Hippocampal glycogen synthase kinase 3β is critical for the antidepressant effect of cyclin-dependent kinase 5 inhibitor in rats.

PubMed

Li, Gang; Liu, Ting; Kong, Xiangqian; Wang, Lei; Jin, Xing

2014-09-01

Cdk5 is a member of cyclin-dependent kinase (Cdk), a proline-directed serine/threonine kinase, and plays a key role in normal neural development and function. Evidence of previous study showed that chronic inhibition of Cdk5 in hippocampal dentate gyrus (DG) blocked the development of depressive-like symptoms, suggesting that Cdk5 plays a role in development of depression. Forced swim test, novelty-suppressed feeding test, and learned helplessness were used to evaluate the cellular and molecular mechanisms underlying the behavioral regulation of Cdk5 inhibitors in rats. Two Cdk5 inhibitors butyrolactone and roscovitine were used to investigate the possible antidepressant-like actions of Cdk5 blockade and the potential mechanisms. Systemic administration of butyrolactone (200 mg/kg, IP) or roscovitine (100 mg/kg, IP) produced effective antidepressant-like actions. Moreover, infusion (5 mM) of GSK3β activator LY294002 into DG abolished the antidepressant-like actions of butyrolactone and roscovitine, suggesting that inhibition of GSK3β might be involved in the antidepressant effect of Cdk5 inhibitors. Moreover, pretreatment of LY294002 (5 mM) blocked the antidepressant-like effect of butyrolactone and roscovitine in learned helplessness. Additionally, inescapable footshock induced a significant increase of GSK3β activity, while butyrolactone and roscovitine decreased GSK3β activity. In contrast, pretreatment of LY294002 prevented the inhibitory effects of butyrolactone and roscovitine on GSK3β activation. Finally, a specific GSK3β inhibitor, SB216763 (1 ng, DG), demonstrated an effective antidepressant-like action. These findings demonstrate that systemic administration of Cdk5 inhibitors produced antidepressant-like actions and that inhibition of GSK3β is involved in behavioral response of Cdk5 inhibitors.

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

PubMed

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

PubMed Central

Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

2017-01-01

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

Glioblastoma adaptation traced through decline of an IDH1 clonal driver and macro-evolution of a double-minute chromosome.

PubMed

Favero, F; McGranahan, N; Salm, M; Birkbak, N J; Sanborn, J Z; Benz, S C; Becq, J; Peden, J F; Kingsbury, Z; Grocok, R J; Humphray, S; Bentley, D; Spencer-Dene, B; Gutteridge, A; Brada, M; Roger, S; Dietrich, P-Y; Forshew, T; Gerlinger, M; Rowan, A; Stamp, G; Eklund, A C; Szallasi, Z; Swanton, C

2015-05-01

Glioblastoma (GBM) is the most common malignant brain cancer occurring in adults, and is associated with dismal outcome and few therapeutic options. GBM has been shown to predominantly disrupt three core pathways through somatic aberrations, rendering it ideal for precision medicine approaches. We describe a 35-year-old female patient with recurrent GBM following surgical removal of the primary tumour, adjuvant treatment with temozolomide and a 3-year disease-free period. Rapid whole-genome sequencing (WGS) of three separate tumour regions at recurrence was carried out and interpreted relative to WGS of two regions of the primary tumour. We found extensive mutational and copy-number heterogeneity within the primary tumour. We identified a TP53 mutation and two focal amplifications involving PDGFRA, KIT and CDK4, on chromosomes 4 and 12. A clonal IDH1 R132H mutation in the primary, a known GBM driver event, was detectable at only very low frequency in the recurrent tumour. After sub-clonal diversification, evidence was found for a whole-genome doubling event and a translocation between the amplified regions of PDGFRA, KIT and CDK4, encoded within a double-minute chromosome also incorporating miR26a-2. The WGS analysis uncovered progressive evolution of the double-minute chromosome converging on the KIT/PDGFRA/PI3K/mTOR axis, superseding the IDH1 mutation in dominance in a mutually exclusive manner at recurrence, consequently the patient was treated with imatinib. Despite rapid sequencing and cancer genome-guided therapy against amplified oncogenes, the disease progressed, and the patient died shortly after. This case sheds light on the dynamic evolution of a GBM tumour, defining the origins of the lethal sub-clone, the macro-evolutionary genomic events dominating the disease at recurrence and the loss of a clonal driver. Even in the era of rapid WGS analysis, cases such as this illustrate the significant hurdles for precision medicine success. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Cyclin-dependent kinase 2 protects podocytes from apoptosis

PubMed Central

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

2016-01-01

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

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

PubMed

Dixon-Clarke, Sarah E; Shehata, Saifeldin N; Krojer, Tobias; Sharpe, Timothy D; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

2017-02-20

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. © 2017 The Author(s).

Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability.

PubMed

Benavides, David R; Quinn, Jennifer J; Zhong, Ping; Hawasli, Ammar H; DiLeone, Ralph J; Kansy, Janice W; Olausson, Peter; Yan, Zhen; Taylor, Jane R; Bibb, James A

2007-11-21

Cyclin-dependent kinase 5 (Cdk5) regulates dopamine neurotransmission and has been suggested to serve as a homeostatic target of chronic psychostimulant exposure. To study the role of Cdk5 in the modulation of the cellular and behavioral effects of psychoactive drugs of abuse, we developed Cre/loxP conditional knock-out systems that allow temporal and spatial control of Cdk5 expression in the adult brain. Here, we report the generation of Cdk5 conditional knock-out (cKO) mice using the alphaCaMKII promoter-driven Cre transgenic line (CaMKII-Cre). In this model system, loss of Cdk5 in the adult forebrain increased the psychomotor-activating effects of cocaine. Additionally, these CaMKII-Cre Cdk5 cKO mice show enhanced incentive motivation for food as assessed by instrumental responding on a progressive ratio schedule of reinforcement. Behavioral changes were accompanied by increased excitability of medium spiny neurons in the nucleus accumbens (NAc) in Cdk5 cKO mice. To study NAc-specific effects of Cdk5, another model system was used in which recombinant adeno-associated viruses expressing Cre recombinase caused restricted loss of Cdk5 in NAc neurons. Targeted knock-out of Cdk5 in the NAc facilitated cocaine-induced locomotor sensitization and conditioned place preference for cocaine. These results suggest that Cdk5 acts as a negative regulator of neuronal excitability in the NAc and that Cdk5 may govern the behavioral effects of cocaine and motivation for reinforcement.

Anticancer effect of cucurbitacin B on MKN-45 cells via inhibition of the JAK2/STAT3 signaling pathway

PubMed Central

Xie, You-Li; Tao, Wen-Hui; Yang, Ti-Xiong; Qiao, Jian-Guo

2016-01-01

The aim of the present study was to investigate the effect of cucurbitacin B on MKN-45 gastric carcinoma cells. Cell proliferation was determined using a cell counting kit-8 assay, and commercial cell cycle and apoptosis analysis kits were used to determine the cell cycle by flow cytometry. The mRNA expression of genes which mediate cell cycle checkpoints and apoptosis was detected using reverse transcription-quantitative polymerase chain reaction, and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine apoptosis rate. Western blot analysis was used to detect the protein expression levels of JAK2/STAT3 signaling pathway-associated proteins. The presented data show that cucurbitacin B significantly inhibited the proliferation of MKN-45 cells in a dose- and time-dependent manner. In accordance with these findings, cucurbitacin B blocked the progression of the cell cycle from G0/G1 to S phase, which was confirmed by the mRNA expression analysis. Cucurbitacin B treatment significantly suppressed the expression of cyclin D1, cyclin E, cyclin-dependent kinase 4 (CDK4) and CDK2, while increasing the expression of p27. Cucurbitacin B also promoted cell apoptosis, as was determined by TUNEL assay and evaluation of mRNA expression. Further experiments suggested that the beneficial effect of cucurbitacin B on blocking the proliferation and inducing the apoptosis of MKN-45 cells may have been associated with suppression of the JAK2/STAT3 signaling pathway. Thus, the present results indicate that cucurbitacin B suppresses proliferation and promoted apoptosis of MKN-45 cells, which may be mediated by inhibition of the JAK2/STAT3 signaling pathway. Cucurbitacin B therefore may warrant further investigation as a feasible therapy for gastric carcinoma. PMID:27698776

Pan-Cancer Analysis of the Mediator Complex Transcriptome Identifies CDK19 and CDK8 as Therapeutic Targets in Advanced Prostate Cancer.

PubMed

Brägelmann, Johannes; Klümper, Niklas; Offermann, Anne; von Mässenhausen, Anne; Böhm, Diana; Deng, Mario; Queisser, Angela; Sanders, Christine; Syring, Isabella; Merseburger, Axel S; Vogel, Wenzel; Sievers, Elisabeth; Vlasic, Ignacija; Carlsson, Jessica; Andrén, Ove; Brossart, Peter; Duensing, Stefan; Svensson, Maria A; Shaikhibrahim, Zaki; Kirfel, Jutta; Perner, Sven

2017-04-01

Purpose: The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches. Experimental Design: We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities ( n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer-specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer ( n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq. Results: Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type-specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19 In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro , inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion. Conclusions: Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829-40. ©2016 AACR . ©2016 American Association for Cancer Research.

Collective synchronization of divisions in Drosophila development

NASA Astrophysics Data System (ADS)

Vergassola, Massimo

Mitoses in the early development of most metazoans are rapid and synchronized across the entire embryo. While diffusion is too slow, in vitro experiments have shown that waves of the cell-cycle regulator Cdk1 can transfer information rapidly across hundreds of microns. However, the signaling dynamics and the physical properties of chemical waves during embryonic development remain unclear. We develop FRET biosensors for the activity of Cdk1 and the checkpoint kinase Chk1 in Drosophila embryos and exploit them to measure waves in vivo. We demonstrate that Cdk1 chemical waves control mitotic waves and that their speed is regulated by the activity of Cdk1 during the S-phase (and not mitosis). We quantify the progressive slowdown of the waves with developmental cycles and identify its underlying control mechanism by the DNA replication checkpoint through the Chk1/Wee1 pathway. The global dynamics of the mitotic signaling network illustrates a novel control principle: the S-phase activity of Cdk1 regulates the speed of the mitotic wave, while the Cdk1 positive feedback ensures an invariantly rapid onset of mitosis. Mathematical modeling captures the speed of the waves and predicts a fundamental distinction between the S-phase Cdk1 trigger waves and the mitotic phase waves, which is illustrated by embryonic ablation experiments. In collaboration with Victoria Deneke1, Anna Melbinger2, and Stefano Di Talia1 1 Department of Cell Biology, Duke University Medical Center 2 Department of Physics, University of California San Diego.

Cooperation of p27Kip1 and p18INK4c in Progestin-Mediated Cell Cycle Arrest in T-47D Breast Cancer Cells

PubMed Central

Swarbrick, Alexander; Lee, Christine S. L.; Sutherland, Robert L.; Musgrove, Elizabeth A.

2000-01-01

The steroid hormone progesterone regulates proliferation and differentiation in the mammary gland and uterus by cell cycle phase-specific actions. The long-term effect of progestins on T-47D breast cancer cells is inhibition of cellular proliferation. This is accompanied by decreased G1 cyclin-dependent kinase (CDK) activities, redistribution of the CDK inhibitor p27Kip1 among these CDK complexes, and alterations in the elution profile of cyclin E-Cdk2 upon gel filtration chromatography, such that high-molecular-weight complexes predominate. This study aimed to determine the relative contribution of CDK inhibitors to these events. Following progestin treatment, the majority of cyclin E- and D-CDK complexes were bound to p27Kip1 and few were bound to p21Cip1. In vitro, recombinant His6-p27 could quantitatively reproduce the effects on cyclin E-Cdk2 kinase activity and the shift in molecular weight observed following progestin treatment. In contrast, cyclin D-Cdk4 was not inhibited by His6-p27 in vitro or p27Kip1 in vivo. However, an increase in the expression of the Cdk4/6 inhibitor p18INK4c and its extensive association with Cdk4 and Cdk6 were apparent following progestin treatment. Recombinant p18INK4c led to the reassortment of cyclin-CDK-CDK inhibitor complexes in vitro, with consequent decrease in cyclin E-Cdk2 activity. These results suggest a concerted model of progestin action whereby p27Kip1 and p18INK4c cooperate to inhibit cyclin E-Cdk2 and Cdk4. Since similar models have been developed for growth inhibition by transforming growth factor β and during adipogenesis, interaction between the Cip/Kip and INK4 families of inhibitors may be a common theme in physiological growth arrest and differentiation. PMID:10713180

Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle–coupled loss of IRF4

PubMed Central

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L.; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C.; Staudt, Louis M.; Niesvizky, Ruben; Moore, Malcolm A. S.

2012-01-01

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy. PMID:22718837

Prolonged early G(1) arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4.

PubMed

Huang, Xiangao; Di Liberto, Maurizio; Jayabalan, David; Liang, Jun; Ely, Scott; Bretz, Jamieson; Shaffer, Arthur L; Louie, Tracey; Chen, Isan; Randolph, Sophia; Hahn, William C; Staudt, Louis M; Niesvizky, Ruben; Moore, Malcolm A S; Chen-Kiang, Selina

2012-08-02

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

Cyclin-dependent kinase 11p110 (CDK11p110) is crucial for human breast cancer cell proliferation and growth

PubMed Central

Zhou, Yubing; Han, Chao; Li, Duolu; Yu, Zujiang; Li, Fengmei; Li, Feng; An, Qi; Bai, Huili; Zhang, Xiaojian; Duan, Zhenfeng; Kan, Quancheng

2015-01-01

Cyclin-dependent kinases (CDKs) play important roles in the development of many types of cancers by binding with their paired cyclins. However, the function of CDK11 larger protein isomer, CDK11p110, in the tumorigenesis of human breast cancer remains unclear. In the present study, we explored the effects and molecular mechanisms of CDK11p110 in the proliferation and growth of breast cancer cells by determining the expression of CDK11p110 in breast tumor tissues and examining the phenotypic changes of breast cancer cells after CDK11p110 knockdown. We found that CDK11p110 was highly expressed in breast tumor tissues and cell lines. Tissue microarray analysis showed that elevated CDK11p110 expression in breast cancer tissues significantly correlated with poor differentiation, and was also associated with advanced TNM stage and poor clinical prognosis for breast cancer patients. In vitro knockdown of CDK11p110 by siRNA significantly inhibited cell growth and migration, and dramatically induced apoptosis in breast cancer cells. Flow cytometry demonstrated that cells were markedly arrested in G1 phase of the cell cycle after CDK11p110 downregulation. These findings suggest that CDK11p110 is critical for the proliferation and growth of breast cancer cells, which highlights CDK11p110 may be a promising therapeutic target for the treatment of breast cancer. PMID:25990212

A novel pyrazolo[1,5-a]pyrimidine is a potent inhibitor of cyclin-dependent protein kinases 1, 2, and 9, which demonstrates antitumor effects in human tumor xenografts following oral administration.

PubMed

Heathcote, Dean A; Patel, Hetal; Kroll, Sebastian H B; Hazel, Pascale; Periyasamy, Manikandan; Alikian, Mary; Kanneganti, Seshu K; Jogalekar, Ashutosh S; Scheiper, Bodo; Barbazanges, Marion; Blum, Andreas; Brackow, Jan; Siwicka, Alekasandra; Pace, Robert D M; Fuchter, Matthew J; Snyder, James P; Liotta, Dennis C; Freemont, Paul S; Aboagye, Eric O; Coombes, R Charles; Barrett, Anthony G M; Ali, Simak

2010-12-23

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Open Source Bayesian Models. 1. Application to ADME/Tox and Drug Discovery Datasets.

PubMed

Clark, Alex M; Dole, Krishna; Coulon-Spektor, Anna; McNutt, Andrew; Grass, George; Freundlich, Joel S; Reynolds, Robert C; Ekins, Sean

2015-06-22

On the order of hundreds of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) models have been described in the literature in the past decade which are more often than not inaccessible to anyone but their authors. Public accessibility is also an issue with computational models for bioactivity, and the ability to share such models still remains a major challenge limiting drug discovery. We describe the creation of a reference implementation of a Bayesian model-building software module, which we have released as an open source component that is now included in the Chemistry Development Kit (CDK) project, as well as implemented in the CDD Vault and in several mobile apps. We use this implementation to build an array of Bayesian models for ADME/Tox, in vitro and in vivo bioactivity, and other physicochemical properties. We show that these models possess cross-validation receiver operator curve values comparable to those generated previously in prior publications using alternative tools. We have now described how the implementation of Bayesian models with FCFP6 descriptors generated in the CDD Vault enables the rapid production of robust machine learning models from public data or the user's own datasets. The current study sets the stage for generating models in proprietary software (such as CDD) and exporting these models in a format that could be run in open source software using CDK components. This work also demonstrates that we can enable biocomputation across distributed private or public datasets to enhance drug discovery.

Open Source Bayesian Models. 1. Application to ADME/Tox and Drug Discovery Datasets

PubMed Central

2015-01-01

On the order of hundreds of absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox) models have been described in the literature in the past decade which are more often than not inaccessible to anyone but their authors. Public accessibility is also an issue with computational models for bioactivity, and the ability to share such models still remains a major challenge limiting drug discovery. We describe the creation of a reference implementation of a Bayesian model-building software module, which we have released as an open source component that is now included in the Chemistry Development Kit (CDK) project, as well as implemented in the CDD Vault and in several mobile apps. We use this implementation to build an array of Bayesian models for ADME/Tox, in vitro and in vivo bioactivity, and other physicochemical properties. We show that these models possess cross-validation receiver operator curve values comparable to those generated previously in prior publications using alternative tools. We have now described how the implementation of Bayesian models with FCFP6 descriptors generated in the CDD Vault enables the rapid production of robust machine learning models from public data or the user’s own datasets. The current study sets the stage for generating models in proprietary software (such as CDD) and exporting these models in a format that could be run in open source software using CDK components. This work also demonstrates that we can enable biocomputation across distributed private or public datasets to enhance drug discovery. PMID:25994950

Osteosarcoma cells induce endothelial cell proliferation during neo-angiogenesis.

PubMed

de Nigris, Filomena; Mancini, Francesco Paolo; Schiano, Concetta; Infante, Teresa; Zullo, Alberto; Minucci, Pellegrino Biagio; Al-Omran, Mohammed; Giordano, Antonio; Napoli, Claudio

2013-04-01

Understanding the mechanisms inducing endothelial cell (EC) proliferation following tumor microenvironment stimuli may be important for the development of antiangiogenic therapies. Here, we show that cyclin-dependent kinase 2 and 5 (Cdk2, Cdk5) are important mediators of neoangiogenesis in in vitro and in vivo systems. Furthermore, we demonstrate that a specific Yin Yang 1 (YY1) protein-dependent signal from osteosarcoma (SaOS) cells determines proliferation of human aortic endothelial cells (HAECs). Following tumor cell stimuli, HAECs overexpress Cdk2 and Cdk5, display increased Cdk2 activity, undergo enhanced proliferation, and form capillary-like structures. Moreover, Roscovitine, an inhibitor of Cdks, blunted overexpression of Cdk2 and Cdk5 and Cdk2 activity induced by the YY1-dependent signal secreted by SaOS cells. Furthermore, Roscovitine decreased HAEC proliferation and angiogenesis (the latter by 70% in in vitro and 50% in in vivo systems; P < 0.01 vs. control). Finally, the finding that Roscovitine triggers apoptosis in SaOS cells as well as in HAECs by activating caspase-3/7 indicates multiple mechanisms for the potential antitumoral effect of Roscovitine. Present work suggests that Cdk2 and Cdk5 might be pharmacologically accessible targets for both antiangiogenic and antitumor therapy. Copyright © 2012 Wiley Periodicals, Inc.

CDK6 inhibits white to beige fat transition by suppressing RUNX1

USDA-ARS?s Scientific Manuscript database

Whereas white adipose tissue depots contribute to the development of metabolic diseases, brown and beige adipose tissue has beneficial metabolic effects. Here we show that CDK6 regulates beige adipocyte formation. We demonstrate that mice lacking the CDK6 protein or its kinase domain (K43M) exhibit ...

CYCLIN-DEPENDENT KINASE8 differentially regulates plant immunity to fungal pathogens through kinase-dependent and -independent functions in Arabidopsis.

PubMed

Zhu, Yingfang; Schluttenhoffer, Craig M; Wang, Pengcheng; Fu, Fuyou; Thimmapuram, Jyothi; Zhu, Jian-Kang; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

2014-10-01

CYCLIN-DEPENDENT KINASE8 (CDK8) is a widely studied component of eukaryotic Mediator complexes. However, the biological and molecular functions of plant CDK8 are not well understood. Here, we provide evidence for regulatory functions of Arabidopsis thaliana CDK8 in defense and demonstrate its functional and molecular interactions with other Mediator and non-Mediator subunits. The cdk8 mutant exhibits enhanced resistance to Botrytis cinerea but susceptibility to Alternaria brassicicola. The contributions of CDK8 to the transcriptional activation of defensin gene PDF1.2 and its interaction with MEDIATOR COMPLEX SUBUNIT25 (MED25) implicate CDK8 in jasmonate-mediated defense. Moreover, CDK8 associates with the promoter of AGMATINE COUMAROYLTRANSFERASE to promote its transcription and regulate the biosynthesis of the defense-active secondary metabolites hydroxycinnamic acid amides. CDK8 also interacts with the transcription factor WAX INDUCER1, implying its additional role in cuticle development. In addition, overlapping functions of CDK8 with MED12 and MED13 and interactions between CDK8 and C-type cyclins suggest the conserved configuration of the plant Mediator kinase module. In summary, while CDK8's positive transcriptional regulation of target genes and its phosphorylation activities underpin its defense functions, the impaired defense responses in the mutant are masked by its altered cuticle, resulting in specific resistance to B. cinerea. © 2014 American Society of Plant Biologists. All rights reserved.

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.

PubMed

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 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Strange Case of CDK4/6 Inhibitors: Mechanisms, Resistance, and Combination Strategies

PubMed Central

Knudsen, Erik S.; Witkiewicz, Agnieszka K.

2016-01-01

CDK4/6 inhibitors have emerged as a powerful class of agents with clinical activity in a number of malignancies. Targeting the cell cycle represents a core attack on a defining feature of cancer. However, the mechanisms through which selective CDK4/6 targeted agents act has few parallels in the current pharmaceutical armamentarium against cancer. Notably, CDK4/6 inhibitors act downstream of most mitogenic signaling cascades, which have implications both related to clinical efficacy and resistance. Core knowledge of cell cycle processes has provided insights into mechanisms of intrinsic resistance to CDK4/6 inhibitors; however, the basis of acquired resistance versus durable response is only beginning to emerge. This review focuses on the mechanism of action and biomarkers to direct the precision use of CDK4/6 inhibitors and rationally-developed combination therapies. PMID:28303264

Cell Cycle Regulators during Human Atrial Development

PubMed Central

Kim, Won Ho; Joo, Chan Uhng; Ku, Ja Hong; Ryu, Chul Hee; Koh, Keum Nim; Koh, Gou Young; Ko, Jae Ki

1998-01-01

Objectives The molecular mechanisms that regulate cardiomyocyte cell cycle and terminal differentiation in humans remain largely unknown. To determine which cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) are important for cardiomyocyte proliferation, we have examined protein levels of cyclins, CDKs and CKIs during normal atrial development in humans. Methods Atrial tissues were obtained in the fetus from inevitable abortion and in the adult during surgery, Cyclin and CDK proteins were determined by Western blot analysis, CDK activities were determined by phosphorylation amount using specific substrate. Results Most cyclins and CDKs were high during the fetal period and their levels decreased at different rates during the adult period. While the protein levels of cyclin D1, cyclin D3, CDK4, CDK6 and CDK2 were still detectable in adult atria, the protein levels of cyclin E, cyclin A, cyclin B, cdc2 and PCNA were not detectable. Interestingly, p27KIP 1 protein increased markedly in the adult period, while p21C IP 1 protein in atria was detectable only in the fetal period. While the activities of CDK6, CDK2 and cdc2 decreased markedly, the activity of CDK4 did not change from the fetal period to the adult period. Conclusion These findings indicate that marked reduction of protein levels and activities of cyclins and CDKs, and marked induction of p27KIP 1 in atria, are associated with the withdrawal of cardiac cell cycle in adult humans. PMID:9735660

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

PubMed

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

2010-12-17

An alternative strategy for inhibition of the cyclin dependent kinases (CDKs) in antitumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation, and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential to generate highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anticancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27(KIP)¹, with cyclin D1. This information has been used to shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 that can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and nonpeptidic compounds have been synthesized in order to explore structure-activity relationship for binding to the cyclin D1 groove, which to date has not been carried out in a systematic fashion. Collectively, the data presented provide new insights into how compounds can be developed that function as chemical biology probes to determine the cellular and antitumor effects of CDK inhibition. Furthermore, such compounds will serve as templates for structure-guided efforts to develop potential therapeutics based on selective inhibition of CDK4/cyclin D activity.

Expression of CDK6 in Oral Squamous Cell Carcinomas

PubMed

Andisheh-Tadbir, Azadeh; Ashraf, Mohammad Javad; Jeiroodi, Naghmeh

2018-04-25

Background: CDK6 is the key factor in regulation of the cell cycle and essential for passage into the G1 phase. It also plays an important role in the development of various tumors. In this cross-sectional study expression of the CDK6 protein in oral squamous cell carcinoma (OSCC) and healthy oral mucosa of controls was assessed to determine relations with malignant transformation and clinicopathologic factors. Method: A total of 60 samples, 45 from OSCCs and 15 from healthy tissue, underwent immunohistochemistry for CDK6. Nuclear and cytoplasmic staining of keratinocytes was considered as positive and the percentages of positive cells were calculated. Results: Expression of CDK6 was detected in 55.6% of OSCC samples (25 cases) and 13.3% of controls (2 cases), the difference being significant. Mean percentage of CDK6 stained cells was 24.2±29.3 in the OSCC cases and 4.33±2.1 in the control group, again statistically significant. No relationship was detected between CDK6 expression and clinicopathologic factors. Conclusion: Overexpression of CDK6 observed in OSCC points to a role for this protein in oral carcinogenesis. Creative Commons Attribution License

Specific activity of cyclin-dependent kinase I is a new potential predictor of tumour recurrence in stage II colon cancer.

PubMed

Zeestraten, E C M; Maak, M; Shibayama, M; Schuster, T; Nitsche, U; Matsushima, T; Nakayama, S; Gohda, K; Friess, H; van de Velde, C J H; Ishihara, H; Rosenberg, R; Kuppen, P J K; Janssen, K-P

2012-01-03

There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer. In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples. Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55-0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ≥11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44-26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours. Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer.

Cdk5 phosphorylation of WAVE2 regulates oligodendrocyte precursor cell migration through nonreceptor tyrosine kinase Fyn.

PubMed

Miyamoto, Yuki; Yamauchi, Junji; Tanoue, Akito

2008-08-13

Myelin formation of the CNS is a complex and dynamic process. Before the onset of myelination, oligodendrocytes (OLs), the myelin-forming glia of the CNS, proliferate and migrate along axons. Little is known about the molecular mechanisms underlying the early myelination processes. Here, we show that platelet-derived growth factor (PDGF), the crucial physiological ligand in early OL development, controls the migration of oligodendrocyte precursor cells (OPCs) through cyclin-dependent kinase 5 (Cdk5). PDGF stimulates Cdk5 activity in a time-dependent manner, whereas suppression of Cdk5 by the specific inhibitor roscovitine or by the retrovirus encoding short-hairpin RNA for Cdk5 impairs PDGF-dependent OPC migration. The activation of Cdk5 by PDGF is mediated by the phosphorylation of the nonreceptor tyrosine kinase, Fyn, whose inhibition reduces PDGF-dependent OPC migration. Furthermore, Cdk5 regulates PDGF-dependent OPC migration through the direct phosphorylation of WASP (Wiskott-Aldrich syndrome protein)-family verprolin-homologous protein 2 (WAVE2). Cdk5 phosphorylates WAVE2 at Ser-137 in vitro. Infection of the WAVE2 construct harboring the Ser-137-to-Ala reduces PDGF-dependent migration. Together, PDGF regulates OPC migration through an as-yet-unidentified signaling cascade coupling Fyn kinase to Cdk5 phosphorylation of WAVE2. These results provide new insights into both the role of Cdk5 in glial cells and the molecular mechanisms controlling the early developmental stage of OLs.

Overexpression of p35 in Min6 pancreatic beta cells induces a stressed neuron-like apoptosis.

PubMed

Zheng, Ya-Li; Hu, Ya-Fang; Zhang, Aiping; Wang, Wei; Li, Bo; Amin, Niranjana; Grant, Philip; Pant, Harish C

2010-12-15

Cdk5 activity has been implicated in brain development and the regulation of many neuronal processes. Recently, the expression of p35 and Cdk5 activity has been reported in pancreatic beta cells. Decreased Cdk5 activity enhanced glucose-stimulated insulin secretion. This suggests that Cdk5 may play an important role in the regulation of insulin secretion. To further understand how Cdk5 regulates insulin secretion in glucose-stimulated pancreatic β cells, we first confirmed the presence of a low level of p35 in pancreatic Min6 cells. Next, in a time-course experiment in high glucose (25 mM) we showed that endogenous p35 increased gradually accompanied by a 3-fold increase in Cdk5 activity by 16 h. Insulin secretion, however, doubled after 2 h followed by progressive downregulation, negatively correlated with Cdk5 activity. On the other hand, overexpression of p35 in these cells resulted in more than a three-fold increase in Cdk5 activity within 2 h coupled to a 50% reduction in insulin secretion in both high and low (3 mM) glucose. Most significantly, cells overexpressing p35, treated with high glucose for 4 h, showed induction of p25, the p35-derived truncated fragment which hyperactivates Cdk5 in neurons. As a result, insulin secretion was inhibited and cells became apoptotic. Roscovitine or co-infection of dominant negative Cdk5 (dnCdk5) with p35 increased insulin secretion and inhibited apoptosis. These results suggest that the model for deregulation and hyperactivation of Cdk5 in neurodegeneration may apply to the pathology seen in type 2 diabetes (T2DM). It is consistent with the view that Alzheimer's disease and T2DM are linked metabolically and pathologically by Cdk5 in a number of ways. Copyright © 2010. Published by Elsevier B.V.

Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors.

PubMed

Németh, Gábor; Greff, Zoltán; Sipos, Anna; Varga, Zoltán; Székely, Rita; Sebestyén, Mónika; Jászay, Zsuzsa; Béni, Szabolcs; Nemes, Zoltán; Pirat, Jean-Luc; Volle, Jean-Noël; Virieux, David; Gyuris, Ágnes; Kelemenics, Katalin; Ay, Eva; Minarovits, Janos; Szathmary, Susan; Kéri, György; Orfi, László

2014-05-22

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

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

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

Zhang, Yue; Li, Hongbo; Hao, Jun

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 notmore » 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 decreased p35 expression in HG-cultured podocytes. • Inhibition of Cdk5 activity alleviated podocyte apoptosis induced by HG or TGF-β1.« less

Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells.

PubMed

Pan, Min-Hsiung; Chen, Wei-Jen; Lin-Shiau, Shoei-Yn; Ho, Chi-Tang; Lin, Jen-Kun

2002-10-01

Tangeretin (5,6,7,8,4'-pentamethoxyflavone) is concentrated in the peel of citrus fruits. DNA flow cytometric analysis indicated that tangeretin blocked cell cycle progression at G1 phase in colorectal carcinoma COLO 205 cells. Over a 24 h exposure to tangeretin, the degree of phosphorylation of Rb was decreased after 12 h and G1 arrest developed. The protein expression of cyclins A, D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that tangeretin inhibited the activities of cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4) in a dose-dependent manner in the cell-free system. As the cells were exposed to tangeretin (50 microM) over 48 h a gradual loss of both Cdk2 and 4 kinase activities occurred. Tangeretin also increased the content of the Cdk inhibitor p21 protein and this effect correlated with the elevation in p53 levels. In addition, tangeretin also increased the level of the Cdk inhibitor p27 protein within 18 h. These results suggest that tangeretin either exerts its growth-inhibitory effects through modulation of the activities of several key G1 regulatory proteins, such as Cdk2 and Cdk4, or mediates the increase of Cdk inhibitors p21 and p27.

Requirement for CDK6 in MLL-rearranged acute myeloid leukemia

PubMed Central

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

2014-01-01

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

Pharmacological targeting of CDK9 in cardiac hypertrophy.

PubMed

Krystof, Vladimír; Chamrád, Ivo; Jorda, Radek; Kohoutek, Jirí

2010-07-01

Cardiac hypertrophy allows the heart to adapt to workload, but persistent or unphysiological stimulus can result in pump failure. Cardiac hypertrophy is characterized by an increase in the size of differentiated cardiac myocytes. At the molecular level, growth of cells is linked to intensive transcription and translation. Several cyclin-dependent kinases (CDKs) have been identified as principal regulators of transcription, and among these CDK9 is directly associated with cardiac hypertrophy. CDK9 phosphorylates the C-terminal domain of RNA polymerase II and thus stimulates the elongation phase of transcription. Chronic activation of CDK9 causes not only cardiac myocyte enlargement but also confers predisposition to heart failure. Due to the long interest of molecular oncologists and medicinal chemists in CDKs as potential targets of anticancer drugs, a portfolio of small-molecule inhibitors of CDK9 is available. Recent determination of CDK9's crystal structure now allows the development of selective inhibitors and their further optimization in terms of biochemical potency and selectivity. CDK9 may therefore constitute a novel target for drugs against cardiac hypertrophy.

CDK-5 regulates the polarized trafficking of neuropeptide-containing dense-core vesicles in C. elegans motor neurons

PubMed Central

Goodwin, Patricia R.; Sasaki, Jennifer M.; Juo, Peter

2012-01-01

The polarized trafficking of axonal and dendritic proteins is essential for the structure and function of neurons. Cyclin-dependent kinase-5 (CDK-5) and its activator CDKA-1/p35 regulate diverse aspects of nervous system development and function. Here, we show that CDK-5 and CDKA-1/p35 are required for the polarized distribution of neuropeptide-containing dense-core vesicles (DCVs) in C. elegans cholinergic motor neurons. In cdk-5 or cdka-1/p35 mutants, the predominantly axonal localization of DCVs containing INS-22 neuropeptides was disrupted and DCVs accumulated in dendrites. Time-lapse microscopy in DB class motor neurons revealed decreased trafficking of DCVs in axons and increased trafficking and accumulation of DCVs in cdk-5 mutant dendrites. The polarized distribution of several axonal and dendritic markers, including synaptic vesicles, was unaltered in cdk-5 mutant DB neurons. We found that microtubule polarity is plus-end out in axons and predominantly minus-end out in dendrites of DB neurons. Surprisingly, cdk-5 mutants had increased amounts of plus-end-out microtubules in dendrites, suggesting that CDK-5 regulates microtubule orientation. However, these changes in microtubule polarity are not responsible for the increased trafficking of DCVs into dendrites. Genetic analysis of cdk-5 and the plus-end-directed axonal DCV motor unc-104/KIF1A suggest that increased trafficking of UNC-104 into dendrites cannot explain the dendritic DCV accumulation. Instead, we found that mutations in the minus-end-directed motor cytoplasmic dynein, completely block the increased DCVs observed in cdk-5 mutant dendrites without affecting microtubule polarity. We propose a model where CDK-5 regulates DCV polarity by both promoting DCV trafficking in axons and preventing dynein-dependent DCV trafficking into dendrites. PMID:22699897

Targeting the cyclin D-cyclin-dependent kinase (CDK) 4/6-retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions.

PubMed

Spring, Laura; Bardia, Aditya; Modi, Shanu

2016-01-01

Dysregulation of the cyclin D-cyclin-dependent kinase (CDK) 4/6-INK4-retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively non-selective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicated promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II-III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D-CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combination strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer.

Selective impact of CDK4/6 suppression on patient-derived models of pancreatic cancer.

PubMed

Witkiewicz, Agnieszka K; Borja, Nicholas A; Franco, Jorge; Brody, Jonathan R; Yeo, Charles J; Mansour, John; Choti, Michael A; McCue, Peter; Knudsen, Erik S

2015-06-30

Pancreatic ductal adenocarcinoma (PDA) harbors an exceedingly poor prognosis, and is generally considered a therapy-recalcitrant disease due to poor response to conventional chemotherapy coupled with non-actionable genetic drivers (e.g. KRAS mutations). However, PDA frequently loses p16ink4a, thereby leading to deregulation of CDK4/6. Surprisingly, in established cell models and xenografts, CDK4/6 inhibition has a modest effect on proliferation and resistance develops rapidly. To determine if such weak response was an intrinsic feature of PDA, we developed primary tumor explants that maintain the tumor environment and recapitulate feuture of primary PDA. The CDK4/6 inhibitor PD-0332991 was highly efficient at suppressing proliferation in 14 of the 15 explants. In the single resistant explant, we identified the rare loss of the RB tumor suppressor as the basis for resistance. Patient-derived xenografts (PDXs) were developed in parallel, and unlike the xenografts emerging from established cell lines, the PDXs maintained the histoarchitecture of the primary tumor. These PDXs were highly sensitive to CDK4/6 inhibition, yielding a complete suppression of PDA proliferation. Together, these data indicate that primary PDA is sensitive to CDK4/6 inhibition, that specific biomarkers can delineate intrinsic resistance, and that established cell line models may not represent an adequate means for evaluating therapeutic sensitivities.

Oligodendrocyte-specific loss of Cdk5 disrupts the architecture of nodes of Ranvier as well as learning and memory.

PubMed

Luo, Fucheng; Zhang, Jessie; Burke, Kathryn; Romito-DiGiacomo, Rita R; Miller, Robert H; Yang, Yan

2018-05-02

Myelination of the central nervous system is important for normal motor and sensory neuronal function and recent studies also link it to efficient learning and memory. Cyclin-dependent kinase 5 (Cdk5) is required for normal oligodendrocyte development, myelination and myelin repair. Here we show that conditional deletion of Cdk5 by targeting with CNP (CNP;Cdk5 CKO) results in hypomyelination and disruption of the structural integrity of Nodes of Ranvier. In addition, CNP;Cdk5 CKO mice exhibited a severe impairment of learning and memory compared to controls that may reflect perturbed neuron-glial interactions. Co-culture of cortical neurons with CNP;Cdk5 CKO oligodendrocyte lineage cells resulted in a significant reduction in the density of neuronal dendritic spines. In short term fear-conditioning studies, CNP;Cdk5 CKO mice had decreased hippocampal levels of immediate early genes such as Arc and Fos, and lower levels of p-CREB and p-cofilin suggested these pathways are affected by the levels of myelination. The novel roles of Cdk5 in oligodendrocyte lineage cells may provide insights for helping understand the cognitive changes sometimes seen in demyelinating diseases such as multiple sclerosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Cyclin-dependent kinase 5 mediates adult OPC maturation and myelin repair through modulation of Akt and GsK-3β signaling.

PubMed

Luo, FuCheng; Burke, Kathryn; Kantor, Christopher; Miller, Robert H; Yang, Yan

2014-07-30

Failure of remyelination in diseases, such as multiple sclerosis (MS), leads to permanent axonal damage and irreversible functional loss. The mechanisms controlling remyelination are currently poorly understood. Recent studies implicate the cyclin-dependent kinase 5 (Cdk5) in regulating oligodendrocyte (OL) development and myelination in CNS. In this study, we show that Cdk5 is also an important regulator of remyelination. Pharmacological inhibition of Cdk5 inhibits repair of lysolecithin lesions. This inhibition is a consequence of Cdk5 disruption in neural cells because remyelination in slice cultures is blocked by Cdk5 inhibitors, whereas specific deletion of Cdk5 in OLs inhibits myelin repair. In CNP-Cre;Cdk5(fl/fl) conditional knock-out mouse (Cdk5 cKO), myelin repair was delayed significantly in response to focal demyelinating lesions compared with wild-type animals. The lack of myelin repair was reflected in decreased expression of MBP and proteolipid protein and a reduction in the total number of myelinated axons in the lesion. The number of CC1(+) cells in the lesion sites was significantly reduced in Cdk5 cKO compared with wild-type animals although the total number of oligodendrocyte lineage cells (Olig2(+) cells) was increased, suggesting that Cdk5 loss perturbs the transition of early OL lineage cell into mature OL and subsequent remyelination. The failure of remyelination in Cdk5 cKO animals was associated with a reduction in signaling through the Akt pathway and an enhancement of Gsk-3β signaling pathways. Together, these data suggest that Cdk5 is critical in regulating the transition of adult oligodendrocyte precursor cells to mature OLs that is essential for myelin repair in adult CNS. Copyright © 2014 the authors 0270-6474/14/3410415-15$15.00/0.

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

NASA Astrophysics Data System (ADS)

Cholko, Timothy; Chen, Wei; Tang, Zhiye; Chang, Chia-en A.

2018-05-01

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein-ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein-ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

PubMed

Strausfeld, U P; Howell, M; Descombes, P; Chevalier, S; Rempel, R E; Adamczewski, J; Maller, J L; Hunt, T; Blow, J J

1996-06-01

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Design, synthesis and biological evaluation of tetrahydronaphthyridine derivatives as bioavailable CDK4/6 inhibitors for cancer therapy.

PubMed

Zha, Chuantao; Deng, Wenjia; Fu, Yan; Tang, Shuai; Lan, Xiaojing; Ye, Yan; Su, Yi; Jiang, Lei; Chen, Yi; Huang, Ying; Ding, Jian; Geng, Meiyu; Huang, Min; Wan, Huixin

2018-03-25

CDK4/6 pathway is an attractive chemotherapeutic target for antitumor drug discovery and development. Herein, we reported the structure-based design and synthesis of a series of novel tetrahydronaphthyridine analogues as selective CDK4/6 inhibitors. Compound 5 was identified as a hit and then systematically structure optimization study was conducted. These efforts led to compound 28, which exhibited excellent in vitro potencies against CDK4/6 enzymatic activity with high selectivity over CDK1, and against Colo-205 cell growth. The compound demonstrated favorable in vitro metabolic and robust mice pharmacokinetic properties. In Colo-205 xenograft models, compound 28 showed potent tumor growth inhibition with acceptable toxic effects, which could serve as a novel anticancer agent for further preclinical study. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Brain Exposure of Two Selective Dual CDK4 and CDK6 Inhibitors and the Antitumor Activity of CDK4 and CDK6 Inhibition in Combination with Temozolomide in an Intracranial Glioblastoma Xenograft.

PubMed

Raub, Thomas J; Wishart, Graham N; Kulanthaivel, Palaniappan; Staton, Brian A; Ajamie, Rose T; Sawada, Geri A; Gelbert, Lawrence M; Shannon, Harlan E; Sanchez-Martinez, Concepcion; De Dios, Alfonso

2015-09-01

Effective treatments for primary brain tumors and brain metastases represent a major unmet medical need. Targeting the CDK4/CDK6-cyclin D1-Rb-p16/ink4a pathway using a potent CDK4 and CDK6 kinase inhibitor has potential for treating primary central nervous system tumors such as glioblastoma and some peripheral tumors with high incidence of brain metastases. We compared central nervous system exposures of two orally bioavailable CDK4 and CDK6 inhibitors: abemaciclib, which is currently in advanced clinical development, and palbociclib (IBRANCE; Pfizer), which was recently approved by the U.S. Food and Drug Administration. Abemaciclib antitumor activity was assessed in subcutaneous and orthotopic glioma models alone and in combination with standard of care temozolomide (TMZ). Both inhibitors were substrates for xenobiotic efflux transporters P-glycoprotein and breast cancer resistant protein expressed at the blood-brain barrier. Brain Kp,uu values were less than 0.2 after an equimolar intravenous dose indicative of active efflux but were approximately 10-fold greater for abemaciclib than palbociclib. Kp,uu increased 2.8- and 21-fold, respectively, when similarly dosed in P-gp-deficient mice. Abemaciclib had brain area under the curve (0-24 hours) Kp,uu values of 0.03 in mice and 0.11 in rats after a 30 mg/kg p.o. dose. Orally dosed abemaciclib significantly increased survival in a rat orthotopic U87MG xenograft model compared with vehicle-treated animals, and efficacy coincided with a dose-dependent increase in unbound plasma and brain exposures in excess of the CDK4 and CDK6 Ki values. Abemaciclib increased survival time of intracranial U87MG tumor-bearing rats similar to TMZ, and the combination of abemaciclib and TMZ was additive or greater than additive. These data show that abemaciclib crosses the blood-brain barrier and confirm that both CDK4 and CDK6 inhibitors reach unbound brain levels in rodents that are expected to produce enzyme inhibition; however, abemaciclib brain levels are reached more efficiently at presumably lower doses than palbociclib and are potentially on target for a longer period of time. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Targeting the cyclin D–cyclin-dependent kinase (CDK)4/6–retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions

PubMed Central

Spring, Laura; Bardia, Aditya; Modi, Shanu

2017-01-01

Dysregulation of the cyclin D–cyclin-dependent kinase (CDK)4/6–INK4–retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively nonselective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicate promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II–III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D–CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combinations strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer. PMID:26896604

p35 Regulates the CRM1-Dependent Nucleocytoplasmic Shuttling of Nuclear Hormone Receptor Coregulator-Interacting Factor 1 (NIF-1)

PubMed Central

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W. Y.; Li, Zhen; Fu, Amy K. Y.; Ip, Nancy Y.

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators. PMID:25329792

p35 regulates the CRM1-dependent nucleocytoplasmic shuttling of nuclear hormone receptor coregulator-interacting factor 1 (NIF-1).

PubMed

Zhao, Xiao-Su; Fu, Wing-Yu; Chien, Winnie W Y; Li, Zhen; Fu, Amy K Y; Ip, Nancy Y

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase, which plays critical roles in a wide spectrum of neuronal functions including neuronal survival, neurite outgrowth, and synapse development and plasticity. Cdk5 activity is controlled by its specific activators: p35 or p39. While knockout studies reveal that Cdk5/p35 is critical for neuronal migration during early brain development, functions of Cdk5/p35 have been unraveled through the identification of the interacting proteins of p35, most of which are Cdk5/p35 substrates. However, it remains unclear whether p35 can regulate neuronal functions independent of Cdk5 activity. Here, we report that a nuclear protein, nuclear hormone receptor coregulator (NRC)-interacting factor 1 (NIF-1), is a new interacting partner of p35. Interestingly, p35 regulates the functions of NIF-1 independent of Cdk5 activity. NIF-1 was initially discovered as a transcriptional regulator that enhances the transcriptional activity of nuclear hormone receptors. Our results show that p35 interacts with NIF-1 and regulates its nucleocytoplasmic trafficking via the nuclear export pathway. Furthermore, we identified a nuclear export signal on p35; mutation of this site or blockade of the CRM1/exportin-dependent nuclear export pathway resulted in the nuclear accumulation of p35. Intriguingly, blocking the nuclear export of p35 attenuated the nuclear accumulation of NIF-1. These findings reveal a new p35-dependent mechanism in transcriptional regulation that involves the nucleocytoplasmic shuttling of transcription regulators.

FBXL19 recruits CDK-Mediator to CpG islands of developmental genes priming them for activation during lineage commitment

PubMed Central

Dimitrova, Emilia; Nakayama, Manabu; Koseki, Yoko; Konietzny, Rebecca; Kessler, Benedikt M; Koseki, Haruhiko

2018-01-01

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment. PMID:29809150

MD simulation of the Tat/Cyclin T1/CDK9 complex revealing the hidden catalytic cavity within the CDK9 molecule upon Tat binding.

PubMed

Asamitsu, Kaori; Hirokawa, Takatsugu; Okamoto, Takashi

2017-01-01

In this study, we applied molecular dynamics (MD) simulation to analyze the dynamic behavior of the Tat/CycT1/CDK9 tri-molecular complex and revealed the structural changes of P-TEFb upon Tat binding. We found that Tat could deliberately change the local flexibility of CycT1. Although the structural coordinates of the H1 and H2 helices did not substantially change, H1', H2', and H3' exhibited significant changes en masse. Consequently, the CycT1 residues involved in Tat binding, namely Tat-recognition residues (TRRs), lost their flexibility with the addition of Tat to P-TEFb. In addition, we clarified the structural variation of CDK9 in complex with CycT1 in the presence or absence of Tat. Interestingly, Tat addition significantly reduced the structural variability of the T-loop, thus consolidating the structural integrity of P-TEFb. Finally, we deciphered the formation of the hidden catalytic cavity of CDK9 upon Tat binding. MD simulation revealed that the PITALRE signature sequence of CDK9 flips the inactive kinase cavity of CDK9 into the active form by connecting with Thr186, which is crucial for its activity, thus presumably recruiting the substrate peptide such as the C-terminal domain of RNA pol II. These findings provide vital information for the development of effective novel anti-HIV drugs with CDK9 catalytic activity as the target.

Preclinical development of G1T38: A novel, potent and selective inhibitor of cyclin dependent kinases 4/6 for use as an oral antineoplastic in patients with CDK4/6 sensitive tumors

PubMed Central

Jordan, Jamie L.; Darr, David D.; Roberts, Patrick J.; Tavares, Francis X.; Strum, Jay C.

2017-01-01

Inhibition of the p16INK4a/cyclin D/CDK4/6/RB pathway is an effective therapeutic strategy for the treatment of estrogen receptor positive (ER+) breast cancer. Although efficacious, current treatment regimens require a dosing holiday due to severe neutropenia potentially leading to an increased risk of infections, as well as tumor regrowth and emergence of drug resistance. Therefore, a next generation CDK4/6 inhibitor that can inhibit proliferation of CDK4/6-dependent tumors while minimizing neutropenia could reduce both the need for treatment holidays and the risk of inducing drug resistance. Here, we describe the preclinical characterization and development of G1T38; a novel, potent, selective, and orally bioavailable CDK4/6 inhibitor. In vitro, G1T38 decreased RB1 (RB) phosphorylation, caused a precise G1 arrest, and inhibited cell proliferation in a variety of CDK4/6-dependent tumorigenic cell lines including breast, melanoma, leukemia, and lymphoma cells. In vivo, G1T38 treatment led to equivalent or improved tumor efficacy compared to the first-in-class CDK4/6 inhibitor, palbociclib, in an ER+ breast cancer xenograft model. Furthermore, G1T38 accumulated in mouse xenograft tumors but not plasma, resulting in less inhibition of mouse myeloid progenitors than after palbociclib treatment. In larger mammals, this difference in pharmacokinetics allowed for 28 day continuous dosing of G1T38 in beagle dogs without producing severe neutropenia. These data demonstrate G1T38 has unique pharmacokinetic and pharmacodynamic properties, which result in high efficacy against CDK4/6 dependent tumors while minimizing the undesirable on-target bone marrow activity, thus potentially allowing G1T38 to be used as a continuous, daily oral antineoplastic agent. PMID:28418845

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases*

PubMed Central

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

2013-01-01

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

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2

PubMed Central

Pisani, Pasquale; Rastelli, Giulio

2016-01-01

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome. PMID:27100206

Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2.

PubMed

Pisani, Pasquale; Caporuscio, Fabiana; Carlino, Luca; Rastelli, Giulio

2016-01-01

Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer

PubMed Central

Dachineni, Rakesh; Kumar, D. Ramesh; Callegari, Eduardo; Kesharwani, Siddharth S.; Sankaranarayanan, Ranjini; Seefeldt, Teresa; Tummala, Hemachand; Bhat, G. Jayarama

2017-01-01

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxy-benzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment. PMID:29075787

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

PubMed

Davis, S T; Benson, B G; Bramson, H N; Chapman, D E; Dickerson, S H; Dold, K M; Eberwein, D J; Edelstein, M; Frye, S V; Gampe, R T; Griffin, R J; Harris, P A; Hassell, A M; Holmes, W D; Hunter, R N; Knick, V B; Lackey, K; Lovejoy, B; Luzzio, M J; Murray, D; Parker, P; Rocque, W J; Shewchuk, L; Veal, J M; Walker, D H; Kuyper, L F

2001-01-05

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Development of mice without Cip/Kip CDK inhibitors.

PubMed

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu; Hara, Eiji; Nakayama, Keiko; Nakayama, Keiichi I

2012-10-19

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(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 normal development (although it is thought to be a key player in the response to DNA damage). Copyright © 2012 Elsevier Inc. All rights reserved.

Memory Enhancement by Targeting Cdk5 Regulation of NR2B

PubMed Central

Plattner, Florian; Hernandéz, Adan; Kistler, Tara M.; Pozo, Karine; Zhong, Ping; Yuen, Eunice Y.; Tan, Chunfeng; Hawasli, Ammar H.; Cooke, Sam F.; Nishi, Akinori; Guo, Ailan; Wiederhold, Thorsten; Yan, Zhen; Bibb, James A.

2014-01-01

SUMMARY Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers due to its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when over-expressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction using a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a novel regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers. PMID:24607229

Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos

PubMed Central

Tavernier, Nicolas; Noatynska, Anna; Panbianco, Costanza; Martino, Lisa; Van Hove, Lucie; Schwager, Françoise; Léger, Thibaut

2015-01-01

The molecular mechanisms governing mitotic entry during animal development are incompletely understood. Here, we show that the mitotic kinase CDK-1 phosphorylates Suppressor of Par-Two 1 (SPAT-1)/Bora to regulate its interaction with PLK-1 and to trigger mitotic entry in early Caenorhabditis elegans embryos. Embryos expressing a SPAT-1 version that is nonphosphorylatable by CDK-1 and that is defective in PLK-1 binding in vitro present delays in mitotic entry, mimicking embryos lacking SPAT-1 or PLK-1 functions. We further show that phospho–SPAT-1 activates PLK-1 by triggering phosphorylation on its activator T loop in vitro by Aurora A. Likewise, we show that phosphorylation of human Bora by Cdk1 promotes phosphorylation of human Plk1 by Aurora A, suggesting that this mechanism is conserved in humans. Our results suggest that CDK-1 activates PLK-1 via SPAT-1 phosphorylation to promote entry into mitosis. We propose the existence of a positive feedback loop that connects Cdk1 and Plk1 activation to ensure a robust control of mitotic entry and cell division timing. PMID:25753036

3D-QSAR and molecular docking study on bisarylmaleimide series as glycogen synthase kinase 3, cyclin dependent kinase 2 and cyclin dependent kinase 4 inhibitors: an insight into the criteria for selectivity.

PubMed

Dessalew, Nigus; Bharatam, Prasad V

2007-07-01

Selective glycogen synthase kinase 3 (GSK3) inhibition over cyclin dependent kinases such as cyclin dependent kinase 2 (CDK2) and cyclin dependent kinase 4 (CDK4) is an important requirement for improved therapeutic profile of GSK3 inhibitors. The concepts of selectivity and additivity fields have been employed in developing selective CoMFA models for these related kinases. Initially, sets of three individual CoMFA models were developed, using 36 compounds of bisarylmaleimide series to correlate with the GSK3, CDK2 and CDK4 inhibitory potencies. These models showed a satisfactory statistical significance: CoMFA-GSK3 (r(2)(con), r(2)(cv): 0.931, 0.519), CoMFA-CDK2 (0.937, 0.563), and CoMFA-CDK4 (0.892, 0.725). Three different selective CoMFA models were then developed using differences in pIC(50) values. These three models showed a superior statistical significance: (i) CoMFA-Selective1 (r(2)(con), r(2)(cv): 0.969, 0.768), (ii) CoMFA-Selective 2 (0.974, 0.835) and (iii) CoMFA-Selective3 (0.963, 0.776). The selective models were found to outperform the individual models in terms of the quality of correlation and were found to be more informative in pinpointing the structural basis for the observed quantitative differences of kinase inhibition. An in-depth comparative investigation was carried out between the individual and selective models to gain an insight into the selectivity criterion. To further validate this approach, a set of new compounds were designed which show selectivity and were docked into the active site of GSK3, using FlexX based incremental construction algorithm.

Molecular Recognition of PPARγ by Kinase Cdk5/p25: Insights from a Combination of Protein-Protein Docking and Adaptive Biasing Force Simulations.

PubMed

Mottin, Melina; Souza, Paulo C T; Skaf, Munir S

2015-07-02

The peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that plays a major role in the regulation of glucose and lipid metabolisms and has, therefore, many implications in modern-life metabolic disorders such as diabetes, obesity, and cardiovascular diseases. Phosphorylation of PPARγ by the cyclin-dependent kinase 5 (Cdk5) has been recently proved to promote obesity and loss of insulin sensitivity. The inhibition of this reaction is currently being pursued to develop PPARγ ligands for type 2 diabetes treatments. The knowledge of the protein-protein interactions between Cdk5/p25 and PPARγ can be an important asset for better understanding of the molecular basis of the Cdk5-meditated phosphorylation of PPARγ and its inhibition. By means of a computational approach that combines protein-protein docking and adaptive biasing force molecular dynamics simulations, we obtained PPARγ-Cdk5/p25 structural models that are consistent with the mechanism of the enzymatic reaction and with overall structural features of the full length PPARγ-RXRα heterodimer bound to DNA. In addition to the active site, our model shows that the interacting regions between the two proteins should involve two distal docking sites, comprising the PPARγ Ω-loop and Cdk5 N-terminal lobe and the PPARγ β-sheet and Cdk5 C-terminal lobe. These sites are related to PPARγ transactivation and directly interact with PPARγ ligands. Our results suggest that β-sheets and Ω-loop stabilization promoted by PPARγ agonists could be important to inhibit Cdk5-mediated phosphorylation.

A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

PubMed

Bagella, L; Sun, A; Tonini, T; Abbadessa, G; Cottone, G; Paggi, M G; De Luca, A; Claudio, P P; Giordano, A

2007-03-22

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

Development of mice without Cip/Kip CDK inhibitors

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

Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu

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 largelymore » 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 normal development (although it is thought to be a key player in the response to DNA damage).« less

Identification and characterization of the BmCyclin L1-BmCDK11A/B complex in relation to cell cycle regulation.

PubMed

Liu, Tai-Hang; Wu, Yun-Fei; Dong, Xiao-Long; Pan, Cai-Xia; Du, Guo-Yu; Yang, Ji-Gui; Wang, Wei; Bao, Xi-Yan; Chen, Peng; Pan, Min-Hui; Lu, Cheng

2017-05-03

Cyclin proteins are the key regulatory and activity partner of cyclin-dependent kinases (CDKs), which play pivotal regulatory roles in cell cycle progression. In the present study, we identified a Cyclin L1 and 2 CDK11 2 CDK11 splice variants, CDK11A and CDK11B, from silkworm, Bombyx mori. We determined that both Cyclin L1 and CDK11A/B are nuclear proteins, and further investigations were conducted to elucidate their spatiofunctional features. Cyclin L1 forms a complex with CDK11A/B and were co-localized to the nucleus. Moreover, the dimerization of CDK11A and CDK11B and the effects of Cyclin L1 and CDK11A/B on cell cycle regulation were also investigated. Using overexpression or RNA interference experiments, we demonstrated that the abnormal expression of Cyclin L1 and CDK11A/B leads to cell cycle arrest and cell proliferation suppression. Together, these findings indicate that CDK11A/B interacts with Cyclin L1 to regulate the cell cycle.

Active Cdk5 Immunoprecipitation and Kinase Assay

PubMed Central

Bankston, Andrew N.; Ku, Li; Feng, Yue

2017-01-01

Cdk5 activity is regulated by the amounts of two activator proteins, p35 and p39 (Tsai et al., 1994; Zheng et al., 1998; Humbert et al., 2000). The p35-Cdk5 and p39-Cdk5 complexes have differing sensitivity to salt and detergent concentrations (Hisanaga and Saito, 2003; Sato et al., 2007; Yamada et al., 2007; Asada et al., 2008). Cdk5 activation can be directly measured by immunoprecipitation of Cdk5 with its bound activator, followed by a Cdk5 kinase assay. In this protocol, buffers for cell lysis and immunoprecipitation are intended to preserve both p35- and p39-Cdk5 complexes to assess total Cdk5 activity. Cells are lysed and protein concentration is determined in the post-nuclear supernatant. Cdk5 is immunoprecipitated from equal amounts of total protein between experimental groups. Washes are then performed to remove extraneous proteins and equilibrate the Cdk5-activator complexes in the kinase buffer. Cdk5 is then incubated with histone H1, a well-established in vitro target of Cdk5, and [γ-32P]ATP. Reactions are resolved by SDS-PAGE and transferred to membranes for visualization of H1 phosphorylation and immunoblot of immunoprecipitated Cdk5 levels. We have used this assay to establish p39 as the primary activator for Cdk5 in the oligodendroglial lineage. However, this assay is amenable to other cell lineages or tissues with appropriate adjustments made to lysis conditions. PMID:28868329

A phosphatase threshold sets the level of Cdk1 activity in early mitosis in budding yeast

PubMed Central

Harvey, Stacy L.; Enciso, Germán; Dephoure, Noah; Gygi, Steven P.; Gunawardena, Jeremy; Kellogg, Douglas R.

2011-01-01

Entry into mitosis is initiated by synthesis of cyclins, which bind and activate cyclin-dependent kinase 1 (Cdk1). Cyclin synthesis is gradual, yet activation of Cdk1 occurs in a stepwise manner: a low level of Cdk1 activity is initially generated that triggers early mitotic events, which is followed by full activation of Cdk1. Little is known about how stepwise activation of Cdk1 is achieved. A key regulator of Cdk1 is the Wee1 kinase, which phosphorylates and inhibits Cdk1. Wee1 and Cdk1 show mutual regulation: Cdk1 phosphorylates Wee1, which activates Wee1 to inhibit Cdk1. Further phosphorylation events inactivate Wee1. We discovered that a specific form of protein phosphatase 2A (PP2ACdc55) opposes the initial phosphorylation of Wee1 by Cdk1. In vivo analysis, in vitro reconstitution, and mathematical modeling suggest that PP2ACdc55 sets a threshold that limits activation of Wee1, thereby allowing a low constant level of Cdk1 activity to escape Wee1 inhibition in early mitosis. These results define a new role for PP2ACdc55 and reveal a systems-level mechanism by which dynamically opposed kinase and phosphatase activities can modulate signal strength. PMID:21849476

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

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

Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young

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.more » 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.« less

Vitex rotundifolia Fruit Suppresses the Proliferation of Human Colorectal Cancer Cells through Down-regulation of Cyclin D1 and CDK4 via Proteasomal-Dependent Degradation and Transcriptional Inhibition.

PubMed

Song, Hun Min; Park, Gwang Hun; Park, Su Bin; Kim, Hyun-Seok; Son, Ho-Jun; Um, Yurry; Jeong, Jin Boo

2018-01-01

Viticis Fructus (VF) as the dried fruit from Vitex rotundifolia L. used as a traditional medicine for treating inflammation, headache, migraine, chronic bronchitis, eye pain, and gastrointestinal infections has been reported to have antiproliferative effects against various cancer cells, including breast, lung and colorectal cancer cells. However, the molecular mechanisms by which VF mediates the inhibitory effect of the proliferation of cancer cells have not been elucidated in detail. In this study, we investigated the molecular mechanism of VF on the down-regulation of cyclin D1 and CDK4 level associated with cancer cell proliferation. VF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 and SW480. VF induced decrease in cyclin D1 and CDK4 in both protein and mRNA levels. However, the protein levels of cyclin D1 and CDK4 were decreased by VF at an earlier time than the change of mRNA levels; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 and CDK4 degradation, we found that Thr286 phosphorylation of cyclin D1 plays a pivotal role in VF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that VF-mediated degradation of cyclin D1 may be dependent on GSK3[Formula: see text] and VF-mediated degradation of CDK4 is dependent on ERK1/2, p38 and GSK3[Formula: see text]. In the transcriptional regulation of cyclin D1 and CDK4, we found that VF inhibited Wnt activation associated with cyclin D1 transcriptional regulation through TCF4 down-regulation. In addition, VF treatment down-regulated c-myc expression associated CDK4 transcriptional regulation. Our results suggest that VF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Downregulation of microRNA-33a promotes cyclin-dependent kinase 6, cyclin D1 and PIM1 expression and gastric cancer cell proliferation

PubMed Central

WANG, YUDONG; ZHOU, XINLIANG; SHAN, BAOEN; HAN, JING; WANG, FEIFEI; FAN, XIAOJIE; LV, YALEI; CHANG, LIANG; LIU, WEI

2015-01-01

Although microRNA-33 (miR-33) family members are known to be involved in the regulation and balancing of cholesterol metabolism, fatty acid oxidation and insulin signaling, their functions in carcinogenesis are controversial and the underlying mechanisms have remained elusive. Gastric cancer is the fourth most common malignancy in the world; however, the dysregulation and function of miR-33 family members in gastric cancer have not been extensively studied. The present study reported that a miR-33 family member, miR-33a, was significantly downregulated in gastric cancer tissues and gastric cancer cell lines. Of note, the expression of miR-33a was inversely correlated with pathological differentiation and metastasis as well as gastric cancer biomarker CA199. A cell-counting kit-8 assay showed that transfection of the SGC-7901 gastric cell line with miR-33a-overexpression plasmid inhibited the capability of the cells to proliferate. Furthermore, overexpression of miR-33a led to cell cycle arrest of SGC-7901 cells in G1 phase. In addition, a luciferase reporter assay showed that miR-33a directly targeted cyclin-dependent kinase 6 (CDK6), cyclin D1 (CCND1) and serine/threonine kinase PIM-1. In gastric cancer specimens, the reduced expression of miR-33a was associated with increased expression of CDK-6, CCND1 and PIM1. However, only PIM1 expression was significantly increased in cancer tissues compared with that in their adjacent tissues. The present study revealed that miR-33a was downregulated in gastric cancer tissues and cell lines, while forced overexpression of miR-33a decreased CDK-6, CCND1 and PIM1 expression to inhibit gastric cancer cell proliferation by causing G1 phase arrest. miR-33a overexpression may therefore resemble an efficient strategy for gastric cancer therapy. PMID:26352175

A Cdk5 inhibitory peptide reduces tau hyperphosphorylation and apoptosis in neurons

PubMed Central

Zheng, Ya-Li; Kesavapany, Sashi; Gravell, Maneth; Hamilton, Rebecca S; Schubert, Manfred; Amin, Niranjana; Albers, Wayne; Grant, Philip; Pant, Harish C

2005-01-01

The extracellular aggregation of amyloid β (Aβ) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Aβ peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Aβ1−42-induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting ‘normal' Cdk5 activity. PMID:15592431

Both p62/SQSTM1-HDAC6-dependent autophagy and the aggresome pathway mediate CDK1 degradation in human breast cancer.

PubMed

Galindo-Moreno, María; Giráldez, Servando; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2017-08-30

Cyclin-dependent kinase 1 (CDK1) is the central mammalian regulator of cell proliferation and a promising therapeutic target for breast cancer. In fact, CDK1 inhibition downregulates survival and induces apoptosis. Due to its essential role, CDK1 expression and activity are strictly controlled at various levels. We previously described that CDK1 stability is also regulated and that SCF(βTrCP) ubiquitinates CDK1, which is degraded via the lysosomal pathway. In addition, in breast tumors from patients, we found a negative correlation between CDK1 accumulation and βTrCP levels, and a positive correlation with the degree of tumor malignancy. This prompted us to study the molecular mechanism involved in CDK1 clearance. In this report, we determine that both chemotherapeutic agents and proteolytic stress induce CDK1 degradation in human breast cancer MCF7 cells through p62/HDAC6-mediated selective autophagy. On the one hand, CDK1 binds to p62/SQSTM1-LC3 and, on the other hand, it interacts with HDAC6. Both complexes are dependent on the presence of an intact βTrCP-binding motif on CDK1. Furthermore, we also show that CDK1 is recruited to aggresomes in response to proteasome inhibition for an extended period. We propose CDK1 clearance as a potential predictive biomarker of antitumor treatment efficacy.

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

PubMed

The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

2015-01-06

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.

Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells

PubMed Central

The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P.; Cristobal, Alba; Prinsen, Martine B. W.; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J. R.; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

2015-01-01

Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/CFZR1 activity as an important determinant in response to CDK4/6-inhibitors. PMID:25562820

Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein.

PubMed

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-05-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G(1) phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21(CIP). Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.

Physical Interaction of Human T-Cell Leukemia Virus Type 1 Tax with Cyclin-Dependent Kinase 4 Stimulates the Phosphorylation of Retinoblastoma Protein

PubMed Central

Haller, Kerstin; Wu, Yalin; Derow, Elisabeth; Schmitt, Iris; Jeang, Kuan-Teh; Grassmann, Ralph

2002-01-01

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein. PMID:11971966

βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy.

PubMed

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

2014-09-15

In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFβTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates βTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to βTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients' tumors shows a negative correlation with βTrCP and a positive correlation with the degree of tumor malignancy.

Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells.

PubMed

Johannessen, Liv; Sundberg, Thomas B; O'Connell, Daniel J; Kolde, Raivo; Berstler, James; Billings, Katelyn J; Khor, Bernard; Seashore-Ludlow, Brinton; Fassl, Anne; Russell, Caitlin N; Latorre, Isabel J; Jiang, Baishan; Graham, Daniel B; Perez, Jose R; Sicinski, Piotr; Phillips, Andrew J; Schreiber, Stuart L; Gray, Nathanael S; Shamji, Alykhan F; Xavier, Ramnik J

2017-10-01

Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.

Deciphering the binding behavior of flavonoids to the cyclin dependent kinase 6/cyclin D complex.

PubMed

Zhang, Jingxiao; Zhang, Lilei; Xu, Yangcheng; Jiang, Shanshan; Shao, Yueyue

2018-01-01

Flavonoids, a class of natural compounds with variable phenolic structures, have been found to possess anti-cancer activities by modulating different enzymes and receptors like CDK6. To understand the binding behavior of flavonoids that inhibit the active CDK6, molecular dynamics (MD) simulations were performed on six inhibitors, chrysin (M01), fisetin (M03), galangin (M04), genistein (M05), quercetin (M06) and kaempferol (M07), complexed with CDK6/cyclin D. For all six flavonoids, the 3'-OH and 4'-OH of B-ring were found to be favorable for hydrogen bond formation, but the 3-OH on the C-ring and 5-OH on the A-ring were unfavorable, which were confirmed by the MD simulation results of the test molecule, 3', 4', 7-trihydroxyflavone (M15). The binding efficiencies of flavonoids against the CDK6/cyclin D complex were mainly through the electrostatic (especially the H-bond force) and vdW interactions with residues ILE19, VAL27, ALA41, GLU61, PHE98, GLN103, ASP163 and LEU152. The order of binding affinities of these flavonoids toward the CDK6/cyclin D was M03 > M01 > M07 > M15 > M06 > M05 > M04. It is anticipated that the binding features of flavonoid inhibitors studied in the present work may provide valuable insights for the development of CDK6 inhibitors.

Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19.

PubMed

Park, Min Ju; Shen, Hailian; Spaeth, Jason M; Tolvanen, Jaana H; Failor, Courtney; Knudtson, Jennifer F; McLaughlin, Jessica; Halder, Sunil K; Yang, Qiwei; Bulun, Serdar E; Al-Hendy, Ayman; Schenken, Robert S; Aaltonen, Lauri A; Boyer, Thomas G

2018-03-30

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of MED12 mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs. © 2018 Park et al.

Molecular dynamics simulations on the inhibition of cyclin-dependent kinases 2 and 5 in the presence of activators.

PubMed

Zhang, Bing; Tan, Vincent B C; Lim, Kian Meng; Tay, Tong Earn

2006-06-01

Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.

CAK-Cyclin-dependent Activating Kinase: a key kinase in cell cycle control and a target for drugs?

PubMed

Lolli, Graziano; Johnson, Louise N

2005-04-01

The Cyclin-dependent kinase (CDK) Activating Kinase (CAK) is responsible for the activating phosphorylation of CDK1, CDK2, CDK4 and CDK6 and regulation of the cell cycle. The kinase is composed of three subunits: CDK7, Cyclin H and MAT1 (ménage a trois). Together with six other subunits, CAK is also part of the general transcription factor TFIIH where it is involved in promoter clearance and progression of transcription from the preinitiation to the initiation stage. CAK is required for cell cycle progression, which suggests that CDK7 could be a target for cancer therapy. However its role in transcription and its ubiquitous presence raise sensible concerns about possible toxicity of its inhibitors. The recently determined structure of CDK7 allows the design of inhibitors with differential specificity for the different CDKs. We review the role of CAK in different biological processes and evaluate the biological evidence for CDK7 as a possible pharmacological target.

βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

PubMed Central

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

2014-01-01

In mammals, cell cycle progression is controlled by cyclin-dependent kinases, among which CDK1 plays important roles in the regulation of the G2/M transition, G1 progression and G1/S transition. CDK1 is highly regulated by its association to cyclins, phosphorylation and dephosphorylation, changes in subcellular localization, and by direct binding of CDK inhibitor proteins. CDK1 steady-state protein levels are held constant throughout the cell cycle by a coordinated regulation of protein synthesis and degradation. We show that CDK1 is ubiquitinated by the E3 ubiquitin ligase SCFβTrCP and degraded by the lysosome. Furthermore, we found that DNA damage not only triggers the stabilization of inhibitory phosphorylation sites on CDK1 and repression of CDK1 gene expression, but also regulates βTrCP-induced CDK1 degradation in a cell type-dependent manner. Specifically, treatment with the chemotherapeutic agent doxorubicin in certain cell lines provokes CDK1 degradation and induces apoptosis, whereas in others it inhibits destruction of the protein. These observations raise the possibility that different tumor types, depending on their pathogenic spectrum mutations, may display different sensitivity to βTrCP-induced CDK1 degradation after DNA damage. Finally, we found that CDK1 accumulation in patients’ tumors shows a negative correlation with βTrCP and a positive correlation with the degree of tumor malignancy. PMID:25149538

Intact follicular maturation and defective luteal function in mice deficient for cyclin- dependent kinase-4.

PubMed

Moons, David S; Jirawatnotai, Siwanon; Tsutsui, Tateki; Franks, Roberta; Parlow, A F; Hales, Dale B; Gibori, Geula; Fazleabas, Asgerally T; Kiyokawa, Hiroaki

2002-02-01

Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4(-/-) females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4(-/-) mice. However, unlike cyclin D2-deficient mice, Cdk4(-/-) mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4(-/-) ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4(-/-) mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27(Kip1). Progesterone treatment rescued implantation and restored fertility in Cdk4(-/-) mice. Serum PRL levels after mating were significantly reduced in Cdk4(-/-) mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.

FGF inhibits the activity of the cyclin B1/CDK1 kinase to induce a transient G₂arrest in RCS chondrocytes.

PubMed

Tran, Tri; Kolupaeva, Victoria; Basilico, Claudio

2010-11-01

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G₁ phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G₂ phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G₁ arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G₂ arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G₂ checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G₂ arrest. Additionally our results indicate that the transient G₂ arrest is induced by FGF in RCS cell through mechanisms that are independent of the G₁ arrest, and that the G₂ block is not strictly required for the sustained G₁ arrest but may provide a pausing mechanism that allows the FGF response to be fully established.

Frequent amplification of receptor tyrosine kinase genes in welldifferentiated/ dedifferentiated liposarcoma.

PubMed

Asano, Naofumi; Yoshida, Akihiko; Mitani, Sachiyo; Kobayashi, Eisuke; Shiotani, Bunsyo; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Chuman, Hirokazu; Morioka, Hideo; Matsumoto, Morio; Nakamura, Masaya; Kubo, Takashi; Kato, Mamoru; Kohno, Takashi; Kawai, Akira; Kondo, Tadashi; Ichikawa, Hitoshi

2017-02-21

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

Synthetic Lethal Therapy for KRAS Mutant Non-small-cell Lung Carcinoma with Nanoparticle-mediated CDK4 siRNA Delivery

PubMed Central

Mao, Cheng-Qiong; Xiong, Meng-Hua; Liu, Yang; Shen, Song; Du, Xiao-Jiao; Yang, Xian-Zhu; Dou, Shuang; Zhang, Pei-Zhuo; Wang, Jun

2014-01-01

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4. PMID:24496383

Neurofilament tail phosphorylation: identity of the RT-97 phosphoepitope and regulation in neurons by cross-talk among proline-directed kinases.

PubMed

Veeranna; Lee, Ju-Hyun; Pareek, Tej K; Jaffee, Howard; Boland, Barry; Vinod, K Yaragudri; Amin, Niranjana; Kulkarni, Ashok B; Pant, Harish C; Nixon, Ralph A

2008-10-01

As axons myelinate, establish a stable neurofilament network, and expand in caliber, neurofilament proteins are extensively phosphorylated along their C-terminal tails, which is recognized by the monoclonal antibody, RT-97. Here, we demonstrate in vivo that RT-97 immunoreactivity (IR) is generated by phosphorylation at KSPXK or KSPXXXK motifs and requires flanking lysines at specific positions. extracellular signal regulated kinase 1,2 (ERK1,2) and pERK1,2 levels increase in parallel with phosphorylation at the RT-97 epitope during early postnatal brain development. Purified ERK1,2 generated RT-97 on both KSP motifs on recombinant NF-H tail domain proteins, while cdk5 phosphorylated only KSPXK motifs. RT-97 epitope generation in primary hippocampal neurons was regulated by extensive cross-talk among ERK1,2, c-Jun N-terminal kinase 1,2 (JNK1,2) and cdk5. Inhibition of both ERK1,2 and JNK1,2 completely blocked RT-97 generation. Cdk5 influenced RT-97 generation indirectly by modulating JNK activation. In mice, cdk5 gene deletion did not significantly alter RT-97 IR or ERK1,2 and JNK activation. In mice lacking the cdk5 activator P35, the partial suppression of cdk5 activity increased RT-97 IR by activating ERK1,2. Thus, cdk5 influences RT-97 epitope generation partly by modulating ERKs and JNKs, which are the two principal kinases regulating neurofilament phosphorylation. The regulation of a single target by multiple protein kinases underscores the importance of monitoring other relevant kinases when the activity of a particular one is blocked.

Suppression of Akt-mediated HDAC3 expression and CDK2 T39 phosphorylation by a bichalcone analog contributes to S phase retardation of cancer cells.

PubMed

Hung, Kuang-Chen; Lin, Meng-Liang; Hsu, Shih-Wei; Lee, Chuan-Chun; Huang, Ren-Yu; Wu, Tian-Shung; Chen, Shih-Shun

2018-06-15

Targeting cell cycle regulators has been a suggested mechanism for therapeutic cancer strategies. We report here that the bichalcone analog TSWU-CD4 induces S phase arrest of human cancer cells by inhibiting the formation of cyclin A-phospho (p)-cyclin-dependent kinase 2 (CDK2, threonine [Thr] 39) complexes, independent of mutant p53 expression. Ectopic expression of CDK2 (T39E), which mimics phosphorylation of the Thr 39 residue of CDK2, partially rescues the cells from TSWU-CD4-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TSWU-CD4-triggered S phase arrest. Decreased histone deacetylase 3 (HDAC3) expression after TSWU-CD4 treatment was demonstrated, and TSWU-CD4 induced S phase arrest and inhibitory effects on cyclin A expression and CDK2 Thr 39 phosphorylation, while cyclin A-p-CDK2 (Thr 39) complex formation was suppressed by ectopic wild-type HDAC3 expression. The co-transfection of CDK2 (T39E) along with HDAC3 completely restored cyclin A expression, Thr 39-phosphorylated CDK2, cyclin A-p-CDK2 (Thr 39) complex formation, and the S phase population to normal levels. Protein kinase B (Akt) inactivation was required for TSWU-CD4-induced S phase cell cycle arrest, because constitutively active Akt1 blocks the induction of S phase arrest and the suppression of cyclin A and HDAC3 expression, CDK2 Thr 39 phosphorylation, and cyclin A-p-CDK2 (Thr 39) complex formation by TSWU-CD4. Taken together, our results indicate that TSWU-CD4 induces S phase arrest by inhibiting Akt-mediated HDAC3 expression and CDK2 Thr 39 phosphorylation to suppress the formation of cyclin A-p-CDK2 (Thr 39) complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Mechanistic insights into avian reovirus p17-modulated suppression of cell-cycle CDK/cyclin complexes and enhancement of p53 and cyclin H interaction.

PubMed

Chiu, Hung-Chuan; Huang, Wei-Ru; Liao, Tsai-Ling; Chi, Pei-I; Nielsen, Brent L; Liu, Jyung-Hurng; Liu, Hung-Jen

2018-06-15

The avian reovirus (ARV) p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remains unclear. This is the first report that ARV p17 possesses broad inhibitory effects on cell-cycle CDKs, cyclins, CDK/cyclin complexes, and CDK activating kinase (CAK) activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK/cyclin complexes, transcriptional downregulation of CDKs, cytoplasmic retention of CDKs and cyclins, and inhibition of CAK activity by promoting p53/cyclin H interaction. p17 binds to CDK/cyclin except for CDK1/cyclin B1 and CDK7/cyclin H complexes. We have determined that the negatively charged 151 LAVxDxDxE/DDGADPN 165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1/cyclin B1, but is required for other CDK/cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif 125 RXL 127 Sequence and mutagenic analyses of p17 indicated that a 140 WXFD 143 motif and residues D113 and K122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication while p17 mutants with low binding ability to cell-cycle CDKs have no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Modulated Expression of Genes Encoding Estrogen Metabolizing Enzymes by G1-Phase Cyclin-Dependent Kinases 6 and 4 in Human Breast Cancer Cells

PubMed Central

Jia, Yi; Domenico, Joanne; Swasey, Christina; Wang, Meiqin; Gelfand, Erwin W.; Lucas, Joseph J.

2014-01-01

G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers. PMID:24848372

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

PubMed

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.

Animal Models for Studying the In Vivo Functions of Cell Cycle CDKs.

PubMed

Risal, Sanjiv; Adhikari, Deepak; Liu, Kui

2016-01-01

Multiple Cdks (Cdk4, Cdk6, and Cdk2) and a mitotic Cdk (Cdk1) are involved in cell cycle progression in mammals. Cyclins, Cdk inhibitors, and phosphorylations (both activating and inhibitory) at different cellular levels tightly modulate the activities of these kinases. Based on the results of biochemical studies, it was long believed that different Cdks functioned at specific stages during cell cycle progression. However, deletion of all three interphase Cdks in mice affected cell cycle entry and progression only in certain specialized cells such as hematopoietic cells, beta cells of the pancreas, pituitary lactotrophs, and cardiomyocytes. These genetic experiments challenged the prevailing biochemical model and established that Cdks function in a cell-specific, but not a stage-specific, manner during cell cycle entry and the progression of mitosis. Recent in vivo studies have further established that Cdk1 is the only Cdk that is both essential and sufficient for driving the resumption of meiosis during mouse oocyte maturation. These genetic studies suggest a minimal-essential cell cycle model in which Cdk1 is the central regulator of cell cycle progression. Cdk1 can compensate for the loss of the interphase Cdks by forming active complexes with A-, B-, E-, and D-type Cyclins in a stepwise manner. Thus, Cdk1 plays an essential role in both mitosis and meiosis in mammals, whereas interphase Cdks are dispensable.

Genetic characterization of the role of the Cip/Kip family of proteins as cyclin-dependent kinase inhibitors and assembly factors.

PubMed

Cerqueira, Antonio; Martín, Alberto; Symonds, Catherine E; Odajima, Junko; Dubus, Pierre; Barbacid, Mariano; Santamaría, David

2014-04-01

The Cip/Kip family, namely, p21(Cip1), p27(Kip1), and p57(Kip2), are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21(Cip1) and p27(Kip1) reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2(-/-) p21(-/-) p27(-/-) mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57(Kip2) downregulation in the absence of p21(Cip1) and p27(Kip1) aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21(-/-) p27(-/-) mice, suggesting partial Cdk2-dependent compensation. However, Cdk2(-/-) p21(-/-) p27(-/-) survivors displayed all phenotypes described for p27(-/-) mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types.

Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

PubMed

Grant, Nicola J; Coates, Philip J; Woods, Yvonne L; Bray, Susan E; Morrice, Nicholas A; Hastie, C James; Lamont, Douglas J; Carey, Francis A; Sutherland, Calum

2015-11-10

Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with oncogenesis in some but not all tumour types, implicating altered CDK5 activity in aspects of pathogenesis. These data identify a novel oncogenic mechanism where CDK5 activation induces CRMP2A phosphorylation in the nuclei of tumour cells.

Cdk1-dependent control of membrane-trafficking dynamics

PubMed Central

McCusker, Derek; Royou, Anne; Velours, Christophe; Kellogg, Douglas

2012-01-01

Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which polarize the actin cytoskeleton for delivery of membrane to growth sites via the secretory pathway. Here we investigate whether Cdk1 plays additional roles in the initiation and maintenance of polarized cell growth. We find that inhibition of Cdk1 causes a cell surface growth defect that is as severe as that caused by actin depolymerization. However, unlike actin depolymerization, Cdk1 inhibition does not result in a massive accumulation of intracellular secretory vesicles or their cargoes. Analysis of post-Golgi vesicle dynamics after Cdk1 inhibition demonstrates that exocytic vesicles are rapidly mistargeted away from the growing bud, possibly to the endomembrane/vacuolar system. Inhibition of Cdk1 also causes defects in the organization of endocytic and exocytic zones at the site of growth. Cdk1 thus modulates membrane-trafficking dynamics, which is likely to play an important role in coordinating cell surface growth with cell cycle progression. PMID:22767578

The expression of intermediate filament protein nestin and its association with cyclin-dependent kinase 5 in the glomeruli of rats with diabetic nephropathy.

PubMed

Liu, Wei; Zhang, Yue; Liu, Shuxia; Liu, Qingjuan; Hao, Jun; Shi, Yonghong; Zhao, Song; Duan, Huijun

2013-06-01

Podocyte injury plays a crucial role in the development of diabetic nephropathy (DN), but its underlying mechanism remains poorly understood. Emerging evidences suggest that the cytoskeleton disruption is related to podocyte injury. The aim of this study was to investigate whether nestin, a cytoskeleton-associated intermediate filament protein, is involved in the development of DN. Rat diabetes was induced by intraperitoneal injection of streptozotocin. The renal histological changes were investigated by light microscopy and transmission electron microscopy. The location of nestin and vimentin in renal tissues was observed by immunohistochemistry. The protein or messenger RNA levels of nestin and cyclin-dependent kinase 5 (Cdk5) were detected by Western blot and real-time polymerase chain reaction. The relationship between nestin and vimentin was detected by co-immunoprecipitation. Compared with controls, diabetic rats showed significant characteristics of renal damage. The expression of nestin and vimentin in the glomeruli was increased at the early stage of diabetes, which then gradually decreased. Co-immunoprecipitation assays demonstrated that nestin disassembled with vimentin in diabetic rats. The expression of Cdk5 was increased in a time-dependent manner in diabetic rats. The degree of albuminuria in diabetic rats was negatively correlated with nestin and positively correlated with Cdk5. Roscovitine, a Cdk5 inhibitor, reduced the degradation of nestin. Moreover, podocyte injuries were significantly ameliorated by treatment with roscovitine. The intermediate filament protein nestin is associated with development of DN. Blockage of Cdk5 increases the level of nestin and attenuates renal damage, which would provide a useful target for DN therapy.

The Cytokinin Requirement for Cell Division in Cultured Nicotiana plumbaginifolia Cells Can Be Satisfied by Yeast Cdc25 Protein Tyrosine Phosphatase. Implications for Mechanisms of Cytokinin Response and Plant Development

PubMed Central

Zhang, Kerong; Diederich, Ludger; John, Peter C.L.

2005-01-01

Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67Cdc25 protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr. PMID:15618425

The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be satisfied by yeast Cdc25 protein tyrosine phosphatase: implications for mechanisms of cytokinin response and plant development.

PubMed

Zhang, Kerong; Diederich, Ludger; John, Peter C L

2005-01-01

Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.

High-resolution array comparative genomic hybridization analysis of human bronchial and salivary adenoid cystic carcinoma.

PubMed

Bernheim, Alain; Toujani, Saloua; Saulnier, Patrick; Robert, Thomas; Casiraghi, Odile; Validire, Pierre; Temam, Stéphane; Menard, Philippe; Dessen, Philippe; Fouret, Pierre

2008-05-01

Adenoid cystic carcinoma (ACC) is a rare but distinctive tumor. Oligonucleotide array comparative genomic hybridization has been applied for cataloging genomic copy number alterations (CNAs) in 17 frozen salivary or bronchial tumors. Only four whole chromosome CNAs were found, and most cases had 2-4 segmental CNAs. No high level amplification was observed. There were recurrent gains at 7p15.2, 17q21-25, and 22q11-13, and recurrent losses at 1p35, 6q22-25, 8q12-13, 9p21, 12q12-13, and 17p11-13. The minimal region of gain at 7p15.2 contained the HOXA cluster. The minimal common regions of deletions contained the CDKN2A/CDKN2B, TP53, and LIMA1 tumor suppressor genes. The recurrent deletion at 8q12.3-13.1 contained no straightforward tumor suppressor gene, but the MIRN124A2 microRNA gene, whose product regulates MMP2 and CDK6. Among unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, MDM2, and JAK2. The CNAs involving CCND1, MDM2, KIT, CDKN2A/2B, and TP53 were validated by FISH and/or multiplex ligation-dependent probe amplification. Although most tumors overexpressed cyclin D1 compared with surrounding glands, the only case to overexpress MDM2 had the corresponding CNA. In conclusion, our report suggests that ACC is characterized by a relatively low level of structural complexity. Array CGH and immunohistochemical data implicate MDM2 as the oncogene targeted at 12q15. The gain at 4q12 warrants further exploration as it contains a cluster of receptor kinase genes (KIT/PDGFRA/KDR), whose products can be responsive to specific therapies.

HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a Novel Cyclin-cdk2 Substrate Whose Expression Blocks S-Phase Progression

PubMed Central

Hall, Caitlin; Nelson, David M.; Ye, Xiaofen; Baker, Kayla; DeCaprio, James A.; Seeholzer, Steven; Lipinski, Marc; Adams, Peter D.

2001-01-01

Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle. PMID:11238922

Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis.

PubMed

Maddika, Subbareddy; Ande, Sudharsana Rao; Wiechec, Emilia; Hansen, Lise Lotte; Wesselborg, Sebastian; Los, Marek

2008-04-01

Here, we show that CDK2, an S-phase cyclin-dependent kinase, is a novel target for Akt during cell cycle progression and apoptosis. Akt phosphorylates CDK2 at threonine 39 residue both in vitro and in vivo. Although CDK2 threonine 39 phosphorylation mediated by Akt enhances cyclin-A binding, it is dispensable for its basal binding and the kinase activity. In addition, for the first time, we report a transient nucleo-cytoplasmic shuttling of Akt during specific stages of the cell cycle, in particular during the late S and G2 phases. The Akt that is re-localized to the nucleus phosphorylates CDK2 and causes the temporary cytoplasmic localization of the CDK2-cyclin-A complex. The CDK2 cytoplasmic redistribution is required for cell progression from S to G2-M phase, because the CDK2 T39A mutant, which lacks the phosphorylation site and is defective in cytoplasmic localization, severely affects cell cycle progression at the transition from S to G2-M. Interestingly, we also show that the Akt/CDK2 pathway is constitutively activated by some anticancer drugs, such as methotrexate and docetaxel, and under these conditions it promotes, rather than represses, cell death. Thus, the constitutive activation of the Akt/CDK2 pathway and changed subcellular localization promotes apoptosis. By contrast, the transient, physiological Akt/CDK2 activation is necessary for cell cycle progression.

The assembly, activation, and substrate specificity of Cyclin D1/Cdk2 complexes

PubMed Central

Jahn, Stephan C.; Law, Mary E.; Corsino, Patrick E.; Rowe, Thomas C.; Davis, Bradley J.; Law, Brian K.

2013-01-01

Previous studies have shown conflicting data regarding Cyclin D1/Cdk2 complexes and, considering the widespread overexpression of Cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown Cyclin D1/Cdk2 complexes to form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with Cyclin D1 and Cdk2 failing to complex in its absence. These p21/Cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct Cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of Cyclin D1/Cdk2 complexes, and that Cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer are shown to be phosphorylated by Cyclin D1/Cdk2 complexes. Additionally, PSF is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either Cyclin E or Cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity. PMID:23627734

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

PubMed

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

2014-04-01

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, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

PubMed Central

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, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

Inhibition of CDK-mediated phosphorylation of Smad3 results in decreased oncogenesis in triple negative breast cancer cells

PubMed Central

Tarasewicz, Elizabeth; Rivas, Lisbi; Hamdan, Randala; Dokic, Danijela; Parimi, Vamsi; Bernabe, Beatriz Penalver; Thomas, Alexandra; Shea, Lonnie D; Jeruss, Jacqueline S

2014-01-01

Breast cancer onset and disease progression have been linked to members of the TGFβ superfamily and their downstream signaling components, the Smads. Alterations in Smad3 signaling are associated with the dichotomous role of TGFβ in malignancy, mediating both tumor suppressant and pro-metastatic behaviors. Overexpression of cell cycle regulators, cyclins D and E, renders cyclin-dependent kinases (CDKs) 4/2 hyperactive. Noncanonical phosphorylation of Smad3 by CDK4/2 inhibits tumor suppressant actions of Smad3. We hypothesized that CDK inhibition (CDKi) would restore Smad3 action and help promote cancer cell regression. Treatment of triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-436, Hs578T) with CDK2i or CDK4i resulted in increased Smad3 activity and decreased cell migration. Transfection with a 5M Smad3 construct containing inhibitory mutations in 5 CDK phosphorylation sites also resulted in decreased TNBC cell migration and invasion. MDA-MB-231 cells treated with CDK2i or CDK4i resulted in decreased Smad3 protein phosphorylation at the CDK phosphorylation T179 site, decreased MMP2 and c-myc expression, and increased p15 and p21 expression. Using a novel transfected cell array, we found that CDK2i treatment decreased activity of the epithelial-to-mesenchymal transition related transcription factors Snail and Twist. In vivo studies in an MDA-MB-231 tumor model showed that individual and combination treatment with paclitaxel and CDK2i resulted in decreased tumor volume and Ki67 staining. Collectively, these data support further investigation of targeted CDK inhibitors as a promising therapeutic strategy for TNBC, a breast cancer subtype with limited treatment options. PMID:25485498

Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

PubMed

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

2015-04-01

Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. © 2014 John Wiley & Sons Ltd.

Inhibition of CDC25B Phosphatase Through Disruption of Protein–Protein Interaction

DOE PAGES

Lund, George; Dudkin, Sergii; Borkin, Dmitry; ...

2014-11-25

CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here in this paper, 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 ofmore » 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.« less

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

PubMed Central

Gérard, Claude; Goldbeter, Albert

2012-01-01

We recently proposed a detailed model describing the dynamics of the network of cyclin-dependent kinases (Cdks) driving the mammalian cell cycle (Gérard and Goldbeter, 2009). 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 (PF) 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. PMID:23130001

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic acid: a Potential Role in Cancer Prevention

PubMed Central

Dachineni, Rakesh; Ai, Guoqiang; Kumar, D. Ramesh; Sadhu, Satya S.; Tummala, Hemachand; Bhat, G. Jayarama

2015-01-01

Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anti-cancer effects are still being elucidated. We hypothesized that aspirin’s chemopreventive actions may involve cell cycle regulation through modulation of the levels or activity of cyclin A2/cyclin dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The down regulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. Firstly, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Secondly, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, pre-incubation of CDK2 with salicylic acid, dose-dependently quenched the fluorescence due to ANS. Thirdly, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid down-regulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. Implications Biochemical and structural studies indicate that the anti-proliferative actions of aspirin are mediated through cyclin A2/CDK2. PMID:26685215

Stem cell factor and interleukin-2/15 combine to enhance MAPK-mediated proliferation of human natural killer cells

PubMed Central

Benson, Don M.; Yu, Jianhua; Becknell, Brian; Wei, Min; Freud, Aharon G.; Ferketich, Amy K.; Trotta, Rossana; Perrotti, Danilo; Briesewitz, Roger

2009-01-01

Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)–cell development. CD34+ hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34+ HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells. PMID:19060242

Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine437.

PubMed

Nandi, Nilay; Tyra, Lauren K; Stenesen, Drew; Krämer, Helmut

2017-12-11

Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health. The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood. Here, we show that in Drosophila melanogaster Cdk5 regulates basal autophagy, a key mechanism suppressing neurodegeneration. In a targeted screen, Cdk5 genetically interacted with Acinus (Acn), a primarily nuclear protein, which promotes starvation-independent, basal autophagy. Loss of Cdk5, or its required cofactor p35, reduces S437-Acn phosphorylation, whereas Cdk5 gain-of-function increases pS437-Acn levels. The phospho-mimetic S437D mutation stabilizes Acn and promotes basal autophagy. In p35 mutants, basal autophagy and lifespan are reduced, but restored to near wild-type levels in the presence of stabilized Acn S437D . Expression of aggregation-prone polyQ-containing proteins or the Amyloid-β42 peptide, but not alpha-Synuclein, enhances Cdk5-dependent phosphorylation of S437-Acn. Our data indicate that Cdk5 is required to maintain the protective role of basal autophagy in the initial responses to a subset of neurodegenerative challenges.

All-atomic Molecular Dynamic Studies of Human CDK8: Insight into the A-loop, Point Mutations and Binding with Its Partner CycC

PubMed Central

Xu, Wu; Amire-Brahimi, Benjamin; Xie, Xiao-Jun; Huang, Liying; Ji, Jun-Yuan

2014-01-01

The Mediator, a conserved multisubunit protein complex in eukaryotic organisms, regulates gene expression by bridging sequence-specific DNA-binding transcription factors to the general RNA polymerase II machinery. In yeast, Mediator complex is organized in three core modules (head, middle and tail) and a separable ‘CDK8 submodule’ consisting of four subunits including Cyclin-dependent kinase CDK8 (CDK8), Cyclin C (CycC), MED12, and MED13. The 3-D structure of human CDK8-CycC complex has been recently experimentally determined. To take advantage of this structure and the improved theoretical calculation methods, we have performed molecular dynamic simulations to study dynamics of CDK8 and two CDK8 point mutations (D173A and D189N), which have been identified in human cancers, with and without full length of the A-loop as well as the binding between CDK8 and CycC. We found that CDK8 structure gradually loses two helical structures during the 50-ns molecular dynamic simulation, likely due to the presence of the full-length A-loop. In addition, our studies showed the hydrogen bond occupation of the CDK8 A-loop increases during the first 20-ns MD simulation and stays stable during the later 30-ns MD simulation. Four residues in the A-loop of CDK8 have high hydrogen bond occupation, while the rest residues have low or no hydrogen bond occupation. The hydrogen bond dynamic study of the A-loop residues exhibits three types of changes: increasing, decreasing, and stable. Furthermore, the 3-D structures of CDK8 point mutations D173A, D189N, T196A and T196D have been built by molecular modeling and further investigated by 50-ns molecular dynamic simulations. D173A has the highest average potential energy, while T196D has the lowest average potential energy, indicating that T196D is the most stable structure. Finally, we calculated theoretical binding energy of CDK8 and CycC by MM/PBSA and MM/GBSA methods, and the negative values obtained from both methods demonstrate stability of CDK8-CycC complex. Taken together, these analyses will improve our understanding of the exact functions of CDK8 and the interaction with its partner CycC. PMID:24754906

[Circadian rhythm variation of the clock genes Per1 and cell cycle related genes in different stages of carcinogenesis of buccal mucosa in animal model].

PubMed

Tan, Xuemei; Ye, Hua; Yang, Kai; Chen, Dan; Tang, Hong

2015-07-01

To investigate the expression and circadian rhythm variation of biological clock gene Per1 and cell cycle genes p53, CyclinD1, cyclin-dependent kinases (CDK1), CyclinB1 in different stages of carcinogenesis in buccal mucosa and its relationship with the development of buccal mucosa carcinoma. Ninety golden hamsters were housed under 12 hours light-12 hours dark cycles, and the model of buccal squamous cell carcinoma was established by using the dimethylbenzanthracene (DMBA) to smear the golden hamster buccal mucosa. Before the DMBA was used and after DMBA was used 6 weeks and 14 weeks respectively, the golden hamsters were sacrificed at 6 different time points (5 rats per time point) within 24 hour, including 4, 8, 12, 16, 20 and 24 hour after lights onset (HALO), and the normal buccal mucosa, precancerous lesions and cancer tissue were obtained, respectively. HE stained sections were prepared to observe the canceration of each tissue. Real time RT-PCR was used to detect the mRNA expression of Per1, p53, CyclinD1, CDK1 and CyclinB1, and a cosine analysis method was applied to determine the circadian rhythm variation of Per1, p53, CyclinD1, CDK1 and CyclinB1 mRNA expression, which were characterized by median, amplitude and acrophase. The expression of Per1, p53, CDK1 and CyclinD1 mRNA in 6 different time points within 24 hours in the tissues of three different stages of carcinogenesis had circadian rhythm, respectively. However, the CyclinB1 mRNA was expressed with circadian rhythm just in normal and cancer tissue (P < 0.05), while in precancerous lesions the circadian rhythm was in disorder (P > 0.05). As the development of carcinoma, the median of Per1 and p53 mRNA expression were significantly decreased (P < 0.05), yet the median of CDK1, CyclinB1 and CyclinD1 mRNA expression were significantly increased (P < 0.05). The amplitude of Per1, p53 and CyclinD1 mRNA expression was significantly decreased as the development of carcinoma (P < 0.05), however the amplitude of CDK1 mRNA expression was significantly increased (P < 0.05). In addition, there was no significant difference in the amplitude of CyclinB1 mRNA expression. The time that the peak expression value of Per1 and CDK1 mRNA appeared (Acrophase) in precancerous lesions was remarkably earlier than that in normal tissues, but the acrophase of p53 and CyclinD1 mRNA expression was remarkably delayed. Moreover, the acrophase of CDK1 and CyclinB1 mRNA expression in cancer tissues was obviously earlier than that in normal tissues, yet there was no significant variation in acrophase of Per1, p53, CyclinD1 mRNA expression between normal tissues and cancer tissues. The circadian rhythm of clock gene Per1 and cell cycle genes p53, CyclinD1, CDK1, CyclinB1 expression remarkably varied with the occurrence and development of carcinoma. Further research into the interaction between circadian and cell cycle of two cycle activity and relationship with the carcinogenesis may providenew ideas and methods of individual treatment and the mechanism of carcinogenesis.

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

PubMed Central

Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

2009-01-01

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

Molecular principle of the cyclin-dependent kinase selectivity of 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine-5-carbonitrile derivatives revealed by molecular modeling studies.

PubMed

Kong, Xiaotian; Sun, Huiyong; Pan, Peichen; Tian, Sheng; Li, Dan; Li, Youyong; Hou, Tingjun

2016-01-21

Due to the high sequence identity of the binding pockets of cyclin-dependent kinases (CDKs), designing highly selective inhibitors towards a specific CDK member remains a big challenge. 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine derivatives are effective inhibitors of CDKs, among which the most promising inhibitor 12u demonstrates high binding affinity to CDK9 and attenuated binding affinity to other homologous kinases, such as CDK2. In this study, in order to rationalize the principle of the binding preference towards CDK9 over CDK2 and to explore crucial information that may aid the design of selective CDK9 inhibitors, MM/GBSA calculations based on conventional molecular dynamics (MD) simulations and enhanced sampling simulations (umbrella sampling and steered MD simulations) were carried out on two representative derivatives (12u and 4). The calculation results show that the binding specificity of 12u to CDK9 is primarily controlled by conformational change of the G-loop and variation of the van der Waals interactions. Furthermore, the enhanced sampling simulations revealed the different reaction coordinates and transient interactions of inhibitors 12u and 4 as they dissociate from the binding pockets of CDK9 and CDK2. The physical principles obtained from this study may facilitate the discovery and rational design of novel and specific inhibitors of CDK9.

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma

PubMed Central

Desai, Shraddha R.; Pillai, Prajit P.; Patel, Rekha S.; McCray, Andrea N.; Win-Piazza, Hla Y.; Acevedo-Duncan, Mildred E.

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G1–S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway. PMID:22021906

Regulation of Cdk7 activity through a phosphatidylinositol (3)-kinase/PKC-ι-mediated signaling cascade in glioblastoma.

PubMed

Desai, Shraddha R; Pillai, Prajit P; Patel, Rekha S; McCray, Andrea N; Win-Piazza, Hla Y; Acevedo-Duncan, Mildred E

2012-01-01

The objective of this research was to study the potential function of protein kinase C (PKC)-ι in cell cycle progression and proliferation in glioblastoma. PKC-ι is highly overexpressed in human glioma and benign and malignant meningioma; however, little is understood about its role in regulating cell proliferation of glioblastoma. Several upstream molecular aberrations and/or loss of PTEN have been implicated to constitutively activate the phosphatidylinositol (PI) (3)-kinase pathway. PKC-ι is a targeted mediator in the PI (3)-kinase signal transduction repertoire. Results showed that PKC-ι was highly activated and overexpressed in glioma cells. PKC-ι directly associated and phosphorylated Cdk7 at T170 in a cell cycle-dependent manner, phosphorylating its downstream target, cdk2 at T160. Cdk2 has a major role in inducing G(1)-S phase progression of cells. Purified PKC-ι phosphorylated both endogenous and exogenous Cdk7. PKC-ι downregulation reduced Cdk7 and cdk2 phosphorylation following PI (3)-kinase inhibition, phosphotidylinositol-dependent kinase 1 knockdown as well as PKC-ι silencing (by siRNA treatment). It also diminished cdk2 activity. PKC-ι knockdown inhibited overall proliferation rates and induced apoptosis in glioma cells. These findings suggest that glioma cells may be proliferating through a novel PI (3)-kinase-/PKC-ι/Cdk7/cdk2-mediated pathway.

Cdk1 and okadaic acid-sensitive phosphatases control assembly of nuclear pore complexes in Drosophila embryos.

PubMed

Onischenko, Evgeny A; Gubanova, Natalia V; Kiseleva, Elena V; Hallberg, Einar

2005-11-01

Disassembly and reassembly of the nuclear pore complexes (NPCs) is one of the major events during open mitosis in higher eukaryotes. However, how this process is controlled by the mitotic machinery is not clear. To investigate this we developed a novel in vivo model system based on syncytial Drosophila embryos. We microinjected different mitotic effectors into the embryonic cytoplasm and monitored the dynamics of disassembly/reassembly of NPCs in live embryos using fluorescently labeled wheat germ agglutinin (WGA) or in fixed embryos using electron microscopy and immunostaining techniques. We found that in live embryos Cdk1 activity was necessary and sufficient to induce disassembly of NPCs as well as their cytoplasmic mimics: annulate lamellae pore complexes (ALPCs). Cdk1 activity was also required for keeping NPCs and ALPCs disassembled during mitosis. In agreement recombinant Cdk1/cyclin B was able to induce phosphorylation and dissociation of nucleoporins from the NPCs in vitro. Conversely, reassembly of NPCs and ALPCs was dependent on the activity of protein phosphatases, sensitive to okadaic acid (OA). Our findings suggest a model where mitotic disassembly/reassembly of the NPCs is regulated by a dynamic equilibrium of Cdk1 and OA-sensitive phosphatase activities and provide evidence that mitotic phosphorylation mediates disassembly of the NPC.

Cyclin-Dependent Kinase 11 (CDK11) is Crucial in the Growth of Liposarcoma Cells

PubMed Central

Jia, Bin; Choy, Edwin; Cote, Gregory; Harmon, David; Ye, Shunan; Kan, Quancheng; Mankin, Henry; Hornicek, Francis; Duan, Zhenfeng

2014-01-01

Liposarcoma is the second most common soft tissue sarcoma in adults, but treatment options have been quite limited thus far. In this study, we investigated the functional and therapeutic relevance of cyclin-dependent kinase 11 (CDK11) as a putative target in liposarcoma. CDK11 knockdown by synthetic siRNA or lentiviral shRNA decreased cell proliferation, and induced apoptosis in liposarcoma cells. Moreover, CDK11 knockdown enhances the cytotoxic effect of doxorubicin to inhibit cell growth in liposarcoma cells. These findings suggest that CDK11 is critical for the growth and proliferation of liposarcoma cells. CDK11 may be a promising therapeutic target for the treatment of liposarcoma patients. PMID:24007862

Cdk5 Phosphorylation of ErbB4 is Required for Tangential Migration of Cortical Interneurons

PubMed Central

Rakić, Sonja; Kanatani, Shigeaki; Hunt, David; Faux, Clare; Cariboni, Anna; Chiara, Francesca; Khan, Shabana; Wansbury, Olivia; Howard, Beatrice; Nakajima, Kazunori; Nikolić, Margareta; Parnavelas, John G.

2015-01-01

Interneuron dysfunction in humans is often associated with neurological and psychiatric disorders, such as epilepsy, schizophrenia, and autism. Some of these disorders are believed to emerge during brain formation, at the time of interneuron specification, migration, and synapse formation. Here, using a mouse model and a host of histological and molecular biological techniques, we report that the signaling molecule cyclin-dependent kinase 5 (Cdk5), and its activator p35, control the tangential migration of interneurons toward and within the cerebral cortex by modulating the critical neurodevelopmental signaling pathway, ErbB4/phosphatidylinositol 3-kinase, that has been repeatedly linked to schizophrenia. This finding identifies Cdk5 as a crucial signaling factor in cortical interneuron development in mammals. PMID:24142862

Cyclin C influences the timing of mitosis in fission yeast

PubMed Central

Banyai, Gabor; Szilagyi, Zsolt; Baraznenok, Vera; Khorosjutina, Olga; Gustafsson, Claes M.

2017-01-01

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II–dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase. PMID:28515143

Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells.

PubMed

Kranenburg, O; Scharnhorst, V; Van der Eb, A J; Zantema, A

1995-10-01

Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.

Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells

PubMed Central

1995-01-01

Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation. PMID:7559779

Cdk5 is required for the neuroprotective effect of transforming growth factor-β1 against cerebral ischemia-reperfusion

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

Zhao, Wenhui; Yan, Jing; Sino-UK Joint Laboratory of Brain Functions and Injury, Science and Technology Department of Henan Province

Transforming Growth Factor β1 (TGF-β1), a well-known neuroprotective and neurotrophic factor in the central nervous system, is also involved in the repair process responses after ischemia-reperfusion injury. Herein, we found that TGF-β1 enhanced Cdk5 expression while decreased Tunel-positive cells compared with the ischemia group, and roscovitine(Cdk5 inhibitor) treatment could blunt these effects. In vitro study, TGF-β1 facilitated Cdk5/p35 complex, the proliferation, neurite growth and differentiation of PC12 cells, effects of which could be blunted by roscovitine and Cdk5 silencing. Moreover, ERK1/2 inhibitor SCH772984 abrogated the effects of TGF- β1 on Cdk5 and Bax levels. Taken together, we conclude that Cdk5 contributes tomore » the neuroprotective function of TGF- β1 via ERK1/2 signaling.« less

Discovery of thienoquinolone derivatives as selective and ATP non-competitive CDK5/p25 inhibitors by structure-based virtual screening

PubMed Central

Chatterjee, Arindam; Doerksen, Robert J.; Khan, Ikhlas A.

2014-01-01

Calpain mediated cleavage of CDK5 natural precursor p35 causes a stable complex formation of CDK5/p25, which leads to hyperphosphorylation of tau. Thus inhibition of this complex is a viable target for numerous acute and chronic neurodegenerative diseases involving tau protein, including Alzheimer’s disease. Since CDK5 has the highest sequence homology with its mitotic counterpart CDK2, our primary goal was to design selective CDK5/p25 inhibitors targeting neurodegeneration. A novel structure-based virtual screening protocol comprised of e-pharmacophore models and virtual screening work-flow was used to identify nine compounds from a commercial database containing 2.84 million compounds. An ATP non-competitive and selective thieno[3,2-c]quinolin-4(5H)-one inhibitor (10) with ligand efficiency (LE) of 0.3 was identified as the lead molecule. Further SAR optimization led to the discovery of several low micromolar inhibitors with good selectivity. The research represents a new class of potent ATP non-competitive CDK5/p25 inhibitors with good CDK2/E selectivity. PMID:25438765

Stress-Induced CDK5 Activation Disrupts Axonal Transport via Lis1/Ndel1/Dynein.

PubMed

Klinman, Eva; Holzbaur, Erika L F

2015-07-21

Axonal transport is essential for neuronal function, and defects in transport are associated with multiple neurodegenerative diseases. Aberrant cyclin-dependent kinase 5 (CDK5) activity, driven by the stress-induced activator p25, also is observed in these diseases. Here we show that elevated CDK5 activity increases the frequency of nonprocessive events for a range of organelles, including lysosomes, autophagosomes, mitochondria, and signaling endosomes. Transport disruption induced by aberrant CDK5 activation depends on the Lis1/Ndel1 complex, which directly regulates dynein activity. CDK5 phosphorylation of Ndel1 favors a high affinity Lis1/Ndel/dynein complex that blocks the ATP-dependent release of dynein from microtubules, inhibiting processive motility of dynein-driven cargo. Similar transport defects observed in neurons from a mouse model of amyotrophic lateral sclerosis are rescued by CDK5 inhibition. Together, these studies identify CDK5 as a Lis1/Ndel1-dependent regulator of transport in stressed neurons, and suggest that dysregulated CDK5 activity contributes to the transport deficits observed during neurodegeneration. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Cdk5-dependent phosphorylation of liprinα1 mediates neuronal activity-dependent synapse development

PubMed Central

Huang, Huiqian; Lin, Xiaochen; Liang, Zhuoyi; Zhao, Teng; Du, Shengwang; Loy, Michael M. T.; Lai, Kwok-On; Fu, Amy K. Y.

2017-01-01

The experience-dependent modulation of brain circuitry depends on dynamic changes in synaptic connections that are guided by neuronal activity. In particular, postsynaptic maturation requires changes in dendritic spine morphology, the targeting of postsynaptic proteins, and the insertion of synaptic neurotransmitter receptors. Thus, it is critical to understand how neuronal activity controls postsynaptic maturation. Here we report that the scaffold protein liprinα1 and its phosphorylation by cyclin-dependent kinase 5 (Cdk5) are critical for the maturation of excitatory synapses through regulation of the synaptic localization of the major postsynaptic organizer postsynaptic density (PSD)-95. Whereas Cdk5 phosphorylates liprinα1 at Thr701, this phosphorylation decreases in neurons in response to neuronal activity. Blockade of liprinα1 phosphorylation enhances the structural and functional maturation of excitatory synapses. Nanoscale superresolution imaging reveals that inhibition of liprinα1 phosphorylation increases the colocalization of liprinα1 with PSD-95. Furthermore, disruption of liprinα1 phosphorylation by a small interfering peptide, siLIP, promotes the synaptic localization of PSD-95 and enhances synaptic strength in vivo. Our findings collectively demonstrate that the Cdk5-dependent phosphorylation of liprinα1 is important for the postsynaptic organization during activity-dependent synapse development. PMID:28760951

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins

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

McGrath, Denise A.; Fifield, Bre‐Anne; Marceau, Aimee H.

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changesmore » to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.« less

Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum.

PubMed

Huber, Robert J; O'Day, Danton H

2011-08-01

The Dictyostelium discoideum homolog of mammalian cyclin dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however, the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions, suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations identified puromycin-sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium.

Cyclin-dependent kinase 5-mediated phosphorylation of CHIP promotes the tAIF-dependent death pathway in rotenone-treated cortical neurons.

PubMed

Kim, Chiho; Lee, Juhyung; Ko, Yeon Uk; Oh, Young J

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase. Its dysregulation has been implicated in various neurodegenerative diseases. We previously reported that phosphorylation of the C-terminus of the Hsc70-interacting protein (CHIP) by Cdk5 promotes truncated apoptosis-inducing factor (tAIF)-mediated neuronal death induced by oxidative stress. Here, we determined whether this Cdk5-dependent cell death signaling pathway is present in experimental models of Parkinson's disease. First, we showed that rotenone activates Cdk5 in primary cultures of cortical neurons and causes tAIF-dependent neuronal cell death. This event was attenuated by negative regulation of endogenous Cdk5 activity by the pharmacological Cdk5 inhibitor, roscovitine, or by lentiviral knockdown of Cdk5. Cdk5 phosphorylates CHIP at Ser20 in rotenone-treated neurons. Consequently, overexpression of CHIP S20A , but not CHIP WT , attenuates tAIF-induced cell death in rotenone-treated cortical neurons. Taken together, these results indicate that phosphorylation of CHIP at Ser20 by Cdk5 activation inhibits CHIP-mediated tAIF degradation, thereby contributing to tAIF-induced neuronal cell death following rotenone treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome.

PubMed

Pitzen, Valentin; Askarzada, Sophie; Gräf, Ralph; Meyer, Irene

2018-04-23

Dictyostelium centrosomes consist of a nucleus-associated cylindrical, three-layered core structure surrounded by a corona consisting of microtubule-nucleation complexes embedded in a scaffold of large coiled-coil proteins. One of them is the conserved CDK5RAP2 protein. Here we focus on the role of Dictyostelium CDK5RAP2 for maintenance of centrosome integrity, its interaction partners and its dynamic behavior during interphase and mitosis. GFP-CDK5RAP2 is present at the centrosome during the entire cell cycle except from a short period during prophase, correlating with the normal dissociation of the corona at this stage. RNAi depletion of CDK5RAP2 results in complete disorganization of centrosomes and microtubules suggesting that CDK5RAP2 is required for organization of the corona and its association to the core structure. This is in line with the observation that overexpressed GFP-CDK5RAP2 elicited supernumerary cytosolic MTOCs. The phenotype of CDK5RAP2 depletion was very reminiscent of that observed upon depletion of CP148, another scaffolding protein of the corona. BioID interaction assays revealed an interaction of CDK5RAP2 not only with the corona markers CP148, γ-tubulin, and CP248, but also with the core components Cep192, CP75, and CP91. Furthermore, protein localization studies in both depletion strains revealed that CP148 and CDK5RAP2 cooperate in corona organization.

Cdk5 inhibitory peptide (CIP) inhibits Cdk5/p25 activity induced by high glucose in pancreatic beta cells and recovers insulin secretion from p25 damage.

PubMed

Zheng, Ya-Li; Li, Congyu; Hu, Ya-Fang; Cao, Li; Wang, Hui; Li, Bo; Lu, Xiao-Hua; Bao, Li; Luo, Hong-Yan; Shukla, Varsha; Amin, Niranjana D; Pant, Harish C

2013-01-01

Cdk5/p25 hyperactivity has been demonstrated to lead to neuron apoptosis and degenerations. Chronic exposure to high glucose (HG) results in hyperactivity of Cdk5 and reduced insulin secretion. Here, we set out to determine whether abnormal upregulation of Cdk5/p25 activity may be induced in a pancreatic beta cell line, Min6 cells. We first confirmed that p25 were induced in overexpressed p35 cells treated with HG and increased time course dependence. Next, we showed that no p25 was detected under short time HG stimulation (4-12 hrs), however was detectable in the long exposure in HG cells (24 hrs and 48 hrs). Cdk5 activity in the above cells was much higher than low glucose treated cells and resulted in more than 50% inhibition of insulin secretion. We confirmed these results by overexpression of p25 in Min6 cells. As in cortical neurons, CIP, a small peptide, inhibited Cdk5/p25 activity and restored insulin secretion. The same results were detected in co-infection of dominant negative Cdk5 (DNCdk5) with p25. CIP also reduced beta cells apoptosis induced by Cdk5/p25. These studies indicate that Cdk5/p25 hyperactivation deregulates insulin secretion and induces cell death in pancreatic beta cells and suggests that CIP may serve as a therapeutic agent for type 2 diabetes.

Dinaciclib Induces Anaphase Catastrophe in Lung Cancer Cells via Inhibition of Cyclin-Dependent Kinases 1 and 2.

PubMed

Danilov, Alexey V; Hu, Shanhu; Orr, Bernardo; Godek, Kristina; Mustachio, Lisa Maria; Sekula, David; Liu, Xi; Kawakami, Masanori; Johnson, Faye M; Compton, Duane A; Freemantle, Sarah J; Dmitrovsky, Ethan

2016-11-01

Despite advances in targeted therapy, lung cancer remains the most common cause of cancer-related mortality in the United States. Chromosomal instability is a prominent feature in lung cancer and, because it rarely occurs in normal cells, it represents a potential therapeutic target. Our prior work discovered that lung cancer cells undergo anaphase catastrophe in response to inhibition of cyclin-dependent kinase 2 (CDK2), followed by apoptosis and reduced growth. In this study, the effects and mechanisms of the multi-CDK inhibitor dinaciclib on lung cancer cells were investigated. We sought to determine the specificity of CDK-dependent induction of anaphase catastrophe. Live cell imaging provided direct evidence that dinaciclib caused multipolar cell divisions resulting in extensive chromosome missegregation. Genetic knockdown of dinaciclib CDK targets revealed that repression of CDK2 and CDK1, but not CDK5 or CDK9, triggered anaphase catastrophe in lung cancer cells. Overexpression of CP110, which is a mediator of CDK2 inhibitor-induced anaphase catastrophe (and a CDK1 and 2 phosphorylation substrate), antagonized anaphase catastrophe and apoptosis following dinaciclib treatment. Consistent with our previous findings, acquisition of activated KRAS sensitized lung cancer cells to dinaciclib-mediated anaphase catastrophe and cell death. Combining dinaciclib with the mitotic inhibitor taxol augmented anaphase catastrophe induction and reduced cell viability of lung cancer cells. Thus, the multi-CDK inhibitor dinaciclib causes anaphase catastrophe in lung cancer cells and should be investigated as a potential therapeutic for wild-type and KRAS-mutant lung cancer, individually or in combination with taxanes. Mol Cancer Ther; 15(11); 2758-66. ©2016 AACR. ©2016 American Association for Cancer Research.

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

PubMed Central

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

2012-01-01

Deregulation of the p16INK4a-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16INK4a-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16INK4a-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16INK4a and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18INK4c (GBM43). In contrast, 2 GBM lines with p16INK4a expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16INK4a expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16INK4a expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991. PMID:22711607

Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone

PubMed Central

Stetz, Gabrielle; Tse, Amanda

2017-01-01

The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired signaling in the client kinase. According to our results, these network features may uniquely define chaperone dependencies of CDK clients. The perturbation response scanning and rigidity decomposition approaches identified regulatory hotspots that mediate differences in stability and cooperativity of allosteric interaction networks in the CDK structures. By combining these synergistic approaches, our study revealed dynamic and network signatures that can differentiate kinase clients and rationalize subtle divergences in the activation mechanisms of CDK family members. The therapeutic implications of these results are illustrated by identifying structural hotspots of pathogenic mutations that preferentially target regions of the increased flexibility to enable modulation of activation changes. Our study offers a network-based perspective on dynamic kinase mechanisms and drug design by unravelling relationships between protein kinase dynamics, allosteric communications and chaperone dependencies. PMID:29095844

Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells

PubMed Central

Bolton, Eric C.

2015-01-01

The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation. PMID:26372468

Expression of cdk4 and p16 in Oral Lichen Planus.

PubMed

Goel, Sinny; Khurana, Nita; Marwah, Akanksha; Gupta, Sunita

2015-01-01

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. 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 squamous cell carcinoma (OSCC) cases with immunohistochemistry. This study demonstrated a significantly increased expression of cytoplasmic cdk4 (80% cases, cells stained - 19.6%), and cytoplasmic p16 (68.3% cases, cells stained - 16.4%) in oral lichen planus (OLP) compared to normal mucosa. cdk4 was much higher in OSCC in both cytoplasm and nuclei compared to normal mucosa. Also, while comparing OLP with positive control, significant difference was noted for cdk4 and p16, with expression being more in OSCC. While comparing EOLP with NOLP; significant differences were seen for cdk4 cytoplasmic staining only, for number of cases with positive staining as well as number of cells stained. Overexpression of cytoplasmic cdk4 and p16 was registered in oral lichen planus, however considerably lower than in squamous cell carcinoma. Erosive oral lichen planus demonstrated overexpression of cytoplasmic cdk4 and premalignant nature compared to non-erosive lesion. Therefore there is an obvious possibility for cytoplasmic expression of cdk4 and p16 to predict malignant potential of oral lichen planus lesions.

Direct binding of the N-terminus of HTLV-1 tax oncoprotein to cyclin-dependent kinase 4 is a dominant path to stimulate the kinase activity.

PubMed

Li, Junan; Li, Hongyuan; Tsai, Ming-Daw

2003-06-10

The involvement of Tax oncoprotein in the INK4-CDK4/6-Rb pathway has been regarded as a key factor for immortalization and transformation of human T-cell leukemia virus 1 (HTLV-1) infected cells. In both p16 -/- and +/+ cells, expression of Tax has been correlated with an increase in CDK4 activity, which subsequently increases the phosphorylation of Rb and drives the infected cells into cell cycle progression. In relation to these effects, Tax has been shown to interact with two components of the INK4-CDK4/6-Rb pathway, p16 and cyclin D(s). While Tax competes with CDK4 for p16 binding, thus suppressing p16 inhibition of CDK4, Tax also binds to cyclin D(s) with concomitant increases in both CDK4 activity and the phosphorylation of cyclin D(s). Here we show that both Tax and residues 1-40 of the N-terminus of Tax, Tax40N, bind to and activate CDK4 in vitro. In the presence of INK4 proteins, binding of Tax and Tax40N to CDK4 counteracts against the inhibition of p16 and p18 and acts as the major path to regulate Tax-mediated activation of CDK4. We also report that Tax40N retains the transactivation ability. These results of in vitro studies demonstrate a potentially novel, p16-independent route to regulate CDK4 activity by the Tax oncoprotein in HTLV-1 infected cells.

Molecular profiling and combinatorial activity of CCT068127: a potent CDK2 and CDK9 inhibitor.

PubMed

Whittaker, Steven R; Barlow, Clare; Martin, Mathew P; Mancusi, Caterina; Wagner, Steve; Self, Annette; Barrie, Elaine; Te Poele, Robert; Sharp, Swee; Brown, Nathan; Wilson, Stuart; Jackson, Wayne; Fischer, Peter M; Clarke, Paul A; Walton, Michael I; McDonald, Edward; Blagg, Julian; Noble, Martin; Garrett, Michelle D; Workman, Paul

2018-03-01

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

EMODIN DOWNREGULATES CELL PROLIFERATION MARKERS DURING DMBA INDUCED ORAL CARCINOGENESIS IN GOLDEN SYRIAN HAMSTERS.

PubMed

Manimaran, Asokan; Buddhan, Rajamanickam; Manoharan, Shanmugam

2017-01-01

Cell-cycle disruption is the major characteristic features of neoplastic transformation and the status of cell-cycle regulators can thus be utilized to assess the prognostic significance in patients with cancer. The PCNA, cyclin D1, CDK4, CDK6 and survivin expression in the buccal mucosa was utilized to evaluate the Emodin efficacy on abnormal cell proliferation during 7,12-dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis in golden Syrian hamsters. Topical application of DMBA, three times a week for 14 weeks, on the hamsters' buccal pouches developed well differentiated squamous cell carcinoma. Cyclin D1 and PCNA over-expression and up-regulation of CDK4, CDK6 and survivin were noticed in the buccal mucosa of hamsters treated with DMBA alone. Emodin administration (50mg/kg b.w) orally to hamsters treated with DMBA down-regulated the expression of cell proliferation markers in the buccal mucosa. The anti-cell proliferative role of Emodin is owing to its modulating efficacy on cell-cycle markers towards the tumor suppression during DMBA induced oral carcinogenesis.

CDK5RAP2 gene and tau pathophysiology in late-onset sporadic Alzheimer's disease.

PubMed

Miron, Justin; Picard, Cynthia; Nilsson, Nathalie; Frappier, Josée; Dea, Doris; Théroux, Louise; Poirier, Judes

2018-06-01

Because currently known Alzheimer's disease (AD) single-nucleotide polymorphisms only account for a small fraction of the genetic variance in this disease, there is a need to identify new variants associated with AD. Our team performed a genome-wide association study in the Quebec Founder Population isolate to identify novel protective or risk genetic factors for late-onset sporadic AD and examined the impact of these variants on gene expression and AD pathology. The rs10984186 variant is associated with an increased risk of developing AD and with a higher CDK5RAP2 mRNA prevalence in the hippocampus. On the other hand, the rs4837766 variant, which is among the best cis-expression quantitative trait loci in the CDK5RAP2 gene, is associated with lower mild cognitive impairment/AD risk and conversion rate. The rs10984186 risk and rs4837766 protective polymorphic variants of the CDK5RAP2 gene might act as potent genetic modifiers for AD risk and/or conversion by modulating the expression of this gene. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2014-01-01

Background The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas. Methods From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR). Results 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). Conclusions Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection. PMID:25121597

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

PubMed

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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Direct Protein Interactions Are Responsible for Ikaros-GATA and Ikaros-Cdk9 Cooperativeness in Hematopoietic Cells

PubMed Central

Bottardi, Stefania; Mavoungou, Lionel; Bourgoin, Vincent; Mashtalir, Nazar; Affar, El Bachir

2013-01-01

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells. PMID:23732910

Direct protein interactions are responsible for Ikaros-GATA and Ikaros-Cdk9 cooperativeness in hematopoietic cells.

PubMed

Bottardi, Stefania; Mavoungou, Lionel; Bourgoin, Vincent; Mashtalir, Nazar; Affar, El Bachir; Milot, Eric

2013-08-01

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

PubMed Central

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

CDK5 A Novel Role in Prostate Cancer Immunotherapy

DTIC Science & Technology

2016-10-01

Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official...We are now discussing our next steps in pursuing this finding; these may include treatment of TRAMP Cdk5 knockout mice to inhibit the putative...Merck and Bayer have terminated clinical development of these compounds. The compounds are still well suited as “tool compounds,” for the

General Methods for Identifying G1-phase Substrates of Cdk Protein Kinases

DTIC Science & Technology

1999-06-01

all eukaryotes, phosphorylation by various cyclin-Cdk complexes controls and orchestrates key cell cycle events. These events include commitment in...kinases, and none of these explain the control of critical cell cycle events. In particular, we do not know what substrates have to be...phosphorylated for commitment to occur (although in mammalian cells, Rb is almost certainly one of the substrates). The purpose of the present work is to develop

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

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

Li, Xuesong; Gong, Xuhai; Chen, Jing

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 defectmore » 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.« less

Putting one step before the other: distinct activation pathways for Cdk1 and Cdk2 bring order to the mammalian cell cycle

PubMed Central

Merrick, Karl A.; Fisher, Robert P.

2010-01-01

Eukaryotic cell division is controlled by the activity of cyclin-dependent kinases (CDKs). Cdk1 and Cdk2, which function at different stages of the mammalian cell cycle, both require cyclin-binding and phosphorylation of the activation (T-) loop for full activity, but differ with respect to the order in which the two steps occur in vivo. To form stable complexes with either of its partners—cyclins A and B—Cdk1 must be phosphorylated on its T-loop, but that phosphorylation in turn depends on the presence of cyclin. Cdk2 can follow a kinetically distinct path to activation in which T-loop phosphorylation precedes cyclin-binding, and thereby out-compete the more abundant Cdk1 for limiting amounts of cyclin A. Mathematical modeling suggests this could be a principal basis for the temporal ordering of CDK activation during S phase, which may dictate the sequence in which replication origins fire. Still to be determined are how: 1) the activation machinery discriminates between closely related CDKs, and 2) coordination of the cell cycle is affected when this mechanism of pathway insulation breaks down. PMID:20139727

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

PubMed

Nahálková, Jarmila; Tomkinson, Birgitta

2014-12-15

Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Inhibitors of cyclin-dependent kinases as cancer therapeutics.

PubMed

Whittaker, Steven R; Mallinger, Aurélie; Workman, Paul; Clarke, Paul A

2017-05-01

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

An essential role for Ink4 and Cip/Kip cell-cycle inhibitors in preventing replicative stress.

PubMed

Quereda, V; Porlan, E; Cañamero, M; Dubus, P; Malumbres, M

2016-03-01

Cell-cycle inhibitors of the Ink4 and Cip/Kip families are involved in cellular senescence and tumor suppression. These inhibitors are individually dispensable for the cell cycle and inactivation of specific family members results in increased proliferation and enhanced susceptibility to tumor development. We have now analyzed the consequences of eliminating a substantial part of the cell-cycle inhibitory activity in the cell by generating a mouse model, which combines the absence of both p21(Cip1) and p27(Kip1) proteins with the endogenous expression of a Cdk4 R24C mutant insensitive to Ink4 inhibitors. Pairwise combination of Cdk4 R24C, p21-null and p27-null alleles results in frequent hyperplasias and tumors, mainly in cells of endocrine origin such as pituitary cells and in mesenchymal tissues. Interestingly, complete abrogation of p21(Cip1) and p27(Kip1) in Cdk4 R24C mutant mice results in a different phenotype characterized by perinatal death accompanied by general hypoplasia in most tissues. This phenotype correlates with increased replicative stress in developing tissues such as the nervous system and subsequent apoptotic cell death. Partial inhibition of Cdk4/6 rescues replicative stress signaling as well as p53 induction in the absence of cell-cycle inhibitors. We conclude that one of the major physiological activities of cell-cycle inhibitors is to prevent replicative stress during development.

Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

PubMed Central

Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

2016-01-01

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

CDK1 promotes nascent DNA synthesis and induces resistance of cancer cells to DNA-damaging therapeutic agents

PubMed Central

Liao, Hongwei; Ji, Fang; Geng, Xinwei; Xing, Meichun; Li, Wen; Chen, Zhihua; Shen, Huahao; Ying, Songmin

2017-01-01

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy. PMID:29207595

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

PubMed Central

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

2011-01-01

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

CBX3 promotes colon cancer cell proliferation by CDK6 kinase-independent function during cell cycle

PubMed Central

Fan, Yao; Li, Haiping; Liang, Xiaolong; Xiang, Zheng

2017-01-01

Heterochromatin protein 1γ (CBX3) links histone methylation marks to transcriptional silence, DNA repair and RNA splicing, but a role for CBX3 in cancer remains largely unknown. In this study, we show that CBX3 in colon cancer cells promotes the progression of the cell cycle and proliferation in vitro and in vivo. Cell cycle (G1 phase to S phase) related gene CDK6 and p21 were further identified as targets of CBX3. In addition, we found that enhancing CDK6 suppresses cell proliferation by upregulating inhibitor p21 in the absence of CBX3, and this function is independent of the kinase activity of CDK6. Our results demonstrate a key role of CBX3 in colon carcinogenesis via suppressing the expression of CDK6/p21, which may disrupt the role of CDK6 in transcriptionally regulating p21, as part of a negative feedback loop to limit CDK6 excessive activation. PMID:28193906

Genomic and protein expression profiling identifies CDK6 as novel independent prognostic marker in medulloblastoma.

PubMed

Mendrzyk, Frank; Radlwimmer, Bernhard; Joos, Stefan; Kokocinski, Felix; Benner, Axel; Stange, Daniel E; Neben, Kai; Fiegler, Heike; Carter, Nigel P; Reifenberger, Guido; Korshunov, Andrey; Lichter, Peter

2005-12-01

Medulloblastoma is the most common malignant brain tumor in children. Despite multimodal aggressive treatment, nearly half of the patients die as a result of this tumor. Identification of molecular markers for prognosis and development of novel pathogenesis-based therapies depends crucially on a better understanding of medulloblastoma pathomechanisms. We performed genome-wide analysis of DNA copy number imbalances in 47 medulloblastomas using comparative genomic hybridization to large insert DNA microarrays (matrix-CGH). The expression of selected candidate genes identified by matrix-CGH was analyzed immunohistochemically on tissue microarrays representing medulloblastomas from 189 clinically well-documented patients. To identify novel prognostic markers, genomic findings and protein expression data were correlated to patient survival. Matrix-CGH analysis revealed frequent DNA copy number alterations of several novel candidate regions. Among these, gains at 17q23.2-qter (P < .01) and losses at 17p13.1 to 17p13.3 (P = .04) were significantly correlated to poor prognosis. Within 17q23.2-qter and 7q21.2, two of the most frequently gained chromosomal regions, confined amplicons were identified that contained the PPM1D and CDK6 genes, respectively. Immunohistochemistry revealed strong expression of PPM1D in 148 (88%) of 168 and CDK6 in 50 (30%) of 169 medulloblastomas. Overexpression of CDK6 correlated significantly with poor prognosis (P < .01) and represented an independent prognostic marker of overall survival on multivariate analysis (P = .02). We identified CDK6 as a novel molecular marker that can be determined by immunohistochemistry on routinely processed tissue specimens and may facilitate the prognostic assessment of medulloblastoma patients. Furthermore, increased protein-levels of PPM1D and CDK6 may link the TP53 and RB1 tumor suppressor pathways to medulloblastoma pathomechanisms.

Altered miRNA expression in aniline-mediated cell cycle progression in rat spleen.

PubMed

Wang, Gangduo; Wang, Jianling; Khan, M Firoze

2017-09-01

Aniline exposure is associated with toxicity to the spleen, however, early molecular events in aniline-induced cell cycle progression in the spleen remain unknown. MicroRNAs (miRNAs) have been implicated in tumor development by modulating key cell cycle regulators and controlling cell proliferation. This study was, therefore, undertaken on the expression of miRNAs, regulation of cyclins and cyclin-dependent kinases (CDKs) in an experimental condition that precedes a tumorigenic response. Male SD rats were treated with aniline (1 mmol/kg/day by gavage) for 7 days, and expression of miRNAs, cyclins and CDKs in rat spleens were analyzed. Microarray and/or qPCR analyses showed that aniline exposure led to significantly decreased miRNA expression of let-7a, miR-24, miR-34c, miR-100, miR-125b, and greatly increased miR-181a. The aberrant expression of miRNAs was associated with significantly increased protein expression of cyclins A, B1, D3 and E. Furthermore, remarkably enhanced expression of CDKs like CDK1, CDK2, CDK4, CDK6, especially p-CDK1 and p-CDK2 as well as alternations in the expression of pRB, p27, and CDC25A in the spleens of aniline-treated rats was also observed. The data suggest that aniline exposure leads to aberrant expression of miRNAs in the spleen which could be important in the regulation of cell cycle proteins. Our findings, thus, provide new insight into the role of miRNAs in cell cycle progression, which may contribute to aniline-induced tumorigenic response in the spleen.

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

PubMed

Shi, Xi-Nan; Li, Hongjian; Yao, Hong; Liu, Xu; Li, Ling; Leung, Kwong-Sak; Kung, Hsiang-Fu; Lin, Marie Chia-Mi

2015-11-01

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

Clinical role and biological function of CDK5 in hepatocellular carcinoma: A study based on immunohistochemistry, RNA-seq and in vitro investigation.

PubMed

Zhang, Rui; Lin, Peng; Yang, Hong; He, Yun; Dang, Yi-Wu; Feng, Zhen-Bo; Chen, Gang

2017-12-12

To investigate the clinical role and biological function of cyclin-dependent kinase 5 (CDK5) in hepatocellular carcinoma (HCC), 412 surgically resected tissue samples (HCC, n=171; non-HCC=241) were obtained and analyzed with immunohistochemistry. The diagnostic and prognostic values of CDK5 expression levels in HCC were clarified. Moreover, RNA-seq data or microarray datasets from The Cancer Genome Atlas (TCGA) (HCC, n=374; normal, n=50) or other public databases (HCC, n=1864; non-tumor=1995) regarding CDK5 in HCC were extracted and examined. Several bioinformatic methods were performed to identify CDK5-regulated pathways. In vitro experiments were adopted to measure proliferation and apoptosis in HCC cells after CDK5 mRNA was inhibited in the HCC cell lines HepG2 and HepB3. Based on immunohistochemistry, CDK5 expression levels were notably increased in HCC tissues (n=171) compared with normal (n=33, P <0.001), cirrhosis (n=37, P <0.001), and adjacent non-cancerous liver (n=171, P <0.001) tissues. The up-regulation of CDK5 was associated with higher differentiation ( P <0.001), metastasis ( P <0.001), advanced clinical TNM stages ( P <0.001), portal vein tumor embolus ( P =0.003) and vascular invasion ( P =0.004). Additionally, TCGA data analysis also revealed significantly increased CDK5 expression in HCC compared with non-cancerous hepatic tissues ( P <0.001). The pooled standard mean deviation (SMD) based on 36 included datasets (HCC, n=2238; non-cancerous, n=2045) indicated that CDK5 was up-regulated in HCC (SMD=1.23, 95% CI: 1.00-1.45, P <0.001). The area under the curve (AUC) of the summary receiver operating characteristic (SROC) curve was 0.88. Furthermore, CDK5 knock-down inhibited proliferation and promoted apoptosis. In conclusion, CDK5 plays an essential role in the initiation and progression of HCC, most likely via accelerating proliferation and suppressing apoptosis in HCC cells by regulating the cell cycle and DNA replication pathways.

LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

PubMed Central

Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

2012-01-01

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib. PMID:22479189

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

PubMed Central

N, Nagasundaram; Zhu, Hailong; Liu, Jiming; V, Karthick; C, George Priya Doss; Chakraborty, Chiranjib; Chen, Luonan

2015-01-01

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. PMID:26252490

Inactivation of CDK2 is synthetically lethal to MYCN over-expressing cancer cells

PubMed Central

Molenaar, Jan J.; Ebus, Marli E.; Geerts, Dirk; Koster, Jan; Lamers, Fieke; Valentijn, Linda J.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.

2009-01-01

Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics. PMID:19525400

Temporal self-organization of the cyclin/Cdk network driving the mammalian cell cycle

PubMed Central

Gérard, Claude; Goldbeter, Albert

2009-01-01

We propose an integrated computational model for the network of cyclin-dependent kinases (Cdks) that controls the dynamics of the mammalian cell cycle. The model contains four Cdk modules regulated by reversible phosphorylation, Cdk inhibitors, and protein synthesis or degradation. Growth factors (GFs) trigger the transition from a quiescent, stable steady state to self-sustained oscillations in the Cdk network. These oscillations correspond to the repetitive, transient activation of cyclin D/Cdk4–6 in G1, cyclin E/Cdk2 at the G1/S transition, cyclin A/Cdk2 in S and at the S/G2 transition, and cyclin B/Cdk1 at the G2/M transition. The model accounts for the following major properties of the mammalian cell cycle: (i) repetitive cell cycling in the presence of suprathreshold amounts of GF; (ii) control of cell-cycle progression by the balance between antagonistic effects of the tumor suppressor retinoblastoma protein (pRB) and the transcription factor E2F; and (iii) existence of a restriction point in G1, beyond which completion of the cell cycle becomes independent of GF. The model also accounts for endoreplication. Incorporating the DNA replication checkpoint mediated by kinases ATR and Chk1 slows down the dynamics of the cell cycle without altering its oscillatory nature and leads to better separation of the S and M phases. The model for the mammalian cell cycle shows how the regulatory structure of the Cdk network results in its temporal self-organization, leading to the repetitive, sequential activation of the four Cdk modules that brings about the orderly progression along cell-cycle phases. PMID:20007375

Mitotic waves in the early embryogenesis of Drosophila: Bistability traded for speed.

PubMed

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

2018-03-06

Early embryogenesis of most metazoans is characterized by rapid and synchronous cleavage divisions. Chemical waves of Cdk1 activity were previously shown to spread across Drosophila embryos, and the underlying molecular processes were dissected. Here, we present the theory of the physical mechanisms that control Cdk1 waves in Drosophila The in vivo dynamics of Cdk1 are captured by a transiently bistable reaction-diffusion model, where time-dependent reaction terms account for the growing level of cyclins and Cdk1 activation across the cell cycle. We identify two distinct regimes. The first one is observed in mutants of the mitotic switch. There, waves are triggered by the classical mechanism of a stable state invading a metastable one. Conversely, waves in wild type reflect a transient phase that preserves the Cdk1 spatial gradients while the overall level of Cdk1 activity is swept upward by the time-dependent reaction terms. This unique mechanism generates a wave-like spreading that differs from bistable waves for its dependence on dynamic parameters and its faster speed. Namely, the speed of "sweep" waves strikingly decreases as the strength of the reaction terms increases and scales as the powers 3/4, -1/2, and 7/12 of Cdk1 molecular diffusivity, noise amplitude, and rate of increase of Cdk1 activity in the cell-cycle S phase, respectively. Theoretical predictions are supported by numerical simulations and experiments that couple quantitative measurements of Cdk1 activity and genetic perturbations of the accumulation rate of cyclins. Finally, our analysis bears upon the inhibition required to suppress Cdk1 waves at the cell-cycle pause for the maternal-to-zygotic transition.

[Effects of polydatin on learning and memory and Cdk5 kinase activity in the hippocampus of rats with chronic alcoholism].

PubMed

Li, Xin-juan; Zhang, Yan; Xu, Chun-yang; Li, Shuang; Du, Ai-lin; Zhang, Li-bin; Zhang, Rui-ling

2015-03-01

To observe the effects of polydatin on learning and memory and cyclin-dependent kinase 5 (Cdk5) kinase activity in the hippocampus of rats with chronic alcoholism. Forty rats were randomly divided into 4 groups: control group, chronic alcoholism group, low and high polydatin group. The rat chronic alcoholism model was established by ethanol 3.0 g/(kg · d) (intragastric administration). The abstinence scoring was used to evaluate the rats withdrawal symptoms; cognitive function was measured by Morris water maze experiment; Cdk5 protein expression in the hippocampus was detected by immunofluorescence; Cdk5 kinase activity in the hippocampus was detected by liquid scintillation counting method. The abstinence score, escape latency, Cdk5 kinase activity in chronic alcoholism group rats were significantly higher than those of control group (P < 0.05). The abstinence score, escape latency in high polydatin group rats were significantly lower than those of chronic alcoholism group (P < 0.05); Cdk5 kinase activity in high and low polydatin group rats was significantly lower than that of chronic alcoholism group( P < 0.05); immunofluorescence showed that the Cdk5 positive cells of chronic alcoholism group were significantly increased compared with control group (P < 0.05), and the Cdk5 positive cells of polydatin groups were significantly decreased compared with chronic alcoholism group ( P < 0.05). Polydatin-reduced the chronic alcoholism damage may interrelate with regulation of Cdk5 kinase activity.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

PubMed

Bettayeb, Karima; Tirado, Oscar M; Marionneau-Lambot, Séverine; Ferandin, Yoan; Lozach, Olivier; Morris, Jonathan C; Mateo-Lozano, Silvia; Drueckes, Peter; Schächtele, Christoph; Kubbutat, Michael H G; Liger, François; Marquet, Bernard; Joseph, Benoît; Echalier, Aude; Endicott, Jane A; Notario, Vicente; Meijer, Laurent

2007-09-01

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Cyclin-Dependent Kinase Inhibitors for the Treatment of Breast Cancer: Past, Present, and Future.

PubMed

DiPippo, Adam J; Patel, Neelam K; Barnett, Chad M

2016-06-01

Treatment of metastatic breast cancer (MBC) that is resistant to endocrine therapy presents a significant clinical challenge. The well-known role of cell cycle dysregulation in these patients is partly mediated by cyclin-dependent kinase (CDK) activity. Specific cyclin and CDK complexes regulate cell cycle progression by managing the transition through the cell cycle, and inhibition of CDKs represents an important target for novel agents. First-generation CDK inhibitors (e.g., flavopiridol) were relatively nonselective and had an unacceptable toxicity profile in early trials. Second-generation CDK inhibitors were designed to target the CDK4 and CDK6 (CDK4/6) pathway and have shown promising clinical activity with an acceptable toxicity profile in patients with MBC. Palbociclib is a first-in-class CDK4/6 inhibitor that was granted accelerated U.S. Food and Drug Administration approval in combination with letrozole for the treatment of MBC in the first-line setting (February 2015) as well as in combination with fulvestrant for MBC that had progressed on previous endocrine therapy (February 2016). Other CDK4/6 inhibitors, including ribociclib and abemaciclib, are under investigation as monotherapy and in combination with endocrine or anti-human epidermal growth receptor 2 therapy for the treatment of MBC. Ongoing clinical trials should provide additional information to guide the appropriate use of these agents and identify patient populations that could derive the most benefit. © 2016 Pharmacotherapy Publications, Inc.

Increased CDK5 expression in HIV encephalitis contributes to neurodegeneration via tau phosphorylation and is reversed with Roscovitine.

PubMed

Patrick, Christina; Crews, Leslie; Desplats, Paula; Dumaop, Wilmar; Rockenstein, Edward; Achim, Cristian L; Everall, Ian P; Masliah, Eliezer

2011-04-01

Recent treatments with highly active antiretroviral therapy (HAART) regimens have been shown to improve general clinical status in patients with human immunodeficiency virus (HIV) infection; however, the prevalence of cognitive alterations and neurodegeneration has remained the same or has increased. These deficits are more pronounced in the subset of HIV patients with the inflammatory condition known as HIV encephalitis (HIVE). Activation of signaling pathways such as GSK3β and CDK5 has been implicated in the mechanisms of HIV neurotoxicity; however, the downstream mediators of these effects are unclear. The present study investigated the involvement of CDK5 and tau phosphorylation in the mechanisms of neurodegeneration in HIVE. In the frontal cortex of patients with HIVE, increased levels of CDK5 and p35 expression were associated with abnormal tau phosphorylation. Similarly, transgenic mice engineered to express the HIV protein gp120 exhibited increased brain levels of CDK5 and p35, alterations in tau phosphorylation, and dendritic degeneration. In contrast, genetic knockdown of CDK5 or treatment with the CDK5 inhibitor roscovitine improved behavioral performance in the water maze test and reduced neurodegeneration, abnormal tau phosphorylation, and astrogliosis in gp120 transgenic mice. These findings indicate that abnormal CDK5 activation contributes to the neurodegenerative process in HIVE via abnormal tau phosphorylation; thus, reducing CDK5 might ameliorate the cognitive impairments associated with HIVE. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Development of Novel p16INK4a Mimetics as Anticancer Therapy

DTIC Science & Technology

2015-10-01

peptide (or substituted peptide) or the crystal structure of the relevant sequence from p16INK4 ( PDB 1BI7) was used as the starting structure . Model...small peptides that interact with CDK4/6. The specific aims are as follows. (1) Determine structure -function relationships of overlapping peptides...Determine structure -function relationships of overlapping peptides derived from p16 INK4a that inhibit the activity of CDK4/6 and identify stabilized

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases

PubMed Central

2010-01-01

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation. PMID:24900195

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.

PubMed

Paruch, Kamil; Dwyer, Michael P; Alvarez, Carmen; Brown, Courtney; Chan, Tin-Yau; Doll, Ronald J; Keertikar, Kerry; Knutson, Chad; McKittrick, Brian; Rivera, Jocelyn; Rossman, Randall; Tucker, Greg; Fischmann, Thierry; Hruza, Alan; Madison, Vincent; Nomeir, Amin A; Wang, Yaolin; Kirschmeier, Paul; Lees, Emma; Parry, David; Sgambellone, Nicole; Seghezzi, Wolfgang; Schultz, Lesley; Shanahan, Frances; Wiswell, Derek; Xu, Xiaoying; Zhou, Quiao; James, Ray A; Paradkar, Vidyadhar M; Park, Haengsoon; Rokosz, Laura R; Stauffer, Tara M; Guzi, Timothy J

2010-08-12

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Treating cancer with selective CDK4/6 inhibitors.

PubMed

O'Leary, Ben; Finn, Richard S; Turner, Nicholas C

2016-07-01

Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting.

SAR study on N2,N4-disubstituted pyrimidine-2,4-diamines as effective CDK2/CDK9 inhibitors and antiproliferative agents† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ra01440j

PubMed Central

Jing, Liandong; Tang, Yanbo; Goto, Masuo; Lee, Kuo-Hsiung

2018-01-01

Cyclin-dependent kinases (CDKs) are pivotal kinases in cell cycle transition and gene transcription. A series of N2,N4-diphenylpyrimidine-2,4-diamines were previously identified as potent CDK2/CDK9 inhibitors. To explore the SAR of this structural prototype, twenty-four novel N2,N4-disubstituted pyrimidine-2,4-diamines were designed and synthesized. Among them, twenty-one compounds exhibited potent inhibitory activities against both CDK2/cyclin A and CDK9/cyclin T1 systems, and the most potent CDK2 and CDK9 inhibitors, 3g and 3c, showed IC50 values of 83 nM and 65 nM respectively. Most of these compounds displayed significant inhibition against the tested tumor cell lines in the SRB assay, and in particular, remained active against the triple-negative breast cancer (TNBC) cell line MDA-MB-231. Flow cytometer analysis of compounds 2a, 2d and 3b in MDA-MB-231 cells indicated that these compounds induced cell cycle arrest in G2/M phase. Docking studies on compound 3g were performed, which provided conducive clues for further molecular optimization. PMID:29682280

Uniting Cheminformatics and Chemical Theory To Predict the Intrinsic Aqueous Solubility of Crystalline Druglike Molecules

PubMed Central

2014-01-01

We present four models of solution free-energy prediction for druglike molecules utilizing cheminformatics descriptors and theoretically calculated thermodynamic values. We make predictions of solution free energy using physics-based theory alone and using machine learning/quantitative structure–property relationship (QSPR) models. We also develop machine learning models where the theoretical energies and cheminformatics descriptors are used as combined input. These models are used to predict solvation free energy. While direct theoretical calculation does not give accurate results in this approach, machine learning is able to give predictions with a root mean squared error (RMSE) of ∼1.1 log S units in a 10-fold cross-validation for our Drug-Like-Solubility-100 (DLS-100) dataset of 100 druglike molecules. We find that a model built using energy terms from our theoretical methodology as descriptors is marginally less predictive than one built on Chemistry Development Kit (CDK) descriptors. Combining both sets of descriptors allows a further but very modest improvement in the predictions. However, in some cases, this is a statistically significant enhancement. These results suggest that there is little complementarity between the chemical information provided by these two sets of descriptors, despite their different sources and methods of calculation. Our machine learning models are also able to predict the well-known Solubility Challenge dataset with an RMSE value of 0.9–1.0 log S units. PMID:24564264

Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates.

PubMed

Daigh, Leighton H; Liu, Chad; Chung, Mingyu; Cimprich, Karlene A; Meyer, Tobias

2018-06-04

Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events. Copyright © 2018. Published by Elsevier Inc.

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

PubMed Central

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

2016-01-01

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 CHIPS20A, but not CHIPWT, 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. PMID:26206088

Climatic droplet keratopathy, exfoliation syndrome, and cataract.

PubMed Central

Resnikoff, S; Filliard, G; Dell'Aquila, B

1991-01-01

During a countrywide survey we assessed the prevalence of climatic droplet keratopathy (CDK) in a randomised sample of 2446 subjects representative of the population of the Republic of Djibouti. The investigation of the relationship between CDK and two diseases considered to be related to exposure to ultra-violet light--namely, exfoliation syndrome and cataract--was planned as a case control study. In the rural area prevalence of CDK was five times higher than in the urban one (2.8% vs 0.5%) and varied according to districts. The highest rates were observed where the inhabitants' activities were related to the sea. The case control study revealed that the opacification of the lens was about three times commoner in patients with CDK than in patients without CDK (p = 0.03) and that the exfoliation syndrome was about six times commoner in patients with CDK than in controls of similar age, sex, climatic conditions, and lens status (p = 0.02). Moreover, we noticed that the sequelae of corneal perforations were about 30 times commoner in patients with CDK than in controls of similar age (p less than 0.00001). PMID:1768663

Developing Save Your Food Kit (Sayofu Kit) to Support Inquiry, Improve Student Learning Outcomes at SMP Plus Hidayatul Mubtadiin and Public Awareness on Food Additives

NASA Astrophysics Data System (ADS)

Astutik, J.

2017-02-01

Food additives are materials that can not be separated from the lives of students and the community. Based on the preliminary questionnaire, it indicates the lack of kit supporting material additives in some schools and communities. The research objectives of this development are (1) to develop Kit experiment (SAYOFU KIT) and supplementary books to improve student learning outcomes in the classroom and public awareness on food additives (2) to describe the feasibility and potential effectiveness of SAYOFU KIT developed (3) to analyze the practice of SAYOFU KIT and benefits for students and the community. This development study uses 4-D models Thiagarajan, et al (1974). Through some stages, they are: defining, designing, developing and disseminating which involes the students and community. The developed SAYOFU KIT includes additives sample kit, borax test kit, curcumin test kit, formaldehyde test kit, modification heater to the identification of dyes and dye test paper. The study is conducted at SMP Plus Hidayatul Mubtadiin, and TKIT Al Uswah. The products are validated by experts and education practitioners. Qualitative data processing uses descriptive method, whereas quantitative data by using the N-gain. The average yield of expert validation of SAYOFU KIT with supplementary books 76.50% teacher’s book and 76.30% student’s book are eligible. The average yield of 96.81% validation of educational practitioners criteria, piloting a small group of 83.15%, and 82.89% field trials are very decent. The average yield on the student questionnaire responses SAYOFU kit and supplementary book is 87.6% with the criteria very well worth it. N-Gain 0:56 cognitive achievement with the criteria enough. The results of the public poll showed 95% feel the benefits SAYOFU kits for testing food. Based from description indicates that SAYOFU Kit developed feasible, practical, useful to support inquiry learning and improve student learning outcomes as well as public awareness of food additives.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

PubMed

Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

2015-04-20

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cdk5 Contributes to Huntington's Disease Learning and Memory Deficits via Modulation of Brain Region-Specific Substrates.

PubMed

Alvarez-Periel, Elena; Puigdellívol, Mar; Brito, Verónica; Plattner, Florian; Bibb, James A; Alberch, Jordi; Ginés, Silvia

2017-12-29

Cognitive deficits are a major hallmark of Huntington's disease (HD) with a great impact on the quality of patient's life. Gaining a better understanding of the molecular mechanisms underlying learning and memory impairments in HD is, therefore, of critical importance. Cdk5 is a proline-directed Ser/Thr kinase involved in the regulation of synaptic plasticity and memory processes that has been associated with several neurodegenerative disorders. In this study, we aim to investigate the role of Cdk5 in learning and memory impairments in HD using a novel animal model that expresses mutant huntingtin (mHtt) and has genetically reduced Cdk5 levels. Genetic reduction of Cdk5 in mHtt knock-in mice attenuated both corticostriatal learning deficits as well as hippocampal-dependent memory decline. Moreover, the molecular mechanisms by which Cdk5 counteracts the mHtt-induced learning and memory impairments appeared to be differentially regulated in a brain region-specific manner. While the corticostriatal learning deficits are attenuated through compensatory regulation of NR2B surface levels, the rescue of hippocampal-dependent memory was likely due to restoration of hippocampal dendritic spine density along with an increase in Rac1 activity. This work identifies Cdk5 as a critical contributor to mHtt-induced learning and memory deficits. Furthermore, we show that the Cdk5 downstream targets involved in memory and learning decline differ depending on the brain region analyzed suggesting that distinct Cdk5 effectors could be involved in cognitive impairments in HD.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

PubMed

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Two Bistable Switches Govern M Phase Entry.

PubMed

Mochida, Satoru; Rata, Scott; Hino, Hirotsugu; Nagai, Takeharu; Novák, Béla

2016-12-19

The abrupt and irreversible transition from interphase to M phase is essential to separate DNA replication from chromosome segregation. This transition requires the switch-like phosphorylation of hundreds of proteins by the cyclin-dependent kinase 1 (Cdk1):cyclin B (CycB) complex. Previous studies have ascribed these switch-like phosphorylations to the auto-activation of Cdk1:CycB through the removal of inhibitory phosphorylations on Cdk1-Tyr15 [1, 2]. The positive feedback in Cdk1 activation creates a bistable switch that makes mitotic commitment irreversible [2-4]. Here, we surprisingly find that Cdk1 auto-activation is dispensable for irreversible, switch-like mitotic entry due to a second mechanism, whereby Cdk1:CycB inhibits its counteracting phosphatase (PP2A:B55). We show that the PP2A:B55-inhibiting Greatwall (Gwl)-endosulfine (ENSA) pathway is both necessary and sufficient for switch-like phosphorylations of mitotic substrates. Using purified components of the Gwl-ENSA pathway in a reconstituted system, we found a sharp Cdk1 threshold for phosphorylation of a luminescent mitotic substrate. The Cdk1 threshold to induce mitotic phosphorylation is distinctly higher than the Cdk1 threshold required to maintain these phosphorylations-evidence for bistability. A combination of mathematical modeling and biochemical reconstitution show that the bistable behavior of the Gwl-ENSA pathway emerges from its mutual antagonism with PP2A:B55. Our results demonstrate that two interlinked bistable mechanisms provide a robust solution for irreversible and switch-like mitotic entry. Copyright © 2016 Elsevier Ltd. All rights reserved.

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

PubMed

Hossain, Manzar; Stillman, Bruce

2012-08-15

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

Dependence of Cisplatin-Induced Cell Death In Vitro and In Vivo on Cyclin-Dependent Kinase 2

PubMed Central

Price, Peter M.; Yu, Fang; Kaldis, Philipp; Aleem, Eiman; Nowak, Grażyna; Safirstein, Robert L.; Megyesi, Judit

2006-01-01

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity. PMID:16914540

Mutations in CDK5RAP2 cause Seckel syndrome.

PubMed

Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

2015-09-01

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 shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

Mutations in CDK5RAP2 cause Seckel syndrome

PubMed Central

Yigit, Gökhan; Brown, Karen E; Kayserili, Hülya; Pohl, Esther; Caliebe, Almuth; Zahnleiter, Diana; Rosser, Elisabeth; Bögershausen, Nina; Uyguner, Zehra Oya; Altunoglu, Umut; Nürnberg, Gudrun; Nürnberg, Peter; Rauch, Anita; Li, Yun; Thiel, Christian Thomas; Wollnik, Bernd

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 shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152. PMID:26436113

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

PubMed Central

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-01-01

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division. PMID:24714560

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression.

PubMed

Landry, Benjamin D; Mapa, Claudine E; Arsenault, Heather E; Poti, Kristin E; Benanti, Jennifer A

2014-05-02

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

Triple negative breast cancer therapy with CDK1 siRNA delivered by cationic lipid assisted PEG-PLA nanoparticles.

PubMed

Liu, Yang; Zhu, Yan-Hua; Mao, Cheng-Qiong; Dou, Shuang; Shen, Song; Tan, Zi-Bin; Wang, Jun

2014-10-28

There is no effective clinical therapy yet for triple-negative breast cancer (TNBC) without particular human epidermal growth factor receptor-2, estrogen and progesterone receptor expression. In this study, we report a molecularly targeted and synthetic lethality-based siRNA therapy for TNBC treatment, using cationic lipid assisted poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PLA) nanoparticles as the siRNA carrier. It is demonstrated that only in c-Myc overexpressed TNBC cells, while not in normal mammary epithelial cells, delivery of siRNA targeting cyclin-dependent kinase 1 (CDK1) with the nanoparticle carrier (NPsiCDK1) induces cell viability decreasing and cell apoptosis through RNAi-mediated CDK1 expression inhibition, indicating the synthetic lethality between c-Myc with CDK1 in TNBC cells. Moreover, systemic delivery of NPsiCDK1 is able to suppress tumor growth in mice bearing SUM149 and BT549 xenograft and cause no systemic toxicity or activate the innate immune response, suggesting the therapeutic promise with such nanoparticles carrying siCDK1 for c-Myc overexpressed triple negative breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

A Clb/Cdk1-mediated regulation of Fkh2 synchronizes CLB expression in the budding yeast cell cycle.

PubMed

Linke, Christian; Chasapi, Anastasia; González-Novo, Alberto; Al Sawad, Istabrak; Tognetti, Silvia; Klipp, Edda; Loog, Mart; Krobitsch, Sylvia; Posas, Francesc; Xenarios, Ioannis; Barberis, Matteo

2017-01-01

Precise timing of cell division is achieved by coupling waves of cyclin-dependent kinase (Cdk) activity with a transcriptional oscillator throughout cell cycle progression. Although details of transcription of cyclin genes are known, it is unclear which is the transcriptional cascade that modulates their expression in a timely fashion. Here, we demonstrate that a Clb/Cdk1-mediated regulation of the Fkh2 transcription factor synchronizes the temporal mitotic CLB expression in budding yeast. A simplified kinetic model of the cyclin/Cdk network predicts a linear cascade where a Clb/Cdk1-mediated regulation of an activator molecule drives CLB3 and CLB2 expression. Experimental validation highlights Fkh2 as modulator of CLB3 transcript levels, besides its role in regulating CLB2 expression. A Boolean model based on the minimal number of interactions needed to capture the information flow of the Clb/Cdk1 network supports the role of an activator molecule in the sequential activation, and oscillatory behavior, of mitotic Clb cyclins. This work illustrates how transcription and phosphorylation networks can be coupled by a Clb/Cdk1-mediated regulation that synchronizes them.

Neuroprotective effects of the anti-cancer drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of neurodegenerative disorders.

PubMed

Wrasidlo, Wolf; Crews, Leslie A; Tsigelny, Igor F; Stocking, Emily; Kouznetsova, Valentina L; Price, Diana; Paulino, Amy; Gonzales, Tania; Overk, Cassia R; Patrick, Christina; Rockenstein, Edward; Masliah, Eliezer

2014-12-01

Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 μM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders. © 2014 The British Pharmacological Society.

Inhibition of herpes simplex virus type 1 by the CDK6 inhibitor PD-0332991 (palbociclib) through the control of SAMHD1.

PubMed

Badia, Roger; Angulo, Guillem; Riveira-Muñoz, Eva; Pujantell, Maria; Puig, Teresa; Ramirez, Cristina; Torres-Torronteras, Javier; Martí, Ramón; Pauls, Eduardo; Clotet, Bonaventura; Ballana, Ester; Esté, José A

2016-02-01

Sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) has been shown to restrict retroviruses and DNA viruses by decreasing the pool of intracellular deoxynucleotides. In turn, SAMHD1 is controlled by cyclin-dependent kinases (CDK) that regulate the cell cycle and cell proliferation. Here, we explore the effect of CDK6 inhibitors on the replication of herpes simplex virus type 1 (HSV-1) in primary monocyte-derived macrophages (MDM). MDM were treated with palbociclib, a selective CDK4/6 inhibitor, and then infected with a GFP-expressing HSV-1. Intracellular deoxynucleotide triphosphate (dNTP) content was determined using a polymerase-based method. CDK6 inhibitor palbociclib blocked SAMHD1 phosphorylation, intracellular dNTP levels and HSV-1 replication in MDM at subtoxic concentrations. Treatment of MDM with palbociclib reduced CDK2 activation, measured as the phosphorylation of the T-loop at Thr160. The antiviral activity of palbociclib was lost when SAMHD1 was degraded by viral protein X. Similarly, palbociclib did not block HSV-1 replication in SAMHD1-negative Vero cells at subtoxic concentrations, providing further evidence for a role of SAMHD1 in mediating the antiviral effect. SAMHD1-mediated HSV-1 restriction is controlled by CDK and points to a preferential role for CDK6 and CDK2 as mediators of SAMHD1 activation. Similarly, the restricting activity of SAMHD1 against DNA viruses suggests that control of dNTP availability is the major determinant of its antiviral activity. This is the first study describing the anti-HSV-1 activity of palbociclib. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Phase II trial of the CDK4 inhibitor PD0332991 in patients with advanced CDK4-amplified well-differentiated or dedifferentiated liposarcoma.

PubMed

Dickson, Mark A; Tap, William D; Keohan, Mary Louise; D'Angelo, Sandra P; Gounder, Mrinal M; Antonescu, Cristina R; Landa, Jonathan; Qin, Li-Xuan; Rathbone, Dustin D; Condy, Mercedes M; Ustoyev, Yelena; Crago, Aimee M; Singer, Samuel; Schwartz, Gary K

2013-06-01

CDK4 is amplified in > 90% of well-differentiated (WDLS) and dedifferentiated liposarcomas (DDLS). The selective cyclin-dependent kinase 4 (CDK4)/CDK6 inhibitor PD0332991 inhibits growth and induces senescence in cell lines and xenografts. In a phase I trial of PD0332991, several patients with WDLS or DDLS experienced prolonged stable disease. We performed an open-label phase II study to determine the safety and efficacy of PD0332991 in patients with advanced WDLS/DDLS. Patients age ≥ 18 years experiencing disease progression while receiving systemic therapy before enrollment received PD0332991 200 mg orally once per day for 14 consecutive days in 21-day cycles. All were required to have CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein (RB) expression by immunohistochemistry (≥ 1+). The primary end point was progression-free survival (PFS) at 12 weeks, with 12-week PFS of ≥ 40% considered promising and ≤ 20% not promising. If ≥ nine of 28 patients were progression free at 12 weeks, PD0332991 would be considered active. We screened 48 patients (44 of 48 had CDK4 amplification; 41 of 44 were RB positive). Of those, 30 were enrolled, and 29 were evaluable for the primary end point. Grade 3 to 4 events included anemia (17%), thrombocytopenia (30%), neutropenia (50%), and febrile neutropenia (3%). At 12 weeks, PFS was 66% (90% CI, 51% to 100%), significantly exceeding the primary end point. The median PFS was 18 weeks. There was one partial response. Treatment with the CDK4 inhibitor PD0332991 was associated with a favorable progression-free rate in patients with CDK4-amplified and RB-expressing WDLS/DDLS who had progressive disease despite systemic therapy.

The BDNF/TrkB signaling pathway is involved in heat hyperalgesia mediated by Cdk5 in rats.

PubMed

Zhang, Hong-Hai; Zhang, Xiao-Qin; Xue, Qing-Sheng; Yan-Luo; Huang, Jin-Lu; Zhang, Su; Shao, Hai-Jun; Lu, Han; Wang, Wen-Yuan; Yu, Bu-Wei

2014-01-01

Cyclin-dependent kinase 5 (Cdk5) has been shown to play an important role in mediating inflammation-induced heat hyperalgesia. However, the underlying mechanism remains unclear. The aim of this study was to determine whether roscovitine, an inhibitor of Cdk5, could reverse the heat hyperalgesia induced by peripheral injection of complete Freund's adjuvant (CFA) via the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling pathway in the dorsal horn of the spinal cord in rats. Heat hyperalgesia induced by peripheral injection of CFA was significantly reversed by roscovitine, TrkB-IgG, and the TrkB inhibitor K252a, respectively. Furthermore, BDNF was significantly increased from 0.5 h to 24 h after CFA injection in the spinal cord dorsal horn. Intrathecal adminstration of the Cdk5 inhibitor roscovitine had no obvious effects on BDNF levels. Increased TrkB protein level was significantly reversed by roscovitine between 0.5 h and 6 h after CFA injection. Cdk5 and TrkB co-immunoprecipitation results suggested Cdk5 mediates the heat hyperalgesia induced by CFA injection by binding with TrkB, and the binding between Cdk5 and TrkB was markedly blocked by intrathecal adminstration of roscovitine. Our data suggested that the BDNF-TrkB signaling pathway was involved in CFA-induced heat hyperalgesia mediated by Cdk5. Roscovitine reversed the heat hyperalgesia induced by peripheral injection of CFA by blocking BDNF/TrkB signaling pathway, suggesting that severing the close crosstalk between Cdk5 and the BDNF/TrkB signaling cascade may present a potential target for anti-inflammatory pain.

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

PubMed

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. © 2016 Höckner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A single cyclin–CDK complex is sufficient for both mitotic and meiotic progression in fission yeast

PubMed Central

Gutiérrez-Escribano, Pilar; Nurse, Paul

2015-01-01

The dominant model for eukaryotic cell cycle control proposes that cell cycle progression is driven by a succession of CDK complexes with different substrate specificities. However, in fission yeast it has been shown that a single CDK complex generated by the fusion of the Cdc13 cyclin with the CDK protein Cdc2 can drive the mitotic cell cycle. Meiosis is a modified cell cycle programme in which a single S-phase is followed by two consecutive rounds of chromosome segregation. Here we systematically analyse the requirements of the different fission yeast cyclins for meiotic cell cycle progression. We also show that a single Cdc13–Cdc2 complex, in the absence of the other cyclins, can drive the meiotic cell cycle. We propose that qualitatively different CDK complexes are not absolutely required for cell cycle progression either during mitosis or meiosis, and that a single CDK complex can drive both cell cycle programmes. PMID:25891897

Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation

PubMed Central

Arsenault, Heather E.; Roy, Jagoree; Mapa, Claudine E.; Cyert, Martha S.; Benanti, Jennifer A.

2015-01-01

Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation. PMID:26269584

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

PubMed

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Virtual screening studies to design potent CDK2-cyclin A inhibitors.

PubMed

Vadivelan, S; Sinha, Barij Nayan; Irudayam, Sheeba Jem; Jagarlapudi, Sarma A R P

2007-01-01

The cell division cycle is controlled by cyclin-dependent kinases (CDK), which consist of a catalytic subunit (CDK1-CDK8) and a regulatory subunit (cyclin A-H). Pharmacophore analysis indicates that the best inhibitor model consists of (1) two hydrogen bond acceptors, (2) one hydrogen bond donor, and (3) one hydrophobic feature. The HypoRefine pharmacophore model gave an enrichment factor of 1.31 and goodness of fit score of 0.76. Docking studies were carried out to explore the structural requirements for the CDK2-cyclin A inhibitors and to construct highly predictive models for the design of new inhibitors. Docking studies demonstrate the important role of hydrogen bond and hydrophobic interactions in determining the inhibitor-receptor binding affinity. The validated pharmacophore model is further used for retrieving the most active hits/lead from a virtual library of molecules. Subsequently, docking studies were performed on the hits, and novel series of potent leads were suggested based on the interaction energy between CDK2-cyclin A and the putative inhibitors.

CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin

PubMed Central

Tarasewicz, Elizabeth; Hamdan, Randala; Straehla, Joelle; Hardy, Ashley; Nunez, Omar; Zelivianski, Stanislav; Dokic, Danijela; Jeruss, Jacqueline S

2014-01-01

Cyclin D1/CDK4 activity is upregulated in up to 50% of breast cancers and CDK4-mediated phosphorylation negatively regulates the TGFβ superfamily member Smad3. We sought to determine if CDK4 inhibition and doxorubicin chemotherapy could impact Smad3-mediated cell/colony growth and apoptosis in breast cancer cells. Parental and cyclin D1-overexpressing MCF7 cells were treated with CDK4 inhibitor, doxorubicin, or combination therapy and cell proliferation, apoptosis, colony formation, and expression of apoptotic proteins were evaluated using an MTS assay, TUNEL staining, 3D Matrigel assay, and apoptosis array/immunoblotting. Study cells were also transduced with WT Smad3 or a Smad3 construct resistant to CDK4 phosphorylation (5M) and colony formation and expression of apoptotic proteins were assessed. Treatment with CDK4 inhibitor/doxorubicin combination therapy, or transduction with 5M Smad3, resulted in a similar decrease in colony formation. Treating cyclin D overexpressing breast cancer cells with combination therapy also resulted in the greatest increase in apoptosis, resulted in decreased expression of anti-apoptotic proteins survivin and XIAP, and impacted subcellular localization of pro-apoptotic Smac/DIABLO. Additionally, transduction of 5M Smad3 and doxorubicin treatment resulted in the greatest change in apoptotic protein expression. Collectively, this work showed the impact of CDK4 inhibitor-mediated, Smad3-regulated tumor suppression, which was augmented in doxorubicin-treated cyclin D-overexpressing study cells. PMID:25006666

Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis.

PubMed

Domingo-Sananes, Maria Rosa; Kapuy, Orsolya; Hunt, Tim; Novak, Bela

2011-12-27

Activation of the cyclin-dependent kinase (Cdk1) cyclin B (CycB) complex (Cdk1:CycB) in mitosis brings about a remarkable extent of protein phosphorylation. Cdk1:CycB activation is switch-like, controlled by two auto-amplification loops--Cdk1:CycB activates its activating phosphatase, Cdc25, and inhibits its inhibiting kinase, Wee1. Recent experimental evidence suggests that parallel to Cdk1:CycB activation during mitosis, there is inhibition of its counteracting phosphatase activity. We argue that the downregulation of the phosphatase is not just a simple latch that suppresses futile cycles of phosphorylation/dephosphorylation during mitosis. Instead, we propose that phosphatase regulation creates coherent feed-forward loops and adds extra amplification loops to the Cdk1:CycB regulatory network, thus forming an integral part of the mitotic switch. These network motifs further strengthen the bistable characteristic of the mitotic switch, which is based on the antagonistic interaction of two groups of proteins: M-phase promoting factors (Cdk1:CycB, Cdc25, Greatwall and Endosulfine/Arpp19) and interphase promoting factors (Wee1, PP2A-B55 and a Greatwall counteracting phosphatase, probably PP1). The bistable character of the switch implies the existence of a CycB threshold for entry into mitosis. The end of G2 phase is determined by the point where CycB level crosses the CycB threshold for Cdk1 activation.

CDK activity provides temporal and quantitative cues for organizing genome duplication

PubMed Central

Perrot, Anthony; Millington, Christopher Lee; Gómez-Escoda, Blanca; Schausi-Tiffoche, Diane

2018-01-01

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome. PMID:29466359

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans

PubMed Central

van Rijnberk, Lotte M.; van der Horst, Suzanne E. M.; van den Heuvel, Sander; Ruijtenberg, Suzan

2017-01-01

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit. PMID:28158315

Tailored first-line and second-line CDK4-targeting treatment combinations in mouse models of pancreatic cancer.

PubMed

Chou, Angela; Froio, Danielle; Nagrial, Adnan M; Parkin, Ashleigh; Murphy, Kendelle J; Chin, Venessa T; Wohl, Dalia; Steinmann, Angela; Stark, Rhys; Drury, Alison; Walters, Stacey N; Vennin, Claire; Burgess, Andrew; Pinese, Mark; Chantrill, Lorraine A; Cowley, Mark J; Molloy, Timothy J; Waddell, Nicola; Johns, Amber; Grimmond, Sean M; Chang, David K; Biankin, Andrew V; Sansom, Owen J; Morton, Jennifer P; Grey, Shane T; Cox, Thomas R; Turchini, John; Samra, Jaswinder; Clarke, Stephen J; Timpson, Paul; Gill, Anthony J; Pajic, Marina

2017-10-28

Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Adaptation of commercial biomarker kits and proposal for 'drug development kits' to support bioanalysis: call for action.

PubMed

Islam, Rafiqul; Kar, Sumit; Islam, Clarinda; Farmen, Raymond

2018-06-01

There has been an increased use of commercial kits for biomarker measurement, commensurate with the increased demand for biomarkers in drug development. However, in most cases these kits do not meet the quality attributes for use in regulated environment. The process for adaptation of these kits can be frustrating, time consuming and resource intensive. In addition, a lack of harmonized guidance for the validation of biomarker poses a significant challenge in the adaptation of kits in a regulated environment. The purpose of this perspective is to propose a tiered approach to commercial drug development kits with clearly defined quality attributes and to demonstrate how these kits can be adapted to perform analytical validation in a regulated environment.

Azolium analogues as CDK4 inhibitors: Pharmacophore modeling, 3D QSAR study and new lead drug discovery

NASA Astrophysics Data System (ADS)

Rondla, Rohini; Padma Rao, Lavanya Souda; Ramatenki, Vishwanath; Vadija, Rajender; Mukkera, Thirupathi; Potlapally, Sarita Rajender; Vuruputuri, Uma

2017-04-01

The cyclin-dependent kinase 4 (CDK4) enzyme is a key regulator in cell cycle G1 phase progression. It is often overexpressed in variety of cancer cells, which makes it an attractive therapeutic target for cancer treatment. A number of chemical scaffolds have been reported as CDK4 inhibitors in the literature, and in particular azolium scaffolds as potential inhibitors. Here, a ligand based pharmacophore modeling and an atom based 3D-QSAR analyses for a series of azolium based CDK4 inhibitors are presented. A five point pharmacophore hypothesis, i.e. APRRR with one H-bond acceptor (A), one positive cationic feature (P) and three ring aromatic sites (R) is developed, which yielded an atom based 3D-QSAR model that shows an excellent correlation coefficient value- R2 = 0.93, fisher ratio- F = 207, along with good predictive ability- Q2 = 0.79, and Pearson R value = 0.89. The visual inspection of the 3D-QSAR model, with the most active and the least active ligands, demonstrates the favorable and unfavorable structural regions for the activity towards CDK4. The roles of positively charged nitrogen, the steric effect, ligand flexibility, and the substituents on the activity are in good agreement with the previously reported experimental results. The generated 3D QSAR model is further applied as query for a 3D database screening, which identifies 23 lead drug candidates with good predicted activities and diverse scaffolds. The ADME analysis reveals that, the pharmacokinetic parameters of all the identified new leads are within the acceptable range.

CDK4/6 Inhibitors: Game Changers in the Management of Hormone Receptor–Positive Advanced Breast Cancer?

PubMed

Shah, Mirat; Nunes, Maria Raquel; Stearns, Vered

2018-05-15

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors palbociclib, ribociclib, and abemaciclib are rapidly transforming the care of patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) advanced breast cancer. Current clinical questions include how to choose among these agents and how to sequence them with other therapies. Areas of active inquiry include identifying predictive biomarkers for CDK4/6 inhibitors, deciding whether to continue CDK4/6 inhibitors after disease progression, creating novel treatment combinations, and expanding use beyond HR+/HER2- advanced breast cancer. Here, we review the current use of and potential next directions for CDK4/6 inhibitors in the treatment of patients with HR+ breast cancer.

Cyclin C influences the timing of mitosis in fission yeast.

PubMed

Banyai, Gabor; Szilagyi, Zsolt; Baraznenok, Vera; Khorosjutina, Olga; Gustafsson, Claes M

2017-07-01

The multiprotein Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Mediator contains the Cdk8 regulatory subcomplex, which directs periodic transcription and influences cell cycle progression in fission yeast. Here we investigate the role of CycC, the cognate cyclin partner of Cdk8, in cell cycle control. Previous reports suggested that CycC interacts with other cellular Cdks, but a fusion of CycC to Cdk8 reported here did not cause any obvious cell cycle phenotypes. We find that Cdk8 and CycC interactions are stabilized within the Mediator complex and the activity of Cdk8-CycC is regulated by other Mediator components. Analysis of a mutant yeast strain reveals that CycC, together with Cdk8, primarily affects M-phase progression but mutations that release Cdk8 from CycC control also affect timing of entry into S phase. © 2017 Banyai et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Cell-cell contact regulates gene expression in CDK4-transformed mouse podocytes.

PubMed

Sakairi, Toru; Abe, Yoshifusa; Jat, Parmijit S; Kopp, Jeffrey B

2010-10-01

We transformed mouse podocytes by ectopic expression of cyclin-dependent kinase 4 (CDK4). Compared with podocytes transformed with a thermo-sensitive SV40 large T antigen mutant tsA58U19 (tsT podocytes), podocytes transformed with CDK4 (CDK4 podocytes) exhibited significantly higher expression of nephrin mRNA. Synaptopodin mRNA expression was significantly lower in CDK4 podocytes and in tsT podocytes under growth-permissive conditions (33°C) compared with tsT podocytes under growth-restricted conditions (37°C), which suggests a role for cell cycle arrest in synaptopodin mRNA expression. Confluent CDK4 podocytes showed significantly higher mRNA expression levels for nephrin, synaptopodin, Wilms tumor 1, podocalyxin, and P-cadherin compared with subconfluent cultures. We carried out experiments to clarify roles of various factors in the confluent podocyte cultures; our findings indicate that cell-cell contact promotes expression of five podocyte marker genes studied, that cellular quiescence increases synaptopodin and podocalyxin mRNA expression, and that soluble factors play a role in nephrin mRNA expression. Our findings suggest that CDK4 podocytes are useful tools to study podocyte biology. Furthermore, the role of cell-cell contact in podocyte gene expression may have relevance for podocyte function in vivo.

Computational study of the inhibitory mechanism of the kinase CDK5 hyperactivity by peptide p5 and derivation of a pharmacophore

NASA Astrophysics Data System (ADS)

Cardone, A.; Brady, M.; Sriram, R.; Pant, H. C.; Hassan, S. A.

2016-06-01

The hyperactivity of the cyclic dependent kinase 5 (CDK5) induced by the activator protein p25 has been linked to a number of pathologies of the brain. The CDK5-p25 complex has thus emerged as a major therapeutic target for Alzheimer's disease (AD) and other neurodegenerative conditions. Experiments have shown that the peptide p5 reduces the CDK5-p25 activity without affecting the endogenous CDK5-p35 activity, whereas the peptide TFP5, obtained from p5, elicits similar inhibition, crosses the blood-brain barrier, and exhibits behavioral rescue of AD mice models with no toxic side effects. The molecular basis of the kinase inhibition is not currently known, and is here investigated by computer simulations. It is shown that p5 binds the kinase at the same CDK5/p25 and CDK5/p35 interfaces, and is thus a non-selective competitor of both activators, in agreement with available experimental data in vitro. Binding of p5 is enthalpically driven with an affinity estimated in the low µM range. A quantitative description of the binding site and pharmacophore is presented, and options are discussed to increase the binding affinity and selectivity in the design of drug-like compounds against AD.

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

PubMed Central

Poon, R Y; Yamashita, K; Adamczewski, J P; Hunt, T; Shuttleworth, J

1993-01-01

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit. Images PMID:8393783

The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.

PubMed

Poon, R Y; Yamashita, K; Adamczewski, J P; Hunt, T; Shuttleworth, J

1993-08-01

Activation of the cyclin-dependent protein kinases p34cdc2 and p33cdk2 requires binding with a cyclin partner and phosphorylation on the first threonine residue in the sequence THEVVTLWYRAPE. We present evidence that this threonine residue, number 160 in p33cdk2, can be specifically phosphorylated by a cdc2-related protein kinase from Xenopus oocytes called p40MO15. Binding to cyclin A and phosphorylation of this threonine are both required to activate fully the histone H1 kinase activity of p33cdk2. In cell extracts, a portion of p40MO15 is found in a high molecular weight complex that is considerably more active than a lower molecular weight form. Wild-type MO15 protein expressed in bacteria does not possess kinase activity, but acquires p33cdk2-T160 kinase activity after incubation with cell extract and ATP. We conclude that p40MO15 corresponds to CAK (cdc2/cdk2 activating kinase) and speculate that, like p33cdk2 and p34cdc2, p40MO15 requires activation by phosphorylation and association with a companion subunit.

Rapid Cdc13 turnover and telomere length homeostasis are controlled by Cdk1-mediated phosphorylation of Cdc13.

PubMed

Tseng, Shun-Fu; Shen, Zih-Jie; Tsai, Hung-Ji; Lin, Yi-Hsuan; Teng, Shu-Chun

2009-06-01

Budding yeast telomerase is mainly activated by Tel1/Mec1 (yeast ATM/ATR) on Cdc13 from late S to G2 phase of the cell cycle. Here, we demonstrated that the telomerase-recruitment domain of Cdc13 is also phosphorylated by Cdk1 at the same cell cycle stage as the Tel1/Mec1-dependent regulation. Phosphor-specific gel analysis demonstrated that Cdk1 phosphorylates residues 308 and 336 of Cdc13. The residue T308 of Cdc13 is critical for efficient Mec1-mediated S306 phosphorylation in vitro. Phenotypic analysis in vivo revealed that the mutations in the Cdc13 S/TP motifs phosphorylated by Cdk1 caused cell cycle delay and telomere shortening and these phenotypes could be partially restored by the replacement with a negative charge residue. In the absence of Ku or Tel1, Cdk1-mediated phosphorylation of Cdc13 showed no effect on telomere length maintenance. Moreover, this Cdk1-mediated phosphorylation was required to promote the regular turnover of Cdc13. Together these results demonstrate that Cdk1 phosphorylates the telomerase recruitment domain of Cdc13, thereby preserves optimal function and expression level of Cdc13 for precise telomere replication and cell cycle progression.

Cyclin-dependent kinase 8 module expression profiling reveals requirement of mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila.

PubMed

Kuuluvainen, Emilia; Hakala, Heini; Havula, Essi; Sahal Estimé, Michelle; Rämet, Mika; Hietakangas, Ville; Mäkelä, Tomi P

2014-06-06

The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. Here we have investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Detailed dissection of the chromosomal region containing the Ph1 locus in wheat Triticum aestivum: with deletion mutants and expression profiling.

PubMed

Al-Kaff, Nadia; Knight, Emilie; Bertin, Isabelle; Foote, Tracie; Hart, Nicola; Griffiths, Simon; Moore, Graham

2008-04-01

Understanding Ph1, a dominant homoeologous chromosome pairing suppressor locus on the long arm of chromosome 5B in wheat Triticum aestivum L., is the core of the investigation in this article. The Ph1 locus restricts chromosome pairing and recombination at meiosis to true homologues. The importance of wheat as a crop and the need to exploit its wild relatives as donors for economically important traits in wheat breeding programmes is the main drive to uncover the mechanism of the Ph1 locus and regulate its activity. Following the molecular genetic characterization of the Ph1 locus, five additional deletion mutants covering the region have been identified. In addition, more bacterial artificial chromosomes (BACs) were sequenced and analysed to elucidate the complexity of this locus. A semi-quantitative RT-PCR was used to compare the expression profiles of different genes in the 5B region containing the Ph1 locus with their homoeologues on 5A and 5D. PCR products were cloned and sequenced to identify the gene from which they were derived. Deletion mutants and expression profiling of genes in the region containing the Ph1 locus on 5B has further restricted Ph1 to a cluster of cdk-like genes. Bioinformatic analysis of the cdk-like genes revealed their close homology to the checkpoint kinase Cdk2 from humans. Cdk2 is involved in the initiation of replication and is required in early meiosis. Expression profiling has revealed that the cdk-like gene cluster is unique within the region analysed on 5B in that these genes are transcribed. Deletion of the cdk-like locus on 5B results in activation of transcription of functional cdk-like copies on 5A and 5D. Thus the cdk locus on 5B is dominant to those on 5A and 5D in determining the overall activity, which will be dependent on a complex interplay between transcription from non-functional and functional cdk-like genes. The Ph1 locus has been defined to a cdk-like gene cluster related to Cdk2 in humans, a master checkpoint gene involved in the initiation of replication and required for early meiosis.

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

PubMed

Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

2016-02-01

Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. Copyright © 2016 by the Genetics Society of America.

Detection of MDM2/CDK4 amplification in lipomatous soft tissue tumors from formalin-fixed, paraffin-embedded tissue: comparison of multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridization (FISH).

PubMed

Creytens, David; van Gorp, Joost; Ferdinande, Liesbeth; Speel, Ernst-Jan; Libbrecht, Louis

2015-02-01

In this study, the detection of MDM2 and CDK4 amplification was evaluated in lipomatous soft tissue tumors using multiplex ligation-dependent probe amplification (MLPA), a PCR-based technique, in comparison with fluorescence in situ hybridization (FISH). These 2 techniques were evaluated in a series of 77 formalin-fixed, paraffin-embedded lipomatous tumors (27 benign adipose tumors, 28 atypical lipomatous tumors/well-differentiated liposarcomas, 18 dedifferentiated liposarcomas, and 4 pleomorphic liposarcomas). Using MLPA, with a cut-off ratio of >2, 36/71 samples (22 atypical lipomatous tumors/well-differentiated liposarcomas, and 14 dedifferentiated liposarcomas) showed MDM2 and CDK4 amplification. Using FISH as gold standard, MLPA showed a sensitivity of 90% (36/40) and a specificity of 100% (31/31) in detecting amplification of MDM2 and CDK4 in lipomatous soft tissue tumors. In case of high-level amplification (MDM2-CDK4/CEP12 ratio >5), concordance was 100%. Four cases of atypical lipomatous tumor/well-differentiated liposarcoma (4/26, 15%) with a low MDM2 and CDK4 amplification level (MDM2-CDK4/CEP12 ratio ranging between 2 and 2.5) detected by FISH showed no amplification by MLPA, although gain of MDM2 and CDK4 (ratios ranging between 1.6 and 1.9) was seen with MLPA. No amplification was detected in benign lipomatous tumors and pleomorphic liposarcomas. Furthermore, there was a very high concordance between the ratios obtained by FISH and MLPA. In conclusion, MLPA proves to be an appropriate and straightforward technique for screening MDM2/CDK4 amplification in lipomatous tumors, especially when a correct cut-off value and reference samples are chosen, and could be considered a good alternative to FISH to determine MDM2 and CDK4 amplification in liposarcomas. Moreover, because MLPA, as a multiplex technique, allows simultaneous detection of multiple chromosomal changes of interest, it could be in the future a very reliable and fast molecular analysis on paraffin-embedded material to test for other diagnostically, prognostically, or therapeutically relevant genomic mutations in lipomatous tumors.

CDK9-Dependent Transcriptional Elongation in the Innate Interferon-Stimulated Gene Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells

PubMed Central

Tian, Bing; Zhao, Yingxin; Kalita, Mridul; Edeh, Chukwudi B.; Paessler, Slobodan; Casola, Antonella; Teng, Michael N.; Garofalo, Roberto P.

2013-01-01

Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections. During infection, the presence of double-stranded RNA (dsRNA) activates the interferon (IFN) regulatory factor 3 (IRF3) transcription factor, an event triggering expression of immediate early, IFN-stimulated genes (ISGs). We examine the role of transcriptional elongation in control of IRF3-dependent ISG expression. RSV infection induces ISG54, ISG56, and CIG5 gene expression in an IRF3-dependent manner demonstrated by IRF3 small interfering RNA (siRNA) silencing in both A549 epithelial cells and IRF3−/− MEFs. ISG expression was mediated by the recruitment of IRF3, CDK9, polymerase II (Pol II), and phospho-Ser2 carboxy-terminal domain (CTD) Pol II to the IFN-stimulated response element (ISRE) binding sites of the IRF3-dependent ISG promoters in native chromatin. We find that RSV infection enhances the activated fraction of cyclin-dependent kinase 9 (CDK9) by promoting its association with bromodomain 4 (BRD4) and disrupting its association with the inhibitory 7SK small nuclear RNA. The requirement of CDK9 activity for ISG expression was shown by siRNA-mediated silencing of CDK9 and by a selective CDK9 inhibitor in A549 cells. In contrast, RSV-induced beta interferon (IFN-β) expression is not influenced by CDK9 inhibition. Using transcript-selective quantitative real-time reverse transcription-PCR (Q-RT-PCR) assays for the ISG54 gene, we observed that RSV induces transition from short to fully spliced mRNA transcripts and that this transition is blocked by CDK9 inhibition in both A549 and primary human small airway epithelial cells. These data indicate that transcription elongation plays a major role in RSV-induced ISG expression and is mediated by IRF3-dependent recruitment of activated CDK9. CDK9 activity may be a target for immunomodulation in RSV-induced lung disease. PMID:23596302

Cyclin-dependent Kinase 5: Novel role of gene variants identified in ADHD.

PubMed

Maitra, Subhamita; Chatterjee, Mahasweta; Sinha, Swagata; Mukhopadhyay, Kanchan

2017-07-28

Cortical neuronal migration and formation of filamentous actin cytoskeleton, needed for development, normal cell growth and differentiation, are regulated by the cyclin-dependent kinase 5 (Cdk5). Attention deficit hyperactivity disorder (ADHD) is associated with delayed maturation of the brain and hence we hypothesized that cdk5 may have a role in ADHD. Eight functional CDK5 gene variants were analyzed in 848 Indo-Caucasoid individuals including 217 families with ADHD probands and 250 healthy volunteers. Only three variants, rs2069454, rs2069456 and rs2069459, predicted to affect transcription, were found to be bimorphic. Significant difference in rs2069456 "AC" genotype frequency was noticed in the probands, more specifically in the males. Family based analysis revealed over transmission of rs2069454 "C" and rs2069456 "A" to the probands. Quantitative trait analysis exhibited association of haplotypes with inattention, domain specific impulsivity, and behavioral problem, though no significant contribution was noticed on the age of onset of ADHD. Gene variants also showed significant association with cognitive function and co-morbidity. Probands having rs2069459 "TT" showed betterment during follow up. It may be inferred from this pilot study that CDK5 may affect ADHD etiology, possibly by attenuating synaptic neurotransmission and could be a useful target for therapeutic intervention.

Overexpression of Cdk5 or non-phosphorylatable retinoblastoma protein protects septal neurons from oxygen-glucose deprivation.

PubMed

Panickar, Kiran S; Nonner, Doris; White, Michael G; Barrett, John N

2008-09-01

Activation of cyclin dependent kinases (Cdks) contributes to neuronal death following ischemia. We used oxygen-glucose deprivation (OGD) in septal neuronal cultures to test for possible roles of cell cycle proteins in neuronal survival. Increased cdc2-immunoreactive neurons were observed at 24 h after the end of 5 h OGD. Green fluorescent protein (GFP) or GFP along with a wild type or dominant negative form of the retinoblastoma protein (Rb), or cyclin-dependent kinase5 (Cdk5), were overexpressed using plasmid constructs. Following OGD, when compared to controls, neurons expressing both GFP and dominant negative Rb, RbDeltaK11, showed significantly less damage using microscopy imaging. Overexpression of Rb-wt did not affect survival. Surprisingly, overexpression of Cdk5-wild type significantly protected neurons from process disintegration but Cdk5T33, a dominant negative Cdk5, gave little or no protection. Thus phosphorylation of the cell cycle regulator, Rb, contributes to death in OGD in septal neurons but Cdk5 can have a protective role.

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

PubMed Central

Costa-Cabral, Sara; Brough, Rachel; Konde, Asha; Aarts, Marieke; Campbell, James; Marinari, Eliana; Riffell, Jenna; Bardelli, Alberto; Torrance, Christopher; Lord, Christopher J.; Ashworth, Alan

2016-01-01

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G1/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation. PMID:26881434

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

PubMed Central

Lee, Janet; Baek, Jeong-Hwa; Choi, Kyu-Sil; Kim, Hyun-Soo; Park, Hye-Young; Ha, Geun-Hyoung; Park, Ho; Lee, Kyo-Won; Lee, Chang Geun; Yang, Dong-Yun; Moon, Hyo Eun; Paek, Sun Ha; Lee, Chang-Woo

2013-01-01

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs. PMID:23324348

Regulation of lipogenesis by cyclin-dependent kinase 8–mediated control of SREBP-1

PubMed Central

Zhao, Xiaoping; Feng, Daorong; Wang, Qun; Abdulla, Arian; Xie, Xiao-Jun; Zhou, Jie; Sun, Yan; Yang, Ellen S.; Liu, Lu-Ping; Vaitheesvaran, Bhavapriya; Bridges, Lauren; Kurland, Irwin J.; Strich, Randy; Ni, Jian-Quan; Wang, Chenguang; Ericsson, Johan; Pessin, Jeffrey E.; Ji, Jun-Yuan; Yang, Fajun

2012-01-01

Altered lipid metabolism underlies several major human diseases, including obesity and type 2 diabetes. However, lipid metabolism pathophysiology remains poorly understood at the molecular level. Insulin is the primary stimulator of hepatic lipogenesis through activation of the SREBP-1c transcription factor. Here we identified cyclin-dependent kinase 8 (CDK8) and its regulatory partner cyclin C (CycC) as negative regulators of the lipogenic pathway in Drosophila, mammalian hepatocytes, and mouse liver. The inhibitory effect of CDK8 and CycC on de novo lipogenesis was mediated through CDK8 phosphorylation of nuclear SREBP-1c at a conserved threonine residue. Phosphorylation by CDK8 enhanced SREBP-1c ubiquitination and protein degradation. Importantly, consistent with the physiologic regulation of lipid biosynthesis, CDK8 and CycC proteins were rapidly downregulated by feeding and insulin, resulting in decreased SREBP-1c phosphorylation. Moreover, overexpression of CycC efficiently suppressed insulin and feeding–induced lipogenic gene expression. Taken together, these results demonstrate that CDK8 and CycC function as evolutionarily conserved components of the insulin signaling pathway in regulating lipid homeostasis. PMID:22684109

Atomistic simulations and network-based modeling of the Hsp90-Cdc37 chaperone binding with Cdk4 client protein: A mechanism of chaperoning kinase clients by exploiting weak spots of intrinsically dynamic kinase domains

PubMed Central

Czemeres, Josh; Buse, Kurt

2017-01-01

A fundamental role of the Hsp90 and Cdc37 chaperones in mediating conformational development and activation of diverse protein kinase clients is essential in signal transduction. There has been increasing evidence that the Hsp90-Cdc37 system executes its chaperoning duties by recognizing conformational instability of kinase clients and modulating their folding landscapes. The recent cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex has provided a framework for dissecting regulatory principles underlying differentiation and recruitment of protein kinase clients to the chaperone machinery. In this work, we have combined atomistic simulations with protein stability and network-based rigidity decomposition analyses to characterize dynamic factors underlying allosteric mechanism of the chaperone-kinase cycle and identify regulatory hotspots that control client recognition. Through comprehensive characterization of conformational dynamics and systematic identification of stabilization centers in the unbound and client- bound Hsp90 forms, we have simulated key stages of the allosteric mechanism, in which Hsp90 binding can induce instability and partial unfolding of Cdk4 client. Conformational landscapes of the Hsp90 and Cdk4 structures suggested that client binding can trigger coordinated dynamic changes and induce global rigidification of the Hsp90 inter-domain regions that is coupled with a concomitant increase in conformational flexibility of the kinase client. This process is allosteric in nature and can involve reciprocal dynamic exchanges that exert global effect on stability of the Hsp90 dimer, while promoting client instability. The network-based rigidity analysis and emulation of thermal unfolding of the Cdk4-cyclin D complex and Hsp90-Cdc37-Cdk4 complex revealed weak spots of kinase instability that are present in the native Cdk4 structure and are targeted by the chaperone during client recruitment. Our findings suggested that this mechanism may be exploited by the Hsp90-Cdc37 chaperone to recruit and protect intrinsically dynamic kinase clients from degradation. The results of this investigation are discussed and interpreted in the context of diverse experimental data, offering new insights into mechanisms of chaperone regulation and binding. PMID:29267381

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

PubMed

Parua, Pabitra K; Booth, Gregory T; Sansó, Miriam; Benjamin, Bradley; Tanny, Jason C; Lis, John T; Fisher, Robert P

2018-06-13

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .

A novel anticancer agent, decursin, induces G1 arrest and apoptosis in human prostate carcinoma cells.

PubMed

Yim, Dongsool; Singh, Rana P; Agarwal, Chapla; Lee, Sookyeon; Chi, Hyungjoon; Agarwal, Rajesh

2005-02-01

We isolated a coumarin compound decursin (C(19)H(20)O(5); molecular weight 328) from Korean angelica (Angelica gigas) root and characterized it by spectroscopy. Here, for the first time, we observed that decursin (25-100 micromol/L) treatment for 24 to 96 hours strongly inhibits growth and induces death in human prostate carcinoma DU145, PC-3, and LNCaP cells. Furthermore, we observed that decursinol [where (CH(3))(2)-C=CH-COO- side chain of decursin is substituted with -OH] has much lower effects compared with decursin, suggesting a possible structure-activity relationship. Decursin-induced growth inhibition was associated with a strong G(1) arrest (P < 0.001) in DU145 and LNCaP cells, and G(1), S as well as G(2)-M arrests depending upon doses and treatment times in PC-3 cells. Comparatively, decursin was nontoxic to human prostate epithelial PWR-1E cells and showed only moderate growth inhibition and G(1) arrest. Consistent with G(1) arrest in DU145 cells, decursin strongly increased protein levels of Cip1/p21 but showed a moderate increase in Kip1/p27 with a decrease in cyclin-dependent kinases (CDK); CDK2, CDK4, CDK6, and cyclin D1, and inhibited CDK2, CDK4, CDK6, cyclin D1, and cyclin E kinase activity, and increased binding of CDK inhibitor (CDKI) with CDK. Decursin-caused cell death was associated with an increase in apoptosis (P < 0.05-0.001) and cleaved caspase-9, caspase-3, and poly(ADP-ribose) polymerase; however, pretreatment with all-caspases inhibitor (z-VAD-fmk) only partially reversed decursin-induced apoptosis, suggesting the involvement of both caspase-dependent and caspase-independent pathways. These findings suggest the novel anticancer efficacy of decursin mediated via induction of cell cycle arrest and apoptosis selectively in human prostate carcinoma cells.

CDC-25.1 controls the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively regulating CDK-1 in Caenorhabditis elegans.

PubMed

Yoon, Sunghee; Kawasaki, Ichiro; Shim, Yhong-Hee

2012-04-01

In Caenorhabditis elegans, cdc-25.1 loss-of-function mutants display a lack of germline proliferation. We found that the proliferation defect of cdc-25.1 mutants was suppressed by wee-1.3 RNAi. Further, among the seven cdk and seven cyclin homologs examined, cdk-1 and cyb-3 RNAi treatment caused the most severe germline proliferation defects in an rrf-1 mutant background, which were similar to those of the cdc-25.1 mutants. In addition, while RNAi of cyd-1 and cye-1 caused significant germline proliferation defects, RNAi of cdk-2 and cdk-4 did not. Compared with the number of germ nuclei in wee-1.3(RNAi) worms, the number in wee-1.3(RNAi);cdk-1(RNAi) and wee-1.3(RNAi);cyb-3(RNAi) worms further decreased to the level of cdk-1(RNAi) and cyb-3(RNAi) worms, respectively, indicating that cdk-1 and cyb-3 are epistatic and function downstream of cdc-25.1 and wee-1.3 in the control of the cell cycle. BrdU labeling of adult worms showed that, while 100% of the wild-type germ nuclei in the mitotic region incorporated BrdU when labeled for more than 12 h at 20°C, a small fraction of the cdc-25.1 mutant germ nuclei failed to incorporate BrdU even when labeled for 68 h. These results indicate that CDC-25.1 is required for maintaining proper rate of germline mitotic cell cycle. We propose that CDC-25.1 regulates the rate of germline mitotic cell cycle by counteracting WEE-1.3 and by positively controlling CDK-1, which forms a complex primarily with CYB-3, but also possibly with CYD-1 and CYE-1.

The cyclin D1-CDK4 oncogenic interactome enables identification of potential novel oncogenes and clinical prognosis

PubMed Central

Jirawatnotai, Siwanon; Sharma, Samanta; Michowski, Wojciech; Suktitipat, Bhoom; Geng, Yan; Quackenbush, John; Elias, Joshua E; Gygi, Steven P; Wang, Yaoyu E; Sicinski, Piotr

2014-01-01

Overexpression of cyclin D1 and its catalytic partner, CDK4, is frequently seen in human cancers. We constructed cyclin D1 and CDK4 protein interaction network in a human breast cancer cell line MCF7, and identified novel CDK4 protein partners. Among CDK4 interactors we observed several proteins functioning in protein folding and in complex assembly. One of the novel partners of CDK4 is FKBP5, which we found to be required to maintain CDK4 levels in cancer cells. An integrative analysis of the extended cyclin D1 cancer interactome and somatic copy number alterations in human cancers identified BAIAPL21 as a potential novel human oncogene. We observed that in several human tumor types BAIAPL21 is expressed at higher levels as compared to normal tissue. Forced overexpression of BAIAPL21 augmented anchorage independent growth, increased colony formation by cancer cells and strongly enhanced the ability of cells to form tumors in vivo. Lastly, we derived an Aggregate Expression Score (AES), which quantifies the expression of all cyclin D1 interactors in a given tumor. We observed that AES has a prognostic value among patients with ER-positive breast cancers. These studies illustrate the utility of analyzing the interactomes of proteins involved in cancer to uncover potential oncogenes, or to allow better cancer prognosis. PMID:25486477

Climatic Droplet Keratopathy in Argentina: Involvement of Environmental Agents in Its Genesis Which Would Open the Prospect for New Therapeutic Interventions

PubMed Central

Suárez, María Fernanda; Correa, Leandro; Crim, Nicolás; Espósito, Evangelina; Monti, Rodolfo; Urrets-Zavalía, Julio Alberto; Serra, Horacio Marcelo

2015-01-01

Climatic droplet keratopathy (CDK) is a degenerative corneal disease of unknown etiology. We described CDK for the first time in Latin America in the Argentinean Patagonia (El Cuy). A deeper knowledge of CDK pathogenic mechanisms will provide new therapeutic strategies. For that reason we investigated the prevalence of CDK in El Cuy and its existence in other 3 provinces with similar climate. Patients eyes were examined, habits throughout lives were inquired about, and serum ascorbate (sAA) was determined. All individuals work outdoors for most of the day. All regions had normal O3 levels. Individuals from regions 1, 2, and 3 had very low consumption of vegetables/fruits and low sAA levels. Conversely, region 4 individuals had balanced diet and higher sAA concentrations. CDK was only found in region 3 where individuals had partial deficiency of sAA and did not use eye protection. No CDK was found in regions 1 and 2 where individuals had similar work activities and dietary habits to those in region 3 but wear eye protection. No disease was found in region 4 where individuals work outdoors, have balanced diet, and use eye protection. To summarize, the CDK existence was related not only to climate but also to the dietary habits and lack of protection from sunlight. PMID:26451372

Climatic Droplet Keratopathy in Argentina: Involvement of Environmental Agents in Its Genesis Which Would Open the Prospect for New Therapeutic Interventions.

PubMed

Suárez, María Fernanda; Correa, Leandro; Crim, Nicolás; Espósito, Evangelina; Monti, Rodolfo; Urrets-Zavalía, Julio Alberto; Serra, Horacio Marcelo

2015-01-01

Climatic droplet keratopathy (CDK) is a degenerative corneal disease of unknown etiology. We described CDK for the first time in Latin America in the Argentinean Patagonia (El Cuy). A deeper knowledge of CDK pathogenic mechanisms will provide new therapeutic strategies. For that reason we investigated the prevalence of CDK in El Cuy and its existence in other 3 provinces with similar climate. Patients eyes were examined, habits throughout lives were inquired about, and serum ascorbate (sAA) was determined. All individuals work outdoors for most of the day. All regions had normal O3 levels. Individuals from regions 1, 2, and 3 had very low consumption of vegetables/fruits and low sAA levels. Conversely, region 4 individuals had balanced diet and higher sAA concentrations. CDK was only found in region 3 where individuals had partial deficiency of sAA and did not use eye protection. No CDK was found in regions 1 and 2 where individuals had similar work activities and dietary habits to those in region 3 but wear eye protection. No disease was found in region 4 where individuals work outdoors, have balanced diet, and use eye protection. To summarize, the CDK existence was related not only to climate but also to the dietary habits and lack of protection from sunlight.

DEVELOPMENT OF AN IDENTIFICATION KIT FOR SPILLED HAZARDOUS MATERIALS

EPA Science Inventory

The Chemical Systems Laboratory (CSL) has developed a field kit to identify spilled hazardous materials in inland waters and on the ground. The Hazardous Materials Spills Identification Kit is a two-component kit consisting of an inverter/shortwave UV lamp unit for photochemical ...

Pirfenidone Induces G1 Arrest in Human Tenon's Fibroblasts In Vitro Involving AKT and MAPK Signaling Pathways.

PubMed

Guo, Xiujuan; Yang, Yangfan; Liu, Liling; Liu, Xiaoan; Xu, Jiangang; Wu, Kaili; Yu, Minbin

2017-06-01

To investigate the underlying mechanism by which pirfenidone blocks the transition from the G1 to S phase in primary human Tenon's fibroblasts. Primary human Tenon's fibroblasts were characterized by immunocytofluorescence staining with vimentin, fibroblast surface protein, and cytokeratin. After treating Tenon's fibroblasts with pirfenidone under proliferation conditions (10% fetal bovine serum), cell proliferation was measured using a WST-1 assay. Progression through the cell cycle was analyzed by flow cytometry. The expression of CDK2, CDK6, cyclinD1, cyclinD3, and cyclinE and the phosphorylation of AKT, ERK1/2/MAPK, JNK/MAPK, and p38 MAPK were estimated using western blot analysis. Under proliferative conditions, pirfenidone inhibited Tenon's fibroblasts proliferation and arrested the cell cycle at the G1 phase; decreased the phosphorylation of AKT, GSK3β, ERK1/2/MAPK, and JNK/MAPK; increased the phosphorylation of p38 MAPK; and inhibited CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E in a dose-dependent manner. Inhibitors of AKT (LY294002), ERK1/2 (U0126), and JNK (SP600125) arrested the G1/S transition, similar to the effect of pirfenidone. The p38 inhibitor (SB202190) decreased the G1-blocking effect of pirfenidone. The expression of CDK2, CDK6, cyclin D1, and cyclin D3 were inhibited by LY294002, U0126, and SP600125. SB202190 attenuated the pirfenidone-induced reduction of CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E. Pirfenidone inhibited HTFs proliferation and induced G1 arrest by downregulating CDKs and cyclins involving the AKT/GSK3β and MAPK signaling pathways.

Genetic Background and Climatic Droplet Keratopathy Incidence in a Mapuche Population from Argentina

PubMed Central

Schurr, Theodore G.; Dulik, Matthew C.; Cafaro, Thamara A.; Suarez, María F.

2013-01-01

Purpose To determine whether the incidence of and susceptibility to climatic droplet keratopathy (CDK), an acquired, often bilateral degenerative corneal disease, is influenced by the genetic background of the individuals who exhibit the disorder. Methods To determine whether the disease expression was influenced by the genetic ancestry of CDK cases in native Mapuche of the northwest area of Patagonia in Argentina, we examined mitochondrial DNA and Y-chromosome variation in 53 unrelated individuals. Twenty-nine of them were part of the CDK (patient) population, while 24 were part of the control group. The analysis revealed the maternal and paternal lineages that were present in the two study groups. Results This analysis demonstrated that nearly all persons had a Native American mtDNA background, whereas 50% of the CDK group and 37% of the control group had Native American paternal ancestry, respectively. There was no significant difference in the frequencies of mtDNA haplogroups between the CDK patient and control groups. Although the Y-chromosome data revealed differences in specific haplogroup frequencies between these two groups, there was no statistically significant relationship between individual paternal genetic backgrounds and the incidence or stage of disease. Conclusions These results indicate a lack of correlation between genetic ancestry as represented by haploid genetic systems and the incidence of CDK in Mapuche populations. In addition, the mtDNA appears to play less of a role in CDK expression than for other complex diseases linked to bioenergetic processes. However, further analysis of the mtDNA genome sequence and other genes involved in corneal function may reveal the more precise role that mitochondria play in the expression of CDK. PMID:24040292

Genetic background and climatic droplet keratopathy incidence in a Mapuche population from Argentina.

PubMed

Schurr, Theodore G; Dulik, Matthew C; Cafaro, Thamara A; Suarez, María F; Urrets-Zavalia, Julio A; Serra, Horacio M

2013-01-01

To determine whether the incidence of and susceptibility to climatic droplet keratopathy (CDK), an acquired, often bilateral degenerative corneal disease, is influenced by the genetic background of the individuals who exhibit the disorder. To determine whether the disease expression was influenced by the genetic ancestry of CDK cases in native Mapuche of the northwest area of Patagonia in Argentina, we examined mitochondrial DNA and Y-chromosome variation in 53 unrelated individuals. Twenty-nine of them were part of the CDK (patient) population, while 24 were part of the control group. The analysis revealed the maternal and paternal lineages that were present in the two study groups. This analysis demonstrated that nearly all persons had a Native American mtDNA background, whereas 50% of the CDK group and 37% of the control group had Native American paternal ancestry, respectively. There was no significant difference in the frequencies of mtDNA haplogroups between the CDK patient and control groups. Although the Y-chromosome data revealed differences in specific haplogroup frequencies between these two groups, there was no statistically significant relationship between individual paternal genetic backgrounds and the incidence or stage of disease. These results indicate a lack of correlation between genetic ancestry as represented by haploid genetic systems and the incidence of CDK in Mapuche populations. In addition, the mtDNA appears to play less of a role in CDK expression than for other complex diseases linked to bioenergetic processes. However, further analysis of the mtDNA genome sequence and other genes involved in corneal function may reveal the more precise role that mitochondria play in the expression of CDK.

Loss of MutL Disrupts CHK2-Dependent Cell-Cycle Control through CDK4/6 to Promote Intrinsic Endocrine Therapy Resistance in Primary Breast Cancer.

PubMed

Haricharan, Svasti; Punturi, Nindo; Singh, Purba; Holloway, Kimberly R; Anurag, Meenakshi; Schmelz, Jacob; Schmidt, Cheryl; Lei, Jonathan T; Suman, Vera; Hunt, Kelly; Olson, John A; Hoog, Jeremy; Li, Shunqiang; Huang, Shixia; Edwards, Dean P; Kavuri, Shyam M; Bainbridge, Matthew N; Ma, Cynthia X; Ellis, Matthew J

2017-10-01

Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen receptor-positive (ER + ) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex ( MLH1/3 , PMS1/2 ), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER + breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER + breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor-resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care. Significance: MutL deficiency in a subset of ER + primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy-resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168-83. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047 . ©2017 American Association for Cancer Research.

Specific CDK4/6 inhibition in breast cancer: a systematic review of current clinical evidence

PubMed Central

Polk, Anne; Kolmos, Ida Lykke; Kümler, Iben; Nielsen, Dorte Lisbeth

2016-01-01

Background Loss of cell cycle control is a hallmark of cancer, and aberrations in the cyclin-dependent kinase-retinoblastoma (CDK-Rb) pathway are common in breast cancer (BC). Consequently, inhibition of this pathway is an attractive therapeutic strategy. The present review addresses efficacy and toxicity of CDK4/6 inhibition in BC. Methods A literature search was carried out using PubMed and EMBASE; data reported at international meetings and clinicaltrials.gov were included. Results Three specific CDK4/6 inhibitors palbociclib, abemaciclib and ribociclib are tested in clinical trials. A randomised phase II trial of palbociclib plus letrozole versus letrozole and a phase III of palbociclib plus fulvestrant versus fulvestrant showed significantly increased progression-free survival when compared with endocrine therapy alone in first-line and second-line treatment for advanced hormone receptor-positive HER2-negative BC. At the moment several phase III studies are ongoing with all three CDK4/6 inhibitors in hormone receptor-positive HER2-negative BC as well as other subtypes of BC. The predominant toxicity of agents was limited neutropenia. Other common adverse events were infections, fatigue and gastrointestinal toxicity. The toxicities seemed manageable. Yet data are too limited to differentiate between the compounds. Retinoblastoma protein (Rb) is considered a promising biomarker. Conclusion CDK4/6 inhibition might represent a substantial advance for patients with hormone receptor-positive HER2-negative BC. Results must be confirmed in phase III trials before any firm conclusions can be made regarding the future influence of CDK4/6 inhibition. There is an urgent need for prospective biomarker-driven trials to identify patients for whom CDK4/6 inhibition is cost-effective. PMID:28848657

Disrupting Cyclin Dependent Kinase 1 in Spermatocytes Causes Late Meiotic Arrest and Infertility in Mice1

PubMed Central

Clement, Tracy M.; Inselman, Amy L.; Goulding, Eugenia H.; Willis, William D.; Eddy, Edward M.

2015-01-01

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes. PMID:26490841

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

PubMed Central

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

2014-01-01

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. PMID:25139855

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

PubMed Central

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. PMID:27068241

MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.

PubMed

Adamczewski, J P; Rossignol, M; Tassan, J P; Nigg, E A; Moncollin, V; Egly, J M

1996-04-15

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.

MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.

PubMed Central

Adamczewski, J P; Rossignol, M; Tassan, J P; Nigg, E A; Moncollin, V; Egly, J M

1996-01-01

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation. Images PMID:8617234

Phosphorylation of caspase-9 at Thr125 directs paclitaxel resistance in ovarian cancer.

PubMed

Byun, Mi Ran; Choi, Jin Woo

2018-01-02

Although paclitaxel is routinely prescribed for the treatment of epithelial ovarian cancer (EOC), paclitaxel resistance is common in EOC and correlates with short survival of patients. A previous pharmacogenomic study revealed the importance of cyclin-dependent kinase 1 (CDK1) activity in a response on paclitaxel. However, a subsequent research showed that the expression level of CDK1 failed to show significant correlation with delayed apoptosis and patient survival. Rather, the expression and phosphorylation of capase-9, the downstream target molecule of CDK1, appeared to determine drug resistance. Our results suggest that treatment with the CDK1 inhibitor alsterpaullone reduces phosphorylation of caspase-9. Its phosphorylation level was dependent on CDK1 activity and it directs paclitaxel resistance. This observation was reproducible in xenografted tumors. Thus, the regulation of caspase-9 may be a novel therapeutic strategy to reverse paclitaxel-induced resistance in ovarian cancer cells.

GridKit

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

Peles, Slaven

2016-11-06

GridKit is a software development kit for interfacing power systems and power grid application software with high performance computing (HPC) libraries developed at National Labs and academia. It is also intended as interoperability layer between different numerical libraries. GridKit is not a standalone application, but comes with a suite of test examples illustrating possible usage.

The Flavonoid Apigenin Downregulates CDK1 by Directly Targeting Ribosomal Protein S9

PubMed Central

Iizumi, Yosuke; Oishi, Masakatsu; Taniguchi, Tomoyuki; Goi, Wakana; Sowa, Yoshihiro; Sakai, Toshiyuki

2013-01-01

Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth. PMID:24009741

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

PubMed

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication

PubMed Central

Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

2015-01-01

Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. DOI: http://dx.doi.org/10.7554/eLife.07519.001 PMID:26297806

CDK6 protects epithelial ovarian cancer from platinum-induced death via FOXO3 regulation.

PubMed

Dall'Acqua, Alessandra; Sonego, Maura; Pellizzari, Ilenia; Pellarin, Ilenia; Canzonieri, Vincenzo; D'Andrea, Sara; Benevol, Sara; Sorio, Roberto; Giorda, Giorgio; Califano, Daniela; Bagnoli, Marina; Militello, Loredana; Mezzanzanica, Delia; Chiappetta, Gennaro; Armenia, Joshua; Belletti, Barbara; Schiappacassi, Monica; Baldassarre, Gustavo

2017-10-01

Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum-based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro , in xenografts in vivo , and in primary tumor cells derived from platinum-treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Mediator can regulate mitotic entry and direct periodic transcription in fission yeast.

PubMed

Banyai, Gabor; Lopez, Marcela Davila; Szilagyi, Zsolt; Gustafsson, Claes M

2014-11-01

Cdk8 is required for correct timing of mitotic progression in fission yeast. How the activity of Cdk8 is regulated is unclear, since the kinase is not activated by T-loop phosphorylation and its partner, CycC, does not oscillate. Cdk8 is, however, a component of the multiprotein Mediator complex, a conserved coregulator of eukaryotic transcription that is connected to a number of intracellular signaling pathways. We demonstrate here that other Mediator components regulate the activity of Cdk8 in vivo and thereby direct the timing of mitotic entry. Deletion of Mediator components Med12 and Med13 leads to higher cellular Cdk8 protein levels, premature phosphorylation of the Cdk8 target Fkh2, and earlier entry into mitosis. We also demonstrate that Mediator is recruited to clusters of mitotic genes in a periodic fashion and that the complex is required for the transcription of these genes. We suggest that Mediator functions as a hub for coordinated regulation of mitotic progression and cell cycle-dependent transcription. The many signaling pathways and activator proteins shown to function via Mediator may influence the timing of these cell cycle events. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Real-time polymerase chain reaction analysis of MDM2 and CDK4 expression using total RNA from core-needle biopsies is useful for diagnosing adipocytic tumors

PubMed Central

2014-01-01

Background Diagnosing adipocytic tumors can be challenging because it is often difficult to morphologically distinguish between benign, intermediate and malignant adipocytic tumors, and other sarcomas that are histologically similar. Recently, a number of tumor-specific chromosome translocations and associated fusion genes have been identified in adipocytic tumors and atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDL), which have a supernumerary ring and/or giant chromosome marker with amplified sequences of the MDM2 and CDK4 genes. The purpose of this study was to investigate whether quantitative real-time polymerase chain reaction (PCR) could be used to amplify MDM2 and CDK4 from total RNA samples obtained from core-needle biopsy sections for the diagnosis of ALT/WDL. Methods A series of lipoma (n = 124) and ALT/WDL (n = 44) cases were analyzed for cytogenetic analysis and lipoma fusion genes, as well as for MDM2 and CDK4 expression by real-time PCR. Moreover, the expression of MDM2 and CDK4 in whole tissue sections was compared with that in core-needle biopsy sections of the same tumor in order to determine whether real-time PCR could be used to distinguish ALT/WDL from lipoma at the preoperative stage. Results In whole tissue sections, the medians for MDM2 and CDK4 expression in ALT/WDL were higher than those in the lipomas (P < 0.05). Moreover, karyotype subdivisions with rings and/or giant chromosomes had higher MDM2 and CDK4 expression levels compared to karyotypes with 12q13-15 rearrangements, other abnormal karyotypes, and normal karyotypes (P < 0.05). On the other hand, MDM2 and CDK4 expression levels in core-needle biopsy sections were similar to those in whole-tissue sections (MDM2: P = 0.6, CDK4: P = 0.8, Wilcoxon signed-rank test). Conclusion Quantitative real-time PCR of total RNA can be used to evaluate the MDM2 and CDK4 expression levels in core-needle biopsies and may be useful for distinguishing ALT/WDL from adipocytic tumors. Thus, total RNA from core-needle biopsy sections may have potential as a routine diagnostic tool for other tumors where gene overexpression is a feature of the tumor. PMID:24965044

The ethanol extract from Artemisia princeps Pampanini induces p53-mediated G1 phase arrest in A172 human neuroblastoma cells.

PubMed

Park, Eun Young; Lee, Kyung-Won; Lee, Heon-Woo; Cho, Young-Wuk; Baek, Nam-In; Chung, Hae-Gon; Jeong, Tae-Sook; Choi, Myung-Sook; Lee, Kyung-Tae

2008-06-01

In the present study, the antiproliferative effects of the ethanol extract of Artemisia princeps Pampanini (EAPP) and the mechanism involved were investigated. Of the various cancer cells examined, human neuroblastoma A172 cells were most sensitive to EAPP, and their proliferation was dose- and time-dependently inhibited by EAPP. DNA flow cytometry analysis indicated that EAPP notably induced the G(1) phase arrest in A172 cells. Of the G(1) phase cycle-related proteins examined, the expressions of cyclin-dependent kinase (CDK) 2, CDK4, and CDK6 and of cyclin D(1), D(2), and D(3) were found to be markedly reduced by EAPP, whereas cyclin E was unaffected. Moreover, the protein and mRNA levels of the CDK inhibitors p16(INK4a), p21(CIP1/WAF1), and p27(KIP1) were increased, and the activities of CDK2, CDK4, and CDK6 were reduced. Furthermore, the expressions of E2F-1 and of phosphorylated pRb were also decreased, and the protein levels of p53 and pp53 (Ser15) were increased. Up-regulation of p21(CIP1/WAF1) was found to be mediated by a p53-dependent pathway in EAPP-induced G(1)-arrested A172 cells. When these data are taken together, the EAPP was found to potently inhibit the proliferation of human neuroblastoma A172 cells via G(1) phase cell cycle arrest.

Cyclin-dependent Kinases Phosphorylate the Cytomegalovirus RNA Export Protein pUL69 and Modulate Its Nuclear Localization and Activity*S⃞

PubMed Central

Rechter, Sabine; Scott, Gillian M.; Eickhoff, Jan; Zielke, Katrin; Auerochs, Sabrina; Müller, Regina; Stamminger, Thomas; Rawlinson, William D.; Marschall, Manfred

2009-01-01

Replication of human cytomegalovirus (HCMV) is subject to regulation by cellular protein kinases. Recently, we and others reported that inhibition of cyclin-dependent protein kinases (CDKs) or the viral CDK ortholog pUL97 can induce intranuclear speckled aggregation of the viral mRNA export factor, pUL69. Here we provide the first evidence for a direct regulatory role of CDKs on pUL69 functionality. Although replication of all HCMV strains was dependent on CDK activity, we found strain-specific differences in the amount of CDK inhibitor-induced pUL69 aggregate formation. In all cases analyzed, the inhibitor-induced pUL69 aggregates were clearly localized within viral replication centers but not subnuclear splicing, pore complex, or aggresome structures. The CDK9 and cyclin T1 proteins colocalized with these pUL69 aggregates, whereas other CDKs behaved differently. Phosphorylation analyses in vivo and in vitro demonstrated pUL69 was strongly phosphorylated in HCMV-infected fibroblasts and that CDKs represent a novel class of pUL69-phosphorylating kinases. Moreover, the analysis of CDK inhibitors in a pUL69-dependent nuclear mRNA export assay provided evidence for functional impairment of pUL69 under suppression of CDK activity. Thus, our data underline the crucial importance of CDKs for HCMV replication, and indicate a direct impact of CDK9-cyclin T1 on the nuclear localization and activity of the viral regulator pUL69. PMID:19179338

CDK2 phosphorylation of Smad2 disrupts TGF-beta transcriptional regulation in resistant primary bone marrow myeloma cells.

PubMed

Baughn, Linda B; Di Liberto, Maurizio; Niesvizky, Ruben; Cho, Hearn J; Jayabalan, David; Lane, Joseph; Liu, Fang; Chen-Kiang, Selina

2009-02-15

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

C-MORE Science Kits as a Classroom Learning Tool

ERIC Educational Resources Information Center

Foley, J. M.; Bruno, B. C.; Tolman, R. T.; Kagami, R. S.; Hsia, M. H.; Mayer, B.; Inazu, J. K.

2013-01-01

To support teachers in enhancing ocean literacy, the Center for Microbial Oceanography: Research and Education (C-MORE) has developed a series of portable, hands-on science kits on selected topics in oceanography. This paper provides an overview of kit content, describes how the kits were developed, and evaluates their efficacy as a curriculum…

Genomic Analyses Reveal Broad Impact of miR-137 on Genes Associated with Malignant Transformation and Neuronal Differentiation in Glioblastoma Cells

PubMed Central

Tamim, Saleh; Vo, Dat T.; Uren, Philip J.; Qiao, Mei; Bindewald, Eckart; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Nakaya, Helder I.; Burns, Suzanne C.; Araujo, Patricia R.; Nakano, Ichiro; Radek, Agnes J.; Kuersten, Scott; Smith, Andrew D.; Penalva, Luiz O. F.

2014-01-01

miR-137 plays critical roles in the nervous system and tumor development; an increase in its expression is required for neuronal differentiation while its reduction is implicated in gliomagenesis. To evaluate the potential of miR-137 in glioblastoma therapy, we conducted genome-wide target mapping in glioblastoma cells by measuring the level of association between PABP and mRNAs in cells transfected with miR-137 mimics vs. controls via RIPSeq. Impact on mRNA levels was also measured by RNASeq. By combining the results of both experimental approaches, 1468 genes were found to be negatively impacted by miR-137 – among them, 595 (40%) contain miR-137 predicted sites. The most relevant targets include oncogenic proteins and key players in neurogenesis like c-KIT, YBX1, AKT2, CDC42, CDK6 and TGFβ2. Interestingly, we observed that several identified miR-137 targets are also predicted to be regulated by miR-124, miR-128 and miR-7, which are equally implicated in neuronal differentiation and gliomagenesis. We suggest that the concomitant increase of these four miRNAs in neuronal stem cells or their repression in tumor cells could produce a robust regulatory effect with major consequences to neuronal differentiation and tumorigenesis. PMID:24465609

Genetic profiling of a rare condition: co-occurrence of albinism and multiple primary melanoma in a caucasian family

PubMed Central

Tommasi, Stefania; Strippoli, Sabino; Pellegrini, Cristina; Concetta Fargnoli, Maria; Pilato, Brunella; Natalicchio, Iole; Guida, Gabriella; Pinto, Rosamaria

2017-01-01

Multiple primary melanoma (MPM) is a rare condition, whose genetic basis has not yet been clarified. Only 8-12% of MPM are due to germline mutations of CDKN2A. However, other genes (POT1, BRCA1/2, MC1R, MGMT) have been demonstrated to be involved in predisposition to this pathology. To our knowledge, this is the first family study based on two siblings with the rare coexistence of MPM and oculocutaneous albinism (OCA), an autosomal recessive disease characterized by the absence or decrease in pigmentation in the skin, hair, and eyes. In this study, we evaluated genes involved in melanoma predisposition (CDKN2A, CDK4, MC1R, MITF, POT1, RB1, MGMT, BRCA1, BRCA2), pathogenesis (BRAF, NRAS, PIK3CA, KIT, PTEN), skin/hair pigmentation (MC1R, MITF) and in immune pathways (CTLA4) to individuate alterations able to explain the rare onset of MPM and OCA in indexes and the transmission in their pedigree. From the analysis of the pedigree, we were able to identify a “protective” haplotype with respect to MPM, including MGMT p.I174V alteration. The second generation offspring is under strict follow up as some of them have a higher risk of developing MPM according to our model. PMID:27776349

The development of the residential Fire H.E.L.P. tool kit: a resource to protect homebound older adults.

PubMed

Diekman, Shane; Huitric, Michele; Netterville, Linda

2010-01-01

This article describes the development of the Fire H.E.L.P. tool kit for training selected Meals On Wheels (MOW) staff in Texas to implement a fire safety program for homebound older adults. We used a formative evaluation approach during the tool kit's development, testing, and initial implementation stages. The tool kit includes instructional curricula on how to implement Fire H.E.L.P., a home assessment tool to determine a residence's smoke alarm needs, and fire safety educational materials. During the tool kit's pilot test, MOW participants showed enhanced fire safety knowledge and high levels of confidence about applying their newfound training skills. After the pilot test, MOW staff used the tool kit to conduct local training sessions, provide fire safety education, and install smoke alarms in the homes of older adults. We believe the approach used to develop this tool kit can be applied to education efforts for other, related healthy home topics.

Studying the Role of the Mitotic Exit Network in Cytokinesis.

PubMed

Foltman, Magdalena; Sanchez-Diaz, Alberto

2017-01-01

In budding yeast cells, cytokinesis is achieved by the successful division of the cytoplasm into two daughter cells, but the precise mechanisms of cell division and its regulation are still rather poorly understood. The Mitotic Exit Network (MEN) is the signaling cascade that is responsible for the release of Cdc14 phosphatase leading to the inactivation of the kinase activity associated to cyclin-dependent kinases (CDK), which drives exit from mitosis and a rapid and efficient cytokinesis. Mitotic CDK impairs the activation of MEN before anaphase, and activation of MEN in anaphase leads to the inactivation of CDK, which presents a challenge to determine the contribution that each pathway makes to the successful onset of cytokinesis. To determine CDK and MEN contribution to cytokinesis irrespectively of each other, here we present methods to induce cytokinesis after the inactivation of CDK activity in temperature sensitive mutants of the MEN pathway. An array of methods to monitor the cellular events associated with the successful cytokinesis is included.

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss.

PubMed

Teitz, Tal; Fang, Jie; Goktug, Asli N; Bonga, Justine D; Diao, Shiyong; Hazlitt, Robert A; Iconaru, Luigi; Morfouace, Marie; Currier, Duane; Zhou, Yinmei; Umans, Robyn A; Taylor, Michael R; Cheng, Cheng; Min, Jaeki; Freeman, Burgess; Peng, Junmin; Roussel, Martine F; Kriwacki, Richard; Guy, R Kiplin; Chen, Taosheng; Zuo, Jian

2018-04-02

Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration-approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing hearing loss. © 2018 Teitz et al.

CDK2 inhibitors as candidate therapeutics for cisplatin- and noise-induced hearing loss

PubMed Central

Teitz, Tal; Fang, Jie; Goktug, Asli N.; Bonga, Justine D.; Diao, Shiyong; Iconaru, Luigi; Morfouace, Marie; Currier, Duane; Zhou, Yinmei; Umans, Robyn A.; Taylor, Michael R.; Cheng, Cheng; Peng, Junmin; Roussel, Martine F.; Kriwacki, Richard; Guy, R. Kiplin; Chen, Taosheng

2018-01-01

Hearing loss caused by aging, noise, cisplatin toxicity, or other insults affects 360 million people worldwide, but there are no Food and Drug Administration–approved drugs to prevent or treat it. We screened 4,385 small molecules in a cochlear cell line and identified 10 compounds that protected against cisplatin toxicity in mouse cochlear explants. Among them, kenpaullone, an inhibitor of multiple kinases, including cyclin-dependent kinase 2 (CDK2), protected zebrafish lateral-line neuromasts from cisplatin toxicity and, when delivered locally, protected adult mice and rats against cisplatin- and noise-induced hearing loss. CDK2-deficient mice displayed enhanced resistance to cisplatin toxicity in cochlear explants and to cisplatin- and noise-induced hearing loss in vivo. Mechanistically, we showed that kenpaullone directly inhibits CDK2 kinase activity and reduces cisplatin-induced mitochondrial production of reactive oxygen species, thereby enhancing cell survival. Our experiments have revealed the proapoptotic function of CDK2 in postmitotic cochlear cells and have identified promising therapeutics for preventing hearing loss. PMID:29514916

Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21

PubMed Central

Wang, Yuefeng; Fisher, John C.; Mathew, Rose; Ou, Li; Otieno, Steve; Sublett, Jack; Xiao, Limin; Chen, Jianhan; Roussel, Martine F.; Kriwacki, Richard W.

2011-01-01

Traditionally, well-defined three-dimensional structure was thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently exhibit “binding diversity” by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and herein report that the cyclin-dependent kinase (Cdk) inhibitor, p21Cip1, adaptively binds to and inhibits the various Cdk/cyclin complexes that regulate eukaryotic cell division. Based on results from NMR spectroscopy, and biochemical and cellular assays, we show that structural adaptability of a helical sub-domain within p21 termed LH enables two other sub-domains termed D1 and D2 to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk/cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes. PMID:21358637

Functional reprogramming of polyploidization in megakaryocytes.

PubMed

Trakala, Marianna; Rodríguez-Acebes, Sara; Maroto, María; Symonds, Catherine E; Santamaría, David; Ortega, Sagrario; Barbacid, Mariano; Méndez, Juan; Malumbres, Marcos

2015-01-26

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process in which megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms in vivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells. Copyright © 2015 Elsevier Inc. All rights reserved.

P‐TEFb goes viral

PubMed Central

Zaborowska, Justyna; Isa, Nur F.

2015-01-01

Positive transcription elongation factor b (P‐TEFb), which comprises cyclin‐dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P‐TEFb is required for productive elongation of transcription of protein‐coding genes by RNA polymerase II (pol II). In addition, P‐TEFb‐mediated phosphorylation of the carboxyl‐terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P‐TEFb could be effective anti‐viral agents. PMID:27398404

CDK regulation of transcription by RNAP II: Not over 'til it's over?

PubMed

Fisher, Robert P

2017-03-15

Transcription by RNA polymerase (RNAP) II is regulated at multiple steps by phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. The CDKs involved in transcription have overlapping substrate specificities, but play largely non-redundant roles in coordinating gene expression. Novel functions and targets of CDKs have recently emerged at the end of the transcription cycle, when the primary transcript is cleaved, and in most cases polyadenylated, and transcription is terminated by the action of the "torpedo" exonuclease Xrn2, which is a CDK substrate. Collectively, various functions have been ascribed to CDKs or CDK-mediated phosphorylation: recruiting cleavage and polyadenylation factors, preventing premature termination within gene bodies while promoting efficient termination of full-length transcripts, and preventing extensive readthrough transcription into intergenic regions or neighboring genes. The assignment of precise functions to specific CDKs is still in progress, but recent advances suggest ways in which the CDK network and RNAP II machinery might cooperate to ensure timely exit from the transcription cycle.

CDK1 enhances mitochondrial bioenergetics for radiation-induced DNA repair

DOE PAGES

Qin, Lili; Fan, Ming; Candas, Demet; ...

2015-12-06

Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy-consuming process. However, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of the cell-cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is reduced significantly in cells harboring CDK1 phosphorylation-deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair aremore » also compromised severely in cells harboring mitochondrially targeted, kinase-deficient CDK1. These findings demonstrate a mechanism governing the communication between mitochondria and the nucleus by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress conditions.« less

CDK regulation of transcription by RNAP II: Not over ‘til it's over?

PubMed Central

Fisher, Robert P.

2017-01-01

ABSTRACT Transcription by RNA polymerase (RNAP) II is regulated at multiple steps by phosphorylation, catalyzed mainly by members of the cyclin-dependent kinase (CDK) family. The CDKs involved in transcription have overlapping substrate specificities, but play largely non-redundant roles in coordinating gene expression. Novel functions and targets of CDKs have recently emerged at the end of the transcription cycle, when the primary transcript is cleaved, and in most cases polyadenylated, and transcription is terminated by the action of the “torpedo” exonuclease Xrn2, which is a CDK substrate. Collectively, various functions have been ascribed to CDKs or CDK-mediated phosphorylation: recruiting cleavage and polyadenylation factors, preventing premature termination within gene bodies while promoting efficient termination of full-length transcripts, and preventing extensive readthrough transcription into intergenic regions or neighboring genes. The assignment of precise functions to specific CDKs is still in progress, but recent advances suggest ways in which the CDK network and RNAP II machinery might cooperate to ensure timely exit from the transcription cycle. PMID:28005463

The role of ventral striatal cAMP signaling in stress-induced behaviors

PubMed Central

Plattner, Florian; Hayashi, Kanehiro; Hernandez, Adan; Benavides, David R.; Tassin, Tara C.; Tan, Chunfeng; Day, Jonathan; Fina, Maggy W.; Yuen, Eunice Y.; Yan, Zhen; Goldberg, Matthew S.; Nairn, Angus C.; Greengard, Paul; Nestler, Eric J.; Taussig, Ronald; Nishi, Akinori; Houslay, Miles D.; Bibb, James A.

2015-01-01

The cAMP/PKA signaling cascade is a ubiquitous pathway acting downstream of multiple neuromodulators. We found that the phosphorylation of phosphodiesterase-4 (PDE4) by cyclin-dependent protein kinase 5 (Cdk5) facilitates cAMP degradation and homeostasis of cAMP/PKA signaling. In mice, loss of Cdk5 throughout the forebrain elevated cAMP levels and increased PKA activity in striatal neurons, and altered behavioral responses to acute or chronic stressors. Ventral striatum- or D1 dopamine receptor-specific conditional knockout of Cdk5, or ventral striatum infusion of a small interfering peptide that selectively targets the regulation of PDE4 by Cdk5, all produced analogical effects on stress-induced behavioral responses. Together, our results demonstrate that altering cAMP signaling in medium spiny neurons of the ventral striatum can effectively modulate stress-induced behavioral states. We propose that targeting the Cdk5 regulation of PDE4 could be a new therapeutic approach for clinical conditions associated with stress, such as depression. PMID:26192746

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases

PubMed Central

Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.

2014-01-01

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440

The expression of MDM2/CDK4 gene product in the differential diagnosis of well differentiated liposarcoma and large deep-seated lipoma

PubMed Central

Pilotti, S; Torre, G Della; Mezzelani, A; Tamborini, E; Azzarelli, A; Sozzi, G; Pierotti, M A

2000-01-01

Ordinary lipomas are cytogenetically characterized by a variety of balanced rearrangements involving chromosome segment 12q13–15, whereas well differentiated liposarcomas (WDL) show supernumerary ring and giant marker chromosomes, known to contain amplified 12q sequences. The tight correlation between the presence of ring chromosomes and both amplification and overexpression of MDM2 and CDK4 genes suggests the exploration of the possibility that immunocytochemistry (ICC) might assist in the differential diagnosis of lipoma-like well differentiated liposarcomas (LL-WDL) and large deep-seated lipomas (LDSL). For this purpose, 21 cases of the former and 19 cases of the latter tumours were analysed by ICC and, according to the availability of material, by molecular and cytogenetic approaches. All lipomas displayed a null MDM2/CDK4 phenotype, whereas all LL-WDL showed MDM2/CDK4 or CDK4 phenotypes. Southern blot analysis performed on 16 suitable cases, complemented by fluorescence in situ hybridization and classical cytogenetic analysis in 11 cases, was consistent with, and further supported the immunophenotyping data. In conclusion, MDM2/CDK4 product-based immunophenotyping appears to represent a valuable method for the categorization of arguable LDSL. © 2000 Cancer Research Campaign PMID:10755400

Hcm1 integrates signals from Cdk1 and calcineurin to control cell proliferation.

PubMed

Arsenault, Heather E; Roy, Jagoree; Mapa, Claudine E; Cyert, Martha S; Benanti, Jennifer A

2015-10-15

Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation. © 2015 Arsenault, Roy, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Selective antitumor activity of roscovitine in head and neck cancer

PubMed Central

Biktasova, Asel; Bellinger, Gary; Yarbrough, Wendell G.; Issaeva, Natalia

2016-01-01

Radiation and chemotherapy that are commonly used to treat human cancers damage cellular DNA. DNA damage appears to be more toxic to cancer cells than normal cells, most likely due to deregulated checkpoint activation and/or deficiency in DNA repair pathways that are characteristics of many tumors. However, unwanted side effects arise as a result of DNA damage to normal cells during the treatment. Here, we show that roscovitine, a cyclin-dependent kinase (CDK) inhibitor that inhibits CDK-1, CDK-2, CDK-5, CDK-7, and CDK-9 due to competitive binding to the ATP site on the kinases, causes significant DNA damage followed by p53-dependent cell death in human papilloma virus (HPV)-positive, but not in HPV-negative, head and neck cancer cells. Since HPV positivity was a molecular marker for increased sensitivity of cells to roscovitine, we reasoned that systemic roscovitine administration would not be toxic to healthy HPV-negative tissue. Indeed, low roscovitine doses significantly inhibited the growth of HPV-associated xenografted tumors in mice without causing any detectable side effects. Given that inhibition of CDKs has been shown to inhibit replication of several viruses, we suggest that roscovitine treatment may represent a selective and safe targeted therapeutic option against HPV-positive head and neck cancer. PMID:27233076

Human Cdc14A regulates Wee1 stability by counteracting CDK-mediated phosphorylation

PubMed Central

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 such as Plk1, triggering Wee1 degradation at the onset of mitosis. Here we report that Cdc14A plays an important role in the regulation of Wee1 stability. Depletion of Cdc14A results in a significant reduction in Wee1 protein levels. Cdc14A binds to Wee1 at its amino-terminal domain and reverses CDK-mediated Wee1 phosphorylation. In particular, we found that Cdc14A inhibits Wee1 degradation through the dephosphorylation of Ser-123 and Ser-139 residues. Thus the lack of phosphorylation of these two residues prevents the interaction with Plk1 and the consequent efficient Wee1 degradation at the onset of mitosis. These data support the hypothesis that Cdc14A counteracts Cdk1–cyclin B1 activity through Wee1 dephosphorylation. PMID:23051732

Cdk5 regulates PSD-95 ubiquitination in neurons

PubMed Central

Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

2011-01-01

The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases.

PubMed

Huang, Yongqi; Yoon, Mi-Kyung; Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W

2015-01-30

The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinase inhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, as well as its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.

PubMed Central

Rossignol, M; Kolb-Cheynel, I; Egly, J M

1997-01-01

The transcription/DNA repair factor TFIIH consists of nine subunits, several exhibiting known functions: helicase/ATPase, kinase activity and DNA binding. Three subunits of TFIIH, cdk7, cyclin H and MAT1, form a ternary complex, cdk-activating kinase (CAK), found either on its own or as part of TFIIH. In the present work, we demonstrate that purified human CAK complex (free CAK) and recombinant CAK (rCAK) produced in insect cells exhibit a strong preference for the cyclin-dependent kinase 2 (cdk2) over a ctd oligopeptide substrate (which mimics the carboxy-terminal domain of the RNA polymerase II). In contrast, TFIIH preferentially phosphorylates the ctd as well as TFIIE alpha, but not cdk2. TFIIH was resolved into four subcomplexes: the kinase complex composed of cdk7, cyclin H and MAT1; the core TFIIH which contains XPB, p62, p52, p44 and p34; and two other subcomplexes in which XPD is found associated with either the kinase complex or with the core TFIIH. Using these fractions, we demonstrate that TFIIH lacking the CAK subcomplex completely recovers its transcriptional activity in the presence of free CAK. Furthermore, studies examining the interactions between TFIIH subunits provide evidence that CAK is integrated within TFIIH via XPB and XPD. PMID:9130708

A cdk1 gradient guides surface contraction waves in oocytes.

PubMed

Bischof, Johanna; Brand, Christoph A; Somogyi, Kálmán; Májer, Imre; Thome, Sarah; Mori, Masashi; Schwarz, Ulrich S; Lénárt, Péter

2017-10-11

Surface contraction waves (SCWs) in oocytes and embryos lead to large-scale shape changes coupled to cell cycle transitions and are spatially coordinated with the cell axis. Here, we show that SCWs in the starfish oocyte are generated by a traveling band of myosin II-driven cortical contractility. At the front of the band, contractility is activated by removal of cdk1 inhibition of the RhoA/RhoA kinase/myosin II signaling module, while at the rear, contractility is switched off by negative feedback originating downstream of RhoA kinase. The SCW's directionality and speed are controlled by a spatiotemporal gradient of cdk1-cyclinB. This gradient is formed by the release of cdk1-cyclinB from the asymmetrically located nucleus, and progressive degradation of cyclinB. By combining quantitative imaging, biochemical and mechanical perturbations with mathematical modeling, we demonstrate that the SCWs result from the spatiotemporal integration of two conserved regulatory modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.Surface contraction waves (SCWs) are prominent shape changes coupled to cell cycle transitions in oocytes. Here the authors show that SCWs are patterned by the spatiotemporal integration of two conserved modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.

Recommendations for adaptation and validation of commercial kits for biomarker quantification in drug development.

PubMed

Khan, Masood U; Bowsher, Ronald R; Cameron, Mark; Devanarayan, Viswanath; Keller, Steve; King, Lindsay; Lee, Jean; Morimoto, Alyssa; Rhyne, Paul; Stephen, Laurie; Wu, Yuling; Wyant, Timothy; Lachno, D Richard

2015-01-01

Increasingly, commercial immunoassay kits are used to support drug discovery and development. Longitudinally consistent kit performance is crucial, but the degree to which kits and reagents are characterized by manufacturers is not standardized, nor are the approaches by users to adapt them and evaluate their performance through validation prior to use. These factors can negatively impact data quality. This paper offers a systematic approach to assessment, method adaptation and validation of commercial immunoassay kits for quantification of biomarkers in drug development, expanding upon previous publications and guidance. These recommendations aim to standardize and harmonize user practices, contributing to reliable biomarker data from commercial immunoassays, thus, enabling properly informed decisions during drug development.

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

PubMed

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.

The Smad3/Smad4/CDK9 complex promotes renal fibrosis in mice with unilateral ureteral obstruction.

PubMed

Qu, Xinli; Jiang, Mengjie; Sun, Yu Bo Yang; Jiang, Xiaoyun; Fu, Ping; Ren, Yi; Wang, Die; Dai, Lie; Caruana, Georgina; Bertram, John F; Nikolic-Paterson, David J; Li, Jinhua

2015-12-01

Transforming growth factor-β1 (TGF-β1)/Smad signaling has a central role in the pathogenesis of renal fibrosis. Smad3 and Smad4 are pro-fibrotic, while Smad2 is anti-fibrotic. However, these Smads form heterogeneous complexes, the functions of which are poorly understood. Here we studied Smad complex function in renal fibrosis using the mouse model of unilateral ureteric obstruction. Mice heterozygous for Smad3/4 (Smad3/4 +/- ) exhibited substantial protection from renal fibrosis through day 7 of obstruction, whereas Smad2/3 +/- and Smad2/4 +/- mice showed only modest protection. Formation of Smad3/Smad4/CDK9 complexes was an early event following obstruction in wild-type mice, which involved nuclear phosphorylation of the linker regions of Smad3. Significantly, Smad3 or Smad4 deficiency decreased the formation of Smad4/CDK9 or Smad3/CDK9 complex, Smad3 linker phosphorylation, and fibrosis but at different degrees. In vitro, TGF-β1 stimulation of collagen I promoter activity involved formation of Smad3/Smad4/CDK9 complexes, and overexpression of each component gave additive increases in collagen promoter activity. Co-administration of a CDK9 inhibitor and Smad3-specific inhibition achieved better protection from TGF-β1-induced fibrotic response in vitro and renal interstitial fibrosis in vivo. Thus formation of Smad3/Smad4/CDK9 complex drives renal fibrosis during ureteral obstruction. Formation of this complex represents a novel target for antifibrotic therapies.

Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.

PubMed

Zhang, Jinfang; Bu, Xia; Wang, Haizhen; Zhu, Yasheng; Geng, Yan; Nihira, Naoe Taira; Tan, Yuyong; Ci, Yanpeng; Wu, Fei; Dai, Xiangpeng; Guo, Jianping; Huang, Yu-Han; Fan, Caoqi; Ren, Shancheng; Sun, Yinghao; Freeman, Gordon J; Sicinski, Piotr; Wei, Wenyi

2018-01-04

Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.

Differential diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma: utility of p16 in combination with MDM2 and CDK4 immunohistochemistry.

PubMed

Kammerer-Jacquet, Solène-Florence; Thierry, Sixte; Cabillic, Florian; Lannes, Morgane; Burtin, Florence; Henno, Sébastien; Dugay, Frédéric; Bouzillé, Guillaume; Rioux-Leclercq, Nathalie; Belaud-Rotureau, Marc-Antoine; Stock, Nathalie

2017-01-01

The differential diagnosis between atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDLPS) and dedifferentiated liposarcoma (DDLPS) from their morphologic counterparts is challenging. Currently, the diagnosis is guided by MDM2 and CDK4 immunohistochemistry (IHC) and is confirmed by the amplification of the corresponding genes. Recently, p16 IHC has been proposed as a useful diagnostic biomarker. The objective was to assess the utility of p16 IHC in the differential diagnosis of ALT/WDLPS and DDLPS. Our series included 101 tumors that were previously analyzed using fluorescence in situ hybridization for MDM2 and CDK4 amplification. We compared sensitivity and specificity of p16 IHC to MDM2 and CDK4 IHC in the differential diagnosis of ALT-WDLPS (n=19) versus benign adipocytic tumors (n=44) and DDLPS (n=18) versus mimicking sarcomas (n=20). In the differential diagnosis of ALT-WDLPS, p16 had a sensitivity of 89.5% but a specificity of 68.2%, which was impaired by false-positive lipomas with secondary changes, especially in biopsies. Likewise, in the differential diagnosis of DDLPS, p16 had a sensitivity of 94.4% and a specificity of 70%, which hampered its use as a single marker. However, adding p16 to MDM2 and/or CDK4 increased diagnostic specificity. Indeed, MDM2+/p16+ tumors were all ALT-WDLPS, and MDM2-/p16- tumors were all benign adipocytic tumors. Moreover, all MDM2+/CDK4+/p16+ tumors were DDLPS, and the MDM2-/CDK4-/p16- tumor was an undifferentiated sarcoma. Although the use of p16 as a single immunohistochemical marker is limited by its specificity, its combination with MDM2 and CDK4 IHC may help discriminate ALT-WDLPS/DDLPS. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi

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 remainsmore » 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.« less

Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

PubMed Central

Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

2014-01-01

Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

PubMed

Egyházi, E; Ossoinak, A; Lee, J M; Greenleaf, A L; Mäkelä, T P; Pigon, A

1998-07-10

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.

YB-1 Is Important for Late-Stage Embryonic Development, Optimal Cellular Stress Responses, and the Prevention of Premature Senescence

PubMed Central

Lu, Zhi Hong; Books, Jason T.; Ley, Timothy J.

2005-01-01

Proteins containing “cold shock” domains belong to the most evolutionarily conserved family of nucleic acid-binding proteins known among bacteria, plants, and animals. One of these proteins, YB-1, is widely expressed throughout development and has been implicated as a cell survival factor that regulates the transcription and/or translation of many cellular growth and death-related genes. For these reasons, YB-1 deficiency has been predicted to be incompatible with cell survival. However, the majority of YB-1−/− embryos develop normally up to embryonic day 13.5 (E13.5). After E13.5, YB-1−/− embryos exhibit severe growth retardation and progressive mortality, revealing a nonredundant role of YB-1 in late embryonic development. Fibroblasts derived from YB-1−/− embryos displayed a normal rate of protein synthesis and minimal alterations in the transcriptome and proteome but demonstrated reduced abilities to respond to oxidative, genotoxic, and oncogene-induced stresses. YB-1−/− cells under oxidative stress expressed high levels of the G1-specific CDK inhibitors p16Ink4a and p21Cip1 and senesced prematurely; this defect was corrected by knocking down CDK inhibitor levels with specific small interfering RNAs. These data suggest that YB-1 normally represses the transcription of CDK inhibitors, making it an important component of the cellular stress response signaling pathway. PMID:15899865

Megakaryocytes compensate for Kit insufficiency in murine arthritis.

PubMed

Cunin, Pierre; Penke, Loka R; Thon, Jonathan N; Monach, Paul A; Jones, Tatiana; Chang, Margaret H; Chen, Mary M; Melki, Imene; Lacroix, Steve; Iwakura, Yoichiro; Ware, Jerry; Gurish, Michael F; Italiano, Joseph E; Boilard, Eric; Nigrovic, Peter A

2017-05-01

The growth factor receptor Kit is involved in hematopoietic and nonhematopoietic development. Mice bearing Kit defects lack mast cells; however, strains bearing different Kit alleles exhibit diverse phenotypes. Herein, we investigated factors underlying differential sensitivity to IgG-mediated arthritis in 2 mast cell-deficient murine lines: KitWsh/Wsh, which develops robust arthritis, and KitW/Wv, which does not. Reciprocal bone marrow transplantation between KitW/Wv and KitWsh/Wsh mice revealed that arthritis resistance reflects a hematopoietic defect in addition to mast cell deficiency. In KitW/Wv mice, restoration of susceptibility to IgG-mediated arthritis was neutrophil independent but required IL-1 and the platelet/megakaryocyte markers NF-E2 and glycoprotein VI. In KitW/Wv mice, platelets were present in numbers similar to those in WT animals and functionally intact, and transfer of WT platelets did not restore arthritis susceptibility. These data implicated a platelet-independent role for the megakaryocyte, a Kit-dependent lineage that is selectively deficient in KitW/Wv mice. Megakaryocytes secreted IL-1 directly and as a component of circulating microparticles, which activated synovial fibroblasts in an IL-1-dependent manner. Transfer of WT but not IL-1-deficient megakaryocytes restored arthritis susceptibility to KitW/Wv mice. These findings identify functional redundancy among Kit-dependent hematopoietic lineages and establish an unanticipated capacity of megakaryocytes to mediate IL-1-driven systemic inflammatory disease.

Education kits for fiber optics, optoelectronics, and optical communications

NASA Astrophysics Data System (ADS)

Hájek, Martin; Švrček, Miroslav

2007-04-01

Our company MIKROKOM, s.r.o. is engaged for many years in development of education equipment and kits for fiber optics, optoelectronics and optical communications. We would like to inform competitors of conference about results of this long-time development. Requirements on education kits and equipment in a modern and dynamic area as is optical communications and fiber optics are quite difficult. The education kits should to clearly introduce students to given issue - the most important physical principles and technical approaches, but it should to introduce also to new and modern technologies, which are quickly changing and developing. On the other hand should be these tools and kits reasonable for the schools. In our paper we would like to describe possible ways of development of this education kits and equipment and present our results of long-time work, which covers very wide range. On the one hand we developed equipment and kits for clear demonstration of physical effects using plastic optical fibers POF, next we prepare kits with a glass fibers, which are the most used fibers in practice and after as much as the kits, which covers broad range of passive and active elements of the optical networks and systems and which makes possible to create complex optical transmission connection. This kind of systems with using corresponding tools and equipment introduce the students to properties, manipulation, measurement and usage of optical fibers, traces and many active and passive components. Furthermore, with using different sorts of optical sources, photodetectors, fiber optics couplers etc., students can get acquainted with all optoelectronics transmission system, which uses different sorts of signals. Special part will be devoted also to effort mentioned before - to implement modern technologies such as e.g. Wavelength Division Multiplex (WDM) into the education kits. Our presentation will inform auditors about development of mentioned education kits and equipment and about their potentials and practical utility at school education.

Discovery of potent and selective CDK8 inhibitors through FBDD approach.

PubMed

Han, Xingchun; Jiang, Min; Zhou, Chengang; Zhou, Zheng; Xu, Zhiheng; Wang, Lisha; Mayweg, Alexander V; Niu, Rui; Jin, Tai-Guang; Yang, Song

2017-09-15

A fragment library screen was carried out to identify starting points for novel CDK8 inhibitors. Optimization of a fragment hit guided by co-crystal structures led to identification of a novel series of potent CDK8 inhibitors which are highly ligand efficient, kinase selective and cellular active. Compound 16 was progressed to a mouse pharmacokinetic study and showed good oral bioavailability. Copyright © 2017 Elsevier Ltd. All rights reserved.

CDK5-A Novel Role in Prostate Cancer Immunotherapy

DTIC Science & Technology

2017-10-01

of the involvement of a T cell antitumor response in impaired growth of prostate cancer in immunocompetent murine models of prostate cancer, and...of immune system activation by Cdk5 deletion in prostate cancer. We will confirm the involvement of a T cell antitumor response in impaired growth of...project? Major Task 1: Involvement of T cell anticancer immune response in impaired growth of TRAMP Cdk5-/- model. Months 1-10. Completed, month 10

Traffic safety for the cell: influence of cyclin-dependent kinase activity on genomic stability.

PubMed

Enders, Greg H; Maude, Shannon L

2006-04-12

Genomic instability has long been considered a key factor in tumorigenesis. Recent evidence suggests that DNA damage may be widespread in early pre-neoplastic states, with deregulation of cyclin-dependent kinase (Cdk) activity a driving force. Increased Cdk activity may critically reduce licensing of origins of DNA replication, drive re-replication, or mediate overexpression of checkpoint proteins, inducing deleterious cell cycle delay. Conversely, inhibition of Cdk activity may compromise replication efficiency, expression of checkpoint proteins, or activation of DNA repair proteins. These vital functions point to the impact of Cdk activity on the stability of the genome. Insight into these pathways may improve our understanding of tumorigenesis and lead to more rational cancer therapies.

Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

PubMed

She, Hua; Mao, Zixu

2017-01-01

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

Recent Advances of Cell-Cycle Inhibitor Therapies for Pediatric Cancer.

PubMed

Mills, Christopher C; Kolb, E A; Sampson, Valerie B

2017-12-01

This review describes the pivotal roles of cell-cycle and checkpoint regulators and discusses development of specific cell-cycle inhibitors for therapeutic use for pediatric cancer. The mechanism of action as well as the safety and tolerability of drugs in pediatric patients, including compounds that target CDK4/CDK6 (palbociclib, ribociclib, and abemaciclib), aurora kinases (AT9283 and MLN8237), Wee1 kinase (MK-1775), KSP (ispinesib), and tubulin (taxanes, vinca alkaloids), are presented. The design of mechanism-based combinations that exploit the cross-talk of signals activated by cell-cycle arrest, as well as pediatric-focused drug development, are critical for the advancement of drugs for rare childhood diseases. Cancer Res; 77(23); 6489-98. ©2017 AACR . ©2017 American Association for Cancer Research.

Development and evaluation of the RT-PCR kit for the rabies virus diagnosis.

PubMed

Dedkov, Vladimir G; Deviatkin, A A; Poleschuk, E M; Safonova, M V; Markelov, M L; Shipulin, G A

To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.

Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

PubMed

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27(kip1) is a critical regulator of cell cycle progression, but the mechanisms by which p27(kip1) controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27(kip1) binding partner. To get more insights into the in vivo significance of this interaction, we generated p27(kip1) and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27(kip1) null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27(kip1) null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27(kip1) to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

Targeting the cell cycle and the PI3K pathway: a possible universal strategy to reactivate innate tumor suppressor programmes in cancer cells.

PubMed

David-Pfeuty, Thérèse; Legraverend, Michel; Ludwig, Odile; Grierson, David S

2010-04-01

Corruption of the Rb and p53 pathways occurs in virtually all human cancers. This could be because it lends oncogene-bearing cells a surfeit of Cdk activity and growth, enabling them to elaborate strategies to evade tumor-suppressive mechanisms and divide inappropriately. Targeting both Cdk activities and the PI3K pathway might be therefore a potentially universal means to palliate their deficiency in cancer cells. We showed that the killing efficacy of roscovitine and 16 other purines and potentiation of roscovitine-induced apoptosis by the PI3K inhibitor, LY294002, decreased with increasing corruption of the Rb and p53 pathways. Further, we showed that purines differing by a single substitution, which exerted little lethal effect on distant cell types in rich medium, could display widely-differing cytotoxicity profiles toward the same cell types in poor medium. Thus, closely-related compounds targeting similar Cdks may interact with different targets that could compete for their interaction with therapeutically-relevant Cdk targets. In the perspective of clinical development in association with the PI3K pathway inhibitors, it might thus be advisable to select tumor cell type-specific Cdk inhibitors on the basis of their toxicity in cell-culture-based assays performed at a limiting serum concentration sufficient to suppress their interaction with undesirable crossreacting targets whose range and concentration would depend on the cell genotype.

Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

PubMed

Guo, Lei; Xiao, Yongsheng; Fan, Ming; Li, Jian Jian; Wang, Yinsheng

2015-01-02

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

A Novel Non-agonist Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand UHC1 Blocks PPARγ Phosphorylation by Cyclin-dependent Kinase 5 (CDK5) and Improves Insulin Sensitivity*

PubMed Central

Choi, Sun-Sil; Kim, Eun Sun; Koh, Minseob; Lee, Soo-Jin; Lim, Donghyun; Yang, Yong Ryoul; Jang, Hyun-Jun; Seo, Kyung-ah; Min, Sang-Hyun; Lee, In Hee; Park, Seung Bum; Suh, Pann-Ghill; Choi, Jang Hyun

2014-01-01

Thiazolidinedione class of anti-diabetic drugs which are known as peroxisome proliferator-activated receptor γ (PPARγ) ligands have been used to treat metabolic disorders, but thiazolidinediones can also cause several severe side effects, including congestive heart failure, fluid retention, and weight gain. In this study, we describe a novel synthetic PPARγ ligand UNIST HYUNDAI Compound 1 (UHC1) that binds tightly to PPARγ without the classical agonism and which blocks cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation. We modified the non-agonist PPARγ ligand SR1664 chemically to improve its solubility and then developed a novel PPARγ ligand, UHC1. According to our docking simulation, UHC1 occupied the ligand-binding site of PPARγ with a higher docking score than SR1664. In addition, UHC1 more potently blocked CDK5-mediated PPARγ phosphorylation at Ser-273. Surprisingly, UHC1 treatment effectively ameliorated the inflammatory response both in vitro and in high-fat diet-fed mice. Furthermore, UHC1 treatment dramatically improved insulin sensitivity in high-fat diet-fed mice without causing fluid retention and weight gain. Taken together, compared with SR1664, UHC1 exhibited greater beneficial effects on glucose and lipid metabolism by blocking CDK5-mediated PPARγ phosphorylation, and these data indicate that UHC1 could be a novel therapeutic agent for use in type 2 diabetes and related metabolic disorders. PMID:25100724

Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

PubMed Central

Berton, Stefania; Pellizzari, Ilenia; Fabris, Linda; D'Andrea, Sara; Segatto, Ilenia; Canzonieri, Vincenzo; Marconi, Daniela; Schiappacassi, Monica; Benevol, Sara; Gattei, Valter; Colombatti, Alfonso; Belletti, Barbara; Baldassarre, Gustavo

2014-01-01

The CDK inhibitor p27kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression. PMID:25486569

ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

PubMed Central

Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

2016-01-01

It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

Regulation of Neuronal Cav3.1 Channels by Cyclin-Dependent Kinase 5 (Cdk5)

PubMed Central

González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels. PMID:25760945

Regulation of neuronal cav3.1 channels by cyclin-dependent kinase 5 (Cdk5).

PubMed

Calderón-Rivera, Aida; Sandoval, Alejandro; González-Ramírez, Ricardo; González-Billault, Christian; Felix, Ricardo

2015-01-01

Low voltage-activated (LVA) T-type Ca2+ channels activate in response to subthreshold membrane depolarizations and therefore represent an important source of Ca2+ influx near the resting membrane potential. In neurons, these proteins significantly contribute to control relevant physiological processes including neuronal excitability, pacemaking and post-inhibitory rebound burst firing. Three subtypes of T-type channels (Cav3.1 to Cav3.3) have been identified, and using functional expression of recombinant channels diverse studies have validated the notion that T-type Ca2+ channels can be modulated by various endogenous ligands as well as by second messenger pathways. In this context, the present study reveals a previously unrecognized role for cyclin-dependent kinase 5 (Cdk5) in the regulation of native T-type channels in N1E-115 neuroblastoma cells, as well as recombinant Cav3.1channels heterologously expressed in HEK-293 cells. Cdk5 and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Our results show that overexpression of Cdk5 causes a significant increase in whole cell patch clamp currents through T-type channels in N1E-115 cells, while siRNA knockdown of Cdk5 greatly reduced these currents. Consistent with this, overexpression of Cdk5 in HEK-293 cells stably expressing Cav3.1channels upregulates macroscopic currents. Furthermore, using site-directed mutagenesis we identified a major phosphorylation site at serine 2234 within the C-terminal region of the Cav3.1subunit. These results highlight a novel role for Cdk5 in the regulation of T-type Ca2+ channels.

MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas

PubMed Central

Grünewald, Inga; Trautmann, Marcel; Busch, Alina; Bauer, Larissa; Huss, Sebastian; Schweinshaupt, Petra; Vollbrecht, Claudia; Odenthal, Margarete; Quaas, Alexander; Büttner, Reinhard; Meyer, Moritz F.; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Stenner, Markus; Hartmann, Wolfgang

2016-01-01

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs. 25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2. In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance. PMID:27662657

MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas.

PubMed

Grünewald, Inga; Trautmann, Marcel; Busch, Alina; Bauer, Larissa; Huss, Sebastian; Schweinshaupt, Petra; Vollbrecht, Claudia; Odenthal, Margarete; Quaas, Alexander; Büttner, Reinhard; Meyer, Moritz F; Beutner, Dirk; Hüttenbrink, Karl-Bernd; Wardelmann, Eva; Stenner, Markus; Hartmann, Wolfgang

2016-11-15

Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.25 of 51 cases were found to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.In summary, we here show that subgroups of SDCs display genomic amplifications of MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.

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

PubMed

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. Copyright © 2014 by the Genetics Society of America.

Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

PubMed Central

Mi, Da; Carr, Catherine B.; Georgala, Petrina A.; Huang, Yu-Ting; Manuel, Martine N.; Jeanes, Emily; Niisato, Emi; Sansom, Stephen N.; Livesey, Frederick J.; Theil, Thomas; Hasenpusch-Theil, Kerstin; Simpson, T. Ian; Mason, John O.; Price, David J.

2013-01-01

Summary The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct. PMID:23622063

Cyclin-dependent kinases: engines, clocks, and microprocessors.

PubMed

Morgan, D O

1997-01-01

Cyclin-dependent kinases (Cdks) play a well-established role in the regulation of the eukaryotic cell division cycle and have also been implicated in the control of gene transcription and other processes. Cdk activity is governed by a complex network of regulatory subunits and phosphorylation events whose precise effects on Cdk conformation have been revealed by recent crystallographic studies. In the cell, these regulatory mechanisms generate an interlinked series of Cdk oscillators that trigger the events of cell division.

Identification of deregulation of apoptosis and cell cycle in neuroendocrine tumors of the lung via NanoString nCounter expression analysis.

PubMed

Walter, Robert Fred Henry; Werner, Robert; Ting, Saskia; Vollbrecht, Claudia; Theegarten, Dirk; Christoph, Daniel Christian; Schmid, Kurt Werner; Wohlschlaeger, Jeremias; Mairinger, Fabian Dominik

2015-09-22

Neuroendocrine tumors of the lung comprise typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine cancer (LCNEC) and small-cell lung cancer (SCLC). Cell cycle and apoptosis are key pathways of multicellular homeostasis and deregulation of these pathways is associated with cancerogenesis. Sixty representative FFPE-specimens (16 TC, 13 AC, 16 LCNEC and 15 SCLC) were used for mRNA expression analysis using the NanoString technique. Eight genes related to apoptosis and ten genes regulating key points of cell cycle were investigated. ASCL1, BCL2, CASP8, CCNE1, CDK1, CDK2, CDKN1A and CDKN2A showed lower expression in carcinoids compared to carcinomas. In contrast, CCNE1 and CDK6 showed elevated expression in carcinoids compared to carcinomas. The calculated BCL2/BAX ratio showed increasing values from TC to SCLC. Between SCLC and LCNEC CDK2, CDKN1B, CDKN2A and PNN expression was significantly different with higher expression in SCLC. Carcinoids have increased CDK4/6 and CCND1 expression controlling RB1 phosphorylation via this signaling cascade. CDK2 and CCNE1 were increased in carcinomas showing that these use the opposite way to control RB1. BAX and BCL2 are antagonists in regulating apoptosis. BCL2 expression increased over BAX expression with increasing malignancy of the tumor from TC to SCLC.

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates

PubMed Central

Sansó, Miriam; Levin, Rebecca S.; Lipp, Jesse J.; Wang, Vivien Ya-Fan; Greifenberg, Ann Katrin; Quezada, Elizabeth M.; Ali, Akbar; Ghosh, Animesh; Larochelle, Stéphane; Rana, Tariq M.; Geyer, Matthias; Tong, Liang; Shokat, Kevan M.; Fisher, Robert P.

2016-01-01

The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5′-to-3′ “torpedo” exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phosphomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2. PMID:26728557

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

PubMed

Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

2014-02-18

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

TFIIH and P-TEFb Coordinate Transcription with Capping Enzyme Recruitment at Specific Genes in Fission Yeast

PubMed Central

Viladevall, Laia; St. Amour, Courtney V.; Rosebrock, Adam; Schneider, Susanne; Zhang, Chao; Allen, Jasmina J.; Shokat, Kevan M.; Schwer, Beate; Leatherwood, Janet K.; Fisher, Robert P.

2009-01-01

Summary Cyclin-dependent kinases (CDKs) are subunits of transcription factor (TF) IIH and positive transcription elongation factor b (P-TEFb). To define their functions, we mutated the TFIIH-associated kinase Mcs6 and P-TEFb homologs Cdk9 and Lsk1 of fission yeast, making them sensitive to bulky purine analogs. Selective inhibition of Mcs6 or Cdk9 blocks cell division, alters RNA polymerase (Pol) II carboxyl-terminal domain (CTD) phosphorylation and represses specific, overlapping subsets of transcripts. At a common target gene, both CDKs must be active for normal Pol II occupancy, and Spt5—a CDK substrate and regulator of elongation—accumulates disproportionately to Pol II when either kinase is inhibited. In contrast, Mcs6 activity is sufficient, and necessary, to recruit the Cdk9/Pcm1 (mRNA cap methyltransferase) complex. In vitro, phosphorylation of the CTD by Mcs6 stimulates subsequent phosphorylation by Cdk9. We propose that TFIIH primes the CTD and promotes recruitment of P-TEFb/Pcm1, serving to couple elongation and capping of select pre-mRNAs. PMID:19328067

Coordinated Expression of Cyclin-dependent Kinase-4 and its Regulators in Human Oral Tumors

PubMed Central

POI, MING J.; KNOBLOCH, THOMAS J.; SEARS, MARTA T.; UHRIG, LANA K.; WARNER, BLAKE M.; WEGHORST, CHRISTOPHER M.; LI, JUNAN

2014-01-01

Background/Aim While aberrant expression of cyclin-dependent kinase-4 (CDK4) has been found in squamous cell carcinoma of the head and neck (SCCHN), the associations between CDK4 and its regulators, namely, cyclin D1, cyclin E, gankyrin, SEI1, and BMI1 in gene expression remain to be explored. Herein we investigated the mRNA profiles of these oncogenes and their interrelations in different oral lesion tissues. Materials and Methods Thirty SCCHN specimens and patient-matched high at-risk mucosa (HARM) and 16 healthy control specimens were subjected to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. Results The mRNA levels of CDK4, cyclin D1, gankyrin, SEI1, BMI1 were significantly elevated in both HARM and SCCHN (in comparison with control specimens), and statistically significant correlations were found among these markers in gene expression. Conclusion Up-regulation of CDK4 and its regulators takes place in oral cancer progression in a coordinate manner, and HARM and SCCHN share a similar molecular signature within the CDK4-pRB pathway. PMID:24982332

PLK1 Activation in Late G2 Sets Up Commitment to Mitosis.

PubMed

Gheghiani, Lilia; Loew, Damarys; Lombard, Bérangère; Mansfeld, Jörg; Gavet, Olivier

2017-06-06

Commitment to mitosis must be tightly coordinated with DNA replication to preserve genome integrity. While we have previously established that the timely activation of CyclinB1-Cdk1 in late G2 triggers mitotic entry, the upstream regulatory mechanisms remain unclear. Here, we report that Polo-like kinase 1 (Plk1) is required for entry into mitosis during an unperturbed cell cycle and is rapidly activated shortly before CyclinB1-Cdk1. We determine that Plk1 associates with the Cdc25C1 phosphatase and induces its phosphorylation before mitotic entry. Plk1-dependent Cdc25C1 phosphosites are sufficient to promote mitotic entry, even when Plk1 activity is inhibited. Furthermore, we find that activation of Plk1 during G2 relies on CyclinA2-Cdk activity levels. Our findings thus elucidate a critical role for Plk1 in CyclinB1-Cdk1 activation and mitotic entry and outline how CyclinA2-Cdk, an S-promoting factor, poises cells for commitment to mitosis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle.

PubMed

Chang, Jeremy B; Ferrell, James E

2013-08-29

Despite the large size of the Xenopus laevis egg (approximately 1.2 mm diameter), a fertilized egg rapidly proceeds through mitosis in a spatially coordinated fashion. Mitosis is initiated by a bistable system of regulatory proteins centred on Cdk1 (refs 1, 2), raising the possibility that this spatial coordination could be achieved through trigger waves of Cdk1 activity. Using an extract system that performs cell cycles in vitro, here we show that mitosis does spread through Xenopus cytoplasm via trigger waves, propagating at a linear speed of approximately 60 µm min(-1). Perturbing the feedback loops that give rise to the bistability of Cdk1 changes the speed and dynamics of the waves. Time-lapse imaging of intact eggs argues that trigger waves of Cdk1 activation are responsible for surface contraction waves, ripples in the cell cortex that precede cytokinesis. These findings indicate that Cdk1 trigger waves help ensure the spatiotemporal coordination of mitosis in large eggs. Trigger waves may be an important general mechanism for coordinating biochemical events over large distances.

The Use of Science Kits in the Professional Development of Rural Elementary School Teachers

ERIC Educational Resources Information Center

Sherman, Ann; MacDonald, A. Leo

2008-01-01

This study reports on a science professional development initiative with elementary school teachers in Canada. Grades 4 and 5 teachers were involved in the implementation and modification of science kits, together with corresponding professional development activities. Each kit was aligned to specific outcomes in the curriculum and provided a…

In vitro toxicity of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons to cetacean cells and tissues

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

Carvan, M.J. III.

1993-01-01

Cetaceans bioaccumulate high aromatic hydrocarbon tissue residues, and elevated levels of PCB residues in tissues are proposed to have occurred concurrently with recent epizootic deaths of dolphins. The objectives of this study were: (1) to develop and characterize an epithelial cell line derived from dolphin tissues, (2) to investigate the effects of hydrocarbon pollutants on those cells, and (3) to analyze the toxicity of hydrocarbon pollutants on cetacean tissues in vitro. An epithelial cell line, Carvan dolphin kidney (CDK), isolated from a spontaneously aborted female bottlenose dolphin, Tursiops truncatus, grew rapidly. These cells were neither transformed nor immortal. Velocity sedimentationmore » analysis showed CDK cells contained nuclear aryl hydrocarbon receptor, suggestive of cytochrome P450 inducibility. BaP inhibited mitosis in CDK cells in a dose-dependent manner. Data indicate that CDK cells metabolize BaP, that BaP metabolites bind to cellular DNA initiating unscheduled DNA synthesis, and that the inhibition of cytochrome P450 metabolism decrease the BaP-associated inhibition of mitosis in dolphin cells. The data also suggest that TCDD acts synergistically to increase the levels of DNA damage by the procarcinogen BaP. Cetacean liver microsomes was isolated and evaluated for the presence of cytochrome P450 proteins by SDS-PAGE, apparent minimum molecular weight determination, and immunoblot analysis. P450 activity was induced in cetacean tissue samples and CDK cells by exposure in vitro to one of several cytochrome P450-inducing chemicals. The data suggest that cetacean tissues and cells can be utilized to study the in vitro induction of cytochrome P450, resultant metabolism of xenobiotic contaminants, and the subsequent cellular and molecular responses. However, the identity of specific P450 isozymes involved in this process will remain undetermined until monoclonal antibodies that recognize cetacean P450s can be generated.« less

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

PubMed Central

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

Evaluation of the first cytostatically active 1-aza-9-oxafluorenes as novel selective CDK1 inhibitors with P-glycoprotein modulating properties.

PubMed

Brachwitz, Kristin; Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Molnár, Josef; Hilgeroth, Andreas

2003-02-27

The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.

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

von Laszewski, G.; Gawor, J.; Lane, P.

In this paper we report on the features of the Java Commodity Grid Kit (Java CoG Kit). The Java CoG Kit provides middleware for accessing Grid functionality from the Java framework. Java CoG Kit middleware is general enough to design a variety of advanced Grid applications with quite different user requirements. Access to the Grid is established via Globus Toolkit protocols, allowing the Java CoG Kit to also communicate with the services distributed as part of the C Globus Toolkit reference implementation. Thus, the Java CoG Kit provides Grid developers with the ability to utilize the Grid, as well asmore » numerous additional libraries and frameworks developed by the Java community to enable network, Internet, enterprise and peer-to-peer computing. A variety of projects have successfully used the client libraries of the Java CoG Kit to access Grids driven by the C Globus Toolkit software. In this paper we also report on the efforts to develop serverside Java CoG Kit components. As part of this research we have implemented a prototype pure Java resource management system that enables one to run Grid jobs on platforms on which a Java virtual machine is supported, including Windows NT machines.« less

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

PubMed

Butz, Henriett; Németh, Kinga; Czenke, Dóra; Likó, István; Czirják, Sándor; Zivkovic, Vladimir; Baghy, Kornélia; Korbonits, Márta; Kovalszky, Ilona; Igaz, Péter; Rácz, Károly; Patócs, Attila

2017-07-01

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.

Personal Computer-less (PC-less) Microcontroller Training Kit

NASA Astrophysics Data System (ADS)

Somantri, Y.; Wahyudin, D.; Fushilat, I.

2018-02-01

The need of microcontroller training kit is necessary for practical work of students of electrical engineering education. However, to use available training kit not only costly but also does not meet the need of laboratory requirements. An affordable and portable microcontroller kit could answer such problem. This paper explains the design and development of Personal Computer Less (PC-Less) Microcontroller Training Kit. It was developed based on Lattepanda processor and Arduino microcontroller as target. The training kit equipped with advanced input-output interfaces that adopted the concept of low cost and low power system. The preliminary usability testing proved this device can be used as a tool for microcontroller programming and industrial automation training. By adopting the concept of portability, the device could be operated in the rural area which electricity and computer infrastructure are limited. Furthermore, the training kit is suitable for student of electrical engineering student from university and vocational high school.

The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription

PubMed Central

Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R.

2016-01-01

Throughout cell cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

Identification of deregulation of apoptosis and cell cycle in neuroendocrine tumors of the lung via NanoString nCounter expression analysis

PubMed Central

Walter, Robert Fred Henry; Werner, Robert; Ting, Saskia; Vollbrecht, Claudia; Theegarten, Dirk; Christoph, Daniel Christian; Schmid, Kurt Werner; Wohlschlaeger, Jeremias; Mairinger, Fabian Dominik

2015-01-01

Background Neuroendocrine tumors of the lung comprise typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine cancer (LCNEC) and small-cell lung cancer (SCLC). Cell cycle and apoptosis are key pathways of multicellular homeostasis and deregulation of these pathways is associated with cancerogenesis. Materials and Methods Sixty representative FFPE-specimens (16 TC, 13 AC, 16 LCNEC and 15 SCLC) were used for mRNA expression analysis using the NanoString technique. Eight genes related to apoptosis and ten genes regulating key points of cell cycle were investigated. Results ASCL1, BCL2, CASP8, CCNE1, CDK1, CDK2, CDKN1A and CDKN2A showed lower expression in carcinoids compared to carcinomas. In contrast, CCNE1 and CDK6 showed elevated expression in carcinoids compared to carcinomas. The calculated BCL2/BAX ratio showed increasing values from TC to SCLC. Between SCLC and LCNEC CDK2, CDKN1B, CDKN2A and PNN expression was significantly different with higher expression in SCLC. Conclusion Carcinoids have increased CDK4/6 and CCND1 expression controlling RB1 phosphorylation via this signaling cascade. CDK2 and CCNE1 were increased in carcinomas showing that these use the opposite way to control RB1. BAX and BCL2 are antagonists in regulating apoptosis. BCL2 expression increased over BAX expression with increasing malignancy of the tumor from TC to SCLC. PMID:26008974

Interaction with Cyclin H/Cyclin-dependent Kinase 7 (CCNH/CDK7) Stabilizes C-terminal Binding Protein 2 (CtBP2) and Promotes Cancer Cell Migration*

PubMed Central

Wang, Yuchan; Liu, Fang; Mao, Feng; Hang, Qinlei; Huang, Xiaodong; He, Song; Wang, Yingying; Cheng, Chun; Wang, Huijie; Xu, Guangfei; Zhang, Tianyi; Shen, Aiguo

2013-01-01

CtBP2 has been demonstrated to possess tumor-promoting capacities by virtue of up-regulating epithelial-mesenchymal transition (EMT) and down-regulating apoptosis in cancer cells. As a result, cellular CtBP2 levels are considered a key factor determining the outcome of oncogenic transformation. How pro-tumorigenic and anti-tumorigenic factors compete for fine-tuning CtBP2 levels is incompletely understood. Here we report that the cyclin H/cyclin-dependent kinase 7 (CCNH/CDK7) complex interacted with CtBP2 in vivo and in vitro. Depletion of either CCNH or CDK7 decreased CtBP2 protein levels by accelerating proteasome-dependent CtBP2 clearance. Further analysis revealed that CCNH/CDK7 competed with the tumor repressor HIPK2 for CtBP2 binding and consequently inhibited phosphorylation and dimerization of CtBP2. Phosphorylation-defective CtBP2 interacted more strongly with CCNH/CDK7 and was more resistant to degradation. Finally, overexpression of CtBP2 increased whereas depletion of CtBP2 dampened the invasive and migratory potential of breast cancer cells. CtBP2 promoted the invasion and migration of breast cancer cells in a CCNH-dependent manner. Taken together, our data have delineated a novel pathway that regulates CtBP2 stability, suggesting that targeting the CCNH/CDK7-CtBP2 axis may yield a viable anti-tumor strategy. PMID:23393140

Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk).

PubMed

Umaña, Angie C; Iwahori, Satoko; Kalejta, Robert F

2018-01-19

Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog-sensitive (AS) kinases. We identified the gatekeeper residues of the v-Cdks encoded by Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) and mutated them to generate AS kinases. The AS-v-Cdks are functional and utilize different ATP derivatives with a specificity closely matching their cellular ortholog, AS-Cdk2. The AS derivative of the EBV v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral Cdks, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of both viral infections and Cdk biology.

MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

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

Xu, Shun; Huang, Haijiao; Li, Nanhong

2016-05-13

MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33more » promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.« less

Development and evaluation of a PCR-based assay kit for authentication of Zaocys dhumnades in traditional Chinese medicine.

PubMed

Zhang, Xiaomei; Zhou, Tingting; Yu, Wenjing; Ai, Jinxia; Wang, Xuesong; Gao, Lijun; Yuan, Guangxin; Li, Mingcheng

2018-01-01

We developed a kind of Zaocys dhumnades DNA test kit and it's indexes including specificity, sensitivity and stability were evaluated and compared with the method recorded in Chinese Pharmacopoeia (2010 edition). The bioinformatics technology was used to design primers, sequencing and blast, in conjunction with PCR technology based on the characteristics of Z. dhumnades cytochrome b (Cyt b) gene. The efficiency of nucleic acid extraction by the kit was done in accordance with Pharmacopoeia method. The kit stability results proved effective after repeated freezing and thawing 20 times. The sensitivity results indicated that the lowest amount detected by the kit was 0. 025 g of each specimen. The specificity test of the kit was 100% specific. All repeatability tests indicated the same results when conducted three times. Compared with the method recorded in Chinese Pharmacopoeia, the PCR-based assay kit by our team developed is accurate, effective in identification of Z. dhumnades, it is simple and fast, demonstrating a broad prospect in quality inspection of Z. dhumnades in the future.

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

von Laszewski, G.; Foster, I.; Gawor, J.

In this paper we report on the features of the Java Commodity Grid Kit. The Java CoG Kit provides middleware for accessing Grid functionality from the Java framework. Java CoG Kit middleware is general enough to design a variety of advanced Grid applications with quite different user requirements. Access to the Grid is established via Globus protocols, allowing the Java CoG Kit to communicate also with the C Globus reference implementation. Thus, the Java CoG Kit provides Grid developers with the ability to utilize the Grid, as well as numerous additional libraries and frameworks developed by the Java community tomore » enable network, Internet, enterprise, and peer-to peer computing. A variety of projects have successfully used the client libraries of the Java CoG Kit to access Grids driven by the C Globus software. In this paper we also report on the efforts to develop server side Java CoG Kit components. As part of this research we have implemented a prototype pure Java resource management system that enables one to run Globus jobs on platforms on which a Java virtual machine is supported, including Windows NT machines.« less

Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA*

PubMed Central

Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

2013-01-01

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing. PMID:23744076

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation.

PubMed

Sansam, Courtney G; Goins, Duane; Siefert, Joseph C; Clowdus, Emily A; Sansam, Christopher L

2015-03-01

S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRR(TESE)) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRR(TESE) does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRR(TESE) does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled. © 2015 Sansam et al.; Published by Cold Spring Harbor Laboratory Press.

Cdk1 Activates Pre-mitotic Nuclear Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells.

PubMed

Baffet, Alexandre D; Hu, Daniel J; Vallee, Richard B

2015-06-22

Dynein recruitment to the nuclear envelope is required for pre-mitotic nucleus-centrosome interactions in nonneuronal cells and for apical nuclear migration in neural stem cells. In each case, dynein is recruited to the nuclear envelope (NE) specifically during G2 via two nuclear pore-mediated mechanisms involving RanBP2-BicD2 and Nup133-CENP-F. The mechanisms responsible for cell-cycle control of this behavior are unknown. We now find that Cdk1 serves as a direct master controller for NE dynein recruitment in neural stem cells and HeLa cells. Cdk1 phosphorylates conserved sites within RanBP2 and activates BicD2 binding and early dynein recruitment. Late recruitment is triggered by a Cdk1-induced export of CENP-F from the nucleus. Forced NE targeting of BicD2 overrides Cdk1 inhibition, fully rescuing dynein recruitment and nuclear migration in neural stem cells. These results reveal how NE dynein recruitment is cell-cycle regulated and identify the trigger mechanism for apical nuclear migration in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

Cdk1 Regulates the Temporal Recruitment of Telomerase and Cdc13-Stn1-Ten1 Complex for Telomere Replication

PubMed Central

Liu, Chang-Ching; Gopalakrishnan, Veena; Poon, Lai-Fong; Yan, TingDong

2014-01-01

In budding yeast (Saccharomyces cerevisiae), the cell cycle-dependent telomere elongation by telomerase is controlled by the cyclin-dependent kinase 1 (Cdk1). The telomere length homeostasis is balanced between telomerase-unextendable and telomerase-extendable states that both require Cdc13. The recruitment of telomerase complex by Cdc13 promotes telomere elongation, while the formation of Cdc13-Stn1-Ten1 (CST) complex at the telomere blocks telomere elongation by telomerase. However, the cellular signaling that regulates the timing of the telomerase-extendable and telomerase-unextendable states is largely unknown. Phosphorylation of Cdc13 by Cdk1 promotes the interaction between Cdc13 and Est1 and hence telomere elongation. Here, we show that Cdk1 also phosphorylates Stn1 at threonine 223 and serine 250 both in vitro and in vivo, and these phosphorylation events are essential for the stability of the CST complexes at the telomeres. By controlling the timing of Cdc13 and Stn1 phosphorylations during cell cycle progression, Cdk1 regulates the temporal recruitment of telomerase complexes and CST complexes to the telomeres to facilitate telomere maintenance. PMID:24164896

Kits in Motion

ERIC Educational Resources Information Center

Gee, Maureen

1975-01-01

Discusses three kits developed by museums in British Columbia for use in rural classrooms. The science kit on marine biology consists of modules which included specimens, books, audiovisual materials and student activities. (BR)

INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions.

PubMed

Cánepa, Eduardo T; Scassa, María E; Ceruti, Julieta M; Marazita, Mariela C; Carcagno, Abel L; Sirkin, Pablo F; Ogara, María F

2007-07-01

The cyclin D-Cdk4-6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The members of INK4 family, comprising p16(INK4a), p15(INK4b), p18(INK4c), and p19(INK4d), block the progression of the cell cycle by binding to either Cdk4 or Cdk6 and inhibiting the action of cyclin D. These INK4 proteins share a similar structure dominated by several ankyrin repeats. Although they appear to be structurally redundant and equally potent as inhibitors, the INK4 family members are differentially expressed during mouse development. The striking diversity in the pattern of expression of INK4 genes suggested that this family of cell cycle inhibitors might have cell lineage-specific or tissue-specific functions. The INK4 proteins are commonly lost or inactivated by mutations in diverse types of cancer, and they represent established or candidate tumor suppressors. Apart from their capacity to arrest cells in the G1-phase of the cell cycle they have been shown to participate in an increasing number of cellular processes. Given their emerging roles in fundamental physiological as well as pathological processes, it is interesting to explore the diverse roles for the individual INK4 family members in different functions other than cell cycle regulation. Extensive studies, over the past few years, uncover the involvement of INK4 proteins in senescence, apoptosis, DNA repair, and multistep oncogenesis. We will focus the discussion here on these unexpected issues.

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity.

PubMed

Veerasami, Maroudam; Venkataraman, K; Karuppannan, Chitra; Shanmugam, Arun Attur; Prudhvi, Mallepaddi Chand; Holder, Thomas; Rathnagiri, Polavarapu; Arunmozhivarman, K; Raj, Gopal Dhinakar; Vordermeier, Martin; Mohana Subramanian, B

2018-03-01

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria . To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit's performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.

Co-amplification of phosphoinositide 3-kinase enhancer A and cyclin-dependent kinase 4 triggers glioblastoma progression | Office of Cancer Genomics

Cancer.gov

Glioblastoma (GBM) is the most common primary brain tumor and has a dismal prognosis. Amplification of chromosome 12q13-q15 (Cyclin-dependent kinase 4 (CDK4) amplicon) is frequently observed in numerous human cancers including GBM. Phosphoinositide 3-kinase enhancer (PIKE) is a group of GTP-binding proteins that belong to the subgroup of centaurin GTPase family, encoded by CENTG1 located in CDK4 amplicon. However, the pathological significance of CDK4 amplicon in GBM formation remains incompletely understood.

A framework for measurement and harmonization of pediatric multiple sclerosis etiologic research studies: The Pediatric MS Tool-Kit.

PubMed

Magalhaes, Sandra; Banwell, Brenda; Bar-Or, Amit; Fortier, Isabel; Hanwell, Heather E; Lim, Ming; Matt, Georg E; Neuteboom, Rinze F; O'Riordan, David L; Schneider, Paul K; Pugliatti, Maura; Shatenstein, Bryna; Tansey, Catherine M; Wassmer, Evangeline; Wolfson, Christina

2018-06-01

While studying the etiology of multiple sclerosis (MS) in children has several methodological advantages over studying etiology in adults, studies are limited by small sample sizes. Using a rigorous methodological process, we developed the Pediatric MS Tool-Kit, a measurement framework that includes a minimal set of core variables to assess etiological risk factors. We solicited input from the International Pediatric MS Study Group to select three risk factors: environmental tobacco smoke (ETS) exposure, sun exposure, and vitamin D intake. To develop the Tool-Kit, we used a Delphi study involving a working group of epidemiologists, neurologists, and content experts from North America and Europe. The Tool-Kit includes six core variables to measure ETS, six to measure sun exposure, and six to measure vitamin D intake. The Tool-Kit can be accessed online ( www.maelstrom-research.org/mica/network/tool-kit ). The goals of the Tool-Kit are to enhance exposure measurement in newly designed pediatric MS studies and comparability of results across studies, and in the longer term to facilitate harmonization of studies, a methodological approach that can be used to circumvent issues of small sample sizes. We believe the Tool-Kit will prove to be a valuable resource to guide pediatric MS researchers in developing study-specific questionnaire.

Conditional Deletion of Kit in Melanocytes: White Spotting Phenotype Is Cell Autonomous.

PubMed

Aoki, Hitomi; Tomita, Hiroyuki; Hara, Akira; Kunisada, Takahiro

2015-07-01

It is well established that cell-intrinsic signaling through the receptor tyrosine kinase KIT is critical for the development of neural crest-derived melanocytes. Nevertheless, it is not entirely clear whether Kit acts exclusively in a melanocyte-autonomous manner or in addition indirectly through other cell types. To address this question in vivo, we generated a targeted allele of Kit that allowed for CRE recombinase-mediated deletion of the transmembrane domain of KIT. Mice carrying one copy of the targeted allele and expressing CRE under the melanoblast/melanocyte-specific tyrosinase promoter exhibited a white spotting phenotype that was even more extensive compared with that found in mice heterozygous for a Kit-null allele. This phenotype is unlikely the result of sequestration of KIT ligand by neighboring cells or by potentially secreted forms of KIT because the spotting phenotype could not be rescued by overexpression of KITL. Likewise, overexpression of endothelin-3 or hepatocyte growth factor was unable to rescue melanocytes in these mice. Although the severity of the observed phenotype remains to be explained, the findings indicate that melanocyte-selective impairment of Kit is sufficient to interfere with normal melanocyte development.

Cloning of cDNAs for H1F0, TOP1, CLTA and CDK1 and the effects of cryopreservation on the expression of their mRNA transcripts in yak (Bos grunniens) oocytes.

PubMed

Niu, Hui-Ran; Zi, Xiang-Dong; Xiao, Xiao; Xiong, Xian-Rong; Zhong, Jin-Cheng; Li, Jian; Wang, Li; Wang, Yong

2014-08-01

We cloned and sequenced four pivotal cDNAs involved in DNA structural maintenance (H1F0 and TOP1) and the cell cycle (CLTA and CDK1) from yak oocytes. In addition, we studied the consequences of freezing-thawing (F/T) processes on the expression of their mRNA transcripts in yak immature and in vitro matured (MII) oocytes. H1F0, TOP1, CLTA and CDK1 cDNAs were cloned from yak oocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) strategy. The expression of their mRNA transcript analyses were performed upon fresh and frozen-thawed immature germinal vesicle (GV) and MII yak oocytes following normalization of transcripts with GAPDH by real-time PCR. The yak H1F0, TOP1, CLTA and CDK1 cDNA sequences were found to consist of CDK1 585, 2539, 740, and 894 bp, respectively. Their coding regions encoded 195, 768, 244, and 298 amino acids, respectively. The homology with that of cattle was very high (95.2%, 98.8%, 93.6%, and 89.5%, respectively nucleotide sequence level, and 94.3%, 98.2%, 87.7%, and 90.9%, respectively at the deduced amino acid level). The overall mRNA expression levels of these four transcripts were reduced by F/T process, albeit at different levels. TOP1 in GV-oocytes, and H1F0 and CDK1 in MII-oocytes of the yak were significantly down-regulated (P<0.05). This is the first isolation and characterization of H1F0, TOP1, CLTA, and CDK1 cDNAs from yak oocytes. The lower fertility and developmental ability of yak oocytes following fertilization after cryopreservation may be explained by the alterations to their gene expression profiles. Copyright © 2014 Elsevier Inc. All rights reserved.

Locomotor conditioning by amphetamine requires cyclin-dependent kinase 5 signaling in the nucleus accumbens

PubMed Central

Singer, Bryan F; Neugebauer, Nichole M; Forneris, Justin; Rodvelt, Kelli R; Li, Dongdong; Bubula, Nancy; Vezina, Paul

2014-01-01

Intermittent systemic exposure to psychostimulants such as amphetamine leads to several forms of long-lasting behavioral plasticity including nonassociative sensitization and associative conditioning. In the nucleus accumbens (NAcc), the protein serine/threonine kinase cyclin-dependent kinase 5 (Cdk5) and its phosphorylation target, the guanine-nucleotide exchange factor kalirin-7 (Kal7), may contribute to the neuroadaptations underlying each of these forms of plasticity. Pharmacological inhibition of Cdk5 in the NAcc prevents the increases in dendritic spine density in this site and enhances the locomotor sensitization normally observed following repeated cocaine. Mice lacking the Kal7 gene display similar phenotypes suggesting that locomotor sensitization and increased NAcc spine density need not be positively correlated. As increases in spine density may relate to the formation of associative memories and both Cdk5 and Kal7 regulate the generation of spines following repeated drug exposure, we hypothesized that either inhibiting Cdk5 or preventing its phosphorylation of Kal7 in the NAcc may prevent the induction of drug conditioning. In the present experiments, blockade in rats of NAcc Cdk5 activity with roscovitine (40 nmol/0.5μl/side) prior to each of 4 injections of amphetamine (1.5 mg/kg; i.p.) prevented the accrual of contextual locomotor conditioning but spared the induction of locomotor sensitization as revealed on tests conducted one week later. Similarly, transient viral expression in the NAcc exclusively during amphetamine exposure of a threoninealanine mutant form of Kal7 [mKal7(T1590A)] that is not phosphorylated by Cdk5 also prevented the accrual of contextual conditioning and spared the induction of sensitization. These results indicate that signaling via Cdk5 and Kal7 in the NAcc is necessary for the formation of context-drug associations, potentially through the modulation of dendritic spine dynamics in this site. PMID:24939858

Toys for Early Development of the Young Blind Child: A Guide for Parents.

ERIC Educational Resources Information Center

Illinois State Office of the Superintendent of Public Instruction, Springfield.

A booklet is presented for parents of blind children. This booklet lists various toys that are useful for the child in his development and cognitive growth. Three kits of toys are presented. Each of these kits was developed for children of approximate age groups. Kit #1 is for the very young blind infants and contains toys that usually interest…

An indigenously developed nitrite kit to aid in the diagnosis of urinary tract infection.

PubMed

Sood, S; Upadhyaya, P; Kapil, A; Lodha, R; Jain, Y; Bagga, A

1999-09-01

To evaluate the utility of an indigenously developed nitrite kit for the rapid diagnosis of urinary tract infection (UTI) METHODS: 1018 urine specimens were collected from all cases where there was clinical suspicion of UTI. Samples were cultured as per standard microbiological protocol. Presence of nitrites was indicated by the development of purple color on addition of color developing solution and compared with the set of graded positive and negative controls also provided in the Kit. The results of the nitrite kit were compared with the semi-quantitative urine culture as the gold standard. The sensitivity, specificity, positive predictive and negative predictive values were 47%, 87%, 31% and 93%, respectively. Nitrite kit as a screening test can decrease the work load in the clinical bacteriology laboratory. More importantly in a field set up that is devoid of culture facilities, it can be used to correctly predict the absence of UTI.

The cyclin-dependent kinase PITSLRE/CDK11 is required for successful autophagy.

PubMed

Wilkinson, Simon; Croft, Daniel R; O'Prey, Jim; Meedendorp, Arenda; O'Prey, Margaret; Dufès, Christine; Ryan, Kevin M

2011-11-01

(Macro)autophagy is a membrane-trafficking process that serves to sequester cellular constituents in organelles termed autophagosomes, which target their degradation in the lysosome. Autophagy operates at basal levels in all cells where it serves as a homeostatic mechanism to maintain cellular integrity. The levels and cargoes of autophagy can, however, change in response to a variety of stimuli, and perturbations in autophagy are known to be involved in the aetiology of various human diseases. Autophagy must therefore be tightly controlled. We report here that the Drosophila cyclin-dependent kinase PITSLRE is a modulator of autophagy. Loss of the human PITSLRE orthologue, CDK11, initially appears to induce autophagy, but at later time points CDK11 is critically required for autophagic flux and cargo digestion. Since PITSLRE/CDK11 regulates autophagy in both Drosophila and human cells, this kinase represents a novel phylogenetically conserved component of the autophagy machinery.

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

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

Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor

2007-02-02

c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2more » activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.« less

Antitumor activity of novel chimeric peptides derived from cyclinD/CDK4 and the protein transduction domain 4.

PubMed

Wang, Haili; Chen, Xi; Chen, Yanping; Sun, Lei; Li, Guodong; Zhai, Mingxia; Zhai, Wenjie; Kang, Qiaozhen; Gao, Yanfeng; Qi, Yuanming

2013-02-01

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

Curcumin: Synthesis optimization and in silico interaction with cyclin dependent kinase.

PubMed

Ahmed, Mahmood; Abdul Qadir, Muhammad; Imtiaz Shafiq, Muhammad; Muddassar, Muhammad; Hameed, Abdul; Nadeem Arshad, Muhammad; Asiri, Abdullah M

2017-09-01

Curcumin is a natural product with enormous biological potential. In this study, curcumin synthesis was revisited using different reaction solvents, a catalyst (n-butylamine) and a water scavenger [(n-BuO)3B], to develop the optimal procedure for its rapid acquisition. During synthesis, solvent choice was found to be an important parameter for better curcumin yield and high purity. In a typical reaction, acetyl acetone was treated with boron trioxide, followed by condensation with vanillin in the presence of tri-n-butyl borate as water scavenger and n-butylamine as catalyst at 80 °C in ethyl acetate to afford curcumin. Moreover, curcumin was also extracted from turmeric powder and spectroscopic properties such as IR, MS, 1H NMR and 13C NMR with synthetic curcumin were established to identify any impurity. The purity of synthetic and extracted curcumin was also checked by TLC and HPLC-DAD. To computationally assess its therapeutic potential against cyclin dependent kinases (CDKs), curcumin was docked in different isoforms of CDKs. It was observed that it did not dock at the active sites of CDK2 and CDK6. However, it could enter into weak interactions with CDK4 protein.

Cyclin A recruits p33cdk2 to the cellular transcription factor DRTF1.

PubMed

Bandara, L R; Adamczewski, J P; Zamanian, M; Poon, R Y; Hunt, T; Thangue, N B

1992-01-01

Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.

Mediator kinase module and human tumorigenesis.

PubMed

Clark, Alison D; Oldenbroek, Marieke; Boyer, Thomas G

2015-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit "kinase" module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways.

Mediator kinase module and human tumorigenesis

PubMed Central

Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

2016-01-01

Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

Advantage of using a home-made ELISA kit for detection of Helicobacter pylori infection over commercially imported kits.

PubMed

Mohammadi, M; Talebkhan, Y; Khalili, G; Mahboudi, F; Massarrat, S; Zamaninia, L; Oghalaei, A

2008-01-01

To evaluate a home-made ELISA kit for detection of Helicobacter pylori (Hp) infection and comparison of its immunologic criteria with those of foreign commercial kits. A home-made IgG ELISA kit was developed using soluble antigenic fractions of Hp proteins. Confirmed sera were tested and serological criteria were evaluated through assessment of 199 serum samples. The accuracy, sensitivity and specificity values of home-made kit were 92, 92 and 90.4%, respectively. These immunologic criteria for Trinity kit were 95.2, 95.2 and 95% in comparison with IBL kit (91.3, 92.2 and 88.5%), BIOHIT kit (72.4, 41.6 and 94.1%) and HelicoBlot2.1 (94.2, 93.4 and 100%). Kappa agreement assessment demonstrated that two of the imported ELISA kits had fair to moderate agreement with the home-made kit while the other one had a poor agreement value. Apart from comparable values between the home-made kit and the most efficient imported kit (Trinity) there was significant cost benefit. Therefore, we recommend the home-made kit as a suitable substitution for detection of Hp infection in the Iranian population.

Convenient Preparation of [(68)Ga]DKFZ-PSMA-11 Using a Robust Single-Vial Kit and Demonstration of Its Clinical Efficacy.

PubMed

Satpati, Drishty; Shinto, Ajit; Kamaleshwaran, K K; Sane, Surekha; Banerjee, Sharmila

2016-06-01

[(68)Ga]DKFZ-PSMA-11 has proved to be an important diagnostic radiotracer for targeting prostate-specific membrane antigen (PSMA) overexpression in both recurrent prostate cancer (PC) and relevant metastatic sites. However, the widespread, routine clinical use of such a potential radiopharmaceutical demands availability of a ready-to-use kit formulation to enable convenient radiopharmaceutical preparation. Herein, we report the development of a freeze-dried kit vial for the formulation of [(68)Ga]DKFZ-PSMA-11 and its clinical use in patients using a "shake-bake-inject" methodology. The freeze-dried kit vial was developed after optimization of ligand content (PSMA-11) and pH conditions. The kit was formulated using (68)Ga from two different commercially available generators. Positron emission tomography/X-ray computed tomography (PET/CT) images of PC patients were obtained using the kit-formulated radiotracer. [(68)Ga]DKFZ-PSMA-11 was prepared in >98 % radiochemical yield and purity using the freeze-dried kit vials. Kits were optimized for the preparation of four patient doses. The clinical utility was evaluated in patients with histologically confirmed prostate cancer, and the images were of good quality as well as conforming to tumor marker and clinical expectations. The development of a simple and ready-to-use freeze-dried DKFZ-PSMA-11 kit for the preparation of Ga-68-based radiotracers constitutes a major step towards the expedition of the widespread and economical screening of PC patients.

Evolution of cyclin-dependent kinases (CDKs) and CDK-activating kinases (CAKs): differential conservation of CAKs in yeast and metazoa.

PubMed

Liu, J; Kipreos, E T

2000-07-01

Cyclin-dependent kinases (CDKs) function as central regulators of both the cell cycle and transcription. CDK activation depends on phosphorylation by a CDK-activating kinase (CAK). Different CAKs have been identified in budding yeast, fission yeast, and metazoans. All known CAKs belong to the extended CDK family. The sole budding yeast CAK, CAK1, and one of the two CAKs in fission yeast, csk1, have diverged considerably from other CDKs. Cell cycle regulatory components have been largely conserved in eukaryotes; however, orthologs of neither CAK1 nor csk1 have been identified in other species to date. To determine the evolutionary relationships of yeast and metazoan CAKs, we performed a phylogenetic analysis of the extended CDK family in budding yeast, fission yeast, humans, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans. We observed that there were 10 clades for CDK-related genes, of which seven appeared ancestral, containing both yeast and metazoan genes. The four clades that contain CDKs that regulate transcription by phosphorylating the carboxyl-terminal domain (CTD) of RNA Polymerase II generally have only a single orthologous gene in each species of yeast and metazoans. In contrast, the ancestral cell cycle CDK (analogous to budding yeast CDC28) gave rise to a number of genes in metazoans, as did the ancestor of budding yeast PHO85. One ancestral clade is unique in that there are fission yeast and metazoan members, but there is no budding yeast ortholog, suggesting that it was lost subsequent to evolutionary divergence. Interestingly, CAK1 and csk1 branch together with high bootstrap support values. We used both the relative apparent synapomorphy analysis (RASA) method in combination with the S-F method of sampling reduced character sets and gamma-corrected distance methods to confirm that the CAK1/csk1 association was not an artifact of long-branch attraction. This result suggests that CAK1 and csk1 are orthologs and that a central aspect of CAK regulation has been conserved in budding and fission yeast. Although there are metazoan CDK-family members for which we could not define ancestral lineage, our analysis failed to identify metazoan CAK1/csk1 orthologs, suggesting that if the CAK1/csk1 gene existed in the metazoan ancestor, it has not been conserved.

miR Profiling Identifies Cyclin-Dependent Kinase 6 Downregulation as a Potential Mechanism of Acquired Cisplatin Resistance in Non-Small-Cell Lung Carcinoma.

PubMed

Bar, Jair; Gorn-Hondermann, Ivan; Moretto, Patricia; Perkins, Theodore J; Niknejad, Nima; Stewart, David J; Goss, Glenwood D; Dimitroulakos, Jim

2015-11-01

To identify the mechanisms of cisplatin resistance, global microRNA (miR) expression was tested. The expression of miR-145 was consistently higher in resistant cells. The expression of cyclin-dependent kinase 6 (CDK6), a potential target of miR-145, was lower in resistant cells, and inhibition of CDK4/6 protected cells from cisplatin. Cell cycle inhibition, currently being tested in clinical trials, might be antagonistic to cisplatin and other cytotoxic drugs. Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Platinum-based chemotherapeutic drugs are the most active agents in treating advanced disease. Resistance to these drugs is common and multifactorial; insight into the molecular mechanisms involved will likely enhance efficacy. A set of NSCLC platinum-resistant sublines was created from the Calu6 cell line. Cell viability was quantified using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentially expressed microRNAs (miRs) in these lines were identified using Affymetrix miR arrays. The potential genes targeted by these miRs were searched using the TargetScan algorithm. The expression levels of miRs and mRNA were tested using real-time polymerase chain reaction. miR-145 was reproducibly elevated in all the resistant sublines tested; however, modulation of miR-145 levels alone in these cells did not affect their response to cisplatin. A potential target of miR-145 is cyclin-dependent kinase 6 (CDK6), an important regulator of cell proliferation. The mRNA and protein levels of CDK6 were both downregulated in the resistant sublines. An inhibitor of CDK4/6 (PD0332991) protected parental NSCLC cells from cisplatin cytotoxicity. In the present study, we identified miRs differentially expressed in cisplatin-resistant cell lines, including miR-145. A predicted target of miR-145 is CDK6, and its expression was found to be downregulated in the resistant sublines, although not directly by miR-145. Inhibition of CDK6 antagonizes cisplatin-induced NSCLC cell cytotoxicity, suggesting that agents that inhibit CDK6 should be avoided during cisplatin therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Implementing and Assessing 4-H Educational Activity Kits for Children

ERIC Educational Resources Information Center

Scheer, Scott D.; Yeske, Janine; Zimmer, Bruce

2011-01-01

Educational activity kits were developed and implemented through a statewide effort for 4-H Youth Development Extension programs serving 5-8 year-old children. The purpose of the kits was to promote life skills in children and assess the learning environment. Data was collected based on the observations of 577 children across 22 counties. Findings…

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

DOEpatents

Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

2003-06-03

The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

Basic Teaching Kit on Consumer Advertising.

ERIC Educational Resources Information Center

Proctor and Gamble Co., Cincinnati, OH.

This advertising kit was developed by Procter and Gamble in response to requests from teachers and consumer educators who asked for materials from business about business. The kit is not intended to cover the entire field of advertising. Rather, it centers on advertising as it is known and practiced by Procter and Gamble. The purpose of the kit is…

Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age-related declines in voluntary physical activity.

PubMed

Ruegsegger, Gregory N; Toedebusch, Ryan G; Childs, Thomas E; Grigsby, Kolter B; Booth, Frank W

2017-01-01

Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases. The nucleus accumbens (the pleasure and reward 'hub' in the brain) influences wheel running behaviour in rodents. RNA-sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age-related reductions in wheel running. Upon completion of follow-up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age-related changes in voluntary running. Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running. The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age-dependent reductions in the motivation to be physically active. Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA-sequencing was used to interrogate transcriptomic changes between 8- and 14-week-old wheel running rats, and select transcripts were later analysed by quantitative RT-PCR in age-matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP-mediated signalling, dopamine- and cAMP-regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8- vs. 14-week-old rats. In depth analysis of these networks showed significant (∼20-30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin-dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14- vs. 8-week-old sedentary rats (P = 0.03). Intriguingly, intra-NAc injection of the Cdk5 inhibitor roscovitine, dose-dependently decreased wheel running. Collectively, these experiments suggest that an age-dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age-related declines in voluntary running behaviour. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age‐related declines in voluntary physical activity

PubMed Central

Ruegsegger, Gregory N.; Toedebusch, Ryan G.; Childs, Thomas E.; Grigsby, Kolter B.

2016-01-01

Key points Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases.The nucleus accumbens (the pleasure and reward ‘hub’ in the brain) influences wheel running behaviour in rodents.RNA‐sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age‐related reductions in wheel running. Upon completion of follow‐up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age‐related changes in voluntary running.Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running.The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age‐dependent reductions in the motivation to be physically active. Abstract Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA‐sequencing was used to interrogate transcriptomic changes between 8‐ and 14‐week‐old wheel running rats, and select transcripts were later analysed by quantitative RT‐PCR in age‐matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP‐mediated signalling, dopamine‐ and cAMP‐regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8‐ vs. 14‐week‐old rats. In depth analysis of these networks showed significant (∼20–30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin‐dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14‐ vs. 8‐week‐old sedentary rats (P = 0.03). Intriguingly, intra‐NAc injection of the Cdk5 inhibitor roscovitine, dose‐dependently decreased wheel running. Collectively, these experiments suggest that an age‐dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age‐related declines in voluntary running behaviour. PMID:27461471

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

PubMed Central

Adeyemi, Richard O.

2012-01-01

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G2/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK. PMID:22623787

Loss of Cyclin-dependent Kinase 2 in the Pancreas Links Primary β-Cell Dysfunction to Progressive Depletion of β-Cell Mass and Diabetes*

PubMed Central

Kim, So Yoon; Lee, Ji-Hyeon; Merrins, Matthew J.; Gavrilova, Oksana; Bisteau, Xavier; Kaldis, Philipp; Satin, Leslie S.; Rane, Sushil G.

2017-01-01

The failure of pancreatic islet β-cells is a major contributor to the etiology of type 2 diabetes. β-Cell dysfunction and declining β-cell mass are two mechanisms that contribute to this failure, although it is unclear whether they are molecularly linked. Here, we show that the cell cycle regulator, cyclin-dependent kinase 2 (CDK2), couples primary β-cell dysfunction to the progressive deterioration of β-cell mass in diabetes. Mice with pancreas-specific deletion of Cdk2 are glucose-intolerant, primarily due to defects in glucose-stimulated insulin secretion. Accompanying this loss of secretion are defects in β-cell metabolism and perturbed mitochondrial structure. Persistent insulin secretion defects culminate in progressive deficits in β-cell proliferation, reduced β-cell mass, and diabetes. These outcomes may be mediated directly by the loss of CDK2, which binds to and phosphorylates the transcription factor FOXO1 in a glucose-dependent manner. Further, we identified a requirement for CDK2 in the compensatory increases in β-cell mass that occur in response to age- and diet-induced stress. Thus, CDK2 serves as an important nexus linking primary β-cell dysfunction to progressive β-cell mass deterioration in diabetes. PMID:28100774

Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

PubMed

Wang, HaiYang; Jo, Yu-Jin; Sun, Tian-Yi; Namgoong, Suk; Cui, Xiang-Shun; Oh, Jeong Su; Kim, Nam-Hyung

2016-12-01

To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

The Cyclin-Dependent Kinase Ortholog pUL97 of Human Cytomegalovirus Interacts with Cyclins

PubMed Central

Graf, Laura; Webel, Rike; Wagner, Sabrina; Hamilton, Stuart T.; Rawlinson, William D.; Sticht, Heinrich; Marschall, Manfred

2013-01-01

The human cytomegalovirus (HCMV)-encoded protein kinase, pUL97, is considered a cyclin-dependent kinase (CDK) ortholog, due to shared structural and functional characteristics. The primary mechanism of CDK activation is binding to corresponding cyclins, including cyclin T1, which is the usual regulatory cofactor of CDK9. This study provides evidence of direct interaction between pUL97 and cyclin T1 using yeast two-hybrid and co-immunoprecipitation analyses. Confocal immunofluorescence revealed partial colocalization of pUL97 with cyclin T1 in subnuclear compartments, most pronounced in viral replication centres. The distribution patterns of pUL97 and cyclin T1 were independent of HCMV strain and host cell type. The sequence domain of pUL97 responsible for the interaction with cyclin T1 was between amino acids 231–280. Additional co-immunoprecipitation analyses showed cyclin B1 and cyclin A as further pUL97 interaction partners. Investigation of the pUL97-cyclin T1 interaction in an ATP consumption assay strongly suggested phosphorylation of pUL97 by the CDK9/cyclin T1 complex in a substrate concentration-dependent manner. This is the first demonstration of interaction between a herpesviral CDK ortholog and cellular cyclins. PMID:24351800

Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study.

PubMed

Taghizadeh, Mohammad; Goliaei, Bahram; Madadkar-Sobhani, Armin

2016-06-01

Protein flexibility, which has been referred as a dynamic behavior has various roles in proteins' functions. Furthermore, for some developed tools in bioinformatics, such as protein-protein docking software, considering the protein flexibility, causes a higher degree of accuracy. Through undertaking the present work, we have accomplished the quantification plus analysis of the variations in the human Cyclin Dependent Kinase 2 (hCDK2) protein flexibility without affecting a significant change in its initial environment or the protein per se. The main goal of the present research was to calculate variations in the flexibility for each residue of the hCDK2, analysis of their flexibility variations through clustering, and to investigate the functional aspects of the residues with high flexibility variations. Using Gromacs package (version 4.5.4), three independent molecular dynamics (MD) simulations of the hCDK2 protein (PDB ID: 1HCL) was accomplished with no significant changes in their initial environments, structures, or conformations, followed by Root Mean Square Fluctuations (RMSF) calculation of these MD trajectories. The amount of variations in these three curves of RMSF was calculated using two formulas. More than 50% of the variation in the flexibility (the distance between the maximum and the minimum amount of the RMSF) was found at the region of Val-154. As well, there are other major flexibility fluctuations in other residues. These residues were mostly positioned in the vicinity of the functional residues. The subsequent works were done, as followed by clustering all hCDK2 residues into four groups considering the amount of their variability with respect to flexibility and their position in the RMSF curves. This work has introduced a new class of flexibility aspect of the proteins' residues. It could also help designing and engineering proteins, with introducing a new dynamic aspect of hCDK2, and accordingly, for the other similar globular proteins. In addition, it could provide a better computational calculation of the protein flexibility, which is, especially important in the comparative studies of the proteins' flexibility.

Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study

PubMed Central

Taghizadeh, Mohammad; Goliaei, Bahram; Madadkar-Sobhani, Armin

2016-01-01

Background Protein flexibility, which has been referred as a dynamic behavior has various roles in proteins’ functions. Furthermore, for some developed tools in bioinformatics, such as protein-protein docking software, considering the protein flexibility, causes a higher degree of accuracy. Through undertaking the present work, we have accomplished the quantification plus analysis of the variations in the human Cyclin Dependent Kinase 2 (hCDK2) protein flexibility without affecting a significant change in its initial environment or the protein per se. Objectives The main goal of the present research was to calculate variations in the flexibility for each residue of the hCDK2, analysis of their flexibility variations through clustering, and to investigate the functional aspects of the residues with high flexibility variations. Materials and Methods Using Gromacs package (version 4.5.4), three independent molecular dynamics (MD) simulations of the hCDK2 protein (PDB ID: 1HCL) was accomplished with no significant changes in their initial environments, structures, or conformations, followed by Root Mean Square Fluctuations (RMSF) calculation of these MD trajectories. The amount of variations in these three curves of RMSF was calculated using two formulas. Results More than 50% of the variation in the flexibility (the distance between the maximum and the minimum amount of the RMSF) was found at the region of Val-154. As well, there are other major flexibility fluctuations in other residues. These residues were mostly positioned in the vicinity of the functional residues. The subsequent works were done, as followed by clustering all hCDK2 residues into four groups considering the amount of their variability with respect to flexibility and their position in the RMSF curves. Conclusions This work has introduced a new class of flexibility aspect of the proteins’ residues. It could also help designing and engineering proteins, with introducing a new dynamic aspect of hCDK2, and accordingly, for the other similar globular proteins. In addition, it could provide a better computational calculation of the protein flexibility, which is, especially important in the comparative studies of the proteins’ flexibility. PMID:28959320

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

PubMed

Laldinsangi, C; Senthilkumaran, B

2018-04-03

C-kit receptor is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analysed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of C-kit was analysed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression in male compared to females during the pre-spawning phase. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression level of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes involved in germ cell development. Decrease in the levels of serum 11-KT and T were also observed after esiRNA silencing. The findings of this study suggest that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish. Copyright © 2018. Published by Elsevier Inc.

Sunitinib: from charge-density studies to interaction with proteins.

PubMed

Malińska, Maura; Jarzembska, Katarzyna N; Goral, Anna M; Kutner, Andrzej; Woźniak, Krzysztof; Dominiak, Paulina M

2014-05-01

Protein kinases are targets for the treatment of a number of diseases. Sunitinib malate is a type I inhibitor of tyrosine kinases and was approved as a drug in 2006. This contribution constitutes the first comprehensive analysis of the crystal structures of sunitinib malate and of complexes of sunitinib with a series of protein kinases. The high-resolution single-crystal X-ray measurement and aspherical atom databank approach served as a basis for reconstruction of the charge-density distribution of sunitinib and its protein complexes. Hirshfeld surface and topological analyses revealed a similar interaction pattern in the sunitinib malate crystal structure to that in the protein binding pockets. Sunitinib forms nine preserved bond paths corresponding to hydrogen bonds and also to the C-H···O and C-H···π contacts common to the VEGRF2, CDK2, G2, KIT and IT kinases. In general, sunitinib interacts with the studied proteins with a similar electrostatic interaction energy and can adjust its conformation to fit the binding pocket in such a way as to enhance the electrostatic interactions, e.g. hydrogen bonds in ligand-kinase complexes. Such behaviour may be responsible for the broad spectrum of action of sunitinib as a kinase inhibitor.

PFTK1 Promotes Gastric Cancer Progression by Regulating Proliferation, Migration and Invasion.

PubMed

Yang, Lei; Zhu, Jia; Huang, Hua; Yang, Qichang; Cai, Jing; Wang, Qiuhong; Zhu, Junya; Shao, Mengting; Xiao, Jinzhang; Cao, Jie; Gu, Xiaodan; Zhang, Shusen; Wang, Yingying

2015-01-01

PFTK1, also known as PFTAIRE1, CDK14, is a novel member of Cdc2-related serine/threonine protein kinases. Recent studies show that PFTK1 is highly expressed in several malignant tumors such as hepatocellular carcinoma, esophageal cancer, breast cancer, and involved in regulation of cell cycle, tumors proliferation, migration, and invasion that further influence the prognosis of tumors. However, the expression and physiological significance of PFTK1 in gastric cancer remain unclear. In this study, we analyzed the expression and clinical significance of PFTK1 by Western blot in 8 paired fresh gastric cancer tissues, nontumorous gastric mucosal tissues and immunohistochemistry on 161 paraffinembedded slices. High PFTK1 expression was correlated with the tumor grade, lymph node invasion as well as Ki-67. Through Cell Counting Kit (CCK)-8 assay, flow cytometry, colony formation, wound healing and transwell assays, the vitro studies demonstrated that PFTK1 overexpression promoted proliferation, migration and invasion of gastric cancer cells, while PFTK1 knockdown led to the opposite results. Our findings for the first time supported that PFTK1 might play an important role in the regulation of gastric cancer proliferation, migration and would provide a novel promising therapeutic strategy against human gastric cancer.

Birth kits for safe motherhood in Bangladesh.

PubMed

Nessa, S; Arco, E S; Kabir, I A

1992-01-01

Tetanus infection remains the leading cause of high neonatal mortality in Bangladesh. Birth kits which instruct and assist in a clean, safe birth are seen as a key measure in reducing the high incidence of neonatal deaths. A multisectoral programme has developed a simple kit and tested its potential for distribution to pregnant women. Initial results are positive and development is continuing.

Hispanic Families' Perspectives on Using a Bilingual Vocabulary Kit to Enhance Their Prekindergarten Children's Vocabulary Development

ERIC Educational Resources Information Center

Query, Rebecca Robinson; Ceglowski, Deborah; Clark, Patricia; Li, Yongmei

2011-01-01

The purpose of this study was to examine Hispanic families' perspectives on using a prepared kit to enhance their preschoolers' vocabulary development at home. Families enrolled in a public prekindergarten program were provided with a bilingual (English/Spanish) home literacy kit that included ways in which to engage their children in activities…

Neutral Buoyancy Simulator - Fluid line repair kit development

NASA Technical Reports Server (NTRS)

1997-01-01

Marshall's Neutral Buoyancy Simulator (NBS) is used to simulate the gravitational fields and buoyancy effects outer space has on astronauts and their ability to perform tasks in this environment. In this example, a diver performs a temporary fluid line repair task using a repair kit developed by Marshall engineers. The analysis will determine the value of this repair kit and its feasibility.

The FLEXTRA kit: a model for instructor support materials.

PubMed

Battles, J B; Sheridan, M M

1989-01-01

The FLEXTRA Kit is a model for the development of resource materials to support instructor-delivered continuing education. Each FLEXTRA Kit consists of camera-ready copy of handout materials; presentation slides, overheads, videotapes, etc.; evaluation instruments; and an instructor's guide. The FLEXTRA Kit is packaged in such a way that it can be easily shipped and stored. Desktop publishing makes the production of FLEXTRA Kits a cost-effective means of providing support to repeated and locally variable training events.

A Take-Home Physics Experiment Kit for On-Campus and Off-Campus Students

ERIC Educational Resources Information Center

Turner, Joanna; Parisi, Alfio

2008-01-01

A take-home experiment kit has been developed to reinforce the concepts in a first year physics course that both on and off campus students from a variety of educational backgrounds can successfully use. The kit is inexpensive and is composed of easy to obtain items. The experiments conducted with the kit are directed experiments that require…

G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

PubMed

Giráldez, Servando; Galindo-Moreno, María; Limón-Mortés, M Cristina; Rivas, A Cristina; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Sáez, Carmen; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2017-07-01

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCF βTrCP /proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G 1 /S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCF βTrCP /proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G 1 -to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G 1 /S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis. © FASEB.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.

PubMed

Neuveut, C; Low, K G; Maldarelli, F; Schmitt, I; Majone, F; Grassmann, R; Jeang, K T

1998-06-01

Human T-cell leukemia virus type 1 is etiologically linked to the development of adult T-cell leukemia and various human neuropathies. The Tax protein of human T-cell leukemia virus type I has been implicated in cellular transformation. Like other oncoproteins, such as Myc, Jun, and Fos, Tax is a transcriptional activator. How it mechanistically dysregulates the cell cycle is unclear. Previously, it was suggested that Tax affects cell-phase transition by forming a direct protein-protein complex with p16(INK4a), thereby inactivating an inhibitor of G1-to-S-phase progression. Here we show that, in T cells deleted for p16(INK4a), Tax can compel an egress of cells from G0/G1 into S despite the absence of serum. We also show that in undifferentiated myocytes, expression of Tax represses cellular differentiation. In both settings, Tax expression was found to increase cyclin D-cdk activity and to enhance pRb phosphorylation. In T cells, a Tax-associated increase in steady-state E2F2 protein was also documented. In searching for a molecular explanation for these observations, we found that Tax forms a protein-protein complex with cyclin D3, whereas a point-mutated and transcriptionally inert Tax mutant failed to form such a complex. Interestingly, expression of wild-type Tax protein in cells was also correlated with the induction of a novel hyperphosphorylated cyclin D3 protein. Taken together, these findings suggest that Tax might directly influence cyclin D-cdk activity and function, perhaps by a route independent of cdk inhibitors such as p16(INK4a).

A current and comprehensive review of cyclin-dependent kinase inhibitors for the treatment of metastatic breast cancer.

PubMed

Bilgin, Burak; Sendur, Mehmet A N; Şener Dede, Didem; Akıncı, Muhammed Bülent; Yalçın, Bülent

2017-09-01

Resistance to endocrine treatment generally occurs over time, especially in the metastatic stage. In this paper, we aimed to review the mechanisms of cyclin-dependent kinase (CDK) 4/6 inhibition and clinical usage of new agents in the light of recent literature updates. A literature search was carried out using PubMed, Medline and ASCO and ESMO annual-meeting abstracts by using the following search keywords; "palbociclib", "abemaciclib", "ribociclib", "cyclin-dependent kinase inhibitors" and "CDK 4/6" in metastatic breast cancer (MBC). The last search was on 10 June 2017. CDKs and cyclins are two molecules that have a key role in cell cycle progression. Today, there are three highly selective CDK4/6 inhibitors in clinical development - palbociclib, ribociclib and abemaciclib. Palbociclib and ribociclib were recently approved by the US FDA in combination with letrozole for the treatment of MBC in a first-line setting, as well as palbociclib in combination with fulvestrant for hormone-receptor (HR)-positive MBC that had progressed while on previous endocrine therapy according to the PALOMA-1, MONALEESA-2 and PALOMA-3 trials, respectively. In the recently published randomized phase III MONARCH 2 trial, abemaciclib plus letrozole had longer progression-free survival and higher objective response rates with less serious adverse events in advanced HR-positive breast cancer previously treated with hormonal treatment. CDK4/6 inhibition is a new and promising target for patients with hormone-receptor-positive MBC. Both palbociclib and ribociclib showed significant additive benefit for patients receiving first-line treatment for HR-positive, epidermal growth factor receptor-2-negative advanced breast cancer. Palbociclib and abemaciclib also had significant activity in combination with fulvestrant for patients with MBC that progressed on previous endocrine therapy.

Progress with palbociclib in breast cancer: latest evidence and clinical considerations

PubMed Central

Rocca, Andrea; Schirone, Alessio; Maltoni, Roberta; Bravaccini, Sara; Cecconetto, Lorenzo; Farolfi, Alberto; Bronte, Giuseppe; Andreis, Daniele

2016-01-01

Deregulation of the cell cycle is a hallmark of cancer, and research on cell cycle control has allowed identification of potential targets for anticancer treatment. Palbociclib is a selective inhibitor of the cyclin-dependent kinases 4 and 6 (CDK4/6), which are involved, with their coregulatory partners cyclin D, in the G1-S transition. Inhibition of this step halts cell cycle progression in cells in which the involved pathway, including the retinoblastoma protein (Rb) and the E2F family of transcription factors, is functioning, although having been deregulated. Among breast cancers, those with functioning cyclin D-CDK4/6-Rb-E2F are mainly hormone-receptor (HR) positive, with some HER2-positive and rare triple-negative cases. Deregulation results from genetic or otherwise occurring hyperactivation of molecules subtending cell cycle progression, or inactivation of cell cycle inhibitors. Based on results of randomized clinical trials, palbociclib was granted accelerated approval by the US Food and Drug Administration (FDA) for use in combination with letrozole as initial endocrine-based therapy for metastatic disease in postmenopausal women with HR-positive, HER2-negative breast cancer, and was approved for use in combination with fulvestrant in women with HR-positive, HER2-negative advanced breast cancer with disease progression following endocrine therapy. This review provides an update of the available knowledge on the cell cycle and its regulation, on the alterations in cyclin D-CDK4/6-Rb-E2F axis in breast cancer and their roles in endocrine resistance, on the preclinical activity of CDK4/6 inhibitors in breast cancer, both as monotherapy and as partners of combinatorial synergic treatments, and on the clinical development of palbociclib in breast cancer. PMID:28203301

α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation.

PubMed

Yang, Lan; Wen, Ya; Lv, Guoqing; Lin, Yuntao; Tang, Junlong; Lu, Jingxiao; Zhang, Manqiao; Liu, Wen; Sun, Xiaojuan

2017-12-09

Alpha lipoic acid (α -LA) is a naturally occurring antioxidant and metabolic enzyme co-factor. Recently, α -LA has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of α -LA on non-small cell lung cancer (NSCLC) development remain unclear. The CCK-8 assay was used to assess cell proliferation in NSCLC cell lines after α -LA treatment. The expression of growth factor receptor-bound protein 2 (Grb2), cyclin-dependent kinase (CDK)-2, CDK4, CDK6, Cyclin D3, Cyclin E1, Ras, c-Raf, epidermal growth factor receptor (EGFR), ERK1/2 and activated EGFR and ERK1/2 was evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in NSCLC cells via specific siRNA-mediated knockdown. α -LA dramatically decreased NSCLC cell proliferation by downregulating Grb2; in contrast, Grb2 overexpression significantly prevented α-LA-induced decrease in cell growth in vitro. Western blot analysis indicated that α-LA decreased the levels of phospho-EGFR, CDK2/4/6, Cyclins D3 and E1, which are associated with the inhibition of G1/S-phase transition. Additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. In addition, α-LA exerted greater inhibitory effects than gefitinib on NSCLC cells by preventing EGF-induced EGFR activation. For the first time, these findings provide the first evidence that α-LA inhibits cell proliferation through Grb2 by suppressing EGFR phosphorylation and that MAPK/ERK is involved in this pathway. Copyright © 2017. Published by Elsevier Inc.

"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.

PubMed

Keith, Matthew C L; Bolli, Roberto

2015-03-27

Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kit(pos) cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes, suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct "string theory" of c-kit(pos) cardiac cells because it reconciles multifarious and sometimes apparently discrepant results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities, or differentiation capacities of specific cardiac progenitors. C-kit(pos) cells derived from the first heart field exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or after birth. The residual c-kit(pos) cells found in the adult heart are probably of proepicardial origin, possess a mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells). If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic potential of first heart field progenitors might be reconciled with recent results of c-kit(pos) cell lineage tracing studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kit(pos) cell administration to ischemically damaged hearts despite the observed paucity of cardiomyogenic differentiation of these cells. © 2015 American Heart Association, Inc.

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

PubMed Central

Cahu, Julie; Olichon, Aurelien; Hentrich, Christian; Schek, Henry; Drinjakovic, Jovana; Zhang, Cunjie; Doherty-Kirby, Amanda; Lajoie, Gilles; Surrey, Thomas

2008-01-01

Background Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. Methodology/Principal Findings We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. Conclusions/Significance These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein. PMID:19079595

[The role of balanced low-protein diet in inhibition of predialysis chronic kidney disease progression in patients with systemic diseases].

PubMed

Milovanov, Iu S; Lysenko, L V; Milovanova, L Iu; Dobrosmyslov, I A

2009-01-01

To evaluate the effects of low-protein diet (LPD) balanced by addition of highly energetic mix and essential keto/amino acids on inhibition of renal failure in patients with systemic diseases with predialysis stages of chronic disease of the kidney (CDK). Forty six patients with stage III--IV of CDK in systemic diseases (33 SLE patients and 13 with systemic vasculitis) were randomized into three groups. Group 1 consisted of 18 patients with CDK (10 with stage III and 8 with stage IV). They received LPD (0.6 g/kg/day) with addition of essential keto/amino acids for 24-48 months. Group 2 of 18 CDK patients with the same stages received the same diet but greater amount of vegetable protein (highly purified soya protein) to 0.3 g/kg/day in highly energetic nutrient mixture. Group 3--10 CDK patients (7 with stage III and 3 with stage IV) received free diet. Group 1 and 2 patients received LPD irrespective of the nutrient status assessed basing on anthropometric and other data. Protein consumption and caloric value were estimated by 3-day food diary. Before diet therapy, out of 46 examinees nutrient status was abnormal in 45.7% patients. Both variants of LPD were well tolerated and nutrient status was corrected while the rate of nutritive disorders in group 3 increased 1.5-fold (from 40 to 60%) with progression of renal failure. Intake of LPD diet for at least a year reduced glomerular filtration rate inhibition, especially in addition of highly energetic mixture. Early (predialysis) restriction of diet protein (0.6 g/kg/day) with addition of highly energetic mixture and essential keto/amino acids improves a nutritive status of CDK patients and inhibits GFR decline.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

PubMed Central

Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

Cyclin-dependent kinase inhibitor Dinaciclib (SCH727965) inhibits pancreatic cancer growth and progression in murine xenograft models

PubMed Central

Bisht, Savita; Karikari, Collins; Garrido-Laguna, Ignacio; Rasheed, Zeshaan; Ottenhof, Niki A; Dadon, Tikva; Alvarez, Hector; Fendrich, Volker; Rajeshkumar, NV; Matsui, William; Brossart, Peter; Hidalgo, Manuel; Bannerji, Rajat

2011-01-01

Pancreatic cancer is one of the most lethal of human malignancies, and potent therapeutic options are lacking. Inhibition of cell cycle progression through pharmacological blockade of cyclin-dependent kinases (CDK) has been suggested as a potential treatment option for human cancers with deregulated cell cycle control. Dinaciclib (SCH727965) is a novel small molecule multi-CDK inhibitor with low nanomolar potency against CDK1, CDK2, CDK5 and CDK9 that has shown favorable toxicity and efficacy in preliminary mouse experiments, and has been well tolerated in Phase I clinical trials. In the current study, the therapeutic efficacy of SCH727965 on human pancreatic cancer cells was tested using in vitro and in vivo model systems. Treatment with SCH727965 significantly reduced in vitro cell growth, motility and colony formation in soft agar of MIAPaCa-2 and Pa20C cells. These phenotypic changes were accompanied by marked reduction of phosphorylation of Retinoblastoma (Rb) and reduced activation of RalA. Single agent therapy with SCH727965 (40 mg/kg i.p. twice weekly) for 4 weeks significantly reduced subcutaneous tumor growth in 10/10 (100%) of tested low-passage human pancreatic cancer xenografts. Treatment of low passage pancreatic cancer xenografts with a combination of SCH727965 and gemcitabine was significantly more effective than either agent alone. Gene Set Enrichment Analysis identified overrepresentation of the Notch and Transforming Growth Factor-β (TGFβ) signaling pathways in the xenografts least responsive to SCH727965 treatment. Treatment with the cyclin-dependent kinase inhibitor SCH727965 alone or in combination is a highly promising novel experimental therapeutic strategy against pancreatic cancer. PMID:21768779

Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

PubMed

Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

2008-01-01

The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1.

PubMed

Xie, Jing; Bai, Jun

2014-07-01

To investigate the antitumor effect of SGI-1776 on human ovarian cancer HO-8910 cells and its molecular mechanism. HO-8910 cells were cultured in vitro, and the proliferation inhibitory effects of SGI- 1776 were determined by MTT assay and colony formation assay. The effect of SGI-1776 on the distribution of cell cycle phase was observed by flow cytometry with propidium iodide (PI) staining. The inhibition rate of migration and invasion were valued by transwell cell assay. Multiple molecular techniques, such as ELISA, Western blot, siRNA and cDNA transfection were used to explore the molecular mechanism. SGI-1776 presented dramatic anti-tumor activity against HO-8910 cells in vitro, inhibited the cells proliferation and colony formation, and attenuated the migration and invasion in a dosedependent manner, accompanied by cell cycle arrest in G1 phase. SGI-1776 caused the proliferation inhibition with concomitant decrease in Pim-1 kinase activity, down-regulated the expression of Pim-1 protein and and its downstream genes, such as CDK6, pCDK6, CDK4, pCDK4, CDK2 and pCDK2, and increased the expression of P21 and P27. Down-regulation expression of Pim-1 by siRNA followed SGI-1776 treatment resulted in enhanced cell proliferation inhibition rate and attenuated migration/invasion. Up-regulation of Pim-1 by cDNA transfection attenuated SGI- 1776-induced cell proliferation inhibition and its migration/invasion. Pim-1 mediates the biological effect of SGI-1776 in human ovarian cancer HO-8910 cells, suggesting Pim-1 might be a novel target for human ovarian cancer.

Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos

PubMed Central

Tsai, Tony Y.-C.; Theriot, Julie A.; Ferrell, James E.

2014-01-01

During the early development of Xenopus laevis embryos, the first mitotic cell cycle is long (∼85 min) and the subsequent 11 cycles are short (∼30 min) and clock-like. Here we address the question of how the Cdk1 cell cycle oscillator changes between these two modes of operation. We found that the change can be attributed to an alteration in the balance between Wee1/Myt1 and Cdc25. The change in balance converts a circuit that acts like a positive-plus-negative feedback oscillator, with spikes of Cdk1 activation, to one that acts like a negative-feedback-only oscillator, with a shorter period and smoothly varying Cdk1 activity. Shortening the first cycle, by treating embryos with the Wee1A/Myt1 inhibitor PD0166285, resulted in a dramatic reduction in embryo viability, and restoring the length of the first cycle in inhibitor-treated embryos with low doses of cycloheximide partially rescued viability. Computations with an experimentally parameterized mathematical model show that modest changes in the Wee1/Cdc25 ratio can account for the observed qualitative changes in the cell cycle. The high ratio in the first cycle allows the period to be long and tunable, and decreasing the ratio in the subsequent cycles allows the oscillator to run at a maximal speed. Thus, the embryo rewires its feedback regulation to meet two different developmental requirements during early development. PMID:24523664

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

PubMed

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

2016-01-01

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

Cdk5: one of the links between senile plaques and neurofibrillary tangles?

PubMed

Lee, Ming-Sum; Tsai, Li-Huei

2003-04-01

The relationship between amyloid plaques and neurofibrillary tangles, the two pathologic hallmarks of Alzheimer's disease (AD), is an unknown and controversial subject. However, emerging evidence from genetic and biochemical studies suggests that accumulation of amyloid beta peptides may play a causative role in AD pathogenesis. This led to the amyloid hypothesis, which proposes that amyloid beta peptides disrupt neuronal metabolic and ionic homeostasis and cause aberrant activation of kinases and/or inhibition of phosphatases. The resulting alteration in kinase and phosphatase activities ultimately leads to hyperphosphorylation of tau and formation of neurofibrillary tangles. Cyclin-dependent kinase 5 (Cdk5) is a tau kinase whose activity is induced by amyloid beta peptides. Its deregulation may represent one of the signal transduction pathways that connect amyloid beta toxicity to tau hyperphosphorylation. This article reviews the functions and regulation of Cdk5. Evidence that suggests deregulation of Cdk5 activity in AD by virtue of calpain cleavage of its activator p35 to p25 will be discussed.

An attempt to evaluate the effect of vitamin K3 using as an enhancer of anticancer agents.

PubMed

Matzno, Sumio; Yamaguchi, Yuka; Akiyoshi, Takeshi; Nakabayashi, Toshikatsu; Matsuyama, Kenji

2008-06-01

The possibility of vitamin K3 (VK3) as an anticancer agent was assessed. VK3 dose-dependently diminished the cell viability (measured as esterase activity) with IC50 of 13.7 microM and Hill coefficient of 3.1 in Hep G2 cells. It also decreased the population of S phase and arrested cell cycle in the G2/M phase in a dose-dependent manner. G2/M arrest was regulated by the increment of cyclin A/cdk1 and cyclin A/cdk2 complex, and contrasting cyclin B/cdk1 complex decrease. Finally, combined application demonstrated that VK3 significantly enhanced the cytotoxicity of etoposide, a G2 phase-dependent anticancer agent, whereas it reduced the cytotoxic activity of irinotecan, a S phase-dependent agent. These findings suggest that VK3 induces G2/M arrest by inhibition of cyclin B/cdk1 complex formation, and is thus useful as an enhancer of G2 phase-dependent drugs in hepatic cancer chemotherapy.

Summary of Research Findings on Children's Developmental Health = Resume des conclusions de la recherche sur la sante developpementale des effants.

ERIC Educational Resources Information Center

Bertrand, Jane

This kit is comprised of bilingual resources for child caregivers related to nourishing and nurturing a child's brain for optimal neurodevelopmental health. The kit is the result of a 30-month project to synthesize research on brain development and to develop resources in support of excellent caregiver practice in Canada. The kit contains the…

CDK5-mediated tau accumulation triggers methamphetamine-induced neuronal apoptosis via endoplasmic reticulum-associated degradation pathway.

PubMed

Xiao, Ning; Zhang, Fu; Zhu, Bofeng; Liu, Chao; Lin, Zhoumeng; Wang, Huijun; Xie, Wei-Bing

2018-08-01

Overexposure to methamphetamine (METH) causes apoptosis in a number of cell types, particularly neuronal cells. However, the underlying mechanisms of METH-induced neuronal apoptosis remain to be elucidated. Accumulation of microtubule-associated protein Tau can lead to activation of multiple neurotoxic pathways, which is closely correlated with neuronal apoptosis. The aim of this study was to determine the role of Tau in METH-induced neuronal apoptosis. We determined the expression of two phosphorylated Tau proteins (serine 396 and threonine 231) in the human neuroblastoma SH-SY5Y cells and in the hippocampus of Sprague-Dawley rats treated with vehicle or METH using western blotting, immunohistochemical staining and immunofluorescence staining. We also measured the expression levels of the phosphorylated Tau protein, ubiquitination proteins, the intermediate products of proteasome degradation pathway, CD3-δ (a substrate of proteasome degradation pathway), endoplasmic reticulum stress signal molecule phosphorylated PERK (pPERK), and endoplasmic reticulum stress-specific apoptotic signal molecule caspase-12 in SH-SY5Y cells and in rats after inhibiting the expression of an upstream regulatory factor of phosphorylated Tau protein (CDK5) using siRNA or virus transfection. The results showed that exposure to METH significantly up-regulated the expression of phosphorylated Tau protein in vivo and in vitro and silencing the expression of CDK5 inhibited the up-regulation of phosphorylated Tau induced by METH exposure. METH exposure also significantly increased the expression of ubiquitination protein and CD3-δ and these effects were blocked by CDK5 silencing. In addition, METH exposure significantly elevated the levels of phosphorylated PERK and caspase-12 and these effects were suppressed after CDK5 silencing, which indicates that blockade of CDK5 expression can mitigate METH-induced neuronal apoptosis. These results suggest that METH can impair the endoplasmic reticulum-associated degradation (ERAD) pathway and induce neuronal apoptosis through endoplasmic reticulum stress, which is mainly mediated by abnormal CDK5-regulated Tau phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen.

PubMed

Wang, Jianling; Wang, Gangduo; Khan, M Firoze

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure.

Disorder of G2-M Checkpoint Control in Aniline-Induced Cell Proliferation in Rat Spleen

PubMed Central

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

2015-01-01

Aniline, a toxic aromatic amine, is known to cause hemopoietic toxicity both in humans and animals. Aniline exposure also leads to toxic response in spleen which is characterized by splenomegaly, hyperplasia, fibrosis and the eventual formation of tumors on chronic in vivo exposure. Previously, we have shown that aniline exposure leads to iron overload, oxidative DNA damage, and increased cell proliferation, which could eventually contribute to a tumorigenic response in the spleen. Despite our demonstration that cell proliferation was associated with deregulation of G1 phase cyclins and increased expression of G1 phase cyclin-dependent kinases (CDKs), molecular mechanisms, especially the regulation of G2 phase and contribution of epigenetic mechanisms in aniline-induced splenic cellular proliferation remain largely unclear. This study therefore, mainly focused on the regulation of G2 phase in an animal model preceding a tumorigenic response. Male Sprague-Dawley rats were given aniline (0.5 mmol/kg/day) in drinking water or drinking water only (controls) for 30 days, and expression of G2 phase cyclins, CDK1, CDK inhibitors and miRNAs were measured in the spleen. Aniline treatment resulted in significant increases in cell cycle regulatory proteins, including cyclins A, B and CDK1, particularly phosphor-CDK1, and decreases in CDK inhibitors p21 and p27, which could promote the splenocytes to go through G2/M transition. Our data also showed upregulation of tumor markers Trx-1 and Ref-1 in rats treated with aniline. More importantly, we observed lower expression of miRNAs including Let-7a, miR-15b, miR24, miR-100 and miR-125, and greater expression of CDK inhibitor regulatory miRNAs such as miR-181a, miR-221 and miR-222 in the spleens of aniline-treated animals. Our findings suggest that significant increases in the expression of cyclins, CDK1 and aberrant regulation of miRNAs could lead to an accelerated G2/M transition of the splenocytes, and potentially to a tumorigenic response on chronic aniline exposure. PMID:26192324

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

PubMed

Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H

2017-12-12

Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.

Chronic Obstructive Pulmonary Disease and Heart Failure Self-Management Kits for Outpatient Transitions of Care.

PubMed

Boylan, Paul; Joseph, Tina; Hale, Genevieve; Moreau, Cynthia; Seamon, Matthew; Jones, Renee

2018-03-01

To develop heart failure (HF) and chronic obstructive pulmonary disease (COPD) self-management kits in an accountable care organization (ACO) to facilitate patients' self-care and prevent hospital readmissions. Pharmacists practice in an outpatient-based ACO. They participate in interprofessional office visits with providers and independently manage maintenance pharmacotherapies. Pharmacists collaborate with an interprofessional team within the ACO including physicians, nurses, case managers, and paramedics. Two commonly encountered diseases are chronic COPD and HF. Reducing preventable readmissions for these conditions are important quality benchmarks and cost-saving strategies. Pharmacists were responsible for developing HF and COPD self-management kits containing patient education materials and prescriptions to facilitate self-care. Prior to kit development, pharmacists performed a literature review to determine the presence of previously published findings on these topics. The interprofessional team continually evaluates the successes and limitations of this initiative. Pharmacists developed training and instructions for ACO allied health professionals in an effort to incorporate the self-management kits in clinical practice. The initial literature search revealed no studies describing the intervention of interest. Innovative programs designed to help reduce preventable readmissions are lacking in primary care. Implementation of the self-management kits was accepted by interprofessional ACO leadership and is currently being integrated into allied health workflow. Patients at risk for having an exacerbation of COPD or HF should receive self-management strategies. Prompt therapy prior to exacerbations reduces hospital admissions and readmissions, speeds recovery, and slows disease progression. Pharmacist-facilitated implementation of self-management kits may be developed by interprofessional health care teams.

Drug synergy screen and network modeling in dedifferentiated liposarcoma identifies CDK4 and IGF1R as synergistic drug targets.

PubMed

Miller, Martin L; Molinelli, Evan J; Nair, Jayasree S; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M; Singer, Samuel; Schwartz, Gary K; Sander, Chris

2013-09-24

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depends on the activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

Drug Synergy Screen and Network Modeling in Dedifferentiated Liposarcoma Identifies CDK4 and IGF1R as Synergistic Drug Targets

PubMed Central

Miller, Martin L.; Molinelli, Evan J.; Nair, Jayasree S.; Sheikh, Tahir; Samy, Rita; Jing, Xiaohong; He, Qin; Korkut, Anil; Crago, Aimee M.; Singer, Samuel; Schwartz, Gary K.; Sander, Chris

2014-01-01

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depend on activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies. PMID:24065146

Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation.

PubMed

Ikegami, Yosuke; Goto, Hidemasa; Kiyono, Tohru; Enomoto, Masato; Kasahara, Kousuke; Tomono, Yasuko; Tozawa, Keiichi; Morita, Akimichi; Kohri, Kenjiro; Inagaki, Masaki

2008-12-26

We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

Nestin is highly expressed in fetal spinal cord isolated from placenta previa patients and promotes inflammation by enhancing NF-κB activity.

PubMed

Li, Ling; Zhang, Jing; Gao, Huahe; Ma, Yuyan

2018-04-26

Purpose Nestin is expressed in various tissues of the embryo in patients with placenta previa, while the regulatory mechanism still unknown. Materials and methods All participants terminated pregnancy. Among them, 75 patients with placenta previa were assigned to the case group and 80 healthy pregnant women with normal placenta were assigned to the control group. Expression of nestin and CDK5 in fetal spinal cord tissues was detected by Western Blot and RT-qPCR methods. The enzyme-linked immunosorbent assay (ELISA) was used to determine the serum expression of some pro-inflammatory cytokines in placenta previa patients. The interaction between nestin and CDK5 was evaluated by immunoprecipitation and siRNA inhibition of nestin was performed to estimate its effect on NF-κB activity in fetal spinal cord tissues. Results Along with increased expression of nestin and CDK5 in fetal spinal cord tissues in the case group, IL-1β, IL-6, TNF-α, and IFN-γ were increased in the serum of placenta previa patients. siRNA inhibition analysis indicated that nestin interacted with CDK5 and regulated NF-κB activity in fetal spinal cord tissues. Conclusions Nestin is highly expressed and the interaction between nestin and CDK5 might lead to the progress of placenta previa through its regulation on NF-κB.

The death effector domain-containing DEDD forms a complex with Akt and Hsp90, and supports their stability

PubMed Central

Kurabe, Nobuya; Mori, Mayumi; Kurokawa, Jun; Taniguchi, Kaori; Aoyama, Hisatoshi; Atsuda, Kazuhiro; Nishijima, Akemi; Odawara, Nariaki; Harada, Saori; Nakashima, Katsuhiko; Arai, Satoko; Miyazaki, Toru

2010-01-01

Insulin secretion and glucose transport are the major mechanisms to balance glucose homeostasis. Recently, we found that the death effector domain-containing DEDD inhibits cyclin-dependent kinase 1 (Cdk1) function, thereby preventing Cdk1-dependent inhibitory phosphorylation of S6 kinase 1 (S6K1), downstream of phosphatidylinositol 3-kinase (PI3K), which overall results in maintenance of S6K1 activity. Here we newly show that DEDD forms a complex with Akt and heat-shock protein 90 (Hsp90), and supports the stability of both proteins. Hence, in DEDD−/− mice, Akt protein levels are diminished in skeletal muscles and adipose tissues, which interferes with the translocation of glucose transporter 4 (GLUT4) upon insulin stimulation, leading to inefficient incorporation of glucose in these organs. Interestingly, as for the activation of S6K1, suppression of Cdk1 is involved in the stabilization of Akt protein by DEDD, since diminishment of Cdk1 in DEDD−/− cells via siRNA expression or treatment with a Cdk1-inhibitor, increases both Akt and Hsp90 protein levels. Such multifaceted involvement of DEDD in glucose homeostasis by supporting both insulin secretion (via maintenance of S6K1 activity) and glucose uptake (via stabilizing Akt protein), may suggest an association of DEDD-deficiency with the pathogenesis of type 2 diabetes mellitus. PMID:20043882

Exploration of cell cycle regulation and modulation of the DNA methylation mechanism of pelargonidin: Insights from the molecular modeling approach.

PubMed

Karthi, Natesan; Karthiga, Arumugasamy; Kalaiyarasu, Thangaraj; Stalin, Antony; Manju, Vaiyapuri; Singh, Sanjeev Kumar; Cyril, Ravi; Lee, Sang-Myeong

2017-10-01

Pelargonidin is an anthocyanidin isolated from plant resources. It shows strong cytotoxicity toward various cancer cell lines, even though the carcinogenesis-modulating pathway of pelargonidin is not yet known. One of our previous reports showed that pelargonidin arrests the cell cycle and induces apoptosis in HT29 cells. Flowcytometry and immunoblot analysis confirmed that pelargonidin specifically inhibits the activation of CDK1 and blocks the G2-M transition of the cell cycle. In addition, DNA fragmentation was observed along with induction of cytochrome c release-mediated apoptosis. Hence, the aim of the present study was to investigate the molecular mechanism of pelargonidin's action on cell cycle regulators CDK1, CDK4, and CDK6 as well as the substrate-binding domain of DNMT1 and DNMT3A, which regulate the epigenetic signals related to DNA methylation. The results of docking analysis, binding free energy calculation, and molecular dynamics simulation correlated with the experimental results, and pelargonidin showed a specific interaction with CDK1. In this context, pelargonidin may also inhibit the recognition of DNA and catalytic binding by DNMT1 and DNMT3A. The HOMO-LUMO analysis mapped the functional groups of pelargonidin. Prediction of pharmacological descriptors suggested that pelargonidin can serve as a multitarget inhibitor for cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparison of Quantifiler(®) Trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints.

PubMed

Goecker, Zachary C; Swiontek, Stephen E; Lakhtakia, Akhlesh; Roy, Reena

2016-06-01

The development techniques employed to visualize fingerprints collected from crime scenes as well as post-development ageing may result in the degradation of the DNA present in low quantities in such evidence samples. Amplification of the DNA samples with short tandem repeat (STR) amplification kits may result in partial DNA profiles. A comparative study of two commercially available quantification kits, Quantifiler(®) Trio and InnoQuant™, was performed on latent fingerprint samples that were either (i) developed using one of three different techniques and then aged in ambient conditions or (ii) undeveloped and then aged in ambient conditions. The three fingerprint development techniques used were: cyanoacrylate fuming, dusting with black powder, and the columnar-thin-film (CTF) technique. In order to determine the differences between the expected quantities and actual quantities of DNA, manually degraded samples generated by controlled exposure of DNA standards to ultraviolet radiation were also analyzed. A total of 144 fingerprint and 42 manually degraded DNA samples were processed in this study. The results indicate that the InnoQuant™ kit is capable of producing higher degradation ratios compared to the Quantifiler(®) Trio kit. This was an expected result since the degradation ratio is a relative value specific for a kit based on the length and extent of amplification of the two amplicons that vary from one kit to the other. Additionally, samples with lower concentrations of DNA yielded non-linear relationships of degradation ratio with the duration of aging, whereas samples with higher concentrations of DNA yielded quasi-linear relationships. None of the three development techniques produced a noticeably different degradation pattern when compared to undeveloped fingerprints, and therefore do not impede downstream DNA analysis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

FUNCTIONAL DEREGULATION OF KIT: LINK TO MAST CELL PROLIFERATIVE DISEASES AND OTHER NEOPLASMS

PubMed Central

Cruse, Glenn; Metcalfe, Dean D.; Olivera, Ana

2014-01-01

SYNOPSIS Signaling through the receptor tyrosine kinase KIT mediates differentiation, proliferation and survival of hematopoietic precursor cells and mast cells. Constitutive KIT signaling due to somatic point mutations in c-Kit is an important occurrence in the development of mast cell proliferation disorders and other hematological malignancies. In this review, we discuss the common gain-of-function mutations found in these malignancies, particularly in mast cell proliferation disorders, and summarize the current understanding of the molecular mechanisms by which transforming point mutations in KIT may affect KIT structure and function and lead to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of these diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided. PMID:24745671

Primary dermal pleomorphic liposarcoma: utility of adipophilin and MDM2/CDK4 immunostainings.

PubMed

Ramírez-Bellver, Jose L; López, Joaquín; Macías, Elena; Alegría-Landa, Victoria; Gimeno, Ignacio; Pérez-Plaza, Alejandra; Kutzner, Heinz; Requena, Luis

2017-03-01

Liposarcoma, usually arises in deep soft tissues and pleomorphic liposarcoma (PL), is the rarest histopathologic variant. However, 15 cases of entirely dermal PL have been reported. We describe a case of a 79-year-old man who developed a rapidly growing nodule on his thorax. Excisional biopsy was performed and immunohistochemical studies were carried. The lesion was a well-circumscribed dermal nodule composed of multivacuolated pleomorphic lipoblasts and atypical mitotic figures. Neoplastic cells expressed CD10 and resulted negative S100 protein, Melan-A, MITF-1, AE1/AE3, CD4, CD68 (PGM1), retinoblastoma gene family protein, pericentrine and lysozyme. Adipophilin stain showed the lipid contents in the cytoplasm of the neoplastic cells. MDM2 and CDK4 resulted both negative. A diagnosis of primary dermal PL was made. This case shows the utility of adipophilin immunostaining to prove the lipid contents in neoplastic cells, which has the advantage of using formalin-fixed paraffin-embedded tissue and making needless frozen sections and ultrastructural studies to show these findings. Negative MDM2/CDK4 staining in our case argues against the possibility of dedifferentiated liposarcoma and further supports the diagnosis of true PL. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Identification of the Key Genes and Pathways in Esophageal Carcinoma.

PubMed

Su, Peng; Wen, Shiwang; Zhang, Yuefeng; Li, Yong; Xu, Yanzhao; Zhu, Yonggang; Lv, Huilai; Zhang, Fan; Wang, Mingbo; Tian, Ziqiang

2016-01-01

Objective . Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods . 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results . A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion . The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

Computation Molecular Kinetics Model of HZE Induced Cell Cycle Arrest

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Ren, Lei

2004-01-01

Cell culture models play an important role in understanding the biological effectiveness of space radiation. High energy and charge (HZE) ions produce prolonged cell cycle arrests at the G1/S and G2/M transition points in the cell cycle. A detailed description of these phenomena is needed to integrate knowledge of the expression of DNA damage in surviving cells, including the determination of relative effectiveness factors between different types of radiation that produce differential types of DNA damage and arrest durations. We have developed a hierarchical kinetics model that tracks the distribution of cells in various cell phase compartments (early G1, late G1, S, G2, and M), however with transition rates that are controlled by rate-limiting steps in the kinetics of cyclin-cdk's interactions with their families of transcription factors and inhibitor molecules. The coupling of damaged DNA molecules to the downstream cyclin-cdk inhibitors is achieved through a description of the DNA-PK and ATM signaling pathways. For HZE irradiations we describe preliminary results, which introduce simulation of the stochastic nature of the number of direct particle traversals per cell in the modulation of cyclin-cdk and cell cycle population kinetics. Comparison of the model to data for fibroblast cells irradiated photons or HZE ions are described.

17β-Estradiol Induces Overproliferation in Adenomyotic Human Uterine Smooth Muscle Cells of the Junctional Zone Through Hyperactivation of the Estrogen Receptor-Enhanced RhoA/ROCK Signaling Pathway.

PubMed

Sun, Fu-Qing; Duan, Hua; Wang, Sha; Li, Jin-Jiao

2015-11-01

Adenomyosis (ADS) is a common estrogen-dependent gynecological disease with unknown etiology. Recent models favor abnormal thickening of the junctional zone (JZ) may be the causative factor in the development of ADS. RhoA, a small guanosine triphosphatase which controls multiple cellular processes, is involved in the control of cell proliferation. Here we demonstrate that treatment of human uterine smooth muscle cells (SMCs) of the JZ with 17β-estradiol (E2) increased expression of RhoA and its downstream effectors (-associated coiled coil containing protein kinase [ROCK] 1 and ROCK2). Compared with non-ADS cells, RhoA, ROCK1, and ROCK2 were overexpressed and hyperactivated in ADS cells. These effects were suppressed in the presence of ICI 182,780, supporting an estrogen receptor (ER)-dependent mechanism. Hyperactivation of ER-enhanced RhoA/ROCK signaling was associated with overproliferation in ADS human uterine SMCs of the JZ. Moreover, E2-induced overproliferation was accompanied by downregulation of cyclin-dependent kinases inhibitors (CKIs; p21(Waf1/Cip1) and p27(Kip1)) and upregulation of cyclin-dependent kinases (CDKs) and cyclins (cyclin D1, cyclin E1, CDK2, CDK4, and CDK6). © The Author(s) 2015.

Tackling Poverty in Rural Mexico: A Case Study of Economic Development. Toward a Better World Series, Learning Kit No. 4.

ERIC Educational Resources Information Center

Baldwin, Harriet; Ross-Larson, Bruce, Ed.

This World Bank (Washington, D.C.) kit is a case study designed to teach secondary school social studies students about an integrated rural development project in Mexico, and how it is helping to raise the standard of living for six million Mexicans in 131 microregions throughout Mexico. The kit contains a pamphlet, a booklet, a sound filmstrip,…

Production of recombinant gG-1 protein of herpes simplex virus type 1 in a prokaryotic system in order to develop a type-specific enzyme-linked immunosorbent assay kit.

PubMed

Zandi, Keivan; Roostaee, Mohammad Hassan; Sadeghizadeh, Majid; Rasaee, Mohammad Javad; Sajedi, Reza Hassan; Soleimanjahi, Hoorieh

2007-08-01

The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.

[Development and evaluation of a rapid PCR detection kit for Ophiocordyceps sinensis].

PubMed

Hou, Fei-Xia; Cao, Jing; Wang, Sha-Sha; Wang, Xi; Yuan, Yuan; Peng, Cheng; Wan, De-Guang; Guo, Jin-Lin

2017-03-01

Ophiocordyceps sinensis is a valuable traditional Chinese medicine. Due to resource shortage, expensive price and huge market demand, there are many adulterants of O. sinensis in markets. Therefore, it is necessary to establish a rapid and effective method for distinguishing O. sinensis. Based on the species-specific PCR of O. sinensis, this study developed a detection kit by optimizing the components and evaluated the specificity, detection limit, repeatability and shelf life of the kit. The results showed that when the quality of O. sinensis accounted for more than 1/200 of that mixture, it could be detected successfully. Moreover, only O. sinensis could be amplified and glowed bright green fluorescence under ultraviolet light. The kit was still in effect when it was placed at 37 ℃ for three days, which indicated that it was stable and effective for one year stored in 4 ℃. The kit in the same batch under different operation conditions, and in different batch under the same operation conditions gave the same result and accuracy, which showed good repeatability of the kit. It is simple, rapid and accurate to distinguish O. sinensis from its adulterants using the kit, and lays the foundation for commercialization of traditional Chinese medicine fast detection kit. Copyright© by the Chinese Pharmaceutical Association.

Rural inland waterways economic impact kit : users guide

DOT National Transportation Integrated Search

2000-08-01

The primary objective of the project was to develop a PC-based kit allowing users to evaluate the economic impact of existing rural inland waterways ports and terminals. By using the Kit, the importance to a community of a port and terminals can be q...

Rural inland waterways economic impact kit : analysis manual

DOT National Transportation Integrated Search

2000-08-01

The primary objective of the project was to develop a PC-based kit allowing users to evaluate the economic impact of existing rural inland waterways ports and terminals. By using the Kit, the importance to a community of a port and terminals can be q...

Land Use as a Driver of Patterns of Rodenticide Exposure in Modeled Kit Fox Populations

PubMed Central

Nogeire, Theresa M.; Lawler, Joshua J.; Schumaker, Nathan H.; Cypher, Brian L.; Phillips, Scott E.

2015-01-01

Although rodenticides are increasingly regulated, they nonetheless cause poisonings in many non-target wildlife species. Second-generation anticoagulant rodenticide use is common in agricultural and residential landscapes. Here, we use an individual-based population model to assess potential population-wide effects of rodenticide exposures on the endangered San Joaquin kit fox (Vulpes macrotis mutica). We estimate likelihood of rodenticide exposure across the species range for each land cover type based on a database of reported pesticide use and literature. Using a spatially-explicit population model, we find that 36% of modeled kit foxes are likely exposed, resulting in a 7-18% decline in the range-wide modeled kit fox population that can be linked to rodenticide use. Exposures of kit foxes in low-density developed areas accounted for 70% of the population-wide exposures to rodenticides. We conclude that exposures of non-target kit foxes could be greatly mitigated by reducing the use of second-generation anticoagulant rodenticides in low-density developed areas near vulnerable populations. PMID:26244655

The Fluid Dynamics Demo Kit: Part I

NASA Astrophysics Data System (ADS)

Flack, Karen; Underhill, Patrick; Prestridge, Kathy

2012-11-01

The goal of this project is to develop a fluid dynamics demonstration/experiment kit that can be used by professors and graduate students at high school outreach events. The demonstrations in the kit will be easy to use and true crowd pleasers in order to inspire understanding and pique curiosity about the physics of flow. The kits will be inexpensive, containing readily available materials so that teachers can duplicate the demonstrations and experiments. The kits will be left with the teachers as a gift from the American Physics Society. The experiments and demonstrations cover the concepts of conservation of mass, momentum, and energy, Bernoulli's equation, frictional losses and the ideal gas law. For each experiment, the teachers will receive presentation material, access to instructional videos, plus a worksheet that can be used in a high school physics classroom. This kit has been developed through the efforts of the APS-DFD Mentoring and Outreach Committee and has received funding from the APS-DFD. Work funded by the APS-DFD.

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

Dumpster Optics: teaching and learning optics without a kit

NASA Astrophysics Data System (ADS)

Donnelly, Judy; Magnani, Nancy; Robinson, Kathleen

2016-09-01

The Next Generation Science Standards (NGSS) and renewed emphasis on STEM education in the U.S. have resulted in the development of many educational kits for teaching science in general and optics in particular. Many teachers do not have funding to purchase kits and practical experience has shown that even costly kits can have poorly written and misleading instructions and may include experiments that would not work in a classroom. Dumpster Optics lessons are designed to use inexpensive, commonly found materials. All lessons have been field-tested with students. We will describe the development of the lessons, provide examples of field testing experiences and outline possible future activities.

Effectiveness of the Peabody Language Development Kits with Educable Mentally Retarded Children: A Report After Two and One-Half Years. IMRID Papers and Reports, Volume V, No. 15.

ERIC Educational Resources Information Center

Dunn, Lloyd M.; And Others

To determine the effectiveness of the Peabody Language Development Kits over an extended period (2.5 years) with educable mentally retarded (EMR) children, daily oral language stimulation lessons using Levels One and Two of the kit were given to 27 classes for the EMR in schools with culturally disadvantaged populations. Results showed the…

Development of a computer-based automated pure tone hearing screening device: a preliminary clinical trial.

PubMed

Gan, Kok Beng; Azeez, Dhifaf; Umat, Cila; Ali, Mohd Alauddin Mohd; Wahab, Noor Alaudin Abdul; Mukari, Siti Zamratol Mai-Sarah

2012-10-01

Hearing screening is important for the early detection of hearing loss. The requirements of specialized equipment, skilled personnel, and quiet environments for valid screening results limit its application in schools and health clinics. This study aimed to develop an automated hearing screening kit (auto-kit) with the capability of realtime noise level monitoring to ensure that the screening is performed in an environment that conforms to the standard. The auto-kit consists of a laptop, a 24-bit resolution sound card, headphones, a microphone, and a graphical user interface, which is calibrated according to the American National Standards Institute S3.6-2004 standard. The auto-kit can present four test tones (500, 1000, 2000, and 4000 Hz) at 25 or 40 dB HL screening cut-off level. The clinical results at 40 dB HL screening cut-off level showed that the auto-kit has a sensitivity of 92.5% and a specificity of 75.0%. Because the 500 Hz test tone is not included in the standard hearing screening procedure, it can be excluded from the auto-kit test procedure. The exclusion of 500 Hz test tone improved the specificity of the auto-kit from 75.0% to 92.3%, which suggests that the auto-kit could be a valid hearing screening device. In conclusion, the auto-kit may be a valuable hearing screening tool, especially in countries where resources are limited.

Ubiquitylation and proteasomal degradation of the p21(Cip1), p27(Kip1) and p57(Kip2) CDK inhibitors.

PubMed

Lu, Zhimin; Hunter, Tony

2010-06-15

The expression levels of the p21(Cip1) family CDK inhibitors (CKIs), p21(Cip1), p27(Kip1) and p57(Kip2), play a pivotal role in the precise regulation of cyclin-dependent kinase (CDK) activity, which is instrumental to proper cell cycle progression. The stabilities of p21(Cip1), p27(Kip1) and p57(Kip2) are all tightly and differentially regulated by ubiquitylation and proteasome-mediated degradation during various stages of the cell cycle, either in steady state or in response to extracellular stimuli, which often elicit site-specific phosphorylation of CKIs triggering their degradation.

Simian virus 40 large T antigen associates with cyclin A and p33cdk2.

PubMed

Adamczewski, J P; Gannon, J V; Hunt, T

1993-11-01

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses.

Identification of Atuveciclib (BAY 1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer

PubMed Central

Scholz, Arne; Lienau, Philip; Siemeister, Gerhard; Kosemund, Dirk; Bohlmann, Rolf; Briem, Hans; Terebesi, Ildiko; Meyer, Kirstin; Prelle, Katja; Denner, Karsten; Bömer, Ulf; Schäfer, Martina; Eis, Knut; Valencia, Ray; Ince, Stuart; von Nussbaum, Franz; Mumberg, Dominik; Ziegelbauer, Karl; Klebl, Bert; Choidas, Axel; Nussbaumer, Peter; Baumann, Matthias; Schultz‐Fademrecht, Carsten; Rühter, Gerd; Eickhoff, Jan; Brands, Michael

2017-01-01

Abstract Selective inhibition of exclusively transcription‐regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY‐958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer. PMID:28961375

Method and platform standardization in MRM-based quantitative plasma proteomics.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

2013-12-16

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics. © 2013.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

PubMed

Gude, Natalie A; Firouzi, Fareheh; Broughton, Kathleen M; Ilves, Kelli; Nguyen, Kristine P; Payne, Christina R; Sacchi, Veronica; Monsanto, Megan M; Casillas, Alexandria R; Khalafalla, Farid G; Wang, Bingyan J; Ebeid, David E; Alvarez, Roberto; Dembitsky, Walter P; Bailey, Barbara A; van Berlo, Jop; Sussman, Mark A

2018-06-22

Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings. © 2018 American Heart Association, Inc.

Planning Systems. SPEC Kit 13.

ERIC Educational Resources Information Center

Association of Research Libraries, Washington, DC. Office of Management Studies.

This kit on planning systems updates a 1974 Management Studies Office Systems and Procedures Exchange Center (SPEC) kit in which developments in planning activities among Association of Research Libraries (ARL) were reviewed. At the time of the original study, in 1972, planning techniques and systems were a subject of much interest because rising…

First Follow Nature, Kit II.

ERIC Educational Resources Information Center

1971

Developing pupils' awareness of their environment, learning to distinguish between what is pleasant and unpleasant, and examining acts of man to determine which are destructive and which are in harmony with nature are the purposes of Scholastic's Earth Corps Environmental Study Kits for Grades 1-6. This kit explores in depth the reasons some…

In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation.

PubMed

Borrell, Víctor

2010-02-15

Ferrets have been extensively used to unravel the neural mechanisms of coding and processing of visual information, and also to identify the developmental mechanisms underlying the emergence of such a complex and fine-tuned neural system. In recent years numerous tools have been generated that allow studying neural systems with unprecedented power. Unfortunately, because many of these tools are genetically encoded, they are having a limited impact on research involving "non-genetic" species, like ferret, cat and monkey. Here I show how in vivo electroporation can be performed in postnatal ferret kits to deliver genetic constructs to pyramidal neurons of the cerebral cortex. Electroporation of GFP- and DsRed-encoding plasmids results in labeling of cortical progenitors first, then migrating neurons, and finally differentiating neurons and their processes. This technique also allows for the genetic manipulation of cortical development in the ferret, as illustrated by electroporation of a dominant-negative form of Cdk5. In the mature brain of electroporated animals, expression of reporter genes reveals the detailed morphological traits of cortical pyramids, including their axonal and dendritic arborization, and dendritic spines. I also show that postnatal electroporation can be used for the transfection of a massive cortical territory, or it can be specifically directed to a subset of cortical areas, and even only to a few scattered pyramids along the cortical mantle. In vivo electroporation of postnatal ferrets is therefore an effective, rapid, simple and highly versatile method for delivering genetic constructs to this animal, optimal for both developmental studies and adult anatomical/functional studies. Copyright 2009 Elsevier B.V. All rights reserved.

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

Treating ER+ Breast Cancer with CDK4/6 Inhibitors.

PubMed

2017-08-01

Data from the MONARCH2, PALOMA-1, and TREnd trials strongly support using CDK4/6 inhibitors alongside standard endocrine therapy for advanced ER-positive breast cancer. Including these targeted agents not only improves progression-free survival but may reverse acquired resistance to hormone treatment. ©2017 American Association for Cancer Research.

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

PubMed Central

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

2012-01-01

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

Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment.

PubMed

Ashraf, Shazia; Kudo, Hiroki; Rao, Jia; Kikuchi, Atsuo; Widmeier, Eugen; Lawson, Jennifer A; Tan, Weizhen; Hermle, Tobias; Warejko, Jillian K; Shril, Shirlee; Airik, Merlin; Jobst-Schwan, Tilman; Lovric, Svjetlana; Braun, Daniela A; Gee, Heon Yung; Schapiro, David; Majmundar, Amar J; Sadowski, Carolin E; Pabst, Werner L; Daga, Ankana; van der Ven, Amelie T; Schmidt, Johanna M; Low, Boon Chuan; Gupta, Anjali Bansal; Tripathi, Brajendra K; Wong, Jenny; Campbell, Kirk; Metcalfe, Kay; Schanze, Denny; Niihori, Tetsuya; Kaito, Hiroshi; Nozu, Kandai; Tsukaguchi, Hiroyasu; Tanaka, Ryojiro; Hamahira, Kiyoshi; Kobayashi, Yasuko; Takizawa, Takumi; Funayama, Ryo; Nakayama, Keiko; Aoki, Yoko; Kumagai, Naonori; Iijima, Kazumoto; Fehrenbach, Henry; Kari, Jameela A; El Desoky, Sherif; Jalalah, Sawsan; Bogdanovic, Radovan; Stajić, Nataša; Zappel, Hildegard; Rakhmetova, Assel; Wassmer, Sharon-Rose; Jungraithmayr, Therese; Strehlau, Juergen; Kumar, Aravind Selvin; Bagga, Arvind; Soliman, Neveen A; Mane, Shrikant M; Kaufman, Lewis; Lowy, Douglas R; Jairajpuri, Mohamad A; Lifton, Richard P; Pei, York; Zenker, Martin; Kure, Shigeo; Hildebrandt, Friedhelm

2018-05-17

No efficient treatment exists for nephrotic syndrome (NS), a frequent cause of chronic kidney disease. Here we show mutations in six different genes (MAGI2, TNS2, DLC1, CDK20, ITSN1, ITSN2) as causing NS in 17 families with partially treatment-sensitive NS (pTSNS). These proteins interact and we delineate their roles in Rho-like small GTPase (RLSG) activity, and demonstrate deficiency for mutants of pTSNS patients. We find that CDK20 regulates DLC1. Knockdown of MAGI2, DLC1, or CDK20 in cultured podocytes reduces migration rate. Treatment with dexamethasone abolishes RhoA activation by knockdown of DLC1 or CDK20 indicating that steroid treatment in patients with pTSNS and mutations in these genes is mediated by this RLSG module. Furthermore, we discover ITSN1 and ITSN2 as podocytic guanine nucleotide exchange factors for Cdc42. We generate Itsn2-L knockout mice that recapitulate the mild NS phenotype. We, thus, define a functional network of RhoA regulation, thereby revealing potential therapeutic targets.

CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

PubMed Central

Mende, Nicole; Kuchen, Erika E.; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D.; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico

2015-01-01

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. PMID:26150472

CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

PubMed

Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

2015-07-27

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. © 2015 Mende et al.

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

PubMed Central

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

2016-01-01

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

Evaluation of the interaction of cyclin-dependent kinase 5 with activator p25 and with p25-derived inhibitor CIP.

PubMed

Cardone, Antonio; Albers, R Wayne; Sriram, Ram D; Pant, Harish C

2010-05-01

A high-affinity inhibitor protein called CIP, produced by small truncations of p35, was experimentally identified. P35 is a physiological activator of the cyclin-dependent kinase cdk5. P25 is derived from proteolytic truncation of p35 within "stressed" neurons, and it is associated with the hyperphosphorylation of specific neuronal proteins, typically occurring in neurodegenerative diseases such as Alzheimer's. Here, we report a study of the binding mechanisms of the cdk5-p25 and cdk5-CIP complexes. This provides a better understanding of the source of the inhibitory activity of the protein CIP. We use a geometry-based technique to test the hypothesis that p25's truncation increases the flexibility of CIP and thus prevents cdk5 from reaching its active conformation. Our study is based on a geometry-based alignment algorithm, which aligns two given protein conformations with respect to their interfaces. Our results support the flexibility hypothesis and will be used as a basis for targeted molecular dynamics simulations.

Molecular Modeling Studies of 4,5-Dihydro-1H-pyrazolo[4,3-h] quinazoline Derivatives as Potent CDK2/Cyclin A Inhibitors Using 3D-QSAR and Docking

PubMed Central

Ai, Yong; Wang, Shao-Teng; Sun, Ping-Hua; Song, Fa-Jun

2010-01-01

CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r2cv values of 0.747 and 0.518 and r2 values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity. PMID:21152296

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

PubMed

Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

2016-04-12

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular modeling studies of 4,5-dihydro-1H-pyrazolo[4,3-h] quinazoline derivatives as potent CDK2/Cyclin a inhibitors using 3D-QSAR and docking.

PubMed

Ai, Yong; Wang, Shao-Teng; Sun, Ping-Hua; Song, Fa-Jun

2010-09-28

CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r(2) (cv) values of 0.747 and 0.518 and r(2) values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity.

Development of Experiment Kits for Processing Biological Samples In-Flight on SLS-2

NASA Technical Reports Server (NTRS)

Jaquez, R.; Savage, P. D.; Hinds, W. E.; Evans, J.; Dubrovin, L.

1994-01-01

The design of the hematology experiment kits for SLS-2 has resulted in a modular, flexible configuration which maximizes crew efficiency and minimizes error and confusion when dealing with over 1200 different components over the course of the mission. The kit layouts proved to be very easy to use and their packaging design provided for positive, secure containment of the many small components. The secondary Zero(Tm) box enclosure also provided an effective means for transport of the kits within the Spacelab and for grouping individual kits by flight day usage. The kits are readily adaptable to use on future flights by simply replacing the inner components as required and changing the labelling scheme to match new mission requirements.

AsMA Medical Guidelines for Air Travel: In-Flight Medical Care.

PubMed

Thibeault, Claude; Evans, Anthony D; Pettyjohn, Frank S; Alves, Paulo M

2015-06-01

Medical Guidelines for Airline Travel provide information that enables healthcare providers to properly advise patients who plan to travel by air. All airlines are required to provide first aid training for cabin crew, and the crew are responsible for managing any in-flight medical events. There are also regulatory requirements for the carriage of first aid and medical kits. AsMA has developed recommendations for first aid kits, emergency medical kits, and universal precaution kits.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases*

PubMed Central

Walkup, Ward G.; Washburn, Lorraine; Sweredoski, Michael J.; Carlisle, Holly J.; Graham, Robert L.; Hess, Sonja; Kennedy, Mary B.

2015-01-01

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons. PMID:25533468

Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell Acute Lymphoblastic Leukemia.

PubMed

Pikman, Yana; Alexe, Gabriela; Roti, Giovanni; Conway, Amy Saur; Furman, Andrew; Lee, Emily S; Place, Andrew E; Kim, Sunkyu; Saran, Chitra; Modiste, Rebecca; Weinstock, David M; Harris, Marian; Kung, Andrew L; Silverman, Lewis B; Stegmaier, Kimberly

2017-02-15

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1 -mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012-24. ©2016 AACR See related commentary by Carroll et al., p. 873 . ©2016 American Association for Cancer Research.

Microtubule plus-end tracking of end-binding protein 1 (EB1) is regulated by CDK5 regulatory subunit-associated protein 2

PubMed Central

Fong, Ka-Wing; Au, Franco K. C.; Jia, Yue; Yang, Shaozhong; Zhou, Liying; Qi, Robert Z.

2017-01-01

Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SXIP (Ser-X-Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SXIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SXIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/β-tubulin binding, and microtubule polymerization. The SXIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SXIP motif alone failed to do so. An SXIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SXIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SXIP-containing chimeras. Our results suggest that disruption of SXIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SXIP-containing +TIPs. PMID:28320860

Development of a forensic evidence protection kit

NASA Astrophysics Data System (ADS)

Acton, Brian; Kelly, Roy

1999-02-01

A kit has been developed for the preservation of vital forensic evidence on a suspect following a serious assault, murder or other offense where contamination may occur. This also includes the handling of firearms, explosives and/or drugs.

R.E.A.D.Y.: Read, Educate and Develop Youth. Reading Plan for Michigan: Parent Information for Infants, Toddlers, and Preschoolers. Learning Begins at Birth. [Kit with Videotape and Audiotape].

ERIC Educational Resources Information Center

Michigan Association for the Education of Young Children, East Lansing.

Noting the important role that parents can play in preparing their child to learn to read, the Read, Educate and Develop Youth (READY) Reading Plan for Michigan provides kits to parents of infants, toddlers, and preschoolers. The kits contain suggestions for age-appropriate activities parents can do with their children to help them learn. In…

Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

PubMed

Ivens, Katherine O; Baumert, Joseph L; Taylor, Steve L

2016-07-01

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions. © 2016 Institute of Food Technologists®

Prognostic value of HMGA2, CDK4, and JUN amplification in well-differentiated and dedifferentiated liposarcomas.

PubMed

Saâda-Bouzid, Esma; Burel-Vandenbos, Fanny; Ranchère-Vince, Dominique; Birtwisle-Peyrottes, Isabelle; Chetaille, Bruno; Bouvier, Corinne; Château, Marie-Christine; Peoc'h, Michel; Battistella, Maxime; Bazin, Audrey; Gal, Jocelyn; Michiels, Jean-François; Coindre, Jean-Michel; Pedeutour, Florence; Bianchini, Laurence

2015-11-01

HMGA2, CDK4, and JUN genes have been described as frequently coamplified with MDM2 in atypical lipomatous tumor, well-differentiated liposarcoma, and dedifferentiated liposarcoma. We studied the frequency of amplification of these genes in a series of 48 dedifferentiated liposarcomas and 68 atypical lipomatous tumors/well-differentiated liposarcomas. We correlated their amplification status with clinicopathological features and outcomes. Histologically, both CDK4 (P=0.007) and JUN (P=0.005) amplifications were associated with dedifferentiated liposarcoma, whereas amplification of the proximal parts of HMGA2 (5'-untranslated region (UTR) and exons 1-3) was associated with atypical lipomatous tumor/well-differentiated liposarcoma (P=0.01). CDK4 amplification was associated with axial tumors. Amplification of 5'-UTR and exons 1-3 of HMGA2 was associated with primary status and grade 1. Shorter overall survival was correlated with: age >64 years (P=0.03), chemotherapy used in first intent (P<0.001), no surgery (P=0.003), grade 3 (P<0.001), distant metastasis (P<0.001), node involvement (P=0.006), and CDK4 amplification (P=0.07). In multivariate analysis, distant metastasis (HR=8.8) and grade 3 (HR=18.2) were associated with shorter overall survival. A shorter recurrence-free survival was associated with dedifferentiated liposarcoma (P<0.001), grade 3 (P<0.001), node involvement (P<0.001), distant metastasis (P=0.02), recurrent status (P=0.009), axial location (P=0.001), and with molecular features such as CDK4 (P=0.05) and JUN amplification (P=0.07). Amplification of 5'-UTR and exons 1-3 (P=0.08) and 3'-UTR (P=0.01) of HMGA2 were associated with longer recurrence-free survival. Distant metastasis was associated with shorter recurrence-free survival (HR=5.8) in multivariate analysis. Dedifferentiated liposarcoma type was associated with axial location, grade 3 and recurrent status. In conclusion, we showed that the amplification of HMGA2 was associated with the atypical lipomatous tumor/well-differentiated liposarcoma histological type and a good prognosis, whereas CDK4 and JUN amplifications were associated with dedifferentiated liposarcoma histology and a bad prognosis. In addition, we also provided the first description of the molecular evolution of a well-differentiated liposarcoma into four successive dedifferentiated liposarcoma relapses, which was consistent with our general observations.

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

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

Irazoqui, Ana P.; Heim, Nadia B.; Boland, Ricardo L.

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, wemore » 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 treatment. • CDK6 change its intracellular localization by 1,25D stimulation.« less

Report on the State of Development, Availability, Evaluation, and Future use of Test Kits for the Measurement of Lead in Paint

EPA Science Inventory

The purpose of this issue paper is to address the availability and performance characteristics of portable lead test kits especially suited for lead in paint, procedures for evaluating the performance of these test kits, and the availability of performance evaluation (PE) materia...

Look Around You, Kit I.

ERIC Educational Resources Information Center

1971

Developing pupils' awareness of their environment, learning to distinguish between what is pleasant and unpleasant, and examining acts of man to determine which are destructive and which are in harmony with nature are the purposes of Scholastic's Earth Corps Environmental Study Kits for grades 1-6. This kit is designed to help the child develop…

Omid Early Intervention Resource Kit for Children with Autism Spectrum Disorders and Their Families

ERIC Educational Resources Information Center

Samadi, Sayyed Ali; Mahmoodizadeh, Ameneh

2014-01-01

Omid early intervention resource kit containing information booklets on autism spectrum disorders (ASD) and related issues, five packs of tangible selected playthings and communication facilitating aids was developed and evaluated with 65 Iranian parents. Beside a pretest before the resource kit deliverance, parents in the control group took part…

Climate change and other stressors change modeled population size and hybridization potential for San Joaquin kit fox

EPA Science Inventory

The San Joaquin kit fox was once widely distributed across the southern San Joaquin Valley, but agriculture and development have replaced much of the endangered subspecies’ habitat. We modeled impacts of climate change, land-use change, and rodenticide exposure on kit fox p...

Teachers' Guide to Curriculum Kit for Understanding Chinese Americans.

ERIC Educational Resources Information Center

Association of Chinese Teachers, San Francisco, CA.

This is a teacher's guide for a multi-media presentation developed using Emergency School Aid Act funds. It was designed for kindergarten to sixth grade students. The purpose of the kit is to increase cross-cultural understanding by providing information about contemporary Chinese American lifestyles. The kit includes a set of three filmstrips…

Mkit: A cell migration assay based on microfluidic device and smartphone.

PubMed

Yang, Ke; Wu, Jiandong; Peretz-Soroka, Hagit; Zhu, Ling; Li, Zhigang; Sang, Yaoshuo; Hipolito, Jolly; Zhang, Michael; Santos, Susy; Hillier, Craig; de Faria, Ricardo Lobato; Liu, Yong; Lin, Francis

2018-01-15

Mobile sensing based on the integration of microfluidic device and smartphone, so-called MS 2 technology, has enabled many applications over recent years, and continues to stimulate growing interest in both research communities and industries. In particular, it has been envisioned that MS 2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction, in this paper, we describe the development of a MS 2 -based cell functional assay for testing cell migration (the M kit ). The system is constructed as an integrated test kit, which includes microfluidic chips, a smartphone-based imaging platform, the phone apps for image capturing and data analysis, and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the M kit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore, neutrophil chemotaxis can be tested from a drop of whole blood using the M kit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the M kit . In addition to research applications, we demonstrated the effective use of the M kit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus, this developed M kit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulatory dephosphorylation of CDK at G₂/M in plants: yeast mitotic phosphatase cdc25 induces cytokinin-like effects in transgenic tobacco morphogenesis.

PubMed

Lipavská, Helena; Masková, Petra; Vojvodová, Petra

2011-05-01

During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics. The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.

Cellular effects of olomoucine, an inhibitor of cyclin-dependent kinases.

PubMed

Abraham, R T; Acquarone, M; Andersen, A; Asensi, A; Bellé, R; Berger, F; Bergounioux, C; Brunn, G; Buquet-Fagot, C; Fagot, D

1995-01-01

Olomoucine (2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine) has been recently described as a competitive inhibitor (ATP-binding site) of the cell cycle regulating p34cdc2/cyclin B, p33cdk2/cyclin A and p33cdk2/cyclin E kinases, the brain p33cdk5/p35 kinase and the ERK1/MAP-kinase. The unusual specificity of this compound towards cell cycle regulating enzymes suggests that it could inhibit certain steps of the cell cycle. The cellular effects of olomoucine were investigated in a large variety of plant and animal models. This compound inhibits the G1/S transition of unicellular algae (dinoflagellate and diatom). It blocks Fucus zygote cleavage and development of Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the Laminaria gametophytes. Stimulated Petunia mesophyl protoplasts are arrested in G1 by olomoucine. By arresting cleavage it blocks the development of Calanus copepod larvae. It reversibly inhibits the early cleavages of Caenorhabditis elegans embryos and those of ascidian embryos. Olomoucine inhibits the serotonin-induced prophase/metaphase transition of clam oocytes; furthermore, it triggers the the release of these oocytes from their meiotic metaphase I arrest, and induces nuclei reformation. Olomoucine slows down the prophase/metaphase transition in cleaving sea urchin embryos, but does not affect the duration of the metaphase/anaphase and anaphase/telophase transitions. It also inhibits the prophase/metaphase transition of starfish oocytes triggered by various agonists. Xenopus oocyte maturation, the in vivo and in vitro phosphorylation of elongation factor EF-1 are inhibited by olomoucine. Mouse oocyte maturation is delayed by this compound, whereas parthenogenetic release from metaphase II arrest is facilitated. Growth of a variety of human cell lines (rhabdomyosarcoma cell lines Rh1, Rh18, Rh28 and Rh30; MCF-7, KB-3-1 and their adriamycin-resistant counterparts; National Cancer Institute 60 human tumor cell lines comprising nine tumor types) is inhibited by olomoucine. Cell cycle parameter analysis of the non-small cell lung cancer cell line MR65 shows that olomoucine affects G1 and S phase transits. Olomoucine inhibits DNA synthesis in interleukin-2-stimulated T lymphocytes (CTLL-2 cells) and triggers a G1 arrest similar to interleukin-2 deprivation. Both cdc2 and cdk2 kinases (immunoprecipitated from nocodazole- and hydroxyurea-treated CTLL-2 cells, respectively) are inhibited by olomoucine. Both yeast and Drosophila embryos were insensitive to olomoucine. Taken together the results of this Noah's Ark approach show that olomoucine arrests cells both at the G1/S and the G2/M boundaries, consistent with the hypothesis of a prevalent effect on the cdk2 and cdc2 kinases, respectively.

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Huang, Yongping; Xie, Mingquan; Luo, Xiaochun; Li, Wenmei; Wang, Jihua

2015-05-30

The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. The cut-off level of detection of the kit was 25 parasites/μl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

Training Educators to Teach the Sun and Space Weather Using a Kit of Tools

NASA Astrophysics Data System (ADS)

Keesee, A. M.; Ensign, T.

2014-12-01

NASA provides a wealth of data from Heliospheric missions to the public, but educators face several challenges to using such data in the classroom. These include the knowledge of what is available and how to use it, a full understanding of the science concepts the data demonstrate, ability to obtain and maintain products to access data, and access to technology (such as computer labs) for anything other than testing. To surmount these challenges, the Educator Resource Center at the NASA Independent Validation and Verification (IV&V) Center in Fairmont, WV has developed an operational model that focuses on housing, maintaining, and lending out kits of necessary equipment along with training educators in the science concepts and use of kit materials. Following this model, we have developed a Sun and Space Weather kit and an educator professional development course that we have presented several times. The kit includes a classroom set of iPads utilized to access data from NASA missions and other sources as well as create video reports for project based outcomes, a set of telescopes for safe solar viewing, and materials to explore magnetic fields and the electromagnetic spectrum. We will present an overview of the training course, the kit materials, and lessons learned.

Taking Aim at Glycolysis with CDK8 Inhibitors.

PubMed

Fisher, Robert P

2018-05-01

Dependence on glycolysis under aerobic conditions, a frequent metabolic derangement in cancer cells, suggests a therapeutic opportunity. Now, through chemical genetics, CDK8, a kinase associated with the Mediator transcriptional coactivator complex, has emerged as an upstream inducer of glycolysis and a possible target for anticancer drug discovery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Simian virus 40 large T antigen associates with cyclin A and p33cdk2.

PubMed Central

Adamczewski, J P; Gannon, J V; Hunt, T

1993-01-01

In this paper we provide evidence that a fraction of large T antigen of simian virus 40 (SV40) interacts with cyclin A and p33cdk2 in both virus-infected and stably transformed cells. Immunoprecipitates of SV40 large T antigen from SV40-infected or SV40 large-T-antigen-transformed cells contain cyclin A, p33cdk2, and histone H1 kinase activity. Conversely, immunoprecipitates of cyclin A from these cells contain SV40 large T antigen. In this respect, SV40 large T antigen has properties similar to those of the E1A oncogene of adenoviruses and the E7 oncogene of human papillomaviruses. Images PMID:8411358

Evaluation of a newly developed quantitative determination kit for tumor marker CA15-3 with chemiluminescent assay.

PubMed

Li, Peihua; Ye, Huiming; Liu, Jiangwu; Jin, Hongwei; Lin, Yongzhi; Yan, Shuidi; Yu, Yang; Gao, Lei; Xu, Feihai; Zhang, Zhongying

2018-01-01

Tumor marker carbohydrate antigen 15-3 (CA15-3) is used as a biomarker to aid to diagnose and monitor the prognosis of breast cancer patients. A new quantitative determination kit for CA15-3 with chemiluminescent assay was developed by Xiamen InnoDx Biotech Co., Ltd, China. Therefore, we conducted the report to evaluate the performance of the kit. According to the "Guiding principles on performance analysis of diagnostic reagents in vitro", the calibration curve, limit of detection, reportable range, accuracy, precision, anti-interference capability, cross-reaction and comparison by measuring EDTA plasma and serum were carried out. In addition, the kit was performed in parallel to electrochemiluminescence immunoassay kit (Roche) to analyze the correlation between the two kits. Regression equation of calibration curve of the kit was Y=0.7914X+4.1032 (R 2 =.990). Limit of detection was 0.0347 U/mL. The reportable range was 0.5-2400 U/mL. Recovery ratio was 100.0%-104.8%. Coefficient of variations (CVs) of within-run and between-run were 4.8%-7.6% and 5.8%-7.4% respectively. No remarkable interferences (all Bias% were less than ±10%) were detected when samples contained hemoglobin ≤183.8 μmol/L, bilirubin ≤340 μmol/L, triglyceride ≤18.1 mmol/L, or rheumatoid factor ≤400 U/mL. No cross-reaction was present in the kit. Moreover, compared with the results from electrochemiluminescence immunoassay kit (Roche) in 345 serum samples, there was a satisfied correlation coefficient of 0.977 (P<.01), and the kit was simultaneously fit for the detection of EDTA plasma and serum samples. The new kit validated satisfactorily, and it can be used for detecting CA15-3 in clinical practice. © 2017 The Authors Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.

FUELS IN SOIL TEST KIT: FIELD USE OF DIESEL DOG SOIL TEST KITS

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

Unknown

2001-05-31

Western Research Institute (WRI) is commercializing Diesel Dog Portable Soil Test Kits for performing analysis of fuel-contaminated soils in the field. The technology consists of a method developed by WRI (U.S. Patents 5,561,065 and 5,976,883) and hardware developed by WRI that allows the method to be performed in the field (patent pending). The method is very simple and does not require the use of highly toxic reagents. The aromatic components in a soil extract are measured by absorption at 254 nm with a field-portable photometer. WRI added significant value to the technology by taking the method through the American Societymore » for Testing and Materials (ASTM) approval and validation processes. The method is designated ASTM Method D-5831-96, Standard Test Method for Screening Fuels in Soils. This ASTM designation allows the method to be used for federal compliance activities. In FY 99, twenty-five preproduction kits were successfully constructed in cooperation with CF Electronics, Inc., of Laramie, Wyoming. The kit components work well and the kits are fully operational. In the calendar year 2000, kits were provided to the following entities who agreed to participate as FY 99 and FY 00 JSR (Jointly Sponsored Research) cosponsors and use the kits as opportunities arose for field site work: Wyoming Department of Environmental Quality (DEQ) (3 units), F.E. Warren Air Force Base, Gradient Corporation, The Johnson Company (2 units), IT Corporation (2 units), TRC Environmental Corporation, Stone Environmental, ENSR, Action Environmental, Laco Associates, Barenco, Brown and Caldwell, Dames and Moore Lebron LLP, Phillips Petroleum, GeoSyntek, and the State of New Mexico. By early 2001, ten kits had been returned to WRI following the six-month evaluation period. On return, the components of all ten kits were fully functional. The kits were upgraded with circuit modifications, new polyethylene foam inserts, and updated instruction manuals.« less

The history and development of NASA survival equipment.

NASA Technical Reports Server (NTRS)

Radnofsky, M. I.

1972-01-01

A research and development program on survival equipment was begun in early 1960 with the Mercury Program. The Mercury survival kit is discussed together with Gemini survival equipment, and Apollo I survival equipment. A study program is conducted to assess potential survival problems that may be associated with future space flights landing in polar waters. Survival kit requirements for applications on the Skylab program are also considered. Other investigations are concerned with the design of a global survival kit in connection with Space Shuttle missions.

The breast cancer patient navigation kit: development and user feedback.

PubMed

Skrutkowski, Myriam; Saucier, Andréanne; Meterissian, Sarkis

2011-12-01

Our interdisciplinary team developed a written cancer patient education tool, the Breast Cancer Navigation Kit, to respond to the information needs of patients and family members and that meet patient literacy levels. A literature review and a focus group provided content development for four modules: "About Breast Cancer," "Body-Mind-Spirit," "After Treatment Ends," and "Practical Information." An evaluation by 31 women showed the kit to be easy to understand, very useful, and informative. However, all agreed that it could not replace the dialogue with health care professionals. An interdisciplinary approach involving patient feedback is key to develop appropriate patient education tools.

Development and Certification of Station Development Test Objective (SDTO) Experiment # 15012-U, "Near RealTime Water Quality Monitoring Demonstration for ISS Biocides Using Colorimetric Solid Phase Extraction (CSPE)"

NASA Technical Reports Server (NTRS)

Gazda, Daniel B.; Nolan, Daniel J.; Rutz, Jeffrey A.; Shcultz, John R.; Siperko, Lorraine M.; Porter, Marc D,; Lipert, Robert J.; Limardo, Jose G.; McCoy, J. Torin

2009-01-01

Scientists and engineers from the Wyle Integrated Science and Engineering Group are working with researchers at the University of Utah and Iowa State University to develop and certify an experimental water quality monitoring kit based on Colorimetric Solid Phase Extraction (CSPE). The kit will be launched as a Station Development Test Objective (SDTO) experiment and evaluated on the International Space Station (ISS) to determine the acceptability of CSPE technology for routine inflight water quality monitoring. Iodine and silver, the biocides used in the US and Russian on-orbit water systems, will serve as test analytes for the technology evaluation. This manuscript provides an overview of the CSPE SDTO experiment and details the development and certification of the experimental water quality monitoring kit. Initial results from reagent and standard solution stability testing and environmental testing performed on the kit hardware are also reported.

Evaluation of Scat Deposition Transects versus Radio Telemetry for Developing a Species Distribution Model for a Rare Desert Carnivore, the Kit Fox.

PubMed

Dempsey, Steven J; Gese, Eric M; Kluever, Bryan M; Lonsinger, Robert C; Waits, Lisette P

2015-01-01

Development and evaluation of noninvasive methods for monitoring species distribution and abundance is a growing area of ecological research. While noninvasive methods have the advantage of reduced risk of negative factors associated with capture, comparisons to methods using more traditional invasive sampling is lacking. Historically kit foxes (Vulpes macrotis) occupied the desert and semi-arid regions of southwestern North America. Once the most abundant carnivore in the Great Basin Desert of Utah, the species is now considered rare. In recent decades, attempts have been made to model the environmental variables influencing kit fox distribution. Using noninvasive scat deposition surveys for determination of kit fox presence, we modeled resource selection functions to predict kit fox distribution using three popular techniques (Maxent, fixed-effects, and mixed-effects generalized linear models) and compared these with similar models developed from invasive sampling (telemetry locations from radio-collared foxes). Resource selection functions were developed using a combination of landscape variables including elevation, slope, aspect, vegetation height, and soil type. All models were tested against subsequent scat collections as a method of model validation. We demonstrate the importance of comparing multiple model types for development of resource selection functions used to predict a species distribution, and evaluating the importance of environmental variables on species distribution. All models we examined showed a large effect of elevation on kit fox presence, followed by slope and vegetation height. However, the invasive sampling method (i.e., radio-telemetry) appeared to be better at determining resource selection, and therefore may be more robust in predicting kit fox distribution. In contrast, the distribution maps created from the noninvasive sampling (i.e., scat transects) were significantly different than the invasive method, thus scat transects may be appropriate when used in an occupancy framework to predict species distribution. We concluded that while scat deposition transects may be useful for monitoring kit fox abundance and possibly occupancy, they do not appear to be appropriate for determining resource selection. On our study area, scat transects were biased to roadways, while data collected using radio-telemetry was dictated by movements of the kit foxes themselves. We recommend that future studies applying noninvasive scat sampling should consider a more robust random sampling design across the landscape (e.g., random transects or more complete road coverage) that would then provide a more accurate and unbiased depiction of resource selection useful to predict kit fox distribution.

KIT Mutations Are Common in Testicular Seminomas

PubMed Central

Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.

2004-01-01

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343

Exacerbated experimental autoimmune encephalomyelitis in mast-cell-deficient Kit W-sh/W-sh mice.

PubMed

Piconese, Silvia; Costanza, Massimo; Musio, Silvia; Tripodo, Claudio; Poliani, Pietro L; Gri, Giorgia; Burocchi, Alessia; Pittoni, Paola; Gorzanelli, Andrea; Colombo, Mario P; Pedotti, Rosetta

2011-04-01

Mast cell (MC)-deficient c-Kit mutant Kit(W/W-v) mice are protected against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggesting a detrimental role for MCs in this disease. To further investigate the role of MCs in EAE, we took advantage of a recently characterized model of MC deficiency, Kit(W-sh/W-sh). Surprisingly, we observed that myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced chronic EAE was exacerbated in Kit(W-sh/W-sh) compared with Kit(+/+) mice. Kit(W-sh/W-sh) mice showed more inflammatory foci in the central nervous system (CNS) and increased T-cell response against myelin. To understand whether the discrepant results obtained in Kit(W-sh/W-sh) and in Kit(W/W-v) mice were because of the different immunization protocols, we induced EAE in these two strains with varying doses of MOG(35-55) and adjuvants. Although Kit(W-sh/W-sh) mice exhibited exacerbated EAE under all immunization protocols, Kit(W/W-v) mice were protected from EAE only when immunized with high, but not low, doses of antigen and adjuvants. Kit(W-sh/W-sh) mice reconstituted systemically, but not in the CNS, with bone marrow-derived MCs still developed exacerbated EAE, indicating that protection from disease could be exerted by MCs mainly in the CNS, and/or by other cells possibly dysregulated in Kit(W-sh/W-sh) mice. In summary, these data suggest to reconsider MC contribution to EAE, taking into account the variables of using different experimental models and immunization protocols.

A national survey of home-based care kits for palliative HIV/AIDS care in South Africa.

PubMed

Mabude, Z A; Beksinska, M E; Ramkissoon, A; Wood, S; Folsom, M

2008-09-01

The objective of this study was to assess home-based care (HBC) kits and programs in South Africa to evaluate the feasibility of scaling up kit production and distribution. South African HBC organizations received structured questionnaires; key informant interviews and a literature review were completed to assess systems for production, distribution and supply of HBC kits. Meetings with stakeholders were held in two Provinces to share and analyze the study findings. The study team distributed questionnaires to 466 organizations and conducted interviews with representatives from 45 organizations, the Provincial Department of Health (DoH) and manufacturers of kits. All identifiable HBC organizations in South Africa were included in the survey. As a result 215 HBC organizations returned questionnaires; including non-governmental organizations (56%), community-based organizations (32%) and organizations affiliated with government health departments. Two types of kits were available: a home kit and a professional kit. The demand for HBC kits exceeded availability, kit contents and availability varied considerably and the supply chain was irregular. Kit production and distribution systems were fragmented. Replenishment of kit items was problematic. End-users are mostly caregivers who have not received adequate training on their use. The study shows that substantial work has been done by HBC organizations in South Africa to respond to the need for palliative care supplies within resource constraints. The growing demand for kits exceeds the supply. There is a need to improve the supply chain management of HBC kits, strengthen referral systems and links between community-based organizations and government departments, expand training opportunities for care givers, and develop monitoring and evaluation systems.

Modifications in cell cycle kinetics and in expression of G1 phase-regulating proteins in human amniotic cells after exposure to electromagnetic fields and ionizing radiation.

PubMed

Lange, S; Viergutz, T; Simkó, M

2004-10-01

Low-frequency electromagnetic fields are suspected of being involved in carcinogenesis, particularly in processes that could be related to cancer promotion. Because development of cancer is associated with deregulated cell growth and we previously observed a magnetic field-induced decrease in DNA synthesis [Lange et al. (2002) Alterations in the cell cycle and in the protein level of cyclin D1p, 21CIP1, and p16INK4a after exposure to 50 HZ. MF in human cells. Radiat. Environ. Biophys.41, 131], this study aims to document the influence of 50 Hz, 1 mT magnetic fields (MF), with or without initial gamma-ionizing radiation (IR), on the following cell proliferation-relevant parameters in human amniotic fluid cells (AFC): cell cycle distribution, expression of the G1 phase-regulating proteins Cdk4, cyclin D1, p21CIP1 and p16INK4a, and Cdk4 activity. While IR induced a G1 delay and a dose-dependent G2 arrest, no discernible changes in cell cycle kinetics were observed due to MF exposure. However, a significant decrease in the protein expression of cyclin D1 and an increase in p21CIP1- and p16INK4a-expression could be detected after exposure to MF alone. IR-exposure caused an augmentation of p21CIP1- and p16INK4a- levels as well, but did not alter cyclin D1 expression. A slight diminution of Cdk4 activity was noticed after MF exposure only, indicating that Cdk4 appears not to act as a mediator of MF- or IR-induced changes in the cell cycle of AFC cells. Co-exposure to MF/IR affected neither cell cycle distribution nor protein expression or kinase activity additionally or synergistically, and therefore MF seems not to modify the mutagenic potency of IR.

The new indirubin derivative inhibitors of glycogen synthase kinase-3, 6-BIDECO and 6-BIMYEO, prevent tau phosphorylation and apoptosis induced by the inhibition of protein phosphatase-2A by okadaic acid in cultured neurons.

PubMed

Martin, Ludovic; Magnaudeix, Amandine; Wilson, Cornelia M; Yardin, Catherine; Terro, Faraj

2011-11-01

Alterations in glycogen synthase kinase-3β (GSK3β) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6-BIDECO (6-bromoindirubin-3'-[O-(N,N-diethylcarbamyl)-oxime] and 6-BIMYEO (6-bromoindirubin-3'-[O-(2-morpholin-1-ylethyl)-oxime] hydrochloride) on OKA-induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6-BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6-BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6-BIDECO and 6-BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. Copyright © 2011 Wiley-Liss, Inc.

Targeted overexpression of Tumor Necrosis Factor-α increases Cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons

PubMed Central

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

2016-01-01

We reported earlier that TNF-α, a proinflammatory cytokine implicated in many inflammatory disorders causing orofacial pain increases Cdk5 activity, a key kinase involved in brain development and function and recently in pain signaling. To investigate a potential mechanism underlying inflammatory pain in trigeminal ganglia (TG), we engineered a transgenic mouse model (TNFglo) that can conditionally overexpresses TNF-α upon genomic recombination by Cre recombinase. TNFglo mice were bred with Nav1.8-Cre mouse line that expresses the Cre recombinase in sensory neurons to obtain TNF-α:Nav1.8-Cre (TNF-α cTg) mice. Although TNF-α cTg mice appeared normal without any gross phenotype, they displayed a significant increase in TNF-α levels after activation of NFκB signaling in the TG. IL-6 and MCP-1 levels were also increased along with intense immunostaining for Iba1 and GFAP in TG, indicating the presence of infiltrating macrophages and the activation of satellite glial cells. TNF-α cTg mice displayed increased trigeminal Cdk5 activity, and this increase was associated with elevated levels of phospho-T407-TRPV1 and capsaicin-evocated Ca2+ influx in cultured trigeminal neurons. Remarkably, this effect was prevented by roscovitine, an inhibitor of Cdk5, suggesting that TNF-α overexpression induced sensitization of the TRPV1 channel. Furthermore, TNF-α cTg mice displayed more aversive behavior to noxious thermal stimulation (45°C) of the face in an operant pain assessment device as compared with control mice. In summary, TNF-α overexpression in the sensory neurons of TNF-α cTg mice results in inflammatory sensitization and increased Cdk5 activity, therefore this mouse model would be valuable for investigating mechanism involved TNF-α in orofacial pain. PMID:26894912

Veterinary drug, 17β-trenbolone promotes the proliferation of human prostate cancer cell line through the Akt/AR signaling pathway.

PubMed

Lee, Hee-Seok; Jung, Da-Woon; Han, Songyi; Kang, Hui-Seung; Suh, Jin-Hyang; Oh, Hyun-Suk; Hwang, Myung-Sil; Moon, Guiim; Park, Yooheon; Hong, Jin-Hwan; Koo, Yong Eui

2018-05-01

Trenbolone acetate (TBA) is a synthetic anabolic steroidal growth factor that is used for rapid muscle development in cattle. The absorbed TBA is hydrolyzed to the active form, 17β-trenbolone (17 TB; 17β-hydroxy-estra-4,9,11-trien-3-one) in meat and milk products, which can cause adverse health effects in humans. Similar to 5α-dihydrotestosterone (DHT), 17 TB was reported to exhibit endocrine disrupting effects on animals and humans due to its androgenic effect via binding to the androgen receptor. The purpose of this study is to investigate the molecular mechanism of cell proliferation in prostate cancer (PCa) cells treated with 17 TB. We found that 17 TB induces AR-dependent cell proliferation in the human prostate cancer cell line, 22Rv1 in a concentration dependent manner. Treatment with 17 TB increased the expression of cell cycle regulatory proteins, cyclin D2/CDK-4 and cyclin E/CDK-2, whereas the expression of p27 was down-regulated. Furthermore, phosphorylation of Rb and activation of E2F were also induced, which suggests the activation of cyclin D2/CDK-4 and cyclin E/CDK-2 in the cells. When 22Rv1 cells were exposed to 30 pM of 17 TB, which is the effective concentration (EC 50 ) value required to observe proliferative effects on 22Rv1 cells, the expression levels of the phosphorylated forms of Akt and GSK3β were increased. This study demonstrates that 17 TB induces AR-dependent proliferation through the modulation of cell cycle-related proteins in the Akt signaling pathway. The present study provides an effective methodology for identifying cell proliferation signaling of veterinary drugs that exert AR agonistic effects. Copyright © 2018 Elsevier Ltd. All rights reserved.

The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

PubMed

Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

2015-01-15

Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

To Kit or Not to Kit? Evaluating and Implementing Science Materials and Resources

ERIC Educational Resources Information Center

Schiller, Ellen; Melin, Jacque; Bair, Mary

2016-01-01

With the release of the "Next Generation Science Standards," many schools are reexamining the science materials they are using. Textbook companies and kit developers are eager to meet the demand for "NGSS"-aligned teaching materials. Teacher may have been asked to serve on a science curriculum committee, or to evaluate current…

Robots in Education: New Trends and Challenges from the Japanese Market

ERIC Educational Resources Information Center

Basoeki, Fransiska; Dalla Libera, Fabio; Menegatti, Emanuele; Moro, Michele

2013-01-01

The paper introduces and compares the use of current robotics kits developed by different companies in Japan for education purposes. These kits are targeted to a large audience: from primary school students, to university students and also up to adult lifelong learning. We selected company and kits that are most successful in the Japanese market.…

INNOVATIVE TECHNOLOGY VERIFICATION REPORT "FIELD MEASUREMENT TECHNOLOGIES FOR TOTAL PETROLEUM HYDROCARBONS IN SOIL" CHEMETRICS, INC., AND AZUR ENVIRONMENTAL LTD REMEDIAID TOTAL PETROLEUM HYDROCARBON STARTER KIT

EPA Science Inventory

The RemediAidTm Total Petroleum Hydrocarbon Starter Kit (RemediAidTm kit) developed by CHEMetries, Inc. (CHEMetrics), and AZUR Environmental Ltd was demonstrated under the U.S. Environmental Protection Agency Superfund Innovative Technology Evaluation Program in June 2000 at the ...

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs.

PubMed

Löschmann, Nadine; Michaelis, Martin; Rothweiler, Florian; Zehner, Richard; Cinatl, Jaroslav; Voges, Yvonne; Sharifi, Mohsen; Riecken, Kristoffer; Meyer, Jochen; von Deimling, Andreas; Fichtner, Iduna; Ghafourian, Taravat; Westermann, Frank; Cinatl, Jindrich

2013-12-01

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.