of various etiology within 8 to 10 months of their life span. The majority of these tumors were found in the pancreas, pituitary, brain, mammary tissue, and skin. In addition, Cdk4(R24C/R24C) mice showed extraordinary susceptibility to carcinogens and developed papillomas within the first 8 to 10 weeks following cutaneous application of the carcinogens 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This report formally establishes that the activation of Cdk4 is sufficient to promote cancer in many tissues. The observation that a wide variety of tumors develop in mice harboring the Cdk4(R24C) mutation offers a genetic proof that Cdk4 activation may constitute a central event in the genesis of many types of cancers in addition to melanoma.

  9. 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...

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  17. 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.

  18. 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.

  19. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    Science.gov (United States)

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle.

  20. 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.

  1. 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...

  2. 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...

  3. 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...

  4. 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.

  5. 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

  6. 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.

  7. 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。

  8. 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.

  9. 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.

  10. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients

    OpenAIRE

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint...

  11. Effects of kaempferol on cell cycle status and CyclinB1,Cdk1 mRNA expressions in CNE-2 cells%山奈酚对CNE-2细胞周期及CyclinB1、Cdk1mRNA表达的影响

    Institute of Scientific and Technical Information of China (English)

    陈育华; 吴国才; 王珍; 周碧云

    2012-01-01

    Aim: To study the effects of kaempferol on cell cycle status and CyclinB1, Cdk1 mRNA expressions in CNE-2 cells. Methods:CNE-2 cells were treated with 0,20,40,60,80,and 100 μmol/L kaempferol. 24,48 and 72 h later, proliferation was determined by MTT assay;24 and 48 h later,cell cycle was detected by flow cytometry;24 h later,the expressions of CyclinBl and Cdkl mRNA were detected by RT-PCR. Results:The CNE-2 cell growth ability was inhibited by kaempferol in a time- and dose-dependent manned Fdose =385. 194,Ftime =237. 324,Finteraetion =13.757,P <0.001 );CNE-2 cells was blocked in G2/M phase ( P<0.05 );the expressions of CyclinB1 and Cdk1 mRNA decreased with the increase of kaempferol dose ( F = 95. 682,154. 871 ,P < 0. 001 ). Conclusion: Kaempferol can block CNE-2 cells in G2/M phase through decreasing the expressions of CyclinBl and Cdkl mRNA,and inhibit the cell proliferation.%目的:观察山奈酚对鼻咽癌CNE-2细胞周期分布及细胞周期素B1(CyclinB1)、细胞周期依赖性蛋白激酶1(Cdk1)表达的影响.方法:分别用0、20、40、60、80和100 μmol/L的山奈酚处理CNE-2细胞.处理24、48和72 h后,应用MTT法测定CNE-2细胞活力;处理24和48 h后用流式细胞术检测细胞周期;处理24 h后用RT-PCR技术检测细胞CyclinB1及Cdk1 mRNA的表达水平.结果:随山奈酚作用剂量的增加和作用时间的延长,CNE-2细胞活力逐渐降低(F浓度=385.194,F时间=237.324,F浓度×时间=13.757,P<0.001,细胞被阻滞于G2/M期(P<0.05);CNE-2细胞中CyclinB1和Cdk1 mRNA的表达量随山奈酚作用浓度的增加而逐渐降低(F=95.682、154.871,P<0.001).结论:山奈酚可能通过下调CNE-2细胞CyclinB1和Cdk1 mRNA的表达水平,诱导G2/M期阻滞,抑制其增殖.

  12. Cdk1 orders mitotic events through coordination of a chromosome-associated phosphatase switch.

    Science.gov (United States)

    Qian, Junbin; Beullens, Monique; Huang, Jin; De Munter, Sofie; Lesage, Bart; Bollen, Mathieu

    2015-01-01

    RepoMan is a scaffold for signalling by mitotic phosphatases at the chromosomes. During (pro)metaphase, RepoMan-associated protein phosphatases PP1 and PP2A-B56 regulate the chromosome targeting of Aurora-B kinase and RepoMan, respectively. Here we show that this task division is critically dependent on the phosphorylation of RepoMan by protein kinase Cyclin-dependent kinase 1 (Cdk1), which reduces the binding of PP1 but facilitates the recruitment of PP2A-B56. The inactivation of Cdk1 in early anaphase reverses this phosphatase switch, resulting in the accumulation of PP1-RepoMan to a level that is sufficient to catalyse its own chromosome targeting in a PP2A-independent and irreversible manner. Bulk-targeted PP1-RepoMan also inactivates Aurora B and initiates nuclear-envelope reassembly through dephosphorylation-mediated recruitment of Importin β. Bypassing the Cdk1 regulation of PP1-RepoMan causes the premature dephosphorylation of its mitotic-exit substrates in prometaphase. Hence, the regulation of RepoMan-associated phosphatases by Cdk1 is essential for the timely dephosphorylation of their mitotic substrates. PMID:26674376

  13. 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

  14. Mutually Unbiased Maximally Entangled Bases for the Bipartite System Cd⊗ C^{dk}

    Science.gov (United States)

    Nan, Hua; Tao, Yuan-Hong; Wang, Tian-Jiao; Zhang, Jun

    2016-10-01

    The construction of maximally entangled bases for the bipartite system Cd⊗ Cd is discussed firstly, and some mutually unbiased bases with maximally entangled bases are given, where 2≤ d≤5. Moreover, we study a systematic way of constructing mutually unbiased maximally entangled bases for the bipartite system Cd⊗ C^{dk}.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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...

  20. Novel optimization of valmerins (tetrahydropyrido[1,2-a]isoindolones) as potent dual CDK5/GSK3 inhibitors.

    Science.gov (United States)

    Ouach, Aziz; Boulahjar, Rajâa; Vala, Christine; Bourg, Stéphane; Bonnet, Pascal; Guguen-Guillouzo, Christiane; Ravache, Myriam; Le Guevel, Rémy; Lozach, Olivier; Lazar, Saïd; Troin, Yves; Meijer, Laurent; Ruchaud, Sandrine; Akssira, Mohamed; Guillaumet, Gérald; Routier, Sylvain

    2016-06-10

    An efficient synthetic strategy able to modulate the structure of the tetrahydropyridine isoindolone (Valmerin) skeleton was developed. A library of more than 30 novel final structures was generated. Biological activities on CDK5 and GSK3 as well as cellular effects on cancer cell lines were measured for each novel compound. Additionally to support the SAR, a docking study was performed. A potent GSK3/CDK5 dual inhibitor (37, IC50 CDK5/GSK3 35/7 nM) was obtained. Best antiproliferative effects were obtained on lung and prostate cell lines with IC50 = 20 nM. PMID:27019296

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

    Science.gov (United States)

    Voit, Renate; Seiler, Jeanette; Grummt, Ingrid

    2015-01-01

    Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAFI110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD+-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAFI110 is prevented. The results demonstrate that reversible phosphorylation of TAFI110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression. PMID:26023773

  2. 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

  3. Hydrophobic Interactions Are a Key to MDM2 Inhibition by Polyphenols as Revealed by Molecular Dynamics Simulations and MM/PBSA Free Energy Calculations.

    Science.gov (United States)

    Verma, Sharad; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Grover, Abhinav

    2016-01-01

    p53, a tumor suppressor protein, has been proven to regulate the cell cycle, apoptosis, and DNA repair to prevent malignant transformation. MDM2 regulates activity of p53 and inhibits its binding to DNA. In the present study, we elucidated the MDM2 inhibition potential of polyphenols (Apigenin, Fisetin, Galangin and Luteolin) by MD simulation and MM/PBSA free energy calculations. All polyphenols bind to hydrophobic groove of MDM2 and the binding was found to be stable throughout MD simulation. Luteolin showed the highest negative binding free energy value of -173.80 kJ/mol followed by Fisetin with value of -172.25 kJ/mol. It was found by free energy calculations, that hydrophobic interactions (vdW energy) have major contribution in binding free energy.

  4. CDK5 Regulates Paclitaxel Sensitivity in Ovarian Cancer Cells by Modulating AKT Activation, p21Cip1- and p27Kip1-Mediated G1 Cell Cycle Arrest and Apoptosis

    OpenAIRE

    Shu Zhang; Zhen Lu; Weiqun Mao; Ahmed, Ahmed A; Hailing Yang; Jinhua Zhou; Nicholas Jennings; Cristian Rodriguez-Aguayo; Gabriel Lopez-Berestein; Roberto Miranda; Wei Qiao; Veera Baladandayuthapani; Zongfang Li; Anil K. Sood; Jinsong Liu

    2015-01-01

    Cyclin-dependent kinase 5 (CDK5) is a cytoplasmic serine/ threonine kinase. Knockdown of CDK5 enhances paclitaxel sensitivity in human ovarian cancer cells. This study explores the mechanisms by which CDK5 regulates paclitaxel sensitivity in human ovarian cancers. Multiple ovarian cancer cell lines and xenografts were treated with CDK5 small interfering RNA (siRNA) with or without paclitaxel to examine the effect on cancer cell viability, cell cycle arrest and tumor growth. CDK5 protein was m...

  5. Schisandrin B inhibits cell proliferation and induces apoptosis in human cholangiocarcinoma cells.

    Science.gov (United States)

    Yang, Xiaohui; Wang, Shuai; Mu, Yunchuan; Zheng, Yixiong

    2016-10-01

    Cholangiocarcinoma (CCA) is the second most common hepatic cancer with high resistance to current chemotherapies and extremely poor prognosis. The present study aimed to examine the effects of schisandrin B (Sch B) on CCA cells both in vitro and in vivo and to examine its underlying mechanism. We found that Sch B inhibited the viability and proliferation of CCA cells in a dose- and time-dependent manner as assessed by MTT and colony formation assays. The flow cytometric assay revealed G0/G1 phase arrest in the Sch B-treated HCCC-9810 and RBE cells. In addition, Sch B induced intrahepatic cholangiocarcinoma apoptosis as shown by the results of Annexin V/PI double staining. Rhodamine 123 staining revealed that Sch B decreased the mitochondrial membrane potential (ΔΨm) in a dose-dependent manner. Mechanistically, western blot analysis indicated that Sch B induced apoptosis by upregulating Bax, cleaved caspase-3, cleaved caspase-9 and cleaved PARP, and by downregulating cyclin D1, Bcl-2 and CDK-4. Moreover, Sch B significantly inhibited HCCC-9810 xenograft growth in athymic nude mice. In summary, these findings suggest that Sch B exhibited potent antitumor activities via the induction of CCA apoptosis and that Sch B may be a promising drug for the treatment of CCA. PMID:27499090

  6. 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

  7. 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.

  8. Cdk5—肿瘤新靶点及其抑制剂研究进展%Progress in the study of new cancer target Cdk5 and its inhibitors

    Institute of Scientific and Technical Information of China (English)

    苗龙星; 杨怡君; 王泽瑜; 李乾斌; 胡高云

    2016-01-01

    细胞周期蛋白依赖性激酶-5 (cyclin-dependent kinase-5,CdkS)是一类丝氨酸/苏氨酸蛋白激酶,参与神经细胞生长发育和信号传导调控.Cdk5的过表达与肿瘤的发生、发展和凋亡有着密切关系.Cdk5抑制剂的研究正成为癌症治疗的热门领域.本文介绍了Cdk5的生物学功能和作用机制,重点阐述以ATP为锚点的小分子抑制剂和介导蛋白-蛋白相互作用的多肽等抑制剂的最新进展.

  9. 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.

  10. EGFR/MEK/ERK/CDK5-dependent integrin-independent FAK phosphorylated on serine 732 contributes to microtubule depolymerization and mitosis in tumor cells.

    Science.gov (United States)

    Rea, K; Sensi, M; Anichini, A; Canevari, S; Tomassetti, A

    2013-01-01

    FAK is a non-receptor tyrosine kinase contributing to migration and proliferation downstream of integrin and/or growth factor receptor signaling of normal and malignant cells. In addition to well-characterized tyrosine phosphorylations, FAK is phosphorylated on several serines, whose role is not yet clarified. We observed that phosphorylated FAK on serine 732 (P-FAKSer732) is present at variable levels in vitro, in several melanoma, ovarian and thyroid tumor cell lines and in vivo, in tumor cells present in fresh ovarian cancer ascites. In vitro P-FAKSer732 was barely detectable during interphase while its levels strongly increased in mitotic cells upon activation of the EGFR/MEK/ERK axis in an integrin-independent manner. P-FAKSer732 presence was crucial for the maintenance of the proliferation rate and its levels were inversely related to the levels of acetylated α-tubulin. P-FAKSer732 localized at the microtubules (MTs) of the spindle, biochemically associated with MTs and contributed to MT depolymerization. The lack of the phosphorylation on Ser732 as well as the inhibition of CDK5 activity by roscovitine impaired mitotic spindle assembly and correct chromosome alignment during mitosis. We also identified, for the first time, that the EGF-dependent EGFR activation led to increased P-FAKSer732 and polymerized MTs. Our data shed light on the multifunctional roles of FAK in neoplastic cells, being involved not only in integrin-dependent migratory signaling but also in integrin-independent MT dynamics and mitosis control. These findings provide a new potential target for inhibiting the growth of tumor cells in which the EGFR/MEK/ERK/CDK5 pathway is active. PMID:24091658

  11. 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.

  12. 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

  13. 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.

  14. Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

    Directory of Open Access Journals (Sweden)

    Li Li

    2008-08-01

    Full Text Available Abstract Background Autosomal Dominant Polycystic Kidney Disease (ADPKD is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2. Methods Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation. Results Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels. Conclusion Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

  15. The cloning of the cdk2 transcript and the localization of its expression during gametogenesis in the freshwater giant prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Chen, Jie; Liu, Ping; Li, Zhen; Chen, Ying; Qiu, Gao-Feng

    2013-08-01

    Cyclin-dependent kinases (cdks) are key regulators of the cell cycle. In mammals, cdk2 plays an essential role in the meiosis of spermatocytes and oocytes. To investigate the role of cdk2 kinase during gametogenesis in crustaceans, we cloned a complete cDNA sequence of cdk2 from the freshwater giant prawn, Macrobrachium rosenbergii, and examined its localization and expression in the developing gonads. The prawn cdk2 cDNA is 1,745 bp in length and encodes a putative protein of 305 amino acids. The deduced protein contains a conserved cyclin binding motif PSTAIRE and shares high homology with reported cdk2 kinases of other species. RT-PCR analysis showed a wide distribution of the cdk2 mRNA in all tested organs including the testis, ovary, heart, muscles, hepatopancreas and gills, and the highest level of expression in the ovary and testis. Localization by in situ hybridization of cdk2 mRNA in the ovary showed high expression in the ooplasm of previtellogenic and the nuclei of late vitellogenic oocytes. In testicular sections, cdk2 transcript is low in spermatogonia, high in spermatocytes, but reduced in spermatids and sperm. The high expression of the cdk2 transcripts in meiotic spermatocytes and oocytes indicated that the cdk2 gene has the conservative function in the germ cells meiosis during gametogenesis.

  16. 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.

  17. 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

  18. Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

    Science.gov (United States)

    Sarenac, Tomislav; Trapecar, Martin; Gradisnik, Lidija; Rupnik, Marjan Slak; Pahor, Dusica

    2016-01-01

    Corneal wound healing is often affected by TGF-β–mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing. PMID:27687492

  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. 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

  1. 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.

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

    Science.gov (United States)

    Gupta, Rupesh K; Meachum, Sharon; Hernández-Ochoa, Isabel; Peretz, Jackye; Yao, Humphrey H; Flaws, Jodi A

    2009-10-01

    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.

  3. 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...

  4. 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...

  5. 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...

  6. Discovery of pyrrolospirooxindole derivatives as novel cyclin dependent kinase 4 (CDK4) inhibitors by catalyst-free, green approach.

    Science.gov (United States)

    Kamal, Ahmed; Mahesh, Rasala; Nayak, V Lakshma; Babu, Korrapati Suresh; Kumar, G Bharath; Shaik, Anver Basha; Kapure, Jeevak Sopanrao; Alarifi, Abdullah

    2016-01-27

    Aiming to develop a new target for the anticancer treatment, a series of 5'H-spiro[indoline-3,4'-pyrrolo [1,2-a]quinoxalin]-2-ones has been synthesized by simple, highly efficient and environmentally friendly method in excellent yields under catalyst-free conditions using ethanol as a green solvent. A simple filtration of the reaction mixture and subsequent drying affords analytically pure products. The synthesized derivatives were evaluated for their antiproliferative activity against five different human cancer cell lines, among the congeners compound 3n showed significant cytotoxicity against the human prostate cancer (DU-145). Flow cytometric analysis revealed that this compound induces cell cycle arrest in the G0/G1 phase and Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell death. This was further confirmed by mitochondrial membrane potential ((ΔΨm), Annexin V-FITC assay and docking experiments. Furthermore, it was observed that there is an increase in expression levels of cyclin dependent kinase inhibitors like Cip1/p21 and Kip1/p27.

  7. Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells

    Directory of Open Access Journals (Sweden)

    Yann Thomas

    2016-04-01

    Full Text Available The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.

  8. 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.

  9. 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.

  10. ALTERATIONS OF pRb/CDK4/p16INK4a PATHWAY IN GASTRIC CARCINOMAS%胃癌中p16INK4a-CDK4-pRb通路蛋白表达异常

    Institute of Scientific and Technical Information of China (English)

    赵英芳; 田新霞; 卢阳

    2005-01-01

    目的:检测胃癌组织中p16INK4a-CDK4-pRb通路p16INK4a、CDK4、pRb蛋白表达状况,探讨蛋白表达与胃癌发生发展以及临床病理指标的关系.方法:采用免疫组织化学方法检测了胃癌组织中p16INK4a、CDK4、pRb蛋白表达.结果:10例正常胃黏膜中相应蛋白表达全部阳性,而肿瘤组织中p16INK4a、pRb蛋白表达阳性率分别为54%(44/81)和90%(73/81),p16INK4a蛋白表达显著低于正常组织(P=0.005),26%(21/81)的肿瘤组织中CDK4过表达.p16INK4a、pRb、CDK4蛋白表达与肿瘤组织学类型、淋巴结转移及性别、年龄均无相关性.结论:p16INK4a、CDK4、pRb蛋白表达异常是胃癌细胞常见的分子事件,p16INK4a-CDK4-pRb细胞周期调控通路异常可能参与了胃癌的发生发展.

  11. 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......, and 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...

  12. 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.

  13. Conditional deletion of Jak2 reveals an essential role in hematopoiesis throughout mouse ontogeny: implications for Jak2 inhibition in humans.

    Directory of Open Access Journals (Sweden)

    Sung O Park

    Full Text Available Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5, at postnatal day 4 (PN4, or at ∼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

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    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.

  20. (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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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。

  11. 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.

  12. Cyclin D and cdk4 Are Required for Normal Development beyond the Blastula Stage in Sea Urchin Embryos

    Science.gov (United States)

    Moore, Jennifer C.; Sumerel, Jan L.; Schnackenberg, Bradley J.; Nichols, Jason A.; Wikramanayake, Athula; Wessel, Gary M.; Marzluff, William F.

    2002-01-01

    cdk4 mRNA and protein are constitutively expressed in sea urchin eggs and throughout embryonic development. In contrast, cyclin D mRNA is barely detectable in eggs and early embryos, when the cell cycles consist of alternating S and M phases. Cyclin D mRNA increases dramatically in embryos at the early blastula stage and remains at a constant level throughout embryogenesis. An increase in cdk4 kinase activity occurs concomitantly with the increase in cyclin D mRNA. Ectopic expression of cyclin D mRNA in eggs arrests development before the 16-cell stage and causes eventual embryonic death, suggesting that activation of cyclin D/cdk4 in cleavage cell cycles is lethal to the embryo. In contrast, blocking cyclin D or cdk4 expression with morpholino antisense oligonucleotides results in normal development of early gastrula-stage embryos but abnormal, asymmetric larvae. These results suggest that in sea urchins, cyclin D and cdk4 are required for normal development and perhaps the patterning of the developing embryo, but may not be directly involved in regulating entry into the cell cycle. PMID:12052892

  13. Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

    Science.gov (United States)

    Chiker, Sara; Pennaneach, Vincent; Loew, Damarys; Dingli, Florent; Biard, Denis; Cordelières, Fabrice P; Gemble, Simon; Vacher, Sophie; Bieche, Ivan; Hall, Janet; Fernet, Marie

    2015-01-01

    Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing. PMID:26237679

  14. 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.

  15. CDK4, pRB and E2F1: connected to insulin

    Directory of Open Access Journals (Sweden)

    Blanchet Emilie

    2010-02-01

    Full Text Available Abstract Pancreatic β-cells are metabolic sensors involved in the control of glucose homeostasis. This particular cell type controls insulin secretion through a fine-tuned process, which dregulation have important pathological consequences, such as observed during type 2 diabetes. We recently implicated E2F1 in the control of glucose homeostasis. First we showed that E2f1-/- mice have decreased pancreatic size, as the result of impaired postnatal pancreatic growth. We observed in this study that E2F1 was highly expressed in non-proliferating pancreatic β-cells, suggesting that E2F1, besides the control of β-cell number could have a role in pancreatic β-cell function. We demonstrate in our recent study, both in vitro and in vivo that E2F1 directly regulates the expression of Kir6.2, a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion in pancreatic β-cells. Expression of Kir6.2 is lost in pancreas of E2f1-/- mice, resulting in insulin secretion defects in these mice. Furthermore, we demonstrated by in tissue chromatin immunoprecipitation analysis that regulation of Kir6.2 expression by E2F1 follows the same regulatory pathway that the classical E2F1 target genes, implicating the participation of CDK4 and retinoblastoma protein. Moreover, in this context, E2F1 transcriptional activity is regulated by glucose and insulin through the CDK4-dependent inactivation of the pRB protein. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis. In our recent study we decipher a new function for these factors in the control of insulin secretion and open up new avenues for the treatment of metabolic diseases, in particular type 2 diabetes.

  16. 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.

  17. Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1

    Directory of Open Access Journals (Sweden)

    Jose-Antonio Rodriguez-Rodriguez

    2016-05-01

    Full Text Available 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.

  18. 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...

  19. 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.

  20. IL12A, MPHOSPH9/CDK2AP1 and RGS1 are novel multiple sclerosis susceptibility loci

    DEFF Research Database (Denmark)

    Sørensen, Per Soelberg

    2010-01-01

    and the 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 MS (P=0.01). Thus, we report three new MS susceptibility loci, including a novel inflammatory disease locus that could affect autoreactive cell proliferation....

  1. Ipl1/Aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis.

    Directory of Open Access Journals (Sweden)

    Louise Newnham

    Full Text Available Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

  2. Desmin phosphorylation by Cdk1 is required for efficient separation of desmin intermediate filaments in mitosis and detected in murine embryonic/newborn muscle and human rhabdomyosarcoma tissues.

    Science.gov (United States)

    Makihara, Hiroyuki; Inaba, Hironori; Enomoto, Atsushi; Tanaka, Hiroki; Tomono, Yasuko; Ushida, Kaori; Goto, Mitsuo; Kurita, Kenichi; Nishida, Yoshihiro; Kasahara, Kousuke; Goto, Hidemasa; Inagaki, Masaki

    2016-09-23

    Desmin is a type III intermediate filament (IF) component protein expressed specifically in muscular cells. Desmin is phosphorylated by Aurora-B and Rho-kinase specifically at the cleavage furrow from anaphase to telophase. The disturbance of this phosphorylation results in the formation of unusual long bridge-like IF structures (IF-bridge) between two post-mitotic (daughter) cells. Here, we report that desmin also serves as an excellent substrate for the other type of mitotic kinase, Cdk1. Desmin phosphorylation by Cdk1 loses its ability to form IFs in vitro. We have identified Ser6, Ser27, and Ser31 on murine desmin as phosphorylation sites for Cdk1. Using a site- and phosphorylation-state-specific antibody for Ser31 on desmin, we have demonstrated that Cdk1 phosphorylates desmin in entire cytoplasm from prometaphase to metaphase. Desmin mutations at Cdk1 sites exhibit IF-bridge phenotype, the frequency of which is significantly increased by the addition of Aurora-B and Rho-kinase site mutations to Cdk1 site mutations. In addition, Cdk1-induced desmin phosphorylation is detected in mitotic muscular cells of murine embryonic/newborn muscles and human rhabdomyosarcoma specimens. Therefore, Cdk1-induced desmin phosphorylation is required for efficient separation of desmin-IFs and generally detected in muscular mitotic cells in vivo. PMID:27565725

  3. 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.

  4. 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.

  5. Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of Xenopus laevis

    Directory of Open Access Journals (Sweden)

    Sible Jill C

    2007-10-01

    Full Text Available Abstract Background The cell cycles of the Xenopus laevis embryo undergo extensive remodeling beginning at the midblastula transition (MBT of early development. Cell divisions 2–12 consist of rapid cleavages without gap phases or cell cycle checkpoints. Some remodeling events depend upon a critical nucleo-cytoplasmic ratio, whereas others rely on a maternal timer controlled by cyclin E/Cdk2 activity. One key event that occurs at the MBT is the degradation of maternal Wee1, a negative regulator of cyclin-dependent kinase (Cdk activity. Results In order to assess the effect of Wee1 on embryonic cell cycle remodeling, Wee1 mRNA was injected into one-cell stage embryos. Overexpression of Wee1 caused cell cycle delay and tyrosine phosphorylation of Cdks prior to the MBT. Furthermore, overexpression of Wee1 disrupted key developmental events that normally occur at the MBT such as the degradation of Cdc25A, cyclin E, and Wee1. Overexpression of Wee1 also resulted in post-MBT apoptosis, tyrosine phosphorylation of Cdks and persistence of cyclin E/Cdk2 activity. To determine whether Cdk2 was required specifically for the survival of the embryo, the cyclin E/Cdk2 inhibitor, Δ34-Xic1, was injected in embryos and also shown to induce apoptosis. Conclusion Taken together, these data suggest that Wee1 triggers apoptosis through the disruption of the cyclin E/Cdk2 timer. In contrast to Wee1 and Δ34-Xic1, altering Cdks by expression of Chk1 and Chk2 kinases blocks rather than promotes apoptosis and causes premature degradation of Cdc25A. Collectively, these data implicate Cdc25A as a key player in the developmentally regulated program of apoptosis in X. laevis embryos.

  6. Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

    Science.gov (United States)

    Siriwardana, Gamini; Seligman, Paul A

    2015-03-01

    Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. PMID:25825542

  7. Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

    Science.gov (United States)

    Siriwardana, Gamini; Seligman, Paul A

    2015-03-01

    Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation.

  8. 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

  9. 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

    of 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...

  10. 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.

  11. 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.

  12. 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。

  13. 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.

  14. Selective inhibition of caspases in skeletal muscle reverses the apoptotic synaptic degeneration in slow-channel myasthenic syndrome.

    Science.gov (United States)

    Zhu, Haipeng; Pytel, Peter; Gomez, Christopher M

    2014-01-01

    Slow-channel syndrome (SCS) is a congenital myasthenic disorder caused by point mutations in subunits of skeletal muscle acetylcholine receptor leading to Ca(2+) overload and degeneration of the postsynaptic membrane, nuclei and mitochondria of the neuromuscular junction (NMJ). In both SCS muscle biopsies and transgenic mouse models for SCS (mSCS), the endplate regions are shrunken, and there is evidence of DNA damage in the subsynaptic region. Activated caspase-9, -3 and -7 are intensely co-localized at the NMJ, and the Ca(2+)-activated protease, calpain, and the atypical cyclin-dependent kinase (Cdk5) are overactivated in mSCS. Thus, the true mediator(s) of the disease process is not clear. Here, we demonstrate that selective inhibition of effector caspases, caspase-3 and -7, or initiator caspase, caspase-9, in limb muscle in vivo by localized expression of recombinant inhibitor proteins dramatically decreases subsynaptic DNA damage, increases endplate area and improves ultrastructural abnormalities in SCS transgenic mice. Calpain and Cdk5 are not affected by this treatment. On the other hand, inhibition of Cdk5 by expression of a dominant-negative form of Cdk5 has no effect on the degeneration. Together with previous studies, these results indicate that focal activation of caspase activity at the NMJ is the principal pathological process responsible for the synaptic apoptosis in SCS. Thus, treatments that reduce muscle caspase activity are likely to be of benefit for SCS patients.

  15. Ndd1 turnover by SCF(Grr1 is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Ellen R Edenberg

    2015-04-01

    Full Text Available In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1, phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1. In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

  16. 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...

  17. 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.

  18. CDK5逆转Sirt1在宫颈癌细胞耐药中的作用机制%CDK5 reverses the multidrug-resistant mechanism of Sirt1 in cervical cancer cells

    Institute of Scientific and Technical Information of China (English)

    陈丽君; 王建; 范春芳

    2014-01-01

    目的:探讨CDK5逆转Sirt1在宫颈癌化疗耐药中的作用机制.方法:体外培养人宫颈癌Hela细胞系和宫颈癌Hela/MMC耐药细胞亚系,Western blotting检测MMC对Hela和Hela/MMC细胞内P-Sirt1蛋白表达的影响;MTT法检测Hela细胞存活率;通过加入CDK5抑制剂Roscovitine来检测CDK5使Sirt1磷酸化的作用;RT-PCR方法检测耐药相关蛋白P-gp的mRNA表达情况.结果:正常情况下,Hela细胞中P-Sirtl的表达显著高于Hela/MMC细胞(P<0.05),MMC处理的Hela细胞中P-Sirt1的表达显著高于未经MMC处理组(P<0.05).超表达P-Sirt1会导致细胞存活率显著下降(P<0.05).CDK5抑制剂Roscovitine可以使Hela细胞的存活率增加,耐药性相关蛋白P-gp的mRNA表达上调(P<0.05).结论:CDK5可以使Sirt1磷酸化,增加了Hela细胞对MMC的敏感性.

  19. 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

  20. Phosphorylation of CRMP2 by Cdk5 Regulates Dendritic Spine Development of Cortical Neuron in the Mouse Hippocampus

    Directory of Open Access Journals (Sweden)

    Xiaohua Jin

    2016-01-01

    Full Text Available Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5 is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2 in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2KI/KI mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

  1. Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity.

    Directory of Open Access Journals (Sweden)

    Indra Tumurbaatar

    Full Text Available Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C-Cdc20 regulation during...

  7. Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

    Science.gov (United States)

    Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

    2015-10-01

    Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1

  8. Cloning and characterization of human IC53-2, a novel CDK5 activator binding protein

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No. AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellularcarcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate thatIC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.

  9. 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.

  10. 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

  11. 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...

  12. 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。

  13. 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

  14. 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.

  15. miR-483 is Down-Regulated in Polycystic Ovarian Syndrome and Inhibits KGN Cell Proliferation via Targeting Insulin-Like Growth Factor 1 (IGF1)

    Science.gov (United States)

    Xiang, Yungai; Song, Yuxia; Li, Yan; Zhao, Dongmei; Ma, Liying; Tan, Li

    2016-01-01

    Background Polycystic ovarian syndrome (PCOS) is a common metabolic disorder in premenopausal woman, characterized by hyperandrogenism, oligoanovulation, and insulin resistance. microRNAs play pivotal roles in regulating key factors of PCOS. However, relevant research remains limited. This study aimed to reveal the role and potential mechanism of miR-483 in PCOS. Material/Methods PCOS patients (n=20) were recruited for detecting miR-483 expression in lesion and normal ovary cortex. Human granulosa-like tumor cell line KGN was used to alter miR-483 expression by cell transfection. Cell viability and proliferation were analyzed by MTT assay and colony formation assay, and cell cycle was detected by flow cytometry. Interaction between miR-483 and IGF1 was verified by luciferase reporter assay. KGN cells were further treated by insulin to investigate the relationship between miR-483 and insulin. Results miR-483 was significantly down-regulated in lesion ovary cortex from PCOS patients (P<0.001). In KGN cells, overexpression of miR-483 inhibited cell viability and proliferation, and induced cell cycle arrest. miR-483 also inhibited CCNB1, CCND1, and CDK2. miR-483 sponge induced the opposite effects. miR-483 directly targeted IGF1 3′UTR, and IGF1 promoted KGN cell proliferation and reversed miR-483-inhibited cell viability. Insulin treatment in KGN cells inhibited miR-483, and promoted IGF1 and cell proliferation. Conclusions These results suggest that miR-483 is a PCOS suppressor inhibiting cell proliferation, possibly via targeting IGF1, and that it is involved in insulin-induced cell proliferation. miR-483 is a potential alternative for diagnosing and treating PCOS. PMID:27662007

  16. 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.

  17. 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

  18. 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;

    2014-01-01

    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 homolog......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...... 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....

  19. 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.

  20. @@%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.

  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.;

    2007-01-01

    mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-DIK2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGF beta), but are relatively resistant to TGF......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...... beta antiproliferative effects. Fibroblasts derived from MMTV-DIK2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70(s6k) and Akt on activating...

  2. 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

    sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGF beta and HGF, resulting in desmoplasia. The MMTV-DIK2 mice should provide a useful model system...... for the 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...

  3. Metabolism and pharmacokinetics of 8-hydroxypiperidinylmethyl-baicalein (BA-j) as a novel selective CDK1 inhibitor in monkey.

    Science.gov (United States)

    Guo, Hong-Min; Sun, Yu-Ming; Zhang, Shi-Xuan; Ju, Xiu-Lan; Xie, Ai-Yun; Li, Jing; Zou, Liang; Sun, Xiao-Dan; Li, Hai-Liang; Zheng, Yang

    2015-12-01

    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. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a Mannich base derivative of baicalein (BA) isolated from Scutellaria baicalensis, as a novel selective CDK1 inhibitor. 12 metabolites of BA-j in the monkey urine were identified by LC-MS-MS and (1)H NMR. The major metabolic pathways of BA-j, by capturing oxygen free radicals ((.)O2(-)) and releasing peroxides (H2O2), are degraded into active intermediate metabolite dihydroflavonol, then into main metabolite M179 by Shiff reaction, second metabolite M264 by sulfation, trace amount of metabolite M559 by glucuronidation UGT1A9, and without metabolism by CYP3A4. The metabolic process of BA-j by regulating intracellular reactive oxygen species (ROS) was related with BA-j selectively inducing apoptosis in cancer cells. Pharmacokinetics of 10mg/kg oral BA-j in monkey by HPLC-UV was best fitted to a two-compartment open model, with t1/2(β) of 4.2h, Cmax 25.4μM at 2h, and Vd 12.6L, meaning the drug distributing widely in body fluids with no special selectivity to certain tissues, and being able to permeate through the blood-brain barrier. The protein binding rate of BA-j was 91.8%. BA-j has excellent druggability for oral administration or injection, and it may be developed into a novel anti-cancer drug as a selective CDK1 inhibitor.

  4. 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...

  5. Metabolism and pharmacokinetics of 8-hydroxypiperidinylmethyl-baicalein (BA-j) as a novel selective CDK1 inhibitor in monkey.

    Science.gov (United States)

    Guo, Hong-Min; Sun, Yu-Ming; Zhang, Shi-Xuan; Ju, Xiu-Lan; Xie, Ai-Yun; Li, Jing; Zou, Liang; Sun, Xiao-Dan; Li, Hai-Liang; Zheng, Yang

    2015-12-01

    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. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a Mannich base derivative of baicalein (BA) isolated from Scutellaria baicalensis, as a novel selective CDK1 inhibitor. 12 metabolites of BA-j in the monkey urine were identified by LC-MS-MS and (1)H NMR. The major metabolic pathways of BA-j, by capturing oxygen free radicals ((.)O2(-)) and releasing peroxides (H2O2), are degraded into active intermediate metabolite dihydroflavonol, then into main metabolite M179 by Shiff reaction, second metabolite M264 by sulfation, trace amount of metabolite M559 by glucuronidation UGT1A9, and without metabolism by CYP3A4. The metabolic process of BA-j by regulating intracellular reactive oxygen species (ROS) was related with BA-j selectively inducing apoptosis in cancer cells. Pharmacokinetics of 10mg/kg oral BA-j in monkey by HPLC-UV was best fitted to a two-compartment open model, with t1/2(β) of 4.2h, Cmax 25.4μM at 2h, and Vd 12.6L, meaning the drug distributing widely in body fluids with no special selectivity to certain tissues, and being able to permeate through the blood-brain barrier. The protein binding rate of BA-j was 91.8%. BA-j has excellent druggability for oral administration or injection, and it may be developed into a novel anti-cancer drug as a selective CDK1 inhibitor. PMID:26474673

  6. Synthesis and preliminary in vitro kinase inhibition evaluation of new diversely substituted pyrido[3,4-g]quinazoline derivatives.

    Science.gov (United States)

    Zeinyeh, Wael; Esvan, Yannick J; Nauton, Lionel; Loaëc, Nadège; Meijer, Laurent; Théry, Vincent; Anizon, Fabrice; Giraud, Francis; Moreau, Pascale

    2016-09-01

    The synthesis of new diversely substituted pyrido[3,4-g]quinazolines is described. The inhibitory potencies of prepared compounds toward a panel of five CMGC protein kinases (CDK5, CLK1, DYRK1A, CK1, GSK3), that are known to play a potential role in Alzheimer's disease, were evaluated. The best overall kinase inhibition profile was found for nitro compound 4 bearing an ethyl group at the 5-position. PMID:27469128

  7. 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.

  8. 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

  9. In vitro growth inhibition of human cancer cells by novel honokiol analogs.

    Science.gov (United States)

    Lin, Jyh Ming; Prakasha Gowda, A S; Sharma, Arun K; Amin, Shantu

    2012-05-15

    Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells.

  10. 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.

  11. The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during the postnatal development in mice hippocampus area CA1

    Directory of Open Access Journals (Sweden)

    Wang Huili

    2007-05-01

    Full Text Available Abstract Background Cell-cycle-related proteins, such as cyclins or cyclin-dependent kinases, may have functions beyond that of cell cycle regulation. The expression and translocation of cyclinD1-CDK4 in post-mitotic neurons indicate that they may have supplementary functions in differentiated neurons that might be associated with neuronal plasticity. Results In the present study, our findings showed that the expression of CDK4 was localized mostly in nuclei and cytoplasm of pyramidal cells of CA1 at postnatal day 10 (P10; whereas at P28 staining of CDK4 could be detected predominantly in the cytoplasm but not nuclei. Basal synaptic transmission was normal in the presence of CDK4 inhibitor. Short-term synaptic plasticity (STP was impaired in CDK4 inhibitor pre-treated slices both from neonatal (P8-15 and adolescent (P21-35 animals; however there was no significant change in paired-pulse facilitation (PPF in slices pre-incubated with the CDK4 inhibitor from adolescent animals. By the treatment of CDK4 inhibitor, the induction or the maintenance of Long-term potentiation (LTP in response to a strong tetanus and NMDA receptor-dependent long-term depression (LTD were normal in hippocampus. However, long-term depression (LTD induced either by group I metabotropic glutamate receptors (mGluRs agonist or by paired-pulse low-frequency stimulation (PP-LFS was impaired in CDK4 inhibitor pretreated slices both from neonatal and adolescent animals. But the effects of the CDK4 inhibitor at slices from adolescent animals were not as robust as at slices from neonatal animals. Conclusion Our results indicated that the activation of cyclinD1-CDK4 is required for short-term synaptic plasticity and mGluR-dependent LTD, and suggested that this cyclin-dependent kinase may have different roles during the postnatal development in mice hippocampus area CA1.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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, ...

  17. 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.

  18. 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

  19. 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.

  20. 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.