Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

PubMed Central

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C.; Nogales, Eva; Taatjes, Dylan J.

2011-01-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly. PMID:21468301

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

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.

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.

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.

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.

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

Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin

PubMed Central

Schwartz, Michal; Travesa, Anna; Martell, Steven W; Forbes, Douglass J

2015-01-01

Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo. Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective “seeds” for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array. PMID:25602437

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays.

PubMed

Windpassinger, Christian; Piard, Juliette; Bonnard, Carine; Alfadhel, Majid; Lim, Shuhui; Bisteau, Xavier; Blouin, Stéphane; Ali, Nur'Ain B; Ng, Alvin Yu Jin; Lu, Hao; Tohari, Sumanty; Talib, S Zakiah A; van Hul, Noémi; Caldez, Matias J; Van Maldergem, Lionel; Yigit, Gökhan; Kayserili, Hülya; Youssef, Sameh A; Coppola, Vincenzo; de Bruin, Alain; Tessarollo, Lino; Choi, Hyungwon; Rupp, Verena; Roetzer, Katharina; Roschger, Paul; Klaushofer, Klaus; Altmüller, Janine; Roy, Sudipto; Venkatesh, Byrappa; Ganger, Rudolf; Grill, Franz; Ben Chehida, Farid; Wollnik, Bernd; Altunoglu, Umut; Al Kaissi, Ali; Reversade, Bruno; Kaldis, Philipp

2017-09-07

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development. Copyright © 2017 American Society of Human Genetics. All rights reserved.

Use of ATP analogs to inhibit HIV-1 transcription

PubMed Central

Narayanan, Aarthi; Sampey, Gavin; Van Duyne, Rachel; Guendel, Irene; Kehn-Hall, Kylene; Roman, Jessica; Currer, Robert; Galons, Hervé; Oumata, Nassima; Joseph, Benoît; Meijer, Laurent; Caputi, Massimo; Nekhai, Sergei; Kashanchi, Fatah

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of “latent cell populations” even after Anti Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells. PMID:22771113

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

PubMed Central

Anandhakumar, Jayamani; Moustafa, Yara W.; Chowdhary, Surabhi; Kainth, Amoldeep S.

2016-01-01

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the “anchor away” (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. PMID:27185874

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.

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

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.

Biogenesis of the yeast cytochrome bc1 complex.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis*

PubMed Central

Li, Lei; Nelson, Clark J.; Carrie, Chris; Gawryluk, Ryan M. R.; Solheim, Cory; Gray, Michael W.; Whelan, James; Millar, A. Harvey

2013-01-01

Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with 15N and isolating mitochondria, we have identified CI subcomplexes through differences in 15N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII2. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII2, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly. PMID:23271729

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

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

Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

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

Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin

Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells withmore » wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.« less

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.

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

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.

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

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.

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.

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.

Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor.

PubMed

Anandhakumar, Jayamani; Moustafa, Yara W; Chowdhary, Surabhi; Kainth, Amoldeep S; Gross, David S

2016-07-15

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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.

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

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.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

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

Cyclin-dependent kinase 9 is a novel specific molecular target in adult T-cell leukemia/lymphoma.

PubMed

Narita, Tomoko; Ishida, Takashi; Ito, Asahi; Masaki, Ayako; Kinoshita, Shiori; Suzuki, Susumu; Takino, Hisashi; Yoshida, Takashi; Ri, Masaki; Kusumoto, Shigeru; Komatsu, Hirokazu; Imada, Kazunori; Tanaka, Yuetsu; Takaori-Kondo, Akifumi; Inagaki, Hiroshi; Scholz, Arne; Lienau, Philip; Kuroda, Taruho; Ueda, Ryuzo; Iida, Shinsuke

2017-08-31

Cyclin-dependent kinase 9 (CDK9), a subunit of the positive transcription elongation factor b (P-TEFb) complex, regulates gene transcription elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). The deregulation of CDK9/P-TEFb has important implications for many cancer types. BAY 1143572 is a novel and highly selective CDK9/P-TEFb inhibitor currently being investigated in phase 1 studies. We evaluated the therapeutic potential of BAY 1143572 in adult T-cell leukemia/lymphoma (ATL). As a result of CDK9 inhibition and subsequent inhibition of phosphorylation at serine 2 of the RNAPII CTD, BAY 1143572 decreased c-Myc and Mcl-1 levels in ATL-derived or human T-cell lymphotropic virus type-1 (HTLV-1)-transformed lines and primary ATL cells tested, leading to their growth inhibition and apoptosis. Median inhibitory concentrations for BAY 1143572 in ATL-derived or HTLV-1-transformed lines (n = 8), primary ATL cells (n = 11), and CD4 + cells from healthy volunteers (n = 5) were 0.535, 0.30, and 0.36 μM, respectively. Next, NOG mice were used as recipients of tumor cells from an ATL patient. BAY 1143572-treated ATL-bearing mice (once daily 12.5 mg/kg oral application) demonstrated significantly decreased ATL cell infiltration of the liver and bone marrow, as well as decreased human soluble interleukin-2 receptor levels in serum (reflecting the ATL tumor burden), compared with untreated mice (n = 8 for both). BAY 1143572-treated ATL-bearing mice demonstrated significantly prolonged survival compared with untreated ATL-bearing mice (n = 7 for both). Collectively, this study indicates that BAY 1143572 showed strong potential as a novel treatment of ATL. © 2017 by The American Society of Hematology.

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.

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.

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.

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.

NEK8 Links the ATR-regulated Replication Stress Response and S-phase CDK Activity to Renal Ciliopathies

PubMed Central

Choi, Hyo Jei Claudia; Lin, Jia-Ren; Vannier, Jean-Baptiste; Slaats, Gisela G.; Kile, Andrew C.; Paulsen, Renee D.; Manning, Danielle K.; Beier, David R.; Giles, Rachel H.; Boulton, Simon J.; Cimprich, Karlene A.

2013-01-01

Summary Renal ciliopathies are a leading cause of kidney failure, but their exact etiology is poorly understood. NEK8/NPHP9 is a ciliary kinase associated with two renal ciliopathies in humans and mice, nephronophthisis (NPHP) and polycystic kidney disease. Here, we identify NEK8 as a key effector of the ATR-mediated replication stress response. Cells lacking NEK8 form spontaneous DNA double-strand breaks (DSBs) which further accumulate when replication forks stall, and they exhibit reduced fork rates, unscheduled origin firing, and increased replication fork collapse. NEK8 suppresses DSB formation by limiting cyclin A-associated CDK activity. Strikingly, a mutation in NEK8 that is associated with renal ciliopathies affects its genome maintenance functions. Moreover, kidneys of NEK8 mutant mice accumulate DNA damage, and loss of NEK8 or replication stress similarly disrupts renal cell architecture in a 3D-culture system. Thus, NEK8 is a critical component of the DNA damage response that links replication stress with cystic kidney disorders. PMID:23973373

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.

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.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

PubMed Central

Kuchin, S; Yeghiayan, P; Carlson, M

1995-01-01

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter. Images Fig. 3 Fig. 4 Fig. 5 PMID:7732022

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.

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.

Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

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

Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin

Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutantmore » (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.« less

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

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

Jun, Do Youn; Institute of Genetic Engineering, Kyungpook National University, Daegu; Kim, Jun Seok

2007-07-15

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

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

PubMed

D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

2016-06-23

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

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.

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.

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.

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.

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

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

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.

The negative cell cycle regulators, p27Kip1, p18Ink4c, and GSK-3, play critical role in maintaining quiescence of adult human pancreatic β-cells and restrict their ability to proliferate

PubMed Central

Stein, Jeffrey; Milewski, Wieslawa M; Dey, Arunangsu

2013-01-01

Adult human pancreatic β-cells are primarily quiescent (G0) yet the mechanisms controlling their quiescence are poorly understood. Here, we demonstrate, by immunofluorescence and confocal microscopy, abundant levels of the critical negative cell cycle regulators, p27(Kip1) and p18(Ink4c), 2 key members of cyclin-dependent kinase (CDK) inhibitor family, and glycogen synthase kinase-3 (GSK-3), a serine-threonine protein kinase, in islet β-cells of adult human pancreatic tissue. Our data show that p27(Kip1) localizes primarily in β-cell nuclei, whereas, p18(Ink4c) is mostly present in β-cell cytosol. Additionally, p-p27(S10), a phosphorylated form of p27(Kip1), which was shown to interact with and to sequester cyclinD-CDK4/6 in the cytoplasm, is present in substantial amounts in β-cell cytosol. Our immunofluorescence analysis displays similar distribution pattern of p27(Kip1), p-p27(S10), p18(Ink4c) and GSK-3 in islet β-cells of adult mouse pancreatic tissue. We demonstrate marked interaction of p27(Kip1) with cyclin D3, an abundant D-type cyclin in adult human islets, and vice versa as well as with its cognate kinase partners, CDK4 and CDK6. Likewise, we show marked interaction of p18(Ink4c) with CDK4. The data collectively suggest that inhibition of CDK function by p27(Kip1) and p18(Ink4c) contributes to human β-cell quiescence. Consistent with this, we have found by BrdU incorporation assay that combined treatments of small molecule GSK-3 inhibitor and mitogen/s lead to elevated proliferation of human β-cells, which is caused partly due to p27(Kip1) downregulation. The results altogether suggest that ex vivo expansion of human β-cells is achievable via increased proliferation for β-cell replacement therapy in diabetes. PMID:23896637

The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

PubMed

Biswas, N; Weller, S K

2001-05-18

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.

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.

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.

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

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.

Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

PubMed

Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

2004-05-01

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due to proteasome-mediated degradation of Cdc25C and Cdk1, and ectopic expression of Bcl-2 fails to confer resistance to PEITC-induced apoptosis. Furthermore, the results of the present study point toward involvement of both caspase-8- and caspase-9-mediated pathways in apoptosis induction by PEITC.

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.

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.

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.

Involvement of atypical transcription factor E2F8 in the polyploidization during mouse and human decidualization.

PubMed

Qi, Qian-Rong; Zhao, Xu-Yu; Zuo, Ru-Juan; Wang, Tong-Song; Gu, Xiao-Wei; Liu, Ji-Long; Yang, Zeng-Ming

2015-01-01

Polyploid decidual cells are specifically differentiated cells during mouse uterine decidualization. However, little is known about the regulatory mechanism and physiological significance of polyploidization in pregnancy. Here we report a novel role of E2F8 in the polyploidization of decidual cells in mice. E2F8 is highly expressed in decidual cells and regulated by progesterone through HB-EGF/EGFR/ERK/STAT3 signaling pathway. E2F8 transcriptionally suppresses CDK1, thus triggering the polyploidization of decidual cells. E2F8-mediated polyploidization is a response to stresses which are accompanied by decidualization. Interestingly, polyploidization is not detected during human decidualization with the down-regulation of E2F8, indicating differential expression of E2F8 may lead to the difference of decidual cell polyploidization between mice and humans.

Involvement of atypical transcription factor E2F8 in the polyploidization during mouse and human decidualization

PubMed Central

Qi, Qian-Rong; Zhao, Xu-Yu; Zuo, Ru-Juan; Wang, Tong-Song; Gu, Xiao-Wei; Liu, Ji-Long; Yang, Zeng-Ming

2015-01-01

Polyploid decidual cells are specifically differentiated cells during mouse uterine decidualization. However, little is known about the regulatory mechanism and physiological significance of polyploidization in pregnancy. Here we report a novel role of E2F8 in the polyploidization of decidual cells in mice. E2F8 is highly expressed in decidual cells and regulated by progesterone through HB-EGF/EGFR/ERK/STAT3 signaling pathway. E2F8 transcriptionally suppresses CDK1, thus triggering the polyploidization of decidual cells. E2F8-mediated polyploidization is a response to stresses which are accompanied by decidualization. Interestingly, polyploidization is not detected during human decidualization with the down-regulation of E2F8, indicating differential expression of E2F8 may lead to the difference of decidual cell polyploidization between mice and humans. PMID:25892397

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

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

A Kinase Independent Role for EGF Receptor in Autophagy Initiation

PubMed Central

Tan, Xiaojun; Thapa, Narendra; Sun, Yue; Anderson, Richard A

2014-01-01

The Epidermal Growth Factor Receptor (EGFR) is upregulated in numerous human cancers. Inhibition of EGFR signaling induces autophagy in tumor cells. Here we report an unanticipated role for the inactive EGFR in autophagy initiation. Inactive EGFR interacts with the oncoprotein LAPTM4B that is required for the endosomal accumulation of EGFR upon serum starvation. Inactive EGFR and LAPTM4B stabilize each other at endosomes and recruit the exocyst subcomplex containing Sec5. We show that inactive EGFR, LAPTM4B, and the Sec5 subcomplex are required for basal and starvation induced autophagy. LAPTM4B and Sec5 promote EGFR association with the autophagy inhibitor Rubicon, which in turn disassociates Beclin 1 from Rubicon to initiate autophagy. Thus, the oncoprotein LAPTM4B facilitates the role of inactive EGFR in autophagy initiation. This pathway is positioned to control tumor metabolism and promote tumor cell survival upon serum deprivation or metabolic stress. PMID:25594178

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.

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.

Long noncoding RNA LINC00858 promotes osteosarcoma through regulating miR-139-CDK14 axis.

PubMed

Gu, Zenghui; Hou, Zhenhai; Zheng, Longbao; Wang, Xinqiang; Wu, Liangbang; Zhang, Cheng

2018-06-23

Long noncoding RNAs (lncRNAs) have been identified to modulate the tumorigenesis of human cancers. The in-depth of lncRNAs on human osteosarcoma oncogenesis is still ambiguous. In present study, functional and mechanism experiments were conducted to investigate the role of long intergenic non-protein coding RNA 00858 (LINC00858) on human osteosarcoma tumorigenesis. Results demonstrated that LINC00858 expression was significantly upregulated in both osteosarcoma tissues and cell lines. Mechanism assays presented that LINC00858 silencing significantly repressed osteosarcoma cells' proliferation and invasion in vitro, and inhibited the tumor growth in vivo. In further experiments, LINC00858 was identified to sponge miR-139 to form RNA-induced silencing complex (RISC) using luciferase reporter assay and RNA immunoprecipitation (RIP). Besides, CDK14 was validated to be the target protein the miR-139. Rescue experiments confirmed the role of LINC00858/miR-139/CDK14 pathway on osteosarcoma cells' phenotype. In summary, these data prove that LINC00858/miR-139/CDK14 axis promotes the tumorigenesis of osteosarcoma, providing a new mechanism or target for osteosarcoma. Copyright © 2018. Published by Elsevier Inc.

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.

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

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.

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

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

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.

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

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

Structure–kinetic relationship study of CDK8/CycC specific compounds

PubMed Central

Schneider, Elisabeth V.; Böttcher, Jark; Huber, Robert; Maskos, Klaus; Neumann, Lars

2013-01-01

In contrast with the very well explored concept of structure–activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure–kinetic relationship. Here, we present a structure–kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif (“DMG” in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket. PMID:23630251

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.

Anti-leukemic effect of a synthetic compound, (±) trans-dihydronarciclasine (HYU-01) via cell-cycle arrest and apoptosis in acute myeloid leukemia.

PubMed

Kim, Seo Ju; Park, Hyun Ki; Kim, Ju Young; Yoon, Jin Sun; Kim, Eun Shil; Cho, Cheon-Gyu; Kim, Byoung Kook; Park, Byeong Bae; Lee, Young Yiul

2012-10-01

(±) trans-Dihydronarciclasine, isolated from Chinese medicinal plant Zephyranthes candida, has been shown to possess quite potent anti-tumoral effect against selected human cancer cell lines. However, little is known about the anti-tumoral effect of (±) trans-dihydronarciclasine in acute myeloid leukemia (AML). This study was performed to investigate the effect of a novel synthetic (±) trans-dihydronarciclasine (code name; HYU-01) in AML. The HYU-01 inhibited the proliferation of various AML cell lines including HL-60 as well as primary leukemic blasts in a dose-dependent manner. To investigate the mechanism of the anti-proliferative effect of HYU-01, cell-cycle analysis was attempted in HL-60 cells, resulting in G1 arrest. The expression levels of CDK2, CDK4, CDK6, cyclin E, and cyclin A were decreased in a time-dependent manner. In addition, HYU-01 up-regulated the expression of the p27, and markedly enhanced the binding of p27 with CDK2, 4, and 6, ultimately resulting in the decrease of their kinase activities. Furthermore, HYU-01 induced the apoptosis through the induction of proapoptotic molecules and reduction of antiapoptotic molecules in association with the activation of caspase-3, -8, and -9. These results suggest that HYU-01 may inhibit the proliferation of HL-60 cells, via apoptosis, as well as G1 block in association with the induction of p27. © 2012 The Authors APMIS © 2012 APMIS.

The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions

PubMed Central

Zuccolo, Michela; Alves, Annabelle; Galy, Vincent; Bolhy, Stéphanie; Formstecher, Etienne; Racine, Victor; Sibarita, Jean-Baptiste; Fukagawa, Tatsuo; Shiekhattar, Ramin; Yen, Tim; Doye, Valérie

2007-01-01

We previously demonstrated that a fraction of the human Nup107–160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107–160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107–160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107–160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore–microtubule attachment defects. Finally, we show that the presence of the Nup107–160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1–RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107–160 complex at kinetochores. PMID:17363900

8. Wayne Chandler, Photographer, May 2000 Photographic copy of engineering ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

8. Wayne Chandler, Photographer, May 2000 Photographic copy of engineering drawings, dated 1911, by U.S. Army Corps of Engineers. Drawings in possession of U.S. Army Corps of Engineers, Sault Ste. Marie, Michigan. General plan of hydraulic accumulator, exhaust reservoir, and pressure pumps. - St. Mary's Falls Canal, Soo Locks, Davis Lock Subcomplex, Southwest Operating Shelter, St. Mary's River at Falls, Sault Ste. Marie, Chippewa County, MI

Fission yeast Csk1 is a CAK-activating kinase (CAKAK).

PubMed Central

Hermand, D; Pihlak, A; Westerling, T; Damagnez, V; Vandenhaute, J; Cottarel, G; Mäkelä, T P

1998-01-01

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK). PMID:9857180

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

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

Conformational Landscape of the p28-Bound Human Proteasome Regulatory Particle.

PubMed

Lu, Ying; Wu, Jiayi; Dong, Yuanchen; Chen, Shuobing; Sun, Shuangwu; Ma, Yong-Bei; Ouyang, Qi; Finley, Daniel; Kirschner, Marc W; Mao, Youdong

2017-07-20

The proteasome holoenzyme is activated by its regulatory particle (RP) consisting of two subcomplexes, the lid and the base. A key event in base assembly is the formation of a heterohexameric ring of AAA-ATPases, which is guided by at least four RP assembly chaperones in mammals: PAAF1, p28/gankyrin, p27/PSMD9, and S5b. Using cryogenic electron microscopy, we analyzed the non-AAA structure of the p28-bound human RP at 4.5 Å resolution and determined seven distinct conformations of the Rpn1-p28-AAA subcomplex within the p28-bound RP at subnanometer resolutions. Remarkably, the p28-bound AAA ring does not form a channel in the free RP and spontaneously samples multiple "open" and "closed" topologies at the Rpt2-Rpt6 and Rpt3-Rpt4 interfaces. Our analysis suggests that p28 assists the proteolytic core particle to select a specific conformation of the ATPase ring for RP engagement and is released in a shoehorn-like fashion in the last step of the chaperone-mediated proteasome assembly. Copyright © 2017 Elsevier Inc. All rights reserved.

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

PubMed

Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

2017-01-01

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

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

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).

The alpha3(betaMet222Ser/Tyr345Trp)3gamma subcomplex of the TF1-ATPase does not hydolyze ATP at a significant rate until the substrate binds to the catalytic site of the lowest affinity.

PubMed

Ren, Huimiao; Bandyopadhyay, Sanjay; Allison, William S

2006-05-16

The alpha(3)(betaM(222)S/Y(345)W)(3)gamma double-mutant subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 (TF(1)), free of endogenous nucleotides, does not entrap inhibitory MgADP in a catalytic site during turnover. It hydrolyzes 100 nM-2 mM ATP with a K(m) of 31 microM and a k(cat) of 220 s(-)(1). Fluorescence titrations of the introduced tryptophans with MgADP or MgATP revealed that both Mg-nucleotide complexes bind to the catalytic site of the highest affinity with K(d)()1 values of less than 1 nM and bind to the site of intermediate affinity with a common K(d)2 value of about 12 nM. The K(d)3 values obtained for the catalytic site of the lowest affinity from titrations with MgADP and MgATP are 25 and 37 microM, respectively. The double mutant hydrolyzes 200 nM ATP with a first-order rate of 1.5 s(-)(1), which is 0.7% of k(cat). Hence, it does not hydrolyze ATP at a significant rate when the catalytic site of intermediate affinity is saturated and the catalytic site of the lowest affinity is minimally occupied. After the addition of stoichiometric MgATP to the alpha(3)(betaM(222)S/Y(345)W)(3)gamma subcomplex, one-third of the tryptophan fluorescence remains quenched after 10 min. The product [(3)H]ADP remains bound when the wild-type and double-mutant subcomplexes hydrolyze substoichiometric [(3)H]ATP. In contrast, (32)P(i) is not retained when the wild-type subcomplex hydrolyzes substoichiometric [gamma-(32)P]ATP. This precludes assessment of the equilibrium at the high-affinity catalytic site when the wild-type TF(1) subcomplex hydrolyzes substoichiometric ATP.

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.

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

PubMed Central

Roychowdhury, Sameek; Iyer, Matthew K.; Robinson, Dan R.; Lonigro, Robert J.; Wu, Yi-Mi; Cao, Xuhong; Kalyana-Sundaram, Shanker; Sam, Lee; Balbin, O. Alejandro; Quist, Michael J.; Barrette, Terrence; Everett, Jessica; Siddiqui, Javed; Kunju, Lakshmi P.; Navone, Nora; Araujo, John C.; Troncoso, Patricia; Logothetis, Christopher J.; Innis, Jeffrey W.; Smith, David C.; Lao, Christopher D.; Kim, Scott Y.; Roberts, J. Scott; Gruber, Stephen B.; Pienta, Kenneth J.; Talpaz, Moshe; Chinnaiyan, Arul M.

2012-01-01

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology. PMID:22133722

Phloretin induces cell cycle arrest and apoptosis of human glioblastoma cells through the generation of reactive oxygen species.

PubMed

Liu, Yuanyuan; Fan, Chenghe; Pu, Lv; Wei, Cui; Jin, Haiqiang; Teng, Yuming; Zhao, Mingming; Yu, Albert Cheung Hoi; Jiang, Feng; Shu, Junlong; Li, Fan; Peng, Qing; Kong, Jian; Pan, Bing; Zheng, Lemin; Huang, Yining

2016-06-01

Phloretin, a flavonoid present in various plants, has been reported to exert anticarcinogenic effects. However, the mechanism of its chemo-preventive effect on human glioblastoma cells is not fully understood. This study aimed to investigate the molecular mechanism of phloretin and its associated chemo-preventive effect in human glioblastoma cells. The results indicate that phloretin inhibited cell proliferation by inducing cell cycle arrest at the G0-G1 phase and induced apoptosis of human glioblastoma cells. Phloretin-induced cell cycle arrest was associated with increased expression of p27 and decreased expression of cdk2, cdk4, cdk6, cyclinD and cyclinE. Moreover, the PI3K/AKT/mTOR signaling cascades were suppressed by phloretin in a dose-dependent manner. In addition, phloretin triggered the mitochondrial apoptosis pathway and generated reactive oxygen species (ROS). This was accompanied by the up-regulation of Bax, Bak and c-PARP and the down-regulation of Bcl-2. The antioxidant agents N-acetyl-L-cysteine and glutathione weakened the effect of phloretin on glioblastoma cells. In conclusion, these results demonstrate that phloretin exerts potent chemo-preventive activity in human glioblastoma cells through the generation of ROS.

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

PubMed Central

2013-01-01

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells PMID:23506616

Chaperoning epigenetics: FKBP51 decreases the activity of DNMT1 and mediates epigenetic effects of the antidepressant paroxetine.

PubMed

Gassen, Nils C; Fries, Gabriel R; Zannas, Anthony S; Hartmann, Jakob; Zschocke, Jürgen; Hafner, Kathrin; Carrillo-Roa, Tania; Steinbacher, Jessica; Preißinger, S Nicole; Hoeijmakers, Lianne; Knop, Matthias; Weber, Frank; Kloiber, Stefan; Lucae, Susanne; Chrousos, George P; Carell, Thomas; Ising, Marcus; Binder, Elisabeth B; Schmidt, Mathias V; Rüegg, Joëlle; Rein, Theo

2015-11-24

Epigenetic processes, such as DNA methylation, and molecular chaperones, including FK506-binding protein 51 (FKBP51), are independently implicated in stress-related mental disorders and antidepressant drug action. FKBP51 associates with cyclin-dependent kinase 5 (CDK5), which is one of several kinases that phosphorylates and activates DNA methyltransferase 1 (DNMT1). We searched for a functional link between FKBP51 (encoded by FKBP5) and DNMT1 in cells from mice and humans, including those from depressed patients, and found that FKBP51 competed with its close homolog FKBP52 for association with CDK5. In human embryonic kidney (HEK) 293 cells, expression of FKBP51 displaced FKBP52 from CDK5, decreased the interaction of CDK5 with DNMT1, reduced the phosphorylation and enzymatic activity of DNMT1, and diminished global DNA methylation. In mouse embryonic fibroblasts and primary mouse astrocytes, FKBP51 mediated several effects of paroxetine, namely, decreased the protein-protein interactions of DNMT1 with CDK5 and FKBP52, reduced phosphorylation of DNMT1, and decreased the methylation and increased the expression of the gene encoding brain-derived neurotrophic factor (Bdnf). In human peripheral blood cells, FKBP5 expression inversely correlated with both global and BDNF methylation. Peripheral blood cells isolated from depressed patients that were then treated ex vivo with paroxetine revealed that the abundance of BDNF positively correlated and phosphorylated DNMT1 inversely correlated with that of FKBP51 in cells and with clinical treatment success in patients, supporting the relevance of this FKBP51-directed pathway that prevents epigenetic suppression of gene expression. Copyright © 2015, American Association for the Advancement of Science.

P21 and p27: roles in carcinogenesis and drug resistance.

PubMed

Abukhdeir, Abde M; Park, Ben Ho

2008-07-01

Human cancers arise from an imbalance of cell growth and cell death. Key proteins that govern this balance are those that mediate the cell cycle. Several different molecular effectors have been identified that tightly regulate specific phases of the cell cycle, including cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors. Notably, loss of expression or function of two G1-checkpoint CDK inhibitors - p21 (CDKN1A) and p27 (CDKN1B) - has been implicated in the genesis or progression of many human malignancies. Additionally, there is a growing body of evidence suggesting that functional loss of p21 or p27 can mediate a drug-resistance phenotype. However, reports in the literature have also suggested p21 and p27 can promote tumours, indicating a paradoxical effect. Here, we review historic and recent studies of these two CDK inhibitors, including their identification, function, importance to carcinogenesis and finally their roles in drug resistance.

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

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

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

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

PubMed

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

PubMed Central

Róna, Gergely; Borsos, Máté; Ellis, Jonathan J; Mehdi, Ahmed M; Christie, Mary; Környei, Zsuzsanna; Neubrandt, Máté; Tóth, Judit; Bozóky, Zoltán; Buday, László; Madarász, Emília; Bodén, Mikael; Kobe, Bostjan; Vértessy, Beáta G

2014-01-01

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle. PMID:25483092

Formononetin promotes cell cycle arrest via downregulation of Akt/Cyclin D1/CDK4 in human prostate cancer cells.

PubMed

Li, Tianyu; Zhao, Xinge; Mo, Zengnan; Huang, Weihua; Yan, Haibiao; Ling, Zhian; Ye, Yu

2014-01-01

Formononetin is an O-methylated isoflavone isolated from the root of Astragalus membranaceus. It has already been reported that formononetin could inhibit cell proliferation and induce cell apoptosis in several cancers, including prostate cancer. This study aimed to further investigate whether cell cycle arrest is involved in formononetin-mediated antitumor effect in human prostate cancer cells, along with the underlying molecular mechanism. Human prostate cancer cells PC-3 and DU145 were respectively treated with various concentrations of formononetin. The inhibitory effect of formononetin on proliferation of prostate cancer cells was determined using MTT assays and flow cytometry. Next, formononetin-induced alterations in cyclin D1, CDK4 and Akt expression in PC-3 cells were detected by real-time PCR and western blot. Formononetin dose-dependently inhibited prostate cancer cell proliferation via the induction of cell cycle arrest at G0/G1 phase in vitro, which was more evident in PC-3 cells. Meanwhile, concomitant with reduced phosphorylation of Akt in PC-3 cells, formononetin remarkably downregulated expression levels of cyclin D1 and CDK4 in a dose-dependent manner. More interestingly, in the in vivo studies, formononetin showed a noticeable inhibition of tumor growth in recipient mice. Formononetin could exhibit inhibitory activity against human prostate cancer cells in vivo and in vitro, which is associated with G1 cell cycle arrest by inactivation of Akt/cyclin D1/CDK4. Therefore, formononetin may be used as a candidate agent for clinical treatment of prostate cancer in the future.

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 .

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

A complex molecular switch directs stress-induced cyclin C nuclear release through SCFGrr1-mediated degradation of Med13

PubMed Central

Stieg, David C.; Willis, Stephen D.; Ganesan, Vidyaramanan; Ong, Kai Li; Scuorzo, Joseph; Song, Mia; Grose, Julianne; Strich, Randy; Cooper, Katrina F.

2018-01-01

In response to oxidative stress, cells decide whether to mount a survival or cell death response. The conserved cyclin C and its kinase partner Cdk8 play a key role in this decision. Both are members of the Cdk8 kinase module, which, with Med12 and Med13, associate with the core mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which thereafter releases cyclin C into the cytoplasm. Cytoplasmic cyclin C associates with mitochondria, where it induces hyperfragmentation and regulated cell death. In this report, we show that residues 742–844 of Med13’s 600–amino acid intrinsic disordered region (IDR) both directs cyclin C-Cdk8 association and serves as the degron that mediates ubiquitin ligase SCFGrr1-dependent destruction of Med13 following oxidative stress. Here, cyclin C-Cdk8 phosphorylation of Med13 most likely primes the phosphodegron for destruction. Next, pro-oxidant stimulation of the cell wall integrity pathway MAP kinase Slt2 initially phosphorylates cyclin C to trigger its release from Med13. Thereafter, Med13 itself is modified by Slt2 to stimulate SCFGrr1-mediated destruction. Taken together, these results support a model in which this IDR of Med13 plays a key role in controlling a molecular switch that dictates cell fate following exposure to adverse environments. PMID:29212878

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.

GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains.

PubMed

Bandyopadhyay, Sanjay; Allison, William S

2004-07-27

In crystal structures of the bovine F(1)-ATPase (MF(1)), the side chains of gammaMet(23), gammaMet(232), and gammaLeu(77) interact in a cluster. Substitution of the corresponding residues in the alpha(3)beta(3)gamma subcomplex of TF(1) with lysine lowers the ATPase activity to 2.3, 11, and 15%, respectively, of that displayed by wild-type. In contrast, TF(1) subcomplexes containing the gammaM(23)C, gammaM(232)C, and gammaL(77)C substitutions display 36, 36, and 130%, respectively, of the wild-type ATPase activity. The ATPase activity of the gammaM(23)C/gammaM(232)C double mutant subcomplex is 36% that of the wild-type subcomplex before and after cross-linking the introduced cysteines, whereas the ATPase activity of the gammaM(23)C/L(77)C double mutant increased from 50 to 85% that of wild-type after cross-linking the introduced cysteines. Only beta-beta cross-links formed when the alpha(3)(betaE(395)C)(3)gammaM(23)C double mutant was inactivated with CuCl(2). The overall results suggest that the attenuated ATPase of the mutant subcomplexes containing the gammaM(23)K, gammaL(77)K, and gammaM(232)K substitutions is caused by disruption of the cluster of hydrophobic amino acid side chains and that the midregion of the coiled-coil comprised of the amino- and carboxyl-terminal alpha helices of the gamma subunit does not undergo unwinding or major displacement from the side chain of gammaLeu(77) during ATP-driven rotation of the gamma subunit.

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.

PPAR{gamma} activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide

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

Heo, Kyung-Sun; Department of Pharmacy, Chungnam National University, Yuseong, Daejeon; Kim, Dong-Uk

Native LDL would be a mitogenic and chemotactic stimulus of VSMC proliferation and differentiation in the atherosclerotic lesion where endothelial disruption occurred. In previous studies, our group investigated the molecular mechanisms by which LDL induces IL-8 production and by which PPAR{alpha} activation abolishes LDL effects in human aortic SMCs (hAoSMCs). Herein is the first report of PPAR{gamma} activation by troglitazone (TG) exerting its inhibitory effects on LDL-induced cell proliferation via generation not of H{sub 2}O{sub 2}, but of O2?-, and the subsequent activation of Erk1/2 in hAoSMCs. Moreover, in this study TG abolished the LDL-accelerated G{sub 1}-S progression to controlmore » levels via down-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and up-regulation of p21{sup Cip1} expression. TG exerted its anti-proliferative effects through the up-regulation of basal superoxide dismutase (SOD) expression. This data suggests that the regulation of O2?- is located at the crossroads between LDL signaling and cell proliferation.« less

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.

Circular RNA hsa_circ_0016788 regulates hepatocellular carcinoma tumorigenesis through miR-486/CDK4 pathway.

PubMed

Guan, Zheng; Tan, Jing; Gao, Wei; Li, Xin; Yang, Yuandong; Li, Xiaogang; Li, Yingchao; Wang, Qiang

2018-06-19

Recent studies have revealed that circular RNAs (circRNAs) play important roles in the tumorigenesis of human cancer, including hepatocellular carcinoma (HCC). In present study, we screen the circular RNA expression profiles in HCC tissue and investigate the molecular roles on HCC tumorigenesis. Human circRNA microarray analysis showed there were total 1,245 differently expressed circular RNAs, including 756 up-regulated circRNAs and 489 down-regulated circRNAs, in three pairs of HCC tissue and adjacent normal tissue. Hsa_circ_0016788 was identified to be up-regulated in both HCC tissue and cell lines. Loss-of-functional experiments in vivo and vitro revealed that hsa_circ_0016788 silencing inhibited the proliferation, invasion and promoted the apoptosis in vitro, and inhibited the tumor growth in vivo. Bioinformatics tools and luciferase reporter assay validated that miR-486 targeted hsa_circ_0016788 and CDK4 accompanying with negatively correlated expression, suggesting the hsa_circ_0016788/miR-486/CDK4 pathway. Receiver operating characteristic (ROC) curve showed that hsa_circ_0016788 had high diagnostic value (AUC = 0.851). In summary, results reveal the role of hsa_circ_0016788/miR-486/CDK4 in HCC tumorigenesis, providing a novel therapeutic target for HCC. © 2018 Wiley Periodicals, Inc.

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

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).

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 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

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

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.

The Saccharomyces cerevisiae Cdk8 Mediator Represses AQY1 Transcription by Inhibiting Set1p-Dependent Histone Methylation.

PubMed

Law, Michael J; Finger, Michael A

2017-03-10

In the budding yeast Saccharomyces cerevisiae , nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus. Copyright © 2017 Law and Finger.

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.

Gene amplification during myogenic differentiation

PubMed Central

Fischer, Ulrike; Ludwig, Nicole; Raslan, Abdulrahman; Meier, Carola; Meese, Eckart

2016-01-01

Gene amplifications are mostly an attribute of tumor cells and drug resistant cells. Recently, we provided evidence for gene amplifications during differentiation of human and mouse neural progenitor cells. Here, we report gene amplifications in differentiating mouse myoblasts (C2C12 cells) covering a period of 7 days including pre-fusion, fusion and post-fusion stages. After differentiation induction we found an increase in copy numbers of CDK4 gene at day 3, of NUP133 at days 4 and 7, and of MYO18B at day 4. The amplification process was accompanied by gamma-H2AX foci that are indicative of double stand breaks. Amplifications during the differentiating process were also found in primary human myoblasts with the gene CDK4 and NUP133 amplified both in human and mouse myoblasts. Amplifications of NUP133 and CDK4 were also identified in vivo on mouse transversal cryosections at stage E11.5. In the course of myoblast differentiation, we found amplifications in cytoplasm indicative of removal of amplified sequences from the nucleus. The data provide further evidence that amplification is a fundamental mechanism contributing to the differentiation process in mammalians. PMID:26760505

Live-cell imaging RNAi screen identifies PP2A–B55α and importin-β1 as key mitotic exit regulators in human cells

PubMed Central

Schmitz, Michael H. A.; Held, Michael; Janssens, Veerle; Hutchins, James R. A.; Hudecz, Otto; Ivanova, Elitsa; Goris, Jozef; Trinkle-Mulcahy, Laura; Lamond, Angus I.; Poser, Ina; Hyman, Anthony A.; Mechtler, Karl; Peters, Jan-Michael; Gerlich, Daniel W.

2013-01-01

When vertebrate cells exit mitosis various cellular structures are re-organized to build functional interphase cells1. This depends on Cdk1 (cyclin dependent kinase 1) inactivation and subsequent dephosphorylation of its substrates2–4. Members of the protein phosphatase 1 and 2A (PP1 and PP2A) families can dephosphorylate Cdk1 substrates in biochemical extracts during mitotic exit5,6, but how this relates to postmitotic reassembly of interphase structures in intact cells is not known. Here, we use a live-cell imaging assay and RNAi knockdown to screen a genome-wide library of protein phosphatases for mitotic exit functions in human cells. We identify a trimeric PP2A–B55α complex as a key factor in mitotic spindle breakdown and postmitotic reassembly of the nuclear envelope, Golgi apparatus and decondensed chromatin. Using a chemically induced mitotic exit assay, we find that PP2A–B55α functions downstream of Cdk1 inactivation. PP2A–B55α isolated from mitotic cells had reduced phosphatase activity towards the Cdk1 substrate, histone H1, and was hyper-phosphorylated on all subunits. Mitotic PP2A complexes co-purified with the nuclear transport factor importin-β1, and RNAi depletion of importin-β1 delayed mitotic exit synergistically with PP2A–B55α. This demonstrates that PP2A–B55α and importin-β1 cooperate in the regulation of postmitotic assembly mechanisms in human cells. PMID:20711181

A Smad action turnover switch operated by WW domain readers of a phosphoserine code

PubMed Central

Aragón, Eric; Goerner, Nina; Zaromytidou, Alexia-Ileana; Xi, Qiaoran; Escobedo, Albert; Massagué, Joan; Macias, Maria J.

2011-01-01

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events—Smad activation first and destruction later—and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers. PMID:21685363

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.

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

Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice.

PubMed

Rowe, Jenny; Greenblatt, Rebecca J; Liu, Dongmei; Moffat, Jennifer F

2010-06-01

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 microM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC(99) doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV. Copyright 2010 Elsevier B.V. All rights reserved.

Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model.

PubMed

Durand, Anne; Bourbon, Marie-Line; Steunou, Anne-Soisig; Khalfaoui-Hassani, Bahia; Legrand, Camille; Guitton, Audrey; Astier, Chantal; Ouchane, Soufian

2018-01-19

The cbb 3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb 3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb 3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex*

PubMed Central

Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S.; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

2014-01-01

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. PMID:25210042

Novel binding motif and new flexibility revealed by structural analyses of a pyruvate dehydrogenase-dihydrolipoyl acetyltransferase subcomplex from the Escherichia coli pyruvate dehydrogenase multienzyme complex.

PubMed

Arjunan, Palaniappa; Wang, Junjie; Nemeria, Natalia S; Reynolds, Shelley; Brown, Ian; Chandrasekhar, Krishnamoorthy; Calero, Guillermo; Jordan, Frank; Furey, William

2014-10-24

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

PubMed Central

Shorey, Lyndsey E.; Hagman, Amanda M.; Williams, David E.; Ho, Emily; Dashwood, Roderick H.; Benninghoff, Abby D.

2012-01-01

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL. PMID:22514694

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells.

PubMed

Park, Hyun Soo; Han, Min Ho; Kim, Gi-Young; Moon, Sung-Kwon; Kim, Wun-Jae; Hwang, Hye Jin; Park, Kun Young; Choi, Yung Hyun

2014-02-01

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway. Copyright © 2013 Elsevier Ltd. 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

Cell Cycle Dependent Regulation of Human Progesterone Receptor in Breast Cancer

DTIC Science & Technology

2004-10-01

wt PR or S400A PR (0.01-1.0 [tg each), CDK2 (ljig), and PRE-2x-TATA-luc reporter plasmid (lig) along with Renilla plasmid (10ng) as a control for...8 hours prior to treatment. Cells were collected following treatment with 10 nM R5020 or ETOH vehicle control for 18 hrs. Luciferase and renilla ...reporter, a renilla reporter construct as a transfection control, either wt PR-B or S400A mutant PR-B, and control parental vector or a vector encoding an

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

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

Identification of differentially expressed genes associated with the enhancement of X-ray susceptibility by RITA in a hypopharyngeal squamous cell carcinoma cell line (FaDu).

PubMed

Luan, Jinwei; Li, Xianglan; Guo, Rutao; Liu, Shanshan; Luo, Hongyu; You, Qingshan

2016-06-01

Next generation sequencing and bio-informatic analyses were conducted to investigate the mechanism of reactivation of p53 and induction of tumor cell apoptosis (RITA)-enhancing X-ray susceptibility in FaDu cells. The cDNA was isolated from FaDu cells treated with 0 X-ray, 8 Gy X-ray, or 8 Gy X-ray + RITA. Then, cDNA libraries were created and sequenced using next generation sequencing, and each assay was repeated twice. Subsequently, differentially expressed genes (DEGs) were identified using Cuffdiff in Cufflinks and their functions were predicted by pathway enrichment analyses. Genes that were constantly up- or down-regulated in 8 Gy X-ray-treated FaDu cells and 8 Gy X-ray + RITA-treated FaDu cells were obtained as RITA genes. Afterward, the protein-protein interaction (PPI) relationships were obtained from the STRING database and a PPI network was constructed using Cytoscape. Furthermore, ClueGO was used for pathway enrichment analysis of genes in the PPI network. Total 2,040 and 297 DEGs were identified in FaDu cells treated with 8 Gy X-ray or 8 Gy X-ray + RITA, respectively. PARP3 and NEIL1 were enriched in base excision repair, and CDK1 was enriched in p53 signaling pathway. RFC2 and EZH2 were identified as RITA genes. In the PPI network, many interaction relationships were identified (e.g., RFC2-CDK1, EZH2-CDK1 and PARP3-EZH2). ClueGO analysis showed that RFC2 and EZH2 were related to cell cycle. RFC2, EZH2, CDK1, PARP3 and NEIL1 may be associated, and together enhance the susceptibility of FaDu cells treated with RITA to the deleterious effects of X-ray.

Role of CDK4 in Breast Development and Cancer

DTIC Science & Technology

2008-04-01

Steward, A. H. Parr, M. D. Thomas, J. A. Henry, B. Angus , T. W. Lennard, C. H. Horne, Oncogene 11, 885 (1995). 12. J. Bartkova, J. Likas, M. Strauss...Cdk6 and Cdk2 in all four of the genotypes studied here. The results presented in Fig. 4A show that in both Cdk4(neo/neo):MMTV-wnt and in Cdk4(neo/neo...contrast, the levels of Cdk6 and Cdk2 were approximately equal in all four genotypes . These results suggest that neither Cdk6 nor Cdk2 compensate for

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

PubMed

Bogdanow, Boris; Weisbach, Henry; von Einem, Jens; Straschewski, Sarah; Voigt, Sebastian; Winkler, Michael; Hagemeier, Christian; Wiebusch, Lüder

2013-10-22

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

PubMed Central

Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

2018-01-01

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base.

PubMed

Funakoshi, Minoru; Tomko, Robert J; Kobayashi, Hideki; Hochstrasser, Mark

2009-05-29

The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here, we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.

[Importance of competition for pollination in formation of the entomophylous plants complex structure].

PubMed

Dlusskiĭ, G M

2013-01-01

Many species of entomophylous plants have a wide range of pollinators, and the same insects visit flowers of many plants. The competition for pollination leads to decreasing in seed production of competing species. However, there exists a variety of adaptations that allow plants to reduce the intensity of competition. A comparative analysis of pollinators spectra has allowed to designate groups (subcomplexes) of plants with regard to dominance of various groups of pollinators: myiophylous (flies from the superfamily Muscomorha dominate), syphidophylous (flies from the family Syrphidae dominate), psychophylous (butterflies dominate), cantharophylous (beetles dominate), nonspecialized and specialized melittophylous (Apidae, mainly bumblebees, dominate). The belonging of plants to a specific subcomplex is defined mainly by the structure of flowers and inflorescences. Modes of mechanical and attractive isolation are discussed that lead to restriction of pollinators composition. Competition abatement between species with similar spectra of pollinators and belonging to the same subcomplex is achieved mainly by spatial (ecological) and temporal (different timing of flowering) isolation.

Assembly and Architecture of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)*

PubMed Central

Lee, Hyung Ho; Nemecek, Daniel; Schindler, Christina; Smith, William J.; Ghirlando, Rodolfo; Steven, Alasdair C.; Bonifacino, Juan S.; Hurley, James H.

2012-01-01

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting. PMID:22203680

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.

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex.

PubMed

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-05-20

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex*

PubMed Central

Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

2016-01-01

Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. PMID:26980730

bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms

PubMed Central

VanderWaal, Kristyn E.; Yamamoto, Ryosuke; Wakabayashi, Ken-ichi; Fox, Laura; Kamiya, Ritsu; Dutcher, Susan K.; Bayly, Phillip V.; Sale, Winfield S.; Porter, Mary E.

2011-01-01

I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability. PMID:21697502

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.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A

PubMed Central

Yoshimoto, Rei; Kaida, Daisuke; Furuno, Masaaki; Burroughs, A. Maxwell; Noma, Shohei; Suzuki, Harukazu; Kawamura, Yumi; Hayashizaki, Yoshihide; Mayeda, Akila; Yoshida, Minoru

2017-01-01

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp. no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5′ splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5′ splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors. PMID:27754875

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

Sulforaphane Induces Cell Death Through G2/M Phase Arrest and Triggers Apoptosis in HCT 116 Human Colon Cancer Cells.

PubMed

Liu, Kuo-Ching; Shih, Ting-Ying; Kuo, Chao-Lin; Ma, Yi-Shih; Yang, Jiun-Long; Wu, Ping-Ping; Huang, Yi-Ping; Lai, Kuang-Chi; Chung, Jing-Gung

2016-01-01

Sulforaphane (SFN), an isothiocyanate, exists exclusively in cruciferous vegetables, and has been shown to possess potent antitumor and chemopreventive activity. However, there is no available information that shows SFN affecting human colon cancer HCT 116 cells. In the present study, we found that SFN induced cell morphological changes, which were photographed by contrast-phase microscopy, and decreased viability. SFN also induced G2/M phase arrest and cell apoptosis in HCT 116 cells, which were measured with flow cytometric assays. Western blotting indicated that SFN increased Cyclin A, cdk 2, Cyclin B and WEE1, but decreased Cdc 25C, cdk1 protein expressions that led to G2/M phase arrest. Apoptotic cell death was also confirmed by Annexin V/PI and DAPI staining and DNA gel electrophoresis in HCT 116 cells after exposure to SFN. The flow cytometric assay also showed that SFN induced the generation of reactive oxygen species (ROS) and Ca[Formula: see text] and decreased mitochondria membrane potential and increased caspase-8, -9 and -3 activities in HCT 116 cell. Western blotting also showed that SFN induced the release of cytochrome c, and AIF, which was confirmed by confocal microscopy examination. SFN induced ER stress-associated protein expression. Based on those observations, we suggest that SFN may be used as a novel anticancer agent for the treatment of human colon cancer in the future.

A naturally occurring mixture of tocotrienols inhibits the growth of human prostate tumor, associated with epigenetic modifications of cyclin-dependent kinase inhibitors p21 and p27.

PubMed

Huang, Ying; Wu, Renyi; Su, Zheng-Yuan; Guo, Yue; Zheng, Xi; Yang, Chung S; Kong, Ah-Ng

2017-02-01

Tocotrienols, members of the vitamin E family, have three unsaturated bonds in their side chains. Recently, it has been suggested that the biological effects of tocotrienols may differ from that of tocopherols. Several in vitro studies have shown that tocotrienols have stronger anticancer effects than tocopherols. VCaP cell line used in this study is from a vertebral bone metastasis from a patient with prostate cancer. Eight-week-old male NCr(-/-) nude mice were subcutaneously injected with VCaP-luc cells in matrigel and then administered a tocotrienol mixture for 8 weeks. The tocotrienol mixture inhibited the growth of human prostate tumor xenografts in a dose-dependent manner. The concentrations of tocotrienols and their metabolites were significantly increased in treatment groups. Tocotrienols inhibited prostate tumor growth by suppressing cell proliferation, which was associated with the induction of the cyclin-dependent kinase (CDK) inhibitors p21 and p27. In addition, tocotrienol treatment was associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27 and with decreased expression of histone deacetylases. Tocotrienols inhibited human prostate tumor growth, associated with up-regulation of the CDK inhibitors p21 and p27. Elevated expression of p21 and p27 could be partly due to the suppressed expression of HDACs. Copyright © 2016 Elsevier Inc. All rights reserved.

Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis

PubMed Central

Schwartz, Justin T.; Bandyopadhyay, Sarmistha; Kobayashi, Scott D.; McCracken, Jenna; Whitney, Adeline R.; DeLeo, Frank R.; Allen, Lee-Ann H.

2013-01-01

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24 h time period relative to the uninfected controls. Of ~47,000 genes analyzed, 3,435 were significantly up- or down-regulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes associated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2, and CASP8) and genes that act via the NF B pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2, and RELA, IL1B, CAST, CDK2, GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that X-linked inhibitor of apoptosis (XIAP) protein remained abundant in PMNs over 48 h of LVS infection, whereas BAX mRNA and protein were progressively down-regulated. These data strongly suggest that antiapoptotic and pro-survival mechanisms collaborate to sustain the viability of F. tularensis infected neutrophils. PMID:22986450

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

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

Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice.

PubMed

Kim, Gyeong-Ji; Jo, Hyeon-Ju; Lee, Kwon-Jai; Choi, Jeong Woo; An, Jeung Hee

2018-05-29

We evaluated oleanolic acid (OA)-induced anti-cancer activity, apoptotic mechanism, cell cycle status, and MAPK kinase signaling in DU145 (prostate cancer), MCF-7 (breast cancer), U87 (human glioblastoma), normal murine liver cell (BNL CL.2) and human foreskin fibroblast cell lines (Hs 68). The IC50 values for OA-induced cytotoxicity were 112.57 in DU145, 132.29 in MCF-7, and 163.60 in U87 cells, respectively. OA did not exhibit toxicity in BNL CL. 2 and Hs 68 cell lines in our experiments. OA, at 100 µg/mL, increased the number of apoptotic cells to 27.0% in DU145, 27.0% in MCF-7, and 15.7% in U87, when compared to control cells. This enhanced apoptosis was due to increases in p53, cytochrome c, Bax, PARP-1 and caspase-3 expression in DU145, MCF-7 and U87 cell lines. OA-treated DU145 cells were arrested in G2 because of the activation of p-AKT, p-JNK, p21 and p27, and the decrease in p-ERK, cyclin B1 and CDK2 expression; OA-treated MCF-7 cells were arrested in G1 owing to the activation of p-JNK, p-ERK, p21, and p27, and the decrease in p-AKT, cyclin D1, CDK4, cyclin E, and CDK2; and OA-treated U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p-AKT, p21, and p27, and the decrease in cyclin D1, CDK4, cyclin E and CDK2. Thus, OA arrested the cell cycle at different phases and induced apoptosis in cancer cells. These results suggested that OA possibly altered the expression of the cell cycle regulatory proteins differently in varying types of cancer.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

PubMed

Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Green tea extract inhibits proliferation of uterine leiomyoma cells in vitro and in nude mice

PubMed Central

ZHANG, Dong; AL-HENDY, Mohamed; RICHARD-DAVIS, Gloria; MONTGOMERY-RICE, Valerie; SHARAN, Chakradhari; RAJARATNAM, Veera; KHURANA, Anjali; AL-HENDY, Ayman

2010-01-01

Objective Investigate the effect of epigallocatechin gallate (EGCG), on rat leiomyoma (ELT3) cells in vitro and in nude mice model. Study Design ELT3 cells were treated with various concentrations of EGCG. Cell proliferation, PCNA and Cdk4 protein levels were evaluated. ELT3 cells were inoculated subcutaneously in female athymic nude mice. Animals were fed 1.25mg EGCG (in drinking water)/mouse/day. Tumors were collected and evaluated at 4 and 8 weeks post-treatment. Results Inhibitory effect of EGCG (200 μM) on ELT3 cells was observed after 24 h treatment (p<0.05). At ≥50μM, EGCG significantly decreased PCNA and Cdk4 protein levels (p<0.05). In vivo, EGCG treatment dramatically reduced the volume and weight of tumors at 4 and 8 weeks post-treatment (p<0.05). The PCNA and Cdk4 protein levels were significantly reduced in EGCG treated group (p<0.05). Conclusion EGCG effectively inhibits the proliferation and induce apoptosis in rat ELT3 uterine leiomyoma cells in vitro and in vivo. PMID:20074693

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

Impaired neurogenesis and neurite outgrowth in an HIV-gp120 transgenic model is reversed by exercise via BDNF production and Cdk5 regulation

PubMed Central

Lee, Myoung-Hwa; Amin, Niranjana D.; Venkatesan, Arun; Wang, Tongguang; Tyagi, Richa; Pant, Harish C.; Nath, Avindra

2013-01-01

Human immunodeficiency virus (HIV) infection associated neurocognitive disorders (HAND) is accompanied with brain atrophy. In these patients, impairment of adult neurogenesis and neurite outgrowth in the hippocampus may contribute to the cognitive dysfunction. Although running exercises can enhance neurogenesis and normalize neurite outgrowth, the underlying molecular mechanisms are not well understood. The HIV envelope protein, gp120, has been shown to impair neurogenesis. Using a gp120 transgenic mouse model, we demonstrate that exercise stimulated neural progenitor cell (NPC) proliferation in the hippocampal dentate gyrus and increased the survival rate and generation of newborn cells. However sustained exercise activity was necessary since the effects were reversed by detraining. Exercise also normalized dendritic outgrowth of neurons. Furthermore, it also increased the expression of hippocampal brainderived neurotrophic factor (BDNF) and normalized hyperactivation of cyclin-dependent kinase 5 (Cdk5). Hyper-activated Cdk5 or gp120 treatment led to aberrant neurite outgrowth and BDNF treatment normalized the neurite outgrowth in NPC cultures. These results suggest that sustained exercise has trophic activity on the neuronal lineage which is mediated by Cdk5 modulation of the BDNF pathway. PMID:23982957

Loss of Cytoplasmic CDK1 Predicts Poor Survival in Human Lung Cancer and Confers Chemotherapeutic Resistance

PubMed Central

Zhang, Chunyu; Elkahloun, Abdel G.; Robertson, Matthew; Gills, Joell J.; Tsurutani, Junji; Shih, Joanna H.; Fukuoka, Junya; Hollander, M. Christine; Harris, Curtis C.; Travis, William D.; Jen, Jin; Dennis, Phillip A.

2011-01-01

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery. PMID:21887332

Alternol induces an S-phase arrest of melanoma B16F0 cells.

PubMed

Liu, Liangliang; Zhang, Bo; Yuan, Xuan; Wang, Penglong; Sun, Xiling; Zheng, Qiusheng

2014-03-01

Alternol is a novel compound purified from the fermentation products of a microorganism in the yew tree bark. This study looks at the effects of alternol on the proliferation and cell cycle distribution of mouse melanoma cells. The inhibition of cell proliferation and changes in cell cycle distribution were analysed by sulforhodamine B and flow cytometry assays, respectively. mRNA expression of cyclin A, cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) and CDK inhibitor1A (p21) were measured by real-time reverse transcription PCR (RT-PCR). The protein levels of cyclin A, CDK2 and PCNA were analysed by Western blot analysis. p21 was measured by ELISA. Alternol treatment caused a significant decrease in the proliferation rate of B16F0 and B16F10 cells, which were significantly arrested in S phase, but this treatment had less effect on normal human embryonic kidney 293T cells. The mechanism by which alternol inhibits B16F0 proliferation in vitro may be associated with the inhibition of CDK2 and PCNA, and the activation of p21. © 2013 International Federation for Cell Biology.

Ablation of cdk4 and cdk6 affects proliferation of basal progenitor cells in the developing dorsal and ventral forebrain.

PubMed

Grison, Alice; Gaiser, Carine; Bieder, Andrea; Baranek, Constanze; Atanasoski, Suzana

2018-03-23

Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin-dependent kinase (cdk) family using cdk-deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018. © 2018 Wiley Periodicals, Inc.

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.

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase

PubMed Central

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2015-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT. PMID:26496927

Establishment of an immortalized cell line derived from the prairie vole via lentivirus-mediated transduction of mutant cyclin-dependent kinase 4, cyclin D, and telomerase reverse transcriptase.

PubMed

Katayama, Masafumi; Kiyono, Tohru; Horie, Kengo; Hirayama, Takashi; Eitsuka, Takahiro; Kuroda, Kengo; Donai, Kenichiro; Hidema, Shizu; Nishimori, Katsuhiko; Fukuda, Tomokazu

2016-01-01

The prairie vole (Microtus ochrogaster) shows social behaviors such as monogamy and parenting of infants with pair bonding. These social behaviors are specific to the prairie vole and have not been observed in other types of voles, such as mountain voles. Although the prairie vole has several unique characteristics, an in vitro cell culture system has not been established for this species. Furthermore, establishment of cultured cells derived from the prairie vole may be beneficial based on the three Rs (i.e., Replacement, Reduction, and Refinement) concept. Therefore, in this study, we attempted to establish an immortalized cell line derived from the prairie vole. Our previous research has shown that transduction with mutant forms of cyclin-dependent kinase 4 (CDK4), cyclin D, and telomerase reverse transcriptase (TERT) could efficiently immortalize cells from multiple species, including humans, cattle, pigs, and monkeys. Here, we introduced these three genes into prairie vole-derived muscle fibroblasts. The expression of mutant CDK4 and cyclin D proteins was confirmed by western blotting, and telomerase activity was detected in immortalized vole muscle-derived fibroblasts (VMF-K4DT cells or VMFs) by stretch PCR. Population doubling analysis showed that the introduction of mutant CDK4, cyclin D, and TERT extended the lifespan of VMFs. To the best of our knowledge, this is the first report describing the establishment of an immortalized cell line derived from the prairie vole through the expression of mutant CDK4, cyclin D, and human TERT.

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

PubMed

Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A; Liu, Kangdong; Kim, Jiyoung; Lim, Soon Sung; Park, Jung Han Yoon; Dong, Zigang; Lee, Ki Won; Lee, Hyong Joo

2013-10-01

Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27(kip1). The elevated p27(kip1) expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. Copyright © 2013 Elsevier Inc. All rights reserved.

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

MicroRNA-188 suppresses G1/S transition by targeting multiple cyclin/CDK complexes.

PubMed

Wu, Jiangbin; Lv, Qing; He, Jie; Zhang, Haoxiang; Mei, Xueshuang; Cui, Kai; Huang, Nunu; Xie, Weidong; Xu, Naihan; Zhang, Yaou

2014-10-11

Accelerated cell cycle progression is the common feature of most cancers. MiRNAs can act as oncogenes or tumor suppressors by directly modulating cell cycle machinery. It has been shown that miR-188 is upregulated in UVB-irradiated mouse skin and human nasopharyngeal carcinoma CNE cells under hypoxic stress. However, little is known about the function of miR-188 in cell proliferation and growth control. Overexpression of miR-188 inhibits cell proliferation, tumor colony formation and G1/S cell cycle transition in human nasopharyngeal carcinoma CNE cells. Using bioinformatics approach, we identify a series of genes regulating G1/S transition as putative miR-188 targets. MiR-188 inhibits both mRNA and protein expression of CCND1, CCND3, CCNE1, CCNA2, CDK4 and CDK2, suppresses Rb phosphorylation and downregulates E2F transcriptional activity. The expression level of miR-188 also inversely correlates with the expression of miR-188 targets in human nasopharyngeal carcinoma (NPC) tissues. Moreover, studies in xenograft mouse model reveal that miR-188 is capable of inhibiting tumor initiation and progression by suppressing target genes expression and Rb phosphorylation. This study demonstrates that miR-188 exerts anticancer effects, via downregulation of multiple G1/S related cyclin/CDKs and Rb/E2F signaling pathway.

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

[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.

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).

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

Protopine, a novel microtubule-stabilizing agent, causes mitotic arrest and apoptotic cell death in human hormone-refractory prostate cancer cell lines.

PubMed

Chen, Chun-Han; Liao, Cho-Hwa; Chang, Ya-Ling; Guh, Jih-Hwa; Pan, Shiow-Lin; Teng, Che-Ming

2012-02-01

In this study, we investigated the anticancer effect of protopine on human hormone-refractory prostate cancer (HRPC) cells. Protopine exhibited an anti-proliferative effect by induction of tubulin polymerization and mitotic arrest, which ultimately led to apoptotic cell death. The data suggest that protopine increased the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex and that contributed to cell apoptosis by modulating mitochondria-mediated signaling pathways, such as Bcl-2 phosphorylation and Mcl-1 down-regulation. In conclusion, the data suggest that protopine is a novel microtubule stabilizer with anticancer activity in HRPC cells through apoptotic pathway by modulating Cdk1 activity and Bcl-2 family of proteins. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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.

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.

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.

Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

PubMed

Lin, Shuw-Yuan; Lai, Wan-Wen; Chou, Chi-Chung; Kuo, Hsiu-Maan; Li, Te-Mao; Chung, Jing-Gung; Yang, Jen-Hung

2006-12-01

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

A Transcriptome-based Perspective of Cell Cycle Regulation in Dinoflagellates.

PubMed

Morse, David; Daoust, Philip; Benribague, Siham

2016-12-01

Dinoflagellates are a group of unicellular and generally marine protists, of interest to many because of their ability to form the large algal blooms commonly called "red tides". The large algal concentrations in these blooms require sustained cell replication, yet to date little is known about cell cycle regulation in these organisms. To address this issue, we have screened the transcriptomes of two dinoflagellates, Lingulodinium polyedrum and Symbiodinium sp., with budding yeast cell cycle pathway components. We find most yeast cell cycle regulators have homologs in these dinoflagellates, suggesting that the yeast model is appropriate for understanding regulation of the dinoflagellate cell cycle. The dinoflagellates are lacking several components essential in yeast, but a comparison with a broader phylogenetic range of protists reveals these components are usually also missing in other organisms. Lastly, phylogenetic analyses show that the dinoflagellates contain at least three cyclin-dependent kinase (CDK) homologs (belonging to the CDK1, CDK5 and CDK8 families), and that the dinoflagellate cyclins belong exclusively to the A/B type. This suggests that dinoflagellate CDKs likely play a limited role outside regulation of the cell cycle. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

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.

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

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

PubMed Central

Fu, Amy KY

2007-01-01

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development. PMID:19270534

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.

The MNS glycophorin variant GP.Mur affects differential erythroid expression of Rh/RhAG transcripts.

PubMed

Hsu, K; Kuo, M-S; Yao, C-C; Cheng, H-C; Lin, H-J; Chan, Y-S; Lin, M

2017-10-01

The band 3 macrocomplex (also known as the ankyrin-associated complex) on the red cell membrane comprises two interacting subcomplexes: a band 3/glycophorin A subcomplex, and a Rh/RhAG subcomplex. Glycophorin B (GPB) is a component of the Rh/RhAG subcomplex that is also structurally associated with glycophorin A (GPA). Expression of glycophorin B-A-B hybrid GP.Mur enhances band 3 expression and is associated with lower levels of Rh-associated glycoprotein (RhAG) and Rh polypeptides. The goal of this study was to determine whether GP.Mur influenced erythroid Rh/RhAG expression at the transcript level. GP.Mur was serologically determined in healthy participants from Taitung County, Taiwan. RNA was extracted from the reticulocyte-enriched fraction of peripheral blood, followed by reverse transcription and quantitative PCR for RhAG, RhD and RhCcEe. Quantification by real-time PCR revealed significantly fewer RhAG and RhCcEe transcripts in the reticulocytes from subjects with homozygous GYP*Mur. Independent from GYP.Mur, both RhAG and RhD transcript levels were threefold or higher than that of RhCcEe. Also, in GYP.Mur and the control samples alike, direct quantitative associations were observed between the transcript levels of RhAG and RhD, but not between that of RhAG and RhCcEe. Erythroid RhD and RhCcEe were differentially expressed at the transcript levels, which could be related to their different degrees of interaction or sensitivity to RhAG. Further, the reduction or absence of glycophorin B in GYP.Mur erythroid cells affected transcript expressions of RhAG and RhCcEe. Thus, GPB and GP.Mur differentially influenced Rh/RhAG expressions prior to protein translation. © 2017 International Society of Blood Transfusion.

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.

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia, Is Essential for Oligodendroglia Differentiation and Myelin Repair*

PubMed Central

Bankston, Andrew N.; Li, Wenqi; Zhang, Hui; Ku, Li; Liu, Guanglu; Papa, Filomena; Zhao, Lixia; Bibb, James A.; Cambi, Franca; Tiwari-Woodruff, Seema K.; Feng, Yue

2013-01-01

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair. PMID:23645679

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

PubMed

Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K

2015-11-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics

PubMed Central

Corsino, Patrick E.; Narayan, Satya

2015-01-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non–ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non–ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. PMID:26018905

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.

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.

Testing of SNS-032 in a Panel of Human Neuroblastoma Cell Lines with Acquired Resistance to a Broad Range of Drugs12

PubMed Central

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

2013-01-01

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-3rCDDP1000 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. PMID:24466371

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.

Nitric oxide-induced cytostasis and cell cycle arrest of a human breast cancer cell line (MDA-MB-231): Potential role of cyclin D1

PubMed Central

Pervin, Shehla; Singh, Rajan; Chaudhuri, Gautam

2001-01-01

DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 μM of NO over a 24-h period. Synchronized population of the cells exposed to DETA-NONOate remained arrested at the G1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G1 phase of the cell cycle. PMID:11248121

Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

PubMed

Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

2015-05-07

The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

PubMed

Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

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

Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae.

PubMed

Shahi, Puja; Gulshan, Kailash; Näär, Anders M; Moye-Rowley, W Scott

2010-07-15

The multiprotein transcriptional Mediator complex provides a key link between RNA polymerase II and upstream transcriptional activator proteins. Previous work has established that the multidrug resistance transcription factors Pdr1 and Pdr3 interact with the Mediator component Med15/Gal11 to drive normal levels of expression of the ATP-binding cassette transporter-encoding gene PDR5 in Saccharomyces cerevisiae. PDR5 transcription is induced upon loss of the mitochondrial genome (rho(0) cells) and here we provide evidence that this rho(0) induction is Med15 independent. A search through other known Mediator components determined that Med12/Srb8, a member of the CDK8 Mediator submodule, is required for rho(0) activation of PDR5 transcription. The CDK8 submodule contains the cyclin C homologue (CycC/Srb11), cyclin-dependent kinase Cdk8/Srb10, and the large Med13/Srb9 protein. Loss of these other proteins did not lead to the same block in PDR5 induction. Chromatin immunoprecipitation analyses demonstrated that Med15 is associated with the PDR5 promoter in both rho(+) and rho(0), whereas Med12 recruitment to this target promoter is highly responsive to loss of the mitochondrial genome. Coimmunoprecipitation experiments revealed that association of Pdr3 with Med12 can only be detected in rho(0) cells. These experiments uncover the unique importance of Med12 in activated transcription of PDR5 seen in rho(0) cells.

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.

Super elongation complex contains a TFIIF-related subcomplex

PubMed Central

Knutson, Bruce A.; Smith, Marissa L.; Walker-Kopp, Nancy; Xu, Xia

2016-01-01

ABSTRACT Super elongation complex (SEC) belongs to a family of RNA polymerase II (Pol II) elongation factors that has similar properties as TFIIF, a general transcription factor that increases the transcription elongation rate by reducing pausing. Although SEC has TFIIF-like functional properties, it apparently lacks sequence and structural homology. Using HHpred, we find that SEC contains an evolutionarily related TFIIF-like subcomplex. We show that the SEC subunit ELL interacts with the Pol II Rbp2 subunit, as expected for a TFIIF-like factor. These findings suggest a new model for how SEC functions as a Pol II elongation factor and how it suppresses Pol II pausing. PMID:27223670

Palbociclib in Hormone-Receptor-Positive Advanced Breast Cancer.

PubMed

Turner, Nicholas C; Ro, Jungsil; André, Fabrice; Loi, Sherene; Verma, Sunil; Iwata, Hiroji; Harbeck, Nadia; Loibl, Sibylle; Huang Bartlett, Cynthia; Zhang, Ke; Giorgetti, Carla; Randolph, Sophia; Koehler, Maria; Cristofanilli, Massimo

2015-07-16

Growth of hormone-receptor-positive breast cancer is dependent on cyclin-dependent kinases 4 and 6 (CDK4 and CDK6), which promote progression from the G1 phase to the S phase of the cell cycle. We assessed the efficacy of palbociclib (an inhibitor of CDK4 and CDK6) and fulvestrant in advanced breast cancer. This phase 3 study involved 521 patients with advanced hormone-receptor-positive, human epidermal growth factor receptor 2-negative breast cancer that had relapsed or progressed during prior endocrine therapy. We randomly assigned patients in a 2:1 ratio to receive palbociclib and fulvestrant or placebo and fulvestrant. Premenopausal or perimenopausal women also received goserelin. The primary end point was investigator-assessed progression-free survival. Secondary end points included overall survival, objective response, rate of clinical benefit, patient-reported outcomes, and safety. A preplanned interim analysis was performed by an independent data and safety monitoring committee after 195 events of disease progression or death had occurred. The median progression-free survival was 9.2 months (95% confidence interval [CI], 7.5 to not estimable) with palbociclib-fulvestrant and 3.8 months (95% CI, 3.5 to 5.5) with placebo-fulvestrant (hazard ratio for disease progression or death, 0.42; 95% CI, 0.32 to 0.56; P<0.001). The most common grade 3 or 4 adverse events in the palbociclib-fulvestrant group were neutropenia (62.0%, vs. 0.6% in the placebo-fulvestrant group), leukopenia (25.2% vs. 0.6%), anemia (2.6% vs. 1.7%), thrombocytopenia (2.3% vs. 0%), and fatigue (2.0% vs. 1.2%). Febrile neutropenia was reported in 0.6% of palbociclib-treated patients and 0.6% of placebo-treated patients. The rate of discontinuation due to adverse events was 2.6% with palbociclib and 1.7% with placebo. Among patients with hormone-receptor-positive metastatic breast cancer who had progression of disease during prior endocrine therapy, palbociclib combined with fulvestrant resulted in longer progression-free survival than fulvestrant alone. (Funded by Pfizer; PALOMA3 ClinicalTrials.gov number, NCT01942135.).

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

A map of protein dynamics during cell-cycle progression and cell-cycle exit

PubMed Central

Gookin, Sara; Min, Mingwei; Phadke, Harsha; Chung, Mingyu; Moser, Justin; Miller, Iain; Carter, Dylan

2017-01-01

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. PMID:28892491

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.

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.

MicroRNA-320c inhibits tumorous behaviors of bladder cancer by targeting Cyclin-dependent kinase 6

PubMed Central

2014-01-01

Background Increasing evidence has suggested that dysregulation of microRNAs (miRNAs) could contribute to human disease including cancer. Previous miRNA microarray analysis illustrated that miR-320c is down-regulated in various cancers. However, the roles of miR-320c in human bladder cancer have not been well elucidated. Therefore, this study was performed to investigate the biological functions and molecular mechanisms of miR-320c in human bladder cancer cell lines, discussing whether it could be a therapeutic biomarker of bladder cancer in the future. Methods Two human bladder cancer cell lines and samples from thirteen patients with bladder cancer were analyzed for the expression of miR-320c by quantitative RT-PCR. Over-expression of miR-320c was established by transfecting mimics into T24 and UM-UC-3. Cell proliferation and cell cycle were assessed by cell viability assay, flow cytometry and colony formation assay. Cell motility ability was evaluated by transwell assay. The target gene of miR-320c was determined by luciferase assay, quantitative RT-PCR and western blot. The regulation of cell cycle and mobility by miR-320c was analyzed by western blot. Results We observed that miR-320c was down-regulated in human bladder cancer tissues and bladder cancer cell lines T24 and UM-UC-3. Over-expression of miR-320c could induce G1 phase arrest in UM-UC-3 and T24 cells, and subsequently inhibited cell growth. We also indentified miR-320c could impair UM-UC-3 and T24 cell motility. In addition, we identified CDK6, a cell cycle regulator, as a novel target of miR-320c. Moreover, we demonstrated miR-320c could induce bladder cancer cell cycle arrest and mobility via regulating CDK6. We also observed that inhibition of miR-320c or restoration of CDK6 in miR-320c-over-expressed bladder cancer cells partly reversed the suppressive effects of miR-320c. Conclusions miR-320c could inhibit the proliferation, migration and invasion of bladder cancer cells via regulating CDK6. Our study revealed that miR-320c could be a therapeutic biomarker of bladder cancer in the future. PMID:25178497

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.

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

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

Influence of chocolate matrix composition on cocoa flavan-3-ol bioaccessibility in vitro and bioavailability in humans.

PubMed

Neilson, Andrew P; George, Judy C; Janle, Elsa M; Mattes, Richard D; Rudolph, Ralf; Matusheski, Nathan V; Ferruzzi, Mario G

2009-10-28

Conflicting data exist regarding the influence of chocolate matrices on the bioavailability of epicatechin (EC) from cocoa. The objective of this study was to assess the bioavailability of EC from matrices varying in macronutrient composition and physical form. EC bioavailability was assessed from chocolate confections [reference dark chocolate (CDK), high sucrose (CHS), high milk protein (CMP)] and cocoa beverages [sucrose milk protein (BSMP), non-nutritive sweetener milk protein (BNMP)], in humans and in vitro. Six subjects consumed each product in a randomized crossover design, with serum EC concentrations monitored over 6 h post consumption. Areas under the serum concentration-time curve (AUC) were similar among chocolate matrices. However, AUCs were significantly increased for BSMP and BNMP (132 and 143 nM h) versus CMP (101 nM h). Peak serum concentrations (C(MAX)) were also increased for BSMP and BNMP (43 and 42 nM) compared to CDK and CMP (32 and 25 nM). Mean T(MAX) values were lower, although not statistically different, for beverages (0.9-1.1 h) versus confections (1.8-2.3 h), reflecting distinct shapes of the pharmacokinetic curves for beverages and confections. In vitro bioaccessibility and Caco-2 accumulation did not differ between treatments. These data suggest that bioavailability of cocoa flavan-3-ols is likely similar from typical commercial cocoa based foods and beverages, but that the physical form and sucrose content may influence T(MAX) and C(MAX).

Role of Cyclin E as an Early Event in Ovarian Carcinogenesis

DTIC Science & Technology

2012-04-01

degradation . P27 is a powerful negative regulator of the cell cycle, preventing activation of cyclin E- cdk2 or cyclin D-cdk4 complexes and cell cycle...Ahmed M, Bavi P, et al. Bortezomib (Velcade) induces p27Kip1 expression through S-phase kinase protein 2 degradation in colorectal cancer. Cancer Res...CA1251CA72·4 CA 125 CA72-4 M-CSF CA 125/CA 72-4/M-CSFICA 15-3 CA125 CA 125/mesothelin CA 125/IL·6JIL·8NEGF;EGF CA 125/IL-6,G-CSFNEGF/EGF Leptin

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

Problem-Solving Test: The Mechanism of Action of a Human Papilloma Virus Oncoprotein

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: human papilloma virus; cervical cancer; oncoproteins; malignant transformation; retinoblastoma protein; cell cycle; quiescent and cycling cells; cyclin/cyclin-dependent kinase (Cdk) complexes; E2F; S-phase genes; enhancer element; proto-oncogenes; tumor suppressor genes; radioactive…

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

Schisantherin A Improves Learning and Memory of Mice with D-Galactose-Induced Learning and Memory Impairment through Its Antioxidation and Regulation of p19/p53/p21/Cyclin D1/CDK4/RB Gene Expressions.

PubMed

Liu, Cong; Sun, Weijing; Li, Ning; Gao, Jiaqi; Yu, Chunyan; Wang, Chunmei; Sun, Jinghui; Jing, Shu; Chen, Jianguang; Li, He

2018-05-31

Schisantherin A (SCA) was evaluated for possible function in restoring the learning and memory impairment induced by D-galactose in mice. ICR mice were treated with D-galactose subcutaneously (220 mg·kg -1 ), and followed by SCA in different doses (1.25, 2.50 and 5.00 mg·kg -1 , administered orally) for 42 days. Effects of SCA on learning and memory were examined by step-through tests and Morris water maze tests. The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) in the peripheral blood and hippocampus of mice were assayed by water-soluble tetrazolium-1 (WST-1) and thiobarbituric acid (TBA) methods. The contents of 8 hydroxy deoxy guanosine (8-OHdG) in the hippocampus of mice were detected by immunosorbent assay methods, respectively. Quantitative real-time PCR and Western Blot were respectively used to detect the expression of p19, p53, p21, cyclin D1, CDK4 and RB genes, and the phosphorylation of RB in the hippocampus of mice. We found that SCA significantly improved the learning and memory impairment induced by D-galactose in mice. After SCA treatment, SOD activity was increased and the content of MDA was decreased in both peripheral blood and hippocampus of mice. 8-OHDG content was also decreased in the hippocampus of mice. Furthermore, the expression of p19, p53 and p21 genes was reduced and the expression of cyclin D1 and CDK4 and the phosphorylation of RB protein were elevated in the hippocampus. SCA may improve the learning and memory impairment induced by D-galactose by enhancing the antioxidant capacity, and regulating the expression of p19/p53/p21/cyclinD1/CDK4 genes, and the phosphorylation of RB protein in the hippocampus of mice.

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.

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

The O-GlcNAc Modification of CDK5 Involved in Neuronal Apoptosis Following In Vitro Intracerebral Hemorrhage.

PubMed

Ning, Xiaojin; Tao, Tao; Shen, Jianhong; Ji, Yuteng; Xie, Lili; Wang, Hongmei; Liu, Ning; Xu, Xide; Sun, Chi; Zhang, Dongmei; Shen, Aiguo; Ke, Kaifu

2017-04-01

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in regulating mammalian CNS development. Studies of CDK5 have focused on its phosphorylation, although the diversity of CDK5 functions in the brain suggests additional forms of regulation. Here we expanded on the functional roles of CDK5 glycosylation in neurons. We showed that CDK5 was dynamically modified with O-GlcNAc in response to neuronal activity and that glycosylation represses CDK5-dependent apoptosis by impairing its association with p53 pathway. Blocking glycosylation of CDK5 alters cellular function and increases neuronal apoptosis in the cell model of the ICH. Our findings demonstrated a new role for O-glycosylation in neuronal apoptosis and provided a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identified a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and apoptosis.

Ribociclib plus letrozole in early breast cancer: A presurgical, window-of-opportunity study.

PubMed

Curigliano, G; Gómez Pardo, P; Meric-Bernstam, F; Conte, P; Lolkema, M P; Beck, J T; Bardia, A; Martínez García, M; Penault-Llorca, F; Dhuria, S; Tang, Z; Solovieff, N; Miller, M; Di Tomaso, E; Hurvitz, S A

2016-08-01

Cyclin D-cyclin-dependent kinase (CDK) 4/6-inhibitor of CDK4/6-retinoblastoma (Rb) pathway hyperactivation is associated with hormone receptor-positive (HR+) breast cancer (BC). This study assessed the biological activity of ribociclib (LEE011; CDK4/6 inhibitor) plus letrozole compared with single-agent letrozole in the presurgical setting. Postmenopausal women (N = 14) with resectable, HR+, human epidermal growth factor receptor 2-negative (HER2-) early BC were randomized 1:1:1 to receive 2.5 mg/day letrozole alone (Arm 1), or with 400 or 600 mg/day ribociclib (Arm 2 or 3). Circulating tumor DNA and tumor biopsies were collected at baseline and, following 14 days of treatment, prior to or during surgery. The primary objective was to assess antiproliferative response per Ki67 levels in Arms 2 and 3 compared with Arm 1. Additional assessments included safety, pharmacokinetics, and genetic profiling. Mean decreases in the Ki67-positive cell fraction from baseline were: Arm 1 69% (range 38-100%; n = 2), Arm 2 96% (range 78-100%; n = 6), Arm 3 92% (range 75-100%; n = 3). Decreased phosphorylated Rb levels and CDK4, CDK6, CCND2, CCND3, and CCNE1 gene expression were observed following ribociclib treatment. Ribociclib and letrozole pharmacokinetic parameters were consistent with single-agent data. The ribociclib plus letrozole combination was well tolerated, with no Grade 3/4 adverse events over the treatment. The results suggest absence of a drug-drug interaction between ribociclib and letrozole and indicate ribociclib plus letrozole may reduce Ki67 expression in HR+, HER2- BC (NCT01919229). Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

Tax-dependent stimulation of G1 phase-specific cyclin-dependent kinases and increased expression of signal transduction genes characterize HTLV type 1-transformed T cells.

PubMed

Haller, K; Ruckes, T; Schmitt, I; Saul, D; Derow, E; Grassmann, R

2000-11-01

Human T cell leukemia virus protein induces T cells to permanent IL-2-dependent growth. These cells occasionally convert to factor independence. The viral oncoprotein Tax acts as an essential growth factor of transformed lymphocytes and stimulates the cell cycle in the G(1) phase. In T cells and fibroblasts Tax enhances the activity of the cyclin-dependent kinases (CDK) CDK4 and CDK6. These kinases, which require binding to cyclin D isotypes for their activity, control the G(1) phase. Coimmunoprecipitation from these cells revealed that Tax associates with cyclin D3/CDK6, suggesting a direct activation of this kinase. The CDK stimulation may account in part for the mitogenic Tax effect, which causes IL-2-dependent T cell growth by Tax. To address the conversion to IL-2-independent proliferation and to identify overexpressed genes, which contribute to the transformed growth, the gene expression patterns of HTLV-1-transformed T cells were compared with that of peripheral blood lymphocytes. Potentially overexpressed cDNAs were cloned, sequenced, and used to determine the RNA expression. Genes found to be up-regulated are involved in signal transduction (STAT5a, cyclin G(1), c-fgr, hPGT) and also glycoprotein synthesis (LDLC, ribophorin). Many of these are also activated during T cell activation and implicated in the regulation of growth and apoptosis. The transcription factor STAT5a, which is involved in IL-2 signaling, was strongly up-regulated only in IL-2-independent cells, thus suggesting that it contributes to factor-independent growth. Thus, the differentially expressed genes could cooperate with the Tax-induced cell cycle stimulation in the maintenance of IL-2-dependent and IL-2-independent growth of HTLV-transformed lymphocytes.

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.

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.

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells.

PubMed

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-02

Previously, we analyzed protein abundance changes across a 'minimally perturbed' cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex.

PubMed

Echalier, Aude; Bettayeb, Karima; Ferandin, Yoan; Lozach, Olivier; Clément, Monique; Valette, Annie; Liger, François; Marquet, Bernard; Morris, Jonathan C; Endicott, Jane A; Joseph, Benoît; Meijer, Laurent

2008-02-28

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

Identification of terpenoids from Rubus corchorifolius L. f. leaves and their anti-proliferative effects on human cancer cells.

PubMed

Chen, Xuexiang; Gao, Zili; Song, Mingyue; Ouyang, Wen; Wu, Xian; Chen, Yunjiao; Zhou, Liping; William, Dixon; Cai, Xiaokun; Cao, Yong; Zhou, Shuangde; Tang, Zhonghai; Xiao, Hang

2017-03-22

The leaves of Rubus corchorifolius L. f. have been consumed as a herbal tea for a long time. In this study, two novel (1 and 5) and four known (2, 3, 4 and 6) terpenoids were isolated from the leaves of Rubus corchorifolius L. f. Structural analysis was performed using various spectroscopic methods ( 1 H NMR, 13 C NMR and MS) to identify the following six compounds: (16α)-16,17,18-trihydroxy-ent-kauran-18-O-β-d-glucoside (1), ent-16β,17-dialkyl-3-oxygen-kaurane (2), ent-kaurane-3α,16β,17-triol (3), ent-kaurane(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol (4), (16R)-16β,17,19-trihydroxy-ent-kaur-3-one (5) and ent-16α,17-dihydroxy-kauran-19-oic-acid (6). These compounds showed different inhibitory effects on various human cancer cells. Compounds 3 and 6 exhibited stronger inhibitory effects on human colon cancer HCT116 cells than the other 4 compounds. Flow cytometry analysis demonstrated that both compounds 3 and 6 caused cell cycle arrest at the G0/G1 phase and induced cellular apoptosis in HCT116 cells. Compounds 3 and 6 modulated the expression levels of key signaling proteins closely related to cell proliferation and apoptosis, i.e., increasing the levels of poly(ADP-ribose) polymerase, p53, and p27, and decreasing the levels of EGFR, cyclin D1, CDK2 and CDK4. Overall, our findings provided insight into the anticancer components of Rubus corchorifolius L. f. leaves, which could facilitate their utilization as functional food ingredients.

Molecular mechanisms of celery seed extract induced apoptosis via s phase cell cycle arrest in the BGC-823 human stomach cancer cell line.

PubMed

Gao, Lin-Lin; Feng, Lei; Yao, Shu-Tong; Jiao, Peng; Qin, Shu-Cun; Zhang, Wei; Zhang, Ya-Bin; Li, Fu-Rong

2011-01-01

Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.

Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

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

Gu, Jun; Department of Orthopaedics, Xishan People's Hospital, Wuxi, Jiangsu; Ni, Yingjie

2016-01-22

Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in themore » spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.« less

MicroRNA-26a is a key regulon that inhibits progression and metastasis of c-Myc/EZH2 double high advanced hepatocellular carcinoma.

PubMed

Zhang, Xiang; Zhang, Xiao; Wang, Ting; Wang, Lei; Tan, Zhijun; Wei, Wei; Yan, Bo; Zhao, Jing; Wu, Kaichun; Yang, Angang; Zhang, Rui; Jia, Lintao

2018-07-10

The transcription factor c-Myc is a key driver for hepatocellular carcinomas (HCCs), while the polycombrepressive complex 2 (PRC2) subunit EZH2 is an essential biomarker of HCC. c-Myc epigenetically silences tumor suppressors by recruiting PRC2 and inducing methylation of histone H3 lysine 27. However, it remains elusive how they are regulated in HCC. We found here that microRNA-26a (miR-26a) suppresses c-Myc, a classical Wnt pathway target gene, by targeting the Wnt pathway coactivator, cyclin-dependent kinase 8 (CDK8); miR-26a also directly targets and inhibits EZH2. The expression of MIR26A2, a predominant origin of miR-26a transcripts in hepatic cells, is repressed by c-Myc/PRC2, thereby forming a c-Myc/miR-26a/CDK8 regulatory circuit in HCC. Meanwhile, miR-26a suppresses migration of HCC by targeting p21-activated kinase 2 (PAK2), a critical kinase linking Rho GTPases to cytoskeleton reorganization. Consequently, in vivo delivery of miR-26a remarkably suppressed the development of xenograft HCC and metastasis of orthotopic HCC by downregulating c-Myc, CDK8 and PAK2. These findings unraveled a novel mechanism of c-Myc and Wnt/β-catenin interplay that dictates HCC pathogenesis, and have implications for the potential applicability of miRNA delivery in targeting the newly identified signaling axis and treating metastatic HCCs. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Mammary Tumors Initiated by Constitutive Cdk2 Activation Contain an Invasive Basal-like Component1

PubMed Central

Corsino, Patrick E; Davis, Bradley J; Nörgaard, Peter H; Teoh Parker, Nicole N; Law, Mary; Dunn, William; Law, Brian K

2008-01-01

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, α smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and β-catenin and p120ctn localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, β-catenin, and p120ctn to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers. PMID:18953433

Xanthorrhizol, a natural sesquiterpenoid, induces apoptosis and growth arrest in HCT116 human colon cancer cells.

PubMed

Kang, You-Jin; Park, Kwang-Kyun; Chung, Won-Yoon; Hwang, Jae-Kwan; Lee, Sang Kook

2009-11-01

Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. In our previous studies, xanthorrhizol suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, inhibited cancer cell growth, and exerted an anti-metastatic effect in an animal model. However, the exact mechanisms for its inhibitory effects against cancer cell growth have not yet been fully elucidated. In the present study, we investigated the growth inhibitory effect of xanthorrhizol on cancer cells. Xanthorrhizol dose-dependently exerted antiproliferative effects against HCT116 human colon cancer cells. Xanthorrhizol also arrested cell cycle progression in the G0/G1 and G2/M phase and induced the increase of sub-G1 peaks. Cell cycle arrest was highly correlated with the downregulation of cyclin A, cyclin B1, and cyclin D1; cyclin-dependent kinase 1 (CDK1), CDK2, and CDK4; proliferating cell nuclear antigen; and inductions of p21 and p27, cyclin-dependent kinase inhibitors. The apoptosis by xanthorrhizol was markedly evidenced by induction of DNA fragmentation, release of cytochrome c, activation of caspases, and cleavage of poly-(ADP-ribose) polymerase. In addition, xanthorrhizol increased the expression and promoter activity of pro-apoptotic non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). These findings provide one plausible mechanism for the growth inhibitory activity of xanthorrhizol against cancer cells.

Cueva Antón: A multi-proxy MIS 3 to MIS 5a paleoenvironmental record for SE Iberia

NASA Astrophysics Data System (ADS)

Zilhão, João; Ajas, Aurélie; Badal, Ernestina; Burow, Christoph; Kehl, Martin; López-Sáez, José Antonio; Pimenta, Carlos; Preece, Richard C.; Sanchis, Alfred; Sanz, Montserrat; Weniger, Gerd-Christian; White, Dustin; Wood, Rachel; Angelucci, Diego E.; Villaverde, Valentín; Zapata, Josefina

2016-08-01

Overlying a palustrine deposit of unknown age (complex FP), and protected from weathering and erosion inside a large cave/rock-shelter cavity, the sedimentary fill of Cueva Antón, a Middle Paleolithic site in SE Spain, corresponds in most part (sub-complexes AS2-to-AS5) to a ca.3 m-thick Upper Pleistocene terrace of the River Mula. Coupled with the constraints derived from the deposit's paleoclimatic proxies, OSL dating places the accumulation of this terrace in MIS 5a, and radiocarbon dates from the overlying breccia cum alluvium (sub-complex AS1) fall in the middle part of MIS 3; the intervening hiatus relates to valley incision and attendant erosion. The two intervals represented remain largely unknown in Iberia, where the archeology of the early-to-middle Upper Pleistocene is almost entirely derived from karst sites; Cueva Antón shows that this dearth of data, often interpreted in demographic terms, has depositional underpinnings ultimately determined by past climate variation. In early MIS 5a, the paleobotanical evidence indicates climate conditions similar to present, albeit wetter, followed by progressive cooling, reflected in the replacement of Aleppo pine by black pine and, at the very end, juniper-dominated landscapes - the latter characterizing also mid-MIS 3 times. The variation in sedimentary facies and composition of the mollusk assemblages reflects the changing position of the river channel relative to the back wall of the cave. Such changes represented the major constraint for the occupation of the site - most of the time inaccessible to terrestrial mammals, it was used throughout by the eagle-owl, explaining the abundance of rabbit bones. Human occupation occurred during a few, short windows of availability, and is reflected in well-preserved living floors defined by hearths, artefact scatters, and the remains of hunted herbivores. The stone tool assemblages are Middle Paleolithic, which, in Europe, implies a Neandertal identity for their makers and, hence, that Neandertals persisted in the region until GI 8. Cueva Antón's high-resolution record provides unique, critical information on the paleoenvironments and adaptations of humans in two short windows of time during which wetter conditions existed in SE Iberia, where arid or semi-arid climates prevailed through most of the Upper Pleistocene and the Holocene.

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.

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.

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.

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.

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

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.

Breast cancer: updates and advances in 2016.

PubMed

Giordano, Sara B; Gradishar, William

2017-02-01

Approximately 1 in 8 US women (12%) will develop invasive breast cancer over the course of her lifetime. In 2016, an estimated 246,660 new cases of invasive breast cancer are expected to be diagnosed and approximately 40,450 would die as a result of it. The global burden of breast cancer exceeds all other cancers and the incidence is increasing. The heterogeneity of breast cancer makes it a challenging solid tumor to diagnose and treat. This review focuses on the recent advances in breast cancer therapy including hormonal treatment of metastatic breast cancer, targeting cyclin-dependent kinases (CDK) 4/6 in breast cancer, updates in targeting human epidermal growth factor receptor 2 (HER2) positive breast cancer, adaptive randomization trial design and cancer genetic risk assessment. Breast cancer is a heterogeneous disease and targeted therapy is improving the outcomes of women. The use of cyclin-dependent kinase inhibitors (CDK) 4/6 have demonstrated a substantial improvement in progression-free survival in the first line setting of metastatic hormone receptor positive breast cancer. And newer agents directed at HER2 continue to revolutionize HER2-positive breast cancer treatment. This review highlights the recent updates in breast cancer treatment, new concepts in clinical trial design and provides a current overview of cancer genetic risk assessment.

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

New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment?

PubMed

Coqueret, Olivier

2003-02-01

Cell division relies on the activation of cyclins, which bind to cyclin-dependent kinases (CDKs) to induce cell-cycle progression towards S phase and later to initiate mitosis. Since uncontrolled cyclin-dependent kinase activity is often the cause of human cancer, their function is tightly regulated by cell-cycle inhibitors such as the p21 and p27 Cip/Kip proteins. Following anti-mitogenic signals or DNA damage, p21 and p27 bind to cyclin-CDK complexes to inhibit their catalytic activity and induce cell-cycle arrest. Interestingly, recent discoveries suggest that p21 and p27 might have new activities that are unrelated to their function as CDK inhibitors. The identification of new targets of Cip/Kip proteins as well as evidence of Cip/Kip cytoplasmic relocalization have revealed unexpected functions for these proteins in the control of CDK activation, in the regulation of apoptosis and in transcriptional activation. This article discusses recent insights into these possible additional functions of p21 and p27.

Transient CDK4/6 inhibition protects hematopoietic stem cells from chemotherapy-induced exhaustion.

PubMed

He, Shenghui; Roberts, Patrick J; Sorrentino, Jessica A; Bisi, John E; Storrie-White, Hannah; Tiessen, Renger G; Makhuli, Karenann M; Wargin, William A; Tadema, Henko; van Hoogdalem, Ewoud-Jan; Strum, Jay C; Malik, Rajesh; Sharpless, Norman E

2017-04-26

Conventional cytotoxic chemotherapy is highly effective in certain cancers but causes dose-limiting damage to normal proliferating cells, especially hematopoietic stem and progenitor cells (HSPCs). Serial exposure to cytotoxics causes a long-term hematopoietic compromise ("exhaustion"), which limits the use of chemotherapy and success of cancer therapy. We show that the coadministration of G1T28 (trilaciclib), which is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6), contemporaneously with cytotoxic chemotherapy protects murine hematopoietic stem cells (HSCs) from chemotherapy-induced exhaustion in a serial 5-fluorouracil treatment model. Consistent with a cell-intrinsic effect, we show directly preserved HSC function resulting in a more rapid recovery of peripheral blood counts, enhanced serial transplantation capacity, and reduced myeloid skewing. When administered to healthy human volunteers, G1T28 demonstrated excellent in vivo pharmacology and transiently inhibited bone marrow (BM) HSPC proliferation. These findings suggest that the combination of CDK4/6 inhibitors with cytotoxic chemotherapy should provide a means to attenuate therapy-induced BM exhaustion in patients with cancer. Copyright © 2017, American Association for the Advancement of Science.

Long Noncoding RNA LINC00958 Accelerates Gliomagenesis Through Regulating miR-203/CDK2.

PubMed

Guo, Erkun; Liang, Chaohui; He, Xin; Song, Guozhi; Liu, Hongjiang; Lv, Zhongqiang; Guan, Jianchao; Yang, Dezhen; Zheng, Jiapeng

2018-05-01

Increasing evidence has indicated that long noncoding RNAs (lncRNAs) play crucial roles in various biological processes, including glioma. However, the underlying mechanism of lncRNAs in gliomagenesis is still ambiguous. In this study, we aim to investigate the role of long intergenic noncoding RNA 00958 (LINC00958) in the tumorigenesis of glioma. Results revealed that LINC00958 was significantly upregulated in glioma tissues and cell lines compared with that of adjacent normal brain tissues and normal human astrocytes. Moreover, the ectopic overexpression of LINC00958 was correlated with poor prognosis of glioma patients. Loss-of-function experiments indicated that LINC00958 knockdown suppressed glioma cell proliferation, invasion, and induced cycle arrest at G0/G1 phase in vitro, and inhibited tumor growth in vivo. Bioinformatics programs and luciferase reporter assay revealed that miR-203 shared complementary binding sites with both 3'-untranslated region of LINC00958 and CDK2. In summary, our study concludes that LINC00958 acts as an oncogenic gene in the gliomagenesis through miR-203-CDK2 regulation, providing a novel insight into glioma tumorigenesis.

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.

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

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

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

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

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

Chemopreventive agents alters global gene expression pattern: predicting their mode of action and targets.

PubMed

Narayanan, Bhagavathi A

2006-12-01

Chemoprevention has the potential to be a major component of colon, breast, prostate and lung cancer control. Epidemiological, experimental, and clinical studies provide evidence that antioxidants, anti-inflammatory agents, n-3 polyunsaturated fatty acids and several other phytochemicals possess unique modes of action against cancer growth. However, the mode of action of several of these agents at the gene transcription level is not completely understood. Completion of the human genome sequence and the advent of DNA microarrays using cDNAs enhanced the detection and identification of hundreds of differentially expressed genes in response to anticancer drugs or chemopreventive agents. In this review, we are presenting an extensive analysis of the key findings from studies using potential chemopreventive agents on global gene expression patterns, which lead to the identification of cancer drug targets. The summary of the study reports discussed in this review explains the extent of gene alterations mediated by more than 20 compounds including antioxidants, fatty acids, NSAIDs, phytochemicals, retinoids, selenium, vitamins, aromatase inhibitor, lovastatin, oltipraz, salvicine, and zinc. The findings from these studies further reveal the utility of DNA microarray in characterizing and quantifying the differentially expressed genes that are possibly reprogrammed by the above agents against colon, breast, prostate, lung, liver, pancreatic and other cancer types. Phenolic antioxidant resveratrol found in berries and grapes inhibits the formation of prostate tumors by acting on the regulatory genes such as p53 while activating a cascade of genes involved in cell cycle and apoptosis including p300, Apaf-1, cdk inhibitor p21, p57 (KIP2), p53 induced Pig 7, Pig 8, Pig 10, cyclin D, DNA fragmentation factor 45. The group of genes significantly altered by selenium includes cyclin D1, cdk5, cdk4, cdk2, cdc25A and GADD 153. Vitamine D shows impact on p21(Waf1/Cip1) p27 cyclin B and cyclin A1. Genomic expression profile with vitamin D indicated differential expression of gene targets such as c-JUN, JUNB, JUND, FREAC-1/FoxF1, ZNF-44/KOX7, plectin, filamin, and keratin-13, involved in antiproliferative, differentiation pathways. The agent UBEIL has a remarkable effect on cyclin D1. Curcumin mediated NrF2 pathway significantly altered p21(Waf1/Cip1) levels. Aromatase inhibitors affected the expression of cyclin D1. Interestingly, few dietary compounds listed in this review also have effect on APC, cdk inhibitors p21(Waf1/Cip1) and p27. Tea polyphenol EGCG has a significant effect on TGF-beta expression, while several other earlier studies have shown its effect on cell cycle regulatory proteins. This review article reveals potential chemoprevention drug targets, which are mainly centered on cell cycle regulatory pathway genes in cancer.

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.

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.

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

The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming.

PubMed

Tang, Hong-Wen; Hu, Yanhui; Chen, Chiao-Lin; Xia, Baolong; Zirin, Jonathan; Yuan, Min; Asara, John M; Rabinow, Leonard; Perrimon, Norbert

2018-05-01

Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

Cdk5 Regulates Activity-Dependent Gene Expression and Dendrite Development.

PubMed

Liang, Zhuoyi; Ye, Tao; Zhou, Xiaopu; Lai, Kwok-On; Fu, Amy K Y; Ip, Nancy Y

2015-11-11

The proper growth and arborization of dendrites in response to sensory experience are essential for neural connectivity and information processing in the brain. Although neuronal activity is important for sculpting dendrite morphology, the underlying molecular mechanisms are not well understood. Here, we report that cyclin-dependent kinase 5 (Cdk5)-mediated transcriptional regulation is a key mechanism that controls activity-dependent dendrite development in cultured rat neurons. During membrane depolarization, Cdk5 accumulates in the nucleus to regulate the expression of a subset of genes, including that of the neurotrophin brain-derived neurotrophic factor, for subsequent dendritic growth. Furthermore, Cdk5 function is mediated through the phosphorylation of methyl-CpG-binding protein 2, a key transcriptional repressor that is mutated in the mental disorder Rett syndrome. These findings collectively suggest that the nuclear import of Cdk5 is crucial for activity-dependent dendrite development by regulating neuronal gene transcription during neural development. Neural activity directs dendrite development through the regulation of gene transcription. However, how molecular signals link extracellular stimuli to the transcriptional program in the nucleus remains unclear. Here, we demonstrate that neuronal activity stimulates the translocation of the kinase Cdk5 from the cytoplasmic compartment into the nucleus; furthermore, the nuclear localization of Cdk5 is required for dendrite development in cultured neurons. Genome-wide transcriptome analysis shows that Cdk5 deficiency specifically disrupts activity-dependent gene transcription of bdnf. The action of Cdk5 is mediated through the modulation of the transcriptional repressor methyl-CpG-binding protein 2. Therefore, this study elucidates the role of nuclear Cdk5 in the regulation of activity-dependent gene transcription and dendritic growth. Copyright © 2015 the authors 0270-6474/15/3515127-08$15.00/0.

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.

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.

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

[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.

Disruption of the G1/S Transition in Human Papillomavirus Type 16 E7-Expressing Human Cells Is Associated with Altered Regulation of Cyclin E

PubMed Central

Martin, Larry G.; Demers, G. William; Galloway, Denise A.

1998-01-01

The development of neoplasia frequently involves inactivation of the p53 and retinoblastoma (Rb) tumor suppressor pathways and disruption of cell cycle checkpoints that monitor the integrity of replication and cell division. The human papillomavirus type 16 (HPV-16) oncoproteins, E6 and E7, have been shown to bind p53 and Rb, respectively. To further delineate the mechanisms by which E6 and E7 affect cell cycle control, we examined various aspects of the cell cycle machinery. The low-risk HPV-6 E6 and E7 proteins did not cause any significant change in the levels of cell cycle proteins analyzed. HPV-16 E6 resulted in very low levels of p53 and p21 and globally elevated cyclin-dependent kinase (CDK) activity. In contrast, HPV-16 E7 had a profound effect on several aspects of the cell cycle machinery. A number of cyclins and CDKs were elevated, and despite the elevation of the levels of at least two CDK inhibitors, p21 and p16, CDK activity was globally increased. Most strikingly, cyclin E expression was deregulated both transcriptionally and posttranscriptionally and persisted at high levels in S and G2/M. Transit through G1 was shortened by the premature activation of cyclin E-associated kinase activity. Elevation of cyclin E levels required both the CR1 and CR2 domains of E7. These data suggest that cyclin E may be a critical target of HPV-16 E7 in the disruption of G1/S cell cycle progression and that the ability of E7 to regulate cyclin E involves activities in addition to the release of E2F. PMID:9444990

Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

PubMed

Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

2013-05-01

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

Berberine and Evodiamine Act Synergistically Against Human Breast Cancer MCF-7 Cells by Inducing Cell Cycle Arrest and Apoptosis.

PubMed

Du, Jia; Sun, Yang; Lu, Yi-Yu; Lau, Eric; Zhao, Ming; Zhou, Qian-Mei; Su, Shi-Bing

2017-11-01

The synergistic combinations of natural products have long been the basis of Traditional Chinese herbal Medicine formulas. In this study, we investigated the synergistic effects of a combination of berberine and evodiamine against human breast cancer MCF-7 cells in vitro and in vivo, and explored its mechanism. Cell survival was measured using the MTT assay. Apoptosis-related proteins were observed using western blot analysis. Apoptosis was detected with flow cytometric analysis and by Hoechst 33258 staining. Tumor xenografts were used in vivo. Compared to berberine or evodiamine treatments alone, the combination treatment of berberine (25 μM) and evodiamine (15 μM) synergistically inhibited the proliferation of MCF-7 cells in a time-dependent manner and resulted in the G 0 /G 1 phase accumulation of cells that exhibited increased expression levels of the CDK inhibitors p21 and p27 with a concomitant reduction in the expression levels of cell-cycle checkpoint proteins cyclin D1, cyclin E, CDK4, and CDK6. Furthermore, the combination treatment induced apoptosis that was accompanied by increased expression levels of p53 and Bax, reduced expression levels of Bcl-2, activation of caspase-7, and caspase-9, and the cleavage of PARP. The combination of berberine and evodiamine synergistically inhibited tumor growth in vivo in MCF-7 human breast cancer xenografts. Combination of berberine and evodiamine acts synergistically to suppress the proliferation of MCF-7 cells by inducing cell cycle arrest and apoptosis, illustrating the potential synergistic and combinatorial application of bioactive natural products. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Base-CP proteasome can serve as a platform for stepwise lid formation

PubMed Central

Yu, Zanlin; Livnat-Levanon, Nurit; Kleifeld, Oded; Mansour, Wissam; Nakasone, Mark A.; Castaneda, Carlos A.; Dixon, Emma K.; Fushman, David; Reis, Noa; Pick, Elah; Glickman, Michael H.

2015-01-01

26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes. PMID:26182356

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.

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.

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.

Movements and survival of black-footed ferrets associated with an experimental translocation in South Dakota

USGS Publications Warehouse

Biggins, D.E.; Godbey, J.L.; Horton, B.M.; Livieri, T.M.

2011-01-01

Black-footed ferrets (Mustela nigripes) apparently were extirpated from all native habitats by 1987, and their repatriation requires a combination of captive breeding, reintroductions, and translocations among sites. Improvements in survival rates of released ferrets have resulted from experience in quasi-natural environments during their rearing. Reestablishment of a self-sustaining wild population by 1999 provided the 1st opportunity to initiate new populations by translocating wild-born individuals. Using radiotelemetry, we compared behaviors and survival of 18 translocated wild-born ferrets and 18 pen-experienced captive-born ferrets after their release into a prairie dog colony not occupied previously by ferrets. Translocated wild-born ferrets moved significantly less and had significantly higher short-term survival rates than their captive-born counterparts. Using markrecapture methods, we also assessed potential impacts to the established donor population of removing 37% of its estimated annual production of kits. Annual survival rates for 30 ferret kits remaining at the donor subcomplex were higher than rates for 54 ferret kits at the control subcomplex (unmanipulated) for males (+82%) and females (+32%). Minimum survival of translocated kits did not differ significantly from survival of those at the control subcomplex. Direct translocation of young, wild-born ferrets from site to site appears to be an efficient method to establish new populations. ?? 2011 American Society of Mammalogists.

Identification of the DotL Coupling Protein Subcomplex of the Legionella Dot/Icm Type IV Secretion System

PubMed Central

Vincent, Carr D.; Friedman, Jonathan R.; Jeong, Kwang Cheol; Sutherland, Molly C.; Vogel, Joseph P.

2012-01-01

Summary Legionella pneumophila, the causative agent of Legionnaires’ disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella T4SS substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. PMID:22694730

Crystal structure of the Csm3-Csm4 subcomplex in the type III-A CRISPR-Cas interference complex.

PubMed

Numata, Tomoyuki; Inanaga, Hideko; Sato, Chikara; Osawa, Takuo

2015-01-30

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

Phase 1/2 study of cyclin-dependent kinase (CDK)4/6 inhibitor palbociclib (PD-0332991) with bortezomib and dexamethasone in relapsed/refractory multiple myeloma.

PubMed

Niesvizky, Ruben; Badros, Ashraf Z; Costa, Luciano J; Ely, Scott A; Singhal, Seema B; Stadtmauer, Edward A; Haideri, Nisreen A; Yacoub, Abdulraheem; Hess, Georg; Lentzsch, Suzanne; Spicka, Ivan; Chanan-Khan, Asher A; Raab, Marc S; Tarantolo, Stefano; Vij, Ravi; Zonder, Jeffrey A; Huang, Xiangao; Jayabalan, David; Di Liberto, Maurizio; Huang, Xin; Jiang, Yuqiu; Kim, Sindy T; Randolph, Sophia; Chen-Kiang, Selina

2015-01-01

This phase 1/2 study was the first to evaluate the safety and efficacy of the cyclin-dependent kinase (CDK) 4/6-specific inhibitor palbociclib (PD-0332991) in sequential combination with bortezomib and dexamethasone in relapsed/refractory multiple myeloma. The recommended phase 2 dose was palbociclib 100 mg orally once daily on days 1-12 of a 21-day cycle with bortezomib 1.0 mg/m2 (intravenous) and dexamethasone 20 mg (orally 30 min pre-bortezomib dosing) on days 8 and 11 (early G1 arrest) and days 15 and 18 (cell cycle resumed). Dose-limiting toxicities were primarily cytopenias; most other treatment-related adverse events were grade≤3. At a bortezomib dose lower than that in other combination therapy studies, antitumor activity was observed (phase 1). In phase 2, objective responses were achieved in 5 (20%) patients; 11 (44%) achieved stable disease. Biomarker and pharmacodynamic assessments demonstrated that palbociclib inhibited CDK4/6 and the cell cycle initially in most patients.

Involvement of PSMD10, CDK4, and Tumor Suppressors in Development of Intrahepatic Cholangiocarcinoma of Syrian Golden Hamsters Induced by Clonorchis sinensis and N-Nitrosodimethylamine

PubMed Central

Uddin, Md. Hafiz; Choi, Min-Ho; Kim, Woo Ho; Jang, Ja-June; Hong, Sung-Tae

2015-01-01

Background Clonorchis sinensis is a group-I bio-carcinogen for cholangiocarcinoma (CCA). Although the epidemiological evidence links clonorchiasis and CCA, the underlying molecular mechanism involved in this process is poorly understood. In the present study, we investigated expression of oncogenes and tumor suppressors, including PSMD10, CDK4, p53 and RB in C. sinensis induced hamster CCA model. Methods Different histochemical/immunohistochemical techniques were performed to detect CCA in 4 groups of hamsters: uninfected control (Ctrl.), infected with C. sinensis (Cs), ingested N-nitrosodimethylamine (NDMA), and both Cs infected and NDMA introduced (Cs+NDMA). The liver tissues from all groups were analyzed for gene/protein expressions by quantitative PCR (qPCR) and western blotting. Principal Findings CCA was observed in all hamsters of Cs+NDMA group with well, moderate, and poorly differentiated types measured in 21.8% ± 1.5%, 13.3% ± 1.3%, and 10.8% ± 1.3% of total tissue section areas respectively. All CCA differentiations progressed in a time dependent manner, starting from the 8th week of infection. CCA stroma was characterized with increased collagen type I, mucin, and proliferative cell nuclear antigen (PCNA). The qPCR analysis showed PSMD10, CDK4 and p16INK4 were over-expressed, whereas p53 was under-expressed in the Cs+NDMA group. We observed no change in RB1 at mRNA level but found significant down-regulation of RB protein. The apoptosis related genes, BAX and caspase 9 were found downregulated in the CCA tissue. Gene/protein expressions were matched well with the pathological changes of different groups except the NDMA group. Though the hamsters in the NDMA group showed no marked pathological lesions, we observed over-expression of Akt/PKB and p53 genes proposing molecular interplay in this group which might be related to the CCA initiation in this animal model. Conclusions/Significance The present findings suggest that oncogenes, PSMD10 and CDK4, and tumor suppressors, p53 and RB, are involved in the carcinogenesis process of C. sinensis induced CCA in hamsters. PMID:26313366

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

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.

Cytogenetic and molecular characterization of plutonium-induced rat osteosarcomas.

PubMed

Roch-Lefevre, Sandrine; Daino, Kazuhiro; Altmeyer-Morel, Sandrine; Guilly, Marie-Noëlle; Chevillard, Sylvie

2010-01-01

The association between ionizing radiation and the subsequent development of osteosarcoma has been well described, but little is known about the cytogenetic and molecular events, which could be involved in the formation of radiation-induced osteosarcomas. Here, we performed comparative genomic hybridization (CGH) to detect chromosomal copy number changes in a series of 16 rat osteosarcomas induced by injection of plutonium-238. Recurrent gains/amplifications were observed at chromosomal regions 3p12-q12, 3q41-qter, 4q41-qter, 6q12-q16, 7q22-q34, 8q11-q23, 9q11-q22, 10q32.1-qter, and 12q, whereas recurrent losses were observed at 1p, 1q, 3q23-q35, 5q21-q33, 8q24-q31, 10q22-q25, 15p, 15q, and 18q. The gained region at 7q22-q34 was homologous to human chromosome bands 12q13-q15/8q24/22q11-q13, including the loci of Mdm2, Cdk4, c-Myc and Pdgf-b genes. The lost regions at 5q21-q33, 10q22-q25 and 15q contained tumor suppressor genes such as p16INK4a/p19ARF, Tp53 and Rb1. To identify potential target gene(s) for the chromosomal aberrations, we compared the expression levels of several candidate genes, located within the regions of frequent chromosomal aberrations, between the tumors and normal osteoblasts by using quantitative RT-PCR analysis. The Cdk4, c-Myc, Pdgf-b and p57KIP2 genes were thought to be possible target genes for the frequent chromosomal gain at 7q22-34 and loss at 1q in the tumors, respectively. In addition, mutations of the Tp53 gene were found in 27% (4 of 15) osteosarcomas. Our data may contribute to further understanding of the molecular mechanisms underlying osteosarcomas induced by ionizing radiation in human.

Proteomic analysis of the response to cell cycle arrests in human myeloid leukemia cells

PubMed Central

Ly, Tony; Endo, Aki; Lamond, Angus I

2015-01-01

Abstract Previously, we analyzed protein abundance changes across a ‘minimally perturbed’ cell cycle by using centrifugal elutriation to differentially enrich distinct cell cycle phases in human NB4 cells (Ly et al., 2014). In this study, we compare data from elutriated cells with NB4 cells arrested at comparable phases using serum starvation, hydroxyurea, or RO-3306. While elutriated and arrested cells have similar patterns of DNA content and cyclin expression, a large fraction of the proteome changes detected in arrested cells are found to reflect arrest-specific responses (i.e., starvation, DNA damage, CDK1 inhibition), rather than physiological cell cycle regulation. For example, we show most cells arrested in G2 by CDK1 inhibition express abnormally high levels of replication and origin licensing factors and are likely poised for genome re-replication. The protein data are available in the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/), an online, searchable resource. DOI: http://dx.doi.org/10.7554/eLife.04534.001 PMID:25555159

New arylated benzo[h]quinolines induce anti-cancer activity by oxidative stress-mediated DNA damage.

PubMed

Yadav, Dharmendra K; Rai, Reeta; Kumar, Naresh; Singh, Surjeet; Misra, Sanjeev; Sharma, Praveen; Shaw, Priyanka; Pérez-Sánchez, Horacio; Mancera, Ricardo L; Choi, Eun Ha; Kim, Mi-Hyun; Pratap, Ramendra

2016-12-06

The anti-cancer activity of the benzo[h]quinolines was evaluated on cultured human skin cancer (G361), lung cancer (H460), breast cancer (MCF7) and colon cancer (HCT116) cell lines. The inhibitory effect of these compounds on the cell growth was determined by the MTT assay. The compounds 3e, 3f, 3h and 3j showed potential cytotoxicity against these human cancer cell lines. Effect of active compounds on DNA oxidation and expression of apoptosis related gene was studied. We also developed a quantitative method to measure the activity of cyclin-dependent kinases-2 (CDK2) by western blotting in the presence of active compound. In addition, molecular docking revealed that benzo[h]quinolines can correctly dock into the hydrophobic pocket of the targets receptor protein aromatase and CDK2, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. These findings reveal that benzo[h]quinolines act as anti-cancer agents by inducing oxidative stress-mediated DNA damage.

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

PubMed

Chiron, David; Di Liberto, Maurizio; Martin, Peter; Huang, Xiangao; Sharman, Jeff; Blecua, Pedro; Mathew, Susan; Vijay, Priyanka; Eng, Ken; Ali, Siraj; Johnson, Amy; Chang, Betty; Ely, Scott; Elemento, Olivier; Mason, Christopher E; Leonard, John P; Chen-Kiang, Selina

2014-09-01

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973. ©2014 American Association for Cancer Research.

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

Lee, Eunjung; Son, Joe Eun; Byun, Sanguine

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

Implication of cyclin-dependent kinase 5 in the development of psychological dependence on and behavioral sensitization to morphine.

PubMed

Narita, Minoru; Shibasaki, Masahiro; Nagumo, Yasuyuki; Narita, Michiko; Yajima, Yoshinori; Suzuki, Tsutomu

2005-06-01

In the present study, we investigated the role of cyclin-dependent kinase 5 (cdk5) in the brain dynamics changed by repeated in vivo treatment with morphine. The level of phosphorylated-cdk5 was significantly increased in the cingulate cortex of mice showing the morphine-induced rewarding effect. Under these conditions, roscovitine, a cdk5 inhibitor, given intracerebroventricularly (i.c.v.) caused a dose-dependent and significant inhibition of the morphine-induced rewarding effect. In addition, the dose-response effect of the morphine-induced rewarding effect was dramatically attenuated in cdk5 heterozygous (+/-) knockout mice. Furthermore, the development of behavioral sensitization by intermittent administration of morphine was virtually abolished in cdk5 (+/-) mice. These findings suggest that the induction and/or activation of cdk5 are implicated in the development of psychological dependence on morphine.

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.

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.

Smad2 and Smad3 phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer.

PubMed

Matsuzaki, Koichi; Kitano, Chiaki; Murata, Miki; Sekimoto, Go; Yoshida, Katsunori; Uemura, Yoshiko; Seki, Toshihito; Taketani, Shigeru; Fujisawa, Jun-ichi; Okazaki, Kazuichi

2009-07-01

Transforming growth factor (TGF)-beta initially inhibits growth of mature epithelial cells. Later, however, autocrine TGF-beta signaling acts in concert with the Ras pathway to induce a proliferative and invasive phenotype. TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also Ras-associated kinases, which differentially phosphorylate the mediators Smad2 and Smad3 to create distinct phosphorylated forms: COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C) and both linker and COOH-terminally phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). In this study, we investigated actions of pSmad2L/C and pSmad3L/C in cancer progression. TGF-beta inhibited cell growth by down-regulating c-Myc oncoprotein through the pSmad2C and pSmad3C pathway; TGF-beta signaling, in turn, enhanced cell growth by up-regulating c-Myc through the cyclin-dependent kinase (CDK) 4-dependent pSmad2L/C and pSmad3L/C pathways in cell nuclei. Alternatively, TbetaRI and c-Jun NH2-terminal kinase (JNK) together created cytoplasmic pSmad2L/C, which entered the nucleus and stimulated cell invasion, partly by up-regulating matrix metalloproteinase-9. In 20 clinical samples, pSmad2L/C and pSmad3L/C showed nuclear localization at invasion fronts of all TGF-beta-producing human metastatic colorectal cancers. In vitro kinase assay confirmed that nuclear CDK4 and cytoplasmic JNK obtained from the tumor tissue could phosphorylate Smad2 or Smad3 at their linker regions. We suggest that CDK4, together with JNK, alters tumor-suppressive TGF-beta signaling to malignant characteristics in later stages of human colorectal cancer. The linker phosphorylation of Smad2 and Smad3 may represent a target for intervention in human metastatic cancer.

A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6 expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent breast cancer cell lines.

PubMed

Brandi, Giorgio; Paiardini, Mirko; Cervasi, Barbara; Fiorucci, Chiara; Filippone, Paolino; De Marco, Cinzia; Zaffaroni, Nadia; Magnani, Mauro

2003-07-15

Indole-3-carbinol (I3C), autolysis product of glucosinolates present in cruciferous vegetables, has been indicated as a promising agent in preventing the development and progression of breast cancer. I3C has been shown to inhibit the growth of human cancer cells in vitro and possesses anticarcinogenic activity in vivo. Because I3C is unstable and may be converted into many polymeric products in the digestive tract, it is not yet clear whether the biological activity observed can be attributed to I3C or some of its polymeric products. In this study we synthesized a stable I3C cyclic tetrameric derivative and investigated its effects on a panel of human breast cancer cell lines. The I3C tetramer suppressed the growth of both estrogen receptor (ER) -positive (MCF-7, 734B, and BT474) and ER-negative (BT20, MDA-MB-231, and BT539) human breast cancer cell lines, and it was found to induce G(1) cell cycle arrest in a dose-dependent manner without evidence of apoptosis, suggesting a growth arrest via a cytostatic mechanism. At the molecular level, the tetramer inhibited cyclin-dependent kinase (CDK) 6 expression and activity, induced an increase in the level of p27(kip1), and reduced the level of retinoblastoma protein expression. Contrarily to CDK6, the level of CDK4, the other kinase involved in the G(1) phase of the cell cycle, remains unchanged. Interestingly, the tetramer resulted about five times more active than I3C in suppressing the growth of human breast cancer cells. On the whole, our data suggest that the I3C tetrameric derivative is a novel lead inhibitor of breast cancer cell growth that may be a considered a new, promising therapeutic agent for both ER+ and ER- breast cancer.

Hormonal Resistance and Metastasis: ER-coregulator-Src Signaling Targeted Therapy

DTIC Science & Technology

2010-09-01

receptors and coregulators The human estrogen receptor (ER) is a key transcriptional regulator in breast cancer biology (Green and Carroll, 2007; Heldring...al, 1991) and over-expression of Cyclin D1 has been noted in over 50% of human breast tumors of all histological types (Gillett et al, 1994; Kenny et...CDK inhibitors Down regulation of p21 has been implicated with Tamoxifen resistant phenotype. Somatic deletion of p21 gene in human breast cancer

Antitumor Effects of Flavopiridol on Human Uterine Leiomyoma In Vitro and in a Xenograft Model

PubMed Central

Lee, Hyun-Gyo; Baek, Jong-Woo; Shin, So-Jin; Kwon, Sang-Hoon; Cha, Soon-Do; Park, Won-Jin; Chung, Rosa; Choi, Eun-Som; Lee, Gun-Ho

2014-01-01

Dysregulated cyclin-dependent kinases (CDKs) are considered a potential target for cancer therapy. Flavopiridol is a potent CDK inhibitor. In this study, the antiproliferative effect of the flavonoid compound flavopiridol and its mechanism in human uterine leiomyoma cells were investigated. The present study focused on the effect of flavopiridol in cell proliferation and cell cycle progression in primary cultured human uterine leiomyoma cells. Cell viability and cell proliferation assays were conducted. Flow cytometry was performed to determine the effect of flavopiridol on cell cycle. The expression of cell cycle regulatory-related proteins was evaluated by Western blotting. Cell viability and proliferation of uterine leiomyoma cells were significantly reduced by flavopiridol treatment in a dose-dependent manner. Flow cytometry results showed that flavopiridol induced G1 phase arrest. Flavopiridol-induced growth inhibition in uterine leiomyoma cells was associated with increased expression of p21cip/wafl and p27kip1 in a dose-dependent manner. Downregulation of CDK2/4 and Cyclin A with a concomitant increase in dephosphorylation of retinoblastoma was observed. This study demonstrates that flavopiridol inhibits cell proliferation by initiating G1 cell cycle arrest in human uterine leiomyoma. We also found that flavopiridol is effective in inhibiting xenografted human uterine leiomyoma growth. These results indicate that flavopiridol could prove to be a promising chemopreventive and therapeutic agent for human uterine leiomyoma. PMID:24572052

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

Metabolic changes associated with metformin potentiates Bcl-2 inhibitor, Venetoclax, and CDK9 inhibitor, BAY1143572 and reduces viability of lymphoma cells.

PubMed

Chukkapalli, Vineela; Gordon, Leo I; Venugopal, Parameswaran; Borgia, Jeffrey A; Karmali, Reem

2018-04-20

Metformin exerts direct anti-tumor effects by activating AMP-activated protein kinase (AMPK), a major sensor of cellular metabolism in cancer cells. This, in turn, inhibits pro-survival mTOR signaling. Metformin has also been shown to disrupt complex 1 of the mitochondrial electron transport chain. Here, we explored the lymphoma specific anti-tumor effects of metformin using Daudi (Burkitt), SUDHL-4 (germinal center diffuse large B-cell lymphoma; GC DLBCL), Jeko-1 (Mantle-cell lymphoma; MCL) and KPUM-UH1 (double hit DLBCL) cell lines. We demonstrated that metformin as a single agent, especially at high concentrations produced significant reductions in viability and proliferation only in Daudi and SUDHL-4 cell lines with associated alterations in mitochondrial oxidative and glycolytic metabolism. As bcl-2 proteins, cyclin dependent kinases (CDK) and phosphoinositol-3- kinase (PI3K) also influence mitochondrial physiology and metabolism with clear relevance to the pathogenesis of lymphoma, we investigated the potentiating effects of metformin when combined with novel agents Venetoclax (bcl-2 inhibitor), BAY-1143572 (CDK9 inhibitor) and Idelalisib (p110δ- PI3K inhibitor). Co-treating KPUM-UH1 and SUDHL-4 cells with 10 mM of metformin resulted in 1.4 fold and 8.8 fold decreases, respectively, in IC-50 values of Venetoclax. By contrast, 3-fold and 10 fold reduction in IC-50 values of BAY-1143572 in Daudi and Jeko-1 cells respectively was seen in the presence of 10 mM of metformin. No change in IC-50 value for Idelalisib was observed across cell lines. These data suggest that although metformin is not a potent single agent, targeting cancer metabolism with similar but more effective drugs in novel combination with either bcl-2 or CDK9 inhibitors warrants further exploration.

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

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

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.

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.

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.

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

Arterial territory-specific phosphorylated retinoblastoma protein species and CDK2 promote differences in the vascular smooth muscle cell response to mitogens

PubMed Central

Lange, Martin; Fujikawa, Tatsuya; Koulova, Anna; Kang, Sona; Griffin, Michael J; Lassaletta, Antonio D; Erat, Anna; Tobiasch, Edda; Bianchi, Cesario; Elmadhun, Nassrene; Sellke, Frank W; Usheva, Anny

2014-01-01

Despite recent advances in medical procedures, cardiovascular disease remains a clinical challenge and the leading cause of mortality in the western world. The condition causes progressive smooth muscle cell (SMC) dedifferentiation, proliferation, and migration that contribute to vascular restenosis. The incidence of disease of the internal mammary artery (IMA), however, is much lower than in nearly all other arteries. The etiology of this IMA disease resistance is not well understood. Here, using paired primary IMA and coronary artery SMCs, serum stimulation, siRNA knockdowns, and verifications in porcine vessels in vivo, we investigate the molecular mechanisms that could account for this increased disease resistance of internal mammary SMCs. We show that the residue-specific phosphorylation profile of the retinoblastoma tumor suppressor protein (Rb) appears to differ significantly between IMA and coronary artery SMCs in cultured human cells. We also report that the differential profile of Rb phosphorylation may follow as a consequence of differences in the content of cyclin-dependent kinase 2 (CDK2) and the CDK4 phosphorylation inhibitor p15. Finally, we present evidence that siRNA-mediated CDK2 knockdown alters the profile of Rb phosphorylation in coronary artery SMCs, as well as the proliferative response of these cells to mitogenic stimulation. The intrinsic functional and protein composition specificity of the SMCs population in the coronary artery may contribute to the increased prevalence of restenosis and atherosclerosis in the coronary arteries as compared with the internal mammary arteries. PMID:24240190

Piperlongumine decreases cell proliferation and the expression of cell cycle-associated proteins by inhibiting Akt pathway in human lung cancer cells.

PubMed

Seok, Jin Sil; Jeong, Chang Hee; Petriello, Michael C; Seo, Han Geuk; Yoo, Hyunjin; Hong, Kwonho; Han, Sung Gu

2018-01-01

Piperlongumine (PL) is an alkaloid of a pepper plant found in Southeast Asia. PL is known to induce selective toxicity towards a variety of cancer cell types. To explore the possible anti-lung cancer effects of PL, A549 cells were treated with PL (0-40 μM) for 24 h. Alterations in the expression of cell cycle-associated proteins (cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and retinoblastoma (Rb)) and intracellular signaling molecules (extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, p38 and nuclear factor-κB (NF-κB)) were examined in cells following treatment of PL using Western blot analysis. Results showed that proliferation of cells were significantly decreased by PL in a dose-dependent manner. Flow cytometry results demonstrated increased number of cells in G1 phase in PL (40 μM)-treated group. Reactive oxygen species was significantly increased in cells treated with PL at 20-40 μM. The expression of cyclin D1, CDK4, CDK6 and p-Rb were markedly decreased in cells treated with PL at 40 μM. Treatment of cells with PL suppressed phosphorylation of Akt but increased ERK1/2 phosphorylation. Treatment of PL significantly decreased nuclear translocation of NF-κB p65 in cells. These results suggest that PL possesses antiproliferative properties in A549 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

The Cdk4-E2f1 pathway regulates early pancreas development by targeting Pdx1+ progenitors and Ngn3+ endocrine precursors

PubMed Central

Kim, So Yoon; Rane, Sushil G.

2011-01-01

Cell division and cell differentiation are intricately regulated processes vital to organ development. Cyclin-dependent kinases (Cdks) are master regulators of the cell cycle that orchestrate the cell division and differentiation programs. Cdk1 is essential to drive cell division and is required for the first embryonic divisions, whereas Cdks 2, 4 and 6 are dispensable for organogenesis but vital for tissue-specific cell development. Here, we illustrate an important role for Cdk4 in regulating early pancreas development. Pancreatic development involves extensive morphogenesis, proliferation and differentiation of the epithelium to give rise to the distinct cell lineages of the adult pancreas. The cell cycle molecules that specify lineage commitment within the early pancreas are unknown. We show that Cdk4 and its downstream transcription factor E2f1 regulate mouse pancreas development prior to and during the secondary transition. Cdk4 deficiency reduces embryonic pancreas size owing to impaired mesenchyme development and fewer Pdx1+ pancreatic progenitor cells. Expression of activated Cdk4R24C kinase leads to increased Nkx2.2+ and Nkx6.1+ cells and a rise in the number and proliferation of Ngn3+ endocrine precursors, resulting in expansion of the β cell lineage. We show that E2f1 binds and activates the Ngn3 promoter to modulate Ngn3 expression levels in the embryonic pancreas in a Cdk4-dependent manner. These results suggest that Cdk4 promotes β cell development by directing E2f1-mediated activation of Ngn3 and increasing the pool of endocrine precursors, and identify Cdk4 as an important regulator of early pancreas development that modulates the proliferation potential of pancreatic progenitors and endocrine precursors. PMID:21490060

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.

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.

A novel strategy of integrated microarray analysis identifies CENPA, CDK1 and CDC20 as a cluster of diagnostic biomarkers in lung adenocarcinoma.

PubMed

Liu, Wan-Ting; Wang, Yang; Zhang, Jing; Ye, Fei; Huang, Xiao-Hui; Li, Bin; He, Qing-Yu

2018-07-01

Lung adenocarcinoma (LAC) is the most lethal cancer and the leading cause of cancer-related death worldwide. The identification of meaningful clusters of co-expressed genes or representative biomarkers may help improve the accuracy of LAC diagnoses. Public databases, such as the Gene Expression Omnibus (GEO), provide rich resources of valuable information for clinics, however, the integration of multiple microarray datasets from various platforms and institutes remained a challenge. To determine potential indicators of LAC, we performed genome-wide relative significance (GWRS), genome-wide global significance (GWGS) and support vector machine (SVM) analyses progressively to identify robust gene biomarker signatures from 5 different microarray datasets that included 330 samples. The top 200 genes with robust signatures were selected for integrative analysis according to "guilt-by-association" methods, including protein-protein interaction (PPI) analysis and gene co-expression analysis. Of these 200 genes, only 10 genes showed both intensive PPI network and high gene co-expression correlation (r > 0.8). IPA analysis of this regulatory networks suggested that the cell cycle process is a crucial determinant of LAC. CENPA, as well as two linked hub genes CDK1 and CDC20, are determined to be potential indicators of LAC. Immunohistochemical staining showed that CENPA, CDK1 and CDC20 were highly expressed in LAC cancer tissue with co-expression patterns. A Cox regression model indicated that LAC patients with CENPA + /CDK1 + and CENPA + /CDC20 + were high-risk groups in terms of overall survival. In conclusion, our integrated microarray analysis demonstrated that CENPA, CDK1 and CDC20 might serve as novel cluster of prognostic biomarkers for LAC, and the cooperative unit of three genes provides a technically simple approach for identification of LAC patients. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

An aza-macrocycle containing maltolic side-arms (maltonis) as potential drug against human pediatric sarcomas

PubMed Central

2014-01-01

Background Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. Methods Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. Results Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. Conclusions Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients. PMID:24575739

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

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.

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

Identification of the DotL coupling protein subcomplex of the Legionella Dot/Icm type IV secretion system.

PubMed

Vincent, Carr D; Friedman, Jonathan R; Jeong, Kwang Cheol; Sutherland, Molly C; Vogel, Joseph P

2012-07-01

Legionella pneumophila, the causative agent of Legionnaires' disease, survives in macrophages by altering the endocytic pathway of its host cell. To accomplish this, the bacterium utilizes a type IVB secretion system to deliver effector molecules into the host cell cytoplasm. In a previous report, we performed an extensive characterization of the L. pneumophila type IVB secretion system that resulted in the identification of a critical five-protein subcomplex that forms the core of the secretion apparatus. Here we describe a second Dot/Icm protein subassembly composed of the type IV coupling protein DotL, the apparatus proteins DotM and DotN, and the secretion adaptor proteins IcmS and IcmW. In the absence of IcmS or IcmW, DotL becomes destabilized at the transition from the exponential to stationary phases of growth, concurrent with the expression of many secreted substrates. Loss of DotL is dependent on ClpA, a regulator of the cytoplasmic protease ClpP. The resulting decreased levels of DotL in the icmS and icmW mutants exacerbates the intracellular defects of these strains and can be partially suppressed by overproduction of DotL. Thus, in addition to their role as chaperones for Legionella type IV secretion system substrates, IcmS and IcmW perform a second function as part of the Dot/Icm type IV coupling protein subcomplex. © 2012 Blackwell Publishing Ltd.

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.

Low-dose radiation modulates human mesenchymal stem cell proliferation through regulating CDK and Rb.

PubMed

Yang, Lei; Liu, Ziling; Chen, Chen; Cong, Xiaofeng; Li, Zhi; Zhao, Shasha; Ren, Meng

2017-01-01

Low-dose radiation (LDR) has been known to stimulate cell proliferation. The effect of LDR on human bone marrow mesenchymal stem cells (BMSCs), however, remains to be determined. The current study, therefore, aimed to investigate the effect of LDR on human BMSC proliferation and its mechanisms. To accomplish this, human BMSCs were isolated from ribs and cultured with or without exposition to LDR (75 mGy) for 24 h. Cell proliferation was assessed by MTT assay, the cytokines secreted by the BMSCs were quantified by ELISA, and the proteins associated with cell proliferation and cell cycle were evaluated by immunoblot analysis. BMSCs isolated from human ribs were capable of differentiating into osteoblasts and adipocytes. LDR stimulated human BMSC proliferation (0.580 ± 0.106 vs 0.419 ± 0.026 on day 4, P < 0.05; 0.794 ± 0.025 vs 0.689 ± 0.047 on day 7, P < 0.05) and increased S-phase proportion. LDR significantly enhanced the production of SCF, GM-CSF, and IL-11. Moreover, BMSCs modulated T-cell proliferation, and LDR further augmented the modulatory effect of BMSCs on T-cell proliferation. Cell cycle-associated proteins, such as Rb, CDK1, and CDC25B, appeared to mediate the stimulatory effect of LDR on BMSC proliferation. The findings of the current study indicate that physical stimulants, such as LDR, could be used for the large-scale expansion of human BMSCs, and thus may be used for MSC cellular therapy in clinic.

D-type Cyclins are important downstream effectors of cytokine signaling that regulate the proliferation of normal and neoplastic mammary epithelial cells

PubMed Central

Zhang, Qian; Sakamoto, Kazuhito; Wagner, Kay-Uwe

2013-01-01

In response to the ligand-mediated activation of cytokine receptors, cells decide whether to proliferate or to undergo differentiation. D-type Cyclins (Cyclin D1, D2, or D3) and their associated Cyclin-dependent Kinases (CDK4, CDK6) connect signals from cytokines to the cell cycle machinery, and they propel cells through the G1 restriction point and into the S phase, after which growth factor stimulation is no longer essential to complete cell division. D-type Cyclins are upregulated in many human malignancies including breast cancer to promote an uncontrolled proliferation of cancer cells. After summarizing important aspects of the cytokine-mediated transcriptional regulation and the posttranslational modification of D-type Cyclins, this review will highlight the physiological significance of these cell cycle regulators during normal mammary gland development as well as the initiation and promotion of breast cancer. Although the vast majority of published reports focus almost exclusively on the role of Cyclin D1 in breast cancer, we summarize here previous and recent findings that demonstrate an important contribution of the remaining two members of this Cyclin family, in particular Cyclin D3, for the growth of ErbB2-associated breast cancer cells in humans and in mouse models. New data from genetically engineered models as well as the pharmacological inhibition of CDK4/6 suggest that targeting the combined functions of D-type Cyclins could be a suitable strategy for the treatment of ErbB2-positive and potentially other types of breast cancer. PMID:23562856

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.

[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.

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.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery.

PubMed

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R; Isaya, Grazia

2016-09-30

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN 42-210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN 42-210 ] 24 ·[NFS1] 24 ·[ISD11] 24 ·[ISCU] 24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN 42-210 ] 24 ·[ISCU] 24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN 42-210 trimer at each of its eight vertices. Binding of 12 [NFS1] 2 ·[ISD11] 2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN 42-210 to ISCU. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Architecture of the Human Mitochondrial Iron-Sulfur Cluster Assembly Machinery*

PubMed Central

Gakh, Oleksandr; Ranatunga, Wasantha; Smith, Douglas Y.; Ahlgren, Eva-Christina; Al-Karadaghi, Salam; Thompson, James R.; Isaya, Grazia

2016-01-01

Fe-S clusters, essential cofactors needed for the activity of many different enzymes, are assembled by conserved protein machineries inside bacteria and mitochondria. As the architecture of the human machinery remains undefined, we co-expressed in Escherichia coli the following four proteins involved in the initial step of Fe-S cluster synthesis: FXN42–210 (iron donor); [NFS1]·[ISD11] (sulfur donor); and ISCU (scaffold upon which new clusters are assembled). We purified a stable, active complex consisting of all four proteins with 1:1:1:1 stoichiometry. Using negative staining transmission EM and single particle analysis, we obtained a three-dimensional model of the complex with ∼14 Å resolution. Molecular dynamics flexible fitting of protein structures docked into the EM map of the model revealed a [FXN42–210]24·[NFS1]24·[ISD11]24·[ISCU]24 complex, consistent with the measured 1:1:1:1 stoichiometry of its four components. The complex structure fulfills distance constraints obtained from chemical cross-linking of the complex at multiple recurring interfaces, involving hydrogen bonds, salt bridges, or hydrophobic interactions between conserved residues. The complex consists of a central roughly cubic [FXN42–210]24·[ISCU]24 sub-complex with one symmetric ISCU trimer bound on top of one symmetric FXN42–210 trimer at each of its eight vertices. Binding of 12 [NFS1]2·[ISD11]2 sub-complexes to the surface results in a globular macromolecule with a diameter of ∼15 nm and creates 24 Fe-S cluster assembly centers. The organization of each center recapitulates a previously proposed conserved mechanism for sulfur donation from NFS1 to ISCU and reveals, for the first time, a path for iron donation from FXN42–210 to ISCU. PMID:27519411

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.

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

2010-01-01

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

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 ...

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.

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.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

IL-35 promotes pancreas cancer growth through enhancement of proliferation and inhibition of apoptosis: evidence for a role as an autocrine growth factor.

PubMed

Nicholl, Michael B; Ledgewood, Chelsea L; Chen, Xuhui; Bai, Qian; Qin, Chenglu; Cook, Kathryn M; Herrick, Elizabeth J; Diaz-Arias, Alberto; Moore, Bradley J; Fang, Yujiang

2014-12-01

Interleukin-35 (IL-35), an IL-12 cytokine family member, mediates the immune inhibitory function of regulatory T cells (Treg). We assayed the presence of IL-35 in paraffin-embedded human pancreas cancer (PCAN) and unexpectedly found IL-35 was expressed mainly by epithelial derived PCAN cells, but not by Treg. We further examined the expression and effect of exogenous IL-35 in human PCAN cell lines and found IL-35 promoted growth and inhibited apoptosis in PCAN cell lines. IL-35 induced proliferation correlated with an increase in cyclin B, cyclin D, cdk2, and cdk4 and a decrease in p27 expression, while inhibition of apoptosis was associated with an increase in Bcl-2 and a decrease in TRAILR1. We conclude IL-35 is produced by PCAN in vivo and promotes PCAN cell line growth in vitro. These results might indicate an important new role for IL-35 as an autocrine growth factor in PCAN growth. Copyright © 2014 Elsevier Ltd. All rights reserved.

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 APC/C E3 Ligase Complex Activator FZR1 Restricts BRAF Oncogenic Function.

PubMed

Wan, Lixin; Chen, Ming; Cao, Juxiang; Dai, Xiangpeng; Yin, Qing; Zhang, Jinfang; Song, Su-Jung; Lu, Ying; Liu, Jing; Inuzuka, Hiroyuki; Katon, Jesse M; Berry, Kelsey; Fung, Jacqueline; Ng, Christopher; Liu, Pengda; Song, Min Sup; Xue, Lian; Bronson, Roderick T; Kirschner, Marc W; Cui, Rutao; Pandolfi, Pier Paolo; Wei, Wenyi

2017-04-01

BRAF drives tumorigenesis by coordinating the activation of the RAS/RAF/MEK/ERK oncogenic signaling cascade. However, upstream pathways governing BRAF kinase activity and protein stability remain undefined. Here, we report that in primary cells with active APC FZR1 , APC FZR1 earmarks BRAF for ubiquitination-mediated proteolysis, whereas in cancer cells with APC-free FZR1, FZR1 suppresses BRAF through disrupting BRAF dimerization. Moreover, we identified FZR1 as a direct target of ERK and CYCLIN D1/CDK4 kinases. Phosphorylation of FZR1 inhibits APC FZR1 , leading to elevation of a cohort of oncogenic APC FZR1 substrates to facilitate melanomagenesis. Importantly, CDK4 and/or BRAF/MEK inhibitors restore APC FZR1 E3 ligase activity, which might be critical for their clinical effects. Furthermore, FZR1 depletion cooperates with AKT hyperactivation to transform primary melanocytes, whereas genetic ablation of Fzr1 synergizes with Pten loss, leading to aberrant coactivation of BRAF/ERK and AKT signaling in mice. Our findings therefore reveal a reciprocal suppression mechanism between FZR1 and BRAF in controlling tumorigenesis. Significance: FZR1 inhibits BRAF oncogenic functions via both APC-dependent proteolysis and APC-independent disruption of BRAF dimers, whereas hyperactivated ERK and CDK4 reciprocally suppress APC FZR1 E3 ligase activity. Aberrancies in this newly defined signaling network might account for BRAF hyperactivation in human cancers, suggesting that targeting CYCLIN D1/CDK4, alone or in combination with BRAF/MEK inhibition, can be an effective anti-melanoma therapy. Cancer Discov; 7(4); 424-41. ©2017 AACR. See related commentary by Zhang and Bollag, p. 356 This article is highlighted in the In This Issue feature, p. 339 . ©2017 American Association for Cancer Research.

Non-Aggregating Tau Phosphorylation by Cyclin-Dependent Kinase 5 Contributes to Motor Neuron Degeneration in Spinal Muscular Atrophy

PubMed Central

Miller, Nimrod; Feng, Zhihua; Edens, Brittany M.; Yang, Ben; Shi, Han; Sze, Christie C.; Hong, Benjamin Taige; Su, Susan C.; Cantu, Jorge A.; Topczewski, Jacek; Crawford, Thomas O.; Ko, Chien-Ping; Sumner, Charlotte J.; Ma, Long

2015-01-01

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35−/− compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration. PMID:25878277

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

HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

PubMed

Tommasino, M; Adamczewski, J P; Carlotti, F; Barth, C F; Manetti, R; Contorni, M; Cavalieri, F; Hunt, T; Crawford, L

1993-01-01

E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

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.

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

Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways.

PubMed

Li, Hui; Yang, Duxiao; Ning, Shanglei; Xu, Yinghui; Yang, Fan; Yin, Rusha; Feng, Taihu; Han, Shouqing; Guo, Lu; Zhang, Pengju; Qu, Wenjie; Guo, Renbo; Song, Chen; Xiao, Peng; Zhou, Chengjun; Xu, Zhigang; Sun, Jin-Peng; Yu, Xiao

2018-01-01

The protein tyrosine phosphatase nonreceptor type 12 (PTPN12) is a multifunctional protein and has elicited much research attention because its decreased protein level has been associated with poor prognosis of several types of cancers. Recently, we have solved the crystal structure of the phosphatase domain of PTPN12, which disclosed a specific PTPN12-insert-loop harboring a cyclin-dependent kinase 2 (CDK2) phosphorylation site. However, the functional significance of this phosphorylation is undefined. In the present study, we found that S19 site phosphorylation of PTPN12 by CDK2 discharged its antitumor activity by down-regulation of its inhibitory role in cell migration, but not affecting its other regulatory functions. Phosphorylation of PTPN12 at the S19 site changed its substrate interface, and by doing so, selectively decreased its activity toward the human epidermal growth factor receptor 2 (HER2)- pY 1196 site, but not other HER2 phosphorylation sites or other known PTPN12 substrates. A further in-depth mechanism study revealed that the phosphorylation of PTPN12 by CDK2 impaired recruitment of the serine/threonine-protein kinase 1 (PAK1) to HER2, resulted in the blockade of the HER2-pY 1196 -PAK1-T 423 signaling pathway, thus increased tumor cell motility. Taken together, our results identified a new phosphorylation-based substrate recognition mechanism of PTPN12 by CDK2, which orchestrated signaling crosstalk between the oncogenic CDK2 and HER2 pathways. The newly identified governing mechanism of the substrate selectivity of a particular phosphatase was previously unappreciated and exemplifies how a phospho-network is precisely controlled in different cellular contexts.-Li, H., Yang, D., Ning, S., Xu, Y., Yang, F., Yin, R., Feng, T., Han, S., Guo, L., Zhang, P., Qu, W., Guo, R., Song, C., Xiao, P., Zhou, C., Xu, Z., Sun, J.-P., Yu, X. Switching of the substrate specificity of protein tyrosine phosphatase N12 by cyclin-dependent kinase 2 phosphorylation orchestrating 2 oncogenic pathways. © FASEB.

Alterations of cyclin-dependent kinase 4 inhibitor (p16INK4A/MTS1) gene structure and expression in acute lymphoblastic leukemias.

PubMed

Delmer, A; Tang, R; Senamaud-Beaufort, C; Paterlini, P; Brechot, C; Zittoun, R

1995-07-01

The cyclin-dependent kinase 4 (cdk4) inhibitor (p16INK4/MTS1/CDKN2) gene has been recently identified as a putative tumor suppressor gene because of the high frequency of homozygous deletion observed in numerous human tumor cell lines, including leukemias. However, results obtained from uncultured tumor samples have led to discussion of the relevance of these findings. Using reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis, we have investigated p16INK4A gene at both RNA and genomic levels in various types of leukemias: acute myeloid leukemia (AML) (n = 23); acute lymphocytic leukemia (ALL) (n = 22) and B cell chronic lymphoproliferative disorders (CLPD) (n = 33). p16INK4A mRNA expression was not found in only 1/20 AML and 2/23 CLPD samples. Conversely, p16INK4A mRNA was not detected in 5/17 ALL cases, and intensity of PCR products were barely detectable in seven additional cases, possibly related to the contamination by normal cells in some cases. By Southern blotting, a homozygous deletion of p16INK4A gene was found in 6/17 ALL cases (35%) among which 4/6 were negative or weakly positive by RT-PCR assay. None of the five AML and 20 CLL samples studied had p16INK4A deletion. Sequence analysis of p16INK4A exon 2 did not show point mutation in two of these cases lacking mRNA expression. Our data provide further evidence that among hematological malignancies, ALL are the most likely to be associated with p16INK4A inactivation, mainly by homozygous gene deletion. Since most hematological malignancies-except ALL-are infrequently associated with p16INK4A and retinoblastoma (Rb) gene alteration it seems worthwhile to explore cdk4 and cdk6 expression to determine whether or not the disruption of the p16INK4A/Rb/cdk4/cdk6 regulatory loop might play a role in their pathogenesis.

Conserved tetramer junction in the kinetochore Ndc80 complex

PubMed Central

Valverde, Roberto; Ingram, Jessica; Harrison, Stephen C.

2016-01-01

Summary The heterotetrameric Ndc80 complex establishes connectivity along the principal longitudinal axis of a kinetochore. Its two heterodimeric subcomplexes, each with a globular end and a coiled-coil shaft, connect end-to-end to create a ∼600 Å long rod spanning the gap from centromere-proximal structures to spindle microtubules. Neither subcomplex has a known function on its own, but the heterotetrameric organization and the characteristics of the junction are conserved from yeast to man. We have determined crystal structures of two shortened (“dwarf”) Ndc80 complexes that contain the full tetramer junction and both globular ends. The junction connects two α-helical coiled coils through regions of four-chain and three-chain overlap. The complexity of its structure depends on interactions among conserved amino-acid residues, suggesting a binding site for additional cellular factor(s) not yet identified. PMID:27851957

MiRNA-124 is a link between measles virus persistent infection and cell division of human neuroblastoma cells.

PubMed

Naaman, Hila; Rall, Glenn; Matullo, Christine; Veksler-Lublinsky, Isana; Shemer-Avni, Yonat; Gopas, Jacob

2017-01-01

Measles virus (MV) infects a variety of lymphoid and non-lymphoid peripheral organs. However, in rare cases, the virus can persistently infect cells within the central nervous system. Although some of the factors that allow MV to persist are known, the contribution of host cell-encoded microRNAs (miRNA) have not been described. MiRNAs are a class of noncoding RNAs transcribed from genomes of all multicellular organisms and some viruses, which regulate gene expression in a sequence-specific manner. We have studied the contribution of host cell-encoded miRNAs to the establishment of MV persistent infection in human neuroblastoma cells. Persistent MV infection was accompanied by differences in the expression profile and levels of several host cell-encoded microRNAs as compared to uninfected cells. MV persistence infection of a human neuroblastoma cell line (UKF-NB-MV), exhibit high miRNA-124 expression, and reduced expression of cyclin dependent kinase 6 (CDK6), a known target of miRNA-124, resulting in slower cell division but not cell death. By contrast, acute MV infection of UKF-NB cells did not result in increased miRNA-124 levels or CDK6 reduction. Ectopic overexpression of miRNA-124 affected cell viability only in UKF-NB-MV cells, causing cell death; implying that miRNA-124 over expression can sensitize cells to death only in the presence of MV persistent infection. To determine if miRNA-124 directly contributes to the establishment of MV persistence, UKF-NB cells overexpressing miRNA-124 were acutely infected, resulting in establishment of persistently infected colonies. We propose that miRNA-124 triggers a CDK6-dependent decrease in cell proliferation, which facilitates the establishment of MV persistence in neuroblastoma cells. To our knowledge, this is the first report to describe the role of a specific miRNA in MV persistence.

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription

PubMed Central

2014-01-01

Background Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. Results To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. Conclusions This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription. PMID:24985467

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription.

PubMed

Kuzmina, Alona; Verstraete, Nina; Galker, Sigal; Maatook, Maayan; Bensaude, Olivier; Taube, Ran

2014-07-01

Human immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II. To map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus. This study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.

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

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

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

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.

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

PubMed

Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

2012-09-01

We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Marquez, Maribel P; Alencastro, Frances; Madrigal, Alma; Jimenez, Jossue Loya; Blanco, Giselle; Gureghian, Alex; Keagy, Laura; Lee, Cecilia; Liu, Robert; Tan, Lun; Deignan, Kristen; Armstrong, Brian; Zhao, Yuanxiang

2017-11-01

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Resveratrol induces cell cycle arrest and apoptosis in human eosinophils from asthmatic individuals.

PubMed

Hu, Xin; Wang, Jing; Xia, Yu; Simayi, Mihereguli; Ikramullah, Syed; He, Yuanbing; Cui, Shihong; Li, Shuang; Wushouer, Qimanguli

2016-12-01

Eosinophils exert a number of inflammatory effects through the degranulation and release of intracellular mediators, and are considered to be key effector cells in allergic disorders, including asthma. In order to investigate the regulatory effects of the natural polyphenol, resveratrol, on eosinophils derived from asthmatic individuals, the cell counting Kit‑8 assay and flow cytometry analysis were used to determine cell proliferation and cell cycle progression in these cells, respectively. Cellular apoptosis was detected using annexin V-fluorescein isothiocyanate/propidium iodide double‑staining. The protein expression levels of p53, p21, cyclin‑dependent kinase 2 (CDK2), cyclin A, cyclin E, Bim, B‑cell lymphoma (Bcl)‑2 and Bcl‑2‑associated X protein (Bax) were measured by western blot analysis following resveratrol treatment. The results indicated that resveratrol effectively suppressed the proliferation of eosinophils from asthmatic patients in a concentration‑ and time‑dependent manner. In addition, resveratrol was observed to arrest cell cycle progression in G1/S phase by increasing the protein expression levels of p53 and p21, and concurrently reducing the protein expression levels of CDK2, cyclin A and cyclin E. Furthermore, resveratrol treatment significantly induced apoptosis in eosinophils, likely through the upregulation of Bim and Bax protein expression levels and the downregulation of Bcl‑2 protein expression. These findings suggested that resveratrol may be a potential agent for the treatment of asthma by decreasing the number of eosinophils.

A novel 3p22.3 gene CMTM7 represses oncogenic EGFR signaling and inhibits cancer cell growth.

PubMed

Li, H; Li, J; Su, Y; Fan, Y; Guo, X; Li, L; Su, X; Rong, R; Ying, J; Mo, X; Liu, K; Zhang, Z; Yang, F; Jiang, G; Wang, J; Zhang, Y; Ma, D; Tao, Q; Han, W

2014-06-12

Deletion of 3p12-22 is frequent in multiple cancer types, indicating the presence of critical tumor-suppressor genes (TSGs) at this region. We studied a novel candidate TSG, CMTM7, located at the 3p22.3 CMTM-gene cluster, for its tumor-suppressive functions and related mechanisms. The three CMTM genes, CMTM6, 7 and 8, are broadly expressed in human normal adult tissues and normal epithelial cell lines. Only CMTM7 is frequently silenced or downregulated in esophageal and nasopharyngeal cell lines, but uncommon in other carcinoma cell lines. Immunostaining of tissue microarrays for CMTM7 protein showed its downregulation or absence in esophageal, gastric, pancreatic, liver, lung and cervix tumor tissues. Promoter CpG methylation and loss of heterozygosity were both found contributing to CMTM7 downregulation. Ectopic expression of CMTM7 in carcinoma cells inhibits cell proliferation, motility and tumor formation in nude mice, but not in immortalized normal cells, suggesting a tumor inhibitory role of CMTM7. The tumor-suppressive function of CMTM7 is associated with its role in G1/S cell cycle arrest, through upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6). Moreover, CMTM7 could promote epidermal growth factor receptor (EGFR) internalization, and further suppress AKT signaling pathway. Thus, our findings suggest that CMTM7 is a novel 3p22 tumor suppressor regulating G1/S transition and EGFR/AKT signaling during tumor pathogenesis.

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.

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis.

PubMed

Jarry, Marie; Lecointre, Céline; Malleval, Céline; Desrues, Laurence; Schouft, Marie-Thérèse; Lejoncour, Vadim; Liger, François; Lyvinec, Gildas; Joseph, Benoît; Loaëc, Nadège; Meijer, Laurent; Honnorat, Jérôme; Gandolfo, Pierrick; Castel, Hélène

2014-11-01

Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Impact of meriolins, a new class of cyclin-dependent kinase inhibitors, on malignant glioma proliferation and neo-angiogenesis

PubMed Central

Jarry, Marie; Lecointre, Céline; Malleval, Céline; Desrues, Laurence; Schouft, Marie-Thérèse; Lejoncour, Vadim; Liger, François; Lyvinec, Gildas; Joseph, Benoît; Loaëc, Nadège; Meijer, Laurent; Honnorat, Jérôme; Gandolfo, Pierrick; Castel, Hélène

2014-01-01

Background Glioblastomas are the most frequent and most aggressive primary brain tumors in adults. The median overall survival is limited to a few months despite surgery, radiotherapy, and chemotherapy. It is now clearly established that hyperactivity of cyclin-dependent kinases (CDKs) is one of the processes underlying hyperproliferation and tumoral growth. The marine natural products meridianins and variolins, characterized as CDK inhibitors, display a kinase-inhibitory activity associated with cytotoxic effects. In order to improve selectivity and efficiency of these CDK inhibitors, a series of hybrid compounds called meriolins have been synthesized. Methods The potential antitumoral activity of meriolins was investigated in vitro on glioma cell lines (SW1088 and U87), native neural cells, and a human endothelial cell line (HUV-EC-C). The impact of intraperitoneal or intratumoral administrations of meriolin 15 was evaluated in vivo on 2 different nude mice-xenografted glioma models. Results Meriolins 3, 5, and 15 exhibited antiproliferative properties with nanomolar IC50 and induced cell-cycle arrest and CDK inhibition associated with apoptotic events in human glioma cell lines. These meriolins blocked the proliferation rate of HUV-EC-C through cell cycle arrest and apoptosis. In vivo, meriolin 15 provoked a robust reduction in tumor volume in spite of toxicity for highest doses, associated with inhibition of cell division, activation of caspase 3, reduction of CD133 cells, and modifications of the vascular architecture. Conclusion Meriolins, and meriolin 15 in particular, exhibit antiproliferative and proapoptotic activities on both glioma and intratumoral endothelial cells, constituting key promising therapeutic lead compounds for the treatment of glioblastoma. PMID:24891448

Using open source computational tools for predicting human metabolic stability and additional absorption, distribution, metabolism, excretion, and toxicity properties.

PubMed

Gupta, Rishi R; Gifford, Eric M; Liston, Ted; Waller, Chris L; Hohman, Moses; Bunin, Barry A; Ekins, Sean

2010-11-01

Ligand-based computational models could be more readily shared between researchers and organizations if they were generated with open source molecular descriptors [e.g., chemistry development kit (CDK)] and modeling algorithms, because this would negate the requirement for proprietary commercial software. We initially evaluated open source descriptors and model building algorithms using a training set of approximately 50,000 molecules and a test set of approximately 25,000 molecules with human liver microsomal metabolic stability data. A C5.0 decision tree model demonstrated that CDK descriptors together with a set of Smiles Arbitrary Target Specification (SMARTS) keys had good statistics [κ = 0.43, sensitivity = 0.57, specificity = 0.91, and positive predicted value (PPV) = 0.64], equivalent to those of models built with commercial Molecular Operating Environment 2D (MOE2D) and the same set of SMARTS keys (κ = 0.43, sensitivity = 0.58, specificity = 0.91, and PPV = 0.63). Extending the dataset to ∼193,000 molecules and generating a continuous model using Cubist with a combination of CDK and SMARTS keys or MOE2D and SMARTS keys confirmed this observation. When the continuous predictions and actual values were binned to get a categorical score we observed a similar κ statistic (0.42). The same combination of descriptor set and modeling method was applied to passive permeability and P-glycoprotein efflux data with similar model testing statistics. In summary, open source tools demonstrated predictive results comparable to those of commercial software with attendant cost savings. We discuss the advantages and disadvantages of open source descriptors and the opportunity for their use as a tool for organizations to share data precompetitively, avoiding repetition and assisting drug discovery.

The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

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

Teng, Chih-Chuan; Institute of Basic Medicine Science, National Cheng Kung University, Tainan, Taiwan; Kuo, Hsing-Chun

2012-08-15

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0 μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr{sup 15} and Cdc25cSer{supmore » 216}. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer{sup 216} expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer{sup 216} in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer{sup 216} cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells. -- Highlights: ► We show the effects of CIL-102 on the G2/M arrest of human astrocytoma cells. ► ROS and the Ras/ERK1/2 triggering pathways are involved in the CIL-102 treatment. ► CIL-102 induces sustained activation of ERK1/2 and Cdc25c and ROS are required.« less

Novel ent-Kaurane Diterpenoid from Rubus corchorifolius L. f. Inhibits Human Colon Cancer Cell Growth via Inducing Cell Cycle Arrest and Apoptosis.

PubMed

Chen, Xuexiang; Wu, Xian; Ouyang, Wen; Gu, Min; Gao, Zili; Song, Mingyue; Chen, Yunjiao; Lin, Yanyin; Cao, Yong; Xiao, Hang

2017-03-01

The tender leaves of Rubus corchorifolius L. f. have been consumed as tea for drinking in China since ancient times. In this study, a novel ent-kaurane diterpenoid was isolated and identified from R. corchorifolius L. f. leaves as ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK). DEK suppressed the growth of HCT116 human colon cancer cells with an IC 50 value of 40 ± 0.21 μM, while it did not cause significant growth inhibition on CCD-18Co human colonic myofibroblasts at up to100 μM. Moreover, DEK induced extensive apoptosis and S phase cell cycle arrest in the colon cancer cells. Accordingly, DEK caused profound effects on multiple signaling proteins associated with cell proliferation, cell death, and inflammation. DEK significantly upregulated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, cleaved caspase-9, cleaved PARP, p53, Bax, and tumor suppressor p21 Cip1/Waf1 , downregulated the levels of cell cycle regulating proteins such as cyclinD1, CDK2, and CDK4 and carcinogenic proteins such as EGFR and COX-2, and suppressed the activation of Akt. Overall, our results provide a basis for using DEK as a potential chemopreventive agent against colon carcinogenesis.

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.

Circular RNA HIPK3 promotes gallbladder cancer cell growth by sponging microRNA-124.

PubMed

Kai, Ding; Yannian, Liao; Yitian, Chen; Dinghao, Gong; Xin, Zhao; Wu, Ji

2018-06-18

Recent studies have implied that circHIPK3, an abundant circular RNA (circRNA), participates in tumorigenesis and cancer progression. Its expression and potential functions in human gallbladder cancer were examined in this study. We show that circHIPK3 is upregulated in human gallbladder cancer cells. But its level is low in gallbladder epithelial cells. circHIPK3 silencing by targeted siRNA potently inhibited survival and proliferation of established and primary human gallbladder cancer cells, while inducing cell apoptosis. Conversely, ectopic over-expression of circHIPK3 can further promote cancer cell proliferation. In gallbladder cancer cells, circHIPK3 sponged the tumor-suppressive microRNA-124 (miR-124) to sequester and inhibit its activity, thereby leading to increased expression of miR-124 targets, including ROCK1 (rho-associated protein kinase 1) and CDK6 (rho-associated protein kinase). Ectopic over-expression of miR-124 b y a lentiviral vector mimicked and abolished actions by circHIPK3 siRNA in gallbladder cancer cells. At last, we show that circHIPK3 is upregulated in human gallbladder cancer tissues, which is correlated with miR-124 downregulation and ROCK1-CDK6 upregulation. Together, we conclude that circHIPK3 promotes gallbladder cancer cell growth possibly by sponging miR-124. The over-expressed circHIPK3 could be a novel therapeutic target and diagnosis marker of human gallbladder cancer. Copyright © 2018. Published by Elsevier Inc.

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.

Sperm associated antigen 9 (SPAG9) a promising therapeutic target of ovarian carcinoma.

PubMed

Jagadish, Nirmala; Fatima, Rukhsar; Sharma, Aditi; Devi, Sonika; Suri, Vitusha; Kumar, Vikash; Suri, Anil

2018-05-01

SPAG9 is a novel tumor associated antigen, expressed in variety of malignancies. However, its role in ovarian cancer remains unexplored. SPAG9 expression was validated in ovarian cancer cells by real time PCR and Western blot. SPAG9 involvement in cell cycle, DNA damage, apoptosis, paclitaxel sensitivity and epithelial- mesenchymal transition (EMT) was investigated employing RNA interference approach. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro. Quantitative PCR and Western blot analysis revealed SPAG9 expression in A10, SKOV-3 and Caov3 compared to normal ovarian epithelial cells. SPAG9 ablation resulted in reduced cellular proliferation, colony forming ability and enhanced cytotoxicity of chemotherapeutic agent paclitaxel. Effect of ablation of SPAG9 on cell cycle revealed S phase arrest and showed decreased expression of CDK1, CDK2, CDK4, CDK6, cyclin B1, cyclin D1, cyclin E and increased expression of tumor suppressor p21. Ablation of SPAG9 also resulted in increased apoptosis with increased expression of various pro- apoptotic molecules including BAD, BID, PUMA, caspase 3, caspase 7, caspase 8 and cytochrome C. Decreased expression of mesenchymal markers and increased expression of epithelial markers was found in SPAG9 ablated cells. Combinatorial effect of SPAG9 ablation and paclitaxel treatment was evaluated in in vitro assays which showed that ablation of SPAG9 resulted in increased paclitaxel sensitivity and caused enhanced cell death. In vivo ovarian cancer xenograft studies showed that ablation of SPAG9 resulted in significant reduction in tumor growth. Present study revealed therapeutic potential of SPAG9 in ovarian cancer.

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.

Polo-like kinase 1 inhibits DNA damage response during mitosis

PubMed Central

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis. PMID:25607646

Polo-like kinase 1 inhibits DNA damage response during mitosis.

PubMed

Benada, Jan; Burdová, Kamila; Lidak, Tomáš; von Morgen, Patrick; Macurek, Libor

2015-01-01

In response to genotoxic stress, cells protect their genome integrity by activation of a conserved DNA damage response (DDR) pathway that coordinates DNA repair and progression through the cell cycle. Extensive modification of the chromatin flanking the DNA lesion by ATM kinase and RNF8/RNF168 ubiquitin ligases enables recruitment of various repair factors. Among them BRCA1 and 53BP1 are required for homologous recombination and non-homologous end joining, respectively. Whereas mechanisms of DDR are relatively well understood in interphase cells, comparatively less is known about organization of DDR during mitosis. Although ATM can be activated in mitotic cells, 53BP1 is not recruited to the chromatin until cells exit mitosis. Here we report mitotic phosphorylation of 53BP1 by Plk1 and Cdk1 that impairs the ability of 53BP1 to bind the ubiquitinated H2A and to properly localize to the sites of DNA damage. Phosphorylation of 53BP1 at S1618 occurs at kinetochores and in cytosol and is restricted to mitotic cells. Interaction between 53BP1 and Plk1 depends on the activity of Cdk1. We propose that activity of Cdk1 and Plk1 allows spatiotemporally controlled suppression of 53BP1 function during mitosis.

Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

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

Orba, Yasuko; Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, 060-8638, Sapporo; Research Fellow of the Japan Society for the Promotion of Science

2008-01-05

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

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.

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

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.

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).

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.

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.

Structure guided inhibitor designing of CDK2 and discovery of potential leads against cancer.

PubMed

Kumar, Arun V A; Mohan, Keshav; Riyaz, Syed

2013-09-01

On the basis of stereo specific information obtained from crystal structures of CDK2, indole and chromene analogues were designed by suitably substituting the pharmacophores on their moiety and docked with target protein for calculating binding affinities. The binding affinities are represented in glide score. (5E)-5-[(1-methyl-1H-indol-3-yl)methylidene]-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I1), (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I2) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C9) were selected for synthesis and biological testing based on vital interactions. (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide(I2) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C9) were proved to be active against MCF-7 and HeLa cell lines.

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.

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.

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.

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

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.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

PubMed Central

Baker, Emma K; Taylor, Scott; Gupte, Ankita; Sharp, Phillip P; Walia, Mannu; Walsh, Nicole C; Zannettino, Andrew CW; Chalk, Alistair M; Burns, Christopher J; Walkley, Carl R

2015-01-01

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS. PMID:25944566

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

PubMed Central

Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

2012-01-01

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

Surface Induced Dissociation Coupled with High Resolution Mass Spectrometry Unveils Heterogeneity of a 211 kDa Multicopper Oxidase Protein Complex

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

Zhou, Mowei; Yan, Jing; Romano, Christine A.

Manganese oxidation is an important biogeochemical process that is largely regulated by bacteria through enzymatic reactions. However, the detailed mechanism is poorly understood due to challenges in isolating and characterizing these unknown enzymes. A manganese oxidase Mnx from Bacillus sp. PL-12 has been successfully overexpressed in active form, unexpectedly, as a protein complex with a molecular weight of 211 kDa with no homology to known proteins in the database. We have recently used surface induced dissociation (SID) and ion mobility – mass spectrometry (IM-MS) to release and detect folded subcomplexes for determining subunit connectivity and quaternary structure. The data frommore » the native mass spectrometry experiment led to a plausible model of this unknown multicopper oxidase which has been difficult to study by conventional structural biology methods. However, because each subunit of Mnx binds copper ions as cofactor at varying ratios, there were remaining ambiguities in assigning some of the observed peaks to metal-binding species because of the sample heterogeneity and limited mass resolution. In this study, we performed SID in a modified Fourier transform – ion cyclotron resonance (FT-ICR) mass spectrometer for obtaining the ultimate resolution on the released subcomplexes of Mnx. The high mass accuracy and resolution unveiled unexpected artificial modifications in the protein that have been previously thought to be iron bound species based on lower resolution data. Additionally, most released subcomplexes were isotopically resolved for defining metal binding stoichiometry at each structural level. This method holds great potential for in-depth characterization of metalloproteins and protein-ligand complexes.« less

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

PubMed Central

Madina, Bhaskara R.; Kumar, Vikas; Metz, Richard; Mooers, Blaine H.M.; Bundschuh, Ralf; Cruz-Reyes, Jorge

2014-01-01

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs. PMID:24865612

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

PubMed

Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

2016-10-01

Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

Cyclin B in mouse oocytes and embryos: importance for human reproduction and aneuploidy.

PubMed

Polański, Zbigniew; Homer, Hayden; Kubiak, Jacek Z

2012-01-01

Oocyte maturation and early embryo development require precise coordination between cell cycle progression and the developmental programme. Cyclin B plays a major role in this process: its accumulation and degradation is critical for driving the cell cycle through activation and inactivation of the major cell cycle kinase, CDK1. CDK1 activation is required for M-phase entry whereas its inactivation leads to exit from M-phase. The tempo of oocyte meiotic and embryonic mitotic divisions is set by the rate of cyclin B accumulation and the timing of its destruction. By controlling when cyclin B destruction is triggered and by co-ordinating this with the completion of chromosome alignment, the spindle assembly checkpoint (SAC) is a critical quality control system important for averting aneuploidy and for building in the flexibility required to better integrate cell cycle progression with development. In this review we focus on cyclin B metabolism in mouse oocytes and embryos and illustrate how the cell cycle-powered clock (in fact cyclin B-powered clock) controls oocyte maturation and early embryo development, thereby providing important insight into human reproduction and potential causes of Down syndrome.

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

PubMed

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

2016-01-27

Aiming to develop a new target for the anticancer treatment, a series of 5'H-spiro[indoline-3,4'-pyrrolo [1,2-a]quinoxalin]-2-ones has been synthesized by simple, highly efficient and environmentally friendly method in excellent yields under catalyst-free conditions using ethanol as a green solvent. A simple filtration of the reaction mixture and subsequent drying affords analytically pure products. The synthesized derivatives were evaluated for their antiproliferative activity against five different human cancer cell lines, among the congeners compound 3n showed significant cytotoxicity against the human prostate cancer (DU-145). Flow cytometric analysis revealed that this compound induces cell cycle arrest in the G0/G1 phase and Western blot analysis suggested that reduction in Cdk4 expression level leads to apoptotic cell death. This was further confirmed by mitochondrial membrane potential ((ΔΨm), Annexin V-FITC assay and docking experiments. Furthermore, it was observed that there is an increase in expression levels of cyclin dependent kinase inhibitors like Cip1/p21 and Kip1/p27. Copyright © 2015 Elsevier Masson SAS. 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.

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.

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.

Inheritance of yeast nuclear pore complexes requires the Nsp1p subcomplex

PubMed Central

Makio, Tadashi; Lapetina, Diego L.

2013-01-01

In the yeast Saccharomyces cerevisiae, organelles and macromolecular complexes are delivered from the mother to the emerging daughter during cell division, thereby ensuring progeny viability. Here, we have shown that during mitosis nuclear pore complexes (NPCs) in the mother nucleus are actively delivered through the bud neck and into the daughter cell concomitantly with the nuclear envelope. Furthermore, we show that NPC movement into the daughter cell requires members of an NPC subcomplex containing Nsp1p and its interacting partners. NPCs lacking these nucleoporins (Nups) were blocked from entry into the daughter by a putative barrier at the bud neck. This selection process could be observed within individual cells such that NPCs containing Nup82p (an Nsp1p-interacting Nup) were transferred to the daughter cells while functionally compromised NPCs lacking Nup82p were retained in the mother. This mechanism is proposed to facilitate the inheritance of functional NPCs by daughter cells. PMID:24165935

The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy

NASA Astrophysics Data System (ADS)

Rao, Yijian; Perna, Marco G.; Hofmann, Benjamin; Beier, Viola; Wollert, Thomas

2016-01-01

Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology.

PubMed

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-05-21

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

PubMed Central

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-01-01

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture. DOI: http://dx.doi.org/10.7554/eLife.06265.001 PMID:25997101

Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex.

PubMed

Qi, Shiqian; Kim, Do Jin; Stjepanovic, Goran; Hurley, James H

2015-10-06

The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

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.

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.

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.

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.

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

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.